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Immunobiology (MCDB) 430/530: Fall 2012 Biology of the Immune System

Lectures in KBT 1214, MWF 9:25 - 10:15 AM Information for Students
Who should take this course? This course is intended to provide a detailed survey of current knowledge of the immune system for advanced undergraduates with a serious commitment to biology and for graduate students in Immunobiology and related programs. Prior knowledge of cell biology and biochemistry is absolutely necessary; Biology (MCDB) 300a provides a good basis. Students who believe that they have equivalent knowledge may take the course, but prior discussion with the course organizer is required. Course content: The course consists of lectures, tutorials, review sessions, and assigned reading material. We provide a syllabus with an outline of the individual lectures. Each lecture is accompanied by assigned reading material. In addition, the students are expected to know the experimental systems (techniques) routinely used to study the immune system as detailed below. All handouts and reading assignments plus supplemental items of interest can be found at the Yale website http://classesv2.yale.edu. Grades: The course grade is based on a mid-term exam (30% of the grade), take-home questions (20% of the grade), participation in tutorial sessions (10% of the grade), and a final exam (40% of the grade). The questions are designed to test the understanding and application of key immunological concepts. Reasoning as well as recall is required. You will also participate in small group tutorial sessions in which primary research articles and questions will be presented and discussed. These sessions will be oriented to discussion and analysis of primary literature, as well as review of the information provided in the lectures. Teachers: The course is taught by the faculty of the Department of Immunobiology. Three Teaching Fellows will assist in the class. They will lead the tutorial as well as the review sessions. Textbook: "Janeway’s Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC.), 8th Edition, 2011 is required for this course. This textbook is available at the Barnes & Noble Bookstore on Broadway. Reading Material: In addition to material covered in the lecture, the assigned readings in “Immunobiology. The Immune System in Health and Disease” are required and will be covered on the examinations. Given the problem solving nature of the course, the students will be expected to know the experimental systems used to study the immune system. These include immunochemical techniques (ELISA, immunoblotting, immunohistochemistry, immunoprecipitation, anti-immunoglobulins, use of antibodies to isolate and identify proteins and genes, monoclonal antibodies); cell isolation techniques (flow cytometry, FACS); manipulation of the immune system (adoptive transfer, cell depletion in vivo, gene knockout, transgenic mice; and analysis of response (RNA expression, microarrays, proliferation, etc.). Information about these experimental procedures is found in Appendix I of the textbook. Course Director: Carla Rothlin, Ph.D. TAC, S624, Phone: 737-4679 carla.rothlin@yale.edu

Teaching Fellows: Will Khoury-Hanold william.khoury-hanold@yale.edu Omotooke Arojo omotooke.arojo@yale.edu Tianxia Guan tianxia.guan@yale.edu

MCDB 430/530 Lecture Schedule & Syllabus
Fall 2012
August 29 August 31 Why Study Immunology? Structure of the Immune system I Rothlin Rothlin

September 3: Labor Day (no class) September 5 September 7 Structure of the Immune System II Receptors and Signaling I Rothlin Rothlin

Evening Tutorial 1: Primary literature discussion – Methods in immunology (week September 3-7) September 10 September 12 September 14 September 17 September 19 Innate Immune System Pattern Recognition Receptors Complement, NK Cells and Acute Phase Responses Innate Immunity to Bacteria, Fungi and Parasites Innate Anti-viral Immunity Iwasaki Medzhitov Medzhitov Medzhitov Medzhitov

Evening Tutorial 2: Primary literature discussion – Innate Immunity (week September 17-21) September 21 September 24 September 26 September 28 Structure and Genetics of MHC Molecules MHC Class I Restricted Antigen Processing MHC Class II Restricted Antigen Processing Dendritic Cells and their Functions/CD1 Molecules Cresswell Cresswell Cresswell Cresswell

Evening Tutorial 3: Primary literature discussion – Antigen presentation (week October 1-5) October 1 October 3 October 5 October 8 October 10 B & T Cell Receptors: Structure and Classes Immunoglobulin and TCR Gene Structure & Rearrangement B & T Cell Development Positioning, Maturation & Trafficking of Lymphocytes Receptors and Signaling II Schatz Schatz Schatz Pereira Rothlin

Evening Tutorial 4: Evening review session for midterm exam (week October 8-12) October 12 MIDTERM EXAM (on materials covered up to Oct. 8th lecture)

October 15 October 17 October 19 October 22

Innate Control and Initiation of Adaptive Immune Responses T Cell Priming and Effector Cell Differentiation I T Cell Priming and Effector Cell Differentiation II Effector Molecules (Function and Signaling)

Craft Craft Craft Craft

Tuesday October 23rd –RECESS BEGINS at 5:30 PM Monday, October 29th – CLASSES RESUME

October 29 October 31 November 2

Primary B Cell Responses Mechanisms of Somatic Hypermutation and Isotype Switch and Their Consequences Humoral Immune Responses

Shlomchik Shlomchik Shlomchik

Evening Tutorial 5: Primary literature discussion – Cell-mediated immunity (week October 29-2) November 5 November 7 November 9 B & T Cell Memory Vaccination Inflammation - Response to Infection Shlomchik Iwasaki Iwasaki

Evening Tutorial 6: Primary literature discussion – Humoral immunity (week November 5-9) November 12 November 14 November 16 Mucosal Immunity Transcriptional Regulation in the Immune System Primary and Acquired Immunodeficiencies Iwasaki Chi Meffre

Evening Tutorial 7: Primary literature discussion – Immunity and infection (week November 12-16) Friday, November 16 – RECESS BEGINS at 5:30 PM Monday, November 26 – CLASSES RESUME November 26 November 28 November 30 December 3 Allergy Peripheral Tolerance Autoimmunity Transplantation and Cancer Immunology Medzhitov Herold Herold Herold

Evening Tutorial 8: Primary literature discussion – The immune system in health and disease (week Dec. 3-7) December 5 December 7 Unresolved Questions in Immunology Review session for final exam (in class) Medzhitov The TFs

Friday, December 7 – CLASSES END Thursday, December 13 – FINAL EXAMS BEGIN Tuesday, December 18 – FINAL EXAMS END

Appendix I: The Immunologist’s Toolbox Evening Tutorial 2: Week of September 17 . Review your class notes and bring questions. but also allow for review of class material. Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. vol. 31(2). D. 294(5545). 303(5664). Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Evening Tutorial 6: Week of November 5 . Nature (2011) vol. et al. Journal of Clinical Investigation (2003) vol. 682-87. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Restoring function in exhausted CD8 T cells during chronic viral infection Nature (2006). et al. Defective antigen processing in GILT-free mice.Evening Tutorial Schedule & Required Readings Evening tutorials are held throughout the semester. Evening Tutorial 7: Week of November 12 . as well as students’ questions.21: Innate immunity tutorial Required reading: Sander et al. pp385-9.7: Methods in immunology tutorial Required reading: Janeway’s Immunobiology. pp. pp. 1361-5.November 9: Humoral immunity tutorial Required reading: Gatto.December 7: The immune system in health and disease tutorial Required reading: Boitard. . vol. 259-69. et al. 474(7351). pp. Evening Tutorial 5: Week of October 29 . pp. Immunity (2009). Evening Tutorial 1: Week of September 3 . Tutorial grades (10% of the final grade) are based on participation and attendance. 851-57. 8th Edition. 111(6). 1662-65. These tutorials focus on a discussion of primary literature.October 12: Review for Midterm No required reading. vol. Science (2001) vol. Evening Tutorial 8: Week of December 3 . Evening Tutorial 3: Week of October 1 . et al. Science (2004). Readings will be posted on Classes*v2 prior to each tutorial.November 16: Immunity & infection tutorial Required reading: Macpherson.October 5: Antigen presentation tutorial Required reading: Maric et al. pp. 439(7077).November 2: Cell mediated immunity tutorial Required reading: Barber. Evening Tutorial 4: Week of October 8 . Acceleration of type 1 diabetes mellitus in proinsulin 2–deficient NOD mice.

a student cannot attend class on the day that a take-home question is due. every student must submit his or her answers by the end of the lecture (10:15 a. her or his answer may be emailed to their Teaching Fellow. However. Take-Home Question 1: Posted on September 19th Due end of class September 26th Take-Home Question 2: Posted October 3rd Due end of class October 10th Take-Home Question 3: Posted October 31st Due end of class November 7th Take-Home Question 4: Posted November 30th Due end of class December 7th . Answers are considered late beginning right after class on the due date and will have two points deducted per day late. there will be four graded take-home questions. Answers are to be typed and must not be longer than one page (one inch margins and 12 point “Times” font). each worth 5% of the final grade.Take-Home Question Schedule Throughout the semester.) on the due date. If.m. for any reason.

and introduce the teaching faculty who are responsible for covering each topic. Rothlin In this introductory lecture I will introduce the topics to be covered by this course. I will also discuss the importance of the immune system in health and disease.Why Study Immunology? August 29 Instructor: Carla V. Sit back. relax and enjoy! . grading schemes. course structure.

germline encoded receptors that recognize conserved and invariant features of microorganisms known as pathogen-associated molecular patterns (PAMPs). Lymphocytes are divided into two classes (T and B lymphocytes) with different recognition receptors and function. altering the behavior of the target cell. The innate immune response is needed to initiate and direct adaptive immunity. Phagocytes releasing chemokines can recruit additional cells to the area of infection to help in clearance. B cells express cell surface immunoglobulins. Importantly. neutrophils. Phagocytes become activated upon interaction with the pathogen. The adaptive immune response is mediated by the activation of lymphocytes. These are known as opportunistic pathogens. Phagocytes express various types of receptors including those for pathogen recognition (pattern recognition receptors. mucus. there are two types of immune recognition. The purpose of the immune system is to recognize invading microorganisms and to contain the infection or clear the microorganisms from the body. viruses. innate and adaptive immunity do not operate independently of each other. While the innate immune system operates early in a response to infection. anti-microbial peptides). The innate immune response. B cells . The mammalian immune response can be classified in three phases: immediate innate responses. fungi and parasites. adaptive immunity produces signals that stimulate and modulate innate immune responses. Adaptive immunity amplifies the protection provided by the innate immune response and provides future immunity to the same pathogen (memory/adaptive immunity). The first one consists of physical and chemical barriers (i. These components of the innate immune system contribute both to recognition of the pathogen and to effector responses. referred to as innate (evolutionarily ancient host defense system) and adaptive (vertebrate host defense system). One class of cytokines is called chemokines that have chemoattractant properties. the immune response consists of two main activities: recognition and effector functions. The adaptive immune response. Activated phagocytes secrete small proteins called cytokines that act by binding receptors on target cells. Activation of the innate immune response leads to the induction of adaptive immunity: some activated phagocytes mature and express molecules that will activate lymphocytes of the adaptive immune system. whereas T cells express T cell receptors. The genes encoding antigen receptors are assembled through somatic recombination from gene segments in the germ line. In vertebrates. For example. • Adaptive immune recognition operates by the clonal selection of individual lymphocytes expressing specific antigen receptors. PRRs) and for complement proteins to aid in pathogen clearance. phagocytes (macrophages.Structure of the Immune System I August 31 Instructor: Carla V. certain microorganisms are only pathogenic in immuno-compromised individuals. Invasion by a microorganism leads to a quick response by the innate immune system. It is important to recognize that the pathogenicity of a particular microorganism depends on the fitness of the host.: physical barriers formed by epithelial surfaces. For recognition. Mechanisms of host defense Multicellular organisms defend themselves against pathogens in multiple ways. Mechanisms of microbial recognition in vertebrates Microorganisms with the potential to cause diseases are called “pathogens”. and natural killer cells. allowing the generation of a diverse repertoire of receptors. changes in pH. The innate and adaptive responses are briefly described below. • Innate immune recognition operates by non-clonal. and dendritic cells). Conversely.e. The innate immune system includes complement proteins. induced innate responses and adaptive immunity. Rothlin Immunology is the study of mechanisms used to defend our body from invasion by microorganisms. the adaptive immune response requires time to develop. Therefore. There are four kinds of pathogens: bacteria.

or fail to be activated. This specialization in T cell function allows the immune system to respond to a wide variety of microorganisms. CD4 T cells can be further sub-divided into various types. by secreting antibodies can eliminate pathogens found extracellularly but not intracellularly. TFH and regulatory T cells that release unique panels of cytokines. Once B cells are activated. T cells can be divided into two major classes. Lymphocytes of the adaptive immune system recognize and respond to pathogens. while maintaining a balanced immune response. whereas Th2 cells induce antibody secretion and immunity against large parasites. they display various activities mediated by the release of specific cytokines. Th1 cells activate macrophages and help eliminate bacteria living intracellularly.: dendritic cells) mature and interact with lymphocytes in specialized lymphatic tissues or organs. CD8 T cells kill virally infected cells. Chapter 1. CD4 T cells and CD8 T cells. which are no longer phagocytic. Taylor & Francis Group. Once activated T cells divide and differentiate into effector T cells.molecules are either eliminated. LLC). there are specialized subsets of T cells. Furthermore. For those pathogens that reside and multiply in cells. known as regulatory T cells is fundamental for restraining auto-reactive immune responses. Once pathogens enter. carry the pathogen to the lymphoid tissues via lymphatic vessels to be recognized by lymphocytes. The induction of adaptive immune responses begins when the antigen presenting cells that are activated during the innate response (e. T cells recognize epitopes (small stretch of amino acids) that are often buried inside the antigen. contributing to its clearance. Most lymphocytes live in specialized lymphoid tissues or organs or can be found recirculating in the blood under normal condition. Th2. but only when instructed to do so by the innate immune system. Also. .recognize regions of antigen (epitope) that is displayed on the surface of antigens. Microorganisms grow and survive in different niches once they have invaded. How is self-reactivity avoided? A variety of mechanisms ensure that the immune system is tolerant of itself. fail to mature. In contrast. The mature dendritic cells. These secreted immunoglobulins (antibodies) can circulate throughout the body and bind to the pathogen. Reading: "Janeway’s Immunobiology". The adaptive immune system uses different effector responses to protect against the four classes of pathogen. the lymphocytes with specific receptors will divide and migrate to the site of infection to combat the pathogens. lymphocytes do not respond to self-components because clones of lymphocytes with receptors that bind to self.g. B cells. For example. a subset of T cells. they divide and differentiate into plasma cells that secrete a soluble form of their surface immunoglobulin receptor. Th17. First. by Kenneth Murphy (Garland Science. the innate immune system discriminates self from non-self by recognizing conserved molecular patterns associated with pathogens. such as Th1. 8th Edition. 2011.

Hematopoiesis is a complex developmental process through which pluripotent stem cells in mammalian fetal liver and subsequently neonatal and adult bone marrow give rise to all terminally differentiated cells of the blood. T cell precursors migrate from the bone marrow and further develop in the thymus. non-phagocytic cells. The route of recirculation differs depending on the activation state of the lymphocyte. immunocompetent T cells. such as engulfing and destroying microorganisms and presenting antigens. Lymphocytes develop in primary lymphoid tissues The primary lymphoid tissues contain microenvironments that support the production of mature lymphocytes from progenitors. myeloid. Infusion of bone marrow stem cells into a lethally irradiated animal can repopulate all of these cell populations. the bone marrow. However these early cells express the antigen Thy 1 and Sca-1 (stem cell antigen. In the human. The maturation of stem cells into different cell types is accompanied by the expression of lineage specific genes. GM. more than half of them T cells. lymphoid). Cells of the immune system White blood cells consist of neutrophils. They can be found as naïve cells if they have not interacted with antigen or effector/memory cells if they have interacted with antigen. B cells. and travel towards lymph nodes. Oct-2 (B lymphocyte). Examples include: GATA-1 (erythroid). as most blood cells have a limited life span. However. The other types of white blood cell are important in various processes. a member of the Ly6 family). Lymphocytes consist of T cells. B lymphocytes develop in the bone marrow. eosinophils. GATA-2 (erythroid. and produces clonally diverse. a thoracic organ anterior to the heart. lymphocytes. Naïve lymphocytes enter lymph nodes via a specialized structure called the high endothelial venules (HEV). Rothlin Hematopoietic stem cells and lineage commitment The cellular subsets of peripheral blood are constantly replenished by the process of hematopoiesis. tissues. Activated or memory lymphocytes extravasate from the vasculature. respiratory and reproductive systems. In all vertebrates. Since they manufacture lymphocytes. and natural killer cells. . monocytes. 2) the expression of all surface molecules needed for activation and differentiation. Most T cells continuously recirculate between blood. In addition to influencing differentiation. enter the lymphatic vessels. a specialized subset of large non-recirculating pool of T cells is found in the epithelia of the gut. Certain transcription factors have been shown by targeted deletion to be essential for particular lineages of cells. mast cells. a milliliter of blood contains around 2-4 x 106 lymphocytes. Resting T and B cells are small. The thymus is seeded by progenitor cells without immunological specificity. and the secondary lymphoid organs (lymph nodes and spleen). Lymphocytes in body fluids Naïve B and T cells circulating in the blood traverse the lymph nodes and the spleen. The production and the maturation of the differentiated cell types from pluripotent precursors is regulated by hematopoietic growth factors. Examples of such regulators include erythropoietin.CSF. and basophils. SCF and various cytokines. skin. Hematopoiesis gives rise to erythrocytes. Some of these T cells appear to be independent of the thymus. platelets and lymphocytes. Ikaros (lymphoid). Lymphocytes are the primary cell of the adaptive immune system. while in birds they originate in a specialized organ. and 3) the expression of cell surface molecules needed for recirculation and localization in tissues in the periphery. Stem cells constitute a very small fraction of cells in the bone marrow. They are morphologically distinct from other cells and do not express lineage specific cell surface markers (Lin-). etc. and enter inflamed tissues. indicating the highly proliferative and differentiation capacity of stem cells. they then percolate through intercellular spaces. granulocytes. Three important events occur in these sites: 1) the expression and selection of cells with antigen-recognition receptors. avian Bursa and thymus are termed primary lymphoid organs.Structure of the Immune System II September 5 Instructor: Carla V. macrophages. the Bursa of Fabricius. these regulators also control the functional activity of the cells by eliciting intracellular responses mediated by cell surface receptors.

. The architecture of the secondary lymphoid tissues fosters interaction between antigen and lymphocytes. Lymph nodes drain (via lymphatic vessels) extracellular fluid (lymph) from most tissues of the body as a means of collecting antigen. The major secondary lymphoid organs are: spleen. tonsils. appendix. The secondary lymphoid tissues have specialized mechanisms to collect antigen and antigen reactive lymphocytes. Taylor & Francis Group. 2011. Lymphocytes enter secondary lymphoid organs from the blood via specialized mechanisms designed to recruit naïve lymphocytes (high endothelial venules or HEV). The spleen filters the blood collecting antigens that enter the vasculature. the microvasculature at the site of infection is specialized to recruit activated lymphocytes and other cells important in defense. This activation includes the clonal selection and expansion of lymphocytes expressing specific antigen receptors. lymph nodes and Peyer’s patches. Naïve T and B cells first encounter antigens in the secondary lymphoid organs and become activated. 8th Edition. Lymphocytes migrate to site of infection (the battleground) Activated lymphocytes leave the secondary lymphoid tissues and travel to the infected tissues via the blood to clear the pathogen. LLC). In addition. They divide and migrate out of the secondary lymphoid organs via peripheral blood and into the site of infection. The structure of the secondary lymphoid tissues varies depending on its function.Lymphocytes are activated in secondary lymphoid tissues Secondary lymphoid tissues foster interactions between antigen presenting cells and lymphocytes. Chapter 1. by Kenneth Murphy (Garland Science. Based on early innate immune responses. The mucosa associated lymphoid tissues (including Peyer’s patches) collect antigens crossing the mucosal epithelia. antigen presenting cells carrying the pathogen-associated antigens migrate from the peripheral tissues to the lymph nodes via the lymphatic vessels. Reading: "Janeway’s Immunobiology".

Classical examples of intracellular signaling pathways involve: • Activation of G proteins. in response to agonists. including but not limited to the following ones: Cell surface receptors • Receptor protein kinases: have intrinsic enzymatic activity and exert their regulatory effects by phosphorylating diverse effector proteins. which confers specific recognition by receptor and effector. e.: activation of inward rectifier K+ (GIRK) channels).g. A few receptor protein kinases phosphorylate serine or threonine residues. Ligand-gated ion channels: are receptors for several neurotransmitters that form agonistregulated ion-selective channels in the plasma membrane that convey their signals by altering the cell's membrane potential.g.. and an associated dimer of b and g subunits that can confer both membrane localization of the G protein (e. G-protein coupled receptors (GPCRs): interact with distinct heterotrimeric GTP-binding regulatory proteins known as G proteins. G proteins are signal transducers that regulate multiple effectors including enzymes such as adenylyl cyclase. NOD like receptors. phospholipase C.: growth factor receptors. Receptors of this group include multiple cytokine receptors and T and B-cell antigen receptors. phosphodiesterases. Activation of the Ga subunit by GTP allows it to both regulate an effector protein . G proteins are composed of a GTP-binding a subunit. Receptors can be classified according to their cellular localization and shared mechanism of action into relatively few functional families. How are these external stimuli sensed? How is the recognition of these stimuli transduced into specific cellular responses in a timely fashion? In this first lecture we will discuss the basic features of signal transduction.g. such as Kit and FLT3. which are expressed on developing lymphocytes).Receptors & Signaling I September 7 Instructor: Carla V. such as the TGF-b receptor. Rothlin Immune cells are able to sense changes in the extracellular environment and respond accordingly. GPCRs respond to agonists by promoting the binding of GTP to the G protein a subunit. 1) Signal transduction is initiated upon recognition of the extracellular stimulus (agonist) by its cognate receptor. the process by which extracellular signals interact with their cognate receptors leading to the activation of biochemical cascades that result in a cellular response. and plasma membrane ion channels selective for Ca2+ and K+. This event is followed by the propagation and amplification of the signal through the engagement of intracellular signaling pathways. Most receptor protein kinases phosphorylate tyrosine residues in their substrates (e. via myristoylation) and direct signaling (e. • • • Intracellular receptors.: nuclear receptors. bind to or activate distinct protein kinases on the cytoplasmic face of the plasma membrane. 2) Recognition of the external stimulus by the receptor provides the first message in signal transduction. Protein kinase–associated receptors: lack the intracellular enzymatic domains but.g.

once activated. resulting in receptor “desensitization”. • • • 3) Activation of signaling pathways is tightly regulated by a variety of molecular mechanisms that efficiently terminate the signaling response. activation of phosphatidylinositol phospholipase C results in the hydrolysis of phosphatidylinositol bisphospate (PIP2) and the generation of inositol 1. Activation of the MAP kinase pathway. Nuclear Translocation of Transcription Factors. some GPCRs can be dephosphorylated and efficiently recycled back to the cell surface in a resensitized state in which the receptors are competent to signal again. DAG binds and activates signaling proteins. Ubiquitin-mediated degradation. and most importantly for nuclear signaling. This leads to conformational changes in the receptor. diacylglycerol (DAG). • • • . As a significant proportion of signaling events depend on protein phosphorylation.and drive the release of Gbg subunits. A classical example of this transcytoplasmic signaling is the JAK/STAT system. leading to the activation of the small GTPases and the recruitment of downstream effectors.5-trisphosphate (IP3) and diacylglycerol (DAG). This is a common feature in GPCR signaling. leading to the activation of transcription factors. phospholipases. because the terminal MAPK. Covalent addition of one or more molecules of the small protein Ubiquitin targets proteins to proteosomal or lysosomal degradation.: Tyrosine phosphatase SHP1) play a key role in shutting down signaling pathways. Endocytosed GPCRs are sorted from endosomes to lysosomes and degraded. receptors can be internalized. which leads to the activation of the STAT transcription factors. protein phosphatases (e. returning the system to the basal state. • Activation of small GTPases. In addition to desensitization. the intrinsic GTPase activity of these proteins leads to the hydrolysis of GTP to GDP and their inactivation. and ion channels that upon activation permit the release of second messenger molecules such as cyclic-AMP (cAMP). The elucidation of the modular MAPK cascade with its three consecutive protein kinases has had immediate implications for understanding signal amplification and switching. results from the prolonged exposure of the receptor to the stimulus. commonly observed in ligand-gated ion channels. Common effector proteins of G-proteins include phosphodiesterases. Within endosomes. IP3 acts as a second messenger to release Ca2+ from intracellular stores.4. a state in which the receptor can still recognize the stimulus but is unable to transduce the signal. a process important for signal termination. Phosphatases. GPCR trafficking has critical functions not only in signal termination but also in receptor resensitization. inositol triphosphate (IP3). cyclic-GMP (cGMP). GTPases such as those from the Ras family. This allows GTP to bind in its place. Receptor signaling activates guanine-nucleotide exchange factors (GEFs). This mechanism. which can. Overtime. in addition to regulating their own group of effectors. can migrate into the nucleus. in which ligand binding to a cytokine receptor activates the associated JAK family protein Tyrosine kinases. These include: • Receptor Desensitization. Thus. and a MAP kinase (MAPK). play major roles in the regulation of gene expression and the rearrangement of the actin cytoskeleton. and Ca2+. reassociate with GDP-liganded Ga. In the canonical phosphoinositide pathway. Phospholipid and ion-based signaling. thus triggering a program of Ca2+-activated events. Receptor Internalization. a MAP kinase kinase kinase (MAPKKK). A complex pathway that consists of a cascade of three protein kinases. a MAP kinase kinase (MAPKK).g. such as Ras-GEF and PKC. These events are fundamental to antigen receptors signaling pathway and will be reviewed in the lecture Receptors and Signaling II. which bind to small GTPases in their inactive state and displace GPD. adenylyl cyclases.

Chapter 1.Reading: Text: "Janeway’s Immunobiology". . 8th Edition. John S. Taylor & Francis Group. Parker. Brunton. 2012. LLC). Chapter 7. by Kenneth Murphy (Garland Science. Laurence L. 11th Edition. Lazo and Keith L. Goodman & Gilman's “The Pharmacological Basis of Therapeutics”.

Nature. Review Articles: Medzhitov. the adaptive immune response is generated to provide a powerful and antigen specific immunity to pathogens. Reading: Text: "Janeway’s Immunobiology". IL-1. Chapter 2 and 3. 2007 Oct 18:449(7164):819-826. The activation of host defense modules is coordinated by different cytokines. Iwasaki The innate immune system is an evolutionarily ancient form of host defense. each designed to protect from different types of infectious challenges. . Different infections induce different combination of host defense modules. IL-8 and KC. phagocytes. These include: Epithelial barriers (skin and mucosal epithelia) Phagocytes (macrophages and neutrophils) Anti-microbial peptides and proteins Complement system and acute phase proteins NK cells and plasmacytoid dendritic cells Mast cells. antimicrobial peptides and proteins and the complement system. eosinophils and basophils Each of these host defense modules can be directly activated by the receptors of the innate immune system upon recognition of different classes of invading pathogens. 8th Edition. For example. IL-6. While the adaptive immune system is found only in vertebrates. Taylor & Francis Group. Many infections are successfully handled by the innate immune system. When innate host defenses are insufficient. including TNF. LLC). and chemokines. Innate immune response can be activated immediately upon infection and helps maintain pathogens prior to the induction of adaptive immunity. bacterial infections will typically activate epithelial defenses. R. The innate immune systesm provides the first line of defense against pathogens. 2011. by Kenneth Murphy (Garland Science. including MCP-1. however. the innate immune system exists in all animals and plants.Innate Immune System September 10 Instructor: A. Recognition of microorganisms and activation of the immune response. Innate immune system consists of several host defense modules.

There are 4 TLR signaling adapters that also contain TIR domain – MyD88. TLR3 is specific for double stranded RNA – a common product of viral replication. Innate immune recognition is mediated by non-clonal. TLR7 and TLR9 recognize viral RNA and DNA. Collectively. . TLR4 is specific for LPS of gram-negative bacteria. Thus. ii) PAMPs are produced only by microbes and not by the host organism. as well as for lipoproteins found in all bacteria. TLR2 – for lipoteichoic acids of gram-positive bacteria. This property of PAMPs allows a limited number of germline-encoded receptors of the host to recognize a wide variety of microorganisms. In addition. The receptors that recognize PAMPs are called Pattern Recognition Receptors (PRRs). and TRIF. PAMPs are chemically distinct from any structure synthesized by the host cells. TLR specificities cover almost entire spectrum of microbial pathogens. Different TLRs utilize different combination of the adapters to induce both overlapping and distinct subsets of cellular responses. PAMPs recognized by PRRs represent the major targets of innate immune recognition and have several characteristics in common: i) PAMPs are relatively invariant structures shared by large groups of microorganisms. These are referred to as Pathogen-Associated Molecular Patterns (PAMPs). because it is also found in the members of the IL-1 receptor family.Pattern Recognition Receptors September 12 Instructor: R. There are about dozen or so TLRs in mammalian species. The best-characterized family of PRRs is the Toll-like receptor (TLR) family. TLRs recognize conserved microbial molecular structures (PAMPs) and trigger activation of host defense responses. serine/threonine kinases IRAK1 and IRAK4. TRAM. Medzhitov The major distinguishing feature of innate immunity is the mechanism of pathogen recognition. but they detect the presence of viral RNA in infected cells. They detect infection and activate the host immune responses. Dectins. therefore. In addition to TLRs. innate host defense responses. TLRs induce signaling pathways that activate NF-kB and MAP kinases. In other words. innate immune recognition can lead to activation of adaptive immunity. Recognition of PAMPs. including inflammatory responses. Upon activation by their microbial ligands. and RIG-I/MDA-5. TLRs play a crucial role in the immune system. respectively. as well as several MAP3 kinases that function downstream of TRAF6 and activate NF-kB and MAP kinases. and fungal glucans. as well as induction of adaptive immune responses. including Nod-like receptors. RIG-I and MDA-5 are also intracellular receptors. several other families of pattern recognition receptors (PRRs) have been recently characterized. these receptors detect majority of pathogens and trigger activation of host defense responses. Dectin is a transmembrane receptor specific for beta-glucans – major components of fungal cell walls. ubiquitin ligase TRAF6. TIRAP. Thus TLRs represent a crucial component of the host defense system. Nod1 and Nod2 are intracellular receptors that detect fragments of bacterial peptidoglycans. TLR5 recognizes bacterial flagellin. This property allows for self/non-self discrimination by the innate immune system. germline-encoded receptors that recognize conserved molecular patterns associated with pathogens. The most familiar examples of PAMPs are bacterial LPS and lipoteichoic acids. Activation of these signaling pathways leads to induction of inflammatory responses. prevents generation of 'escape mutants' because such mutations are lethal for the microbes. All TLRs share a conserved cytoplasmic domain called TIR domain. TLRs function as sensors of microbial infection. The canonical pathway shared by all TLRs includes MyD88. iii) PAMPs are conserved molecules essential for the survival of the microbes.

LLC). 2008 Jun 12. Host innate immune receptors and beyond: making sense of microbial infections. Review Article: Ishii KJ. 2011. Cell Host Microbe. by Kenneth Murphy (Garland Science.Reading: Text: "Janeway’s Immunobiology". 8th Edition. . Akira S. Koyama S.3(6):352-63. Taylor & Francis Group. Chapter 2 and 3. Coban C. Nakagawa A.

Complement has two functions – lysis of microbial cells and opsonization of microbial cells to promote their elimination by phagocytes. 2002. mannan binding lectin) function as opsonins – they bind to the pathogen’s surface and facilitate pathogens uptake by phagocytes. but selectively inhibited on host cells. . Early response to infection includes the induction of local inflammation. Other acute phase proteins have direct antimicrobial activity. The classical pathway of complement activation is triggered by antibodies bound to the pathogens’ surface. Thus the complement and the phagocytic systems (as well as the acute phase response) are functionally linked. R. 8th Edition.Complement. 20: 197-216. Taylor & Francis Group. Medzhitov. Some acute phase (C-reactive protein. depending on the mechanism of pathogen recognition. bacteriocidal permeability increasing protein (BPI) can bind to the cell walls of gram negative bacteria and destabilize the bacterial membranes. The inflammatory cytokines IL-6 and IL-1 can also act at a distance and trigger expression of a variety of genes in the hepatocytes. Reading: Text: "Janeway’s Immunobiology". Annu Rev Immunol. thus allowing for specific attack on pathogens.. NK cells and Acute Phase Proteins September 14 Instructor: R. in that it binds to pathogens’ surface and activates the protease cascade much like the antibodies do. The alternative pathway is activated constitutively. The function of acute phase proteins is to help combat the infection at the system level. The function of MBL is reminiscent of the antibodies. The concentration of the acute phase proteins in circulation increases markedly. 2011. CA Jr. The complement system plays a critical role in host defense against a variety of extracellular pathogens. These same proteins also activate the complement cascade. Review Articles: Janeway. which are secreted by lives into circulation. These genes encode so-called acute phase proteins. Innate immune recognition. by Kenneth Murphy (Garland Science. Chapter 2 and 3. Yet other acute phase proteins regulate the coagulation cascades. Finally. For example. There are several pathways of complement activation. Medzhitov The innate immune system utilizes a diverse set of mechanisms to combat infection. the lectin pathway is activated by the acute phase protein mannan binding lectin (MBL). surfactant proteins. LLC).

In addition. Defense against multicellular parasites. in part through the production of potent anti-microbial peptides (defensins) and production of mucus. including ROS and NO generating enzymes. In the case of bacteria and fungi. Text: "Janeway’s Immunobiology". Chapter 2 and 3. 2008 Jan. 8th Edition. Trends Microbiol. Epithelial cells also play an important role in antibacterial and anti-fungal defense.Innate immunity to Bacteria. LLC). . 2011. Taylor & Francis Group. including helminthes (parasitic worms) is mediated by eosinophils and mucosal epithelia. Brown GD. Review Articles: Willment JA. complement has direct anti-microbial activity. Fungi and Parasites September 17 Instructor: R. Eosinophils produce several toxic molecules that can directly target parasites.16(1):27-32. These cells are endowed with powerful antimicrobial defenses. by Kenneth Murphy (Garland Science. The function of phagocytes is aided by the complement system through opsonization. C-type lectin receptors in antifungal immunity. fungal and parasitic infections. anti-microbial peptides and lysosomal enzymes. Phagocytosis and killing by neutrophils and macrophages is the main form of defense against bacteria and fungi. Mucosal epithelial produce mucins that help prevent parasite entry and promote their expulsion. Medzhitov The innate immune system uses a number of mechanisms to protect against bacterial. Mast cells produce several inflammatory mediators that help orchestrate anti-parasitic defenses and promote parasite expulsion through their effects on smooth muscles and endothelium. the main host defenses rely on the function of phagocytes: macrophages and neutrophils.

23:225-74. plasmacytoid dendritic cells (pDCs) use the Toll-like receptors (TLR) 7 and 9 to detect ssRNA and dsDNA viruses. Viruses contain genetic information within the capsid. When the action of these anti-viral proteins is insufficient. Medzhitov Viruses represent the simplest forms of pathogens. In most cells. Taylor & Francis Group. Innate recognition of viral PAMPs occurs by two distinct mechanisms depending on the cell types. In contrast. 2005. The pDCs. Natural killer (NK) cells do just that and play an important role in the anti-viral host defense. . Innate Immunity to Viruses By far the most important innate immune mechanisms against viruses involve the type I interferon system. replicate genomes and assemble using host cell machineries. and possess surface proteins that bind to and fuse with host cell receptors. The combination of the activating and inhibitory ligands generally determines whether or not a given cell would be eliminated by NK cells. IFN-I is induced upon infection in most cell types and triggers expression of over hundred anti-viral proteins which interfere with every aspect of viral infection cycle. The inhibitory receptors recognize ligands that are constitutively expressed on normal healthy cells and inhibit the cytotoxic response against these cells. Complement can also deposit onto the virus surface and neutralize or lyse the virus. Reading: Text: "Janeway’s Immunobiology". the most efficient anti-viral defense is to get rid the infected host cell. Upon entry into the host cells. LLC). Annu Rev Immunol. respectively. and upon binding to the receptor. 8th Edition. NK cells are capable of recognizing and lysing cells that lack surface MHC class I molecules. upon sensing viruses by TLR7 and TLR9 in the lysosomes. secrete large amounts of type I IFNs and IL-12.Innate Anti-viral Immunity September 19 Instructor: R. viruses synthesize proteins. as retinoic acid inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (mda-5). Chapter 2 and 3. Review Articles: Lanier LL. 2011. The key cell types involved in innate clearance of viruses and virus-infected cells include pDCs and Natural killer (NK) cells. NK cells express a number of receptors that can either activate or inhibit their function. as both TLR7 and TLR9 are localized in this compartment. This form of recognition occurs within the lysosome instead of in the cytosol. by Kenneth Murphy (Garland Science. Viruses are obligate intracellular pathogens that can be roughly categorized into DNA and RNA viruses. NK cell recognition. Recent studies have elucidated the mechanisms by which viral PAMPs can trigger the innate IFN production pathways. ssRNA virus infection is detected by a cytosolic sensor. Type I interferons can be secreted by most cells of the body. The activating receptors recognize molecules expressed on the surface of infected cells and induce the cytotoxic response directed at the unwanted infected cell. induce a myriad of antiviral genes that inhibit productive replication of viruses.

21-hydroxylase). the defined extracytoplasmic domains. Cresswell MHC class I molecules are composed of two subunits. B. The β2m gene is not in the MHC. but the peptides bound are longer and more variable in length. 6. As a result of this variability. Individual αβ pairs (e. in which bind small peptides of between 8 and 10 amino acids. Also in the MHC are two genes. Class I and class II molecules can be regarded as similar structures. and genes encoding heat shock proteins (Hsp70). The class I heavy chain and the two class II subunits show allelic variability in amino acid sequence. For class I. the genes for tumor necrosis factors. 5. C4. Genes relevant to antigen processing functions are also tightly linked to the MHC. and a gene encoding tapasin. 3. the first and second domains of the large subunit fold to generate a cleft or groove between two a-helices. Structural studies show that the variable residues generally line the peptide binding groove. These include two genes (TAP. and L in the mouse. encoding three different ab dimers in humans (HLA-DR. One. which encode a molecule structurally similar to class II molecules 8. the membrane proximal domain of the large subunit is homologous to Ig-constant region domains.1 and TAP. the membrane proximal domains of both the α and β-subunits are Ig-like. has a role in presenting N-formylated peptides derived from bacteria. P. 4.. is the product of an unlinked gene and has a molecular weight of 12kDa. LMP. DQ and DP) and two different ab dimers in the mouse (I-A and I-E). which is involved in generating peptides in the cytosol. have also been defined. Three other human class I genes. The MHC contains multiple class I and class II genes. These include genes encoding complement components (C2.g. Factor B). and the transmembrane regions. I-Ab and I-Ek) are encoded by very tightly linked genes. Both are transmembrane glycoproteins and both are products of genes within the MHC. at fixed positions in the peptide sequence. The T-cell receptor recognizes a surface consisting of residues in the a-helices and surface exposed residues in the associated peptide. F and G. Genes encoding class II α and β-subunits are both present in the MHC. H-2M3 from the Qa region. These encode molecules which have a limited tissue distribution. The invariant chain gene (see below) is not in the MHC. . The small subunit. generally in hematopoietic cells. D. and C in the human. In class II. MHC class II molecules are also composed of two non-covalently associated subunits. the peptide binding groove of each allele has an affinity for a different subset of peptides. which facilitates MHC class I peptide binding. Also two genes. In the mouse there are multiple additional class I genes. called anchor residues. Genes not obviously related to antigen processing are present in the MHC.2) encoding a peptide transporter involved in transferring peptides derived from cytosolic proteins into the endoplasmic reticulum where they bind class I molecules. In class I. HLA-E. The large subunit (44kDa) is a transmembrane glycoprotein which is the actual product of the MHC-linked gene.g.Structure and Genetics of MHC Molecules September 21 Instructor: 1. The membrane distal domains of the α and β-subunits of class II generate a similar peptidebinding groove. as is β2m. encode subunits of a cytosolic macromolecular protease called the proteasome. H2-K. with preferred interacting amino acids. Both human and mouse MHC class I and class II genes have a similar organization at the intron/exon level. Some of these have been suggested to have an antigen processing function. β2-microglobulin (β2m). 7.. called α and β. The "classical" class I genes are HLA-A. genes encoding various enzymes (e. DMA and DMB.2. Additional exons encode the cytoplasmic regions. 2. with individual exons encoding the leader sequence.1 and LMP.

Garland Science. 2012. 8th Edition.called H2-M or H2-DM in mice and HLA-DM in humans. Chapter 6. Reading: Janeway’s Immunobiology. These genes are required for efficient class IIrestricted antigen processing. Ltd. . 217-230..

which facilitates peptide binding. ERp57 is permanently disulfide-linked to tapasin. presumably to protect the cells they infect from recognition by CD8-positive T-cells.2 and LMP. or. Animals infected with a virus generate virus-specific. b) incubated with a defined short (8-10 amino acid) peptide which corresponds to a specific sequence in the particular viral protein recognized by the CTL. peptides may be “trimmed” at the N-terminus by ER Amino Peptidases (ERAAP in mice.g. Certain viruses (e. 8th Edition. d) transport of the class I peptide complex to the cell surface. Such proteasomes are often called immunoproteasomes. Garland Science. Cresswell MHC class I-restricted antigen processing 1. The TAP transporter is a member of a structurally homologous family of transporters that use ATP-hydrolysis to translocate small molecules across membranes. Proteolysis of cytosolic proteins is predominantly mediated by the proteasome.. serving as a bridge. and a third. ERp57. the chaperone calreticulin and the thiol oxidoreductase. 3. Many of these CTL prove to be specific for viral proteins that are only expressed intracellularly. class I-restricted CD8-positive cytotoxic T-lymphocytes (CTL). Reading: Text: Janeway’s Immunobiology. with a third protein. 6. 4. Ltd. Virus-derived peptides generated in infected cells are superimposed on this background of normal host peptides. called MECL-1. called LMP. b) transport of the peptide into the endoplasmic reticulum (ER) of the cell. Herpes simplex virus I and II.2) are encoded in the MHC.7. Transport of peptides into the ER is mediated by a heterodimeric transporter. Both subunits of this transporter (TAP. TAP molecules physically associate with newly synthesized "empty" class I molecules in the ER.. . These proteins constitute the Peptide Loading Complex. the normal mechanism which generates the class I-peptide complex involves: a) proteolysis of the cytosolic viral protein. c) association of the peptide with the class I molecule and. 202-217. Chapter 6.MHC Class I Restricted Antigen Processing September 24 Instructor: P. the "loaded" class I molecules dissociate from the PLC and are transported to the cell surface where they are available for T-cell recognition. SV40 T-antigen (nuclear) or influenza virus matrix protein (cytosolic). 2. e. Target cells expressing the appropriate class I allele are not killed unless they are: a) infected with the virus.g. 5. Thus in normal circumstances class I molecules are full of peptides derived from normal cell proteins. After translocation into the ER. 7.. Note that all cytosolic proteins can feed into this system. Thus. human cytomegalovirus) have developed strategies that interfere with MHC class I peptide loading. tapasin. ERAP1 and ERAP2 in humans) to facilitate binding to class I molecules. 2012. incorporates two subunits encoded in the MHC. peptide can bind to class I molecules at the cell surface in vitro and generate a class I-peptide complex that the CTL can recognize. in Antigen Presenting Cells (APCs) or cells treated with interferon-g. However.1 and TAP. a multisubunit protease which. or PLC. After peptide binds. Two additional proteins are also associated.

Primary Literature: The International HIV Controllers Study. 2010. Wearsch. . P. Current Opinion in Cell Biology: 20:1-8. 2008. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. P. Science 330: 1551-1557. The quality control of MHC class I peptide loading. and Cresswell.

the invariant chain. Internalization followed by proteolysis of the antigen is essential. CD4-positive T-cells respond to class II-positive cells which have internalized protein antigens. it contains a cytoplasmic targeting signal that drives class II-invariant chain complexes to the endocytic pathway. Note that. First. 4. These are usually derived from membrane proteins or internalized serum proteins undergoing lysosomal degradation. Garland Science. GILT. Like for class I.MHC Class II Restricted Antigen Processing September 26 Instructor: P. Here the invariant chain is removed by proteolysis. Primary Literature: Sadegh-Nasseri. a class II-like αβ dimer. B-cells are up to 10. Trends in Immunology 29: 141-147. Cresswell MHC class II-restricted antigen processing 1. peptides corresponding to specific short segments of the antigenic proteins can directly bind class II molecules at the cell surface in vitro and can stimulate T-cells. Class II α and β subunits associated with a third glycoprotein. 202-217. immediately upon biosynthesis in the ER. Ltd. The invariant chain is trimeric and associates with three αβ dimers to form a nonameric structure.. For antigens that contain disulfide bonds a lysosomal thiol reductase. Second. S. Invariant chain regulates antigen processing in two ways. The DO α and β subunits are also encoded in the MHC. . They accumulate class II molecules associated with a single peptide (CLIP).. 2. Internalization can occur by phagocytosis (in dendritic cells and macrophages) or by endocytosis. derived from the invariant chain. The specifically endocytosed antigen is degraded and peptides derived from it presented on the surface in association with class II molecules. Mutant cells lacking DM molecules. Class IIpeptide complexes are then transported to the cell surface where they can be recognized by T-cells. 8th Edition. chloroquine. freeing the class II peptide-binding groove to associate with peptides generated from internalized protein antigens. 2012. facilitates protein unfolding and peptide generation. Inhibitors of lysosomal proteolysis (e. some dendritic cells. cannot generate normal class II-peptide complexes. DM function is down-regulated by its association with another class II-like molecule. DM functions by catalyzing the displacement of CLIP and facilitating the binding of the normal complement of peptides derived from internalized proteins. which neutralizes the normally acidic lysosome) inhibit antigen processing and subsequent presentation to T-cells. Chapter 6. The B-cell/T-cell interaction is critical for making antigen-specific antibody responses. The Ig antigen receptor on the surface of B-cells acts to enhance presentation of specific protein antigens to CD4-positive T-cells by binding to the antigen and internalizing it. it prevents class II molecules from binding unfolded proteins or peptides in the early stages of biosynthesis. et al. The biosynthesis of class II molecules is important for proper function. as in the class I system. Recognition of this peptide-class II complex by a CD4-positive T-cell induces activation of the B-cell which can differentiate into a plasma cell secreting antibodies specific for the antigen.000 times more efficient at processing and presenting the antigen for which they are specific than are non-specific B-cells. which inhibits DM function. In B-cells.g. 2008.. 5. The convergent roles of tapasin and HLA-DM in antigen presentation. even in the absence of pathogenic proteins the class II molecules are still occupied with peptides. Reading: Text: Janeway’s Immunobiology. DO. and in thymic epithelial cells. 3.

which recognize CD1d complexes containing self lipids and which play an important role linking innate and adaptive immunity. CD1b. Current evidence suggests that the CD1-lipid complexes are generated in the endocytic pathway and that saposins. At this stage. express an additional type of antigen presenting molecule. 2009. Current Oopinions in Immunology. 342-353. Instead. and Villadangos J. CD1 antigen presentation: how it works. 7: 929-941. Activation of dendritic cells by innate immune mechanisms.. Subsets of dendritic cells have been described and one of them.. CD1d and CD1e. called CD1a. Cresswell Dendritic cells Dendritic cells are the “first line of defense” in the antigen presenting cell world.. Thus. Dendritic cells are also the major antigen presenting cell type capable of cross-presentation. Recent data suggests that recruitment of ER membrane to the phagosome may contribute to this process by introducing a mechanism for protein transfer to the cytosol and by making the MHC class I peptide loading machinery accessible to peptides from the internalized antigens. CD1d molecules are detected by a T cell subset referred to as NKT cells. Barral D. CD1 molecules Certain Antigen Presenting Cells. 2007. Ltd. . in the mouse the CD8a-positive dendritic cell. In peripheral tissues they are highly active in macropinocytosis and phagocytosis.Dendritic Cells and Their Functions/CD1 Molecules September 28 Instructor: P. At this stage they migrate to lymph nodes. they have high levels of intracellular class II molecules. Work in humans has found that cytotoxic T-cells specific for Mycobacterium tuberculosis or Mycobacterium leprae are restricted by CD1 molecules. catalyze lipid binding to CD1 molecules. causes their maturation to a stage with high expression of surface class II-peptide complexes. including dendritic cells. allowing efficient non-specific uptake of soluble and particulate protein antigens.g. is the primary cross-presenting subset. The crystal structures of CD1b and CD1d molecules reveal a binding groove deeper and more hydrophobic than that of the MHC class I molecules. forming similar dimers with β2m. This is a mechanism whereby extracellular antigens can be internalized by endocytosis or phagocytosis and peptides derived from them presented by MHC class I molecules to CD8+ T cells. 21:105-110. A.B. 8th Edition.C and Brenner M. but it is not CD1-peptide complexes that are recognized. co-factors involved in lysosomal lipid degradation. Primary Literature: Segura E. Mice only express CD1d. These are CD1 molecules. dendritic cells are thought to perform antigen processing in peripheral tissues while antigen presentation to antigen-specific CD4-positive T-lymphocytes occurs in the lymph nodes.. They are not the products of MHC genes but are homologues of the classical class I molecules. mycobacterial lipids are bound by CD1 molecules and are recognized by these T-cells. Antigen presentation by dendritic cells in vivo. In humans there are five types of CD1. e. by exposure to bacterial lipopolysaccharides. CD1c. Nature Reviews in Immunology. 2012. Reading: Text: Janeway’s Immunobiology: Garland Science. Chapter 9.

In mammals there are subclasses of IgG antibodies with different biological properties. . Therefore the hypervariable regions are known as complementary determining regions (CDRs). Each immunoglobulin domain consists of two layers of polypeptide chains (which are β-pleated sheets) linked by a disulfide bond.g. e. Much low-affinity serum Ig is IgM. Antibody classes Antibody molecules come in subclasses defined by the H chain.. IgM antibodies dominate early in immune responses. giving it an overall Y shape. The affinity of binding of a particular antibody for its antigen depends on the multiplicity and strength of these interactions. The domains at the N-terminal end of one heavy and light chain make up the antigen binding site and therefore each monomeric antibody contains two such binding sites. The major function of IgA is to protect mucosal surfaces and it is the main antibody secreted into the gut. The use of different heavy chain constant regions produces antibodies of differing molecular weight. Treatment with proteases separates the antigen binding domain (Fab) from the effector domain (Fc). to form a barrel structure. The binding of antibodies to foreign molecules results in their inactivation by effectively targeting the foreign molecules for clearance by other mechanisms such as phagocytosis or complement. some don't. All antibody molecules are similar in overall structure. These distinct forms of antibody are termed isotypes. milk etc. Schatz The Antibody. or Immunoglobulin Antibodies are molecules that are either present in a membrane bound form on the surface of B cells or are secreted by B cells into body fluids. while the remaining domains are relatively invariant. The variable domains are known as V domains or regions and the constant domains are known as C domains or regions. as are the light chains to the heavy chains. The constant domain of the heavy chain determine the isotype of the antibody and therefore the functional characteristics of the antibody molecule. IgG: a monomeric. The structure of immunoglobulins has been elucidated using X-ray crystallography. detergents or extremes of pH. divalent form of antibody which predominates in serum. The variable region of each antibody chain contains three regions of hypervariability and the hypervariable regions of the heavy and light chain together form the antigen binding site. anatomical location. Most high-affinity serum Ig is IgG. these allow pentamerization to form a high avidity structure with 10 combining sites. Antibodies initiate their biological functions by binding to antigen with high affinity. Antigens bind to antibodies through non-covalent interactions that can be disrupted by high salt. IgA: a dimeric. The N-terminal domains of both heavy and light chains are variable in sequence. non-covalent interactions. Both the heavy and light chains contain a repeated 110 amino acid motif known as the immunoglobulin domain. degree of oligomerization. and is the main component of late and memory systemic responses. tetravalent form of antibody in which two Ig units are joined by a "secretory piece" which is a separate protein. The two heavy chains are joined to one another by disulfide bonds.The B Cell and T Cell Receptors: Structure & Classes October 1 Instructor: D. some fix complement. The isotypes found in mammals are (more on this in a later lecture): IgM: has an extra domain formed by two extra β-barrels at the C termini of the H chains. and biological function. respiratory track. The 3-dimensional structure of antibodies reveals that it consists of three globular domains linked by a flexible hinge region. They have a common core that is comprised of 2 heavy and 2 light chains.

to grass pollen. The γδ TCR is probably structurally similar to the Alpha β TCR. which is fundamentally different from B cells which recognize only the antigen. The 3-dimensional structure of the alphabeta TCR has been determined. termed the α and β chains. or fungal spores. ζ chains. and the structure shows that the overall architecture is very similar to that of immunoglobulins although surprisingly the Cα domain does not form an immunoglobulin-like domain. As discussed in detail in the reference below. involved in B cell activation. ε. The antibodies immunoprecipitated a heterodimer with two disulfide linked chains. Of particular note is that the diversity of the CDR3 region of TCRs is much greater than the diversity of CDR1 or 2. Minute quantities in serum. beta. e. The variable regions contain hypervariable regions (positioned much as they are in immunoglobulin V regions) that are functionally analogous to the CDRs of immunoglobulin chains. Involved in many human allergies. gamma and delta chains reveal that they are similar to the chains of the immunoglobulin molecule. Each chain of the TCR contains a variable N-terminal region and a constant C-terminal region. between genetically identical individuals of an inbred strain. Alpha β T cells: Most T cells have alpha beta TCRs on their surface (>95%) and these alpha beta T cells account for the major functional classes of T cells (helper T cells and cytotoxic T cells). γδ T cells: A smaller population of T cells has a TCR on its surface composed of gamma and delta chains. Antibodies may recognize epitopes encoded by allelic differences between antibodies of the same isotype of unrelated members of the same species. Antibodies may recognize epitopes on the combining site. in other members of the same species.g. TCRs were first identified by raising monoclonal antibodies against T cell clones. TCR and BCR signaling complexes: Immunoprecipitation of the TCR from T cells in non-ionic detergents precipitates a complex of associated proteins that are known as the CD3 complex. with the overall structure of the TCR being analogous to an Fab region of the immunoglobulin molecule. BCR: The sequence of the alpha. The CD3 complex consists of delta. TCR vs. and their specificity is clonally distributed... it is not surprising that T cell receptors (TCRs) are similar to antibody molecules.IgE: a monomeric antibody produced in response to macroscopic parasites such as worms. probably without a function there. which are distinct for antibodies of different specificity. goat anti-mouse IgM. this reflects the fact that CDR3 makes critical contacts with the antigenic peptide while the other two CDRs are primarily responsible for contacting the MHC molecule. crosslinking of this receptor results in mast cell degranulation with release of histamine. The CD3 proteins are invarient and their function is to allow signal transduction upon . etc. theta. However. or in autoimmunity within one individual. these are anti-ISOTYPE antibodies.g. IgD: found on the membranes of mature B cells. Triggers severe inflammatory reactions by binding to a specific IgE receptor on mast cells. since T cells recognize specific antigens. these are anti-ALLOTYPE antibodies. Antigenic epitopes on antibody molecules Immunoglobulin molecules may be immunogenic in unrelated species. these are anti-IDIOTYPE antibodies. e. Antibodies may recognize epitopes on the distinct H chains of Ig isotypes. The functional significance of gamma deltaT cells is less understood. The T Cell Receptor T cells recognize antigen as processed fragments in association with MHC molecules.

A.F.. and Wilson. A. Science 293. CD4 positive T cells recognize antigen presented by MHC class II molecules.L.** Review Article: Williams. (2001)...M. 1155-1159.B. please carefully study the beautiful diagrams in the book. MHC class I or II restricted. Primary literature: Saphire. Dwek.12. by K. 5. and Barclay. Reading: Text: "Janeway’s Immunobiology". 2011.M.. Zwick. Rudd. E. Stanfield. **This is a very structure-oriented topic and pictures are worth a thousand words.. Annual Review of Immunology 6:381-405.N. Ig molecules on the surface of B cells are non-covalently associated with two signal transduction proteins known as Iga and Igb.. I. CD8 or CD4 expressing T cells: Of critical importance to the interaction between the TCR and peptide:MHC are the CD4 and CD8 co-receptors found on the surface of T cells. CD4 is a single chain polypeptide that interacts with MHC class II molecules.O.activation of T cells. Chapter 4 and Chapter 5.16.. R. The immunoglobulin superfamily-domains for cell surface recognition (1988). Burton. P. Pantophlet. A. Murphy (Garland Science). while CD8 is a disulfide linked heterodimer that binds to MHC class I molecules.. G.R.13. while CD8 positive T cells recognize antigens presented by MHC class I molecules. and 5. they send a much more potent signal than if TCR or co-receptor alone is involved in the binding.. Parren. . In like manner. R. P. D. Like CD3. M. 8th Edition. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.A. the function of CD4 and CD8 co-receptors is in sending signals to the interior of the T cell: when the TCR:CD3 complex and the co-receptor bind to the same MHC:peptide molecule. R. sections 5. Morris.W.

the dogma of "one gene/one polypeptide" won't work due to the limited number of genes in the genome. The answer: Ig and TCR genes are assembled from component gene segments. the first three of which are shared by TCRs. resulting in a . The random rearrangement of V. D. (D) and J gene segments are joined by a site-specific recombination reaction known as V(D)J recombination. D. The Igk locus contains about 150 Vs and 4 Js (no Ds). the recombination machinery binds to the RSSs flanking the two participating gene segments and then cuts the DNA immediately adjacent to each gene segment. Gene segment combinatorial diversity results from the presence in the germline of multiple different copies of each type (e. one D and one J are used to assemble the Igh gene: with. arising from the many possible different combinations of heavy. and different combinations of these gene segments can be used in different rearrangement events. Schatz The problem: how to encode the millions of different immunoglobulin (Ig) and T cell receptor (TCR) molecules that are made by B and T cells.and light-chain V regions (or TCRa chain and TCRb chain) that pair to form the antigen-binding site in these molecules. Ig and TCR Genes The variable region gene exon is assembled from gene segments called V (variable). chain pairing.000 for Igk). Generation of antigen receptor diversity The diversity of Igs is generated by four main processes. Such added/subtracted nucleotides often disrupt the reading frame of the coding sequence beyond the joint (called nonproductive rearrangements). 100. there are 5200 different V-D-J combinations (100x13x4). For each antigen receptor locus. Somatic hypermutation is the fourth source of diversity. or RSS). This reaction takes place in two phases. Non-germline encoded (N-) nucleotides can be added by the enzyme terminal deoxynucleotidyl transferase (TdT). Antigen receptor loci can be very large (spanning as much as several megabases). D. D (diversity) and J (joining). Junctional diversity. while exonucleases delete nucleotides. D. D. V(D)J recombination V. A third source of diversity. The Ig heavy chain locus (Igh) and TCRb and TCRd loci contain V.g. and J segments is guided by short. J) of gene segment. the theoretical number of possible Igh-Igk pairs is well over one trillion. and J genes have in common (recombination signal sequence. V. is also combinatorial. Junctional diversity dramatically increases the number of possible proteins encoded by these loci (to more than a billion for Igh. In the first. for a combinatorial potential of 600 different genes. which is particularly concentrated in the third hypervariable region (CDR3 region). and J gene segments while the two Ig light chain loci (Igk and Igl) and the TCRa and TCRg loci contain only V and J gene segments. these gene segments and the constant (C) region all lie on a single chromosome.. leading to a nonfunctional protein.Immunoglobulin and TCR Gene Structure and Rearrangement October 3 Instructor: D. and diversity is generated combinatorially. Example: diversity in Igh and Igk Combinatorial diversity: One V. Chain pairing: Assuming that each Igh chain can pair with each Igk chain to form an antibody. 13 Ds and 4 Js. it affects only Ig genes and is considered in a later lecture. 100 Vs. is introduced at the joints between the different V. flanking DNA sequences that all V. and hence junctional diversity is achieved only at the expense of considerable wastage. say. and J segments as a result of addition and subtraction of nucleotides in the recombination process. See figure at bottom of the notes for this lecture for diagrams of the loci.

This transmembrane domain is absent from the secreted form (antibody). gene segments that are far apart on the chromosome will only recombine if they are brought into close proximity. Deletion + The first phase of V(D)J recombination (DNA binding and cleavage) is performed by the RAG1 and RAG2 Inversion proteins. TCRb. the same VH exon can associate with different CH genes in the course of an immune response. Such "opening up" of the chromatin is facilitated by activating histone modifications as well as transcription of the unrearranged gene segments—so called "germline" transcripts. Two V(D)J recombination events are required to assemble the Igh. IgA. whose C-terminus is a hydrophilic secretory tail. 1) RAG1 and RAG2 are expressed only in developing lymphocytes and hence V(D)J recombination can only occurs in these cells. Initially only the first (most 5') of these genes. RAG1 and RAG2 are expressed only in developing lymphocytes and are both essential for V(D)J recombination. like V and J— rectangles in the diagram) and a signal joint (the fusion of the two RSSs—triangles in the diagram). Membrane and secreted forms of Ig molecules Immunoglobulins of all heavy-chain isotypes can be produced either in secreted form or as a membrane-bound receptor. The second phase of V(D)J recombination (end processing and joining) is performed by a group of ubiquitously expressed DNA repair proteins together with TdT (mentioned above). To reiterate. Class Switch Recombination (Isotype Switching) Immunoglobulins can be made in several different forms. respectively) only when they are secreted.g. or isotypes (e. during the course of an antibody response activated B cells often switch to express a different downstream CH gene by a process of somatic recombination known as class switch recombination (CSR) or isotype switching. However. In the second. this structural variation is generated by linking different heavy-chain constant regions to the same Igh variable region.chromosome that has been cut in two places. IgA and IgM polymerize (into dimers and pentamers. IgG. 3) Through the control of long range chromosome "looping". IgM. and IgE). 2) Through the control of the "accessibility" of the DNA substrates. In its membrane-bound form the immunoglobulin heavy chain has a hydrophobic transmembrane domain at the C-terminus which anchors it to the surface of the B lymphocyte. Cm. IgD. CSR is unlike V(D)J recombination in several ways. The membrane forms of all isotypes are monomers comprised of two light and two heavy chains. and TCRd loci (D-to-J and V-to-DJ) and one event (V-to-J) for the other loci.. Regulation of V(D)J Recombination V(D)J recombination is regulated at several levels. The CH regions are encoded in separate genes located downstream of the V genes at the heavy-chain locus. . RAG1/RAG2 can only bind to DNA substrates that are in an "open" chromatin structure. and production of the two forms is achieved by alternative RNA processing. the four free DNA ends are processed and joined to form two new junctions (see diagram): a coding joint (the fusion of the two coding elements. is expressed in conjunction with an assembled V gene. The two different C-termini (transmembrane and secreted) of the heavy chains are encoded in separate exons.

by K.H. repair factors. and Davis. N.. 2011. (2002). and regulation.. M.. Kavaler. Murphy (Garland Science).Reading: Text: "Janeway’s Immunobiology". 101132. Y. Lee. 8th Edition. J.M.E. Gascoigne. Nature 309. 322-326. Somatic recombination in a murine T-cell receptor gene. . Annu Rev Biochem 71. V(D)J recombination: RAG proteins. Chapter 5. pp 157-173.R. (1984).. Primary literature: Chien. Review Article: Gellert. M. N.

Factors and forces controlling V(D)J recombination. 169-232. Constant regions are depicted as single rectangles. Adv. Taken from Hesslein.3 E#3 E#2 C#2 J#2 V#1. Gene segments are depicted as white rectangles. with no attempt made to indicate individual exons. D.. and Vd5 and Vb14 are known to rearrange by inversion. G. respectively. and 12-RSSs and 23-RSSs as white and black triangles. Ig and TCR Loci IgH VH(100-1000) DH(13) PDQ52 JH Eiµ CH(µ.". Enhancers and promoters are represented by gray circles and rectangles. Immunol.$.#. Not drawn to scale.1 J#4C#4 HsA .2 V#1.%) 3'Eµ Ig! V!(100-300) KI/KII J! Ei! C! 3'E! Ig& V&2 V&X J&2 C&2 E&2-4 V&1 J&3 C&3 J&1 C&1 E&3-1 TCR' V'(50) D'1 PD'1 J'1(6) C'1 D'2 J'2(6) C'2 E' V'14 TCR%/" V%/"(100) D" J" E" C" V"5 J%(61) TEA C% E% LCR BEAD-1 TCR# V#5 V#2V#4V#3 J#1 C#1 E#1 V#1. (2001). respectively. The two gray rectangles within the Igk locus represent the two start sites for Jk sterile transcripts (one of which is the KI/KII element). G. and Schatz. Some Vk genes. but pseudogenes have been omitted. D.Figure Legend Schematic representation of the murine antigen receptor loci. 78. Va and Vd gene segments are interspersed.

B Cell and T Cell Development
October 5
Instructor: Overview The overall goal of lymphocyte development is to efficiently generate lymphocytes that have functional receptors and that are not self-reactive. Lymphocytes arise from hematopoietic stem cells in the bone marrow. The HSC gives rise to more differentiated progenitors that either migrate to the thymus, where they develop into T cells, or remain in the bone marrow, where they generate B cells. The local environment—defined by so-called stromal cells—provides the signals (e.g., cytokines such as IL-7) that induce proper differentiation and also that guide lymphoid cells through subsequent steps. Thymocyte development takes three weeks to complete. The first week is spent expanding slowly in the outer cortex of the thymus, the second in rearranging their receptor genes and undergoing positive selection and negative selection, and the third week as maturing thymocytes in the medulla of the thymus. B lymphocytes undergo a similar differentiation process in the bone marrow. The first phase of lymphocyte development is concerned with the generation of functional antigen receptor genes by V(D)J recombination, which creates “in-frame” joints that encode the desired protein 1/3 of the time. TCRs and BCRs have two chains encoded by two separate loci. Rearrangements occur on only one locus at a time. If the rearrangement is successful, the developing lymphocyte will express the protein product of the first locus to rearrange (either TCRb or the IgH locus), along with another invariant protein that mimics the TCRa or IgL chain. Cells that express this “pre-receptor” receive signals that cause them to divide a few times and move to the next stage of differentiation. Cells that do not express the pre-receptor can continue to rearrange (i.e. on the other of the two chromosomal alleles). If neither allele forms a productive rearrangement, the cell dies. The progeny of cells that did successfully rearrange then go on to rearrange the other locus, and if successful, they will express a complete TCR or BCR, again allowing them to proceed in differentiation. Once the B and T cell receptor gene rearrangements have occurred, the receptor must be tested for whether it is harmful (i.e. anti-self) and useful (i.e. can be stimulated by Ag). This occurs via two processes termed negative and positive selection. Negative selection mainly involves interaction by the antigen receptor with antigens presented in the microenvironment in which they have undergone their development, the thymus for T cells and the bone marrow for B cells. Any immature lymphocyte that is strongly stimulated by a self-antigen is prevented from further development before it becomes fully functional. This is called negative selection. Since T cells need to see self MHC:self peptide complexes in order to be stimulated, they will only be useful if they have some affinity for self MHC. Cells are tested for this in the thymus during interactions with thymic epithelial cells in the cortex. T cells that receive a weak but definite signal are selected for survival—so called positive selection. Cells that are not positively selected die while those that are selected also undergo the negative selection process. B cells also undergo both positive and negative selection, but in this case negative selection comes first. Positive selection of B cells is much less well understood. Finally, lymphocytes that pass both negative and positive selection turn off expression of RAG1 and RAG2 and migrate, under the influence of chemokines and “homing receptors” to their respective locations in the peripheral organs, through which they continue to recirculate. D. Schatz

Step 1: Igh or TCRb gene rearrangement in pro-B or pro-T cells In both the T and B cell lineages, the first rearrangement is in the genes that contain V, D, and J gene segments (TCRb locus in T cells, the Igh locus in B cells), in a cell termed either a pro-T or pro-B cell. The earliest pro-T and pro-B cells turn on expression of RAG1 and RAG2 and perform D-to-J recombination. They then perform V-to-DJ recombination. When recombination occurs "in frame" (i.e., produces a continuous reading frame that can be translated into the full length TCRb or Igh protein), these molecules are expressed on the cell surface with surrogates which stand in for the TCRa or Ig light chain protein (which the cell can't make yet because it hasn't assembled these genes yet). These receptors, called the pre-T cell receptor and the pre-B cell receptor, drive enlargement of the cell and its rapid expansion through cell division, so that one properly rearranged locus can generate at least 100 identical progeny. As they finish this burst of proliferation, the cells are called pre-T or pre-B cells. Note: pro-T cells are also referred to as "double negatives" because they do not express CD4 or CD8; pre-T cells are also referred to as "double positives" because they express both CD4 and CD8.

Step 2: IgL or TCRa gene rearrangement in pre-B or pre-T cells The second phase of gene rearrangement involves genes that lack D gene segments (TCRa locus or Ig light chain loci--k or l), and thus can occur multiple times. This is particularly true of T cells, in which the TCRa locus consists of large arrays (~60 gene segments) in which V to J joining can occur several times. This means that most cells that have expanded from the original V—D—J rearrangement are provided with a functional receptor.

Step 3: Selection Positive selection: Once cells express a functional receptor, the suitability of that receptor can be tested. There is negative selection against self-reactive lymphocytes (see below) as well as positive selection for T cells that are going to be functional in the host. Positive selection of T cells selects for cells whose receptors bind some selfantigen weakly but detectably, which is critical to achieve MHC-restriction of recognition by T cells. The positive selection signal is critical for T cell survival and continued development. It occurs at the CD4+/CD8+ (“double positive”) thymocyte stage and is mediated by interactions with MHC I/II+ thymic epithelial cells. T cells which do not recognize a ligand remain developmentally arrested and continue to rearrange their TCRα chains, thus potentially replacing one protein chain with another; if the cell still fails to make a positively-selectable receptor, it will die. Cells which are positively selected by MHC I recognition mature into CD8+ (and CD4-) T cells while those selected by MHC II mature into CD4+ (and CD8-) T cells. Positive selection of B cells is less well understood. Negative selection: Developing B or T cells whose receptors bind self-antigens too well either die (in the case of T cells in the thymus) or undergo developmental arrest (B cells in the bone marrow). Such developmentally arrested B cells, like the T cells that have failed positive selection, will continue to rearrange their light chains and may make a new Ig receptor (a process called "receptor editing"). If the new receptor is not self-reactive, development will proceed, else the cell will die by apoptosis. In any case, both the T cell and B cell populations are thereby purged of self-reactive cells. In order for T cells to be negatively selected, they must see their cognate peptides displayed by antigen presenting cells (such as dendritic cells and macrophages) within the thymus. Many of these antigens are expressed only in certain cell types not present in the thymus (for example, insulin, which is produced by cells of the pancreas). Thymic antigen presenting cells express a gene called autoimmune regulator (AIRE) that enables many such proteins to be expressed at low levels in thymus.

Numbers In the mouse, cortical thymocytes are created at a rate of 20-40 x 106/day, yet only 1-2 x 106 leave the thymus each day. The rest must therefore die in the thymus, almost certainly as a result of negative selection or a failure to receive a positive selection signal (called "death by neglect"). About 80% of the B cells that are produced in the bone marrow fail to find a proper survival "niche" in the periphery and hence die quickly (≈ 3 days). The other 20% become mature naïve B cells and live for about two months. Why some B cells are selected into the long-lived pool and others are not is not understood, but this has been termed B cell "positive selection". A mouse contains about 108 mature peripheral B cells, about 2% (2 x 106) of which die each day. Mouse bone marrow produces about 107 new B cells per day; the 20% that are "positively selected" to become long lived mature B cells replace those that die each day. Reading: Text: "Janeway’s Immunobiology", by K. Murphy (Garland Science), 2011, 8th Edition, Chapter 5, pp 162-173; Chapter 8, pp 275-316. Review Article: Schebesta, M., Heavey, B., and Busslinger, M. (2002). Transcriptional control of B-cell development, Curr. Opin. Immunol. 14, 216-223. Primary Literature : He, X., He, X., Dave, V.P., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B.A., and Kappes, D.J. (2005). The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433, 826-833.

Positioning, Maturation & Trafficking of Lymphocytes
October 8
Instructor: I. J. Pereira

Overview. Lymphocytes are continuously moving within and between lymphoid organs since early stages of their differention. Their movement is largely controlled by three types of transmembrane receptors: G proteincoupled receptor, integrins and selectins. These family of receptors recognize specific extracellular signals and adhesion molecules that guide and retain lymphocytes in specific locations during their development in primary lymphoid organs, and maturation in secondary lymphoid organs. This lecture explains how lymphocyes are organized within primary and secondary lymphoid organs, and why such compartmentalization is important for the immune system to function apropriately. Thymus Structure and Thymocyte Positioning During Development a. DN (CD4-CD8-) cells develop in the cortex and subcapsular region. b. DP (CD4+CD8+) thymocytes are positively selected at the cortico-medullary junction. c. SP (CD4+ or CD8+) lymphocytes are negatively selected in the medulla. d. Non-autoreactive SP-lymphocytes egress via thymic blood vessels at the cortico-medullary junction. Egress is mediated by S1P1 expressed on lymphocytes, and its ligand S1P, which is abundant in blood. B Cell Development in bone marrow niches. a. CXCR4 is fundamental for bone marrow (BM) derived hematopoiesis. Retention of hematopoietic cells in the BM requires CXCR4 and its ligand CXCL12. b. The bone marrow cellular organization is poorly understood. It is characterized by two distinct compartments: parenchymal tissue composed by developing hematopoietic cells, progenitors, stromal cells, adipocytes, osteoblasts, osteoclasts, and chondrocytes; and by a network of small sinusoids that perfuse the marrow and anastomose into large collecting and central sinusoid. In late stages of B cell deleopment, immature B lymphocytes become distributed between parenchyma and sinusoids before exiting into the spleen for further maturation c. In the parenchyma, immature B lymphocytes are negatively selected against membrane-bound selfantigens. Secondary lymphoid organs (SLOs). a. SLOs include Spleen (Sp), Lymph Nodes (LN), Peyer’s Patches (PP), Mucosal-associated lymphoid tissue (MALT) such as bronchial (BALT), nasal (NALT) and gut (GALT), peritoneal and pleural cavity “milky spots”. Spleen is divided in two compartments: white pulp and red pulp. b. Main functions: i. to filter antigens from body fluids; ii. bring together antigen, antigen-presenting cells and antigen-specific lymphocytes iii. support lymphocyte activation and differentiation events c. SLOs are mainly populated by B and T lymphocyte subsets, and smaller numbers of antigen-presenting cells (e.g. interdigitating dendritic cells (IDCs), follicular dendritic cells (FDCs), macrophages), and stromal cells. d. The peripheral B cell compartment is made by B1 cells (mostly in the peritoneal cavity), B2 cells (often called follicular B cells), and marginal zone B cells (MZB, found in the mouse spleen, and in human spleen and LNs). In mice, MZB are located at the interface between Red and White pulp, are exposed to incoming blood, and respond vigorously to blood-borne pathogens. B1 cells populate body cavities exposed to potential pathogens. Both MZB and B1 cells respond faster than B2 cells to antigens of microbial origin (e.g. LPS). e. B2 lymphocytes reside in follicles. FDCs predominate at the center follicle. Specialized follicular stromal cells pave the entire follicle. f. T lymphocytes are distributed in the T cell zone around central arterioles. IDCs, and T-zone stromal cells also populate t-zone.

II.

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Chemokines and Integrins. a. Chemokines are chemoattractant proteins: these are the “scents” that guide cells to specific locations. b. Chemokines are categorized structurally by location of cysteine residues (CC, CXC, C, CX3C). c. Chemokine receptors belong to the G protein-coupled receptor family and are characterized by 7 transmembrane domains that serpentine through the membrane. Induction of chemotaxis depends predominantly on receptor coupling to Gαi proteins. d. Chemokines play two fundamental roles: i. lymphoid or homeostatic chemokines organize the lymphoid organ compartments: CCL19 and CCL21 and their shared receptor CCR7; CXCL13 and its receptor CXCR5; and CXCL12 and its receptor CXCR4. ii. proinflammatory: RANTES, MCP-1, IL-8 e. Integrins are obligate heterodimers composed of an alpha and beta subunits both of which containing a single transmembrane domain. f. Integrins can be in an inactive (non-sticky) and active (sticky) states. Integrin activation requires GPCR signaling. g. Integrins play two basic functions: i. attachment of the cell to the extracellular matrix (ECM) ii. signaling from ECM to cell h. Lymphocytes mainly express 3 integrin heterpdimers: α4β1, αLβ2, and α4β7. Cues organizing SLOs: Integrins, Chemokines and receptors. a. B and T lymphocytes require integrins (αLβ2 and α4β1) for entry into splenic white pulp cords. b. B cells require CXCR5 for homing into follicles; FDCs and follicular stromal cells express the CXCR5 ligand, CXCL13 (a.k.a. BLC – B Lymphocyte Chemoattractant). c. CCR7, and its ligands CCL19 and CCL21 produced by T-zone stromal cells guide cells to the T-zone. d. T cells migrate within the T-zone with an average speed of 12µm/min. As many as 5000 T cells can interact with a single DC per hour. e. B cells move within follicles at about 6µm/min where they survey for antigen displayed on FDCs. d. MZB cells utilize S1P1 and CXCR5 to position at the interface between the red (S1P is abundant in blood) and white pulp (CXCL13 is abundant in white pulp follicles). e. Retention of MZB cells in the MZ also depends on integrins αLβ2 and α4β1 and their ligands ICAM1 and VCAM1, respectively. f. MZB and B1 cell development requires Gαi coupled CXCR5 and CXCL13. Entry in SLOs a. Entry in LNs occurs through specialized blood vessels called High Endothelial Venules (HEVs). HEVs express vascular addressins (GlyCAM-1, PECAM-1, PNAd); recognized by L-selectin expressed on lymphocytes. This event slows down lymphocytes circulating in blood. b. Lymphocyte entry also requires αLβ2 and α4 integrins, and integrin ligands (VCAM-1, ICAM-1 and -2, MadCAM) expressed on HEVs. Activated integrins bring rolling lymphocytes to full arrest and allow transendothelial migration and movement into the lymphoid tissue. c. Entry in the spleen is not via HEV but through terminal open arterioles. Cells are in fact released into the red pulp. e. CCR7, CCL19 and CCL21 are required for T cell entry in LNs; while CCR7, CXCR4, CCL19, CCL21, and CXCL12 promote B cell entry in LNs. B cell homing to PPs is dependent upon CXCR5 and CXCL13. Exit from SLOs a. Egress of B and T lymphocytes requires S1P1 expression, and a gradient of its ligand S1P. S1P is kept at low concentrations in lymphoid organs by the S1P-degrading enzyme S1P lyase, whereas blood and lymph contain high concentrations of S1P. Egress from spleen occurs via blood; LN exit is via the lymphatics. b. Current model hypothesizes that the time spent in lymphoid organs is dictated by a “tug of war” between lymphoid organ retention cues (e.g. CXCL13, CCL19, CCL21) and egress promoting cues (S1P at exit sites).

VI.

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Janeway. 8th Edition. Walport and M.” 2005.. Immunobiology: The immune system in health and disease. Garland Science.. “Chemokines. Annu.A. Travers.c. Immunol . Activated lymphocytes rapidly up-regulate CD69 that counteracts S1P1 function. JG. Rev. S1P and Cell migration in lymphoid organs. This prevents activated T and B lymphocytes from exiting SLOs during an immune response. Reading: Text: C. Chapter 8. 2012. P. Shlomchik. Jr. Lymphoid organ shutdown and blocked egress. M. Review articles: Cyster. Ltd.

In particular. Cytokine Receptor induced JAK/STAT signaling pathway Type I and Type II cytokine receptors are typically non-covalently associated with protein tyrosine kinases of the Janus kinase (JAK) family. and fyn and lck for the TCR. through the activation of death receptors (extrinsic pathway). FADD contains a death effector domain (DED) that allows for the recruitment of the initiator caspase.g. . d. This leads to a conformational change that allows for STATs homo or heterodimerization. The death receptors (e. Antigen receptor signaling The capacity of T cells and B cells to specifically recognize and respond to antigens is fundamental to the activation of the adaptive immune system. leading to cell proliferation and differentiation. Jak3 and Tyk2. Activation of the death receptors by their cognate ligands leads to clustering of the death domains and the recruitment of specific adaptor proteins. Apoptosis can also occur through the activation of an intrinsic pathway that involves the release of cytochrome c from the mitochondria upon exposure to a noxious stimulus. For example. Activation of these tyrosine kinases and the subsequent phosphorylation of downstream targets initiates a signaling cascade that culminates in the activation of the transcription factors NFkB. such as the enzymatic fragmentation of chromosomal DNA. while the TCRa:b heterodimer is associated with three CD3 chains (g. Activation of cytokine receptors leads to the trans-phosphorylation and consequent activation of the associated JAKs. Aggregation leads to the activation of tyrosine kinases that are associated with these cell surface molecules. and CD4 or CD8 molecule in T-cells. Common to both pathways is the activation of specialized proteases called caspases. it has an important role in the termination of the immune responses and removal of potentially autoreactive lymphocytes. Surface immunoglobulins are associated with two polypeptides termed Iga and Igb. The tyrosine kinases that have been found associated with antigen receptors include the Src family members: blk. activation of the Fas receptor leads to the recruitment of FADD (Fas-associated via death domain). Apoptosis can be induced by extracellular ligands. Jak2. Assembly of the antigenbinding chains with these accessory proteins not only provides adequate signaling capacity. Death receptor induced signaling pathway Programmed cell death or apoptosis plays a key role in the regulation of the immune function. their translocation to the nucleus and induction of gene expression. The activated JAKs then phosphorylate their associated cytokine receptors on specific tyrosine residues that generate binding sites for the transcription factors known as signal transducers and activators of transcription (STATs). Optimal signaling also requires co-aggregation of coreceptors such as CD19 in B-cells. This step is followed by the activation of effector caspases leading to a series of events that are hallmarks of apoptosis. Rothlin In this lecture we will review signaling pathways that are central to lymphocytes’ biology. Therefore. fyn and lyn for the B-cell receptor. syk in B-cells and ZAP-70 in T-cells are recruited to the signaling complex upon activation.Receptors & Signaling II October 10 Instructor: Carla V. Recruitment of the STATs to the activated cytokine receptor brings them to proximity to the activated JAKs. NFAT and AP-1 to induce specific gene expression. e) and two z chains. Pro-caspase 8 is self-activated by proteolytic cleavage and subsequently released from the receptor complex. This family is formed by four members: Jak1.: Fas and TNFR-I) are members of the large TNF receptor family and contain a conserved cytoplasmic domain known as death domain (DD). B-cell and T-cell antigen receptors are made up of variable antigen-binding chains (immunoglobulin chains in the B-cell receptor and TCRa:b chains in the T-cell receptor) that lack intrinsic signaling activity. In addition two soluble tyrosine kinases. which induce STAT phosphorylation. but also allows the correct transport of the complex to the plasma membrane. they are associated with invariant proteins that possess the capacity to activate downstream signaling pathways. procaspase 8.

Reading: Text: "Janeway’s Immunobiology". 8th Edition. Chapter 7. Taylor & Francis Group. 2012. . by Kenneth Murphy (Garland Science. LLC).

CD4 and CD8 T cells. CD4 T cells serve many roles in the immune response. quickly respond to eliminate infected cells and pathogen replication. OVERVIEW All adaptive immune responses are mediated by the activation and clonal expansion of antigen-specific T and B lymphocytes. 3. The T cells become activated in the draining lymph nodes when they encounter their cognate antigen on the dendritic cells that have migrated into the lymph nodes from the infected tissue. 4. (2) Contraction (i.Innate Control and Initiation of Adaptive Immune Responses October 15 Instructor: J. the types of signals elicited by the innate immune system. ANAMTOMY OF THE FIRST KISS: Naïve T cells specific for a given virus are very rare and present at very low frequency in the LNs. These memory T cells can mount rapid secondary responses to reinfection and help to protect against reoccurring disease and illness. Expression of chemokine receptors on T cells and DCs regulate their localization in the lymphoid organs to ensure they find each other. PHASES OF THE T CELL RESPONSE: The T cell response can be characterized by three distinct stages (1) Activation and Clonal Expansion. that recognize small peptides presented on MHC class II and class I. Following removal of the pathogen. upon encountering their specific antigen in the periphery. CD8+ T cells see antigen presented by the ubiquitous MHC class I molecules and induce the death of any cell that they detect as infected. INNATE RECOGNITION of INFECTION by DENDRITIC CELLS PERMITS T CELL ACTIVATION: Tissue resident dendritic cells (DCs) sense the invading pathogens at the site of infection and become “activated”. Naive Activation/ Expansion Contraction Maintenance of Memory CD8 T cell response Virus 1 5 8 15 30 60 days post infection . 2. but a minority persist as long-lived memory T cells. respectively. Craft MAIN POINTS 1. T CELL TYPES: There are two primary types of T cells.. in accordance. The effector functions expressed by antigen-specific T cells differ depending on the type of infection at hand and.e. death of the activated T cells) and (3) Formation of memory T cells. This switches them into professional APCs that upregulate lymph node (LN) homing receptors and causes them to migrate into the draining LNs. the majority of the activated T cells die. This is followed by their differentiation into effector cells that. whereas CD4+ T cells recognize antigen presented by MHC class II molecules that are restricted in their distribution in the periphery.

Primary Literature: Kearney ER. Homing to lymph nodes and other secondary lymphoid tissues is also mediated by specific chemokines. 8th Edition. 2011. Pape KA. as well as the importance of the trafficking of lymphocytes through the lymph nodes in search of their specific antigen. It also causes the DCs to mature into cells that have lost the ability to take up antigen. mediated initially by pairs of cell adhesion molecules. the specific recognition of pathogen associated molecular patterns (PAMPs) by the tissue resident DCs and Mφ has two very critical effects for immunity—the first is that this causes the innate immune cells to directly release cytokines and other molecules that will directly attack and kill microbes. highly phagocytic cells with little ability to activate T cells. T cells re-circulate actively from the blood into the lymph nodes. Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. 1(4):327-39. produced by endothelial cells. In summary. which is a highly organized structure of proteins between the adhesion of a DC and an antigen-specific T cell. via binding to endothelial cells by adhesion molecules. but have gained the ability to present antigens to T cells. such as the role of tissue dendritic cells in bringing antigens to the local lymph nodes. in those rare instances when recognition occurs. and subsequent migration through the endothelial cell barrier. LLC). we will review concepts that were introduced earlier. and costimulatory molecules for T cell activation. although it does receive weak signals through its receptor that allow it to survive. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. However. 1994 Jul. Taylor & Francis Group.In this lecture. especially in sites of infection. Once in these secondary lymphoid organs. Immunity. Dendritic cells acquire antigen in the peripheral tissues. which upregulates expression of lymph node homing receptors. Regulation of this switch in DC function is critical to prevent autoimmunity as it is the primary measure that ensure T cells are only activated when a pathogen is present and requires a T cell response for its removal. The encounter with pathogens. The second is that it switches a DC from a “tolerogenic” DC into an “immunogenic” DC. . after pathogen invasion. Chapter 9 (9-1 through 9-9) and Chapter 3 (3-13 through 3-19). Loh DY. where they reside as immature. the lymphocyte does not encounter its specific antigen. naïve T cells then survey the surfaces of dendritic cells for their specific antigen. This leads to formation of an immunological synapse. MHC:antigen complexes. stromal cells and dendritic cells that bind receptors on T cells. via specific receptors of the innate immune system that bind common molecules on the surfaces of pathogens (called pathogen recognition receptors (PRRs)) induce signal transduction events that lead to their secretion of Type I IFNs. the T cell ceases to re-circulate and sets up stable interactions with the antigen-presenting dendritic cell. antimicrobial peptides and chemokines to attract other leukocytes to the site of infection. In the vast majority of cases. Jenkins MK. They also begin to express new molecules that cause their migration to the local lymph nodes (such as CCR7) and increase expression of both classes of MHC molecules and co-stimulatory molecules CD80 and CD86 to activate both CD4+ and CD8+ T cells against the pathogen.

T Cell Priming & Effector Cell Differentiation I October 17 Instructor: J. ICOSL:ICOS). 41BBL:41BB. 2. they receive 3 types of critical signals that are necessary for T cell activation and differentiation into effector T cells. IL-4. Such inflammatory cytokines are IL12. As the T cells are engaged in a tight adhesion with the DCs. OVERVIEW The immunological synapse is composed of Central-SMAC (SupraMolecular Adhesion Complex) that contains the TCR/Ag:MHC. Several types of costimulatory ligand:receptor pairs exist. costimulatory receptors and signaling molecules. The immunological synapse directs the movement of the MTOC (microtubule organizing center) to the site of contact. IL-21. in which inflammatory cytokines produce by the innate immune DCs. signal 1 (antigen). die or will persist in an undifferentiated state with little-to-no effector functions. If T cells do not receive all 3 signals or only interact with the DC very briefly. The 3rd signal is inflammation. C4 or CD8 coreceptors. The second signal comes from costimulatory ligands expressed by activated DCs that activate costimlatory receptors on T cells and enhance TCR signaling. leading to production of the T cell growth factor IL-2 and synthesis of its receptor. IFNγ. signal 2 (costimulation) and signal 3 (inflammation) invoke signal transduction. (B7:CD28. CD70:CD27. that all 3 signals are dependent on . IL-6+TGFβ. ORGANIZATION and DURATION of an IMMUNOLOGICAL SYNAPSE: The inner C-SMAC and outer PSMAC and can persist for more than 7hrs. The first is the direct recognition of antigen (MHC-peptide complexes) by the T Cell Receptor (TCR) that leads to its activation. Craft MAIN POINTS 1. signal 2 (costimulation) and signal 3 (effector-determining inflammatory cytokines). CD40L:CD40. events leading to clonal expansion and eventually to clonal differentiation into effector T cells. NK cells and macrophages help to skew the differentiation of the T cells to develop the appropriate type of effector functions to fight the present infection. The Peripheral-SMAC contains adhesion molecules such as LFA-1 and talin. Collectively. Recent evidence suggests that the first T cell division is likely to be asymmetric leading the unequal distribution of molecules that influence effector T cell differentiation. T CELL PRIMING: THE 3 SIGNAL HYPOTHESIS: Effector T cell clonal expansion and differentiation is governed by three major signals: Signal 1(antigen:TCR). Note. the T cells will either become anergic.

Taylor & Francis Group. LLC). the innate immune system ultimately determines whether a T cell response will be triggered or not. The activation of T cells is balanced by internalization of TCR. Kupfer A. 2011. . Kupfer H. Sciaky N. Primary literature: Monks CR. Freiberg BA. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. Thus. 8th Edition. Bystander innate immune cell Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. 1998 Sep 3.the proper sensing of PAMPs and activation of the innate immune cells. TGF-β and activation induced cell death (AICD) to ensure activated T cells to not grow out of control. Chapter 9 (9-10 through 9-31). expression inhibitory receptors such as CTLA4. 395(6697):82-6.

these cytokines cause activated B cells to produce IgE. which activates Mφ to become highly anti-microbial. TYPES of EFFECTOR and MEMORY T CELLS PRODUCED: CTLs. CCL10 produced at the sites of infection to enable their recruitment to these sites. TH1 cells also help CD8 T cell activation and activate B cells to produce IgG2a and IgG2b. which via production of IL-17 and IL-22 lead to the recruitment of neutrophils to eliminate the bacterium. CTLs secrete the antiviral cytokine IFNγ and can kill on contact any cell in the body that harbors a cytosolic pathogen.T Cell Priming & Effector Cell Differentiation II October 19 Instructor: J. mast cells and macrophages to attack and phagocytose the large foreign bodies. TFH and TREG 3. lung. Th2 cells primarily produce IL-4. In addition. CD8 kill cells via the expression cytolytic molecules called perforins and granzymes. (iv) Other types of T cells that need to be discussed are the formation of follicular helper T cells (T FH) which migrate into the B cell follicle to help B cells divide. adhesion receptors and many other genes. CD8 T cells recognize antigen presented on MHC class I (a ubiquitously expressed molecule) and differentiate into cytotoxic T lymphocytes (CTLs) that can kill infected cells upon direct contact by delivering cytotoxic granules or Fas ligand. TREGS can also be induced to balance the activation of T cells to reduce tissue destruction and immunopathology. Many other changes occur in the activated T cells as they begin to express new effector molecules. A Th2 response is mainly aimed at destroying large. (ii) Infection of helminths (worms) and certain parasites and extracellular bacteria induce a TH2 CD4 T cell response. IL-5 an IL-13 to activate eosinophils. Th1 CD4 T cells are also induced during these types of infections and secrete IFNγ. CCL9. Both CTLs and TH1 cells express Fas ligand and can kill cells expessing the death receptor. class switch and mutate. These cytokines turn on key transcription factors of T cell differentiation that then induce the T cells to adopt particular traits to fight the infection. basophils. TH1. (iii) Some types of extracellular bacterial infections can also induce TH17 responses. T cells leave the APC and downregulate the LN homing molecules and depart into the peripheral tissues via the blood. When CD4+ T cells recognize antigen they can differentiate into a variety of different types of cells that perform specific functions depending on the nature of the infection or stimulus at hand: (i) Viral and intracellular bacterial infections induce the formation CTLs and TH1 CD4 T cells to enable direct killing of infected cells and intracellular pathogens. Craft MAIN POINTS 1. They also migrate to most peripheral tissues including liver. Most regulatory T cells (TREGS) form in the thymus and suppress the responses of any “escapee” CD8 or CD4 T cells in the periphery. and gut. TH2. TH17. Fas. . But during some infections. The different types of T cells are formed in response to particular innate immune cytokines that “specify” the appropriate type of T cell to form according to the pathogen that is infecting the host. The simultaneously upregulate chemokine receptors that detect inflammatory chemokines such as CCL5. Activated T cells leave the LN and go to sites of inflammation/ infection 2. These activated T cells divide at an amazingly fast rate (every 4-6 hrs) and they upregulate telomerase to permit the cells to divide a massive number of times (~15-20 times within a 5-7 days). extracellular pathogens and thus. OVERVIEW Upon activation.

This is how signal 3 (inflammation) controls the type of T cells produced to ensure that the right type of T cell is produced for the right type of infection. . immunity. Figure from Anton M. Jetten. e003. circadian rhythm. These major TFs are shown in the figure. Retinoid-related orphan receptors (RORs): critical roles in development.The differentiation of these different lineages of T cells is determined by key transcription factors (TFs) that are induced by the appropriate types of inflammatory cytokines. and cellular metabolism. Nuclear Receptor Signaling (2009) 7.

T-bet. Fathman CG and Glimcher LH. A novel transcription factor. Chapter 9 (9-10 through 9-31).IL-2 IFNTNF Anti-Viral Anti-Bacterial (intracellular) Activate M IL-4 IL-5 IL-13 IL-10 TGFParasite Infections Allergy (IgE) Reading: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. 2000 Mar 17. Primary literature: Szabo SJ. 2011. Costa GL. LLC). Kim ST. directs Th1 lineage commitment. Taylor & Francis Group. Cell. Zhang X. 8th Edition. 100(6):655-69. .

By reorienting the MTOC to the immunological synapse. Perforin/ Granzymes are stored in granules in the T cells as preformed proteins that can be immediately expelled upon TCR triggering. b. Consequences of sloppiness would be excessive immunopathology and disease or even death of host. Sometimes they even use proteins similar to our own as decoys to their advantage. Secreted cytokines can act on the cell that produced them (autocrine) or on another cell (paracrine). The molecules are precisely delivered to target cells and only upon TCR recognition (ON-OFF-ON) OVERVIEW As T cells differentiate into effector cells they become armed with the appropriate weapons that aide in pathogen eradication.Effector Molecules – Function & Signaling October 22 Instructor: J.CCL3. 2. .directly kill infected cells (or other antigen bearing cells such as APCs) a. The T cells quickly shut off synthesis of these proteins in the absence of TCR engagement. They can work locally and for short periods of time. The effector mechanisms used to clear an infection depend on the infectious agent. Type I interferons (IFN-α/β) produced by DCs. Pathogens have devised many ways to evade the attack of a T cell. IL-5. Thus. Cytokinesa. the pathogens have devised numerous ways to circumvent or avoid these attacks. Fas Ligand (expressed by T cell)/ Fas (expressed on Target cell) T cells use extreme stringency and precision to direct the effector molecules to the infected cells and avoid uninfected cells. IL-21) such as B cells b. stimulator of Mφ. Interleukins. 2.T and B cell factors that act as growth factors (IL-2) or modulate other lymphocytes (IL-4. these potential harmful proteins are only being made when there is antigen and infected cells present. there is polarized delivery of the effector molecules to the target cells. This occurs in an ON-OFF-ON type of mode. In general. Cytotoxic Molecules. 4. 5. Interferon gamma (IFNγ ). Through millions of years of evolution. Mφ and infected cells are also anti-viral. our T cells have become quite adept at producing the molecules that the pathogens we encounter in our environments are most vulnerable too. Craft MAIN POINTS 1. TNFα 3. T cells produce 3 main category of molecules: 1. Cytokines can be membrane bound or secreted. c. now the T cells are interacting with infected cells that are not necessarily professional APCs (don’t receive costimulation). The TCR is still required for release of effector molecules.anti-viral factor. Chemokines and vasodilators. through their own evolutionary process. Likewise. however.

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Viruses and other pathogens have created clever ways of inhibiting effector molecule function to keep the infected cell alive and functioning and some of these will be discussed. Sacco MG. Giliani S. Resolution of Immune Response: After pathogen clearance the macrophages “clean up” and engulf dead cells and debris. LLC). The effector T cells begin to die (death by neglect) and a few survive to become long live d memory T cells. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Candotti F.377(6544):65-8. 1995 Sep 7. Fas/Fas Ligand. Primary literature: Macchi P. . Ugazio AG. 8th Edition.activates Caspase cascade in target cell upon receiving FAS Ligand signal from effector cell. Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. 2011. Frattini A.Cytotoxic Functions of Effector Cells Granzyme/ Perforins: Enter cells and activate Caspase cascade that induces rapid apoptosis of target cell. Villa A. O'Shea JJ. Porta F. Formation of these memory T cells are critical to long-term immunity and the ultimate goal of vaccination. Chapter 9 (9-10 through 9-31) and Chapter 7 (7-19 through 7-22). Memory T cells can rapidly expand and exert effector functions to more rapidly combat secondary infection. et al. Johnston JA. Nature. Taylor & Francis Group.

just outside the marginal sinus. M. called EBI-2 is expressed. Upon binding Ag. enabling the B cells to migrate outward from the center of the follicle.Shlomchik marginal sinus T zone (pe ria rter iolar lymphoid sheathPALS) 1° B cell follicle Germinal center central arteriole Upon encounter with Ag. which allows B cells to migrate to follicles. In addition. differentiation and migration to constitute the primary B cell immune response. which is the receptor for chemokines secreted in the T cell zone. B cell trafficking and splenic architecture B cells traffic through blood. lymph nodes. where T cells and B cells interact and begin proliferating. This is accomplished in part by changes in chemokine receptor expression that control where B cells migrate. Most B cells are in spleen. B cells in the follicle recirculate to blood occasionally. B cells stop migrating and arrest at the border of the T and B zone. not shown below). is increased. (Some B cells remain resident and nonrecirculating in the marginal zone. where most splenic B cells reside. . allowing B cells to arrest near the T cell zone. Expression of CCR7. exiting in the marginal sinus from where they migrate to the B cell follicle. Expression of CXCR5. a chemokine receptor (for the newly identified ligands termed “oxysterols”). and blood. There are defined sequences of events and these occur in defined microanatomical locations within spleen or lymph node. B cells undergo proliferation.Primary B Cell Immune Responses October 29 Instructor: Overview: B cells recognize intact antigens (Ags). and upon interaction with activated T cells. They encounter and respond to Ags that have reached these compartments. is reduced.

but some will migrate to the red pulp areas of the spleen. the entire B cell activation and differentiation program is highly dependent on T cell signals. and these too take on a unique identity as a “T follicular helper” cell. Here they will continue proliferating and will form the “germinal center” (GC). IL5. IL5. nonphagocytic dendritiform cells with uncertain but intriguing function (see below). After this initial encounter. responding to signals from T cells that include cytokines such as IL4.Molecular nature of the T-B interaction There is a complicated interplay between T and B cell. the same factor is required for T cells to enter the GC. one exception is certain highly repetitive antigenic surfaces or antigens that also contain B cell mitogens. 8 or 9. as well as a variety of other molecular pairs that interact. and ICOS (inducible costimulator) and ICOS-ligand. Interestingly. involving T cell recognition of processed peptide Ag fragments that are present on the B cell’s MHC Class II molecules. B cells can also secrete cytokines that may affect themselves and T cells. IL21 and IFN-γ. (Isotype switch will be covered in detail in the next lecture). These pairs include CD40L and CD40. IL6. It is thought that the very high amount of cross-linking that these latter antigens provide to the BCR are sufficient to bypass the need for T cell derived signals. which continue to divide and differentiate at the same time. where they stop dividing and can live for several weeks. Differentiation and migration are the next steps. CD80/86 molecules and CD28. Some B cells. each descended from an initial Ag-specific B cell accumulate at the border of the T-B zones. and may express IgG. In the GC three areas can thus be discerned: a “dark zone” (DZ) with many proliferating cells and which is located closer to the T cell zone. Although most B cells at this stage will continue to express IgM isotype. and a surrounding marginal zone that includes the surrounding B cells. In the follicles are other cells called follicular dendritic cells which are unusual. Typically. some cells will begin the process of isotype switch. B cells can be activated by certain antigens even without T cell help Although typically. These cells and increase the production of Ig secreted mRNA by 100 to 1000-fold. IL6. causing the B cells to adapt a unique “GC” phenotype and suppressing their ability to become plasma cells. the germinal center grows and tends to push out surrounding non-proliferating B cells. They will also lose expression of many of the surface molecules that characterize B lineage cells—they become mini-factories for Ab secretion. such as a T-dendritic cell interaction. they migrate to the border of the T cell zone and the red pulp. Recent data indicates that this transcription factor is expressed very early on by the cells that will go on to become GC B cells and T follicular helper cells and that this factor controls expression of genes essential for GC B and T cell function. T-dependent B cell activation and differentiation at the T-B border Proliferation is the initial consequence of Ag and T cell encounter. where they continue to proliferate. These so-called T-independent responses can be divided into two types: TI-1 and TI-2. The details of this T-B interaction have been covered elsewhere in the course as they are typical of any T cell-Antigen Presenting Cell (APC) interaction. As the initial proliferating cells generate more progeny. these TI responses undergo limited isotype switching and they do not form germinal center and memory B cells. Germinal centers (GC) are comprised of highly proliferative B cells (mainly) along with some T cells. . TI-1 responses are elicited by molecules with inherent mitogenic ability for B cells. Other B cells along with some T cells will migrate away from the T zone-red pulp border—at this point downregulating expression of EBI-2—into the center of the B cell follicles. the roles of B cell cytokine secretion are not well understood yet. Small clones of B cells. cytokines are released by activated T cells that have effects on both B and T cells. and IL21. Germinal Center Overview Architecture: “Germinal” means “growing”. where it controls the expression of a large number of genes. They evolve in the “B cell zones” or “follicles” of lymphoid organs such as spleen and lymph node. The transcriptional repressor factor Bcl6 is essential for the formation of GCs. Bcl6 is turned on in nascent GC B cells. TI-2 responses on the other hand are elicited by highly repetitive antigenic structures such as bacterial polysaccharide cell wall or certain viral coat structures. Most of these cells live only a few days. a “light zone” (LZ) which is less densely packed and contains many FDCs and most of the T cells. processes that will be described below. become antibody-forming or “plasmablasts”. such as bacterial lipopolysaccharide or nucleic acids that bind TLR7. These include IL2. these are said to be relatively “T-cell independent”. IL4. In addition.

and d) isotype switching. 2011. 387 – 407. c) cellular selection. an t ig en CD4+ T cell * B cell T-B zone interface (outer PALS) Primary B cell follicle Late Germinal Center plasma cell differentiation migration to follicle Follicular Dendritic cell (FDC) memory B cell differentiation mutation. plasma cell differentiation (which means the cell begins making and secreting more Ig. stops dividing and probably leaves the GC). Chapter 10 pp. Reading: Text: "Janeway’s Immunobiology". peak at day 10 to 14 and are mainly over by 4-8 weeks. These will be dealt with in the upcoming lecture on memory. light zone dark zone memory B cell maintenance Figure 2: Diagram of the B cell immune response. in which point mutations are introduced into the V region. Events in the GC: Four important events occur in the GC: a) clonal expansion and differentiation. 8th Edition. and leaves the GC probably for the bone marrow). since most are unsuccessful in competition and selection. . Taylor & Francis Group. LLC). where most differentiation takes place. stops dividing. Proliferation: B cells divide as quickly as every 6 to 8 hrs. perhaps due to deleterious somatic mutations or just low affinity). the initial seeding may be by 50 to 100 cells.Clonal Expansion Initiation: Initial seeding of the GC comes from cells that were originally activated at the T-B zone interface. Initial proliferation may occur in the “dark zone” while cells in the “light zone” proliferate but at a somewhat lower rate. with a net flow of cells from the DZ to the LZ. Exactly how cells migrate between dark and light zone is not entirely clear but most likely cells recirculate within their respective zones. with emphasis on the GC reaction. in a GC. isotype switch and differentiation The last three will be covered in the next lecture. by Kenneth Murphy (Garland Science. In mouse spleen GC responses begin about 5-6 days after immunization. Fate of cells in the GC: Cells that survive in the GC can have at least three fates: memory cell differentiation (which means the cell becomes long-lived. Although GC are ultimately comprised of the descendents of only a few cells. b) somatic mutation. or apoptosis (which is probably the result of failure to get sIg or T cell signals.

Okada. Haberman. A.1991. Krummel..Primary literature: Kerfoot. Germinal center B cell and T follicular helper cell development initiates in the interfollicular zone. Jacob. Yaari. O'Garra. Hartle. Rajewsky and U. Kelsoe. Miller. K. Nature 354: 389-392. M.R. G. 2005.. T.M. Parker.H. Patel. Kleinstein. Immunity. G. S.. . Cahalan.M. S. D. and A. PLOS Biology 3 (6): e150. J.L. Johnson. K. Intraclonal generation of antibody mutants in germinal centres. Cyster. S. Gonzalez. Antigen engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. Weiss. J. M. M. 2011.G. M. Neighbors. and J. I. 34:947-60.

How mutation is so specifically targeted is not totally clear. The discovery led to two theories of how AID might work. The biological impact of isotype switch will be covered in the next lecture on humoral immune responses. so named because the gene had strong homology to a family of enzymes that are know to modify RNA (RNA editing enzymes) by deamination. Indeed. Nonetheless. In the first theory. albeit at substantially lower rates than in the V region (10-100 fold lower). However. recent data indicates that SHM occurs in many genes in a B cell. mutations outside of V regions can promote cellular transformation and indeed have been implicated in B cell lymphomas. a gene was discovered that is absolutely required for somatic hypermutation to occur. The basis for relative targeting of the SHM machinery is still not clear. Activated B cells can also rearrange the DNA in the IgH constant region leading to a switch in the isotype expressed. Mechanism: General features of SHM: Somatic mutations are introduced into V regions of dividing B cells at the rate of approximately 0. there is less somatic point mutation and instead a process of gene conversion in which small regions of one gene are replaced by the sequences of a nearby homologous but different gene via a process of unequal recombination. Enzymology of hypermutation: Several years ago.5 mutations per cell per generation. these mutations are repaired and their existence was only revealed when repair pathways were genetically disabled. the V region can undergo point mutations (and in some animals gene conversion) in the germinal center. The final appearance of mutation is likely a balance between the introduction of mutations and the efficiency of their repair. nor is it clear why some genes have efficient mutation repair and others not. They are mainly single point mutations. Transcription is also required for mutation to take place. recent discoveries indicate that mutation can also occur in chronic immune responses that occur in other areas of lymphoid and even non-lymphoid tissue. Somatic Hypermutation Control of mutation: Somatic mutation is not a constitutive process in B cells. This rate is approximately 10 times higher than the background mutation rate in normal mitosis. as can other signals that may include simultaneous BCR. Though we won’t discuss it much. It is likely that signals through the BCR are absolutely required. but never reaching the downstream constant region.Mechanisms of Somatic Hypermutation and Isotype Switch and Their Consequences October 31 Instructor: M. Shlomchik Introduction: B cells are unique in the capability to modify their antigen receptors in response to activating signals. although experiments have shown that any promoter that controls transcription can support mutation. but CD40 signals can do this. with a predilection for 7 transitions (purine-> purine and pyrimidine -> pyrimidine). In this lecture. in some species like chickens and rabbits. TLR and cytokine signaling. As already discussed. The answers are not entirely clear. as correction of mutations is an ongoing process in all cells. This gene was called Activation Induced Deaminase (AID). Somatic hypermutation takes place in germinal centers and until recently this was thought to be the exclusive site of mutation. We will also briefly cover the biological impact of somatic hypermutation on the germinal center reaction. AID modifies an RNA so that it now . as nonspecific activators do not appear to induce mutation. This raises the question of what signals turn the mutation process on in B cells. but sequences in or near the immunoglobulin transcriptional enhancers are important. it is turned on only after a period of B cell activation. Location and targeting: Mutations occur only in a limited area from a few hundred bp downstream of the promoter region extending for about 1kb. not just the V region promoter. In some cases. we will cover how both mutation and switch are controlled and induced and the molecular mechanisms of both of these processes.

Moreover. which are not direct targets of AID. These include uracil deglycosylase. leading in turn to a variety of mutations. leaving the problem of how one gene can be involved in such seemingly disparate molecular processes. seem to occur via error-prone repair involving especially DNA poymerase eta. The initial lesions created by AID in the direct model are repaired in a variety of ways by DNA repair enzymes and polymerases. it seems to be a cytidine deaminase that converts C to U. which removes U base pairs as a mechanism of repairing lesions that have had a base removed. is that AID itself is a DNA-modifying enzyme that directly acts as a mutator. A summary of how AID might work was published by Neuberger and colleagues: . Indeed. which in turn helps to explain the higher frequency of transition type mutations (particularly at G=C base pairs). it has been known since the discovery of AID that it is required also for isotype switching. coli. Perhaps most remarkably. Evidence in favor of this model includes that AID in fact does modify DNA in vitro and also in E. AID is critical for gene conversion. Mutations at adjacent A//T base pairs. which is now widely accepted. The second theory.encodes an enzyme that can carry out or otherwise induce somatic hypermutation—this is the “indirect” model. Other repair enzymes like XRCC2 promote gene recombination (leading to gene conversion).

99 .104 (04 July 2002)) . V. AP endonuclease. DNA-PK. (From Petersen-Mahrt et al. deoxyribophosphodiesterase. variable. KU70/80 are nonhomologous end-joining proteins. For details. Nature 418. dRPase. DNA-dependent protein kinase.Figure 1: DNA deamination model of immunoglobulin gene diversification. see text.. apyrimidic endonuclease.

as seen in the figure above. It may also play a mechanistic role in the actual switch rearrangement. just 5’ to the “switch region” (see below). Via unknown mechanisms (but possibly in part because they are preferred targets of AID. With deglycosylation of the U bases. these short sequences promote nonhomologous recombination between switch regions of different isotypes. as well as a specific (but as yet uncharacterized) enzymatic machinery are required for the switch DNA rearrangement to take place. The intervening DNA is deleted. As with somatic hypermutation. as will be discussed: Molecular mechanism: DNA rearrangement leads to the deletion of intervening C regions and the juxtaposition of a new C region near the VDJ join (fig. but this is not totally clear.Isotype switch Control of switching: Cells initially express IgM. Both transcription (i. as mutant switch loci that lack the promoter region do not undergo switching. which are known to be preferentially introduced into certain sequence motifs. Transcription may “open” the locus to recombination enzymes and for alignment with other switch regions. For example. However. accessibility) of the downstream switch and C region. This transcription is essential. thus setting the stage for switching. possibly with unique secondary structure that are preferred targets for AID. in particular G=C-rich sequences such as GAGCT and GGGGGT. AID is thought to play a role by targeting DNA repair mechanisms to the switch sites. the switch region upstream of IgM might recombine with the switch region of IgG3. along with CD40 signals. IFN-α TGF-β Type of switch IgG1 and IgE IgG2a IgA These cytokines work in part by inducing transcription from promoters that lie just upstream of each of the Ig C region genes. IgG or IgA) isotypes. a lesion is created that could lead to a single or double stranded break in the DNA. IL21 IFN-γ. 2). This DNA contains many repeats of a few short sequences. The pseudohomology between the repeat sequences shared by all of the switch regions may help in aligning the two regions to be joined. Specific cytokines tend to promote specific switches. see below).g. Recent evidence suggests that transcription serves to create regions of single stranded DNA. This is even more difficult to explain at this point than somatic hypermutation. Upstream (5’) of every constant region lies a stretch of DNA known as the “switch region”.) . it is thought that AID targets the DNA in switch regions that is particularly G=C rich (and thus has many cytidine residues for deamination). Isotype switch is driven by cytokines and T cells.e. Now the cell will express IgG3. switching also requires transcription through the region to be switched. but “switch” to expressing non-IgM (e. (A similar mechanism may apply in targeting somatic hypermutations. In the mouse: Cytokine IL4.

Mutations can be classified according to where in the V region coding sequence they occur: complementarity determining region or CDR (i.e. Repetitive DNA sequences that guide isotype switching are found upstream of each of the immunoglobulin constant-region genes. Clones evolve. with deletion of the intervening DNA. How mutations affect the cell’s fate: Many mutations (perhaps about 1/2) in FR might destroy the structure of the overall molecule. or switch signals. part of the protein that contacts antigen) and framework region or FR (part that generally does not contact antigen but maintains the overall immunoglobulin structure). with the exception of the δ gene.Figure 2: Isotype switching involves recombination between specific signals. The initial switching event takes place from the µ switch region. 3. Switching occurs by recombination between these repetitive sequences. S. fig.26) Selection The overall result of somatic hypermutation is that among clonal descendents. meaning that no amino acid substitution occurs. These mutations will . (reproduced from Janeway text. All mutations can be divided into replacement (R) meaning that the base substitution leads to an amino acid substitution in the resulting protein—or silent (S). switch region. Only R mutations will influence the fate of the cell. and indeed the GC can be thought of as a miniature evolution system that occurs in real time. switching to other isotypes can take place subsequently from the recombinant switch region formed after µ-γ switching. nearly every cell is different from every other cell.

. more linear rate. Cells with these mutations will be positively selected and preserved in the GC. Molecular mechanisms of antibody somatic hypermutation. So if there is negative selection—for example in a FR—the R/S ratio will be lower. 2011.M.5.. and M. M. Altering the pathway of immunoglobulin hypermutation by inhibiting uracilDNA glycosylase. 30:173-81. 2002. some R mutations will be enriched and the ratio will be higher. exponential rate. LLC). Taylor & Francis Group.H. Nature 419:43-48. 20:683-94. higher affinity cells may be better able to capture antigen and present it to T follicular helper cells. if there is positive selection. meaning that about 1/2 of all R mutations are selected against. Neuberger. the affinity of a BCR determines the strength or frequency of positive signals a cell gets: higher affinity cells will get more/better signals. 8th Edition. Shlomchik. M. This can result in either more proliferation or less death or both among the higher affinity cells. Schatz.G. Liu. 2008. 2007.. Reviews: Di Noia.S. 2009. Such skewing of R/S ratios are actually seen in real Abs. Negative selection is thus a frequent event. M. Only such R mutations change the cell’s phenotype and thus can be selected. and indeed many B cells die in the GC. and thereby gain survival or proliferative signals from the T cells. U. and M. The average R/S ratio in FR regions is about 1. The consequence of this is that clonal expansion is not at the ideal. J. Trends Immunol. Some mutations in the CDR might also destroy Ag-binding and would be selected against. J.J. But some are likely to improve binding to the Ag. In the absence of any selection. Reading: Text: "Janeway’s Immunobiology". Balancing AID and DNA repair during somatic hypermutation. and S. Chapter 5 pp. increasing the affinity of the B cell. Kleinstein. How does selection occur in the GC?: Presumably. Primary literature: Di Noia. Neuberger.be selected against (“negative selection”) and cells that have them undergo apoptosis. Conversely. by Kenneth Murphy (Garland Science.S.. but follows a much slower. Alternatively. 173-186. since some R mutations will be eliminated. and D. Hershberg. which is determined by the codon translation table in that most mutations do cause an amino acid substitution. Improved methods for detecting selection by mutation analysis of Ig V region sequences. Negative and positive selection can be inferred from the ratio of replacement type (R) to silent type (S) mutations (R/S ratio). this ratio is about 3. Annu Rev Biochem 76:1-22. Uduman. Int Immunol.

those that have already recovered from infection) antibodies protect mucosal surfaces and the blood from new infections. During the course of an immune response. and also to which compartments the Ab molecule itself gains access (for example. which in turn controls whether the Ab can bind to certain receptors for the Fc portion (FcR’s). activate or “fix” complement. which can result in lysis. such as macrophages. IgM is too large to diffuse into most tissues). other effector functions of Abs depend on the soluble complement system. M. B cells rearrange their DNA to allow secretion of isotypes other than IgM. in immune animals. prevent the establishment of new infections. Complement activation mediated by antibody binding to the surface of a pathogen can lead to both the covalent binding of fragments of C3 and C4 to the pathogen— in a form of opsonization—but also to the activation of the terminal pathways of complement. They prevent the spread of established infections and. Shlomchik . Opsonization is the process of coating the surface of a pathogen with Ab or complement fragments (i. b) opsonization and c) activation of complement. C3b). Antibodies that bind to the pathogen can prevent this and are said to neutralize the pathogen. Antibodies are secreted by plasma cells. particularly of eukaryotic pathogens. Effector functions of Abs are dictated by the Fc portion of the Ab. such as NK cells or macrophages. Similarly. and under the direction of signals from T cells. which are diverse and mediate a variety of effects upon FcR binding of Abs. Neutralization by antibodies is also important in preventing bacterial toxins from entering cells. that bear receptors for the Fc part of antibodies and for complement fragments. Since antibody titers are long-lasting in immune animals (i. To enter cells. These other isotypes have unique properties and effector functions. viruses and intracellular bacteria bind to specific molecules on the target cell surface. Medzhitov). Thus the effects of Abs provide yet another link between the adaptive and innate immune system. The effector functions of Abs are in turn linked to the cells that express FcR’s. which in turn enhances phagocytosis by cells.e.e. Most of these effects are mediated by cells that can be classified as part of the innate immune system. which are the terminally differentiated products of activated B cells. The effector functions of Abs Antibodies carry out their effects via: a) neutralization. another element that also functions in the innate immune system (see earlier lectures by Dr.Humoral Immune Responses November 2 Instructor: Overview: Humoral responses are important to protect the extracellular spaces from pathogens.

as will be discussed below. that can expel infectious agents. IgM is mainly found in the blood and. The pentameric structure of IgM makes it especially effective in activating the complement system. and most of the B cells expressing these isotypes have been selected for increased affinity of antigen-binding in germinal centers. The distribution and main functions of antibodies of the different isotypes are summarized here (from Janeway text): . sneezing. each with the same combining site. and IgE: These Ab molecules are smaller in size and diffuse easily out of the blood into the tissues. IgA is a less potent opsonin and a weak activator of complement. it triggers mast cells to release powerful chemical mediators that induce reactions. however. Although IgA can form dimers. such as coughing. and vomiting. as a result of the large size of the pentamers. the most important being those of the mucus epithelium of the intestinal and respiratory tracts. IgG is the principal isotype in the blood and extracellular fluid. This compensates for the relatively low affinity of the IgM monomers. as IgG operates mainly in the body tissues. the progeny of a single B cell can produce antibodies. IgM: The first antibodies to be produced in a humoral immune response are always IgM. whereas IgA is the principal isotype in secretions. Mast cells are found just beneath the skin and mucosa. IgA. and along blood vessels in connective tissue. to a lesser extent. but is bound avidly by high affinity FcR’s specific for IgE (Fc R) on mast cells. Whereas IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system. However. IgE antibody is present only at very low levels in blood or extracellular fluid. Via isotype switching. IgG. When Ag is bound by this mast-cell associated IgE. whereas IgA operates mainly on epithelial surfaces where complement and phagocytes are not normally present. where accessory cells and molecules are available. IgM molecules. the lymph. yet expressing a variety of different Ig isotypes.Ab isotypes have different properties and effector functions Antibodies of different isotypes are adapted to function in different compartments of the body. IgG and IgE are always monomeric. This distinction is not surprising. The affinity of the individual antigenbinding sites for their antigen is therefore critical for the effectiveness of these antibodies. form pentamers whose 10 antigen-binding sites can bind simultaneously to multivalent antigens such as bacterial capsular polysaccharides. These early IgM antibodies are produced before B cells have undergone somatic hypermutation and therefore tend to be of low affinity. and therefore functions chiefly as a neutralizing antibody.

there must be several molecules in proximity. A key feature of FcRs for IgG is that they generally bind multivalent IgG that is part of an immune complex much better than they bind free IgG. can activate C1q. In the case of IgG. Another is a series of receptors for the Fc portion of Abs (FcRs). neutrophils. via complement receptors (CRs) that are on red blood cells as well as on a variety of immune system cells.It is critical that soluble Ab molecules. B cells. and mast cells. These are on the surface of many hematopoietic cell types. Complement activation is one adjunct for antibody-mediated protection. this may be . It also leads to the release of soluble inflammatory mediators. As described earlier in the course. which circulate at mg/ml concentrations. A single molecule IgM. nor are they capable of destroying many kinds of pathogens. because C1q must bind at least two IgG Fc regions in order to be activated and start the cascade. but only when that IgM is bound to a surface and undergoes a conformational change allowing C1q activation. they do not by themselves remove pathogens from the body. eosinophils and basophils which all have receptors for IgE (see above). which is already a pentamer. NK cells. as this would lead to uncontrolled inflammation. complement activation leads to binding of C3b and C4b to nearby protein structures. the ability of Abs to activate complement is controlled by a requirement for Ab to be bound to a surface. and are much more sensitive to BCR crosslinking when the CRs are also bound. do not activate the complement cascade. which also have CRs. NK cells and dendritic cells Although high affinity Abs can neutralize. Instead. These two outcomes of complement activation lead to more efficient removal of immune-complex-bound pathogens. dendritic cells. resulting in a form of opsonization. including macrophages and dendritic cells. including: macrophages. FcR’s mediate the destruction of antibody coated pathogens and result in activation of macrophages. Complement coated pathogens are also especially potent at stimulating B cells.

Cell types like NK cells are not phagocytic but instead are triggered to release stored mediators. dendritic cells and neutrophils). binding of FcRs activates the cells (for example to undergo oxidative burst) and in particular to enhance the phagocytosis of the bound IgG-coated pathogen or molecule. perforin and granzyme B (that mediate cell lysis).due to affinity as well as steric reasons. from Janeway text. don’t memorize it!): . like cytokines. when their FcR’s are bound. A summary of the properties of the different types of FcRs is given here (FYI. In the case of the phagocytic cell types (macrophages. Phagocytosis in turn leads to destruction of the pathogen as well as enhanced presentation of its antigenic peptides to T cells. and inflammatory agents. This process is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). The cells are also activated to undergo new protein synthesis again leading to subsequent secretion of cytokines. Thus only IgG that is complexed to Ags will effectively activate these receptors.

. Ravetch. 8th Edition. 408 . . 2005.Reading Text: "Janeway’s Immunobiology". Primary literature: Nimmerjahn. Taylor & Francis Group. and J. F. LLC).424. Chapter 10 pp. Divergent Immunoglobulin G subclass activity through selective Fc receptor binding. 2011. by Kenneth Murphy (Garland Science. Science 310: 1510 -1512.

one must isolate putative memory precursors and do a functional assay. A system shows memory when. Memory in the immune system is defined functionally. However. the IgG positive. one expects a cellular basis for memory. compared to the first response. in which cowpox infection (a recoverable disease) was deliberately induced and later protected against fatal smallpox. an animal would quickly fill up with memory cells and have no "room" left for new cells to respond to new antigens. faster and qualitatively different. nor do we know whether all high affinity cells become memory cells. For example: memory cells carry . they probably do not need a GC reaction. This form of specific enhanced immunity to reinfection occurs because of immunologic memory. and treatments that block the GC reactions. Secondary response Abs have higher affinity than primary response Abs. Memory is a property of the immune system as a whole. Of the many cells which initially respond to antigen and enter the GC. For example: sequencing of V genes of such expanded cells isolated during a late primary or secondary immune response led to the discovery that these are somatically mutated. differentiation to a more "potent" type of cell. and the V genes of cells participating in a secondary response are somatically mutated. Similar studies showed that these cells are also isotype switched. Memory T cells seem to need a period in which there is little Ag exposure in order to form memory cells—too much persistent Ag or too much inflammation drives T cell differentiation towards an effector phenotype rather than a memory phenotype. Jenner was perhaps the first to intentionally take advantage of this phenomenon to protect naive individuals. Shlomchik Introduction It has been known for a long time that people who recover from a particular infection often are not susceptible to reinfection by the same infectious agent. since if all responding cells were retained. These cells are too infrequent to observe in unimmunized animals but can be seen at low frequency in immunized animals due to clonal expansion following Ag exposure. To prove this. Presumably. He invented vaccination. with higher amounts of cytokine production and secretion of “polarized” cytokines such as IL-4 and IFN-g without delay. Another approach is to use a property associated with memory cells as a surrogate marker for those cells. but since the system is composed of cells. we do believe that the GC reaction is necessary for the formation of high affinity memory cells. The requirements for the formation of a memory T cell are less clear. This approach is a descriptive one and doesn't really prove that these "secondary cells" are really the precursors to long-lived memory cells. B cells that had already responded to Ag (“secondary cells”) were also characterized by the expression or lack of certain surface molecules such as CD44 (elevated) and J11D (reduced). Memory cell development The precursors of memory B cells form mainly in the germinal center (GC). This could work in a number of (nonexclusive) ways: clonal expansion of Agspecific cells. How to track down a memory cell: Insight has been gained into the identity of memory cells by the study of cells specific for a particular Ag after the animal has been immunized with that Ag. but instead an interaction with a professional APC such as a dendritic cell. This is termed the secondary immune response. or increased longevity of a cell and/or its clonal descendants. IgM negative cells contain memory precursors though we don’t know that all IgG positive cells are memory precursors. Similarly. the affinity for the antigen is higher. This has been easiest to do for the cell surface markers. Memory B cells are reactivated upon reexposure to the Ag. B cell memory responses are qualitatively different from primary responses in three main ways: the antibody formed is IgG or IgA (not IgM). We do not know which is true. only a small subset are retained as memory cells.B and T Cell Memory November 5 Instructor: M. memory T cell responses occur faster. higher affinity cells (possibly created through beneficial somatic mutations) are selected in some way to become memory cells and this underlies the affinity maturation. This makes sense. will block memory formation. a second or later response is characteristically different: usually stronger. These cells could be better competitors for antigen or might receive a different or stronger signal to differentiate because of their higher affinity. a phenomenon termed "affinity maturation". For example. such as blocking the CD40-CD40L interaction by infusing anti-CD40L Abs into mice.

For example.e. However.mutated Ig V regions. Are Memory cells heterogeneous? Memory cells have to carry out multiple functions including providing a variety of effector functions upon secondary challenge (see below) as well as to renew the memory cell compartment. Memory B cells and T cells both probably exist for long times and at stable overall levels without undergoing cell division. activated. is not needed to maintain memory B cells. Among memory B cells. Surface marker (phenotype) comparison among naïve. this might be a site where memory cells are formed. principally IL-15 and IL-7. The role of cytokines in maintaining B memory cells has yet to be elucidated. Are Memory cells long-lived in the absence of continuous antigen stimulation? The answer to this question would seem to be an automatic "Yes". Memory T cells are also heterogeneous in their ability to make various cytokines upon restimulation. via multiparameter flow cytometry. Property IgM/sIgD (frequency of + cells) IgG (frequency of + cells) Secrete Ig Lifespan (without division) Naive +++ +/6-8 wk Activated (GC) +/++ + Hours Effector (plasma cell) Memory (?) +/+++ +/Indefinite? +++ Weeks to months Surface Markers MHC Class II CD80 PD-L2 (mouse only) CD27 (human only) Bcl-2 (survival protein) Fas (death receptor) CXCR4 (chemokine receptor) CXCR5 + +/+ + + ++ ++ +/+ +/+/++ + + +/+++ +/++ +/+ ++ (subset) ++ (subset) ++ ++ + ? ++ Table 1. In addition. If all of the memory cells became terminally differentiated effectors (i. plasma cells or effector T cells) upon Ag reexposure. The signals that stimulate this division and the consequences are unclear. These diverse requirements are probably dealt with by heterogeneity among memory cells—not all are created equally. In fact. while effector cells that are destined to “burn out” do not upregulate this cytokine receptor. although it seems likely that antigen goes away after a period of time. . If we could know where mutations occur. that not all memory cells expressed all markers. developing memory T cells can be distinguished by their expression of the IL-7 receptor. replicating antigens (especially certain viruses) may remain at low levels which are inconsequential for the host but which maintain memory. which is required to maintain the survival of naïve B cells. Second. Emerging data indicates that these different memory B cell subsets carry out different functions in a secondary immune response. there is good evidence that the body can store it in special cells (follicular dendritic cells) for long periods. the cells themselves may not be long-lived even though the clone from which they descend is. effector and memory B cells. This reasoning was one way in which the GC was implicated as the site of memory B cell development. this would actually deplete memory and impair any subsequent responses. it is known that the TNF-family cytokine called BAFF (B Cell Activator of the TNF Family). without these. the discovery of surface proteins that mark memory cells revealed. the diagram below shows that PD-L2 and CD80 expression defines distinct subsets of murine memory B cells. However. memory CD8 responses appear to decay. It appears that cytokines are important for maintaining T cells. But it needn't be so for two reasons: first. Memory cells thus may divide every so often yielding two memory daughters. it is possible to observe memory cells dividing infrequently. In the T cell compartment. heterogeneity is seen in terms of expression of surface proteins that control migration.

In general. Surface marker (phenotype) comparison among naïve. as mentioned. The degree to which all of these are present also influences the nature of the secondary response. secondary responses are characterized by large bursts of extra-follicular B and T cell proliferation. depending on the timing. early differentiation into plasma cells at these sites. d) preexisting Ab. which was also used to measure expression of CD80 and PD-L2. but there is .IgG1 memory cells specific for the hapten NP were generated in a transfer and immunization model. that Ab responses are more prompt. c) non-limiting Ag-specific T cell help. Ag dose and nature of the Ag. the B cells that seed secondary response GC’s are memory cells or “new” primary B cells. then identified by flow cytometry. It is not even clear if. effector and memory T cells. generally. and are isotype switched and that T cell responses feature prompt induction of cytokines and/or cytotoxicity. are of higher affinity. activated. b) more sensitive “memory” differentiated B or T cells. We do know.or + (two subsets) Table 2. in some circumstances it does appear that memory B cells can undergo a second round of somatic mutation. Secondary immune responses Much less is known about the nature of the secondary immune response than the primary. But the cellular and microanatomic basis of this are less clear. Property TCR expression Cytokine secretion Ag recognition for maintenance/activation Cytokine polarization Lifespan (without division) Surface Markers LFA-3/CD58 CD44 L-selectin (CD62L) Bcl-2 (survival protein) Fas (death receptor) IL-7 receptor CD45RA CD45RO CD69 CXCR5 CCR7 Naive +++ Self-MHC Not 6-8 wk + + ++ + + ++ ++ + +/++ Activated/ Effector +/+++ MHC+peptide Evolving Hours to days ++ +++ ++ +/++ ++ ++ ++ ++ (subset) ++ Memory +++ +/No MHC Definite Indefinite? ++ ++ +/(heterogeneous) ++ + +++ + +++ ? . There are several key differences with primary responses: a) higher B or T cell precursor frequency. Secondary immune responses seem to be highly variable. and a paradoxically attenuated GC response.

247. 15517-15522. Travers. V. Proc Natl Acad Sci U S A 105. 2005. Gourley.J. K.M. M. P. T. 2000. Current opinion in immunology 18.E. Ltd. M. (2012). P.. and R.. O'Neill. Articles: Maruyama. Garland Science. and Weisel.. J. Nat Immunol. Tomlinson. Immunol. 407:636-642..clearly early clonal expansion without further mutation as well. C.H... and Ahmed.P.. M. B.. Immunobiology: The immune system in health and disease.T. M. Long. Walport. M. Rajewsky. R. Crowley. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells.L. 3rd. Steinel. Zamoyska. Nature.. Scholz. 52-63. Germinal center selection and the development of memory B and plasma cells. In any case. F.. Reading: Text: Janeway. Sections 10:22-10:26. R. Y. 4:680-686. Goenka. Quinn. Shlomchik. 2003. 255-264.. Tomayko. N. . Rouse. (2008). Reviews: Kalia. W... B. and Shlomchik. Differentiation of memory B and T cells. Cho. Rev. (2006)... V. Memory B-cell persistence is independent of persisting immunizing antigen. the consequence is a vigorous and high affinity IgG and effector T cell immune response that functions well to protect the host against reinfection. and K. S.J. J.S. Miller.. BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact.J.... et al. Seddon. Sarkar.P. J. Lam. 6th ed. P..

Killed viruses are also commonly used as these are very safe. 2011. generating blocking antibodies to toxins. It needs to provide sustained and long-lived immunity. They do so by generating long-lived. Reading: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. 2011 Jun. Other ways of achieving “protection” from pathogens is to target the molecules that actually cause disease. Despite the great advances made in the field of vaccine research. attenuated virus because it will mimic the natural infection the best. and what immunologists might do to tackle these problems in the future. cause disease by secreting toxins. Ahmed R. but not the bacteria. Chapter 16 (16-19 through 16-31). (3) herd immunity— this is where individuals in a population are protected from the pathogen without direct vaccination because the frequency of vaccinated people is so high that spread of infection and risk of exposure drops to such a significant degree that they are.. Taylor & Francis Group. these are not available for many viruses. For example. a single dose). illness. Many types of vaccines are being used today with success or are in clinical trials.e. Many critical elements need to be encompassed by a “good” vaccine. protective memory T cells that provide cell-mediated protection and remove infected cells or pathogens. LLC). The most effective vaccine is always a live. but surprisingly to date. There are 3 major ways in which vaccines can work: (1) prophylatic— these are the most common form of vaccination and are preventive vaccines to protect the host prior to exposure to the pathogen (2) therapeutic—these are vaccines designed to treat individuals already infected with the pathogen (common for chronic viral infections). HIV-1. are lacking. Likewise protective T cells should also be induced. and memory B cells and long-lived plasma cells that provide humoral immunity. When a pathogen jumps hosts.12(6):509-17. whereby the pathogen causes no undo harm to the host to maximize its replication and spread. small pox and yellow fever vaccines have demonstrated protection for more than 50 years in humans. But. For instance. Nat Immunol. Immunological mechanisms of vaccination. The ultimate goal of vaccination therefore is to prevent the viruses and bacteria that cause disease in humans and other animals from infecting the host. 8th Edition. malaria and TB. . protected. much needed vaccines for major killers. Iwasaki True co-evolution between pathogen and host can be found when a “carrier” state is produced. disease and death can result. certain bacteria. A protective vaccine needs to produce neutralizing antibody to prevent immediate infection and contain spread. For such pathogens. such as Vibrio cholerae. Pulendran B. very few vaccines have been formulated in a way that rely only on T cell responses. We will discuss some of the obtacles of vaccine development against these agents. An ideal vaccine would also be low cost and biologically stable for shipping and storage and have an ease of administration (i. in essence. It must be safe with minimal side effects and protective against the live pathogen when encountered naturally. is a viable strategy. Sometimes “sterilizing” immunity is met by some vaccines that have extremely potent antibody responses.Vaccination November 7 Instructor: A.

Regulation of inflammation Although inflammatory response is an essential component of host defense. IL-1b processing requires the formation of inflammasome complex in the cytosol. recruitment and activation of neutrophils. which include surfactant proteins (which promote phagocytosis in the lung). C-reactive protein (binds to phosophocholine portion of bacterial and fungal cell walls for opsonization and C’ activation). TLRs in turn trigger production of inflammatory cytokines. which leads to production of inflammatory cytokines and chemokines. most importantly TNF. Inflammasomes NOD-like receptors (NLRs) are pattern recognition receptors that reside in the cytosol. Molecular mediators of inflammation include proinflammatory cytokines. Fever is beneficial to the host because most pathogens grow better at lower temperatures. They do so by inducing the sythesisi of prostaglandin E2 (PGE2) by the enzyme cyclooxygenase-2 (COX2). neuropeptides and amines (histamine and serotonin). are particularly important for the negative regulation of inflammation. Inflammasomes are induced by sensing cellular stress. such as TNF. A typical inflammatory response is initiated by macrophages resident in peripheral tissues. Fever TNF. expression of which is induced by these cytokines. . The inflammatory response to infection is designed to optimize the elimination of the invading pathogen. as well as nitric oxide and reactive oxygen species (ROS). it can be highly detrimental if it becomes excessive. IL-1 and IL-6. IL-1 and IL-6. expression of adhesion molecules). persistent of systemic. Collectively. several NLRs play a key role in the activation of caspase-1 by forming a multi-protein complex known as the ‘inflammasome’. IL-1 and IL-6 also act on the hepatocytes to induce the acute phase response. MBL (binds to carbohydrate moiety on pathogens and induce opsonization). The inflammatory response includes the following physiological components: vasodialtion. Caspase-1 is an essential mediator of inflammatory response through its capacity to cleave and generate active forms of IL-1b and IL-18.Inflammation – Response to Infection November 9 Instructor: A. Indeed. PGE2 act on hypothalamus resulting in increase in heat production by brown fat and increased vasoconstriction decreased in the loss of excess heat through the skin. Pathogens activate TLRs on macrophages. this results in local changes in endothelium (vasodialtion and increased vascular permeability. The so-called antiinflammatory cytokines. The cellular component of the acute inflammatory response includes activation of neutrophils. This response is triggered most commonly by TLRs activated by infectious agents. acute phase response proteins. eosinophils. Iwasaki Inflammation is a complex response to infection and injury that destroys the infecting agent and restores the injured tissues. Liver cells start to produce acute phase proteins. Acute phase response TNF. lipid mediators. IL-10 and TGF-beta. In particular. dysregulated inflammatory response leads to a wide variety of pathological conditions and is normally prevented by several anti-inflammatory mechanisms. IL-1 and IL-6 are called endogenous pyrogens because they case fever. and fever. increased vascular permeability. such as prostaglandins and leukotrienes. platelets and endothelial cells. plasma proteases (complement and coagulation protease cascades). Although the functions of many of the NLRs are largely unknown. monocytes and macrophages. damage or bacterial products. These cytokines orchestrate the inflammatory response at the local and systemic levels. which promotes recruitment of leukocytes to the site of infection.

LLC).420(6917):846-52. Nature. 2002 Dec 19-26. by Kenneth Murphy (Garland Science.Reading: Review Articles: Nathan C. 8th Edition. Taylor & Francis Group. 2011. . Text: "Janeway’s Immunobiology". Points of control in inflammation. Chapter 3.

pathogen specific T and B cells are found within the disorganized lymphoid tissues of the lamina propria. In addition. the upper female reproductive tract.). digestive. First. mucosal tissues have developed unique features not found in other types of tissues to 1) combat pathogens. Third. Type I: Mucosal Surfaces Covered by Simple Epithelia Type I mucosal surfaces are covered by simple epithelia of one cell–layer thickness. excretory. to combat pathogens. 2) induce rapid immune responses. 4) prevent unnecessary Th1 immunity to maintain the physiological functions of the mucosal tissues. Consequently. all pathogens must enter the host through the mucosal surfaces.Mucosal Immunity November 12 Instructor: A. Iwasaki Infection in real life With the exception of vector-borne diseases. respiratory. These surfaces are represented by those that cover the small and large intestine. The primary role of these surfaces is to perform absorptive. 3) provide a reservoir of effector and memory cells just beneath the surface epithelial cells for rapid protection. Common features of type I mucosal surfaces include the presence of mucus-secreting cells (goblet cells) and the expression of pIgR on the basolateral surface of the . and reproductive functions that are vital to the life of the host. Two types of mucosal lining. Forth. to provide the most efficient form of protection. mucosal tissues contain specialized dendritic cells and regulatory T cells that suppress Th1 immune to harmless antigens to maintain the physiological functions of the organs (respiratory. mucosal surfaces are lined with mucus and other physical barriers that act as a first line of defense against pathogens. Second to induce rapid immune responses. and the pseudostratified epithelia of the respiratory tract. one can find organized lymphoid structures just beneath the mucosal lining. reproductive…etc. mucosal lining contains specialized cells that secrete antimicrobial peptides into the lumen.

based on the observation that mucosal sensitization at one site provide primed cells selectively to other mucosal sites. many studies have now confirmed this theory and provided molecular mechanisms for the homing pattern of effector lymphocytes. Certain chemokines. In the gut and lung. Immune Inductive Sites Organized lymphoid tissue (inductive sites) Tonsil. releasing the secretory IgA (SIgA) into the lumen. Cell migration mechanisms behind the CMIS have been elucidated recently. Unique Components of the Mucosal Immune System Innate Immunity Mucosal epithelial cells and Paneth cells produce a variety of antimicrobial peptides (defensins. These lymphocytes detect non-classical MHC I molecules induced by infection. and tonsils. The main function of type II mucosa is to provide a physical barrier. such as CCL25 and CCL28 are expressed by the small and large intestinal epithelial cells and are known to recruit CD8 T cells expressing the receptors CCR9 and CCR10. There are no pIgR on the type II mucosal surface and thus the dominant protective immunoglobulin at these surfaces is IgG. . and IgA secreting plasma cells can be found in the lamina propria of these tissues. which share many common features with the skin. appendix. IgA is the most important isotype that provides protection against pathogens. Type II: Mucosal Surfaces Covered by Stratified Epithelia Type II mucosal surfaces are covered by stratified squamous epithelia (keratinocytes). FAE contains M cells capable of transporting luminal antigens across by transcytosis. The pIgR binds to polymeric IgA (pIgA) secreted by the plasma cells in the LP and exports the pIgA transepithelially. These observations were originally described for gut induction leading to bronchus effector functions.epithelia. Further. • Prevention of bacterial colonization • Prevention of viral adherence • Neutralization of bacterial toxins • Prevention of intracellular spread of microbes within the epithelial cells Common mucosal immune system The concept of the common mucosal immune system (CMIS) was pioneered by Bienenstock and McDermott in 1978. FcRn is expressed by vaginal epithelium and can transport antibodies from the tissue and back into the tissue. Type II mucosal surfaces are devoid of MALT structures but are drained by regional lymph nodes. bactericidal/permeability increasing protein) and bacteriolytic enzymes (lysozyme. cathelicidins. group IIA phospholipase A2) that protect mucosal surfaces and crypts containing intestinal stem cells against invading microbes. IgA provides its effector functions in multiple ways. Effector Immune Reservoir Diffuse lymphoid tissue of the lamina propria contain effector and memory T and B cells that provide immediate protection against invasion by pathogens. This concept has been the main force behind the efforts to develop “mucosal vaccines” in which immunogen delivery is targeted to various mucosal surfaces. appendix Follicle Associated Epithelium (FAE) The FAE is a specialized epithelial layer that cover the dome region of the Peyer’s patch. Another important cell type that provides early non-specific immune responses are the intraepithelial lymphocytes. Peyer's patches. who proposed that the immune system of the mucosal tissues is somehow connected. and the keratinocytes that cover these surfaces do not have absorptive or respiratory functions. other studies have provided evidence that distinct branches exist within the CMIS. Since the original description of the CMIS. respectively. injury or stress and destroy infected cells.

This phenomenon is known as “oral tolerance”. The loss of tolerance in the intestinal mucosa can result in inflammatory bowel diseases. 8th Edition. Review Article: Iwasaki. When soluble protein antigen is given orally in the absence of an adjuvant. Science. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. but it likely involves mucosal dendritic cells and regulatory T cells that suppress Th1 immune responses to antigens. Uhr T. 2004 Mar 12. The mechanism of oral tolerance is not completely understood.303(5664):1662-5. Annual Review of Immunology 25:381-418 Primary Literature: Macpherson AJ. Questions Q1: How do we distinguish between commensal bacteria and pathogenic bacteria? Reading: Text: “Janeway’s Immunobiology." Kenneth Murphy. (2007) Mucosal dendritic cells. Mounting protective immune responses against harmful pathogens while preventing excessive responses to harmless Ags (including commensal bacteria) is the difficult task achieved by the mucosal immune system to maintain homeostasis (a disease-free state). 2011.Immune Regulation Mucosal tissues are constantly exposed to tremendous amounts of exogenous antigens that are either resident or ingested/inhaled. A. . Chapter 12. the animal becomes unresponsive to that antigen upon secondary encounter.

What are the biological “purposes”. immune responses to pathogens and other stimuli rely heavily on transcriptional regulation in the responding cells. 5) Epigenetic memory. Trans-mechanism. Th2. Chromatin structure is tightly regulated in part by covalent modifications of DNA and histones. Developmental cues are known to direct lineage commitment and differentiation by controlling transcription. For example. transcription defects underlie diverse immunological disorders. sometimes. such as T-bet and GATA3. and such modifications. The latent factors are often constitutively localized in the cytoplasm until activation. which are necessary and sufficient for directing the differentiation of naïve CD4 cells into Th1 and Th2 cells.Transcriptional Regulation in the Immune System November 14 Instructor: T. when they move to the nucleus to regulate target genes. which involves chromatin. because incorporation of DNA into chromatin inhibits access of transcription factors. how cyclosporin A and corticosteroids work). 4) Epigenetic memory is achieved by two mechanisms: a. b. Chromatin is a focal point of transcription regulation. in which the induced transcription factors auto-regulate their own expression after the termination of the transient signals. 2) The most important target genes turned on by the latent factors are “master transcription factors” specifying cell identity/critical immune functions. There are 5 general principles underlying transcriptional responses to environmental signals. 3) Environmental signals are transient. Similarly. Th17 and Tregs) are interconvertable to some extents. just as our brains’ memory of our experiences. respectively. which has important clinical implications. respectively. which underlies phenomena such as trans-differentiation. as if cells can “remember” things of the past. However. typically called epigenetic memory. from the autoimmune disease IPEX to the immune deficiency disease DiGeorge’s syndrome. Although this state is stable. but rather is a functional state of genes. while transcription factors constitute effective targets for immune therapies (consider. Chi The development and function of the immune system are controlled to a large extent at the level of transcription. various CD4 cell subsets (Th1. is at the heart of lineage differentiation and immunological memory. is not hardwired or coded in the DNA sequence. retrodifferentiation and epigenetic reprogramming. the latter being a special form of lineage differentiation. can sometimes be sustained and propagated to daughter cells independently of the initiating signals. for example. and what might be the underlying mechanisms? . Cis-mechanism. and so can be the corresponding transcriptional responses they elicit. it is also intrinsically plastic. This cellular memory. with the optimal adaptive immune responses achievable only after further transcription-mediated differentiation of the responding naïve lymphocytes into effector/memory cells. Consequently. Questions: The effects of transient signals on target gene functions can be reversible or irreversible. which is best understood in the context of antigen-induced differentiation of naïve CD4 cells into effector cells: 1) Signaling molecules directly activate latent transcription factors such as NFAT and Stat1 that are activated by TCR signals and IFNγ. once established. the transient signals can cause irreversible or long-lasting changes in gene function that persist after the termination of the signaling events.

W. by Kenneth Murphy (Garland Science. Chapter 8-17 to 8-19 (pages 308-11) Primary Literature: O’Shea. 8th Edition. . 327. & Paul. J. pp. 1098-1102. Mechanisms Underlying Lineage Commitment and Plasticity of Helper CD4+ T Cells. 2011.. Science (2010) vol.Reading: Text: “Janeway’s Immunobiology”. Taylor & Francis Group. LLC).

They will be segregated in two main categories: -Those resulting from intrinsic B cell defects and characterized by gene mutations encoding component of the isotype switching machinery (AID deficiency) -Those characterize by defects extrinsinc to B cells. We will review three major kinds of SCID: . In this section. we will present a few syndromes such as IPEX and APECED. Meffre Primary immunodeficiencies are a set of rare diseases usually resulting from defects in a single gene. Indeed. Actually.Those resulting from defects of in the components of the V(D)J recombination machinery. As a consequence. NK and other antigen presenting cell functions. we will report on common variable immunodeficiency diseases (CVID). Interestingly. We will review in this section the different types of mutations that can affect the production of memory B cells and gammaglobulin secretion. these cells are very important for the establishment and/or maintenance of peripheral tolerance. . As a consequence. Primary immunodeficiencies affecting late B cell development and the generation of memory cells There are many examples of primary immunodeficiencies affecting the production of isotype switched B cells and the production of gammaglobulins.Primary and Acquired Immunodeficiencies November 16 Instructor: E. In this case. B and NK cells in the patient’s blood. demonstrating that we have still a lot to learn from the analysis of primary immunodeficiencies. which are characterized by the impaired generation of regulatory T cells. In these cases. More classical forms of such defects are called HyperIgM syndromes and still allow IgM secretion but impair IgG and IgA production. while patients are usually unable to mount proper immune reactions against pathogens. Indeed. B. the first reviewed case of primary immunodeficiency was reported in 1952 and affected a boy who displayed no gammaglobulins in his serum. Severe Combined Immunodeficiency diseases (SCID) SCIDs are a set of primary immunodeficiencies that are diagnosed during the first year of life. we will discuss the case of the common g chain gene mutation. Symptoms often occur during the months/first years of life and affect diverse components of the immune systems. patients have no or very few T and B cells. the lack of expression of molecules expressed on T cells and essential to activate B cells such as CD40 ligand result in the first identified form of hyperIgM syndrome. A classification of these diseases is based on the presence of T. We will present in this lecture a group of primary immunodeficiencies and discus their impact on T. . it is very common to identify specific immune responses targeting the patient’s own body. . early T and B cell precursors are unable to recombine their specific receptor genes (T cell receptor and immunoglobulin genes.Those resulting from mutations in genes encoding B cell receptor components and inducing specific blocks in B cell development Primary immunodeficiencies affecting the maintenance of tolerance Patients with primary immunodeficiencies often suffer from paradoxical autoimmune syndromes. the analysis of primary immunodeficiencies provides unique opportunities to determine the impact of gene mutations on the development and/or function of the immune system in humans. It took more than forty years before gene mutations associated with this syndrome called X-linked agammaglobulinemia (XLA). These lifethreatening diseases usually affect several arms of the immune system.Those characterized by an absence of T cells and resulting from either T cell specific gene mutations (CD3 component) or cytokine receptors. On a scientific aspect. -Finally. patients suffer from recurrent infections that can be life threatening. which are the most common of all immunodeficiencies and whose gene defects are mostly unknown. respectively). which in humans results in an absence of T cells and normal B cell counts whereas similar mutations result in an opposite phenotype in mice (no B cells and normal T cell numbers).

” Nat Rev Immun (2005). vol 5. et al. “Molecular Defects in T and B cell Primary Immunodeficiency disease. pp. 880-892 .Reading: Primary Literature: Cunningham-Rundles.

IL-13 and IgE exists to provide protective immunity to parasites. There are two main types of mast cells in the body. the inhaled allergen can be engulfed by the lung resident dendritic cells.Allergy November 26 Instructor: R. neurotoxins leukotrienes) that are intended to kill parasites but are also very toxic to the host. Basophils can amplify the response stated by mast cells. Genetic factors Several genetic loci have been identified to be associated with atopy. Most human allergens are relatively small proteins that are inhaled or ingested in very small quantities. • Chr. However. Thus. IL-13. Unlike mast cells and basophils. . IL-4. They tend to have enzymatic activity (cystein protease) and are stable and soluble. collagenase. Basophils release histamine and other substances toxic to parasites but also to the host. the activation of the eosinophils is tightly regulated at the level of 1) total cell number. 11: Encodes the β subunit of FceR. contraction of bronchial smooth muscle leading to asthma. β2-adrenergic receptor. those associated with vascularized connective tissues (connective tissue mast cells) and those found in submucosal layers of the gut and lung (mucosal mast cells). Predisposition to allergy The prevalence of atopy in the industrialized countries is on the rise in recent years. • Chr. Antigens that evoke Th2 cells that drive an IgE response are known as “allergens”. 2) location and 3) threshold of activation. Recent studies indicated that basophils. Systemic encounter of allergen (via the blood stream) triggers general release of histamine by the connective tissue mast cells leading to anaphylaxis and even death. 6: HLA-DR • Chr. IL-5. all of these mast cells have prebound IgE on their FceR. and depending on the route of allergen encounter. Th2 responses to innocuous antigens cause allergic diseases. basophils also constitutively express FceR and can be recruited to the site of IgE-triggered reactions via inflammatory lipid mediators cytokines and chemokines. IL-9. in the industrialized countries where parasite infection is rare. 5: cluster of Th2 genes (IL-3. Medzhitov Although Th2 immunity characterized by IL-4. Basophils Like mast cells. eosinophils only express surface FceR upon activation by Th2 cytokines and chemokines. Food allergens that are ingested survive the acidic environment of the stomach and reach the small intestine where they are taken up by dendritic cells. Once taken up into the airway mucosa. can prime Th2 responses to parasites and allergens. Eosinophils The eosinophils are found in tissues within the connective tissue beneath the mucosal epithelium. In allergic individual. Cellular mediators of allergy Mast cells Mast cells express high affinity FceR on their cell surface. Ingestion of allergens triggers activation of the gut mucosal mast cells inducing contraction of intestinal smooth muscle (vomiting) and outflow of fluid into the intestine (diarrhea). Inhaled allergens trigger lung mucosal mast cells to release their granules and cause increased mucus production. causes different diseases. IgE can bind to the FceR in the absence of bound antigen. the precise mechanism by which the allergens induce Th2 immunity by dendritic cells is unknown. but not dendritic cells. eosinophils release contents of preformed granules containing many toxic substances (peroxidase. GM-CSF). Atopic individuals possess higher levels of IgE and eosinophils in circulation. Upon activation.

2001 Jan 11.Environmental factors • Early exposure to infectious diseases reduce the prevalence of allergy (“Hygiene hypothesis”) • Pollution Allergic diseases Asthma Skin allergy (urticaria. 2001 Jan 4.344(1):30-7. Celiac disease Treatment of allergy Desensitization: shift the preexisting Th2 response to Th1 Antihistamine: block histamine H1 receptor Steroids: anti-inflammatory Blockade of chitinase? Reading: Text: "Janeway’s Immunobiology". . Primary Literature: Kay AB. Chapter 13. Taylor & Francis Group. Allergy and allergic diseases N Engl J Med. 8th Edition. chronic eczema) Food allergy. and N Engl J Med. LLC). by Kenneth Murphy (Garland Science. 2011.344(2):109-13.

b. d. 2. K. c. mechanisms are needed in the periphery to contain the immune response or to divert it from a pathogenic response = peripheral tolerance. b. In the event that central tolerance is not complete. Generation of a diverse repertoire will lead to production of T and B cell receptors that are reactive with self. A failure of self tolerance results in autoimmunity. Mechanisms needed to eliminate most autoreactive cells at the time and location of development – this is referred to as central tolerance. B cell tolerance a. Death by apoptosis (clonal deletion b. Central B cell tolerance in the Bone marrow: a. Fate of autoreactive B cells depends on the strength of the BCR signal: 1. for example for antigens that are not expressed in the central compartments at the time of development. Herold Definition of tolerance: The lack of an immune response to an antigen. 2. Mechanisms of tolerance – over view – note that it is difficult to precisely define mechanisms for an immune response that doesn’t happen! a.Peripheral Tolerance November 28 Instructor: 1. Lymphocyte repertoire must be diverse. Receptor editing . In the case of self tolerance it refers to the state in which an immune response against self antigens is not generated under normal conditions in spite of the fact that MHC molecules normally are binding self antigens. somatic hypermutation of Ig will generate self reactive T cells.

Antigen specificity of Tregs? Treg function is not antigen specific c. Proteins are expressed in the medullary thymic epithelial cells. Central Tolerance i. Oral tolerance: mechanisms of tolerance at the gut mucosal surface 4. et al) a. Foxp3: Foxp3 can reprogram CD4+ T cell function. IL-10 c. brain. Infectious tolerance c. IL-5. TGF-beta. 3. d. Effects of cytokines in the environment b. A number of different phenotypes have been described. Regulation/regulatory T cells a. Adaptive Tregs: T cells that do not emerge from the thymus as regulatory T cells can acquire regulatory function. Leads to negative selection. Ignorance 2. Immune privileged sites: eye. Some of these cells also express Foxp3. testes. Tolerance limited to antigens expressed in the thymus ii. Lack of costimulation or negative costimulation 4.2. It can inhibit NFAT activation at the IL2 promoter preventing transcriptional activation of the IL-2 gene. Outside of the bone marrow Immunologic ignorance T cell tolerance a. 3. uterus. Mutations are associated with IPEX (immune dysfunction/polyendocrinopathy/enteropathy/ enteropathy/X-linked. b. TGF-β Environmental tolerance (Cobbold. Civil service model 5. and other cytokines been described. . Cells that produce IL-10. Cytokines a. APECED = autoimmune polyendocrinopathy candidiasis ectodermal dystrophy In the periphery 1. Inhibits AP-1 RORγt – a transcription factor involved in Th17 cell differentiation. IL-4. Naturally occurring Tregs b. The identification of the molecular basis of the IPEX syndrome was a key finding in understanding the role of Foxp3 in regulatory T cells. 3. c. Bystander suppression b. The AIRE gene promotes the expression of several genes in the thymus. iii. Waldmann.

" Kenneth Murphy. Sprent J. Fazekas de St Groth B. Vinuesa CG.435(7042):590-7. Nature. Paul Travers and Mark Walport.Reading: Text: “Janeway’s Immunobiology. 2005 Jun 2. . 8th Edition. Cellular and genetic mechanisms of self tolerance and autoimmunity. Chapter 15 and Chapter 8 section 8-19 Review Article: Goodnow CC.

In certain cases cellular mechanisms are thought to be predominant mediators but studies of immune interventions in man have suggested that both humoral and cellular arms of the adaptive immune response are involved. C. Indeed. e. g. 3. In certain cases (e. CTLA-4. Certain drugs can also initiate autoimmunity. X-linked disease c. Herold Autoimmunity A. enteropathy. targets of autoimmune responses such as DNA can activate TLRs (e. polyendocrinopathy. Scurfy/IPEX: Immune dysregulation. Rates of concordance in identical twins B.g. Other genetic loci. The major genetic determinants of autoimmunity are genes within the major histocompatibility complex i.Autoimmunity November 30 Instructor: 1. Mechanisms that may account for the relationship d. etc. some of which are associated with immune responses. the precise immunologic mechanisms are not certain. K. Molecular mimicry is one postulated mechanism to account for the relationship between environmental events and autoimmunity – specific examples f. Please refer to these mechanisms discussed in the lecture on tolerance. There are examples of specific genetic mutations that lead to autoimmune diseases i. Examples of MHC-disease relationships ii. Innate immune responses may also be involved in initiation of autoimmune responses. The immune nature of several diseases now identified as autoimmune was based on the finding of autoantibodies but in most cases. B. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy ii. e. Therefore consideration of the causes and treatments of these diseases is closely linked to understanding of mechanisms that maintain tolerance. Autoimmunity is the ultimate example of loss of tolerance to self: horror autotoxicus. Adaptive immune responses are involved in autoimmune diseases . have also been associated with autoimmune diseases. The initiation of autoimmune diseases. IL-23R.g TLR9). involves a combination of environmental and genetic factors A. 2. Autoimmune diseases affect every organ system.g. hence loss of self tolerance. anti-CTLA-4 antibodies) this can occur through known mechanisms. C.

2008 Jun 19. antigens.453(7198):1051-7. Examples include: d. intra and intermolecular spreading of the immune response 4. iii. Pathogenic autoreactive T cells can clearly cause autoimmune disease in animal models and are found in human autoimmune diseases C. Eisenbarth G. Graves’ disease d. . Unstable Tregs? D. Examples of specific autoimmune diseases: Most organ specific autoimmune diseases are T cell mediated. Induction and effector functions of T(H)17 cells. Therapies for autoimmune diseases: anti-T cell/anti-B cell/anti-cytokine/blockade of cell migration. Genetics.A. pathogenesis and clinical interventions in type 1 diabetes. Chapter 15. Nature. Chronicity is a feature of autoimmune diseases: i. Herold K. Myasthenia gravis 5." Kenneth Murphy. modification of self proteins as a mechanism of generating new epitopes. Role of B cells: as antibody producing cells. development of tertiary lymphoid organs as sites of activation of adaptive immune responses. 2010 Apr 29. Examples include: a. Nature. Reading: Text: “Janeway’s Immunobiology. Multiple sclerosis But others are mediated by autoantibodies. Rheumatoid arthritis c. 8th Edition. Autoimmune hemolytic anemia e. Tolerance is the ultimate goal. as antigen presenting cells B.464(7293):1293-300. animal models. and Kuchroo. VK et al. cellular mechanisms b. ii. Review Articles: Bettelli E. Type 1 diabetes (as an example): MHC association. Paul Travers and Mark Walport. Bluestone JA.

b.g. It can be transferred with T cells. Mechanisms whereby tumors avoid immune recognition a. Acute i. Immunization with tumor specific antigens/Adoptive transfer of dendritic cells pulsed with peptides b. Autologous b. CD8+ T cells Tumor antigens: a. 2. Grafts a. 5. 3. 4.Transplantation & Cancer Immunology December 3 Instructor: K. 2. most tumor antigens are peptides that are over-expressed in tumor cells but area also expressed at lower levels in normal tissues d. Costimulation enhancers e. Proteins selectively expressed in tumors are candidate tumor rejection antigens c. Failure to express antigen: immune selection of antigen loss variants e. Syngeneic: same strain Allogeneic: different strain. Hyperacute: preformed antibodies against a graft – often against blood group or polymorphic MHC antigens. These proteins are generally presented by Class I MHC molecules Paradox –T cells infiltrate a tumor but do not destroy the tumor . Herold Cancer Immunology 1. BCG c. 6. same species c. Alloreactivity occurring days and weeks after transplantation. recruitment of regulatory T cells Immune surveillance may normally prevent the outgrowth of variant cells: role of NK cells. Blocking negative signals: anti-CTLA-4 Biologics against antigens expressed on tumor cells e.tumor infiltrating lymphocytes (TILs) Enhancing immunogenicity to tumor antigens and cells a.g. Transplant Immunology and Immune therapy 1. a. 7. However. Rituximab (anti-CD20 mAb). Adjuvant e. HER-2/Neu Therapy with engineered effector cells. . Xenogeneic: different species Classification of Rejection: Graft rejection is mediated by T cells. Failure to present antigen including loss of Class I MHC expression b. Failure to activate T cells c. B7-1 d. Poor access to the tissue d. Inhibitory factors: TGF-β. Tumor rejection antigens may arise by point mutations in self proteins b.g.

Presentation of graft antigens to the host a. Chronic: i. Adoptive immune therapy with regulatory T cells? g. Mycophenolate mofetil f. anti-IL-6. ii. Campath (anti-CD52) v. Direct: recognition of donor cells by recipient’s T cells b. Indirect: Recognition of processed antigens of the donor by recipients APC’s c. Janus kinase inhibitors d. Glucocorticoids b.g. anti-IL-1. Rapamycin e. Examples of Biologics: Human Abs vs humanized abs vs murine abs vs other animals i. Chronic organ rejection is caused by inflammatory vascular injury to the graft. Nataluzimab binds to α4β1 (on central memory and effector T cells) and α4β3 integrins iii. . Anti-CD3 vi. Cytotoxic drugs: i.2. Rituximab (anti-CD20) iv. Drugs to treat rejection and autoimmunity: a. Azathioprine: ii. Alloreactivity can occur months to years after transplantation and is associated with gradual loss of graft function. Infliximab (anti-TNF) Etanercept (the p75 TNF receptor on a Fc portion of IgG. Calcineurin inhibitors c. Anti-lymphocyte serum ii. Anti-Cytokines: e. CTLA4Ig vii. Cyclophosphamide iii.

Ferrone S. Lancet. 8th Edition. Clin Exp Immunol.Reading: Text: “Janeway’s Immunobiology. Weiner LM." Kenneth Murphy. Monoclonal antibodies for cancer immunotherapy. Allison JP. Chapter 15. 2009 Jul. Quezada SA. 2009 Mar 21.157(1):9-19.373(9668):1033-4 . Dhodapkar MV. Paul Travers and Mark Walport. Cancer immunotherapy: co-stimulatory agonists and co-inhibitory antagonists. Review Articles: Peggs KS.

there are many fundamental questions that remain unresolved. Why some infections are so common? Why some infections are so deadly? Why the majority of symptoms of infectious diseases are caused by the immune system itself? Why do we react to allergens? We now know a fair amount of details about activation of the immuen response. What parameters dictate the generation of a protective immune response? Does the immune system play a role outside of host defense from infections? These and other questions of fundamental scientific and medical importance will be discussed in this lecture. . and yet we still cannot design successful vaccines for most pathogens. Medzhitov Despite tremendous advances in understanding the function of the immune system.Unresolved Questions in Immunology December 5 Instructor: R.