1. IMMUNOLOGICAL TOLERANCE

2. BASIC FACTS ABOUT TOLERANCE Tolerance – a state of unresponsiveness specific for a given antigen It is specific (negative) immune response Self tolerance – prevents the body to elicit an immune attack against its own tissues Mechanisms of active tolerance prevent inflammatory reactions to many innocuous airborne and food antigens found at mucosal surfaces

3. Tolerance mechanisms are in place to protect individual from self-reactive lymphocytes Central tolerance – deleting T or B clones before maturity if they have receptors that recognize self-antigens with great affinity Peripheral tolerance – kills lymphocytes in secondary lymphoid tissue Also, life span of lymphocytes regulated by apoptosis

4. Features of self-tolerance Self-non-self discrimination is learned during development Tolerance is NOT genetically programmed The time of first encounter is critical in determining responsiveness

5. FACTORS IMPORTANT IN THE INDUCTION OF TOLERANCE 1.The stage of differentiation of lymphocytes at the time of antigen confrontation 2.The site of encounter 3.The nature of cells presenting antigenic epitopes 4.The number of lymphocytes able to respond 5.Microenvironment of encounter (expression of cell adhesion molecules, influence of cytokines etc.)

6. TOLERANCE – GENERAL PROPERTIES 1.Immature or developing lymphocyte is more susceptible to tolerance induction than mature one 2.Tolerance to foreign antigens is induced even in mature lymphocytes under special conditions 3.Tolerance of T lymphocytes is a particularly effective for maintaining long-lived unresponsiveness to self antigens

7. POSSIBLE WAYS OF PREVENTION OF SELF-REACTIVITY Clonal deletion – physical elimination of cells from the repertoire during their lifespan Clonal anergy – downregulating the intrinsic mechanism of the immune response such as lack of costimulatory molecules or insufficient second signal for cell activation Suppression – inhibition of cellular activation by interaction with other cells

8. DIVISION OF TOLERANCE Central The site for T cells is the thymus The site for B cells is the bone marrow The mechanism – clonal deletion Peripheral The site – everywhere in the body Cells – both T and B Mechanisms – anergy, cell death, supression

9. T cell maturation in thymus In thymus developing T cells (thymocytes) proliferate and differentiate to mature T cells. Maturation involves 1.rearrangements of the germ-lineTCR genes and 2. the expression of various membrane markers. CD3complex,CD4,CD8 and TCR

10. Development of αβ T cells

11. LOCATION: BONE MARROW TO BLOOD

12. Formation of precursor of pro Tcells T cell precursor in bone marrow lacks detectable markers (CD4 and CD 8) called double negative (DN) T cells These precursor T cells are called Pro T cells Differentiating Pro T cells express some specific markers 1.C-Kit 2.CD 44 3.CD 25

13. c-Kit- the receptor for stem-cell growth factor CD44-an adhesion molecule involved in homing CD25-the - the IL-2 receptor required for T cell growth and differentiation

14. LOCATION :THYMUS

15. Formation of DN Pre T cell receptor C-Kit expression stop Reduce expression of CD44 T cell proliferation stop and expression of RAG 1 and RAG 2 genes initiate RAG genes code for recombinase enzymes required for TCR gene rearrangement These T cells are DN Pre T cells having 1.V β -D β -J β arrangement of β chain 2.Pre α-chain rearrangement V α Pre T cell 3.CD3 arrangement receptor

16. Successful association of these three arrangement called Pre T cell receptor the DN Pre T cells, failed to productively rearrange and express β chain -died

17. Formation of Double positive T cells Pre TCR receives signal of intra thymus ligands and activates signal transduction pathways for 1.Indicating cell has made productive β chains 2.Suppressing further arrangement of β chains – allelic exclusion 3.induce development of CD4 and CD8 (DP T cells) 4.Renders the cell permissive for α chain rearrangement

18. Structure and activity of the pre– T-cell receptor (pre-TCR)

19. Rearrangement of α chain to form mature T cell DP T cells begin to proliferate again RAG 1 and RAG 2 genes activated earlier RAG 2 proteins start degrading as soon as T cell proliferation start After optimum proliferation clones of T cells produce with single β chain

20. As T cell proliferation stops RAG 2 protein level start increasing those help to arrange the α chain Mature much more diverse population of T cells produced.

21. THYMIC SELECTION OF T CELL REPERTOIRE

22. In T cell development process 98% Thrombocytes don't mature They died by apoptosis due to 1.Fail to produce productive TCR rearrangement 2.Fail to survive thymic selection

23. Central tolerance in thymus Possible outcome: Clonal Deletion Positive selection for thymocytes bearing receptors capable of binding self-MHC molecules, which results in MHC restriction. Cells that fail positive selection are eliminated within the thymus by apoptosis. Negative selection that eliminates thymocytes bearing high-affinity receptors for self-MHC molecules alone or self-antigen presented by self- MHC, which results in self-tolerance.

24. Positive selection Immature thrombocytes TCRs interact with MHC molecules of thymic cortical epithelial cells The interraction allows immature thrombocytes to receive a positive signal prevents cells under going apoptosis Cells not interacting with cortical epithelial cells fail to get positive signals undergo apoptosis

26. Negative selection The thrombocytes surviving positive selection scanned by dendritic cells and macrophages having self MHC I and MHC II molecules alone or with self peptides Thrombocytes interracting with these cells undergo negative selection and died by apoptosis

27. Peripheral Tolerance of mature naïve T cells Naïve T cells are activated in lymph nodes and spleen. Dendritic cells are key antigen presenting cells for naïve T cells. Naïve T cells require MHC/peptide plus antigen-presenting cell "costimulation" in the form of B7 molecules. Only "professional" antigen presenting cells express B7 molecules. And these only express B7 when activated. Antigen stimulation without costimulation can lead to loss of T cell responsiveness and immune tolerance– PERIPHERAL TOLERANCE.

28. Appropriate activation of naïve T cells requires in addition to a ligand for the T cell receptor a second interaction with CD28. This second signal is called "costimulation.” Costimulation by signaling through CD28

29. Only activated antigen presenting cells express B7 molecules which are among the most important costimulatory signals.

30. inactivated In the absence of appropriate costimulation, T cells can become inactivated (anergic)

31. APC TCR T cell CD28 Activated T cells APC TCR Functional unresponsiveness Normal T cell response Anergy Apoptosis (activation-induced cell death) APC Deletion APC Block in activation Suppression Peripheral tolerance Off signals Activated T cell

32. B cell maturation in bone marrow Lymphoid stem cells differentiate and give rise to progenitor B cells (Pro B cells) The marker for Pro B cells is CD 45 R which has tyrosine phosphatase activity Pro B cells proliferate in bone marrow stromal cells to form Pre B cells

33. Function of BM Stromal Cells Directly interact with pro B cells Release IL7 that is required for B cell developement

34. Development of Pro B cells to Pre B cells In Pro B cell stage the Ig heavy chain gene (V H -D H -J H ) rearrangement occurs Interaction of Pro B cells with stromal cells by means of cell adhesion molecules (VLA 4 on Pro B cells and VCAM 1 on stromal cells) After this interaction Pro B cells express a receptor c-Kit

35. C-Kit binds to specific GF ligands from stem cells Initiating signal transduction pathway causing Pro B cells begin to differentiate to Pre B cells

36. Development of Pre B cells to immature B cells Development of Pre B cells to immature B cells required Ig Light chain gene (κ/λ) rearrangement Immature B cells having mIgM (This B cells are immature soimmunologically unresponsive –this state is also called CLONAL ANERGIC)

37. Development of Mature B cells Change in RNA processing of heavy chain primary transcript of immature B cells Co-expression of mIgM and mIgD Fully matured naive B cells, where mIgD are the markers for Naïve matured B cells

38. BONE MARROW SELECTION OF B CELL REPERTOIRE

39. Only 10% of total B cell pool is recruited in the circulation 90% died

40. Central tolerance in BM Possible outcome: Clonal Deletion Negative selection: immature B cell receptor that recognizes self Ag Positive selection: immature B cells those produce Auto Ab