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ORIGINS OF IMMUNE RESPONSE Mehtap KAÇAR KOÇAK MD. PhD. 2009

ORIGINS OF IMMUNE RESPONSE Mehtap KAÇAR KOÇAK MD. PhD. 2009. Points to be discussed. Monoclonal antibodies CD (Cluster Determinants) classification Origin and subsets of B cells Origin and subsets of T cells NK cells Cytokines and chemokines Cell adhesion molecules

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ORIGINS OF IMMUNE RESPONSE Mehtap KAÇAR KOÇAK MD. PhD. 2009

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  1. ORIGINS OF IMMUNE RESPONSE Mehtap KAÇAR KOÇAK MD. PhD. 2009

  2. Points to be discussed • Monoclonal antibodies • CD (Cluster Determinants) classification • Origin and subsets of B cells • Origin and subsets of T cells • NK cells • Cytokines and chemokines • Cell adhesion molecules • Patterns and mechanisms of cell migration • Immunoglobulins and generation of diversity

  3. CD (CLUSTER DETERMINANTS) CLASSIFICATION • Based on the identification of single epitopes by monoclonal antibodies • Involves mainly differentiation antigens of cells and cell receptors • Other CD markers include various proteins, enzymes, complex lipids, adhesion molecules, cell receptors etc. • Most, but not all, CD markers are at cell surface • CD markers cover mainly hemopoetic cells • Actual number of CD markers is about 250.

  4. EXAMPLES OF CD MARKERS • CD3 TCR signalling complex: T cells • CD4 MHC class II receptor: T cells • CD8 MHC class I receptor: T cells • CD10 neutral endopeptidase: ALL cells • CD19 co-receptor subunit: B cells • CD45 LCA (tyrosine phosphatase): leukocytes • CD62L L-selectin: T cells, mono-, granulocytes • CD247 zeta chain of TCR : T cells, NK cells

  5. MARKERS AND SUBSETS OF B CELLS • CD markers: CD19, CD20, CD21, CD22, CD35, CD40, CD72, CD80, CD86 • B1 cells: B1a (CD5+) and B1b (CD5-) • B1 cells: comprise about 20% B cells in blood and spleen of healthy people, secrete IgM only • B2 cells: majority of fully competent B cells

  6. CYTOKINES – KEY CONCEPTS • Cytokines have pleiotropic effects – they often have more than one receptor • Cytokines can be redundant – their receptors often share subunits • Cytokines can have specific and unique functions – their receptors have ligand specific subunits as well

  7. CYTOKINES – KEY CONCEPTS -2 • Many immunologically relevant cytokines are made by non-lymphoid cells • Interleukins – may also act on nonhematopoetic cells • Most of them act in autocrine or paracrine fashion

  8. CYTOKINESSIGNAL TRANSFERRING MOLECULES Interleukins • directing other cells to divide and differentiate Interferons • type I (alpha/beta), type 2-gamma Colony stimulating factors (CSF) • directing bone marrow stem cells Chemokines • directing cell movement Other • TNF, TNF, TGF – involved in inflammation, cytotoxicity and immunosuppression respectively

  9. Main properties of some interleukins • IL-1 – proinflammatory, pleotropic • IL-2 – growth factor for T, B and NK cells • IL-4 – maturation and differentiation of B cells • IL-5 – maturation and differentiation of eosinophils • IL-6 – proinflammatory, differentiating agent for B cells

  10. Main properties of some interleukins-2 • IL-10 – immunosuppressive • IL-12 – strong activator of cellular immune response • IL-15 – maturation of NK cells in bone marrow • IL-17 – proinflammatory, pleotropic • IL-18 – stimulates of interferon-γ production by NK and T cells

  11. CHEMOKINES – CHEMOTACTIC CYTOKINES • Ch. are small, soluble heparin-binding important proteins that regulate leukocyte trafficking • Some of them are strategically located on vascular endothelium, participating in the adhesion cascade • They are divided into four families defined by a cysteine motif – CXC, CC, C and CX3C (C is cysteine, X - any aminoacid residue)

  12. CHEMOKINES – CHEMOTACTIC CYTOKINES - 2 • Different leukocyte subsets bear alternative sets of receptors to respond to chemokine • SLC – secondary lymphoid ch. is located on HEV, attracts T lymphocytes bearing SLC receptor CCR7

  13. EXAMPLES OF CHEMOKINES • Interleukin-8 (IL-8) – from monocytes • RANTES – from T cells • Eotaxin – from monocytes • MCP-1 – from monocytes, epithelia • MIP-1alpha – from T cells, white cells • IP-10 – from monocytes

  14. CELL ADHESION MOLECULES (CAM) Integrins: adhesion to endothelium and extracellular matrix (VLA-1 to 6, LFA-1, LPAM, CR3, CR4) CAM of the immunoglobulin supergene family: various (ICAM-1-3, VCAM-1, PECAM-1, NCAM, CEA) Selectins: molecules on leucocytes and endothelium which bind to carbohydrate (E, P, L-selectins, )

  15. CELL ADHESION MOLECULES (CAM)-2 Cadherins: bind to catenins, cytoskeleton elements in calcium dependent manner (E, N,T-cadherins) CD44 and it variants: cell hyaluronate receptor involved in cell-to-cell and cell-to-matrix interactions

  16. CD4 Th1 and Th2 T cells:profile of produced cytokines Th1Th2 IL-2IL-4 IFN-gammaIL-5 TNF-alpha IL-6 LT-alpha IL-10 IL-13

  17. REGULATORY (SUPRESSOR) T CELLS (Treg) • Th2 cells – secrete IL-4 • Th3 cells – secrete TGF-beta • Tr1 cells – secrete IL-10 • CD4+, CD25+, Foxp3 T cells • CD8+, CD28- T cells • Some cytotoxic T cells • Some TCR gamma/delta T cells

  18. CYTOTOXIC T CELL SUBSETS(Tc) • TCR alpha/beta CD8+ T cells • TCR alpha/beta CD4+ Th1 cells • TCR gamma/delta T cells (CD3+, CD4-, CD8-, CD16+) • NKT (NT) cells (CD3+, CD4-+, CD8- CD56+) • NK cells (CD2+, CD3-, CD4-, CD8-, CD56+ CD16+)

  19. FEATURES OF NK CELLS • Belong to so called large granular lymphocytes (LGL) • Comprise about 10% lymphocytes in human blood • Show spontaneous cytotoxic activity against infected and tumor cells

  20. FEATURES OF NK CELLS -2 • Their cytotoxicity is inhibited by conventional MHC antigen expression on target cells • Incidence of tumor formation is lower in individuals with high NK cell content • Beige mice lack NK cells – high incidence of tumors

  21. T CELL ACTIVATION-EARLY STEPS • Formation of immunological synapse – lymphocyte polarization, adhesion to APC, maturation of synapse • Microdomains (lipid rafts) – regions of cell membranes rich in lipids: contain several proteins able for fast signal transduction, kinases from Src family, and other

  22. T CELL ACTIVATION-EARLY STEPS-2 • Lymphocyte activation leads to microdomain grouping- so called supramolecular activation clusters-SMAC • First (Ag-TCR) and second signal (CD28-CD80, CD86, CD58-CD2) concept; naive lymphocytes need 2 signals, activated cells – only the first one

  23. INTRACELLULAR SIGNALING IN T CELL ACTIVATION • Involves transduction of signals from both T cell receptor and CD28 • CD4 bound lck kinases become activated by CD45 phosphatase • ITAM domains of CD3 (zeta chains) become phosphorylated by lck

  24. INTRACELLULAR SIGNALING IN T CELL ACTIVATION • ITAMs associate with other kinases such as ZAP-70 and fyn • Fyn activates phopholipase C (PLC) which cause release of intracellular calcium (calcium flux) • Calcium binds to calcineurin and activates transcription factors (NF-AT, NF-kappa B, AP-1)

  25. SIGNALING IN B CELL ACTIVATION • Tyrosine kinases lck, lyn ,fyn become activated via Ig and Ig of B cell receptor • They phosphorylate BCR ITAM domains • These can then bind Syk, another kinase, which activates phospholipase C (PLC-)

  26. SIGNALING IN B CELL ACTIVATION-2 • PLC and three other pathways (Ras, RhO, PI-3K) lead to induction of transcription factors such as NF-AT, AP-1 and NF-kappa B • Co-stimulators:CD40, CD19/CD21, CD22, CD32

  27. WHAT ARE TRANSCRIPTION FACTORS? • Answer: transcription factors are complex protein molecules residing in cytoplasm, which after stimulation and assembly are able to enter cell nucleus and induce several genes transcription.

  28. LEUCOCYTE-ENDOTHELIAL CELL INTERACTIONS • Leucocytes interact with the vessel wall in multistep fashion, using several leucocyte surface molecules that recognize their counter-receptors on endothelial cells • The rolling and tethering of leucocytes on vessel wall is mediated by selectins

  29. LEUCOCYTE-ENDOTHELIAL CELL INTERACTIONS -2 • Chemokines and their receptors are needed to activate leucocyte integrins • Only activated integrins are able to mediate firm adhesion between leucocytes and endothelium • The transmigration of leucocytes into the tissues requires proteinases and repair mechanisms

  30. LYMPHOCYTE RECIRCULATION • Lymphocytes recirculate continuously between blood and lymphoid organs • 80% of lymphocytes enter the lymph nodes via specialized vessels called high endothelial venules (HEV) • The remaining lymphocytes enter the lymph nodes together with dendritic cells and antigens via afferent lymphatics

  31. LYMPHOCYTE RECIRCULATION - 2 • Lymphocytes leave the lymph nodes via efferent lymphatics • Lymphocyte recirculation allows the lymphocytes to meet their cognate antigens and other leukocyte subsets to evoke an efficient immune response

  32. IMMUNOGLOBULINS KEY CONCEPTS • Isotype: antigenic differences between classes, subclasses and types • Allotype: antigenic differences between Ig constant domains of various individuals • Idiotype: antigenic differences within variable domains reflecting antigen binding site

  33. IMMUNOGLOBULINS KEY CONCEPTS - 2 • Isotype (class)switching: the change of produced Ig (from IgM to other Ig), usually in secondaryimmune response • Polyclonal Ig: a mixture of Igs having either kappa or lambda chains (3:1 ratio in humans) • Monoclonal Ig: either kappa or lambda light chain – incidence in tumors such as myeloma

  34. Immunoglobulin classes • IgG – the most abundant Ig. Exists in 4 subclasses (IgG1, IgG2, IgG3, IgG4) • IgA – exists as serum and secretory Ig present on mucosal surfaces, 2 subclasses (IgA1 and IgA2). • IgM – present in bloodstream is composed of 5 molecules forming pentamer. Protects from sepsis. Produced mainly in spleen.

  35. Immunoglobulin classes - 2 • IgD –with IgM forms antigen receptor on B cells. In serum in trace amounts. • IgE – anti-parasitic. Participates in allergic reactions. Very short lifetime when free, but stable when bound to cell surface such as mast cells.

  36. COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENS similarities • Members of Ig superfamily • Heterodimeric antigen-binding site • Divided into variable and constant domains • Variable domains constructed by V(D)J rearrangements

  37. COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENS Similarities -2 • Nongermline-encoded N-nucleotide additions at V(D)J junctions • Exhibit allelic exclusion • Mature T and B cells display receptors of one and only one antigenic specificity • Negative selection for receptors with self-antigen specific.

  38. COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENS differences • Ig binds native antigen in solution; TCR binds processed antigen when presented by APC • Ig can be secreted; TCR is not • Somatic mutation of Ig genes; TCR genes – never

  39. COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENS -2 Differences - 2 • Isotype class switching of Ig genes (from IgM to IgG or IgA or IgE) • Positive selection of TCR for self-MHC recognition (MHC restriction)

  40. FEATURES OF IMMUNOGLOBULIN SUPERFAMILY • Large family of ancestrally related genes (probably >100) • Most products involved in immune system function or other cell – cell interactions

  41. FEATURES OF IMMUNOGLOBULIN SUPERFAMILY - 2 • Proteins exhibit domain structure of ca 110 amino acids, usually translated from a single exon and with a single intradomain disulfide bond, helping to stabilize the structure • Examples include MHC class I and class II molecules, TCR, cytokine receptors, some cell adhesion molecules and others

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