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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 firstname.lastname@example.org
Teaching Fellows: Will Khoury-Hanold email@example.com Omotooke Arojo firstname.lastname@example.org Tianxia Guan email@example.com
MCDB 430/530 Lecture Schedule & Syllabus
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
Evening Tutorial 5: Week of October 29 .November 2: Cell mediated immunity tutorial Required reading: Barber. 31(2). 1361-5.October 5: Antigen presentation tutorial Required reading: Maric et al. pp. pp. 111(6). Readings will be posted on Classes*v2 prior to each tutorial. vol.December 7: The immune system in health and disease tutorial Required reading: Boitard. Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Evening Tutorial 8: Week of December 3 .21: Innate immunity tutorial Required reading: Sander et al. Tutorial grades (10% of the final grade) are based on participation and attendance. Defective antigen processing in GILT-free mice. 294(5545). . vol. pp. Science (2004). Evening Tutorial 3: Week of October 1 .October 12: Review for Midterm No required reading. Evening Tutorial 6: Week of November 5 . 439(7077). 851-57. et al. Nature (2011) vol. but also allow for review of class material.Evening Tutorial Schedule & Required Readings Evening tutorials are held throughout the semester.November 9: Humoral immunity tutorial Required reading: Gatto. et al.7: Methods in immunology tutorial Required reading: Janeway’s Immunobiology. pp. Immunity (2009). 259-69. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Journal of Clinical Investigation (2003) vol. Acceleration of type 1 diabetes mellitus in proinsulin 2–deficient NOD mice. Appendix I: The Immunologist’s Toolbox Evening Tutorial 2: Week of September 17 . as well as students’ questions. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Evening Tutorial 7: Week of November 12 . 303(5664). et al. Evening Tutorial 1: Week of September 3 . 474(7351). Evening Tutorial 4: Week of October 8 . D. Science (2001) vol. pp. pp385-9. Review your class notes and bring questions. 1662-65. et al.November 16: Immunity & infection tutorial Required reading: Macpherson. vol. 8th Edition. 682-87. These tutorials focus on a discussion of primary literature. Restoring function in exhausted CD8 T cells during chronic viral infection Nature (2006).
her or his answer may be emailed to their Teaching Fellow. a student cannot attend class on the day that a take-home question is due. each worth 5% of the final grade. 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 to be typed and must not be longer than one page (one inch margins and 12 point “Times” font). Answers are considered late beginning right after class on the due date and will have two points deducted per day late. However.Take-Home Question Schedule Throughout the semester. every student must submit his or her answers by the end of the lecture (10:15 a.) on the due date. If. for any reason. there will be four graded take-home questions.m.
and introduce the teaching faculty who are responsible for covering each topic. course structure.Why Study Immunology? August 29 Instructor: Carla V. I will also discuss the importance of the immune system in health and disease. grading schemes. Sit back. relax and enjoy! . Rothlin In this introductory lecture I will introduce the topics to be covered by this course.
It is important to recognize that the pathogenicity of a particular microorganism depends on the fitness of the host. • Innate immune recognition operates by non-clonal.Structure of the Immune System I August 31 Instructor: Carla V. The innate immune response. adaptive immunity produces signals that stimulate and modulate innate immune responses. phagocytes (macrophages. germline encoded receptors that recognize conserved and invariant features of microorganisms known as pathogen-associated molecular patterns (PAMPs). altering the behavior of the target cell. Phagocytes express various types of receptors including those for pathogen recognition (pattern recognition receptors. mucus. Phagocytes become activated upon interaction with the pathogen. The mammalian immune response can be classified in three phases: immediate innate responses. the immune response consists of two main activities: recognition and effector functions. changes in pH. The genes encoding antigen receptors are assembled through somatic recombination from gene segments in the germ line.e. The adaptive immune response. neutrophils. referred to as innate (evolutionarily ancient host defense system) and adaptive (vertebrate host defense system). While the innate immune system operates early in a response to infection. The purpose of the immune system is to recognize invading microorganisms and to contain the infection or clear the microorganisms from the body. fungi and parasites. there are two types of immune recognition. For recognition. For example. These components of the innate immune system contribute both to recognition of the pathogen and to effector responses. certain microorganisms are only pathogenic in immuno-compromised individuals. induced innate responses and adaptive immunity. the adaptive immune response requires time to develop. The innate and adaptive responses are briefly described below. viruses. Activated phagocytes secrete small proteins called cytokines that act by binding receptors on target cells. B cells . Therefore. anti-microbial peptides). Adaptive immunity amplifies the protection provided by the innate immune response and provides future immunity to the same pathogen (memory/adaptive immunity). The adaptive immune response is mediated by the activation of lymphocytes. The first one consists of physical and chemical barriers (i. Conversely. • Adaptive immune recognition operates by the clonal selection of individual lymphocytes expressing specific antigen receptors. In vertebrates. Lymphocytes are divided into two classes (T and B lymphocytes) with different recognition receptors and function. 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. Phagocytes releasing chemokines can recruit additional cells to the area of infection to help in clearance. whereas T cells express T cell receptors. One class of cytokines is called chemokines that have chemoattractant properties. The innate immune response is needed to initiate and direct adaptive immunity. Importantly. PRRs) and for complement proteins to aid in pathogen clearance. Invasion by a microorganism leads to a quick response by the innate immune system. Rothlin Immunology is the study of mechanisms used to defend our body from invasion by microorganisms. Mechanisms of host defense Multicellular organisms defend themselves against pathogens in multiple ways. and dendritic cells). These are known as opportunistic pathogens. There are four kinds of pathogens: bacteria. and natural killer cells. innate and adaptive immunity do not operate independently of each other. allowing the generation of a diverse repertoire of receptors. The innate immune system includes complement proteins. Mechanisms of microbial recognition in vertebrates Microorganisms with the potential to cause diseases are called “pathogens”. B cells express cell surface immunoglobulins.: physical barriers formed by epithelial surfaces.
Once pathogens enter. lymphocytes do not respond to self-components because clones of lymphocytes with receptors that bind to self. CD4 T cells and CD8 T cells. B cells. Reading: "Janeway’s Immunobiology". T cells recognize epitopes (small stretch of amino acids) that are often buried inside the antigen. LLC). but only when instructed to do so by the innate immune system. the innate immune system discriminates self from non-self by recognizing conserved molecular patterns associated with pathogens.g. . a subset of T cells. The mature dendritic cells. Furthermore. CD4 T cells can be further sub-divided into various types. 8th Edition. T cells can be divided into two major classes. TFH and regulatory T cells that release unique panels of cytokines. by Kenneth Murphy (Garland Science.: dendritic cells) mature and interact with lymphocytes in specialized lymphatic tissues or organs. by secreting antibodies can eliminate pathogens found extracellularly but not intracellularly. First. which are no longer phagocytic. while maintaining a balanced immune response. whereas Th2 cells induce antibody secretion and immunity against large parasites. Th17. fail to mature. they display various activities mediated by the release of specific cytokines. The adaptive immune system uses different effector responses to protect against the four classes of pathogen. Also. 2011. Once B cells are activated. carry the pathogen to the lymphoid tissues via lymphatic vessels to be recognized by lymphocytes. In contrast. known as regulatory T cells is fundamental for restraining auto-reactive immune responses. Most lymphocytes live in specialized lymphoid tissues or organs or can be found recirculating in the blood under normal condition.molecules are either eliminated. Once activated T cells divide and differentiate into effector T cells. Th1 cells activate macrophages and help eliminate bacteria living intracellularly. Chapter 1. Taylor & Francis Group. These secreted immunoglobulins (antibodies) can circulate throughout the body and bind to the pathogen. contributing to its clearance. Th2. or fail to be activated. Lymphocytes of the adaptive immune system recognize and respond to pathogens. Microorganisms grow and survive in different niches once they have invaded.recognize regions of antigen (epitope) that is displayed on the surface of antigens. the lymphocytes with specific receptors will divide and migrate to the site of infection to combat the pathogens. The induction of adaptive immune responses begins when the antigen presenting cells that are activated during the innate response (e. How is self-reactivity avoided? A variety of mechanisms ensure that the immune system is tolerant of itself. there are specialized subsets of T cells. This specialization in T cell function allows the immune system to respond to a wide variety of microorganisms. CD8 T cells kill virally infected cells. For example. For those pathogens that reside and multiply in cells. they divide and differentiate into plasma cells that secrete a soluble form of their surface immunoglobulin receptor. such as Th1.
Naïve lymphocytes enter lymph nodes via a specialized structure called the high endothelial venules (HEV). T cell precursors migrate from the bone marrow and further develop in the thymus. more than half of them T cells. B lymphocytes develop in the bone marrow. a milliliter of blood contains around 2-4 x 106 lymphocytes. Examples include: GATA-1 (erythroid). In all vertebrates. and basophils. granulocytes. as most blood cells have a limited life span. the bone marrow. Oct-2 (B lymphocyte). myeloid. lymphoid). Some of these T cells appear to be independent of the thymus. the Bursa of Fabricius. 2) the expression of all surface molecules needed for activation and differentiation. monocytes. In the human. 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. Lymphocytes are the primary cell of the adaptive immune system. Infusion of bone marrow stem cells into a lethally irradiated animal can repopulate all of these cell populations. The maturation of stem cells into different cell types is accompanied by the expression of lineage specific genes. a member of the Ly6 family). The thymus is seeded by progenitor cells without immunological specificity. GM. 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. Cells of the immune system White blood cells consist of neutrophils. B cells. Activated or memory lymphocytes extravasate from the vasculature. . eosinophils. avian Bursa and thymus are termed primary lymphoid organs. and 3) the expression of cell surface molecules needed for recirculation and localization in tissues in the periphery. Resting T and B cells are small.Structure of the Immune System II September 5 Instructor: Carla V. GATA-2 (erythroid. immunocompetent T cells. The other types of white blood cell are important in various processes. while in birds they originate in a specialized organ. Ikaros (lymphoid). skin. they then percolate through intercellular spaces. etc. The production and the maturation of the differentiated cell types from pluripotent precursors is regulated by hematopoietic growth factors. However these early cells express the antigen Thy 1 and Sca-1 (stem cell antigen. The route of recirculation differs depending on the activation state of the lymphocyte. respiratory and reproductive systems. a thoracic organ anterior to the heart. a specialized subset of large non-recirculating pool of T cells is found in the epithelia of the gut. and natural killer cells. macrophages. Since they manufacture lymphocytes. lymphocytes. platelets and lymphocytes. Three important events occur in these sites: 1) the expression and selection of cells with antigen-recognition receptors. and the secondary lymphoid organs (lymph nodes and spleen). and produces clonally diverse. SCF and various cytokines. these regulators also control the functional activity of the cells by eliciting intracellular responses mediated by cell surface receptors. non-phagocytic cells. Examples of such regulators include erythropoietin. They are morphologically distinct from other cells and do not express lineage specific cell surface markers (Lin-). tissues. Rothlin Hematopoietic stem cells and lineage commitment The cellular subsets of peripheral blood are constantly replenished by the process of hematopoiesis. Most T cells continuously recirculate between blood.CSF. Stem cells constitute a very small fraction of cells in the bone marrow. Certain transcription factors have been shown by targeted deletion to be essential for particular lineages of cells. In addition to influencing differentiation. Hematopoiesis gives rise to erythrocytes. and travel towards lymph nodes. mast cells. and enter inflamed tissues. However. They can be found as naïve cells if they have not interacted with antigen or effector/memory cells if they have interacted with antigen. enter the lymphatic vessels. Lymphocytes in body fluids Naïve B and T cells circulating in the blood traverse the lymph nodes and the spleen. indicating the highly proliferative and differentiation capacity of stem cells. Lymphocytes consist of T cells.
The mucosa associated lymphoid tissues (including Peyer’s patches) collect antigens crossing the mucosal epithelia. The spleen filters the blood collecting antigens that enter the vasculature. 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. They divide and migrate out of the secondary lymphoid organs via peripheral blood and into the site of infection. Lymph nodes drain (via lymphatic vessels) extracellular fluid (lymph) from most tissues of the body as a means of collecting antigen. Based on early innate immune responses. lymph nodes and Peyer’s patches. Reading: "Janeway’s Immunobiology". The major secondary lymphoid organs are: spleen. LLC). Taylor & Francis Group. antigen presenting cells carrying the pathogen-associated antigens migrate from the peripheral tissues to the lymph nodes via the lymphatic vessels. The secondary lymphoid tissues have specialized mechanisms to collect antigen and antigen reactive lymphocytes. The architecture of the secondary lymphoid tissues fosters interaction between antigen and lymphocytes. The structure of the secondary lymphoid tissues varies depending on its function. Lymphocytes enter secondary lymphoid organs from the blood via specialized mechanisms designed to recruit naïve lymphocytes (high endothelial venules or HEV). Naïve T and B cells first encounter antigens in the secondary lymphoid organs and become activated. This activation includes the clonal selection and expansion of lymphocytes expressing specific antigen receptors. the microvasculature at the site of infection is specialized to recruit activated lymphocytes and other cells important in defense. In addition. 2011. tonsils. appendix.Lymphocytes are activated in secondary lymphoid tissues Secondary lymphoid tissues foster interactions between antigen presenting cells and lymphocytes. 8th Edition. by Kenneth Murphy (Garland Science. . Chapter 1.
Activation of the Ga subunit by GTP allows it to both regulate an effector protein .. in response to agonists. and an associated dimer of b and g subunits that can confer both membrane localization of the G protein (e. GPCRs respond to agonists by promoting the binding of GTP to the G protein a subunit.Receptors & Signaling I September 7 Instructor: Carla V. via myristoylation) and direct signaling (e. such as Kit and FLT3. the process by which extracellular signals interact with their cognate receptors leading to the activation of biochemical cascades that result in a cellular response. A few receptor protein kinases phosphorylate serine or threonine residues.: nuclear receptors.g. G proteins are composed of a GTP-binding a subunit.g. G-protein coupled receptors (GPCRs): interact with distinct heterotrimeric GTP-binding regulatory proteins known as G proteins. Classical examples of intracellular signaling pathways involve: • Activation of G proteins. phospholipase C. 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. • • • Intracellular receptors.: growth factor receptors. 2) Recognition of the external stimulus by the receptor provides the first message in signal transduction. 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. Receptors can be classified according to their cellular localization and shared mechanism of action into relatively few functional families. e. 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.g. 1) Signal transduction is initiated upon recognition of the extracellular stimulus (agonist) by its cognate receptor. which are expressed on developing lymphocytes). Rothlin Immune cells are able to sense changes in the extracellular environment and respond accordingly. Protein kinase–associated receptors: lack the intracellular enzymatic domains but. Most receptor protein kinases phosphorylate tyrosine residues in their substrates (e.: activation of inward rectifier K+ (GIRK) channels). which confers specific recognition by receptor and effector. and plasma membrane ion channels selective for Ca2+ and K+. Receptors of this group include multiple cytokine receptors and T and B-cell antigen receptors. This event is followed by the propagation and amplification of the signal through the engagement of intracellular signaling pathways. NOD like receptors. bind to or activate distinct protein kinases on the cytoplasmic face of the plasma membrane. G proteins are signal transducers that regulate multiple effectors including enzymes such as adenylyl cyclase. such as the TGF-b receptor. phosphodiesterases.
protein phosphatases (e.and drive the release of Gbg subunits. A classical example of this transcytoplasmic signaling is the JAK/STAT system. Receptor signaling activates guanine-nucleotide exchange factors (GEFs). can migrate into the nucleus. IP3 acts as a second messenger to release Ca2+ from intracellular stores. and ion channels that upon activation permit the release of second messenger molecules such as cyclic-AMP (cAMP). results from the prolonged exposure of the receptor to the stimulus. The elucidation of the modular MAPK cascade with its three consecutive protein kinases has had immediate implications for understanding signal amplification and switching.5-trisphosphate (IP3) and diacylglycerol (DAG). Common effector proteins of G-proteins include phosphodiesterases. diacylglycerol (DAG). which can. These include: • Receptor Desensitization. leading to the activation of the small GTPases and the recruitment of downstream effectors. cyclic-GMP (cGMP). in addition to regulating their own group of effectors. Ubiquitin-mediated degradation. a state in which the receptor can still recognize the stimulus but is unable to transduce the signal. This leads to conformational changes in the receptor. This mechanism.: Tyrosine phosphatase SHP1) play a key role in shutting down signaling pathways. Thus. because the terminal MAPK. Nuclear Translocation of Transcription Factors. in which ligand binding to a cytokine receptor activates the associated JAK family protein Tyrosine kinases.4. once activated. such as Ras-GEF and PKC. • Activation of small GTPases. a MAP kinase kinase (MAPKK). and Ca2+. This allows GTP to bind in its place. activation of phosphatidylinositol phospholipase C results in the hydrolysis of phosphatidylinositol bisphospate (PIP2) and the generation of inositol 1. DAG binds and activates signaling proteins. inositol triphosphate (IP3). resulting in receptor “desensitization”. • • • 3) Activation of signaling pathways is tightly regulated by a variety of molecular mechanisms that efficiently terminate the signaling response. play major roles in the regulation of gene expression and the rearrangement of the actin cytoskeleton. returning the system to the basal state. Endocytosed GPCRs are sorted from endosomes to lysosomes and degraded. • • • . Phosphatases. In the canonical phosphoinositide pathway. Covalent addition of one or more molecules of the small protein Ubiquitin targets proteins to proteosomal or lysosomal degradation. Phospholipid and ion-based signaling. receptors can be internalized. reassociate with GDP-liganded Ga. phospholipases. a MAP kinase kinase kinase (MAPKKK). leading to the activation of transcription factors. which bind to small GTPases in their inactive state and displace GPD. which leads to the activation of the STAT transcription factors. These events are fundamental to antigen receptors signaling pathway and will be reviewed in the lecture Receptors and Signaling II. GPCR trafficking has critical functions not only in signal termination but also in receptor resensitization. As a significant proportion of signaling events depend on protein phosphorylation. Within endosomes. the intrinsic GTPase activity of these proteins leads to the hydrolysis of GTP to GDP and their inactivation. a process important for signal termination. adenylyl cyclases. and a MAP kinase (MAPK). and most importantly for nuclear signaling. Receptor Internalization. Activation of the MAP kinase pathway. commonly observed in ligand-gated ion channels. This is a common feature in GPCR signaling.g. A complex pathway that consists of a cascade of three protein kinases. 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. Overtime. thus triggering a program of Ca2+-activated events. In addition to desensitization. GTPases such as those from the Ras family.
Chapter 7.Reading: Text: "Janeway’s Immunobiology". Goodman & Gilman's “The Pharmacological Basis of Therapeutics”. . Laurence L. 2012. Chapter 1. Parker. Lazo and Keith L. LLC). John S. 8th Edition. Brunton. Taylor & Francis Group. 11th Edition. by Kenneth Murphy (Garland Science.
including TNF. antimicrobial peptides and proteins and the complement system. by Kenneth Murphy (Garland Science. however. Many infections are successfully handled by the innate immune system. LLC). IL-8 and KC. R. Iwasaki The innate immune system is an evolutionarily ancient form of host defense. 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. Nature. 8th Edition. Innate immune response can be activated immediately upon infection and helps maintain pathogens prior to the induction of adaptive immunity. 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. 2007 Oct 18:449(7164):819-826. IL-1. Reading: Text: "Janeway’s Immunobiology". phagocytes. Recognition of microorganisms and activation of the immune response. bacterial infections will typically activate epithelial defenses. each designed to protect from different types of infectious challenges. the adaptive immune response is generated to provide a powerful and antigen specific immunity to pathogens. Chapter 2 and 3. . The innate immune systesm provides the first line of defense against pathogens. The activation of host defense modules is coordinated by different cytokines. including MCP-1. 2011. IL-6. Review Articles: Medzhitov. While the adaptive immune system is found only in vertebrates. For example. the innate immune system exists in all animals and plants.Innate Immune System September 10 Instructor: A. and chemokines. Different infections induce different combination of host defense modules. Innate immune system consists of several host defense modules. Taylor & Francis Group. When innate host defenses are insufficient.
as well as several MAP3 kinases that function downstream of TRAF6 and activate NF-kB and MAP kinases. including Nod-like receptors. as well as for lipoproteins found in all bacteria. TIRAP. serine/threonine kinases IRAK1 and IRAK4. There are about dozen or so TLRs in mammalian species. Nod1 and Nod2 are intracellular receptors that detect fragments of bacterial peptidoglycans. In addition to TLRs. because it is also found in the members of the IL-1 receptor family. TLRs function as sensors of microbial infection. Collectively. TLR5 recognizes bacterial flagellin. ii) PAMPs are produced only by microbes and not by the host organism. TLRs induce signaling pathways that activate NF-kB and MAP kinases. TLRs play a crucial role in the immune system. This property of PAMPs allows a limited number of germline-encoded receptors of the host to recognize a wide variety of microorganisms.Pattern Recognition Receptors September 12 Instructor: R. The best-characterized family of PRRs is the Toll-like receptor (TLR) family. They detect infection and activate the host immune responses. prevents generation of 'escape mutants' because such mutations are lethal for the microbes. innate host defense responses. Medzhitov The major distinguishing feature of innate immunity is the mechanism of pathogen recognition. innate immune recognition can lead to activation of adaptive immunity. iii) PAMPs are conserved molecules essential for the survival of the microbes. These are referred to as Pathogen-Associated Molecular Patterns (PAMPs). There are 4 TLR signaling adapters that also contain TIR domain – MyD88. Different TLRs utilize different combination of the adapters to induce both overlapping and distinct subsets of cellular responses. germline-encoded receptors that recognize conserved molecular patterns associated with pathogens. Activation of these signaling pathways leads to induction of inflammatory responses. but they detect the presence of viral RNA in infected cells. Dectin is a transmembrane receptor specific for beta-glucans – major components of fungal cell walls. The receptors that recognize PAMPs are called Pattern Recognition Receptors (PRRs). Thus TLRs represent a crucial component of the host defense system. as well as induction of adaptive immune responses. ubiquitin ligase TRAF6. and TRIF. Thus. Dectins. The most familiar examples of PAMPs are bacterial LPS and lipoteichoic acids. The canonical pathway shared by all TLRs includes MyD88. Innate immune recognition is mediated by non-clonal. In addition. TLR7 and TLR9 recognize viral RNA and DNA. Upon activation by their microbial ligands. TLR specificities cover almost entire spectrum of microbial pathogens. TRAM. TLR4 is specific for LPS of gram-negative bacteria. TLR3 is specific for double stranded RNA – a common product of viral replication. respectively. therefore. PAMPs are chemically distinct from any structure synthesized by the host cells. TLRs recognize conserved microbial molecular structures (PAMPs) and trigger activation of host defense responses. and RIG-I/MDA-5. and fungal glucans. In other words. Recognition of PAMPs. . 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. including inflammatory responses. TLR2 – for lipoteichoic acids of gram-positive bacteria. All TLRs share a conserved cytoplasmic domain called TIR domain. several other families of pattern recognition receptors (PRRs) have been recently characterized. RIG-I and MDA-5 are also intracellular receptors. these receptors detect majority of pathogens and trigger activation of host defense responses. This property allows for self/non-self discrimination by the innate immune system.
Coban C. . Akira S.Reading: Text: "Janeway’s Immunobiology". Nakagawa A. Cell Host Microbe. Koyama S. 8th Edition. Host innate immune receptors and beyond: making sense of microbial infections.3(6):352-63. Chapter 2 and 3. LLC). by Kenneth Murphy (Garland Science. Taylor & Francis Group. Review Article: Ishii KJ. 2011. 2008 Jun 12.
The function of acute phase proteins is to help combat the infection at the system level. Medzhitov The innate immune system utilizes a diverse set of mechanisms to combat infection. thus allowing for specific attack on pathogens. depending on the mechanism of pathogen recognition. The alternative pathway is activated constitutively. Reading: Text: "Janeway’s Immunobiology". Finally. in that it binds to pathogens’ surface and activates the protease cascade much like the antibodies do. Other acute phase proteins have direct antimicrobial activity. 20: 197-216. LLC). These same proteins also activate the complement cascade.. CA Jr. Early response to infection includes the induction of local inflammation.Complement. Yet other acute phase proteins regulate the coagulation cascades. The function of MBL is reminiscent of the antibodies. Taylor & Francis Group. Innate immune recognition. The complement system plays a critical role in host defense against a variety of extracellular pathogens. bacteriocidal permeability increasing protein (BPI) can bind to the cell walls of gram negative bacteria and destabilize the bacterial membranes. Complement has two functions – lysis of microbial cells and opsonization of microbial cells to promote their elimination by phagocytes. R. These genes encode so-called acute phase proteins. 2011. Annu Rev Immunol. 8th Edition. Some acute phase (C-reactive protein. 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. surfactant proteins. There are several pathways of complement activation. For example. the lectin pathway is activated by the acute phase protein mannan binding lectin (MBL). Chapter 2 and 3. Review Articles: Janeway. mannan binding lectin) function as opsonins – they bind to the pathogen’s surface and facilitate pathogens uptake by phagocytes. Medzhitov. which are secreted by lives into circulation. by Kenneth Murphy (Garland Science. . NK cells and Acute Phase Proteins September 14 Instructor: R. 2002. but selectively inhibited on host cells. The concentration of the acute phase proteins in circulation increases markedly. 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.
including ROS and NO generating enzymes. Epithelial cells also play an important role in antibacterial and anti-fungal defense. Fungi and Parasites September 17 Instructor: R. LLC). In the case of bacteria and fungi. In addition. including helminthes (parasitic worms) is mediated by eosinophils and mucosal epithelia. Taylor & Francis Group. Mucosal epithelial produce mucins that help prevent parasite entry and promote their expulsion. Defense against multicellular parasites. Brown GD. 2011.16(1):27-32. These cells are endowed with powerful antimicrobial defenses. 2008 Jan. Chapter 2 and 3. Text: "Janeway’s Immunobiology". the main host defenses rely on the function of phagocytes: macrophages and neutrophils. 8th Edition.Innate immunity to Bacteria. anti-microbial peptides and lysosomal enzymes. by Kenneth Murphy (Garland Science. complement has direct anti-microbial activity. Phagocytosis and killing by neutrophils and macrophages is the main form of defense against bacteria and fungi. Eosinophils produce several toxic molecules that can directly target parasites. in part through the production of potent anti-microbial peptides (defensins) and production of mucus. fungal and parasitic infections. C-type lectin receptors in antifungal immunity. Mast cells produce several inflammatory mediators that help orchestrate anti-parasitic defenses and promote parasite expulsion through their effects on smooth muscles and endothelium. Review Articles: Willment JA. Medzhitov The innate immune system uses a number of mechanisms to protect against bacterial. Trends Microbiol. The function of phagocytes is aided by the complement system through opsonization. .
ssRNA virus infection is detected by a cytosolic sensor. LLC). Viruses contain genetic information within the capsid. In most cells. viruses synthesize proteins. replicate genomes and assemble using host cell machineries.23:225-74. 2005. as both TLR7 and TLR9 are localized in this compartment. NK cell recognition. Upon entry into the host cells. Chapter 2 and 3. Annu Rev Immunol. plasmacytoid dendritic cells (pDCs) use the Toll-like receptors (TLR) 7 and 9 to detect ssRNA and dsDNA viruses. and possess surface proteins that bind to and fuse with host cell receptors. by Kenneth Murphy (Garland Science. Viruses are obligate intracellular pathogens that can be roughly categorized into DNA and RNA viruses.Innate Anti-viral Immunity September 19 Instructor: R. Review Articles: Lanier LL. as retinoic acid inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (mda-5). and upon binding to the receptor. In contrast. Natural killer (NK) cells do just that and play an important role in the anti-viral host defense. respectively. The key cell types involved in innate clearance of viruses and virus-infected cells include pDCs and Natural killer (NK) cells. Reading: Text: "Janeway’s Immunobiology". Innate Immunity to Viruses By far the most important innate immune mechanisms against viruses involve the type I interferon system. upon sensing viruses by TLR7 and TLR9 in the lysosomes. When the action of these anti-viral proteins is insufficient. The combination of the activating and inhibitory ligands generally determines whether or not a given cell would be eliminated by NK cells. Type I interferons can be secreted by most cells of the body. the most efficient anti-viral defense is to get rid the infected host cell. 8th Edition. Medzhitov Viruses represent the simplest forms of pathogens. 2011. The pDCs. Complement can also deposit onto the virus surface and neutralize or lyse the virus. Recent studies have elucidated the mechanisms by which viral PAMPs can trigger the innate IFN production pathways. Taylor & Francis Group. . Innate recognition of viral PAMPs occurs by two distinct mechanisms depending on the cell types. This form of recognition occurs within the lysosome instead of in the cytosol. 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. secrete large amounts of type I IFNs and IL-12. induce a myriad of antiviral genes that inhibit productive replication of viruses. The activating receptors recognize molecules expressed on the surface of infected cells and induce the cytotoxic response directed at the unwanted infected cell. NK cells are capable of recognizing and lysing cells that lack surface MHC class I molecules. NK cells express a number of receptors that can either activate or inhibit their function. The inhibitory receptors recognize ligands that are constitutively expressed on normal healthy cells and inhibit the cytotoxic response against these cells.
In class I. Class I and class II molecules can be regarded as similar structures. the membrane proximal domains of both the α and β-subunits are Ig-like.1 and TAP.2) encoding a peptide transporter involved in transferring peptides derived from cytosolic proteins into the endoplasmic reticulum where they bind class I molecules. Cresswell MHC class I molecules are composed of two subunits. have also been defined. These encode molecules which have a limited tissue distribution. H2-K. and L in the mouse. which facilitates MHC class I peptide binding. The large subunit (44kDa) is a transmembrane glycoprotein which is the actual product of the MHC-linked gene. called anchor residues. HLA-E. at fixed positions in the peptide sequence. F and G. the genes for tumor necrosis factors. 3. The MHC contains multiple class I and class II genes. and the transmembrane regions. encoding three different ab dimers in humans (HLA-DR. genes encoding various enzymes (e. has a role in presenting N-formylated peptides derived from bacteria. 21-hydroxylase). The class I heavy chain and the two class II subunits show allelic variability in amino acid sequence.g. The membrane distal domains of the α and β-subunits of class II generate a similar peptidebinding groove. B. 4. H-2M3 from the Qa region. which encode a molecule structurally similar to class II molecules 8. and C in the human. The T-cell receptor recognizes a surface consisting of residues in the a-helices and surface exposed residues in the associated peptide. LMP. Genes relevant to antigen processing functions are also tightly linked to the MHC. Both human and mouse MHC class I and class II genes have a similar organization at the intron/exon level. 7. In class II. but the peptides bound are longer and more variable in length. Also in the MHC are two genes. 5. C4. P. I-Ab and I-Ek) are encoded by very tightly linked genes. as is β2m. the first and second domains of the large subunit fold to generate a cleft or groove between two a-helices.. is the product of an unlinked gene and has a molecular weight of 12kDa. Also two genes. which is involved in generating peptides in the cytosol. the defined extracytoplasmic domains. As a result of this variability. .Structure and Genetics of MHC Molecules September 21 Instructor: 1. called α and β. These include genes encoding complement components (C2. β2-microglobulin (β2m). Individual αβ pairs (e. Additional exons encode the cytoplasmic regions. For class I. and genes encoding heat shock proteins (Hsp70). The β2m gene is not in the MHC. Factor B).. Some of these have been suggested to have an antigen processing function. Both are transmembrane glycoproteins and both are products of genes within the MHC. One. Genes encoding class II α and β-subunits are both present in the MHC. In the mouse there are multiple additional class I genes. The invariant chain gene (see below) is not in the MHC.2.1 and LMP. MHC class II molecules are also composed of two non-covalently associated subunits. Structural studies show that the variable residues generally line the peptide binding groove. encode subunits of a cytosolic macromolecular protease called the proteasome. and a gene encoding tapasin. with individual exons encoding the leader sequence. 6. the peptide binding groove of each allele has an affinity for a different subset of peptides. with preferred interacting amino acids. D. The small subunit. the membrane proximal domain of the large subunit is homologous to Ig-constant region domains. 2. generally in hematopoietic cells. in which bind small peptides of between 8 and 10 amino acids. DMA and DMB. Three other human class I genes. The "classical" class I genes are HLA-A. DQ and DP) and two different ab dimers in the mouse (I-A and I-E). Genes not obviously related to antigen processing are present in the MHC. These include two genes (TAP.g.
These genes are required for efficient class IIrestricted antigen processing. Reading: Janeway’s Immunobiology. 2012. Garland Science.. . 217-230. Ltd. 8th Edition. Chapter 6.called H2-M or H2-DM in mice and HLA-DM in humans.
7. which facilitates peptide binding. 2. Proteolysis of cytosolic proteins is predominantly mediated by the proteasome. Thus. in Antigen Presenting Cells (APCs) or cells treated with interferon-g. Transport of peptides into the ER is mediated by a heterodimeric transporter. ERp57.g. 8th Edition.. ERAP1 and ERAP2 in humans) to facilitate binding to class I molecules.g. ERp57 is permanently disulfide-linked to tapasin. or PLC.. The TAP transporter is a member of a structurally homologous family of transporters that use ATP-hydrolysis to translocate small molecules across membranes. Both subunits of this transporter (TAP. After peptide binds. Reading: Text: Janeway’s Immunobiology. Note that all cytosolic proteins can feed into this system.2 and LMP. serving as a bridge. peptides may be “trimmed” at the N-terminus by ER Amino Peptidases (ERAAP in mice.MHC Class I Restricted Antigen Processing September 24 Instructor: P. Ltd. Certain viruses (e.. tapasin. SV40 T-antigen (nuclear) or influenza virus matrix protein (cytosolic). the normal mechanism which generates the class I-peptide complex involves: a) proteolysis of the cytosolic viral protein. After translocation into the ER. or. 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. incorporates two subunits encoded in the MHC. b) transport of the peptide into the endoplasmic reticulum (ER) of the cell. 4. Virus-derived peptides generated in infected cells are superimposed on this background of normal host peptides. called MECL-1. Cresswell MHC class I-restricted antigen processing 1. and a third. 5. Target cells expressing the appropriate class I allele are not killed unless they are: a) infected with the virus. Two additional proteins are also associated. e. 6. However. 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. the "loaded" class I molecules dissociate from the PLC and are transported to the cell surface where they are available for T-cell recognition.2) are encoded in the MHC. These proteins constitute the Peptide Loading Complex. 202-217. Thus in normal circumstances class I molecules are full of peptides derived from normal cell proteins. presumably to protect the cells they infect from recognition by CD8-positive T-cells. Such proteasomes are often called immunoproteasomes. TAP molecules physically associate with newly synthesized "empty" class I molecules in the ER.1 and TAP. . the chaperone calreticulin and the thiol oxidoreductase. d) transport of the class I peptide complex to the cell surface. class I-restricted CD8-positive cytotoxic T-lymphocytes (CTL). 3. with a third protein. Herpes simplex virus I and II. c) association of the peptide with the class I molecule and. Many of these CTL prove to be specific for viral proteins that are only expressed intracellularly.7. Chapter 6. Garland Science. 2012. a multisubunit protease which. called LMP. Animals infected with a virus generate virus-specific. human cytomegalovirus) have developed strategies that interfere with MHC class I peptide loading.
Primary Literature: The International HIV Controllers Study. P. and Cresswell. P. 2008. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Current Opinion in Cell Biology: 20:1-8. Wearsch. Science 330: 1551-1557. The quality control of MHC class I peptide loading. . 2010.
The specifically endocytosed antigen is degraded and peptides derived from it presented on the surface in association with class II molecules. even in the absence of pathogenic proteins the class II molecules are still occupied with peptides. Garland Science. et al. DM functions by catalyzing the displacement of CLIP and facilitating the binding of the normal complement of peptides derived from internalized proteins. Mutant cells lacking DM molecules. and in thymic epithelial cells. 2008. Internalization followed by proteolysis of the antigen is essential. Cresswell MHC class II-restricted antigen processing 1. DO. cannot generate normal class II-peptide complexes. 202-217. a class II-like αβ dimer. First. S. They accumulate class II molecules associated with a single peptide (CLIP). For antigens that contain disulfide bonds a lysosomal thiol reductase. In B-cells. Ltd. Internalization can occur by phagocytosis (in dendritic cells and macrophages) or by endocytosis.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. B-cells are up to 10. Class II α and β subunits associated with a third glycoprotein. The invariant chain is trimeric and associates with three αβ dimers to form a nonameric structure.g. CD4-positive T-cells respond to class II-positive cells which have internalized protein antigens. GILT. some dendritic cells.. 2012. 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. Inhibitors of lysosomal proteolysis (e. it contains a cytoplasmic targeting signal that drives class II-invariant chain complexes to the endocytic pathway. 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. The biosynthesis of class II molecules is important for proper function. The DO α and β subunits are also encoded in the MHC. immediately upon biosynthesis in the ER. Chapter 6.. 4. Reading: Text: Janeway’s Immunobiology. Primary Literature: Sadegh-Nasseri. Here the invariant chain is removed by proteolysis. These are usually derived from membrane proteins or internalized serum proteins undergoing lysosomal degradation. the invariant chain. which neutralizes the normally acidic lysosome) inhibit antigen processing and subsequent presentation to T-cells. it prevents class II molecules from binding unfolded proteins or peptides in the early stages of biosynthesis. DM function is down-regulated by its association with another class II-like molecule. 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. The convergent roles of tapasin and HLA-DM in antigen presentation. Invariant chain regulates antigen processing in two ways. Class IIpeptide complexes are then transported to the cell surface where they can be recognized by T-cells. as in the class I system. freeing the class II peptide-binding groove to associate with peptides generated from internalized protein antigens..MHC Class II Restricted Antigen Processing September 26 Instructor: P. chloroquine. Like for class I. derived from the invariant chain. 8th Edition. Second. 2. 3. facilitates protein unfolding and peptide generation. Trends in Immunology 29: 141-147. 5. Note that. The B-cell/T-cell interaction is critical for making antigen-specific antibody responses.
Instead. Reading: Text: Janeway’s Immunobiology: Garland Science. which recognize CD1d complexes containing self lipids and which play an important role linking innate and adaptive immunity. CD1d and CD1e. 2007. A. At this stage. Thus. Cresswell Dendritic cells Dendritic cells are the “first line of defense” in the antigen presenting cell world. 2012. Dendritic cells are also the major antigen presenting cell type capable of cross-presentation.C and Brenner M..B. allowing efficient non-specific uptake of soluble and particulate protein antigens. Work in humans has found that cytotoxic T-cells specific for Mycobacterium tuberculosis or Mycobacterium leprae are restricted by CD1 molecules. Chapter 9. co-factors involved in lysosomal lipid degradation. CD1c. 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. At this stage they migrate to lymph nodes. 21:105-110. CD1b. 2009. Ltd. is the primary cross-presenting subset. They are not the products of MHC genes but are homologues of the classical class I molecules. called CD1a. and Villadangos J.. 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. These are CD1 molecules. 342-353. Antigen presentation by dendritic cells in vivo. Nature Reviews in Immunology. Mice only express CD1d. by exposure to bacterial lipopolysaccharides. express an additional type of antigen presenting molecule. Current evidence suggests that the CD1-lipid complexes are generated in the endocytic pathway and that saposins. In humans there are five types of CD1. 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. causes their maturation to a stage with high expression of surface class II-peptide complexes.Dendritic Cells and Their Functions/CD1 Molecules September 28 Instructor: P. Activation of dendritic cells by innate immune mechanisms. Barral D. mycobacterial lipids are bound by CD1 molecules and are recognized by these T-cells. e. Primary Literature: Segura E. catalyze lipid binding to CD1 molecules. 8th Edition. CD1 antigen presentation: how it works. in the mouse the CD8a-positive dendritic cell. CD1 molecules Certain Antigen Presenting Cells. Subsets of dendritic cells have been described and one of them.. In peripheral tissues they are highly active in macropinocytosis and phagocytosis.. they have high levels of intracellular class II molecules.g. but it is not CD1-peptide complexes that are recognized. 7: 929-941. forming similar dimers with β2m. CD1d molecules are detected by a T cell subset referred to as NKT cells. . including dendritic cells. The crystal structures of CD1b and CD1d molecules reveal a binding groove deeper and more hydrophobic than that of the MHC class I molecules. Current Oopinions in Immunology.
They have a common core that is comprised of 2 heavy and 2 light chains. The constant domain of the heavy chain determine the isotype of the antibody and therefore the functional characteristics of the antibody molecule.. The structure of immunoglobulins has been elucidated using X-ray crystallography. 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. The affinity of binding of a particular antibody for its antigen depends on the multiplicity and strength of these interactions. These distinct forms of antibody are termed isotypes. Most high-affinity serum Ig is IgG. IgM antibodies dominate early in immune responses. divalent form of antibody which predominates in serum. giving it an overall Y shape. The 3-dimensional structure of antibodies reveals that it consists of three globular domains linked by a flexible hinge region. these allow pentamerization to form a high avidity structure with 10 combining sites. The use of different heavy chain constant regions produces antibodies of differing molecular weight. non-covalent interactions. as are the light chains to the heavy chains. e. degree of oligomerization. The two heavy chains are joined to one another by disulfide bonds. respiratory track. to form a barrel structure. Antibody classes Antibody molecules come in subclasses defined by the H chain. Antigens bind to antibodies through non-covalent interactions that can be disrupted by high salt. 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. Treatment with proteases separates the antigen binding domain (Fab) from the effector domain (Fc). . while the remaining domains are relatively invariant. IgG: a monomeric. Schatz The Antibody. and is the main component of late and memory systemic responses. The variable domains are known as V domains or regions and the constant domains are known as C domains or regions. detergents or extremes of pH. The major function of IgA is to protect mucosal surfaces and it is the main antibody secreted into the gut. Each immunoglobulin domain consists of two layers of polypeptide chains (which are β-pleated sheets) linked by a disulfide bond.g. some don't. 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. 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. IgA: a dimeric.The B Cell and T Cell Receptors: Structure & Classes October 1 Instructor: D. Antibodies initiate their biological functions by binding to antigen with high affinity. and biological function. anatomical location. Both the heavy and light chains contain a repeated 110 amino acid motif known as the immunoglobulin domain. Therefore the hypervariable regions are known as complementary determining regions (CDRs). Much low-affinity serum Ig is IgM. All antibody molecules are similar in overall structure. some fix complement. 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 N-terminal domains of both heavy and light chains are variable in sequence. milk etc. In mammals there are subclasses of IgG antibodies with different biological properties. tetravalent form of antibody in which two Ig units are joined by a "secretory piece" which is a separate protein.
these are anti-ISOTYPE antibodies. TCRs were first identified by raising monoclonal antibodies against T cell clones. 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. e. it is not surprising that T cell receptors (TCRs) are similar to antibody molecules. which is fundamentally different from B cells which recognize only the antigen. 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. The functional significance of gamma deltaT cells is less understood. Minute quantities in serum. ζ chains. The CD3 proteins are invarient and their function is to allow signal transduction upon . Antibodies may recognize epitopes on the distinct H chains of Ig isotypes. The γδ TCR is probably structurally similar to the Alpha β TCR. since T cells recognize specific antigens. γδ T cells: A smaller population of T cells has a TCR on its surface composed of gamma and delta chains. probably without a function there.. The antibodies immunoprecipitated a heterodimer with two disulfide linked chains. these are anti-IDIOTYPE antibodies. which are distinct for antibodies of different specificity. in other members of the same species. crosslinking of this receptor results in mast cell degranulation with release of histamine.g. The T Cell Receptor T cells recognize antigen as processed fragments in association with MHC molecules. Of particular note is that the diversity of the CDR3 region of TCRs is much greater than the diversity of CDR1 or 2. IgD: found on the membranes of mature B cells. The CD3 complex consists of delta. The 3-dimensional structure of the alphabeta TCR has been determined. with the overall structure of the TCR being analogous to an Fab region of the immunoglobulin molecule. or fungal spores. Involved in many human allergies. etc. e. 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. between genetically identical individuals of an inbred strain. Antigenic epitopes on antibody molecules Immunoglobulin molecules may be immunogenic in unrelated species. beta. TCR vs. Triggers severe inflammatory reactions by binding to a specific IgE receptor on mast cells. termed the α and β chains. and their specificity is clonally distributed.g.IgE: a monomeric antibody produced in response to macroscopic parasites such as worms. involved in B cell activation. gamma and delta chains reveal that they are similar to the chains of the immunoglobulin molecule. or in autoimmunity within one individual.. theta. Each chain of the TCR contains a variable N-terminal region and a constant C-terminal region. ε. 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). goat anti-mouse IgM. Antibodies may recognize epitopes encoded by allelic differences between antibodies of the same isotype of unrelated members of the same species. these are anti-ALLOTYPE antibodies. BCR: The sequence of the alpha. As discussed in detail in the reference below. Antibodies may recognize epitopes on the combining site. However. to grass pollen. 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.
P.B. they send a much more potent signal than if TCR or co-receptor alone is involved in the binding.. In like manner.. 5. I. Reading: Text: "Janeway’s Immunobiology". D. Morris. Science 293. (2001).M. R.R. Chapter 4 and Chapter 5. Ig molecules on the surface of B cells are non-covalently associated with two signal transduction proteins known as Iga and Igb. M.M...F. .N. A. A. 1155-1159. 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. 8th Edition. please carefully study the beautiful diagrams in the book. MHC class I or II restricted. and Barclay.12. while CD8 positive T cells recognize antigens presented by MHC class I molecules.16. while CD8 is a disulfide linked heterodimer that binds to MHC class I molecules.activation of T cells. and 5. R.. G. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. R. 2011. by K.. sections 5.W.A. Primary literature: Saphire. Like CD3. Annual Review of Immunology 6:381-405. 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. P. Parren..O. CD4 is a single chain polypeptide that interacts with MHC class II molecules. E. Stanfield. Dwek. The immunoglobulin superfamily-domains for cell surface recognition (1988). **This is a very structure-oriented topic and pictures are worth a thousand words. CD4 positive T cells recognize antigen presented by MHC class II molecules. Rudd.13.. Murphy (Garland Science).. and Wilson.A.L. Pantophlet.** Review Article: Williams. Zwick. Burton.
the first three of which are shared by TCRs. For each antigen receptor locus. D. resulting in a . which is particularly concentrated in the third hypervariable region (CDR3 region). 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. V. and J genes have in common (recombination signal sequence. The answer: Ig and TCR genes are assembled from component gene segments. while exonucleases delete nucleotides. D.. and hence junctional diversity is achieved only at the expense of considerable wastage. 100 Vs. there are 5200 different V-D-J combinations (100x13x4). for a combinatorial potential of 600 different genes. chain pairing. This reaction takes place in two phases. The Ig heavy chain locus (Igh) and TCRb and TCRd loci contain V. J) of gene segment. D (diversity) and J (joining). Somatic hypermutation is the fourth source of diversity. A third source of diversity. say. 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. one D and one J are used to assemble the Igh gene: with.000 for Igk). V(D)J recombination V. Non-germline encoded (N-) nucleotides can be added by the enzyme terminal deoxynucleotidyl transferase (TdT). D. and different combinations of these gene segments can be used in different rearrangement events. See figure at bottom of the notes for this lecture for diagrams of the loci. 13 Ds and 4 Js. leading to a nonfunctional protein.Immunoglobulin and TCR Gene Structure and Rearrangement October 3 Instructor: D. and J segments is guided by short. Ig and TCR Genes The variable region gene exon is assembled from gene segments called V (variable). the recombination machinery binds to the RSSs flanking the two participating gene segments and then cuts the DNA immediately adjacent to each gene segment. Junctional diversity dramatically increases the number of possible proteins encoded by these loci (to more than a billion for Igh. it affects only Ig genes and is considered in a later lecture. flanking DNA sequences that all V. 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. and J segments as a result of addition and subtraction of nucleotides in the recombination process. is also combinatorial. Junctional diversity. D. arising from the many possible different combinations of heavy. the theoretical number of possible Igh-Igk pairs is well over one trillion.and light-chain V regions (or TCRa chain and TCRb chain) that pair to form the antigen-binding site in these molecules. The Igk locus contains about 150 Vs and 4 Js (no Ds). In the first. or RSS). Antigen receptor loci can be very large (spanning as much as several megabases). (D) and J gene segments are joined by a site-specific recombination reaction known as V(D)J recombination. these gene segments and the constant (C) region all lie on a single chromosome. Generation of antigen receptor diversity The diversity of Igs is generated by four main processes. is introduced at the joints between the different V. 100.g. the dogma of "one gene/one polypeptide" won't work due to the limited number of genes in the genome. Gene segment combinatorial diversity results from the presence in the germline of multiple different copies of each type (e. The random rearrangement of V. Such added/subtracted nucleotides often disrupt the reading frame of the coding sequence beyond the joint (called nonproductive rearrangements).
IgM. IgA. RAG1/RAG2 can only bind to DNA substrates that are in an "open" chromatin structure. IgD. or isotypes (e. 2) Through the control of the "accessibility" of the DNA substrates. Initially only the first (most 5') of these genes. In the second. Cm. The membrane forms of all isotypes are monomers comprised of two light and two heavy chains. The CH regions are encoded in separate genes located downstream of the V genes at the heavy-chain locus. Regulation of V(D)J Recombination V(D)J recombination is regulated at several levels. 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. is expressed in conjunction with an assembled V gene. and production of the two forms is achieved by alternative RNA processing. IgG. 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. and IgE). Deletion + The first phase of V(D)J recombination (DNA binding and cleavage) is performed by the RAG1 and RAG2 Inversion proteins. CSR is unlike V(D)J recombination in several ways. 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. To reiterate. TCRb. 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). gene segments that are far apart on the chromosome will only recombine if they are brought into close proximity.chromosome that has been cut in two places. respectively) only when they are secreted.g. RAG1 and RAG2 are expressed only in developing lymphocytes and are both essential for V(D)J recombination. . The two different C-termini (transmembrane and secreted) of the heavy chains are encoded in separate exons. and TCRd loci (D-to-J and V-to-DJ) and one event (V-to-J) for the other loci. like V and J— rectangles in the diagram) and a signal joint (the fusion of the two RSSs—triangles in the diagram). whose C-terminus is a hydrophilic secretory tail. this structural variation is generated by linking different heavy-chain constant regions to the same Igh variable region. 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. IgA and IgM polymerize (into dimers and pentamers. Two V(D)J recombination events are required to assemble the Igh. 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.. the same VH exon can associate with different CH genes in the course of an immune response. Class Switch Recombination (Isotype Switching) Immunoglobulins can be made in several different forms. 1) RAG1 and RAG2 are expressed only in developing lymphocytes and hence V(D)J recombination can only occurs in these cells. This transmembrane domain is absent from the secreted form (antibody). 3) Through the control of long range chromosome "looping".
M.E. N. 322-326.. Chapter 5. . 2011. N.. Y. Primary literature: Chien. Nature 309. 101132. and Davis. Murphy (Garland Science). repair factors. Review Article: Gellert. M. Somatic recombination in a murine T-cell receptor gene.H.R. V(D)J recombination: RAG proteins. (1984). by K. (2002). Gascoigne...Reading: Text: "Janeway’s Immunobiology". pp 157-173. and regulation. 8th Edition. Kavaler.M. Lee. J. Annu Rev Biochem 71.
78. Enhancers and promoters are represented by gray circles and rectangles.#. Not drawn to scale.Figure Legend Schematic representation of the murine antigen receptor loci. Some Vk genes. Ig and TCR Loci IgH VH(100-1000) DH(13) PDQ52 JH Eiµ CH(µ. Factors and forces controlling V(D)J recombination. respectively.". (2001). G. and Schatz. G. with no attempt made to indicate individual exons. D. and 12-RSSs and 23-RSSs as white and black triangles. D. Constant regions are depicted as single rectangles. 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).%) 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. Immunol. and Vd5 and Vb14 are known to rearrange by inversion. Taken from Hesslein..$. Va and Vd gene segments are interspersed.1 J#4C#4 HsA . Adv. 169-232. but pseudogenes have been omitted.3 E#3 E#2 C#2 J#2 V#1.2 V#1. Gene segments are depicted as white rectangles. respectively.
B Cell and T Cell Development
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
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.
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).
Jr. Immunol .. Annu. Travers. Ltd..c. Lymphoid organ shutdown and blocked egress. “Chemokines. This prevents activated T and B lymphocytes from exiting SLOs during an immune response. Janeway. Immunobiology: The immune system in health and disease. M. Shlomchik.A. Chapter 8. Review articles: Cyster. S1P and Cell migration in lymphoid organs. 2012. Reading: Text: C. P.” 2005. JG. Activated lymphocytes rapidly up-regulate CD69 that counteracts S1P1 function. Rev. Garland Science. Walport and M. 8th Edition.
and CD4 or CD8 molecule in T-cells. it has an important role in the termination of the immune responses and removal of potentially autoreactive lymphocytes. and fyn and lck for the TCR. This step is followed by the activation of effector caspases leading to a series of events that are hallmarks of apoptosis. . through the activation of death receptors (extrinsic pathway). 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. Therefore. such as the enzymatic fragmentation of chromosomal DNA. 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. Activation of cytokine receptors leads to the trans-phosphorylation and consequent activation of the associated JAKs. they are associated with invariant proteins that possess the capacity to activate downstream signaling pathways. Jak2.Receptors & Signaling II October 10 Instructor: Carla V. Common to both pathways is the activation of specialized proteases called caspases. Assembly of the antigenbinding chains with these accessory proteins not only provides adequate signaling capacity. 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. Jak3 and Tyk2. Rothlin In this lecture we will review signaling pathways that are central to lymphocytes’ biology.: Fas and TNFR-I) are members of the large TNF receptor family and contain a conserved cytoplasmic domain known as death domain (DD). but also allows the correct transport of the complex to the plasma membrane. Pro-caspase 8 is self-activated by proteolytic cleavage and subsequently released from the receptor complex. Optimal signaling also requires co-aggregation of coreceptors such as CD19 in B-cells. 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). This leads to a conformational change that allows for STATs homo or heterodimerization. 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). their translocation to the nucleus and induction of gene expression. Recruitment of the STATs to the activated cytokine receptor brings them to proximity to the activated JAKs. FADD contains a death effector domain (DED) that allows for the recruitment of the initiator caspase. In particular. This family is formed by four members: Jak1. syk in B-cells and ZAP-70 in T-cells are recruited to the signaling complex upon activation. procaspase 8. which induce STAT phosphorylation. Death receptor induced signaling pathway Programmed cell death or apoptosis plays a key role in the regulation of the immune function. In addition two soluble tyrosine kinases. NFAT and AP-1 to induce specific gene expression. d.g. Surface immunoglobulins are associated with two polypeptides termed Iga and Igb. Apoptosis can be induced by extracellular ligands. while the TCRa:b heterodimer is associated with three CD3 chains (g. For example. e) and two z chains. 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. The death receptors (e. Activation of the death receptors by their cognate ligands leads to clustering of the death domains and the recruitment of specific adaptor proteins. 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. leading to cell proliferation and differentiation. fyn and lyn for the B-cell receptor. Aggregation leads to the activation of tyrosine kinases that are associated with these cell surface molecules.
Taylor & Francis Group. by Kenneth Murphy (Garland Science. Chapter 7. LLC). 8th Edition. 2012. .Reading: Text: "Janeway’s Immunobiology".
Following removal of the pathogen. 4. Craft MAIN POINTS 1. 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. This switches them into professional APCs that upregulate lymph node (LN) homing receptors and causes them to migrate into the draining LNs. upon encountering their specific antigen in the periphery. 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”. 3. Naive Activation/ Expansion Contraction Maintenance of Memory CD8 T cell response Virus 1 5 8 15 30 60 days post infection . quickly respond to eliminate infected cells and pathogen replication. the types of signals elicited by the innate immune system. 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. whereas CD4+ T cells recognize antigen presented by MHC class II molecules that are restricted in their distribution in the periphery. death of the activated T cells) and (3) Formation of memory T cells. 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. in accordance. (2) Contraction (i. The effector functions expressed by antigen-specific T cells differ depending on the type of infection at hand and. 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. respectively. This is followed by their differentiation into effector cells that. These memory T cells can mount rapid secondary responses to reinfection and help to protect against reoccurring disease and illness. CD4 T cells serve many roles in the immune response. 2. the majority of the activated T cells die. T CELL TYPES: There are two primary types of T cells.e. but a minority persist as long-lived memory T cells.. that recognize small peptides presented on MHC class II and class I. OVERVIEW All adaptive immune responses are mediated by the activation and clonal expansion of antigen-specific T and B lymphocytes.Innate Control and Initiation of Adaptive Immune Responses October 15 Instructor: J. CD4 and CD8 T cells.
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. 1(4):327-39. Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. produced by endothelial cells. 2011. Jenkins MK. where they reside as immature. In summary. Once in these secondary lymphoid organs. which upregulates expression of lymph node homing receptors. 8th Edition. In the vast majority of cases. stromal cells and dendritic cells that bind receptors on T cells. Taylor & Francis Group. Immunity. Loh DY. 1994 Jul. and costimulatory molecules for T cell activation. in those rare instances when recognition occurs.In this lecture. 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. via binding to endothelial cells by adhesion molecules. which is a highly organized structure of proteins between the adhesion of a DC and an antigen-specific T cell. antimicrobial peptides and chemokines to attract other leukocytes to the site of infection. the T cell ceases to re-circulate and sets up stable interactions with the antigen-presenting dendritic cell. T cells re-circulate actively from the blood into the lymph nodes. naïve T cells then survey the surfaces of dendritic cells for their specific antigen. but have gained the ability to present antigens to T cells. It also causes the DCs to mature into cells that have lost the ability to take up antigen. Dendritic cells acquire antigen in the peripheral tissues. and subsequent migration through the endothelial cell barrier. Homing to lymph nodes and other secondary lymphoid tissues is also mediated by specific chemokines. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. as well as the importance of the trafficking of lymphocytes through the lymph nodes in search of their specific antigen. after pathogen invasion. . The encounter with pathogens. we will review concepts that were introduced earlier. the lymphocyte does not encounter its specific antigen. highly phagocytic cells with little ability to activate 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. although it does receive weak signals through its receptor that allow it to survive. This leads to formation of an immunological synapse. 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. mediated initially by pairs of cell adhesion molecules. MHC:antigen complexes. such as the role of tissue dendritic cells in bringing antigens to the local lymph nodes. Pape KA. LLC). The second is that it switches a DC from a “tolerogenic” DC into an “immunogenic” DC. However. especially in sites of infection. Primary Literature: Kearney ER. Chapter 9 (9-1 through 9-9) and Chapter 3 (3-13 through 3-19).
Note. As the T cells are engaged in a tight adhesion with the DCs. CD70:CD27. 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. that all 3 signals are dependent on . die or will persist in an undifferentiated state with little-to-no effector functions. 2. IFNγ. The second signal comes from costimulatory ligands expressed by activated DCs that activate costimlatory receptors on T cells and enhance TCR signaling. the T cells will either become anergic. costimulatory receptors and signaling molecules. The Peripheral-SMAC contains adhesion molecules such as LFA-1 and talin. signal 2 (costimulation) and signal 3 (inflammation) invoke signal transduction. Several types of costimulatory ligand:receptor pairs exist. signal 2 (costimulation) and signal 3 (effector-determining inflammatory cytokines). in which inflammatory cytokines produce by the innate immune DCs. The 3rd signal is inflammation. ORGANIZATION and DURATION of an IMMUNOLOGICAL SYNAPSE: The inner C-SMAC and outer PSMAC and can persist for more than 7hrs. 41BBL:41BB.T Cell Priming & Effector Cell Differentiation I October 17 Instructor: J. C4 or CD8 coreceptors. Collectively. Craft MAIN POINTS 1. IL-4. IL-21. ICOSL:ICOS). leading to production of the T cell growth factor IL-2 and synthesis of its receptor. OVERVIEW The immunological synapse is composed of Central-SMAC (SupraMolecular Adhesion Complex) that contains the TCR/Ag:MHC. 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. events leading to clonal expansion and eventually to clonal differentiation into effector T cells. The immunological synapse directs the movement of the MTOC (microtubule organizing center) to the site of contact. CD40L:CD40. The first is the direct recognition of antigen (MHC-peptide complexes) by the T Cell Receptor (TCR) that leads to its activation. (B7:CD28. If T cells do not receive all 3 signals or only interact with the DC very briefly. they receive 3 types of critical signals that are necessary for T cell activation and differentiation into effector T cells. Such inflammatory cytokines are IL12. T CELL PRIMING: THE 3 SIGNAL HYPOTHESIS: Effector T cell clonal expansion and differentiation is governed by three major signals: Signal 1(antigen:TCR). IL-6+TGFβ. signal 1 (antigen).
Thus. the innate immune system ultimately determines whether a T cell response will be triggered or not. TGF-β and activation induced cell death (AICD) to ensure activated T cells to not grow out of control. Kupfer H. 395(6697):82-6. Bystander innate immune cell Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. Freiberg BA. Taylor & Francis Group. Chapter 9 (9-10 through 9-31). 1998 Sep 3. Nature. LLC). . Three-dimensional segregation of supramolecular activation clusters in T cells. Primary literature: Monks CR. expression inhibitory receptors such as CTLA4. 2011. 8th Edition.the proper sensing of PAMPs and activation of the innate immune cells. The activation of T cells is balanced by internalization of TCR. Sciaky N. Kupfer A.
Many other changes occur in the activated T cells as they begin to express new effector molecules. Fas. TH2. TFH and TREG 3. and gut. which activates Mφ to become highly anti-microbial. OVERVIEW Upon activation. 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. class switch and mutate. Th2 cells primarily produce IL-4. They also migrate to most peripheral tissues including liver. TYPES of EFFECTOR and MEMORY T CELLS PRODUCED: CTLs. IL-5 an IL-13 to activate eosinophils. 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. (ii) Infection of helminths (worms) and certain parasites and extracellular bacteria induce a TH2 CD4 T cell response. . (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. Activated T cells leave the LN and go to sites of inflammation/ infection 2. Both CTLs and TH1 cells express Fas ligand and can kill cells expessing the death receptor. But during some infections. which via production of IL-17 and IL-22 lead to the recruitment of neutrophils to eliminate the bacterium. A Th2 response is mainly aimed at destroying large. Craft MAIN POINTS 1. The simultaneously upregulate chemokine receptors that detect inflammatory chemokines such as CCL5. TH17. basophils. adhesion receptors and many other genes. mast cells and macrophages to attack and phagocytose the large foreign bodies. 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. extracellular pathogens and thus. CCL10 produced at the sites of infection to enable their recruitment to these sites. CTLs secrete the antiviral cytokine IFNγ and can kill on contact any cell in the body that harbors a cytosolic pathogen. 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. (iii) Some types of extracellular bacterial infections can also induce TH17 responses. CCL9. In addition. Most regulatory T cells (TREGS) form in the thymus and suppress the responses of any “escapee” CD8 or CD4 T cells in the periphery. lung. T cells leave the APC and downregulate the LN homing molecules and depart into the peripheral tissues via the blood. CD8 kill cells via the expression cytolytic molecules called perforins and granzymes. these cytokines cause activated B cells to produce IgE. 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). TH1 cells also help CD8 T cell activation and activate B cells to produce IgG2a and IgG2b. Th1 CD4 T cells are also induced during these types of infections and secrete IFNγ. TH1.T Cell Priming & Effector Cell Differentiation II October 19 Instructor: J.
e003. Nuclear Receptor Signaling (2009) 7. . Figure from Anton M. immunity. and cellular metabolism.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. circadian rhythm. Jetten. These major TFs are shown in the figure. Retinoid-related orphan receptors (RORs): critical roles in development. 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.
8th Edition. Kim ST.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. Cell. Taylor & Francis Group. A novel transcription factor. Chapter 9 (9-10 through 9-31). Costa GL. Zhang X. directs Th1 lineage commitment. Primary literature: Szabo SJ. 2011. LLC). 2000 Mar 17. T-bet. . Fathman CG and Glimcher LH. 100(6):655-69.
By reorienting the MTOC to the immunological synapse. Interferon gamma (IFNγ ). Thus. They can work locally and for short periods of time. 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.directly kill infected cells (or other antigen bearing cells such as APCs) a. Type I interferons (IFN-α/β) produced by DCs. This occurs in an ON-OFF-ON type of mode. In general.anti-viral factor. Chemokines and vasodilators. The TCR is still required for release of effector molecules. Cytotoxic Molecules. Likewise. Perforin/ Granzymes are stored in granules in the T cells as preformed proteins that can be immediately expelled upon TCR triggering. Sometimes they even use proteins similar to our own as decoys to their advantage.Effector Molecules – Function & Signaling October 22 Instructor: J. 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. the pathogens have devised numerous ways to circumvent or avoid these attacks. c. Consequences of sloppiness would be excessive immunopathology and disease or even death of host. Cytokinesa. 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.CCL3. 2. T cells produce 3 main category of molecules: 1. however. Pathogens have devised many ways to evade the attack of a T cell. . these potential harmful proteins are only being made when there is antigen and infected cells present. Secreted cytokines can act on the cell that produced them (autocrine) or on another cell (paracrine). 4. Interleukins. now the T cells are interacting with infected cells that are not necessarily professional APCs (don’t receive costimulation). 2. through their own evolutionary process. stimulator of Mφ. b. TNFα 3. The T cells quickly shut off synthesis of these proteins in the absence of TCR engagement. IL-5. Cytokines can be membrane bound or secreted. IL-21) such as B cells b. Craft MAIN POINTS 1. The effector mechanisms used to clear an infection depend on the infectious agent.T and B cell factors that act as growth factors (IL-2) or modulate other lymphocytes (IL-4. 5. there is polarized delivery of the effector molecules to the target cells.
Cytotoxic Functions of Effector Cells Granzyme/ Perforins: Enter cells and activate Caspase cascade that induces rapid apoptosis of target cell. . Resolution of Immune Response: After pathogen clearance the macrophages “clean up” and engulf dead cells and debris. 8th Edition. The effector T cells begin to die (death by neglect) and a few survive to become long live d memory T cells. Memory T cells can rapidly expand and exert effector functions to more rapidly combat secondary infection. O'Shea JJ. Taylor & Francis Group. Johnston JA. LLC). 2011. Candotti F. Porta F. 1995 Sep 7.activates Caspase cascade in target cell upon receiving FAS Ligand signal from effector cell. Formation of these memory T cells are critical to long-term immunity and the ultimate goal of vaccination. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Fas/Fas Ligand. Giliani S. Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. et al. Primary literature: Macchi P. Ugazio AG. Villa A. Frattini A. Sacco MG. 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.377(6544):65-8. Chapter 9 (9-10 through 9-31) and Chapter 7 (7-19 through 7-22). Nature.
and upon interaction with activated T cells. There are defined sequences of events and these occur in defined microanatomical locations within spleen or lymph node. . Expression of CCR7. enabling the B cells to migrate outward from the center of the follicle. B cells in the follicle recirculate to blood occasionally. allowing B cells to arrest near the T cell zone. not shown below).Shlomchik marginal sinus T zone (pe ria rter iolar lymphoid sheathPALS) 1° B cell follicle Germinal center central arteriole Upon encounter with Ag. M. is reduced. lymph nodes. Upon binding Ag. and blood. where most splenic B cells reside. This is accomplished in part by changes in chemokine receptor expression that control where B cells migrate. B cell trafficking and splenic architecture B cells traffic through blood. is increased. exiting in the marginal sinus from where they migrate to the B cell follicle. Expression of CXCR5. Most B cells are in spleen. just outside the marginal sinus.Primary B Cell Immune Responses October 29 Instructor: Overview: B cells recognize intact antigens (Ags). They encounter and respond to Ags that have reached these compartments. where T cells and B cells interact and begin proliferating. differentiation and migration to constitute the primary B cell immune response. called EBI-2 is expressed. B cells undergo proliferation. which allows B cells to migrate to follicles. B cells stop migrating and arrest at the border of the T and B zone. In addition. a chemokine receptor (for the newly identified ligands termed “oxysterols”). which is the receptor for chemokines secreted in the T cell zone. (Some B cells remain resident and nonrecirculating in the marginal zone.
(Isotype switch will be covered in detail in the next lecture). these TI responses undergo limited isotype switching and they do not form germinal center and memory B cells. IL5. Typically. where it controls the expression of a large number of genes. which continue to divide and differentiate at the same time. IL6. Here they will continue proliferating and will form the “germinal center” (GC). IL5. 8 or 9. In addition. T-dependent B cell activation and differentiation at the T-B border Proliferation is the initial consequence of Ag and T cell encounter. 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. responding to signals from T cells that include cytokines such as IL4. These cells and increase the production of Ig secreted mRNA by 100 to 1000-fold. Small clones of B cells. These so-called T-independent responses can be divided into two types: TI-1 and TI-2. IL6. 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. Although most B cells at this stage will continue to express IgM isotype. After this initial encounter. the entire B cell activation and differentiation program is highly dependent on T cell signals. become antibody-forming or “plasmablasts”. and a surrounding marginal zone that includes the surrounding B cells. the roles of B cell cytokine secretion are not well understood yet. As the initial proliferating cells generate more progeny. Interestingly. They evolve in the “B cell zones” or “follicles” of lymphoid organs such as spleen and lymph node. Germinal centers (GC) are comprised of highly proliferative B cells (mainly) along with some T cells. Some B cells. a “light zone” (LZ) which is less densely packed and contains many FDCs and most of the T cells. These pairs include CD40L and CD40. IL4. where they continue to proliferate. B cells can be activated by certain antigens even without T cell help Although typically. They will also lose expression of many of the surface molecules that characterize B lineage cells—they become mini-factories for Ab secretion. the germinal center grows and tends to push out surrounding non-proliferating B cells. These include IL2. The transcriptional repressor factor Bcl6 is essential for the formation of GCs. Most of these cells live only a few days. causing the B cells to adapt a unique “GC” phenotype and suppressing their ability to become plasma cells. 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. Bcl6 is turned on in nascent GC B cells. processes that will be described below. as well as a variety of other molecular pairs that interact. cytokines are released by activated T cells that have effects on both B and T cells. and these too take on a unique identity as a “T follicular helper” cell. IL21 and IFN-γ. where they stop dividing and can live for several weeks. each descended from an initial Ag-specific B cell accumulate at the border of the T-B zones. . and may express IgG. Differentiation and migration are the next steps.Molecular nature of the T-B interaction There is a complicated interplay between T and B cell. In the follicles are other cells called follicular dendritic cells which are unusual. such as a T-dendritic cell interaction. some cells will begin the process of isotype switch. TI-1 responses are elicited by molecules with inherent mitogenic ability for B cells. 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. CD80/86 molecules and CD28. such as bacterial lipopolysaccharide or nucleic acids that bind TLR7. one exception is certain highly repetitive antigenic surfaces or antigens that also contain B cell mitogens. 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. Germinal Center Overview Architecture: “Germinal” means “growing”. but some will migrate to the red pulp areas of the spleen. B cells can also secrete cytokines that may affect themselves and T 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. nonphagocytic dendritiform cells with uncertain but intriguing function (see below). involving T cell recognition of processed peptide Ag fragments that are present on the B cell’s MHC Class II molecules. they migrate to the border of the T cell zone and the red pulp. these are said to be relatively “T-cell independent”. and IL21. the same factor is required for T cells to enter the GC. and ICOS (inducible costimulator) and ICOS-ligand.
by Kenneth Murphy (Garland Science. isotype switch and differentiation The last three will be covered in the next lecture. in a GC. Proliferation: B cells divide as quickly as every 6 to 8 hrs. and d) isotype switching. 387 – 407. 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. In mouse spleen GC responses begin about 5-6 days after immunization. in which point mutations are introduced into the V region. Taylor & Francis Group. stops dividing and probably leaves the GC). perhaps due to deleterious somatic mutations or just low affinity). c) cellular selection. and leaves the GC probably for the bone marrow). stops dividing. with a net flow of cells from the DZ to the LZ. where most differentiation takes place. 2011. Although GC are ultimately comprised of the descendents of only a few cells. LLC). . Initial proliferation may occur in the “dark zone” while cells in the “light zone” proliferate but at a somewhat lower rate. 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. since most are unsuccessful in competition and selection.Clonal Expansion Initiation: Initial seeding of the GC comes from cells that were originally activated at the T-B zone interface. b) somatic mutation. Reading: Text: "Janeway’s Immunobiology". plasma cell differentiation (which means the cell begins making and secreting more Ig. peak at day 10 to 14 and are mainly over by 4-8 weeks. or apoptosis (which is probably the result of failure to get sIg or T cell signals. 8th Edition. with emphasis on the GC reaction. the initial seeding may be by 50 to 100 cells. Exactly how cells migrate between dark and light zone is not entirely clear but most likely cells recirculate within their respective zones. Chapter 10 pp. 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. Events in the GC: Four important events occur in the GC: a) clonal expansion and differentiation.
Cahalan. Immunity. M. and J. Patel.H. S. Kelsoe.L. Kleinstein. Johnson. S. Intraclonal generation of antibody mutants in germinal centres. S. 2005. Haberman. Yaari.Primary literature: Kerfoot. PLOS Biology 3 (6): e150.1991. .R. K. Neighbors.G. 2011. Rajewsky and U. M. T.. Weiss. G. Parker. M. M. Hartle.. Jacob. J. and A. Nature 354: 389-392. Miller. Gonzalez. K. Cyster. I.M. O'Garra. Antigen engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. Krummel. D. Germinal center B cell and T follicular helper cell development initiates in the interfollicular zone. J. Okada. A.. 34:947-60. G.M.
albeit at substantially lower rates than in the V region (10-100 fold lower).Mechanisms of Somatic Hypermutation and Isotype Switch and Their Consequences October 31 Instructor: M. Location and targeting: Mutations occur only in a limited area from a few hundred bp downstream of the promoter region extending for about 1kb. Shlomchik Introduction: B cells are unique in the capability to modify their antigen receptors in response to activating signals. Somatic Hypermutation Control of mutation: Somatic mutation is not a constitutive process in B cells. Indeed. We will also briefly cover the biological impact of somatic hypermutation on the germinal center reaction. as can other signals that may include simultaneous BCR. 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. but CD40 signals can do this. The discovery led to two theories of how AID might work. In this lecture. Though we won’t discuss it much. nor is it clear why some genes have efficient mutation repair and others not. so named because the gene had strong homology to a family of enzymes that are know to modify RNA (RNA editing enzymes) by deamination. with a predilection for 7 transitions (purine-> purine and pyrimidine -> pyrimidine). recent discoveries indicate that mutation can also occur in chronic immune responses that occur in other areas of lymphoid and even non-lymphoid tissue. Activated B cells can also rearrange the DNA in the IgH constant region leading to a switch in the isotype expressed.5 mutations per cell per generation. although experiments have shown that any promoter that controls transcription can support mutation. Transcription is also required for mutation to take place. The basis for relative targeting of the SHM machinery is still not clear. They are mainly single point mutations. The biological impact of isotype switch will be covered in the next lecture on humoral immune responses. In the first theory. This raises the question of what signals turn the mutation process on in B cells. In some cases. but never reaching the downstream constant region. a gene was discovered that is absolutely required for somatic hypermutation to occur. AID modifies an RNA so that it now . recent data indicates that SHM occurs in many genes in a B cell. we will cover how both mutation and switch are controlled and induced and the molecular mechanisms of both of these processes. Somatic hypermutation takes place in germinal centers and until recently this was thought to be the exclusive site of mutation. This gene was called Activation Induced Deaminase (AID). As already discussed. It is likely that signals through the BCR are absolutely required. Nonetheless. 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. However. these mutations are repaired and their existence was only revealed when repair pathways were genetically disabled. Mechanism: General features of SHM: Somatic mutations are introduced into V regions of dividing B cells at the rate of approximately 0. it is turned on only after a period of B cell activation. in some species like chickens and rabbits. TLR and cytokine signaling. as nonspecific activators do not appear to induce mutation. but sequences in or near the immunoglobulin transcriptional enhancers are important. not just the V region promoter. The answers are not entirely clear. Enzymology of hypermutation: Several years ago. mutations outside of V regions can promote cellular transformation and indeed have been implicated in B cell lymphomas. How mutation is so specifically targeted is not totally clear. as correction of mutations is an ongoing process in all cells. This rate is approximately 10 times higher than the background mutation rate in normal mitosis.
Evidence in favor of this model includes that AID in fact does modify DNA in vitro and also in E. The initial lesions created by AID in the direct model are repaired in a variety of ways by DNA repair enzymes and polymerases. seem to occur via error-prone repair involving especially DNA poymerase eta. is that AID itself is a DNA-modifying enzyme that directly acts as a mutator. it has been known since the discovery of AID that it is required also for isotype switching. which are not direct targets of AID. A summary of how AID might work was published by Neuberger and colleagues: . coli. The second theory. Mutations at adjacent A//T base pairs. AID is critical for gene conversion. Other repair enzymes like XRCC2 promote gene recombination (leading to gene conversion). which removes U base pairs as a mechanism of repairing lesions that have had a base removed. leading in turn to a variety of mutations.encodes an enzyme that can carry out or otherwise induce somatic hypermutation—this is the “indirect” model. These include uracil deglycosylase. Indeed. Moreover. leaving the problem of how one gene can be involved in such seemingly disparate molecular processes. which in turn helps to explain the higher frequency of transition type mutations (particularly at G=C base pairs). which is now widely accepted. it seems to be a cytidine deaminase that converts C to U. Perhaps most remarkably.
(From Petersen-Mahrt et al.Figure 1: DNA deamination model of immunoglobulin gene diversification. 99 . AP endonuclease. For details. see text. dRPase. variable. DNA-dependent protein kinase. Nature 418. DNA-PK.. V. deoxyribophosphodiesterase.104 (04 July 2002)) . KU70/80 are nonhomologous end-joining proteins. apyrimidic endonuclease.
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. The pseudohomology between the repeat sequences shared by all of the switch regions may help in aligning the two regions to be joined. In the mouse: Cytokine IL4. IgG or IgA) isotypes. Via unknown mechanisms (but possibly in part because they are preferred targets of AID. This transcription is essential. see below). This is even more difficult to explain at this point than somatic hypermutation. 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). The intervening DNA is deleted. accessibility) of the downstream switch and C region. For example. but this is not totally clear. Isotype switch is driven by cytokines and T cells. but “switch” to expressing non-IgM (e. which are known to be preferentially introduced into certain sequence motifs. Now the cell will express IgG3. the switch region upstream of IgM might recombine with the switch region of IgG3. However. along with CD40 signals. 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. (A similar mechanism may apply in targeting somatic hypermutations. as well as a specific (but as yet uncharacterized) enzymatic machinery are required for the switch DNA rearrangement to take place. IL21 IFN-γ.Isotype switch Control of switching: Cells initially express IgM. Recent evidence suggests that transcription serves to create regions of single stranded DNA. these short sequences promote nonhomologous recombination between switch regions of different isotypes. switching also requires transcription through the region to be switched. This DNA contains many repeats of a few short sequences. in particular G=C-rich sequences such as GAGCT and GGGGGT. It may also play a mechanistic role in the actual switch rearrangement. Both transcription (i.e. as mutant switch loci that lack the promoter region do not undergo switching. Specific cytokines tend to promote specific switches. just 5’ to the “switch region” (see below).) . With deglycosylation of the U bases. a lesion is created that could lead to a single or double stranded break in the DNA.g. thus setting the stage for switching. AID is thought to play a role by targeting DNA repair mechanisms to the switch sites. Upstream (5’) of every constant region lies a stretch of DNA known as the “switch region”. As with somatic hypermutation. Transcription may “open” the locus to recombination enzymes and for alignment with other switch regions. as seen in the figure above. possibly with unique secondary structure that are preferred targets for AID. 2).
(reproduced from Janeway text. nearly every cell is different from every other cell.Figure 2: Isotype switching involves recombination between specific signals. and indeed the GC can be thought of as a miniature evolution system that occurs in real time.e. with deletion of the intervening DNA. S. with the exception of the δ gene. 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.26) Selection The overall result of somatic hypermutation is that among clonal descendents. 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). meaning that no amino acid substitution occurs. 3. These mutations will . switching to other isotypes can take place subsequently from the recombinant switch region formed after µ-γ switching. fig. Repetitive DNA sequences that guide isotype switching are found upstream of each of the immunoglobulin constant-region genes. The initial switching event takes place from the µ switch region. switch region. 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). Only R mutations will influence the fate of the cell. Switching occurs by recombination between these repetitive sequences. Mutations can be classified according to where in the V region coding sequence they occur: complementarity determining region or CDR (i. Clones evolve.
U. So if there is negative selection—for example in a FR—the R/S ratio will be lower.. 2002. 2007. M. 30:173-81. Negative and positive selection can be inferred from the ratio of replacement type (R) to silent type (S) mutations (R/S ratio). Cells with these mutations will be positively selected and preserved in the GC. since some R mutations will be eliminated. In the absence of any selection. Int Immunol. Reviews: Di Noia. higher affinity cells may be better able to capture antigen and present it to T follicular helper cells. and S. 20:683-94. increasing the affinity of the B cell. Neuberger.. 173-186. 2008. Balancing AID and DNA repair during somatic hypermutation. and indeed many B cells die in the GC. Altering the pathway of immunoglobulin hypermutation by inhibiting uracilDNA glycosylase. Improved methods for detecting selection by mutation analysis of Ig V region sequences. Hershberg. This can result in either more proliferation or less death or both among the higher affinity cells. Conversely. 2011. M. Some mutations in the CDR might also destroy Ag-binding and would be selected against. this ratio is about 3.M. Kleinstein. Molecular mechanisms of antibody somatic hypermutation. and M. some R mutations will be enriched and the ratio will be higher.H. . more linear rate. by Kenneth Murphy (Garland Science. Neuberger. Liu. Uduman.. J. The consequence of this is that clonal expansion is not at the ideal. Trends Immunol. 8th Edition. Only such R mutations change the cell’s phenotype and thus can be selected. How does selection occur in the GC?: Presumably. Nature 419:43-48.G. Alternatively.S. exponential rate.J. but follows a much slower. Taylor & Francis Group. Shlomchik.5. Negative selection is thus a frequent event. LLC). if there is positive selection. M.S. which is determined by the codon translation table in that most mutations do cause an amino acid substitution. the affinity of a BCR determines the strength or frequency of positive signals a cell gets: higher affinity cells will get more/better signals. The average R/S ratio in FR regions is about 1.be selected against (“negative selection”) and cells that have them undergo apoptosis. Primary literature: Di Noia. Chapter 5 pp. Reading: Text: "Janeway’s Immunobiology". Annu Rev Biochem 76:1-22. 2009. Schatz. meaning that about 1/2 of all R mutations are selected against. and D. But some are likely to improve binding to the Ag. Such skewing of R/S ratios are actually seen in real Abs. J. and thereby gain survival or proliferative signals from the T cells. and M.
such as macrophages. b) opsonization and c) activation of complement. Antibodies that bind to the pathogen can prevent this and are said to neutralize the pathogen. and also to which compartments the Ab molecule itself gains access (for example. Antibodies are secreted by plasma cells. and under the direction of signals from T cells. in immune animals. viruses and intracellular bacteria bind to specific molecules on the target cell surface.e. Similarly. These other isotypes have unique properties and effector functions. another element that also functions in the innate immune system (see earlier lectures by Dr. which in turn enhances phagocytosis by cells. M. Since antibody titers are long-lasting in immune animals (i. particularly of eukaryotic pathogens. which are diverse and mediate a variety of effects upon FcR binding of Abs. those that have already recovered from infection) antibodies protect mucosal surfaces and the blood from 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. which can result in lysis. The effector functions of Abs are in turn linked to the cells that express FcR’s. such as NK cells or macrophages. The effector functions of Abs Antibodies carry out their effects via: a) neutralization. B cells rearrange their DNA to allow secretion of isotypes other than IgM. other effector functions of Abs depend on the soluble complement system. IgM is too large to diffuse into most tissues). Opsonization is the process of coating the surface of a pathogen with Ab or complement fragments (i. Shlomchik . which in turn controls whether the Ab can bind to certain receptors for the Fc portion (FcR’s). Medzhitov). that bear receptors for the Fc part of antibodies and for complement fragments. To enter cells. which are the terminally differentiated products of activated B cells.Humoral Immune Responses November 2 Instructor: Overview: Humoral responses are important to protect the extracellular spaces from pathogens.e. C3b). activate or “fix” complement. During the course of an immune response. prevent the establishment of new infections. Effector functions of Abs are dictated by the Fc portion of the Ab. They prevent the spread of established infections and. Most of these effects are mediated by cells that can be classified as part of the innate immune system. Neutralization by antibodies is also important in preventing bacterial toxins from entering cells. Thus the effects of Abs provide yet another link between the adaptive and innate immune system.
Whereas IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system. and therefore functions chiefly as a neutralizing antibody. such as coughing. the most important being those of the mucus epithelium of the intestinal and respiratory tracts. form pentamers whose 10 antigen-binding sites can bind simultaneously to multivalent antigens such as bacterial capsular polysaccharides. This distinction is not surprising. The pentameric structure of IgM makes it especially effective in activating the complement system. but is bound avidly by high affinity FcR’s specific for IgE (Fc R) on mast cells. When Ag is bound by this mast-cell associated IgE. and vomiting. IgA is a less potent opsonin and a weak activator of complement. to a lesser extent. however. as a result of the large size of the pentamers. whereas IgA operates mainly on epithelial surfaces where complement and phagocytes are not normally present. the progeny of a single B cell can produce antibodies. whereas IgA is the principal isotype in secretions. sneezing. IgM molecules. IgE antibody is present only at very low levels in blood or extracellular fluid. and most of the B cells expressing these isotypes have been selected for increased affinity of antigen-binding in germinal centers.Ab isotypes have different properties and effector functions Antibodies of different isotypes are adapted to function in different compartments of the body. Via isotype switching. IgG and IgE are always monomeric. each with the same combining site. Although IgA can form dimers. The distribution and main functions of antibodies of the different isotypes are summarized here (from Janeway text): . IgM: The first antibodies to be produced in a humoral immune response are always IgM. Mast cells are found just beneath the skin and mucosa. IgG. IgG is the principal isotype in the blood and extracellular fluid. The affinity of the individual antigenbinding sites for their antigen is therefore critical for the effectiveness of these antibodies. it triggers mast cells to release powerful chemical mediators that induce reactions. that can expel infectious agents. as IgG operates mainly in the body tissues. and along blood vessels in connective tissue. and IgE: These Ab molecules are smaller in size and diffuse easily out of the blood into the tissues. as will be discussed below. the lymph. where accessory cells and molecules are available. This compensates for the relatively low affinity of the IgM monomers. These early IgM antibodies are produced before B cells have undergone somatic hypermutation and therefore tend to be of low affinity. IgA. However. IgM is mainly found in the blood and. yet expressing a variety of different Ig isotypes.
including: macrophages. These two outcomes of complement activation lead to more efficient removal of immune-complex-bound pathogens. as this would lead to uncontrolled inflammation. and are much more sensitive to BCR crosslinking when the CRs are also bound. NK cells. dendritic cells. because C1q must bind at least two IgG Fc regions in order to be activated and start the cascade. neutrophils. A single molecule IgM. complement activation leads to binding of C3b and C4b to nearby protein structures. 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. they do not by themselves remove pathogens from the body. Instead. eosinophils and basophils which all have receptors for IgE (see above). there must be several molecules in proximity. but only when that IgM is bound to a surface and undergoes a conformational change allowing C1q activation.It is critical that soluble Ab molecules. Complement activation is one adjunct for antibody-mediated protection. and mast cells. which is already a pentamer. this may be . Complement coated pathogens are also especially potent at stimulating B cells. As described earlier in the course. B cells. FcR’s mediate the destruction of antibody coated pathogens and result in activation of macrophages. the ability of Abs to activate complement is controlled by a requirement for Ab to be bound to a surface. Another is a series of receptors for the Fc portion of Abs (FcRs). NK cells and dendritic cells Although high affinity Abs can neutralize. which circulate at mg/ml concentrations. including macrophages and dendritic cells. These are on the surface of many hematopoietic cell types. which also have CRs. resulting in a form of opsonization. via complement receptors (CRs) that are on red blood cells as well as on a variety of immune system cells. In the case of IgG. It also leads to the release of soluble inflammatory mediators. nor are they capable of destroying many kinds of pathogens. can activate C1q. do not activate the complement cascade.
and inflammatory agents.due to affinity as well as steric reasons. dendritic cells and neutrophils). don’t memorize it!): . Cell types like NK cells are not phagocytic but instead are triggered to release stored mediators. In the case of the phagocytic cell types (macrophages. 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. from Janeway text. A summary of the properties of the different types of FcRs is given here (FYI. when their FcR’s are bound. This process is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). Phagocytosis in turn leads to destruction of the pathogen as well as enhanced presentation of its antigenic peptides to T cells. The cells are also activated to undergo new protein synthesis again leading to subsequent secretion of cytokines. like cytokines. Thus only IgG that is complexed to Ags will effectively activate these receptors. perforin and granzyme B (that mediate cell lysis).
408 . LLC).Reading Text: "Janeway’s Immunobiology".. 2011. Taylor & Francis Group. F. Chapter 10 pp. and J. by Kenneth Murphy (Garland Science. Ravetch. 8th Edition.424. Primary literature: Nimmerjahn. Science 310: 1510 -1512. 2005. Divergent Immunoglobulin G subclass activity through selective Fc receptor binding. .
Another approach is to use a property associated with memory cells as a surrogate marker for those cells. B cell memory responses are qualitatively different from primary responses in three main ways: the antibody formed is IgG or IgA (not IgM). in which cowpox infection (a recoverable disease) was deliberately induced and later protected against fatal smallpox. Secondary response Abs have higher affinity than primary response Abs. higher affinity cells (possibly created through beneficial somatic mutations) are selected in some way to become memory cells and this underlies the affinity maturation. the IgG positive. Memory B cells are reactivated upon reexposure to the Ag. or increased longevity of a cell and/or its clonal descendants. one must isolate putative memory precursors and do a functional assay. we do believe that the GC reaction is necessary for the formation of high affinity memory cells. This makes sense. only a small subset are retained as memory cells. memory T cell responses occur faster. Of the many cells which initially respond to antigen and enter the GC. a phenomenon termed "affinity maturation". differentiation to a more "potent" type of cell. but instead an interaction with a professional APC such as a dendritic cell. For example. faster and qualitatively different. This form of specific enhanced immunity to reinfection occurs because of immunologic memory. an animal would quickly fill up with memory cells and have no "room" left for new cells to respond to new antigens. Jenner was perhaps the first to intentionally take advantage of this phenomenon to protect naive individuals. Memory in the immune system is defined functionally. with higher amounts of cytokine production and secretion of “polarized” cytokines such as IL-4 and IFN-g without delay. 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. but since the system is composed of cells. We do not know which is true. To prove this.B and T Cell Memory November 5 Instructor: M. IgM negative cells contain memory precursors though we don’t know that all IgG positive cells are memory precursors. Similar studies showed that these cells are also isotype switched. 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. they probably do not need a GC reaction. compared to the first response. and the V genes of cells participating in a secondary response are somatically mutated. These cells could be better competitors for antigen or might receive a different or stronger signal to differentiate because of their higher affinity. He invented vaccination. will block memory formation. nor do we know whether all high affinity cells become memory cells. 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. However. Memory is a property of the immune system as a whole. and treatments that block the GC reactions. one expects a cellular basis for memory. A system shows memory when. such as blocking the CD40-CD40L interaction by infusing anti-CD40L Abs into mice. This is termed the secondary immune response. since if all responding cells were retained. 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. the affinity for the antigen is higher. 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). This approach is a descriptive one and doesn't really prove that these "secondary cells" are really the precursors to long-lived memory cells. Similarly. Memory cell development The precursors of memory B cells form mainly in the germinal center (GC). For example: memory cells carry . This has been easiest to do for the cell surface markers. The requirements for the formation of a memory T cell are less clear. Presumably. 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. a second or later response is characteristically different: usually stronger.
which is required to maintain the survival of naïve B cells. For example. there is good evidence that the body can store it in special cells (follicular dendritic cells) for long periods. It appears that cytokines are important for maintaining T cells. 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. However. This reasoning was one way in which the GC was implicated as the site of memory B cell development. 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. heterogeneity is seen in terms of expression of surface proteins that control migration. The signals that stimulate this division and the consequences are unclear.mutated Ig V regions. this might be a site where memory cells are formed. The role of cytokines in maintaining B memory cells has yet to be elucidated. replicating antigens (especially certain viruses) may remain at low levels which are inconsequential for the host but which maintain memory. Emerging data indicates that these different memory B cell subsets carry out different functions in a secondary immune response. is not needed to maintain memory B cells. effector and memory B cells. memory CD8 responses appear to decay. although it seems likely that antigen goes away after a period of time. principally IL-15 and IL-7. But it needn't be so for two reasons: first. plasma cells or effector T cells) upon Ag reexposure. In addition. it is known that the TNF-family cytokine called BAFF (B Cell Activator of the TNF Family).e. If we could know where mutations occur. Memory T cells are also heterogeneous in their ability to make various cytokines upon restimulation. the cells themselves may not be long-lived even though the clone from which they descend is. it is possible to observe memory cells dividing infrequently. These diverse requirements are probably dealt with by heterogeneity among memory cells—not all are created equally. If all of the memory cells became terminally differentiated effectors (i. developing memory T cells can be distinguished by their expression of the IL-7 receptor. the discovery of surface proteins that mark memory cells revealed. . Among memory B cells. Second. However. In fact. Memory cells thus may divide every so often yielding two memory daughters. the diagram below shows that PD-L2 and CD80 expression defines distinct subsets of murine memory B cells. this would actually deplete memory and impair any subsequent responses. via multiparameter flow cytometry. activated. that not all memory cells expressed all markers. In the T cell compartment. 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 B cells and T cells both probably exist for long times and at stable overall levels without undergoing cell division. Surface marker (phenotype) comparison among naïve. while effector cells that are destined to “burn out” do not upregulate this cytokine receptor. without these.
Surface marker (phenotype) comparison among naïve. that Ab responses are more prompt. c) non-limiting Ag-specific T cell help. Secondary immune responses Much less is known about the nature of the secondary immune response than the primary. activated. generally. and a paradoxically attenuated GC response. We do know. There are several key differences with primary responses: a) higher B or T cell precursor frequency. Secondary immune responses seem to be highly variable. b) more sensitive “memory” differentiated B or T cells. which was also used to measure expression of CD80 and PD-L2. effector and memory T cells. but there is . depending on the timing. as mentioned. the B cells that seed secondary response GC’s are memory cells or “new” primary B cells. In general. It is not even clear if. Ag dose and nature of the Ag. secondary responses are characterized by large bursts of extra-follicular B and T cell proliferation. in some circumstances it does appear that memory B cells can undergo a second round of somatic mutation. But the cellular and microanatomic basis of this are less clear. early differentiation into plasma cells at these sites.or + (two subsets) Table 2. then identified by flow cytometry. d) preexisting Ab.IgG1 memory cells specific for the hapten NP were generated in a transfer and immunization model. are of higher affinity. and are isotype switched and that T cell responses feature prompt induction of cytokines and/or cytotoxicity. 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) ++ + +++ + +++ ? . The degree to which all of these are present also influences the nature of the secondary response.
Immunol.. 255-264. Sections 10:22-10:26. B.. Differentiation of memory B and T cells. R. B.. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells. 3rd. Walport. V. BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact.J..clearly early clonal expansion without further mutation as well.. Immunobiology: The immune system in health and disease. Rev. Current opinion in immunology 18. Quinn. Articles: Maruyama. J. Seddon. Memory B-cell persistence is independent of persisting immunizing antigen. Nature. Ltd.. and R. and Ahmed. In any case.J. Gourley. Travers.. M.. Reading: Text: Janeway. Steinel.. O'Neill.. 2000.M.. P.J. N.E.H. R. Garland Science. W. 407:636-642. J. 247. 6th ed. 4:680-686. Rouse. V.. Miller. et al. and K.. S. M.. 15517-15522.. M. (2012). Scholz. Germinal center selection and the development of memory B and plasma cells. P. Cho. P. 2003. M.. 2005. . and Weisel.. and Shlomchik. Crowley. Tomlinson.. Reviews: Kalia. Goenka. K. Sarkar.T. Long. Zamoyska. F.L.. 52-63. Lam. M. (2008). C. Y. the consequence is a vigorous and high affinity IgG and effector T cell immune response that functions well to protect the host against reinfection. Tomayko.P.P. T. Rajewsky. Proc Natl Acad Sci U S A 105. Shlomchik. (2006). J..S. Nat Immunol.
whereby the pathogen causes no undo harm to the host to maximize its replication and spread. 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. such as Vibrio cholerae. illness. Taylor & Francis Group. But. When a pathogen jumps hosts. HIV-1. in essence. We will discuss some of the obtacles of vaccine development against these agents. but surprisingly to date. and memory B cells and long-lived plasma cells that provide humoral immunity. very few vaccines have been formulated in a way that rely only on T cell responses. It must be safe with minimal side effects and protective against the live pathogen when encountered naturally.12(6):509-17. certain bacteria. Sometimes “sterilizing” immunity is met by some vaccines that have extremely potent antibody responses. Immunological mechanisms of vaccination. Ahmed R. Many types of vaccines are being used today with success or are in clinical trials. protective memory T cells that provide cell-mediated protection and remove infected cells or pathogens. 2011 Jun. It needs to provide sustained and long-lived immunity. malaria and TB. is a viable strategy. (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. Iwasaki True co-evolution between pathogen and host can be found when a “carrier” state is produced. 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. small pox and yellow fever vaccines have demonstrated protection for more than 50 years in humans.e. They do so by generating long-lived. The most effective vaccine is always a live. protected. Pulendran B. Chapter 16 (16-19 through 16-31). For example. disease and death can result. LLC).. For such pathogens. A protective vaccine needs to produce neutralizing antibody to prevent immediate infection and contain spread. 2011. Many critical elements need to be encompassed by a “good” vaccine. cause disease by secreting toxins. much needed vaccines for major killers. Reading: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science. Killed viruses are also commonly used as these are very safe. a single dose). Nat Immunol. are lacking.Vaccination November 7 Instructor: A. and what immunologists might do to tackle these problems in the future. generating blocking antibodies to toxins. attenuated virus because it will mimic the natural infection the best. but not the bacteria. 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). Likewise protective T cells should also be induced. For instance. . these are not available for many viruses. 8th Edition. An ideal vaccine would also be low cost and biologically stable for shipping and storage and have an ease of administration (i.
TLRs in turn trigger production of inflammatory cytokines. several NLRs play a key role in the activation of caspase-1 by forming a multi-protein complex known as the ‘inflammasome’. lipid mediators. expression of which is induced by these cytokines. platelets and endothelial cells. which leads to production of inflammatory cytokines and chemokines. which include surfactant proteins (which promote phagocytosis in the lung). Fever is beneficial to the host because most pathogens grow better at lower temperatures. it can be highly detrimental if it becomes excessive. neuropeptides and amines (histamine and serotonin). IL-10 and TGF-beta. The inflammatory response to infection is designed to optimize the elimination of the invading pathogen. MBL (binds to carbohydrate moiety on pathogens and induce opsonization). They do so by inducing the sythesisi of prostaglandin E2 (PGE2) by the enzyme cyclooxygenase-2 (COX2). recruitment and activation of neutrophils. C-reactive protein (binds to phosophocholine portion of bacterial and fungal cell walls for opsonization and C’ activation). damage or bacterial products. IL-1 and IL-6 are called endogenous pyrogens because they case fever. monocytes and macrophages. as well as nitric oxide and reactive oxygen species (ROS). 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. These cytokines orchestrate the inflammatory response at the local and systemic levels. Molecular mediators of inflammation include proinflammatory cytokines.Inflammation – Response to Infection November 9 Instructor: A. The inflammatory response includes the following physiological components: vasodialtion. Acute phase response TNF. eosinophils. Pathogens activate TLRs on macrophages. This response is triggered most commonly by TLRs activated by infectious agents. and fever. dysregulated inflammatory response leads to a wide variety of pathological conditions and is normally prevented by several anti-inflammatory mechanisms. increased vascular permeability. IL-1 and IL-6. IL-1b processing requires the formation of inflammasome complex in the cytosol. Caspase-1 is an essential mediator of inflammatory response through its capacity to cleave and generate active forms of IL-1b and IL-18. Inflammasomes are induced by sensing cellular stress. The so-called antiinflammatory cytokines. Inflammasomes NOD-like receptors (NLRs) are pattern recognition receptors that reside in the cytosol. A typical inflammatory response is initiated by macrophages resident in peripheral tissues. Liver cells start to produce acute phase proteins. which promotes recruitment of leukocytes to the site of infection. most importantly TNF. Fever TNF. are particularly important for the negative regulation of inflammation. Indeed. plasma proteases (complement and coagulation protease cascades). this results in local changes in endothelium (vasodialtion and increased vascular permeability. expression of adhesion molecules). IL-1 and IL-6. IL-1 and IL-6 also act on the hepatocytes to induce the acute phase response. such as TNF. persistent of systemic. In particular. Although the functions of many of the NLRs are largely unknown. . Regulation of inflammation Although inflammatory response is an essential component of host defense. Collectively. acute phase response proteins. The cellular component of the acute inflammatory response includes activation of neutrophils. such as prostaglandins and leukotrienes. Iwasaki Inflammation is a complex response to infection and injury that destroys the infecting agent and restores the injured tissues.
Chapter 3. Text: "Janeway’s Immunobiology". Nature. by Kenneth Murphy (Garland Science. Taylor & Francis Group. 2011.420(6917):846-52. 8th Edition. Points of control in inflammation. LLC).Reading: Review Articles: Nathan C. . 2002 Dec 19-26.
digestive.Mucosal Immunity November 12 Instructor: A. to provide the most efficient form of protection. Type I: Mucosal Surfaces Covered by Simple Epithelia Type I mucosal surfaces are covered by simple epithelia of one cell–layer thickness. Third. Second to induce rapid immune responses. pathogen specific T and B cells are found within the disorganized lymphoid tissues of the lamina propria. 3) provide a reservoir of effector and memory cells just beneath the surface epithelial cells for rapid protection. mucosal lining contains specialized cells that secrete antimicrobial peptides into the lumen.). Iwasaki Infection in real life With the exception of vector-borne diseases. Two types of mucosal lining. Consequently. In addition. and reproductive functions that are vital to the life of the host. the upper female reproductive tract. excretory. respiratory. First. to combat pathogens. all pathogens must enter the host through the mucosal surfaces. The primary role of these surfaces is to perform absorptive. reproductive…etc. 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 . Forth. one can find organized lymphoid structures just beneath the mucosal lining. and the pseudostratified epithelia of the respiratory tract. These surfaces are represented by those that cover the small and large intestine. 4) prevent unnecessary Th1 immunity to maintain the physiological functions of the mucosal tissues. mucosal surfaces are lined with mucus and other physical barriers that act as a first line of defense against pathogens. 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. 2) induce rapid immune responses. mucosal tissues have developed unique features not found in other types of tissues to 1) combat pathogens.
respectively. FcRn is expressed by vaginal epithelium and can transport antibodies from the tissue and back into the tissue. appendix Follicle Associated Epithelium (FAE) The FAE is a specialized epithelial layer that cover the dome region of the Peyer’s patch. Certain chemokines. 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. group IIA phospholipase A2) that protect mucosal surfaces and crypts containing intestinal stem cells against invading microbes. The pIgR binds to polymeric IgA (pIgA) secreted by the plasma cells in the LP and exports the pIgA transepithelially. based on the observation that mucosal sensitization at one site provide primed cells selectively to other mucosal sites. • 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. Since the original description of the CMIS. This concept has been the main force behind the efforts to develop “mucosal vaccines” in which immunogen delivery is targeted to various mucosal surfaces. and tonsils. and the keratinocytes that cover these surfaces do not have absorptive or respiratory functions. Cell migration mechanisms behind the CMIS have been elucidated recently. injury or stress and destroy infected cells. FAE contains M cells capable of transporting luminal antigens across by transcytosis. Immune Inductive Sites Organized lymphoid tissue (inductive sites) Tonsil. Further. Another important cell type that provides early non-specific immune responses are the intraepithelial lymphocytes. Peyer's patches. The main function of type II mucosa is to provide a physical barrier. releasing the secretory IgA (SIgA) into the lumen. IgA is the most important isotype that provides protection against pathogens. cathelicidins. bactericidal/permeability increasing protein) and bacteriolytic enzymes (lysozyme. These observations were originally described for gut induction leading to bronchus effector functions. who proposed that the immune system of the mucosal tissues is somehow connected. which share many common features with the skin. appendix. many studies have now confirmed this theory and provided molecular mechanisms for the homing pattern of effector lymphocytes. These lymphocytes detect non-classical MHC I molecules induced by infection. In the gut and lung.epithelia. IgA provides its effector functions in multiple ways. . other studies have provided evidence that distinct branches exist within the CMIS. and IgA secreting plasma cells can be found in the lamina propria of these tissues. Type II: Mucosal Surfaces Covered by Stratified Epithelia Type II mucosal surfaces are covered by stratified squamous epithelia (keratinocytes). Type II mucosal surfaces are devoid of MALT structures but are drained by regional lymph nodes. Unique Components of the Mucosal Immune System Innate Immunity Mucosal epithelial cells and Paneth cells produce a variety of antimicrobial peptides (defensins. There are no pIgR on the type II mucosal surface and thus the dominant protective immunoglobulin at these surfaces is IgG. 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.
The mechanism of oral tolerance is not completely understood. (2007) Mucosal dendritic cells. 2011. but it likely involves mucosal dendritic cells and regulatory T cells that suppress Th1 immune responses to antigens. the animal becomes unresponsive to that antigen upon secondary encounter. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria.303(5664):1662-5. 8th Edition." Kenneth Murphy. Review Article: Iwasaki. The loss of tolerance in the intestinal mucosa can result in inflammatory bowel diseases. 2004 Mar 12. Annual Review of Immunology 25:381-418 Primary Literature: Macpherson AJ. 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). This phenomenon is known as “oral tolerance”.Immune Regulation Mucosal tissues are constantly exposed to tremendous amounts of exogenous antigens that are either resident or ingested/inhaled. When soluble protein antigen is given orally in the absence of an adjuvant. . Questions Q1: How do we distinguish between commensal bacteria and pathogenic bacteria? Reading: Text: “Janeway’s Immunobiology. Chapter 12. Uhr T. Science. A.
but rather is a functional state of genes. the latter being a special form of lineage differentiation. as if cells can “remember” things of the past. This cellular memory. Trans-mechanism.Transcriptional Regulation in the Immune System November 14 Instructor: T. For example. can sometimes be sustained and propagated to daughter cells independently of the initiating signals. the transient signals can cause irreversible or long-lasting changes in gene function that persist after the termination of the signaling events. and what might be the underlying mechanisms? . b. Developmental cues are known to direct lineage commitment and differentiation by controlling transcription. 2) The most important target genes turned on by the latent factors are “master transcription factors” specifying cell identity/critical immune functions. and such modifications. once established. for example. Chromatin structure is tightly regulated in part by covalent modifications of DNA and histones. 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. Similarly. which underlies phenomena such as trans-differentiation. respectively. such as T-bet and GATA3. in which the induced transcription factors auto-regulate their own expression after the termination of the transient signals. when they move to the nucleus to regulate target genes. 5) Epigenetic memory. Questions: The effects of transient signals on target gene functions can be reversible or irreversible. is not hardwired or coded in the DNA sequence. and so can be the corresponding transcriptional responses they elicit. The latent factors are often constitutively localized in the cytoplasm until activation. typically called epigenetic memory. which has important clinical implications. various CD4 cell subsets (Th1. Although this state is stable. Chi The development and function of the immune system are controlled to a large extent at the level of transcription. which involves chromatin. Consequently. from the autoimmune disease IPEX to the immune deficiency disease DiGeorge’s syndrome. while transcription factors constitute effective targets for immune therapies (consider. respectively. immune responses to pathogens and other stimuli rely heavily on transcriptional regulation in the responding cells. it is also intrinsically plastic. because incorporation of DNA into chromatin inhibits access of transcription factors. However. transcription defects underlie diverse immunological disorders. which are necessary and sufficient for directing the differentiation of naïve CD4 cells into Th1 and Th2 cells. with the optimal adaptive immune responses achievable only after further transcription-mediated differentiation of the responding naïve lymphocytes into effector/memory cells. 4) Epigenetic memory is achieved by two mechanisms: a. how cyclosporin A and corticosteroids work). What are the biological “purposes”. Cis-mechanism. Th2. just as our brains’ memory of our experiences. is at the heart of lineage differentiation and immunological memory. Th17 and Tregs) are interconvertable to some extents. There are 5 general principles underlying transcriptional responses to environmental signals. 3) Environmental signals are transient. sometimes. retrodifferentiation and epigenetic reprogramming. Chromatin is a focal point of transcription regulation.
2011. Chapter 8-17 to 8-19 (pages 308-11) Primary Literature: O’Shea. by Kenneth Murphy (Garland Science. pp. J.Reading: Text: “Janeway’s Immunobiology”. 1098-1102. & Paul. LLC). 327. W. Science (2010) vol. Taylor & Francis Group.. . Mechanisms Underlying Lineage Commitment and Plasticity of Helper CD4+ T Cells. 8th Edition.
Severe Combined Immunodeficiency diseases (SCID) SCIDs are a set of primary immunodeficiencies that are diagnosed during the first year of life. patients suffer from recurrent infections that can be life threatening. 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). respectively). we will report on common variable immunodeficiency diseases (CVID). Indeed. Indeed. . we will discuss the case of the common g chain gene mutation. 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. which are the most common of all immunodeficiencies and whose gene defects are mostly unknown. In these cases.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. Actually. Interestingly. these cells are very important for the establishment and/or maintenance of peripheral tolerance.Those resulting from defects of in the components of the V(D)J recombination machinery. On a scientific aspect. -Finally. As a consequence. In this case. early T and B cell precursors are unable to recombine their specific receptor genes (T cell receptor and immunoglobulin genes.Primary and Acquired Immunodeficiencies November 16 Instructor: E. It took more than forty years before gene mutations associated with this syndrome called X-linked agammaglobulinemia (XLA). 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. More classical forms of such defects are called HyperIgM syndromes and still allow IgM secretion but impair IgG and IgA production. We will review in this section the different types of mutations that can affect the production of memory B cells and gammaglobulin secretion. We will review three major kinds of SCID: . Meffre Primary immunodeficiencies are a set of rare diseases usually resulting from defects in a single gene. while patients are usually unable to mount proper immune reactions against pathogens. which are characterized by the impaired generation of regulatory T 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. 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. As a consequence. demonstrating that we have still a lot to learn from the analysis of primary immunodeficiencies. the first reviewed case of primary immunodeficiency was reported in 1952 and affected a boy who displayed no gammaglobulins in his serum. These lifethreatening diseases usually affect several arms of the immune system. In this section. we will present a few syndromes such as IPEX and APECED. 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. . . B and NK cells in the patient’s blood. it is very common to identify specific immune responses targeting the patient’s own body.Those characterized by an absence of T cells and resulting from either T cell specific gene mutations (CD3 component) or cytokine receptors. B. NK and other antigen presenting cell functions.
pp.Reading: Primary Literature: Cunningham-Rundles.” Nat Rev Immun (2005). 880-892 . et al. vol 5. “Molecular Defects in T and B cell Primary Immunodeficiency disease.
the inhaled allergen can be engulfed by the lung resident dendritic cells. Basophils Like mast cells. Thus. eosinophils only express surface FceR upon activation by Th2 cytokines and chemokines. Upon activation. but not dendritic cells. 6: HLA-DR • Chr. Most human allergens are relatively small proteins that are inhaled or ingested in very small quantities. Basophils release histamine and other substances toxic to parasites but also to the host. Th2 responses to innocuous antigens cause allergic diseases. Recent studies indicated that basophils. IL-5. basophils also constitutively express FceR and can be recruited to the site of IgE-triggered reactions via inflammatory lipid mediators cytokines and chemokines. IL-13 and IgE exists to provide protective immunity to parasites. 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. Predisposition to allergy The prevalence of atopy in the industrialized countries is on the rise in recent years. . Antigens that evoke Th2 cells that drive an IgE response are known as “allergens”. • Chr. Medzhitov Although Th2 immunity characterized by IL-4. the activation of the eosinophils is tightly regulated at the level of 1) total cell number. Unlike mast cells and basophils. and depending on the route of allergen encounter.Allergy November 26 Instructor: R. They tend to have enzymatic activity (cystein protease) and are stable and soluble. There are two main types of mast cells in the body. IgE can bind to the FceR in the absence of bound antigen. IL-13. Atopic individuals possess higher levels of IgE and eosinophils in circulation. those associated with vascularized connective tissues (connective tissue mast cells) and those found in submucosal layers of the gut and lung (mucosal mast cells). can prime Th2 responses to parasites and allergens. Basophils can amplify the response stated by mast cells. However. IL-9. • Chr. In allergic individual. causes different diseases. Eosinophils The eosinophils are found in tissues within the connective tissue beneath the mucosal epithelium. neurotoxins leukotrienes) that are intended to kill parasites but are also very toxic to the host. in the industrialized countries where parasite infection is rare. collagenase. all of these mast cells have prebound IgE on their FceR. 2) location and 3) threshold of activation. eosinophils release contents of preformed granules containing many toxic substances (peroxidase. Genetic factors Several genetic loci have been identified to be associated with atopy. 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. β2-adrenergic receptor. 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). the precise mechanism by which the allergens induce Th2 immunity by dendritic cells is unknown. IL-4. 11: Encodes the β subunit of FceR. Once taken up into the airway mucosa. Cellular mediators of allergy Mast cells Mast cells express high affinity FceR on their cell surface. 5: cluster of Th2 genes (IL-3. contraction of bronchial smooth muscle leading to asthma. GM-CSF). Inhaled allergens trigger lung mucosal mast cells to release their granules and cause increased mucus production.
2001 Jan 11.Environmental factors • Early exposure to infectious diseases reduce the prevalence of allergy (“Hygiene hypothesis”) • Pollution Allergic diseases Asthma Skin allergy (urticaria.344(1):30-7. Allergy and allergic diseases N Engl J Med. . chronic eczema) Food allergy. by Kenneth Murphy (Garland Science. Primary Literature: Kay AB. and N Engl J Med. 2011.344(2):109-13. 2001 Jan 4. LLC). 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". Chapter 13. 8th Edition. Taylor & Francis Group.
c. Herold Definition of tolerance: The lack of an immune response to an antigen. Receptor editing . K.Peripheral Tolerance November 28 Instructor: 1. Generation of a diverse repertoire will lead to production of T and B cell receptors that are reactive with self. Mechanisms of tolerance – over view – note that it is difficult to precisely define mechanisms for an immune response that doesn’t happen! a. b. Lymphocyte repertoire must be diverse. b. mechanisms are needed in the periphery to contain the immune response or to divert it from a pathogenic response = peripheral tolerance. In the event that central tolerance is not complete. Mechanisms needed to eliminate most autoreactive cells at the time and location of development – this is referred to as central tolerance. somatic hypermutation of Ig will generate self reactive T cells. A failure of self tolerance results in autoimmunity. d. 2. Death by apoptosis (clonal deletion b. Central B cell tolerance in the Bone marrow: a. B cell tolerance a. 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. for example for antigens that are not expressed in the central compartments at the time of development. Fate of autoreactive B cells depends on the strength of the BCR signal: 1. 2.
Civil service model 5. IL-10 c. 3. Effects of cytokines in the environment b. . Leads to negative selection. Lack of costimulation or negative costimulation 4. Bystander suppression b. TGF-beta. IL-4. The identification of the molecular basis of the IPEX syndrome was a key finding in understanding the role of Foxp3 in regulatory T cells. APECED = autoimmune polyendocrinopathy candidiasis ectodermal dystrophy In the periphery 1. Foxp3: Foxp3 can reprogram CD4+ T cell function. 3. brain. Antigen specificity of Tregs? Treg function is not antigen specific c. uterus. Oral tolerance: mechanisms of tolerance at the gut mucosal surface 4. Infectious tolerance c. IL-5. Inhibits AP-1 RORγt – a transcription factor involved in Th17 cell differentiation. b. Naturally occurring Tregs b. Immune privileged sites: eye. et al) a. TGF-β Environmental tolerance (Cobbold. and other cytokines been described. Mutations are associated with IPEX (immune dysfunction/polyendocrinopathy/enteropathy/ enteropathy/X-linked. Cytokines a.2. It can inhibit NFAT activation at the IL2 promoter preventing transcriptional activation of the IL-2 gene. Waldmann. Regulation/regulatory T cells a. Proteins are expressed in the medullary thymic epithelial cells. Cells that produce IL-10. c. Tolerance limited to antigens expressed in the thymus ii. testes. A number of different phenotypes have been described. The AIRE gene promotes the expression of several genes in the thymus. 3. iii. Outside of the bone marrow Immunologic ignorance T cell tolerance a. Ignorance 2. Adaptive Tregs: T cells that do not emerge from the thymus as regulatory T cells can acquire regulatory function. Central Tolerance i. d. Some of these cells also express Foxp3.
" Kenneth Murphy. Cellular and genetic mechanisms of self tolerance and autoimmunity.435(7042):590-7. . Vinuesa CG. 2005 Jun 2. Fazekas de St Groth B. Sprent J.Reading: Text: “Janeway’s Immunobiology. Nature. Paul Travers and Mark Walport. Chapter 15 and Chapter 8 section 8-19 Review Article: Goodnow CC. 8th Edition.
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy ii. Indeed. Other genetic loci. 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. Innate immune responses may also be involved in initiation of autoimmune responses. C. targets of autoimmune responses such as DNA can activate TLRs (e. have also been associated with autoimmune diseases. some of which are associated with immune responses. In certain cases (e. anti-CTLA-4 antibodies) this can occur through known mechanisms.Autoimmunity November 30 Instructor: 1. Autoimmune diseases affect every organ system. Therefore consideration of the causes and treatments of these diseases is closely linked to understanding of mechanisms that maintain tolerance. IL-23R.g. Certain drugs can also initiate autoimmunity. polyendocrinopathy. Mechanisms that may account for the relationship d. e.g. g. involves a combination of environmental and genetic factors A. Rates of concordance in identical twins B. the precise immunologic mechanisms are not certain. K. Scurfy/IPEX: Immune dysregulation. The immune nature of several diseases now identified as autoimmune was based on the finding of autoantibodies but in most cases. The initiation of autoimmune diseases. 3. CTLA-4. Examples of MHC-disease relationships ii. The major genetic determinants of autoimmunity are genes within the major histocompatibility complex i. etc. Molecular mimicry is one postulated mechanism to account for the relationship between environmental events and autoimmunity – specific examples f. Adaptive immune responses are involved in autoimmune diseases .g TLR9). Please refer to these mechanisms discussed in the lecture on tolerance. Autoimmunity is the ultimate example of loss of tolerance to self: horror autotoxicus. e. hence loss of self tolerance. Herold Autoimmunity A. 2. X-linked disease c. There are examples of specific genetic mutations that lead to autoimmune diseases i. B. C. enteropathy.
ii. Induction and effector functions of T(H)17 cells. pathogenesis and clinical interventions in type 1 diabetes. Therapies for autoimmune diseases: anti-T cell/anti-B cell/anti-cytokine/blockade of cell migration. Myasthenia gravis 5. Nature. and Kuchroo. Graves’ disease d. Examples include: a. Eisenbarth G." Kenneth Murphy. intra and intermolecular spreading of the immune response 4.A. Chronicity is a feature of autoimmune diseases: i. Herold K. development of tertiary lymphoid organs as sites of activation of adaptive immune responses. Paul Travers and Mark Walport. VK et al. as antigen presenting cells B. animal models. iii. Examples include: d. Tolerance is the ultimate goal. Nature.464(7293):1293-300. Unstable Tregs? D. 2010 Apr 29. Bluestone JA. Genetics. Examples of specific autoimmune diseases: Most organ specific autoimmune diseases are T cell mediated. 2008 Jun 19. antigens. Role of B cells: as antibody producing cells. Reading: Text: “Janeway’s Immunobiology. Rheumatoid arthritis c. Type 1 diabetes (as an example): MHC association.453(7198):1051-7. Autoimmune hemolytic anemia e. Multiple sclerosis But others are mediated by autoantibodies. 8th Edition. Review Articles: Bettelli E. modification of self proteins as a mechanism of generating new epitopes. Chapter 15. cellular mechanisms b. Pathogenic autoreactive T cells can clearly cause autoimmune disease in animal models and are found in human autoimmune diseases C. .
Immunization with tumor specific antigens/Adoptive transfer of dendritic cells pulsed with peptides b. HER-2/Neu Therapy with engineered effector cells. BCG c. 5. 4. Poor access to the tissue d. Grafts a. . b. Hyperacute: preformed antibodies against a graft – often against blood group or polymorphic MHC antigens. B7-1 d. Mechanisms whereby tumors avoid immune recognition a. 6. Adjuvant e. Inhibitory factors: TGF-β. Tumor rejection antigens may arise by point mutations in self proteins b. CD8+ T cells Tumor antigens: a. Costimulation enhancers e. Rituximab (anti-CD20 mAb). Failure to activate T cells c. 7. Syngeneic: same strain Allogeneic: different strain. Xenogeneic: different species Classification of Rejection: Graft rejection is mediated by T cells. Alloreactivity occurring days and weeks after transplantation. Proteins selectively expressed in tumors are candidate tumor rejection antigens c. These proteins are generally presented by Class I MHC molecules Paradox –T cells infiltrate a tumor but do not destroy the tumor .g. It can be transferred with T cells. a. Acute i. Herold Cancer Immunology 1. Transplant Immunology and Immune therapy 1. 2.g. 3. Blocking negative signals: anti-CTLA-4 Biologics against antigens expressed on tumor cells e. However. recruitment of regulatory T cells Immune surveillance may normally prevent the outgrowth of variant cells: role of NK cells.tumor infiltrating lymphocytes (TILs) Enhancing immunogenicity to tumor antigens and cells a.Transplantation & Cancer Immunology December 3 Instructor: K. most tumor antigens are peptides that are over-expressed in tumor cells but area also expressed at lower levels in normal tissues d. Autologous b. Failure to express antigen: immune selection of antigen loss variants e. Failure to present antigen including loss of Class I MHC expression b.g. same species c. 2.
anti-IL-6. CTLA4Ig vii. Alloreactivity can occur months to years after transplantation and is associated with gradual loss of graft function.g. Nataluzimab binds to α4β1 (on central memory and effector T cells) and α4β3 integrins iii. Presentation of graft antigens to the host a. Drugs to treat rejection and autoimmunity: a. ii. Cytotoxic drugs: i. Chronic organ rejection is caused by inflammatory vascular injury to the graft. anti-IL-1. Infliximab (anti-TNF) Etanercept (the p75 TNF receptor on a Fc portion of IgG. Calcineurin inhibitors c. Anti-CD3 vi. Rapamycin e. Anti-lymphocyte serum ii. Direct: recognition of donor cells by recipient’s T cells b. Mycophenolate mofetil f. Anti-Cytokines: e. Adoptive immune therapy with regulatory T cells? g.2. . Examples of Biologics: Human Abs vs humanized abs vs murine abs vs other animals i. Azathioprine: ii. Chronic: i. Indirect: Recognition of processed antigens of the donor by recipients APC’s c. Janus kinase inhibitors d. Rituximab (anti-CD20) iv. Cyclophosphamide iii. Glucocorticoids b. Campath (anti-CD52) v.
Ferrone S." Kenneth Murphy. 8th Edition. Paul Travers and Mark Walport. Quezada SA. Cancer immunotherapy: co-stimulatory agonists and co-inhibitory antagonists. Clin Exp Immunol.373(9668):1033-4 . Weiner LM.157(1):9-19. 2009 Mar 21. Allison JP. 2009 Jul. Monoclonal antibodies for cancer immunotherapy. Lancet. Chapter 15. Review Articles: Peggs KS. Dhodapkar MV.Reading: Text: “Janeway’s Immunobiology.
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. 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.Unresolved Questions in Immunology December 5 Instructor: R. and yet we still cannot design successful vaccines for most pathogens. Medzhitov Despite tremendous advances in understanding the function of the immune system.
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