Cancer Induction

Cause of Cancer History of Cancer Biology

 The accumulation of genetic and epigenetic defects in key cellular pathways regulating Hippocrates (~400 B.C.) – one
of the first to describe tumors
proliferation, differentiation, and death
 ~1761: Giovanni Magagni
o Epigenetic: cause changes in gene expression without altering the DNA sequence. related patient’s illness to
o Ex) DNA methylation or histone acetylation pathologic finding after death
What is Cancer?  ~1761: John Hill – rarely saw
 Breakdown of proliferative control (display uncontrolled growth) nasal polyps in patients who did
 A control system failure at a cellular level not use snuff
 Breakdown of failsafe systems (e.g. apoptosis)  ~1775: Percival Pott – scrotal
 Loss of proliferative control or apoptosis can lead to a cancerous cell! cancer seen exclusively in
Prolif control Solid tumor
chimney sweeps
Cancerous cell Metastasis
Apoptosis growth  Rudolph Virchow – said “omni
cellula e cellula” – all cells
come from cells (including
Tumor cancer cells)
 Cells that continue to replicate, fail to differentiate into specialized cells, and become immortal.
 1870’s: Wilhelm von Waldeyer
 Grow by being independent from growth factors – first serial sections of tumor
 Errors on the DNA level; genomic instability tissue

Muscle, nerve, bone,

blood
Etiology of Cancer: Factors contributing to cancer development
1. Genetic factors (mutations, translocation, amplifications).
 Ex. conversion of proto-oncogenes to oncogenes; alteration in tumor suppressor genes; thus, cell cycle affected
2. Environmental factors: UV, chemicals, viral infections (HPV)
3. Immune dysfunction: if immune system is not functioning properly there is nothing to fight the cancer so
it continues to grow
Genetic Factors: Cell Cycle
Phases of the cell cycle! G0=no
 G1- (Gap 1) the phase in which cells can
rest (G0),
differentiate, or grow and prepare for DNA
synthesis and cell division. G0 considered as
“post-mitotic”.
 S- the phase in which DNA is replicated
(most important)
 G2 - (Gap 2) preparation for mitosis
The mammalian In tumors the cell cycle is deregulated!
cell cycle
Genetic Factors: Mutations
Three types of mutations contribute to carcinogenesis in
pre- cancerous
Normal Cell Mutation
cell
Mutation Cancer humans:
Carcinogens DNA 1. Point mutations, deletions, insertions
adducts with guanine,  Examples: Proto-oncogenes: EGF receptor
leading to misincorporation (epidermal growth factors), Src, Ras: proteins
of adenosine (mismatch that cannot be switched off
base-pair)  Tumor suppressors: pRb, p53: inactive proteins
Increase in growth factor= increase in possible mutations
2. Amplifications
 Proto-oncogenes  “on”  Examples: c-Myc, Mdm2: strongly
 Tumor suppressors  “off” overexpressed normal proteins
3. Translocations
 Examples: Breakpoint between genes: c-Myc,
Cyclins and cyclin-dependent kinases (Cdks) constitute the core components of the cell cycle clock!

 Quiescence: Quiescence is a state of quietness or inactivity, that is the state of a cell when it is not
dividing. But they remain metabolic active and replication competent.
 Senescence: Senescence means "to grow old”. They are metabolic active but replication incompetent.
 (Postmitotic: Mature terminally differentiated cells that are replication incompetent.)

Overexpression of cyclin D1 tied to gene amplification has been identified in breast, esophageal, bladder, lung, and
squamous cell carcinomas. Other cyclin amplifications are less prevalent, e.g. cyclin E was reported to be
overexpressed in breast and colon cancer, while cyclin A and cyclin E are amplified in some cases of lung carcinomas.

Coupling the cell cycle to DNA damage

 Initiation of DNA
Early G1 synthesis
Restriction
point
Late G1 Early S

Checkpoint to stop
Mitogenic Signals: Growth factors and environment exert
cycle
 Growth Factors mitogenic effects that trigger the production
- Cyclin D of cyclin D, which then binds to Cdk4/6 to
Cell Matrix Cyclin D begin the process of activating the kinase
Junctions Synthesis - CDK 4/6
(Kinase)

Inhibition by p16lnk4
Stimulatory Kinases CDK2 Cyclin
CDK2 + Cyclin
(CAK) E A
Positive
feedback DNA
Dna Master Switch loop
Replication
(Tumor Suppressor)
Hyperphosphorylation
!
DNA replication
E2F
Factors
(Transcription Factor)

Full activation of Cdk4/6 Full phosphorylation of Rb

requires phosphorylation releases sufficient E2F to
by additional stimulatory elevate the production of
kinases. Phosphorylation of cyclin A, which also binds
Rb by activated Cdk4/6 and activates Cdk2.
initiates release of E2F, the Activated Cdk2 maintains
transcription factor family

The Master Switch

“R”point:
Rb inactivation
by hyper-
phosphorylation
results in release pRb is the Gatekeeper of Cell Cycle
of various E2F Entry Hyperphosphorylated
transcription Throughout cell cycle
factors. ---Once progressing through G1/S
boundary, E2Fs are degraded but Rb

G1 Transition:

G1 transition starts at Early effects of Late effects of mitogenic

mitogenic stimulation mitogenic stimulation: stimulation:

The mitogenic signals early response gene cyclinD induced

activates the ‘neo- transcriptional activation of Cdk4/6.
production’ of cyclin D activation for cyclin D The activated Cdks
followed by activating production. work in sequence in
the Cdk4/6; that Cyclin D is the direct triggeing the G1/S
subsequently turn on product of growth transition and
the ‘Master Switch’ factor signal activation. initiation of DNA
pRb by synthesis.
hyperphosphrylation.
The pRb
phosphorylation is the
key event for cell cycle
entry.
 Summary: G1 – S transition

Cell Kinases that are activated in late G1 and early S enable the initiation and maintenance of DNA
divisi synthesis. There are two groups:
on CDK4/6 and CDK2.
kinas (These CDKs are analogous to the CDK1 kinase that pairs with cyclin B to initiate mitosis.)
es
(CDK
s)
There are three: cyclins D, E, and A. They bind specifically to one of the CDKs and, like cyclin B, help to
activate their CDK partner. They pair up in the following way:
CDK4/6cyclin D
G1 CDK2cyclin E (The G1 CDKs are activated in G1-S transition
cyclin CDK2cyclin A. in the order in which they are listed above.)
s

The master switch at R; also acts as a tumor suppressor. Rb stands for “retinoblastoma”
- major role is to regulate the G1S transition
- encoded by a tumor supresspr gene
- Hyper-phosphorylated Rb protein E2F (transcription factor is released and cell cycle starts again
Rb - it was first discovered in a mutated, inactive form in children with retinoblastoma tumors. (found in the retina)

A family of transcription factors that are released by Rb to turn on the last genes needed for the start of
DNA replication.
- required for expression of various enzymes involved in synthesis of dNTPs and DNA
E2F

A transcription factor that blocks progression through the cell cycle and initiates DNA repair if DNA replication
is not proceeding faithfully. It can also initiate cell death (apoptosis) if DNA damage is too severe to be
repaired. p53 is a tumor suppressor.
"guardian" of genomic integrity - tries to repair the DNA during the cell cycle and if it does not repair induces
apapoptosis
- activates DNA repair proteins
p53 - induces growth arrest (holds cell cycle at G1/S phase to allow for repair of DNA)
- initiates apoptosis if the DNA damage proves to be irreparable
 controls the "kill switch" of cell cycle progression

In a normal cell, p53 level is low due to the continuous turnover by its regulator Mdm2. During
cellular stress such as DNA damage, the DNA repair checkpoint signal will lead to the
dissociation of the p53 and mdm2 complex. Once activated, p53 will induce a cell cycle arrest
Defects Leading to Tumor Formation  Regardless of whether the defects
Two types of Defects that lead to cancer: are genetic or epigenetic in nature, a
 Gain-of-function alterations affecting oncogenes common net consequence is
o Gain-of-function = promoting growth factors dysregulation of gene expression in
 Loss-of function alterations affecting tumor suppressor genes.
Genes associated with cancer:
 Oncogenes and tumor suppressor genes that are recurrently mutated in cancer cells of various types include:
o RAS, PIK3CA, EGFR, RAF, β-catenin, and MYC oncogene proteins, the p53, p16Ink4a, ARF, RB1, PTEN,
APC, and NF1 tumor suppressor proteins.
Genetic Factors
1) Proto-oncogenes Normal version of the gene; oncogene is a mutated or overexpressed form of the gene; These are the genes that
promote cancer. Code for proteins that promote cell growth and division.
 First discovered in their oncogenic
 a mutation mimicking a growth signal
form in retroviruses causing tumors
 activation of one cellular proliferation oncogene (K-ras)
in animals
 Gene translocations (ex. Lymphomas: c-myc translocation to the
immunoglobulin H-chain locus) Activated by:
 requires a gain-of-function mutation  EGF/PDGF signal transduction
o mutations are genetically dominant pathway contains several classical
 Proto-oncogenes are often involved in signal transduction and execution proto-oncogenes – this pathway is
of mitogenic signals, usually through their protein products activated in many tumors and is a
 Examples of proto-oncogenes include RAS, WNT, MYC, ERK, and target for intervention (Growth
TRK factors may act oncogenic if their
Oncogenes: myc; ras; BCABL; ERK; WNT, etc. expression is deregulated; the
 Immunoglobulin heavy chain has a constant portion and a variable malignant cell expresses both
portion. The variable portion binds to a pathogen/tumor growth factor and receptor: an
autocrine feedback loop)
2) Tumor suppressor genes  Genes that protect against cancer. Code for
proteins that stop or regulate cell growth and division Chromosomal abnormalities
 Chromosomal abnormalities: involve inactivation or loss of tumor-suppressor genes
 requires a loss-of-function mutation;
o mutations are genetically recessive Platelet derived growth
 Examples of tumor suppressor genes include RB, p53, BRCA1, BRCA2, APC and DCC factor
that codes for a protein that functions as a "master brake" in the cell cycle.  When you have a
cut the platelets
Tumor Suppressor Gene: activate and come
APC (adenomatosis polyposis
coli)
close together. This
APC protein helps secretes the
control how often a cell pathway to make
divides, how it attaches the cells proliferate
to other cells within a and heal/seal the
wound
Epidermal growth
3) Cell cycle  multiple genetic changes can affect cell cycle factor
 Has 2 chains
DNA Repair Checkpoint
1. 2. 3.
DNA damage activates ATM/ATR, p53 up-regulates p21 transcription DNA repairs enzymes such as BRCA1 & 2
Chk1/Chk2 which stabilizes phosphorylate which inhibits CDK2 and blocks further repair the damage.
and activate p53 phosphorylation of Rb, stop the cycle
until DNA repair is completed.

Don’t need to know specific tumor suppressors ( ex-p27 ) Multistep in cancer

 Genetic Factors
Conversion of proto-oncogenes to oncogenes:
 requires a gain-of-function mutation; mutations are genetically dominant Tumors- not a single
 Proto-oncogenes are often involved in signal transduction and execution of event (it accumulates
mitogenic signals, usually through their protein products over time)
o Examples of proto-oncogenes include: RAS, WNT, MYC, BCR/ABL,ERK,  Tumors
andmakeTRKtheir
 amplification of c-erbB2 in breast cancer
 point mutation of c-ras in kidney and bladder cancers
 conversion of
 chromosome translocation of c-myc in Burkitt’s lymphomaProto-oncogenes to
 altered tumor- Uncontrolled cell growth/
suppressor genes Cancer
oncogenes
Altered tumor-suppressor genes:
 requires a loss-of-function mutation; mutations are genetically recessive
o examples of tumor suppressor genes include: p53, BRCA1, BRCA2, RB that codes for a protein that functions as
a "master brake" in the cell cycle.
 p53 mutation in prostate cancer: failure in cell cycle arrest or apoptosis of prostate tumors
 Rb mutation: fail to prevent mitosis
Environmental Factors
UV-induced Cancers:
 Damage or mutation of DNA
o Melanoma  metastatic, highly immunogenic, spontaneous rejection
 Non-melanoma cancers:
o Basal cell carcinoma: rarely spreads (localized)
o Squamous cell carcinoma: can spread (can metastasize)
Chemically-induced cancers:
 Free radicals and other oxidants steal electron from DNA and cause cancer; anti-oxidants (vitamins A, C) protect
against damage
Viruses:
 DNA viruses: papova (papilloma, SV40), hepatitis, EBV  DNA viruses are more
 RNA viruses: retroviruses---> Human T-lymphotropic viruses (HTLV-I and HTLV-II)likely
cause to
T cause tumors
cell leukemia
 Highly immunogenic because of viral antigens  Within RNA viruses
Epigenetic Factors only the retrovirus can
cause tumors
Effect of growth factors on cell proliferation
You need mutations in both sets of
No growth factors= no proliferation
Growth factors/serum= proliferation chromosomes for it to result in
retinoblastoma.

(tumor suppressor= genetically recessive)

3 ways Cancer cells are independent from growth factor requirement
Does not need growth
Epidermal growth
factors
factors

1. Autocrine stimulation 2. Upregulated receptors 3. Truncated receptors

- “Stimulates itself” - Many growth factor
- External growth Receptors are close
factors together and trans-
phosphorylate each other 
send signals to cells to
proliferate
- No external growth factor
necessary
- Tumor cell thinks there is a
growth factor available and
starts to proliferate
- fire constitutively signals
- No external growth factor

Cell Growth
 What are growth factors? Ex- PDGF dimer;
activates fibroblasts in the tissue and
proliferate.
Serum obtained from blood {anti-coagulate
blood=not clot} there is a clump of red cells at
the bottom and the clear liquid on top is the

Multistage process of Carcinogenesis

Initiation Promotion Progression

damage of DNA clonal expansion of altered expression of enzymes

mutation induction in initiated stem cells proteolysis ( breakdown of bonds)
critical target genes development of benign adhesion & deadhesion
activation of proto- tumor invasion
oncogenes migration
inactivation of tumor metastasis
supressor genes
cell replication and
fixation of mutation Stem cell leads to tumor cells
because stem cells can lead to
many different types of cells

mutation
Mutation Mutation of
Mutation inactivates
inactivates proto-
Cells inactivates several more
tumor oncogene Cancer
proliferate DNA repair tumor
suppressor creates an
gene supressor
gene onco-gene
genes
Why does it take a very long time for tumor development?
Normal Hyperplastic
Dysplasia
epithelium epithelium *** each of these steps
takes a long time to occur
Adenomatous Invasion &
Carcinoma
polyp metastasis

Human Breast Hyperplasia

Hyperplasia: Any increase in cell number without cytologic changes in cellular morphology
Rules for Naming Tumors
 Site of origin:
o Carcinoma – from epithelial
cells
o Sarcoma – supporting tissues,
e.g. bone cartilege, blood
vessels, fat, fibrous tissue and
muscle
o Lymphomas and leukemias –
from cells of lymphatic and
blood origin
 Prefix to identify cell type, e.g. cancer of
a gland cell is called adenocarcinoma
 Suffix, -oma without the carcino is used
 Multistage process of Tumorigenesis

Precursor Basics
 Metaplasia: Replacement of one cell type with a better suited cell type in response to an injurious stimulus
o May lead to dysplasia if the stimulus is not removed
 Dysplasia (“Disordered growth”): Predominantly used when describing premalignant epithelial cell changes
o Cells exhibit progressive changes similar to those seen in malignancy (mild, moderate, or severe)
o Some of the same mutations in cancer can be seen in low grade dysplasia (the continuum)
o Loss of maturation, increased number of mitoses (can divide mor/ more proliferation), pleomorphism, increased
N/C ratio
o Low to moderate grade dysplasia may reverse if stimulus is removed
 Carcinoma in situ: Severe, full-thickness dysplasia
o Cancer is intraepithelial (has not breached the basement membrane); often seen near invasive cancer
o Very likely to progress to invasive cancer, but that may take years to accumulate the next round of mutations
required for frank malignancy
o Interesting exceptions for nature of risk include lobular breast carcinoma in situ: If found in one breast, the risk
applies to both breasts
Malignant vs. Benign Tumors
Benign tumors ( not cancer)tumor cells grow only locally and cannot spread by invasion or metastasis
Malignant ( cancer)cells invade neighboring, enter blood vessels, and metastasize to different sites
 Benign Tumors:
Characteristics Benign Malignant Remain localized, with
Differentiation/ Well differentiated; structure Some lack of differentiation exceptions including:
anaplasia sometimes typical of tissue of (anaplasia); structure often o Intravenous
origin atypical leiomyomatosis
[anaplasia= lack of
differentiation]  Malignant Tumors:
Can to invade adjacent
Rate of growth Usually progressive and slow; Erratic, may be slow to tissues and spread to
may come to a standstill or rapid; mitotic figures may be other sites
regress; mitotic figures rare numerous and abnormal  Differentiation: Degree
and normal of structural and
functional resemblance
Local invasion Usually cohesive, expansile, Locally invasive, infiltrating to normal cell
well-demarcated masses that surrounding tissue;  Anaplasia: Absence of
do not invade or infiltrate sometimes may be differentiation
surrounding normal tissues misleadingly cohesive and o Marked
expansile pleomorphism and
Metastasis Absent Frequent; more likely with
large undifferentiated
primary tumors

Benign tumors can still be bad

 if you have a lot of them they
can disrupt function

Malignant Characteristics Local Spread & Seeding:

 Loss of differentiation (e.g. no mucin, keratin)  Cavity seeding occur when cancer breaks
o Contrast anaplasia: Lack of differentiation through a barrier into an open space
  Ovarian cancer  peritoneal carcinomatosis,
Pleomorphism: Variation in size and shape of the cell or the nucleus
 Increased nucleus to cytoplasm (N/C) ratio omental caking (ddx extraovarian primary
 Higher mitotic rate compatible with malignancy peritoneal carcinomatosis)
  Pseudomyxoma peritonei due to rupture of
Atypical mitoses (tripolar, ring etc) – separation of genes is uneven
appendiceal or ovarian mucinous carcinomas
 Nuclear changes (epigenetics!)
o Hyperchromasia
o Clumped and/or cleared chromatin
o Nucleoli
 Loss of polarity and/or discohesion, invasion
o Ex) Lymphovascular invasion

Acquired Capabilities of Cancer

Acquired Capability Example of Mechanismto induce
cancer
Self-sufficiency in growth signals Activate H-Ras oncogene
Insensitivity to anti-growth signals Lose retinoblastoma suppressor
Evading apoptosis Produce IGF survival factors
Limitless replicative potential Turn off telomerase
Sustained angiogenesis Produce VEGF inducer
Tissue invasion & metastasis Inactivate E-cadherin
Stages in Cancer
Stage 1: Stage 2: Stage 3: Stage 4:
Cancer in situ Local growth within Invasion of other Metastasis, to secondary
original organ adjacent organs sites

Primary focus, confined Invasion through Invasion through Vascular/lymphatic

to original site, displace basement organ ‘capsule’ to spread to lung, liver,
normal tissue during membranes in organ. adjacent tissues brain.
slow growth, no
invasion.

“Hallmarks of Cancer” are 8 physiologic changes related to the molecular alterations present in all cancers:
 Autonomous proliferation via oncogenes
 Ability to ignore signals that inhibit growth
 Utilize aerobic glycolysis to synthesize cell material needed for rapid growth (Warburg effect)
 Evade programmed cell death
 Stem cell-like replicative potential
 Induction of angiogenesis to support growth
 Ability to invade locally and travel far (metastasize)
o Loss of contact inhibition
 Ability to hide from the immune system that seeks to eliminate cells with abnormal antigens (oncoproteins)
How does Cancer Spread?
 Barriers to dissemination include cell-cell contact & capsules (slow-growing tumors)
 Cell-cell contact: Intercellular junctions, intercellular adhesion molecules (like E-cadherin), and ECM proteins
 Capsule: Rim of fibrous tissue that forms in response to pressure-induced hypoxic injury to stromal cells
 Cancer is often unencapsulated (or pseudoencapsulated)
 Margins may be microscopically infiltrative even when they appear circumscribed
 Invasion (locally and across tissue boundaries including body cavities) is facilitated by mechanisms including
 Mutations leading to loss of E-cadherin function
 Overexpression of proteases like matrix metalloproteinases by tumor cell
 Stimulation by matrix metalloproteinases  release of VEGF, angiogenesis
 Signaling that promotes survival of free tumor cells over apoptosis
 Solid tumor metastasis begins with invasion of blood and lymphatic vessels
 Metastases ? more likely in poorly differentiated solid neoplasms
 Liquid tumors (leukemias, lymphomas) travel through the blood vessels regularly because they are hematopoietic, so
by definition, are disseminated at diagnosis
Site of circulating tumor cell deposition is based on:
 Pathway of drainage (e.g. closest nodes and organs)
 Tropism for tissue types (unclear...? adhesion molecules, chemokines)
Three pathways of spread:
 Lymphatic spread
 Hematogenous spread
 Direct seeding of body cavities and surfaces

Metastasis
 Hematogenous spread: In blood vessels
o Cancer cells may deposit in the first capillary bed in the drainage (liver, lung, paravertebral plexus)
o Venous propagation  heart: RCC, HCC
 Lymphatic spread: In lymph vessels
o Anatomic drainage routes
o Sentinel lymph node: The first node to receive lymph from the primary tumor
o Skip metastases: Bypassing local nodes (sampling error vs collateral pathways)
 Multiple cancer lesions usually metastasize
o Exceptions include synchronous and/or metachronous primary cancers
Normal cell Cancer cell Implications of cancer immunity

Stable genome Multiple genetic alterations Multiple neoantigens arising

Stable transcriptome Major epigenetic instability Altered levels of antigen density

Potential induction of inflammation

No tissue invasion Invasion and metastasis: a hallmark activating innate and adaptive immunity

Stable pattern of cytokine and Abnormal expression of growth Potential local inhibitory effects on innate
growth factor expression factors such as VEGF and TGF-b and adaptive immunity
Concepts to review:
 Carcinogenesis: The stepwise process that turns normal cell types to cancer
 The accumulation of mutations that allow the cell to achieve the eight hallmarks of malignancy
 Stepwise Accumulation of Complementary Mutations
 Initiating mutation: The first of many mutations; initiated cells can persist for years and self-renew and proliferate in a
stem cell-like manner
 Driver mutations: Allow the transforming alterations that define cancer cells (cancer hallmarks)
 Loss of function mutations in genes that help the cell maintain genomic integrity  accumulation of many driver and
passenger (phenotypically inconsequential but antigenic) mutations over time
 Neoplastic transformation (genetic alterations) – expression of cell surface antigens, i.e. nonself antigens seen by the
immune system
 Tumors arise when there is a loss of proliferative control or apoptosis
 Benign tumors are localized but metastatic tumors invade and spread to distant sites
 Genetic, environmental, and viral factors as well as immune dysfunctions contribute to tumor formation
 Induction of cancer is a multistep process that involves oncogenes and tumor suppressor genes alterations
Immune Response
Immune response can be affected by microbes such as: Bacteria, Viruses, Parasites, Fungi and Cancer( not a microbe)
Innate Immunity Vs. Adaptive Immunity First line of
Innate immune response- first response to a pathogen defense
Cytoki Adaptive immune response
 Induced early – 2nd line of defense.
nes  Responses start several days later- Occurs 0-4 hrs
 4 hrs- 4 days
 After 4 days until pathogen cleared
 Cellular components
Cellular components:
Phagocytosis= eats any
Cellular components:
 Macrophages
 Neutrophils/Granulocytes (PMN) Adaptive immunity
 Dendritic cells Each lymphocyte has unique
receptors that recognize specific
 Natural killer cells
molecule on pathogen
 Mast cells/basophils Kill viral infected cells and
 Eosinophils tumor
Allergic cells
responses Innate immunity  macrophages
Soluble Mediators: Parasite defense Molecules on pathogens:
 Complement proteins o PAMPs
 Defensins & pentraxins How do they recognize pathogens?
- Via: PRRs & TLRs
 Cytokines/chemokines Pathogen recognition receptorsthese bind to PAMPS
 Interferons: anti-viral
Complement: (soluble mediator)
What is a complement system?
 Complement (5-10% of plasma proteins) present in plasma as zymogens and on cell surfaces; C proteins interact
with each other to produce biologically active inflammatory mediators that promote cell and tissue injury
o Why do complement need to be in an inactive form? So it doesn’t attack your own cells when there is no pathogen
present
Zymogen an inactive precursor of an enzyme
Who makes them?
 Produced constantly without stimulus “constitutively” by the liver and at sites of inflammation by macrophages &
dendritic cells
What effect?
 Complement products mark the pathogen for destruction. Final outcome of C activation: killing of pathogen
(directly or by phagocytosis) and inducing inflammatory response
Components of acute phase response
 i.e. concentrations increase during infections, injuries and trauma – recent studies have shown increased levels of
complement degradation products in plasma of cancer patients
Complement= heat labile
What is the role of complement in Immune Response
Removal of immune
Inflamation Opsonization Lysis by MAC
complexes
Recruitment of inflamatory and Opsonization of pathogens Killing of pathogens
immunocpmetent cells "getting ready to eat" MAC = membrane attack complex
when you recruit these cells they When bacteria is "opsonized" aka Terminal pathway (shared by
can destroy the cell in an infection macrophages can "eat" better. all 3 pathways)
or destroy tumor cells
e.g. Macrophages, neutrophils,
and other cells

3 pathways of complement activation pathways!all converge @ C3

 Alternate
o Bacteria, bacterial toxins
o Always active even without the presence of bacteria but with bacteria present can continue the process all the way
o The water in the plasma cleaves the C3
 Lectin
o Manose residues (MBL)- bind to mannose associated serine proteases; can cleave C4+C2 to activate C3
complement activation
o MBL: circulates in plasma as a complex with 2 serine proteases
 MBL-associated serine protease (MASP) 1 & 2
 binding of MBL complex to mannose residues on pathogen induces MASP-2 to be enzymatically active &
MASP-2 is cleaved
 enzymatically active MASP-2 cleaves C4 & C2 forming C3 convertase: C4b2a
 Classical When C3 is activated, it is cleaved resulting in C3b:
o IgM + IgG immune complexes Anytime you have antibodies 1. Complement fixation: C3b is bound to
 (think classic carsGM?) it uses the classical pathway! pathogen covalently
o Serine proteases  At C3 cite
2. Effectors of inflammation: leukocyte
o Regulation: C1-INH – regulates classical pathway. Deficiency of C1 inhibitor is
recruitment
 Cleaved products of C3 + C5 C3a +C5a
 associated with hereditary angioneurotic edema.
When C1 is activated what is the C3 convertase?
For both Classical + Lectin Pathway: C4bC2a
For Alternative pathway: C3bBb
Macrophages
 Fc gamma receptor What is recognized?
 CR1stimulates phagocytosis Initial complement components recognize
( requires C5a) features of microbial surface and mark them for
o Erythrocyte transport of immune destruction by coating them with C3b
complexes
 CR2 part of B-cell co-receptor
Complement control
Pathogen Surface Soluble (plasma) proteins:
• Properdin (Factor P) – stabilizes C3 convertase (C3bBb) & prevents degradation by proteases
• Factor H - binds to C3b and aids in further cleavage to iC3b by Factor I;
- decrease the number of C3 convertase molecules on pathogen surface; essential for controlling alternative
pathway
Host Surface (Membrane proteins):
• Decay-accelerating factor (DAF) binds to C3b of alternative convertase & inactivates alternative C3 convertase
• Membrane cofactor protein (MCP) – binds to alternative C3 convertase & Factor I then cleaves to inactivate it

Anaphylatoxins: C3a, C5a, C4a (degradation products) Important know 2!

 Promote degranulation of mast cells
 Chemotaxis of PMNs
 Act on endothelial cells of blood vessels increasing permeability
Regulation of Complement: Terminal complement (C5-C9) control proteins
 Soluble proteins:
o S protein Prevent the soluble
o Clusterin complex of C5 with C6 +
o Factor J C7 from associating with
 At the cell surface:
Prevent
o HRF (homologous restriction factor)
o CD59 (protectin) recruitment of
C8 or C9
3 major types of effectors are generated by activation of C:
1. Anaphylatoxins
2. Opsonins
3. MAC

1. 2. 3. 4.
How does complement work in terms of Cancer:
 C can act to kill antibody-coated tumor cells
 C can support chronic inflammation (bad for host; good for tumor)
 Conversely, C can hamper anti-tumor immune response and thus enable tumor progression
 In presence of malignancy, the balance between concentrations and proportions of complement components in body
fluids is lost – tumor cells produce C3
 C system is pathologically activated in the tumor microenvironment (this can promote tumor growth because of
dysregulated inflammatory response, effect on cell proliferation, EMT, migration and invasiveness of tumor cells
 Various complement components were shown to increase tumor growth (e.g. C3a), angiogenesis (C1q, C3a, C5a)
 C proteins activate and recruit macrophages to tumor tissue (tumor cell derived C3a promotes the accumulation &
immunosuppressive activity)
 C can enhance tumor cell proliferation (C3 & C5)
 Mediates epithelial to mesenchymal transition in cancer cells
 Imbalanced C activation was shown to trigger metastatic pathways by enhancing motility of cancer cells and
disrupting tissue barriers
 Enhance immunosuppression by upregulating molecules such as PDL-1, Arg-1, IL-10, and TGF-b1
Complement activation in the TME (tumor microenvironment)

Important!
Complement damage is generally limited to the
immediate area in which complement is activated
because of the:
 Short half-lives of the activated complement
components and their rapid inactivation.

Important!
B cell receptors can not pick up signals on it’s own
because it needs alpha and beta immunoglobulins

Cellular Response
Life-styles:
 Extracellular – Bacteria (e.g. staphylococcus, Bacterial toxin from Clostridium t.)
 Intracellular:
o Cytosolic Viruses (Influenza; HIV; Smallpox)
o Vesicular Bacteria (M. tuberculosis)

Adaptive immune response: B-cell vs. T-cell

B-cell:
 B  bone marrow
 Humoral secretion of antibody molecules
 Extracellular  bacteria, toxin molecules
 When B-cells get activated they become plasma
cells and makes antibodies (immunoglobulin)
specific for molecule/pathogen
 Produce immunoglobulins (neutralize pathogens &
toxins)
 Previously thought to inhibit T cell response vs.
cancer; new studies indicate anti-tumor B cell
responses
  2 types of receptors:
o αβ chains  95% of human T lymphocytes;
T-cell: recognize peptide antigens presented by self
 T  Thymus MHC molecules
 Cell mediated o γδ chains  5%of human T lymphocytes;
 Intracellular  Viruses frequent in some epithelial tissues; do not
 Forms viral peptides which can be shown in the recognize MHC-associated peptide antigens
MHC T-cell sees this on the MHC and then goes  Recognizes foreign peptide in groove of MHC
through cytolysis (cytotoxic T-cell)  Never secreted

Hypervariable regions or complementarity CDRs (3 on a & 3 on b)

determining regions (CDRs): 3 sites on each of CDRs (3 on H & 3 on L)
polypeptide chains (BCR or TCR) that actually
make contact with the antigen

B-cells are secreted but T-cells are not

because B-cells lose the transmembrane
region becoming an antibody.
B-cells can bind to carbohydrates, because
they see extracellular envi. They are
hydrophilic while T-cells are hydrophobic
B-cell receptor B-Cell Effector
Variable region what meets the antigen
( antigen binding site) Secreted! NOT secreted!!!

αβ T-cells  MHC
Adaptive immunity Features:
 Recognize antigens in the groove of either MCH I or MHC II
 It takes time (lag phase)
o T cells that recognize peptides in the groove of MHC I are CD8+ T cells
 Exquisitely specific for
 All cells in body (except red blood cells) are represented by MHC1
immunizing antigen
 Intracellular antigen
 Memory
 Binds to α 3 domain
o T cells that recognized peptides in the groove of MHC II are CD4+ T cells  Lack of immunity to self antigens
 Extracellular antigen
 Binds to β 2 domain
 MHC = major histocompatibility
Epitopes seen by BCR + TCR  Epitope=
antigen
BCR  Antibody epitopes (lysozyme)
 On the surface or outside of the protein
 Hydrophilic regions of the protein, i.e. are accessible to the solvent water
Important!!
TCR epitopes (lysozyme)  What is clonal selection theory?
 Peptide fragments derived from pathogens displayed on MHC o Each clone will respond to a specific
 Embedded within the folds of the protein, not accessible to the solvent water antigen and then expand/proliferate.
The clones have memory cells which
Effector functions: TH cells  help macrophages + B cells lead to recognition of the antigen if it
CD4 cells: Help MØs, T cells, B By secreting cytotoxins and inducing cell- was preciously used
cell contact
cells
Cytotoxic T cells  major function is to kill
CD8 T cells: kill infected cells  How does is contribute to 10 & 2 0 immune
response?
“Cytotoxic” “Helper” “Regulatory” o In primary response, the clone is picked
by the antigen and then expanded. This
+ is still a pretty slow response (1 week)
CD4 T Cell o In secondary response, the clones are
Reg
already expanded so each clone will
expand/proliferate even more leading to
a faster response (shorter lag phase)
Self
Antigen  Why doesn’t it respond against your own
cells?
o Some of the self-reacting cells/antigens
are deleted
 Adaptive response
How is it possible for B and T cells to recognize so many different types of antigens?  Because of somatic gene rearrangement; use RAG enzymes
1 gene  1 polypeptide for immunoglobulin or TCR
T-cell Steps:
1. Coming out of the bone marrow the T-cells are called “Naïve” 1. B/T cells “see” antigen in LN (lymph
2. To get activated it needs to see the antigen (signal 1) and co-stimulation (signal 2) if not they continue to
node);
3. Once activated goes through proliferation and differentiation recirculate
4. To do it’s function; becomes Effector/memory cells 2. B cells that see antigen undergo
Where do immune reactions occur? activation by Ag plus T-help
Lymphocyte Trafficking 3. Activated B cells go to follicles and
1. Transport of antigen (from local site to form germinal centers; activated T cells
2. Recognition of antigen by lymphocyte (B-cell or T-cell) occurs in lymphoid tissues
go to the site of infection
3. Effector lymphocytes move from lymphoid tissues into lymphatics and to 4. infected tissues differentiated plasma cells
Terminally
4. Memory lymphocytes continue to circulate, “monitoring” antigens in lymphoid tissues
go to the medullary cords and leave the

Cellular Adhesion Molecules ( CAMs)

 Guide T cells into lymphoid tissue such as LN, PP (are not antigen specific; does not matter
if there is an antigen present)
 Responsible for interactions of naïve lymphocytes with APC
 Responsible for effector cell interactions with target cells
4 major groups of CAMs:
 Selectins: important molecules for lymphocyte trafficking
o CD62L: expressed on all naïve T cells
 binds to carbohydrates (CHO) on vascular addressins (mucin-like).
 Vascular addressins: molecules expressed on endothelial cells in blood vessels
o CD34: on HEV
o GLYCAM 1: on HEV
o MAdCAM: on capillary endothelial cells in mucosa
 Integrins: expressed on all T cells, macrophages, neutrophils, and dendritic cells
o LFA-1 (Lymphocyte Function Associated) also known as CD11a (defect in beta chain of LFA-1 results in
leukocyte adhesion defect – difficulty in getting rid of extracellular bacteria)
o CR3 (Mac-1)
 Immunoglobulin Super Gene Family: present on endothelium, dendritic cells, or resting leukocytes
o ICAM-1, -2, -3: ICAM-1, -2 are on ECs & ICAM-3 is on lymphocytes
o VCAM-1
o CD2 (LFA-2) – on T cells and binds to LFA-3
o CD 58 (LFA-3) – on APCs and lymphocytes
T-cell signaling
 Recognition of antigen occurs through TCR (a and b chains); TCR is monovalent, i.e. 1 binding site; ~30,000
TCRs/cell
 CD3 molecules enable signaling (because of ITAM residues); 3 polypeptides (g,e,d)
 Zeta chains
 ITAMs: ImmunoTyrosine Activation Motifs
How are T lymphocytes stimulated when they recognize an antigen?
 Signal 1: TCR + Ag in groove of MHC
o required for a naïve T cell to become activated (TCR binds to
Naïve T cell activation need an
peptide in the groove of MHC molecule). The co-receptor (e.g. CD4)
antigen presenting cells ( APC)
binds to the constant region of MHC Class II molecules  Dendritic cells (the best)
 Signal 2: Co-stimulatory molecule provided by an APC  Macrophages
o required for a naïve T cell to become activated. Signal 2 consists
B cells
Only these cells represent BOTH
of CD28 on T lymphocyte binding to B7 molecule on an APCMHC class 1 and class 2
 Induces transcription factors (ex. AP-1, NF-kB) that increase
transcription of the IL-2 gene and mRNA (~3x)
 Stabilizes mRNA that encodes IL-2 protein (20-30x) Where do signal 1+2 take place?
 Result: Amount of IL-2 secreted is increased ~100X - In lymph nodes; not at site
 Signal 3: Provided by cytokinesSame APC must deliver
of infection
both signal 1 and signal 2

Interleukin (IL-2)
 Produced primarily by T cells
 Required for T cells to proceed through differentiation and proliferation
 Signals T cells through its receptor IL-2R
 Weak on naïve T cells (dimer); strong on mature T cells (trimer)
o Naïve T cells express only the β and γ chains
o The γ chain is a component of a number of interleukin receptor and is often referred to as “the common γ chain”
o Activated T cells express α (CD25), β, and γ chains
 Required to signal T cell differentiation & proliferation Important!
 IL-2 can function in autocrine (self-stimulation) and paracrine (stimulate nearby
TH1tIFN-γ ( manner
cells) interferon gamma) crucial for
TH1! Activates macrophages and aids in CD8.
What determines the type of T effectors generated?
TH2 clonal expansion of B cells {IL-4 & IL-5}

IL-2 Essential for T cell proliferation & in

development of adaptive immune response

How are memory and effector cells different from naive cells?
 Recirculate to peripheral tissue (memory + effector cells) instead of recirculation to lymphoid tissue (naïve cells)
 Can be activated directly at the site of infection by DCs and macrophages presenting Ags
 Activation requirements are not as stringent (e.g. do not require co-stimulation through CD28)
 Memory cells: decreased expression of L-selectin; increased expression of CD44; express CD45RO instead of
CD45RA
“Braking” molecules: CTLA-4 (CD152) & PD-1
 CTLA-4:
o Expressed after T cell is activated
o Interacts with B7
o Affinity of CTLA-4 for B7 is 20x greater than that of CD28 for B7
o Effect of CTLA-4:B7 interaction: opposes that of CD28:B7; signals lead to “braking” instead of activation
 PD-1 (Programmed Death Receptor 1:
o Member of B7 family of costimulatory molecules
o “Braking’ molecule which is expressed on T effector (i.e. after activation)
o Ligands: PDL-1 & PDL-2
Key points need to know!!
 Innate and adaptive immune systems
recognize many different types of
pathogens (intracellular vs. extracellular)
and destroy them.

 The innate immune system composed of

 TH1 Effector cells direct + amplify Cellular Responses
via Cytokine Production:
 GM-CSF + IL3Increase # of monocytes/ DC
 CCl2Recruits macrophages
 TNFα + IFNγ Activate macrophages
 TNFα Activate endothelial cells

Chronic Granulomatous Disease: Mutations in genes encoding NADPH proteins

- Unable to clear pathogens and chronic stimulation by T cells
Cells that kill:
 Macrophages
 Cytotoxic T lymphocytes
 Natural killer cells
What do they target?
 Normal cells infected with pathogens
 Tumor (malignant) cells
 Cells from other (transplant) individuals
Apoptosis vs Necrosis
Apoptosis
 Natural cell death
 DNA cleavage; nuclear condensation and fragmentation
 Plasma membrane leading to phagocytosis
Necrosis
 Cells explodes
 No fragmentation of DNA
 Leakage of cellular contents
Consequences of macrophage activation
 Increased levels of lysosomal enzymes (50X)
 Increased oxygen consumption (“respiratory burst”) resulting in the generation of O2 free radicals, ions,
superoxides and nitric oxide
Activated B-cells go to follicles and form germinal centers; activated T-cells go to the site of infection