ALLAH does not want from you to be the BEST ALLAH wants from you to do your BEST

1. بسم الله الرحمن الرحيم ALLAHdoes not want from you to be theBEST ALLAHwants from you to do yourBEST ALLAHwill take care of theREST AND

2. Cell-Mediated Immunity( CMI ) Hazem M. Abu-Eisha Ph.D , MD Assistant Professor of Immunology Pathology Department King Saud University King Khaled University Hospital E - mail : hmt149@yahoo.com

3. Outlines • General considerations • Effector cells of cell mediated immunity • T - lymphocytes & Natural killer cells • Mechanism of cytotoxicity • Antibody dependent cell - mediated cytotoxicity • Delayed type hypersensitivity reaction • Assessment of cell mediated cytotoxicity

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5. General Considerations

6. Principal role of cell mediated immunity ( CMI )is to eliminate cells that harbor intra-cellular pathogens • Target cells are • Two types of cells contributing to CMI

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10. T - cell subsets About 90 - 95 % of T - cell are αβ which are divided into two distinct populations 1.One carries CD4 marker ( CD4+ T helper - cells ) mainly help or induce the immune response & recognize antigens with MHC class II molecules . CD4 TH - cells can be subdivided into two subsets depending on cytokines profiles they produce : CD4 TH1 - cells : Secrete IL-2 & IFN-γ CD4 TH2 - cells : Secrete IL-4, IL-5, IL-6 & IL-10 2. The other set carries CD8 marker (CD8+ T cytotoxic - cells ) & mainly cytotoxic that recognize antigens with MHC class I molecules

11. NK - cell Lineage • NK - cells are lymphoid cells derived from HSC in BM • It is large granular lymphocytes & represent about 5 - 10 % of circulating lymphocyte population • NK cells express CD16 & CD56 ( CD16 is a membrane receptor of IgG so, they can attach to these Abs & destroy the target cells ) This process is called ( ANTIBODY DEPENDENT CELL - MEDIATED CYTOTOXICITY : ADCC ) • It is considered the first line of defense against viral infection until complete activation , proliferation and differentiation of pCTL - cells into functional CTLs after about 7 - 10 days.

12. Natural killer (NK) and natural killer T (NKT) cells bridge both adaptive and innate immune systems. NK cells are characteristically large granular lymphocytes that express neither TCRs nor BCRs and bear receptors for stress molecules (killer activation receptors or KARs) and for MHC class I molecules (killer inhibition receptors or KIRs). Unlike NK cells, NKT cells express low levels of TCRs with extremely limited repertoires

13. NK - cells differences from CTLs • No expression of antigen specific T - cell receptors or CD3 molecules • Recognition is not MHC - restricted • No immunological memory

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15. General Properties Of Effector T- Cells • Activation ( Three signals ) • Cell adhesion molecules • Effector molecules ( Soluble & Membrane - bound )

17. Cell-Adhesion Molecules FacilitateTCR-Mediated Interactions

19. CTL Generation Generation of mature CTLs from CTL-Ps needs at least three sequential signals : • An antigen - specific signal 1 transmitted by TCR complex following recognition of MHC class I peptide complex • A co-stimulatory signal transmitted by CD28-B7 interaction of CTL-P and APC • A signal induced by the interaction of IL-2 with high- affinity IL-2 receptor that leads to proliferation & differentiation of antigen- activated CTL-P into an effector CTL

21. Mechanism of Target Cell Destruction By CTL Primary events include : • Conjugate formation • Membrane attack • CTL dissociation • Target cell destruction

22. 2 2 1 1 4 3 3 5 4 Target cell destruction

23. Features of CTLs - Mediated Lysis • CTL killing is : ANTIGEN - SPECIFIC The target cell must bear the same MHC class I associated antigen which trigger pre-CTL differentiation • CTL requires : CELL CONTACT Adjacent cells lacking the appropriate target MHC-antigen are not affected • CTLS are : NOT AFFECTED BY LYSIS OF TARGET CELLS They are capable of killing another numerous target cells

24. The main two mechanisms responsible for CTL- mediated apoptotic death of target cells are : • Delivery of cytotoxic proteins ( Granzymes & perforins ) released from CTLs to target cells • Interaction of membrane bound FAS ligand on CTLs with FAS receptor on the surface of target cells

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28. Antibody – Dependent Cell - Mediated Cytotoxicity ( ADCC ) • Certain types of cells : Neutrophils, Eosinophils, NK cells & macrophages have the ability to lyse target cells express membrane receptors for Fc region of antibody molecule • These cells are not specific against this antigen ( Virus or parasite ) • ADCC does not involve complement - mediated lysis but appears to involve a number of different cytotoxic mechanisms similar to that performed by cytotoxic T - cells including release of lytic enzymes, perforin , TNF - α & granzymes

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31. Delayed Type Hypersensitivity ( DTH ) It was first described by Robert Koch in 1890 when he noticed that persons infected with Mycobacterium Tuberculosis developed a localized inflammatory reaction after intradermal injection of purified protein derivatives ( PPD ) of Tuberculin ( Tuberculin Reaction )

32. Phases of DTH Response • Sensitization phase • Initial sensitization phase of 1-2 weeks after 1ry contact with Ag. During this period , TH cells are activated & clonally expanded by Ag presented with MHC class II on APC • CD4 - TH1 cells are the main cells activated in this phase which are designated T DTH cells . In few cases CD8+ T -cells have been shown to induce a DTH response

33. Phases of DTH Response 2. Effector phase • Subsequent exposure to this Ag induce the effector phase of DTH reaction where T DTH cells secrete various cytokines that leads to activation & recruitment of macrophages • The reaction is usually manifested after 24 hr and reaches its maximum after 48 - 72 hr following exposure which reflect the time needed for cytokines to induce localized influx of macrophages and their activation

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35. Cytokines involved in DTH Reaction • Cytokines released in DTH reaction suggest that TDTH - cells are mainly of TH1 - cell type • It includes { IL-2 , which acts in autocrine fashion, IL-3 , GM-CSF, IFN-γ & TNF-β in addition to Macrophages derived TNF-α & IL-1 }

36. Activated macrophages secrete IL-1 , IL-12 & TNF-α with increasing expression of MHC class II , degradative enzymes & reactive oxygen species Activated macrophages are highly efficient in antigen presentation than resting one which in turn activate more T - cells that secrete more cytokines which recruit and activate more macrophages ( VICIOUS CIRCLE ) The activated macrophages act as a killing machine destroying everything that comes within its “ GRASP ”

37. Various intracellular pathogens & contact Ags can induce DTH reaction • Cells harboring intracellular pathogens are destroyed by lytic enzymes released from activated macrophages

38. Protective role of DTH The response to M. tuberculosis is the best example of DOUBLE EDGED NATURE of DTH RESPONSE where : Macrophages wall off the organism in the lung and contain it within a granuloma type lesion called TUBERCLE ; but the concentrated & extensive release of lytic enzymes within tubercles leads to EXTEVNSIVE DAMAGAE OF LUNG TISSUES

39. Pathologic DTH Responses • In certain cases, DTH response to intracellular pathogens can cause extensive tissue damage that represent a hypersensitivity reaction • Example : Tissue damage that occurred in Tuberculosis due to accumulation of activated macrophages with destruction of lung tissue due to release of lysosomal enzymes

40. Detection of DTH Reaction • Experimentally , intradermal injection ( ID ) of Ag & observing for presence of characteristic skin lesion at the site of injection • Positive skin - test reaction indicates that the person has a population of sensitized TDTH cells specific against this Ag • Example : PPD Test ( Tuberculin Test ) Disadvantages : The test can not differentiate if the response due to previous immunization or due to exposure to a pathogenic form of M. Tuberculosis

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42. Assessment of Cell - Mediated Cytotoxicity • Mixed Lymphocyte Reaction ( MLR ) • Cell - Mediated Lympholysis ( CML ) Chromium release assay

43. Chromium Release Assay • ( Cell - Mediated Lympholysis ) • This test assesses the functions of cytotoxic T lymphocyte (CTL), natural killer (NK), and NK-like T (NKT) cells • Target cells are incubated with radioactive heavy metal (e.g., Na251CrO4) • Na251CrO4 strongly binds to cytosolic proteins within the cells • Test or effector cells are coincubated with radiolabeled target cells at different effector-to-target cell ratios • If present, CTL, NK, or NKT activity causes lysis of the target cells and release of radioactivity into the medium • Cells are separated, and the amount of radioactivity released into the medium is measured to indicate lytic activity

44. TEXT BOOK FOR ADVANCED READING Kuby Immunology 4th edition Richard A. Goldsby , Thomas J. Kindt and Barbara A. Osborne ISBN 0 - 7167 - 3331 - 5

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