Immuno Coverage

Introduction. The role of the immune system. Components of the immune system.

Morphology of lymphoid organs. Basic definitions. The structure of antibodies,

generation of diversity.

Topics

1. Introduction, basic definitions (innate/natural/native immunity, adaptive/specific/acquired immune response, cell-mediated, DTH - Th, humoral immune response, cytokines, receptors, antibodies, antigens, epitopes)

2. Components the immune system (primary/central/generative lymphoid organs, secondary/peripheral lymphoid organs, lymphatic vessels, cells)

3. Sign and symptoms related with activation of the immune system

4. Phases of the immune response: recognition, activation, effector, decline, memory phase, clonal selection hypothesis, two-signal hypotesis, clonal expansion

5. Features of the immune response: specificity, diversity, memory Primary/secondary immune response (naive/mature lymphocytes)

Topics

6. Generation, components and localization of the humoral immune

response (against complex antigens, heterogenous response, effector

functions of antibodies)

7. Antigens (proteins, glycoproteins, lipids, glycolipids), immunogenicity,

antigenicity (hapten, carrier, hapten-carrier conjugate), antigenic

determinant (epitope), adjuvant

8. Structure of an antibody (heterodimers, four glycopeptide chains, amino-

terminal variable regions/segments, CDR/hypervariable regions/antigen-

binding site, FR, constant regions, proline-rich hinge region, homologous

domains/units forming immunoglobulin folds, immunoglobulin-like

domain, quaternary organization of the immuoglobulin protein)

9. Definitions: valence, affinity, avidity, specificity, cross-reactivity,

monoclonal, polyclonal

Topics

12. Classes of immunoglobulins and biological activities (two light-chain

subtypes, λ 4 subtypes, five heavy-chains isotypes/classes,

subisotypes/subclasses, Immunoglobulin superfamily of receptors

14. Secreted immunoglobulins (sIg), membrane-bound immunoglobulins (mIg), carboxyl-terminal domain (extracellular hydrophilic “spacer” sequence, hydrophobic transmembrane sequence, cytoplasmic tail)

16. Immunoglobulin genes

17. Generation of diversity (combinatorial V-D-J joining , junctional diversity, hypermutations)

18. Clinical application of antibody-based therapeutics (IVIg)

Overview of the immune respone

BLOOD CELLS Pluripotent haemopoietic stem cell

Red blood cells 5x1012/l (5x106/mm3) 120 days

Platelets 200x109/l (2x105/mm3) 8-12 days

White blood cells 5x109/l (2x103/mm3) <hours – years

Neutrophils 50-70%

Lymphocytes 20-40%

Monocytes 1-6%

Eosinophils 1-3%

Basophils <1%

BLOOD PLASMAClotting factorsProteins of innate and adaptive immunity

Blood cells

Human red blood cells (red), activated platelets (purple) white blood cells - monocyte (green) T lymphocyte (orange).

Colorized-SEM (scanning electron micrograph)

Magnification:-1200x--(Based on an image size of 1 inch in the narrow dimension)

©Dennis Kunkel Microscopy, Inc., www.DennisKunkel.com

http://www.denniskunkel.com/

WHITE BLOOD CELLS

Basophil mast cellEosinophil phagocyteNeutrophil phagocyte

Monocyte macrophage phagocyteLangerhans / dendritic cell family

Natural killer cell

B lymphocyteT lymphocyte

Neutrophils - phagocytes that form firstline of defence against bacteria thatproliferate in the extracellular fluidsDeficiency in numbers or quality of neutrophils:-

Skin infectionsAbscess formation (staphylococci)

Invasive bacterial infections (rapidly dividing extracellularly replicating)SepticaemiaInvasive fungal infection

Death within 1-2 weeks if no neutrophils

The clonal-selection theory

• Polyclonal and Monoclonal Antibodies• An individual lymphocyte expresses membrane receptors that are

specific for a distinct antigen. • This unique receptor specificity is determined before the lymphocyte

is exposed to the antigen.• Binding of antigen to its specific receptor activates the cell, causing

it to proliferate into a clone of cells that have the same immunologic specificity as the parent cell.

Antigen-antibody interaction

• Globular protein antigens and small peptide antigens interact with antibody in different ways

• The B-cell epitopes on native proteins generally are composed of hydrophilic amino acids on the protein surface that are topographically accessible to membrane-bound or free antibody.

• B-cell epitopes can contain sequential or nonsequential amino acids.

• B-cell epitopes tend to be located in flexible regions of an immunogen and display site mobility.

• Complex proteins contain multiple overlapping B-cell epitopes, some of which are immunodominant.

• The ability to function as a B-cell epitope is determined by the nature of the antigen-binding site on the antibody molecules displayed by B cells.

Definitions

• Ab = glycoprotein molecules which combine specifically with antigens

• Valence• Antigenic determinant (epitope)• Affinity• Avidity• Specificity• Cross-reactivity• Combining site• Monoclonal/polyclonal• Isotypic, allotypic, idiotypic determinants

The immunoglobulin superfamily

• Ig-α/Ig-β heterodimer of the B-cell receptor• Poly-Ig receptor, which contributes the secretory component to secretory

IgA and IgM• T-cell receptor• T-cell accessory proteins, including CD2, CD4, CD8,• CD28, and the γ, δ and ε chains of CD3• Class I and class II MHC molecules• β2-microglobulin, an invariant protein associated with class I MHC

molecules• Various cell-adhesion molecules, including VCAM-1, ICAM-1, ICAM-2, and

LFA-3• Platelet-derived growth factor

Organization and expression of immunoglobulin genes

• Multigene Organization of Ig Genes• Variable-Region Gene Rearrangements• Mechanism of Variable-Region DNA Rearrangements• Generation of Antibody Diversity• Class Switching among Constant-Region Genes• Expression of Ig Genes• Synthesis, Assembly, and Secretion of Immunoglobulins• Regulation of Ig-Gene Transcription• Antibody Genes and Antibody Engineering

Origin of Antibody Diversity

• Multiple germ-line gene segments and combinatorial V-(D)-J joining

• Junctional flexibility

• Junctional diversity (N-region insertions - TdT, addition of P-region

nucleotides – repair enzymes)

• Somatic hypermutations

• Combinatorial association of light and heavy chains

• Multispecificity

Characteristics of the immune system

• Diversity – ability to recognize and respond to many different microbes

• Specificity – responses to distinct microbes are optimized for defense against these microbes

• Memory – enhanced response to recurrent or persistent infections

• Nonreactivity to self antigens – prevents injurious immune respones against host cells and tissues

Current polyvalent antibody based therapeutics

• Antivenins

• Antitoxins

• Antichemical

• Anti-infectives

• Broad spectrum immunoglobulins

Antivenins

• used as antidotes for local snake (Rattlesnake, copperheads, cottonmouth moccasins, Fer-de-lance, Cantil and Bushmaster) and spider bites (Latrodectus mactans - Black-widow spider)

• generally polyvalent and are usually isolated from horse(Horses are immunized with venoms in aluminum hydroxide gels. Plasma from immunized animals is pooled, precipitated and dialyzed. Major form IgG.)

• Immediate administration within the first 4 hours, rate of infusion must take into account the age, weight and cardiac status of the patient; may need to administer more for bites in fingers and toes; IV

• Adverse Reactions - Shock and anaphylaxis may occur within the first 30 mins. Also serum sickness may occur in association with nausea, fever, edema, collapse and pain in limbs.

Antitoxins

• Diphtheria Antitoxin, Botulinum antitoxin

• used to decrease the toxic effects of bacterial infections – For treatment of botulism, neutralization of circulating toxins produced

by the organism; the toxin is one of the most lethal toxins known. Treatment does not prevent the spread of the disease. Antibiotics are effective against the infection.

– For transient protection from the toxic effects of Diphtheria infections.

• Best result given if a very large dose of the antitoxin is given early in the disease, IV and IM

• generally polyvalent and originate from horse

Antichemical

• used as antidotes for intoxication due to drug overdose or chemical exposure (Digoxin Immune FAB)

• polyclonal antigen binding (FAb) fragments from sheep

• The dose amount varies according to the amount of digoxin or digitoxin to be neutralized. For adults about 240 mg is enough to reverse most cases of toxicity. For children as little as 40 mg can reverse toxicity.; IV

• Based on recent clinical trials, roughly 90% of patients undergoing life-threatening intoxication responded favorably.

Anti-infectives

• Generally polyclonal mixture from humans• Hepatitis B Immune Globulin• Rabies Immune Globulin• Cytomegalovirus Immune Globulin• Varicella-Zoster Immune Globulin

• Plasma is pooled from a small group of well-monitored individuals that were hyperimmunized with the appropriate antigen

• For post-exposure prevention. Given immediately after exposure, IM or for prevention of infection associated with kidney, bone and liver transplants.

• Pain and tenderness at the infection site and mild elevation of temperature. Anaphylaxis is rare

Monoclonal antobodies and their derivatives. Immunological tests and techniques employing monoclonal antibodies. Monoclonal antiodies in

therapy. Structure and function of T cell receptor.

Seminar 2

Topics 1

1. Structure of an antibody (heterodimers, four glycopeptide chains, amino-terminal variable

regions/segments, CDR/hypervariable regions/antigen-binding site, FR, constant regions,

proline-rich hinge region, homologous domains/units forming immunoglobulin folds,

immunoglobulin-like domain, quaternary organization of the immuoglobulin protein)

2. Definitions: valence, affinity, avidity, specificity, cross-reactivity, monoclonal, polyclonal

3. Classes of immunoglobulins and biological activities (two light-chain subtypes, λ 4

subtypes, five heavy-chains isotypes/classes, subisotypes/subclasses, Immunoglobulin

superfamily of receptors

4. Secreted immunoglobulins (sIg), membrane-bound immunoglobulins (mIg), carboxyl-

terminal domain (extracellular hydrophilic “spacer” sequence, hydrophobic

transmembrane sequence, cytoplasmic tail)

5. Effector functions of antibodies

6. Immunoglobulin genes

7. Generation of diversity (combinatorial V-D-J joining , junctional diversity, hypermutations)

8. Clinical application of antibody-based therapeutics (IVIg)

Topics 2

• Production of monoclonal antibodies• Nomenclature• Derivatives of monoclonal antibodies

- immunotoxin

- fused with radionuclides, enzymes

- abzymes (catalytic antibodies)

- fused with liposomes, viruses• Engineered antibodies (monovalent fragments, diabody, minibody, triabody,

tetrabody, monovalent fragments – Fab, Fv, scFv, Fab conjugates – dimers and trimers)

• Application of monoclonal antibodies

- Diagnosis (detecting pregnancy, diagnosing numerous pathogenic microorganisms, measuring the blood levels of various drugs, matching histocompatibility antigens, and detecting tumor antigens, radiolabeled monoclonal antibodies can also be used in vivo for detecting or locating tumor antigens, permitting earlier diagnosis of some primary or metastatic tumors in patients).

- Tests employing monoclonal antibodies (ELISA, WB)

• TCR

The clonal-selection theory• An individual lymphocyte expresses membrane receptors that are specific for a

distinct antigen. • This unique receptor specificity is determined before the lymphocyte is exposed to

the antigen.• Binding of antigen to its specific receptor activates the cell, causing it to proliferate

into a clone of cells that have the same immunologic specificity as the parent cell.

Definitions

• Ab = glycoprotein molecules which combine specifically with antigens

• Valence• Antigenic determinant (epitope)• Affinity• Avidity• Specificity• Cross-reactivity• Combining site• Monoclonal/polyclonal• Isotypic, allotypic, idiotypic determinants

Nomenclature

• „- omab” (mouse mAb) • „- amab” (rat mAb)• „- ximab” (chimeric mAb)• „- zumab” (humanized mAb)• „- umab” (human mAb)

Monoclonal antibodies approved for therapeutic use in the USAProduct name / antibody name Product type Antigen Mechanisms of action Approved indication

Orthoclone OKT3

muromonab-CD3

Mouse IgG2a CD3 Blocking of function of T-cell expressed

CD3; reversal of graft rejection

Prophylaxis of acute

kidney-transplant rejection

Zenapax

daclizumab

Humanized IgG1 CD25 Receptor binding and antagonism Prophylaxis of acute


Simulect

basiliximab

Chimeric

IgG1

CD25 Receptor binding and antagonism Prophylaxis of acute


Herceptin trastuzumab Humanized

IgG1

ERBB2 Sensitization of cells to chemotherapy;

inhibition of angiogenesis and proliferation, induction of ADCC

Metastatic breast cancer

that overexpresses ERBB2

Erbitux

cetuximab

Chimeric IgG1 EGFR Receptor binding and antagonism; inhibition of cell proliferation; induction of

apoptosis; sensitization of cells to chemotherapy and radiotherapy; inhibition of

angiogenesis, invasion and metastasis; induction of ADCC

Metastatic colorectal cancer, and head and

neck cancer

Avastin; bevacizumab Humanized

IgG1

VEGF Ligand binding and receptor antagonism; inhibition of angiogenesis and metastatic

disease progression

Metastatic colorectal cancer

Campath; alemtuzumab Humanized

IgG1

CD52 Induction of ADCC and CDC B-cell chronic lymphocytic

leukaemia

Mylotarg;

Gemtuzumab ozogamicin

Humanized IgG4,

Calicheamicin conjugated

CD33 Induction of double-stranded DNA breaks

and cell death (caused by calicheamicin)

Acute myeloid leukaemia

that expresses CD33

Zevalin

ibritumomab tiuxetan

Mouse IgG1,

90Y-labelledCD20 Induction of cell death by radiation;

induction of apoptosis

Non-Hodgkin’s lymphoma

Bexxar; 131I-tositumomab Mouse IgG2a,

131I-labelled

CD20 Induction of cell death by radiation;

induction of apoptosis, ADCC and CDC

Non-Hodgkin’s lymphoma

Rituxan, MabThera; rituximab Chimeric

IgG1

CD20 Sensitization of cells to chemotherapy;

induction of apoptosis, ADCC and CDC

Non-Hodgkin’s lymphoma and

rheumatoid arthritis

Remicade; infliximab Chimeric

IgG1

TNF Ligand binding and receptor antagonism Crohn’s disease, rheumatoid and psoriatic arthritis,

Ulcerative colitis and ankylosing spondylitis

Humira; adalimumab Human IgG1 TNF Ligand binding and receptor antagonism;

induction of CDC

Rheumatoid and psoriatic arthritis

Raptiva; efalizumab Humanized

IgG1

CD11a Receptor binding and antagonism; inhibition of leukocyte adhesion to

other cells

Plaque psoriasis

Tysabri; natalizumab Humanized

IgG4

α4-Subunit of α4β1-integrin

And α4β7-integrin

Receptor binding and antagonism;

inhibition of leukocyte adhesion to their counter receptors

Multiple sclerosis

Xolair; omalizumab Humanized

IgG1

IgE Ligand binding and receptor antagonism; reduction in release of allergic-response

mediators from mast cells and basophils

Persistent asthma

Synagis; palivizumab Humanized

IgG1

RSV gpF Binding and neutralization of RSV;

inhibition of viral fusion and replication

Prophylaxis against RSV

infection in children at high risk

ReoPro; abciximab Chimeric Fab gpIIb–gpIIIa and

αvβ3-Integrin

Receptor binding and antagonism;

inhibition of platelet aggregation

Prevention of plateletmediated

clots in coronary angioplasty

DigiFab Ovine Fab Digoxin Drug binding and neutralization Digoxin overdose

CroFab Ovine Fab Snake venom Toxin binding and neutralization Rattle snake antidote






Immune response, its induction and development. Lymphocyte activation. Subpopulations of

lymphocytes.

Seminar 4

Topics

1. Subpopulations of lymphocytes – B cells (B1a – CD5(+), B1b, B2, Breg) – T cells (Tαβ – CD4(+) or CD8(+), or DN, Tγδ)

• Th – Th1(cellular, Ma, Tc, IgG1, IgG3), Th2 (humoral, IgA, IgE, IgG4, MC, Eos), Th17, Th0, • Tc • Treg (Tr1, Th3)

– NK, NKT 2. General pattern of B and T cell development - maturation, recombination of genes, selection,

characteristics of mature/naïve lymphocytes:– B cells (BCR, MHC I-II, CD19, CD20, CD21 (CR2), CD22, CD32 (FcRII), CD35 (CR1), CD40,

CD72, CD80, CD86)– T cells (TCR, MHC I, CD2, CD3, CD4, CD5, CD8, CD28 i CD154 (CD40L))

3. Lymphocyte circulation/migration in a healthy individual (cell-adhesion molecules, chemokines)4. Inflammatory response (pathogens, route of invasion, innate mechanisms: mast cells, macrophages,

dendritic cells, PAMP)5. Generation of the immune response in the secondary lymphoid organs

– presentation of antigens (DC, MHC)– recognition of antigen by TCR – I signal, co-stimulation – II signal, T cell

activation/anergy/apoptosis – intracellular signaling, proliferation, differentiation, Th1/Th2 balance, immune deviation, split

tolerance – example tuberculoid leprosy and lepromatous leprosy)6. Migration, inflammation (effector mechanisms, DTH), memory

T cell populations

1. αβ T cells – express CD4 or CD8 markers; comprise 90-95% of circulating T cells; share same pool of Vα and Vβ gene segments.

• Naïve T cells- restng; not encountered antigen; circulate between blood and lymph; life span 5-7 weeks

• Effector T cells- antigen-activated; helper, cytotoxic or DTH activity; dispersed from lymph nodes to antigen-containing sites; little recirculation; life span 2-3 days

• Memory T cells- resting cells derived following antigen encounter; easily activated by second antigen challenge; recirculate between blood and lymph; long-lived (30 years)

2. γδ T cells – discovered in 1986• approximately 1-3% of T cell population in lymphoid organs of the mouse• highly concentrated in epithelial tissues-skin, intestine and pulmonary tract• V gene usage appears different in different tissues• may be involved in “surveillance” of epithelium as first line of defense

against pathogens

Cytokines-functions and clinical applications. Regulation of the immune response.

Effector mechanisms of the immune response. Mechanisms of cytotoxicity.

Seminar 5

Effector mechanisms of humoral and cell-mediated immunity - summary

• The effectors of the humoral branch - secreted antibodies - ADCC, CDC (classical pathway), neutralization, opsonization, immunophagocytosis

• Both antigen-specific and -nonspecific cells contribute to the cell-mediated immune response

• Specific cells include antigen specific CD8+ cytotoxic T lymphocytes (Tc cells or CTLs) and cytokine secreting CD4+ T cells

• Nonspecific cells include NK cells and nonlymphoid cell types such as macrophages, neutrophils, and eosinophils.

• Effector cells - less stringent activation requirements, increased expression of cell adhesion molecules, production of both membrane bound and soluble effector molecules, able to respond to TCR-mediated signals with little, if any co-stimulation

• The target cells to which these effectors are directed include allogeneic cells, malignant cells, virus-infected cells, and chemically conjugated cells.

• Naive and effector T cells express different isoforms of CD45 (CD45RA – naive T cells and CD45RO – effector cells, memory cells have both population – CD45RO, predominating). This membrane molecule mediates TCR signal transduction by catalyzing dephosphorylation of a tyrosine residue on the protein tyrosine kinases Lck and Fyn, activating these kinases and triggering the subsequent steps in T-cell activation.

• Memory and effector T cells are more sensitive to TCR-mediated activation by a peptide-MHC complex, second signal not necessary, co-stimulation not necessary

• Effector cytokine-secreting CD4+ T cells mediate delayed type hypersensitivity reactions

Generation of the immune response

Effector mechanisms of the immune system

Th1/Th2 ballance

Role of TH1/TH2 balance in determining disease outcomes

• Balance of two subset determines response to disease

• Leprosy

– Tuberculoid (TH1, CMI response, patient lives)

– Lepromatous (TH2, humoral response, patient dies)

CTL=Tc

• Lytic capacity – recognition and elimination of altered self-cells (virus-infected, tumor) and grafted

• MHC I restricted• Kills in two ways (perforin/granzyme B, FasL)• Naive • Effector

– CD45RA (binds MHC-peptide complex)– CD2 (binds with LFA-3)– LFA-1 (binds with ICAM)– FasL (binds Fas/CD95)– TNF-β (binds LT-βR, TNFR3)– CD40L=CD154 (binds CD40)

NK cells

• 5-10% of recirculating lymphocyte population• derived from bone marrow• the same common progenitor as T cells• produce IFN-γ• express some membrane markers that are found on monocytes and granulocytes, as

well as some of T cells• NK activity stimulated by IFN-α, IFN-β, IL-12, IL-15, TNF• two main families of HLA-class I-specific inhibitory surface receptors

– killer immunoglobulin-like receptors (KIRs, the immunoglobulin superfamily), which are specific for allelic determinants expressed by HLA-A, -B or -C allotypes,

– CD94–NKG2A – the heterodimer (C-type lectin), specific for the non-classical HLA class I molecule HLA-E (CLIR – c-type lectin inhibitory rec)

• triggering receptors/natural cytotoxicity receptors (NCRs):– NKp46, NKp30 and NKp44, restricted to NK cells and are thus the most reliable

markers for human NK-cell identification – NKG2D, also expressed by most cytolytic T cells and is specific for the stress-

inducible MICA/B or UL16 binding proteins (ULBPs)– Other: 2B4, NTB-A, NKp80, CD59 [14] and CD226

• expression of PRR’s (TLR3, 9), CD2, the subunit of the IL-2 receptor, CD16 (FcγRIII),• Similar killing mechanisms to CTLs (FasL, perforin, granzymes)

The effector functions of antibodies

What are cytokines?

• A group of proteins used for communications between cells

• Play role similar to hormones (messengers of the endocrine system)

– Hormones usually act at a distance– Cytokines act locally

• Differ from growth factors that are produced constitutively, while cytokine production is carefully regulated

Cytokine-mediated effects

• Cell growth, proliferation• Cell differentiation• Cell death• Non-responsiveness to other cytokines/cells• Responsiveness to other cytokines/cells• Secretion of other cytokines• Development of cellular and humoral immune

responses, induction of the inflammatory response, regulation of hematopoiesis, and the healing of wounds

Cytokine actions

• Pleiotropy– Act on more than one cell type (INF-α/β)

• Redundancy– More than one cytokine can do the same thing

(IFN-α/β and IFN-γ)• Synergy

– Two or more cytokines cooperate to produce an effect that is different or greater than the combined effect of the two cytokines when functioning separately (IL-12 and IL-8)

• Antagonism– Two or more cytokines work against each other

(IL-4 and IL-12)

How can non-specific cytokines act specifically?

• Only cells expressing receptors for specific cytokines can be activated by them

• Many cytokines have very short half-lives– Only cells in close proximity will be activated

• High concentrations of cytokines are needed for activation

– Only cells in close proximity will be activated– May require cell-to cell contact

Cytokine nomenclature

• Interleukins (IL-1 – 27)• Hematopoietins (GM-CSF, G-CSF, EPO, Flt3)• Interferons (I – α, β, κ, ω, II – γ)• Chemokines (C, CC, CXC, CX3C)• TNF-superfamily (TNF, LT-α, FasL, TRAIL)• Others (common names)

Five cytokine receptor families

• Immunoglobulin superfamily receptors• Class I cytokine receptor family (hematopoietin

receptors)– Binds most of the cytokines in the immune and

hematopoietin systems• Class II cytokine receptor family• TNF receptor family• Chemokine receptor family

Signal Transduction by cytokine receptors

• Cytokine receptors on different cell types trigger different events

• How do you get the message from the outside of the cell to the machinery inside?

Major functions of cytokines

• INNFLAMMATION, ACUTE PHASE REACTION, FEVER• HEMATOPOIESIS• INHIBITION OF THE IMMUNE RESPONSE• ACTIVATION OF B CELS, Ig CLASS SWITCHING• CELLULAR IMMUNE RESPONSE ACTIVATION (CTL, NK, NKT,

MACROPHAGES)• IMMUNE CELL MIGRATION AND CIRCULATION

Early mediators

• Interferons α/β– Induced by dsRNA, etc.– Induced by CD40/CD40L pathway– IFNs can induce more of themselves– Directly interferes with viral replication– Activation of T and NK cells

• IL-12, IL-15, IL-18, IFN-γ (from NK cells), IL-10• Proinflammatory mediators• Produced by cell associated with innate immunity (macrophages,

NK, etc.)• Mediate direct effects• Promote inflammation• Shape downstream responses

Interferon action

• Viral replication stimulates the infected host cell to produce interferon

• Interferon induces uninfected cells to – produce antiviral proteins that prevent translation

of viral mRNA– degrade viral nucleic acid

• Viral replication is blocked in uninfected cells

Chemokines

• Recruit to sites of infection• MIP-1a (NK and T cells)• MIG, RANTES (CD4+T cells)• IL-8 (neutrophils)• Eotaxin (eosinophils)

Late mediators

• IL-2, IL-4, IL-5, IFN-γ, TNF, IL-6, IL-10• Produced by cells of the adaptive immune

response (T and B cells)• Direct effects• More immunoregulatory functions

Down regulators

• IL-10, IL-11, TGF-β• Inhibit proliferation, cytokine production• Produced by both innate and adaptive cells

Maintenance cytokines

• GM-CSF, IL-3, IL-7, IL-9• Induce cell differentiation, cell growth

Cytokine-related diseases

• Bacterial septic shock

– Blood pressure drops, clots form, hypoglycemia, MOF, death

– LPS triggers TNF release

– TNF induces IL-1 which induces IL-6 and IL-8

• Bacterial toxic shock and related diseases

– Superantigens trigger large numbers of T cells which release massive amounts of cytokines (Super antigens are bacterial toxins that bridge CD4 T cell receptors and the MHC class II molecules on APC’s, bypassing the need for antigen)

• Lymphoid and myeloid cancers

– Some cancer cells secrete cytokines

• Chagas’ disease

– Trypanosoma cruzi infection results in severe immune suppression

– Depression of IL-2 receptor production

• XSCID

– Defect in the γ-chain gene of IL-2 subfamily of receptors

Innate immunity. The complement system. The phagocytic system.

Interferons-role and clinical applications. Immunity to infectious diseases.

Seminar 7

Topics

• The differences between innate and adaptive immunty• Components of the innate immunity• Receptors of innate immunity, exogenous and endogenous ligands (HSP), signaling pathways

– secreted – opsonins, CRP, MBL– scavengers, lectins (CLR), integrins - phagocytosis, – activatory – surface (TLR, TREM) / intracellular (TLR, NLR)

• The sequence of the innate mechanisms activation• Macrophages and granulocytes

– chemotaxis (C5a, LT, FMLP, IL-8)– activation (MDP, IFN-γ, chemokines)– Phagocytosis, opsonization (Ab – FcR, C – CR, microbial structures – PAMPR) – klilling mechanisms (reactive oxygen species, enzymes – catepsines, lysozyme, defensins, TNF family)

• Complement - >30 soluble and cell-bound proteins/glycoproteins (source – liver, WBC, epithelial cells –, proenzymes = zymogens, pathways of activation – classical (IgM, IgG1-3, physiologic role, diseases releted to complement disfunction), alternative (spontaneous hydrolysis of C3, sialic acid), lectin (MBL, MASP)

Complement activation

Topics

• The problem of infectious diseases• Types of pathogens (viruses, procaryotes, fungi, parasites) • Types of infections – course of the disease• General rules of the immune response to infectious agents• General immune evasion mechanisms of microbes• Antiviral immune response• Immune response to bacteria• Immune response to parasites• Types of vaccines:

– monovalent/polyvalent– alive(attenuated)/killed

• Vaccine strategies – dependent on antibody mediated immunity (cholera vaccine, DTP,

HIB, pneumococcal vaccine, plague vaccine, infuenza virus vaccine)

– depndent on cell-mediated immunity

Viruses

• DNA– HPV– Herpes Viruses - HHV (HSV1, 2, VZV, EBV, CMV, HHV-6,

HHV-8)– Pox virus (Variola-smallpox, Vaccinia-cowpox)– HBV

• RNA– dsRNA (reovirus, rotavirus)– ssRNA (rhino, coxsackie, polio, HAV, Rubella)– negative dsRNA (Influenza A, B, C, RSV, Rabies)– Retrovirus (HIV-1, 2)

Prokaryotes

• Bacteria (gram+/gram-, aerobic/anaerobic)– Gram +VE (red)

• cocci – Staphylococci, Streptococci ,

• bacili – Bacillus anthracis, Corynebacterium diphteriae, Listeria, Clostridium botulinum, tetani, perfringens

– Gram –VE (violet)• cocci – Neisseria

• bacili – Escherichia coli, Salmonella, Yersinia, Haemophilus, Pseudomonas, Vibrio cholerae, Legionella, Mycobacterum tuberculosis, leprae, Treponema, Borrelia, Helicobacter pylori

• Rickettsia• Chlamydia• Mycoplasma

Fungi

• Candida• Aspergillus• Cryptococcus• Histoplasma

Parasites

• Protozoa – Plasmodium, Toxoplasma– Entamoeba, Cryptosporidium– Trypanosoma, Trichomonas, Leishmania, Giardia

• Invertebrates– Helminths (Nematodes, Cestodes, Trematodes)– Arthropods (scrabies)

General immune evasion mechanisms Reduction of antigenicity

– growing within host cells

– shedding their surcace/membrane antigens

– mimicking the surface of host cells

• expressing molecules with amino acid sequences similar to those of host cell-membrane molecules

• acquiring a covering of host membrane molecules

– suppression of the immune response

• production of immunosuppressive products

• switching the type of response e.g. Th1/Th2

– continual variation in surface antigens (genetic shift/draft)

Anti-viral immune response.

1. Viruses consist of nucleic acid (RNA or DNA) surrounded by a protein coat (Capsid).

2. Some virus are surrounded by an added lipoprotein envelope.

3. Proteins associated with the nucleic acids, capsid, and envelope are all potentially immunogenic.

4. B cells recognize accessible, hydrophilic, and mobile protein antigens associated with the envelope or released from the nucleocapsid.

5. T cells recognize processed viral peptides.

6. In most viral infections, specific CTL activity arises within 3–4 days after infection, peaks by 7–10 days, and then declines.

7. Within 7–10 days of primary infection, most virions have been eliminated, paralleling the development of CTLs.

Anti-viral immune response.Innate immunity.

• mechanical clearance - fluid movement, muco-ciliary blanket

• production of type I interferons (IFN-α, -β) by infected cells, macrophages, monocytes and fibroblasts – blockade of viral replication and activation of NK cells

• activation of NK cells

• Production of proteins that interfere at various levels with specific or nonspecific host defenses

• Inhibition of antigen presentation by infected host cells (HSV), down-rerulation of class I and II MHC molecules (CMV)

• Evasion of complement-mediated destruction (vaccinia, HSV)• Continual antigenic variation (HIV, rhinoviruses)• Induction of immunosupression (measles virus, EBV, CMV, HIV) –

secretion of immunosupressive agents, destruction of lymphatic system

Evasion of cell-mediated immunity by viruses

Anti-bacterial immune response.Innate immunity.

• Biochemical mediators - lysozyme and other protective enzymes.• Acute phase proteins - eg. fibronectin, CRP have opsonic activity.• INF-γ - can be induced directly by Staphlococcal protein A.• monocytes and PMN are particularly relevant when inoculum size

and virulence are both low

• Antibody: Extremely important in control of extracellular bacteria e.g. E. coli, Streptococci and Staphylococci

– Toxin neutralization (Sufficient for complete protection against some pathogens (eg. Clostridia and Corynebacteria diptheria, not usually sufficient for complete protection against Pasteurella, Shigella, Bordetella, or Anthrax)

– Complement - mediated lysis

– Opsonization and phagocytosis (IgG and IgM can opsonize, but the Fc receptors for IgM are rare on phagocytic cells. However IgM is more efficient at C’ activation)

– Anaphylatoxin - mediated mast cell degranulation

– Anti-adhesin antibody - prevents or limits colonization

Anti-bacterial immune response.Acquired immunity.

Anti-bacterial immune response.Acquired immunity.

• DTH: Most important in control of intracellular bacteria eg. Mycobacteria tuberculosis (M.tb) and Mycobacterium leprae

– Macrophage activation

• Via Antibody + Complement

• Via IFN-γ from NK cells

• Via cytokines from T-helper cells

– Cytotoxic T-cell or NK lysis of infected targets exposing bacteria to antibody and complement.

– Granuloma formation - to contain effects of chronic antigen (eg. leprosy, tubercolsis).

Anti-bacterial immune response.Pathogenesis of infectious diseases.

– The response to the pathogen often causes disease symptoms– Overproduction of cytokines - Endotoxin causes release of

large amounts of TNF & IL-1 resulting in septic shock– Chronic antigen - results in long-term activation of T-DTH cells

leading to severe tissue damage and granuloma formation - M.tb tissue damage is due to DTH reaction

– Antibody of the wrong type or class - can actually lead to enhanced uptake of intracellular bacteria - Pasteurella haemolytica, also antibody to flagella of Borellia is not protective and leads to format of destructive Ab-Ag complexes i.e. release of IL-1, rash, joint damage and fever.

– Disregulation of Th1/Th2 ballance - Tuberculoid form (DTH) vs Lepromatous form (Ab)

Characteristics of a good vaccine

• promotes effective immunity• long lasting• safe, i.e., minimum of side effects• convenient: stable, non-traumatic, minimum boosters• produces correct type of immunity (i.e., protective)• produces immunity in the correct places• produces immunity against antigens involved in infection

Types of vaccines

• Vaccines based on antibody mediated immunity:– inactivated (dead or non-infectious) virus or bacteria– antigens either isolated from the organism or produced by

recombinant DNA technology• Vaccines based on cell-mediated immunity:

– generally a live non-pathological (attenuated) virus is administered

– the growing virus induces an immune response, which can result both in CMI and antibody production

Transplantation immunology. Mechanisms of allogeneic transplant rejection, induction of transplantation

tolerance

Seminar 11

Types of transplants

syngeneic = autograftcells/tissue/organ transferred from one part of the body to another in

the same individual

allogeneic = allograftcells/tissue/organ transferred between genetically different

members of the same species

xenotransplant = xenograftcells/tissue/organ transferred between members of different

species

isogeneic = isograftcells/tissue/organ transplanted between genetically identical

individuals

Types of rejection

TYPE OF REJECTION

TIME TEKEN CAUSE

Hyperacute minutes/hours preformed anti-donor antibodies

Accelerated days reactivation of sensitized T cells

Acute days/weeks primary activation of T cells

Chronic months/years antibodies, immune complexes, cellular reaction, recurrence of disease

Hyper-acute rejection

• Pre-existing antibodies cause hyper-acute rejection

• Antibodies to HLA (eg.in mothers or recipients of a previous graft) or ABO blood group antigens can cause rejection of a graft within minutes.

• The antibodies bind to the graft and initiate the complement and clotting cascades, resulting in vasoconstriction leading to deoxygenation of thegraft.

Acute rejection – direct and indirect presentation

Chronic rejection

• develops slowly over a period of years and leads to loss of graft function (usually in the first year post transplantation).

• luminal narrowing and occlusion of arteries and arterioles due to the proliferation of intimal smooth-muscle cells – vascular rejection (stenosis of vessels)

• interstitial fibrosis – parenchymal cell rejection (progressive immune mediated replacement fibrosis of graft parenchyma)

Tests:

• AB0 matching – important with blood transfusion and all vascularized organs-except liver-because the vascular endothelium, also epithelial cells and RBC expresses the blood group antigens. After binding an appropriate antigen antibodies induce complement mediated cell lysis. Antibodies may attack endothelial antigens causing HAR

• Crossmatch• Serologic determinations of MHC class I molecules• MLC (mixed lymphocyte culture) MLR (mixed

lymphocyte reaction) correlates with AR (acute rejection). ImDC are week stimulators of MLReaction.

• PCR determination of MHC molecules

Crossmatch

measuring cell lysis

suspension of donor lymphocytes

recipient’s serumcomplement

Mixed lymphocyte reaction

• The test lymphocytes are mixed with irradiated or mitomycin-C treated homozygous leukocytes, containing B-lymphocytes and monocytes (stimulator cells). In culture (over 4-6 days), T-cells (responder cells) recognize the foreign class II antigen and undergo transformation (DNA synthesis and enlargement: blastogenesis) and proliferation (mitogenesis). These changes are recorded by the addition of radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA.

• Most modern laboratories are switching to the PCR technology for tissue typing using specific probes for MHC specificities.

• a piece of skull from a dog, 1682• frog skin to heal burns, late XIXc.• slices of rabbit kidney into a child, 1905• tissues from testicles of monkeys, 1920• transplantation of chimpanzee kidneys, 1963-1965• transplantation of chimpanzee heart, 1964• baboon to human liver transplantation 1993• porcine fetal or islets cells xenotransplantation to diabetic patients,

1994 • baboon bone marrow for “baby Fae” and an AIDS patient, 1995 • foetal pig neural cells transplantation into human with Parkinson’s

disease 1997 and Huntington’s disease 2000• extracorporeal perfusion through porcine liver 1994, 2000

History of xenotransplantation in men

1. Transgenic procedures eliminating or reducing expression of Gal(1,3)Gal (1,3)galactosyltransferase – knockout pigs, • intracellular expression of recombinant single-chain Fv (ScFv) fragments in pig cells

2. Reduction of complement activation• cobra venom factor,• soluble form of complement receptor (sCR1)• C1 inhibitor • Expression of human cell surface regulators (CD46, CD55, CD59) on the surface of

pig cells.

CRP (complement regulatory proteins):CD55 – DAF (decay accelerating factor) – promotes the dissociation of C3 convertaseCD46 – MCP (membrane cofactor protein) CD59 – membrane inhibitor of reactive lysis, inhibits assembly of the membrane attack complex)

3. Removal of anti-Gal antibodies • plasmapheresis• blocking of anti-Gal antibodies with soluble Gal molecules

Xenotransplantation. Attempts to avoid hyperacute rejection in xenotransplantation

Bone marrow transplantation1. Indications• some types of leukaemias• severe combined immunodeficiency diseases• severe aplastic anaemia• some congenital defects of the hemopoietic system• breast cancer and other solid tumors 2. Source of hematopoietic stem cells• peripheral blood• bone marrow biopsy• umbilical cord blood3. Preconditioning4. GVHD• acute• chronic5. Microchimerism – the presence of donor cells in a variety of peripheral sites and in

the thymus of patients following bone marrow transplantation or organ transplantation, after liver transplantation (there are many bone marrow derived cells in the liver. Spread of donor cells in the periphery of recipient may be vital for induction of graft tolerance.

Immunosuppressive treatment

1. Antibodies• ALG / ALS – heterologous antisera • OKT3 – mouse monoclonal antibodies • Basiliximab/Daclizumab

2. Calcineurin inhibitors• Cyclosporine• Tacrolimus (FK-506)

3. Rapamycin (Sirolimus)

4. Antimetabolites• Azathioprine• Mycophenolate mofetil

5. Corticosteroids

Problems of current transplantology

• Rejection - especially chronic rejection – 5 years after transplantation 30% of grafts from living donors are

lost,– 5 years after transplantation 40% of grafts from cadaver are lost, – after 10 years 50% of grafts from living donors are lost, – after 10 years nearly 70% of grafts from cadaver donors are lost.– 3%-5% of grafts are lost each year due to many causes.

• Immunosuppression side effects– secondary neoplasms,– infections, – specific side effects of each of the drug

• What immunology can help: tolerance induction• Lack of organs – organ shortage (xenotransplantation, stem cell

manipulations) - the number of patients on waiting lists for transplants and the number of transplants performed in US in 1998, 100000 potential candidates die before they are placed on the waiting list.

Hypersensitivity types. Atopic diseases – pathogenesis and current treatment,

immunotherapy.

Seminar 12

Topics

• Definition of hypersensitivity• Types of hypersensitivity reactions• Type I hypersensitivity

– Definitions (atopy, anaphylaxis)– Types of allergens– Pathogensis (– Examples (anaphylactic shock, atopic dermatitis, asthma,

urticaria, hay fever)• Type II hypersensitivity

• Type III hypersensitivity

• Type IV hypersensitivity

HYPERSENSITIVITY

NON-SELF ANTIGEN

AUTOIMMUNITY

SELF ANTIGEN

IMMUNE RESPONSE

TOLERANCE

IgE antibodies

• Serum IgE levels in normal individuals: 0.1–0.4 g/ml;• The most severely allergic individuals rarely have IgE levels greater

than 1 g/ml. • IgE is composed of two heavy and two light chains • Molecular weight of 190,000. • The higher molecular weight as compared with IgG (150,000) is due

to the presence of an additional constant-region domain. • This additional domain (CH4) contributes to an altered conformation

of the Fc portion of the molecule that enables it to bind to glycoprotein receptors on the surface of basophils and mast cells.

• Half-life of IgE in the serum is only 2–3 days,• IgE remains bound to its receptor on mast cells and basophils, it is

stable in that state for a number of weeks.

Characteristics of type II hypersensitivity - cytotoxic hypersensitivity

• Directed against cell surface or tissue antigen• Characterized by complement cascade activation and various

effector cells• Examples

– Blood transfusion reactions– Hemolytic disease of the newborn (Rh disease)– Autoimmune hemolytic anemias– Drug reactions– Hyperacute graft rejection– Myasthenia gravis (acetylcholine receptor)

Type III hypersensitivity – immune complex reaction

• 3-10 h after exposure to the antigen• generalized (serum sicknes, systemic lupus erythematosus) or

affecting specific organs – skin (discoid lupus erythematosus, Arthus reaction), – kidneys (lupus nephritis, glomerulonephritis), – lungs („farmer’s lungs”), – blood vessels (polyarteritis),– joints (rheumatoid arthritis).

• persistent infection– microbial antigens– deposition of immune complexes in kidneys, joints

• autoimmunity– self antigens– deposition of immune complexes in kidneys, joints, arteries and skin

• extrinsic factors– environmental antigens– deposition of immune complexes in lungs

Type IV hypersensitivity

Major histocompatibility complex. Antigen presentation and recognition.

Seminar 4

Topics• The immunoglobulin superfamily (immunoglobulin-fold structure)• TCR (Tαβ, Tγδ, CD3 – γε,δε,ζζ)• The structure of the MHC molecules (glycoproteins)

– MHC I: transmembrane heavy α-chain, light chain β2-microglobulin, peptide-binding cleft/groove – α1, α2 domains, α3 domain – binding CD8, peptides 8-10 (9) amino acids, anchor aminoacids/residues – hydrophobic aminoacids, tetramers

– MHC II: transmembrane α-,β-chains, peptides 13-18 aminoacids, dimers • Expression of the MHC gene products, cellular distribution (membrane-bound, 105 copies of each

class I molecule, secreted MHC) lymphocytes>fibroblsts>muscle cells>hepatocytes>neurons• Organization of the MHC genes – HLA complex

– class I MHC genes • classical class I genes - A, B, C locus, • non-classical class I genes – E, F, G, MIC A, MIC B

– class II MHC genes• classical class II genes – DP, DQ, DR regions• non-classical class II genes – DM, DO, TAP,

– class III MHC genes (comlement components genes, cytokines (TNF-α,-β) genes, heat-shock proteins genes)

• Inheritance of the MHC genes (polymprphism: 240 A alleles, 470 B alleles, and 110 C alleles, locus, allel = form of gene, haplotype, codominant expression – both maternal and paternal gene products are expressed in the same cell, heterozygous, homozygous, inbred mouse strains, linkage disequilibrium, )

• Function of MHC molecules (presentation of antigens, broad specificity, immune responsiveness, disease susceptibility)

• Antigen processing and presentation (MHC restiction, cytosolic processing pathway, endogenous compartment, endocytic processing pathway, exogenous compartment)






Cytosolic pathway

Endocytic pathway

Introduction to tumor biology. Immunology and immunothrapy of tumors.

Seminar 12

Cancer pathways

Cancer screening

Cancer therapeutic agents

Tolerance, autoimmunity, mechanisms preventing autoimmune response. Prospects for specific therapies.

Seminar 13

IMMUNE TOLERANCE

A state of unresponsiveness that is specific for a particular antigen and which is induced by prior exposure to the antigen (tolerogen).

EXAMPLESDizygotic twin calves can exchange haematopoietic stem cells as a result of placental fusion. The twins are thus tolerised to each other’s MHC molecules and once born can accept skin grafts/organ transplants without rejecting the tissue despite being non-identical.

Induction of tolerance in a mice model

FEATURES OF THE IMMUNE TOLERANCE

• consequence of antigen recognition

applies only to B and T cells (parallel to immunological memory), tolerised antigens, definition of self

• it is not absolute and it is not for ever

can be acquired and broken naturally (autoimmune diseases) or artificially (experimental animals models)

• immunologically specific

like immune response, directed against some or all epitopes on an antigen

• lack of response tolerance• lack of response = immunosupression• tolerance = active antigen dependent process in response to the

antigen

SIGNIFICANCE

• healthy individuals are tolerant to their own antigens (self tolerance) and this status is maintained throughout their life despite presence of cells capable of recognising self antigens. Lack of self tolerance leads to autoimmune diseases.

• foreign antigens administered in particular ways may induce tolerance – immunological tolerance may be utilised to treat hypersensitivity disorders and prevent graft rejection

• some microbes and tumours may evade the immune response by inducing specific tolerance

• tolerance induction is potential approach to vaccination (murine leishmaniasis, lymphatic filariasis in humans)

• immune tolerance may be required in gene therapy approaches (immune response to newly expressed gene products could be an important barrier to gene therapy)

MECHANISMS OF TOLERANCE CENTRAL TOLERANCE

T Cells

• Positive selection - selection of the T cells with T cell receptors (TCR’s) that are able to interact with MHC class I and II molecules on thymic epithelial cells (death by neglect, MHC – restriction, expression of self antigens in the thymus)

• Negative selection - deletion of the cells that recognise self antigens expressed in conjunction with MHC class I or II molecules on thymic dendritic cells or macrophages. If the interaction is of a high affinity, the T cells will be deleted, if low affinity the T cells may escape negative selection (generation of regulatory T cells, presence of autoreactive T cells in the periphery – significance)

MECHANISMS OF TOLERANCE CENTRAL TOLERANCE

B cells

• Exposure of developing B cells to high levels of multivalent self antigens leads to deletion of the B cells - clonal deletion (negative selection)

• Low levels of antigen will render the B cells unresponsive by down-regulation of surface IgM expression; these B cells are short lived - clonal anergy (positive selection)

TWO SIGNAL HYPOTHESIS

MECHANISMS OF PERIPHERAL TOLERANCE – T cells

T-cell intrinsic mechanisms

• Ignorance – sequestration of self antigens in sites not accessible for the cells of the immune system or the amount of antigen does not reach the threshold required to trigger a T cell response

• Anergy – TCR ligation (1st signal) in the absence of costimulation (2nd signal) or signalling through alternative inhibitory receptors – CTLA-4 and PD-1 that overcomes stimulatory 2nd signal.

• Apoptosis – activation-induced cell death (AICD), repetitive engagement of TCR causes increased expression of FasL, role of IL-2 (STAT-5, FLIP)

• Phenotypic skewing – despite recognition of self antigen (tolerogen) and activation T cell remains tolerant (non-pathogenic phenotype, defective migration, chemokine receptors)


T-cell extrinsic mechanisms

• Tolerogenic dendritic cells – The nature of DC regulates whether tolerance or immune

response will be initiated.– Immature and/or tolerogenic DC stimulates tolerance– Mature DC induces T cell activation– Signals for maturation (the pattern recognition model vs the

danger model)


T-cell extrinsic mechanisms

• Regulatory T cells – Two major populations:

1. Produced in the thymus CD4+CD25+2. Generated in the periphery (from naive T cells, after

presentation of self antigen by immature/tolerogenic DC)– Mode of action:

1. Cell – cell contact 2. Soluble mediators – cytokines: IL-10, TGF- (infectious

tolerance)

Immune tolerance.

MODIFICATION OF IMMUNE TOLERANCE

APPLICATIONS

induction/enhancement

autoimmune diseaseshypersensitivity disordersgraft rejection certain microbial infections

debilitation/impairment

certain tumoursimmunodeficiency syndromesmost of microbial infections

STRATEGIES OF TOLERANCE INDUCTION IN TRANSPLANTATION, AUTOIMMUNE DISEASES and

HYPERSENSITIVITY DISORDERS

• Targeting signal I– Non-mitogenic anti-CD3 mAbs– Anti-CD3 toxin-conjugate– Anti-CD4 mAbs– Campath-1H (anti-CD52)– Systemic and/or oral peptide therapies/altered peptide ligands (Copaxone and MHC

peptides)– DNA vaccination for allergy therapy (i.e. ragweed)

• Targeting signal II– Blockade of CD28/B7– Blockade of CD40L/CD40 interactions– Anti-ICAM/Anti-LFA1

• Targeting clonal anergy and clonal deletion – Extrathymic T cell deletion using mAbs– Anti-CTLA4 agonists– Induction of mixed chimerism (intrathymic injection of donor cells, donor bone marrow

transplantation)– Pro-apoptotic therapies (Fas, TNF, TRANCE pathways)

• In utero and/or neonatal tolerance induction with donor antigen• Injection of antigen presenting cells (Immature APC’s, Gentically modified DC)• Cytokine manipulation

– Viral – IL-10, TGF-– Anti – TNF – IFN-, IL-4

Primary and secondary immunodeficiencies. Prospects for treatment and prevention of AIDS

Seminar 15

Technology

Immuno Coverage