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  CD3TCR signalling complex:T cells  CD4MHC class II receptor:T cells  CD8MHC class I receptor:T cells  CD10neutral endopeptidase:ALL cells  CD19co-receptor subunit:B cells  CD45LCA (tyrosine phosphatase): leukocytes  CD62LL-selectin: T cells, mono-, granulocytes  CD247zeta 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 have pleiotropic effects – they often have more than one receptor Cytokines can be redundant – their receptors often share subunits Cytokines can be redundant – their receptors often share subunits Cytokines can have specific and unique functions – their receptors have ligand specific subunits as well 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 Many immunologically relevant cytokines are made by non-lymphoid cells Interleukins – may also act on nonhematopoetic cells Interleukins – may also act on nonhematopoetic cells Most of them act in autocrine or paracrine fashion Most of them act in autocrine or paracrine fashion

8. CYTOKINES SIGNAL 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-alphaIL-6 LT-alpha IL-10 IL-13 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.  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 INTRACELLULAR SIGNALING IN T CELL ACTIVATION

24.  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) INTRACELLULAR SIGNALING IN T CELL ACTIVATION

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 secondary immune 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