8. HOW REPERTOIRES OF ANTIGEN SPECIFIC RECEPTORS ARISE

DIFFERENTIATION OF B LYMPHOCYTES

BASIC FEATURES OF FORMATION OF THE ANTIGEN-SPECIFIC Ig (BCR):

Rearrangement of genes H (D-J; V-DJ); N – segments !

1. Rearrangement of genes

2. Primary synthesis of IgM, IgD

3. Elimination resp. silencing of autoreactive clones (binding of (auto)antigen by immature B-lymphocytes → apoptosis, anergy).

4. Missing of TH: many potentially autoreactive B-cells are preserved (“cryptic“ determinants)

5. After the contact with antigen (+ help from T-lymphocytes) → somatic mutations of V-regions, selection of best mutants (“affinity maturation“). Repeatedly ! Isotype switch - cytokines

SOURCES OF REPERTOIR VARIABILITY:

1) Random combinations V(D)J; N

2) Pairing H--L

3) Mutations VH, VL

Even without mutations > 107 different clones.

- Model of “ready-made clothes store“

- Principle of “anticipatory“ system

DEVELOPMENT AND SELECTION OF T LYMPHOCYTES IN THYMUS

BASIC FEATURES OF T LYMPHOCYTE (DEVELOPMENT (THYMUS)

1. Rearrangement of genes (D-J; V-DJ) N-segments !

2. Rearrangement of genes

3. Expression on immature thymocytes (CD4+, CD8+)

4. Elimination of autoreactive clones (if they recognize in thymus anything with sufficiently high affinity – negative selection)

5. Elimination of “useless“ clones, i.e. those unable to bind MHC at all – positive selection. Switching off either CD4 or CD8

6. No afinity maturation

7. Mature T: either CD8+ (MHC I) or CD4+ (MHC II)

PROTECTION AGAINST AUTOREACTIVE LYMPHOCYTES:

1. Elimination (immature B; negative selection of thymocytes)

2. Ignorance – of autoantigens occurring in too low amounts or hidden (potential danger)

3. Mostly necessity of 2 signals (B: help from TH T: professional APC)

without the 2nd (costimulatory) signal - negative stimulation, anergy

4. Active mechanisms of tolerance (supressor T cells)

CRITICAL MOMENTS IN B CELL DEVELOPMENT

1. Successful rearrangement of H

2. Successful rearrangement of event. , formation of

functional surface IgM

3. Elimination of autoreactive (immature B)

4. Somatic mutations, afinity maturation

↓

plasma cells, memory cells

9. IMMUNE RESPONSES BASED ON T LYMPHOCYTES AND

NK CELLS

9A T LYMPHOCYTES

APC!!!

DENDRITIC CELLS

T LYMPHOCYTES: IMPORTNAT FUNCTIONAL SUBPOPULATIONS

ANOTHER RECENTLY DISCOVERED IMPORTANT SUBPOPULATION :

Th17 (somewhat similar to Th1)

9B ADHESION MOLECULES

STRUCTURAL GROUPS OF ADHESION MOLECULES

- INTEGRINS (INTERCELLULAR MATRIX, SURFACE

LIGANDS)

- SELECTINS (SACCHARIDE LIGANDS)

- ADHESION MOLECULES OF IMMUNOGLOBULIN FAMILY

- MUCINS (LIGANDS OF SELECTINS)

- MANY OTHERS (CD5, CD44, TNF-R FAMILY ETC.)

9C SIGNALING

PHOSPHORYLATION!!!

RECEPTORS ASSOCIATED WITH TRIMERIC G-PROTEINS

PHOSPHATASES – CD45

Src KINASES (Lck, Fyn, Lyn…)

REGULATION OF ACTIVITY

9D CYTOKINES

CYTOKINES (LYMPHOKINES)

Proteins secreted by leukocytes and other cells. Also membrane forms.

Act on leukocytes and other cells through surface receptors as “tissue hormones“

Typically pleiotropic and redundant.

- Interleukins 1-35 (IL-1 …)

- Chemokines (IL-8 a related molecules)

- Interferons , , (IFN- …)

- Transforming growth factors (TGF-, )

- Colony stimulating factors (G-CSF, M-CSF, GM-CSF)

- Tumor necrosis factors (TNF-, )

- Growth factors (SCF, EPO, FGF …)

EXAMPLES OF IMPORTANT CYTOKINES

- PLEIOTROPIC: IL-1, IL-3, IL-6, IL-11

- ACTIVATION OF T: IL-2, IL-15, IL-12

- ACTIVATION OF B: IL-4, (IL-5, IL-6)

- ACTIVATION OF NK: IL-12, IL-2

- DIFFERENTIATION OF THYMOCYTES, PRE-B: IL-7

- ACTIVATION OF MACROPHAGES, EXPRESSION OF MHC: IFN-, TNF-

- DIFFERENTIATION OF MYELOID CELLS: G-, M-, GM-CSF, IL-3

- DIFFERENTIATION OF STEM CELLS: SCF, (IL-1, IL-3, IL-7)

- REGULATION OF TH DIFFERENTIATION IL-4, IFN-, IL-10, TGF-

RECEPTORS OF CYTOKINES MOSTLY – ASSOCIATION WITH INTRACELLULAR ENZYMES (PROTEIN KINASES; PHOSPHORYLATING OTHER PROTEINS)

BINDING OF A CYTOKINE:

AGGREGATION OF RECEPTORS

APPROXIMATION AND MUTUAL PHOSPHORYLATION OF THE ASSOCIATED KINASES – THEIR ACTIVATION

PHOSPHORYLATION OF SPECIFIC SUBSTRATES STARTS SIGNALING CASCADES: THE RESULTS DEPEND ON CELL TYPE, SIGNAL TYPE, CONTEXT (PROLIFERATION, EFFECTOR MECHANISMS, BLOCK OF CELL DIVISION, APOPTOSIS …)

USUALLY 2 RECEPTOR SUBUNITS: BINDING AND SIGNALING. THE SIGNALING SUBUNIT IS USUALLY SHARED BY SEVERAL RECEPTORS (COMMON SUBUNIT).

Receptor LigandS (chemokines) Expression on cells

CCR1RANTES, MIP-1a, MCP-3, MCP-4

MF, activated T, neutrophils, bazofils

CCR2 MCP-2, -3, -4, -5 MF, activ. T, bazofils

CCR3Eotaxin, eotaxin-2, MCP-2, -3, -4

Eosinofils, activ. T (TH2), bazofils

CCR4TARC, MDC, RANTES, MIP-1a, MCP-1

Activ. T (TH2), bazofils

CCR5RANTES, MIP-1a, MIP-1b

MF, activ. T (TH1)

CCR7 6Ckine, MIP-3b DC, T, B

CCR8 I-309 Activ. T (TH2), NK

CCR9 TECK DC, thymocytes

CCR10 CTACK T (skin)

CCR11 MCP-1 Various cells

CXCR1 IL-8, GCP-2 Neutrofils, NK

CXCR2IL-8, GCP-2, GRO, ENA-78, NAP-2

Neutrofils, NK

CXCR3 IP-10, Mig, I-TAC Activ T (TH1), NK

CXCR4 SDF-1 MF, T

CXCR5 BLC B

CX3CR1 Fractalkine MF, activ. T, NK

XCR1 Lymfotaktin a, b Activ. T, NK

SUPERANTIGENS

NK CELLS

STIMULATING vs. INHIBITORY RECEPTORS INHIBITORY RECEPTORS RECOGNIZE MHC I (HUMAN – KIR; MOUSE – Ly-49) NK CELLS KILL CELLS LACKING MHC I

10. IMMUNE RESPONSES BASED ON B LYMPHOCYTES

DIFFERENTIATION OF B LYMPHOCYTES

T-CELL INDEPENDENT ANTIGENS

- REPEATED EPITOPES (E.G. POLYSACCHARIDES)

- INTENSIVE “CROSS-LINKING“ OF SURFACE Ig SUBSTITUTES FOR THE HELPER SIGNALS. NO NEED FOR CONTACT WITH TH – SUFFICIENT JUST CYTOKINES FROM NK, T

- IMPORTANT! FIRST LINE OF DEFENCE; RECOGNIZE SURFACE BIOPOLYMERS OF MICROBES

- NO AFFINITY MATURATION

- NO MEMORY

HUMORAL RESPONSE IN VIVO IS STRONGLY POLYCLONAL (“ANTISERUM“)

MONOCLONAL ANTIBODIES:

- IMMORTALIZATION OF B-CELL CLONES

- FUSION WITH MYELOMA CELLS:

HYBRIDOMAS

IMMUNE MECHANISMS “IN ACTION“

- IMMUNITY AGAINST INFECTIONS AND PARASITES

- MUCOSAL IMMUNITY

- TRANSPLANTATION

- ANTI-TUMOR IMMUNITY

- REGULATION OF IMMUNE MECHANISMS (INCLUDING TOLERANCE)

- ALLERGY

- AUTOIMMUNITY

- IMMUNODEFICIENCY (INBORN, ACQUIRED)

- IMMUNOPROPHYLAXIS AND IMMUNOTHERAPY

11. ANTI-INFECTION IMMUNITY

IMMUNITY AGAINST BACTERIA

- Bacteria colonizing mucosal surfaces or intercellular spaces (staphylococci, streptococci, pneumococci, gut bacteria, haemophilus etc.)

- Bacteria living inside cells (macrophages, epithelial cells) (mycobacteria, Yersinia, Brucella, Listeria)

NON-ADAPTIVE, “NON-SPECIFIC“ MECHANISMS (PHAGOCYTOSIS, ALTERNATIVE PATHWAY OF COMPLEMENT, LECTINS, ”ACUTE PHASE PROTEINS”

- Some bacteria are not phagocytosed without opsonisation (e.g. Hemophilus, Escherichia, Salmonella, Treponema)

- Most bactria are not sensitive to complement lysis (sensitive e.g. gonococci (Neisseria))

- Many bacteria are resistant to destruction in phagosomes (e.g. secrete toxins killing phagocytes, survive in aggressive environment of phagolysosomes (resistant cell walls) and even proliferate there)

- Some bactreia inhibit fusion of phagosome with lysosome and live in vacuoles of phagocytes (mycobacteria)

- Some bacteria penetrate phagosome membrane into cytoplasm and live there (e.g. Listeria)

PROTECTION AGAINST YEAST AND MOULDS

- Non-adaptive (phagocytosis)

- Cell-mediated (TH1 – inflammation, killing of pathogens by activated macrophages)

- Humoral – antibodies contribute to opsonization and phagocytosis

- (Direct killing - TC - without MHC !?)

PROTECTION AGAINST VIRUSES

- Non-adaptive (interferons, NK)

- Humoral (blocking antibodies, opsonisation, complement)

-

- TC; also TH1 inflammation, mobilization of adaptive mechanisms)

PROTECTION AGAINST PROTOZOAL

PARASITES

- Intracellular(Leishmania, Plasmodium, Trypanosoma)

- Essential importance of TH1 response (inflammation,

activated macrophages), partially also TC and humoral

- ADCC

Problems:

Variability of surface glycoprotein (Trypanosoma)

PROTECTION AGAINST MULTICELLULAR PARASITES (WORMS)

Most important probably humoral humorální IgE response in collaboration with mastocytes, eosinophils and basophils

Direct toxicity of some substatces secreted by stimulated mastocytes/basophils/eosinophils

Inflammation – participation of other components (makrophages)

Expulsion (stimulation of smooth muscle contraction, mucus

secretion)

MEDIATORS RELEASED BY MASTOCYTES, BASOPHILS A EOSINOPHILS AND THEIR ACTIVITIES

Histamin Prostaglandin D2

Heparin Leukotriens

Hydrolytic enzymes

- Stimulation of nerve endings - cough, vomiting

- Contraction of smooth muscles - expulsion

- Vasodilatation - swelling, influx of antibodies,

cells; exsudation, inflammation

RELATIONSHIP

PATHOGEN - IMMUNE SYSTEM

1. STRATEGY “HIT AND RUN“ (Cholera, smallpox, influenza)

2. CHRONIC INFECTION; PARTIAL COEXISTENCE WITH IMMUNE SYSTEM

STRATEGY OF PATHOGENS

- “Hiding“ – integration into geonome, latence (HSV)

- Variability (influenza, HIV, Trypanosoma)

- Suppression of MHC expression (adenoviruses)

- Suppression of antigen presentation on MHC II (mycobacteria)

- Suppression of inflammation:

EBV - analog of IL10 Vaccinia - antagonist of IL-1 Poxviruses - inhibitor of TNF Adenoviruses - inhibitor of IFN

STRATEGY OF PATHOGENS cont.

- Inhibition of complement (HSV, Vaccinia)

- Exploitation of cytokines (TNF – stimulation of HIV replication, stimulation of egg laying by Schistosoma)

- Unclear role – Protein A, G (binding of Ig; Staphylococus aureus); superantigens (enterotoxins)