Mechanism of immune response ami and cmi

Immunology

1. Mechanism of Immune Response: Antibody mediated Immunity and Cell Mediated Immunity UNDER GARADUATE STUDENT’S PRACTICAL ON BY GUNJAL PN ASSIST. PROF. DEPT OF MICROBIOLOGY DVVPF’S MEDICAL COLLEGE & HOSPITAL AHMENDAGAR 2/17/2021 Department of Microbiology 1

2. Competency MI 1.8 – Describe the Mechanisms of Immune response of the host immune system to infections. 2/17/2021 Department of Microbiology 2 At the end of the session, the students will be able to understand: Antigen Presentation Helper T cells – Activation and Differentiation Cell-mediated Immune Response Humoral/Antibody-mediated Immune Response Learning objectives

3. What is Immune Response? The reaction of the cells and fluids of the body to the presence of a substance which is not recognized as a constituent of the body itself . These are highly coordinated reactions of the cells of immune system and their products. It is divided into two types of mechanisms: Humoral Immune Response OR Antibody Mediated Immune Response (AMI) & Cell Mediated Immune Response (CMI). INTRODUCTION 2/17/2021 Department of Microbiology 3

4. Antibodies provide protection against antigens circulating free or present on surface of host cells and in extracellular spaces. No role against intracellular antigens. Humoral or Antibody-mediated Immune Response (AMI) 2/17/2021 Department of Microbiology 4

5. Cell immediate immunity (CMI) plays an important role in protecting the host against intracellular pathogens as well as tumor cells. Although CMI is mainly T cell mediated (Cytotoxic T cells) and other effector cells such as NK cells (Natural Killer Cells), Macrophages, Granulocytes are various components of CMI. Cell-mediated Immune Response (CMI) 2/17/2021 Department of Microbiology 5

6. CMI and AMI are highly dependent on each other can not function individually. Cytokines released from T cells stimulate B cells to produce antibodies. Many effector cells of CMI, use antibodies as a receptor to recognize target cells to eliminate.(NK, Macrophages) CMI also regulates AMI by releasing cytokines from activated T cells. Which stimulates B cells to transform into antibody secreting plasma cells. 2/17/2021 Department of Microbiology 6 CMI and AMI are Interdependent

7. Common events for induction CMI or AMI. Antigen presentation to Helper T cells. Activation and differentiation of Helper T cells into either T H 1 or T H 2 subsets. Helper T cells are central key decides which immune response to mount. Activated T helper cells differentiated into either T H 1 or T H 2 subsets. T H 1 cells secrets Cytokines – stimulates CMI. T H 2 cells secrets certain cytokines – induce the B cells to produce antibodies. 2/17/2021 Department of Microbiology 7 CMI and AMI are Interdependent

8. Antigen Presentation To stimulate immune responses, reorganization of antigen by T cells is important. T cells can not recognize the naïve or free antigens, but can do so only after antigen is processed into smaller antigenic peptides containing specific epitopes. These epitopes are combined with specific Major Histocompatibility (MHC) molecules (Class I and Class II) and are present on host cell surface. 2/17/2021 Department of Microbiology 8

9. Antigen Presenting Cells (APC’s) Antigen presentation refers to presentation of antigenic peptide to both T H (helper T cell) and T C (cytotoxic T cells) by complexing with MHC – II and I respectively. APC’s implies to those cells only that can present the antigenic peptide along with MHC class II to T H cells. 2/17/2021 Department of Microbiology 9

10. Cells presenting antigenic peptide with MCH-I to T C are not included under APC’s. These are mostly virus infected or tumor cells. Often referred as Target cells as the activated T C cells causes lysis of these cells. Dendritic cells, macrophages and B cells are the major APC’s and known as Professional APC’s. Some are non-professional APC’s can also present antigen to helper T cells. 2/17/2021 Department of Microbiology 10 Antigen Presenting Cells (APC’s)

11. For induction of immune response any CMI or AMI antigen must be presented to T H cells. For induction of CMI, antigen presentation to T C cell is important. Two well defined pathways used by immune system for this. These pathways differ in their mechanism and target antigen. 2/17/2021 Department of Microbiology 11 Antigen Processing Pathways

12. 2/17/2021 Department of Microbiology 12 Antigen Processing Pathways Cytosolic pathway : Endogenous (intracellular) antigens (viral antigens and tumor antigens) are processed Presented along with MHC class I molecules to CD8 T cells. Endocytic pathway: Exogenous antigens (extracellular microbes and their products, e.g. toxins) are processed. Complexed with MHC class II molecules and presented to T H cells. Cells included: APCs such as macrophages, dendritic cells & B cells

13. 2/17/2021 Department of Microbiology 13 Differences between cytosolic and endocytic pathways of antigen presentation Property Cytosolic pathway Endocytic pathway Antigen processed Endogenous Exogenous Antigen is complexed with MHC I molecules MHC II molecules Antigen is presented to T C cells T H cells

14. 2/17/2021 Department of Microbiology 14 Differences between cytosolic and endocytic pathways of antigen presentation

15. Helper T cell (T H ) activation and differentiation is the central event that regulates both the components of immune response; CMI and AMI . Activation of T H cells requires generation of three specific signals. Antigen-specific signal: Binding of antigenic peptide present in the groove of MHC-II on APCs to TCR (T cell receptor) present on surface of T H cells. CD4 molecules of T H cells also interact with β2 domain of MHC-II. 2/17/2021 Department of Microbiology 15 Helper T cells (activation and differentiation)

16. Activation of T H cells requires generation of three specific signals. Co stimulatory signal - Binding of CD28 molecule on T H cells to B7 molecules on APCs. Cytokine signal - APCs (macrophages) secrete interleukin-1 (IL-1) which acts on T H cells. 2/17/2021 Department of Microbiology 16

17. Essential for T H cell activation. Signal transduction is initiated at CD4 molecule which interacts with CD3 complex which in turn transmit the signal leading to activation of T H cells. 2/17/2021 Department of Microbiology 17 Signal Transduction

18. Activated T H cells secrete increased amount of IL-2 as well as IL-2 receptor (IL2R or CD25). IL-2 binds to its receptors on the same T H cell and also on other T H cells and induces the naive T H cells to proliferate and differentiate. T H cells get activated and become lymphoblast cells which subsequently differentiate into memory and effector T H cells. 2/17/2021 Department of Microbiology 18 Differentiation of Helper T cells

19. Derived either from the naive T H cells or pre-existing memory T H cells following antigenic stimulus. Short lived (few days to weeks). Further differentiate into either T H 1 or T H 2 subsets (crucial as they secrete distinct cytokines). Cytokines secreted by T H 1 – stimulate cytotoxic T cells – CMI Cytokines secreted by T H 2 – stimulates B cells producing different class of Abs. (AMI). IL- 12 secreted by macrophages plays an imp. role in differentiation of T H cells and promotes T H 1 subset proliferation. 2/17/2021 Department of Microbiology 19 subs Effector T H cells

20. 2/17/2021 Department of Microbiology 20 Role of cytokines secreted by TH1 and TH2 cells T H 1 cytokines & their functions IL-2 Promotes activation of T H and T C cells. 2. Activates NK cells to become Lymphokine-Activated Killer (LAK) cells. IFN-γ 1. Activates the resting macrophages into activated macrophage. 2. Activates B cells to produce IgG. 3. Promotes inflammation of delayed type of hypersensitivity (along with TNF-β ). 4. Inhibits T H 2 cell proliferation. TNF-β Enhances phagocytic activity of macrophage

21. 2/17/2021 Department of Microbiology 21 T H 2 cytokines & their functions IL-4 1. Inhibits T H 1 cell differentiation. 2. Stimulates B cells to produce IgE and also IgG4 & IgG1 IL-5 1. Enhances proliferation of eosinophils. 2. Both IL-4 & IL-5 together provide protection against helminthic infections and also mediate allergic reaction. IL-6 Promotes B cell proliferation and antibody production. IL-10 Inhibits T H 1 cell differentiation. Role of cytokines secreted by TH1 and TH2 cells

22. Derived from activated T H cell. Longer life span (months to years). They are in resting stage but following subsequent antigenic stimulus, they become activated and differentiated into effector T H cells. Express CD45RO isoform of common leukocyte antigen CD45 as compared to naive T cells which express CD45RA. 2/17/2021 Department of Microbiology 22 Memory T Cells

23. 2/17/2021 Department of Microbiology 23 CELL-MEDIATED IMMUNE RESPONSE

24. Refers to destruction of cells carrying intracellular microbes and other abnormal cells such as tumor cells by various specific and non-specific cells of immune system, of which the most important is cytotoxic T (T C )cells. 2/17/2021 Department of Microbiology 24 Cell-mediated immune response

25. Provides immunity against microbes residing in intracellularly – For obligate intracellular organisms , CMI remains the only effective immune response. (viruses, some bacteria- MTB, Chlamydia and Rickettsia, parasite – Plasmodium, Leishmania, Trypanosoma & Cryptosporidum, Fungi – Pneumocystis) . For facultative intracellular organisms - AMI is active as long as organism is extracellular, once become intracellular CMI takes leading role (bacteria like Listeria, Salmonella & Yersinia and fungi such as Histoplasma and Cryptococcus ). Immunity against tumor cells and other damaged & altered cells. Mediate delayed hypersensitivity (type IV hypersensitivity ). Plays key role in transplantation immunity and graft-versus- host (GVH) reaction. 2/17/2021 Department of Microbiology 25 Role of CMI

26. CMI can be mediated by both Antigen specific and nonspecific effector cells. Perform function by direct killing of target cells (for e.g. virus infected cells or tumor cells). Most imp mediator of CMI- Cytotoxic T cell- which is Ag specific. Non-specific effector cells – Macrophages, NK cells, neutrophils and eosinophils also contribute to CMI. Although CMI has many features different from AMI but not completely independent. The non specific effector cells uses antibodies as receptors to recognize the target cells for killing. 2/17/2021 Department of Microbiology 26 Effector Cells of CMI

27. 2/17/2021 Department of Microbiology 27 Effector Cells of CMI Effector cells of CMI Antigen specificity Cytotoxic T cells Specific NK cells Non-specific Cells performing Antibody Dependent Cell Mediated Cytotoxicity (ADCC) NK cells, macrophages, neutrophil & eosinophils Non-specific

28. Principal effector cells of CMI, involved in the destruction of target cells such as virus infected host cells and tumor cells. Naive T C cells (or CTL precursors) respond to viral or tumor peptide antigens which are processed by the target host cells (by cytosolic pathway) and presented along with MHC class I molecules. Activated T C in turn secretes cytotoxic enzymes that lyse the target cells. 2/17/2021 Department of Microbiology 28 Cytotoxic T Lymphocytes

29. Generation of activated CTL from naïve T C cells requires induction of atleast 3 signals: Antigen-specific signal: Induced by binding of TCR-CD3 complex of naive T C cells to MHC I -peptide complex of target cells. CD8 of T C cells also interacts with α3 domain of MHC-I . Co-stimulatory signal- CD28 of naive T C cells interacts with B7 molecule on target cells. Third signal – IL-2 (secreted by T H cell) acts on high-affinity IL-2 receptor on T C cells. After induction transmission of signals occurs similarly as in TH cells. 2/17/2021 Department of Microbiology 29 Activation of CTL

30. 2/17/2021 Department of Microbiology 30 Activation and differentiation of T C cells

31. Activated T C cells produce two types of lethal enzymes: called Perforins and Granzymes . Perforins produce pores in the target cell membrane; through which granzymes are released inside. Granzymes are serine proteases; they induce cell death by apoptosis, through caspase pathway. 2/17/2021 Department of Microbiology 31 Functions of CTL (Target Cell Lysis)

32. Large granular lymphocytes that constitute 10-15% of peripheral blood lymphocytes. NK cells are cytotoxic but antigen non-specific. Part of innate immunity - act as first line of defense and do not require prior contact with the antigen. Act against virus infected cells & tumor cells till the T C cells are activated and take over the function. 2/17/2021 Department of Microbiology 32 Natural Killer Cells

33. NK Cells Differ from T C cells NK Cell marker: NK cells lacks the T cell markers such as CD3, CD4 or CD8 molecules (hence also know as Null Cells ) instead have specific surface markers such as CD16 and CD56. No MHC Restriction: NK cells can recognise the antigens without MHC presentation. Innate Immunity: These are part of innate immunity. Does not require prior exposure to microbial antigen. No Memory: NK cells do not differentiate into memory cells. 2/17/2021 Department of Microbiology 33

34. 2/17/2021 Department of Microbiology 34 Comparison between NK cells and T C cells Property NK cells T C cells Surface markers CD16 & CD56 CD3, CD8 MHC restriction No MHC-I restricted Memory No Yes Immunity Part of Innate immunity Part of acquired immunity Target cell Virus infected cells Tumor cells Same as NK cells Mechanism of destruction Perforins and granzymes (Constitutive) Same as NK cells (Inducible) Immune response CMI CMI

35. Receptor interaction: Not MHC restricted. Directly recognize certain Ag (e.g. glycoproteins) present on the surface of altered host cells like virus-infected cells or tumor cells. Such Ag (ligands) are also present on normal cells. NK cells - capable of distinguishing normal host cells from the altered cells. Mediated by two types of receptors present on NK cell surface (theory of opposing-signals model). 2/17/2021 Department of Microbiology 35 Mechanism of NK Cell-mediated Cytotoxicity

36. 2/17/2021 Department of Microbiology 36 Mechanism of NK Cell-mediated Cytotoxicity (Cont..) NK cell-mediated cytotoxicity. A. In normal cell; B. In virus infected cell

37. 2/17/2021 Department of Microbiology 37 Mechanism of NK Cell-mediated Cytotoxicity (Cont..) Activation receptors (e.g. NKR-P1, CD16): When these receptors are engaged with Ag (ligands) present on the target cells; NK cells become activated . Inhibitory receptors such as C-type lectin inhibitory receptors: Recognize a part of MHC I molecule (HLA-E) which is present on the surface of all normal nucleated cells. In virus infected cells and tumor cells - MHC-I expression is remarkably reduced. In such cases, there would not be any inhibitory signal . Hence , binding of activation receptor to its ligand leads to activation of NK cells.

38. 2/17/2021 Department of Microbiology 38 Mechanism of NK Cell-mediated Cytotoxicity (Cont..) Target cell destruction: Similar to that of T C cells i.e. via secreting perforins and granzymes. Granzymes forms pore on target cells, through which perforins enter and lyse the target cells . Only difference is that - enzymes are constitutively expressed in NK cell cytoplasm (i.e. they are cytotoxic all the time, even without exposure to the antigen).

39. Alternative Mechanisms of NK Cell Activity : NK cells respond to interleukin-12 (IL-12) produced by macrophages and secrete interferon-γ (IFN-γ), which in turn activates the macrophages. Activated macrophages phagocytose and kill the microbes. NK cells also mediate their function via ADCC. 2/17/2021 Department of Microbiology 39

40. ADCC is exhibited by : NK cells (secrete perforins, and granzymes ) Macrophages ( produce lytic enzymes and TNF ) Monocytes ( produce lytic enzymes and TNF ) Neutrophils (releases lytic enzymes ) Eosinophils (lytic enzymes and perforins and immunity against helminths). 2/17/2021 Department of Microbiology 40 Antibody-dependent Cell-mediated Cytotoxicity (ADCC)

41. 2/17/2021 Department of Microbiology 41 Cytotoxic factors released by various cells in ADCC

42. Mixed-lymphocyte reaction (MLR) – In-vitro system for assaying T-cell proliferation in a cell mediated response . Cell-mediated lympholysis (CML) – In-vitro assay for testing the cytotoxic function of effector cells of CMI . The graft-versus host reaction (GVH) in experimental animals provides an in vivo system for studying cell-mediated cytotoxicity. 2/17/2021 Department of Microbiology 42 Assessment/detection of CMI

43. 2/17/2021 Department of Microbiology 43 HUMORAL/ANTIBODY-MEDIATED IMMUNE RESPONSE

44. AMI provides protection to the host by secreting antibodies – Antibodies prevent against – invasion by microbes present on surface of host cells. Against Ag present in extracellular environments. No role against intracellular microbes. 2/17/2021 Department of Microbiology 44 Humoral/antibody-mediated immune response

45. AMI occurs through the following three sequential steps: Activation of B cells following contact with the microbial antigen (B cells act as APCs). Proliferation and differentiation of B cells into effector cells and memory cells . Effector function- Secreted antibodies by plasma cells which in turn counter act with the microbes in many ways such as neutralization, opsonization, complement activation, etc. 2/17/2021 Department of Microbiology 45 Humoral/antibody-mediated immune response

46. Antigens that activate B cells fall into two categories: Most antigens are thymus dependent (TD ): Activate B cells indirectly via activation of T cells. TD antigens are processed by APCs  presented to T H cells following which the activated T H cells secrets  cytokines that in turn activate the B cells. Thymus independent (TI) antigens (e.g. bacterial capsule) are not processed by APC. They can directly activate B cells without the help of T cell induced cytokines. 2/17/2021 Department of Microbiology 46 Activation of B Cells

47. First step is - Recognition of microbial antigen (TD antigen) by B cell membrane immunoglobulin receptors ( mIg ). Followed by receptor mediated endocytosis of antigen. Then the antigen is processed into smaller antigenic peptides. That are presented in complex with MHC-II to activated T H cells (by endocytic pathway). This leads to induction of signals – signal induction and transduction. 2/17/2021 Department of Microbiology 47 Antigen Presentation of B Cells to Activated T H Cells

48. The naïve B cells are in resting phase activation requires induction of 3 signals - Signal-1 - Induced by the cross linking of B cell membrane immunoglobulin receptor ( mIg ) on B cell membrane with the microbial antigen . Signal-2 - provided by binding of CD40 on B cell with CD40L (ligand) on activated T H cells. Signal-3- Cytokine stimulus. Cytokines produced by the activated T H cells bind to specific cytokine receptor on B cells. 2/17/2021 Department of Microbiology 48 Signal Induction

49. 2/17/2021 Department of Microbiology 49 Antigen presentation of B cells to activated TH cells and signal induction

50. Following the induction of signal, it’s transmission is essential for activation of B cell. Initiated by the B-cell receptor (BCR). The BCR comprises of two parts: Antigen-binding membrane Ig Ig-α/Ig-β heterodimer Following antigen cross linkage to membrane Ig, the Ig-α/Ig-β heterodimer is activated and in turn transmits the signal, ultimately leading to activation of B cells. 2/17/2021 Department of Microbiology 50 Signal Transduction

51. 2/17/2021 Department of Microbiology 51 B cell signal transduction

52. Naive B cells, released from bone marrow go and house in the B cell areas of peripheral lymphoid organs (e.g. cortex of lymph node and marginal zone of spleen)  there, N aive B cells are organized to form primary lymphoid follicles. Following antigenic exposure, naïve B cells activates and proliferates. Eventually, primary lymphoid follicles transform into secondary lymphoid follicles. Secondary lymphoid follicles bear a germinal centre which in turn has two areas; dark zone and light zone. 2/17/2021 Department of Microbiology 52 Proliferation and Differentiation of B Cells

53. Events in Dark zone of Germinal Center The activated B cells differentiated into large dividing cells called centroblasts. Which transform into small non-dividing cells called as centrocytes- expressess mIg membrane Immunoglobulin. 2/17/2021 Department of Microbiology 53

54. Special type of FDC, which differ from the other types of dendritic cells by : Do not act as APCs and do not express MHC class II. Bear Fc receptors that recognize Ag-Ab complex. Antigen is unable to move and is retained in the lymphoid follicle for prolonged periods so that the centrocytes can come and bind to the antigens present in Ag-Ab complex . Allows the FDCs to interact with the centrocytes - results in the selection of the centrocytes with high affinity membrane Ig. 2/17/2021 Department of Microbiology 54 Follicular Dendritic Cells (FDC)

55. 2/17/2021 Department of Microbiology 55 Differentiation of B cells in secondary lymphoid follicles

56. 2/17/2021 Department of Microbiology 56 Cytokines secreted by T H cells and the respective Ig class/subclass they induce Cytokine(s) Ig class produced IFN-γ IgG2a or IgG3 IL-5 + TGF-β IgA or IgG2b IL-4 IgE or IgG1 or IgG4 IL-2,4,5 IgM IL-4,5,6 + IFN-γ IgG

57. Abs secreted from plasma cells mediates a number of immunological functions through their Fc portions that binds to Fc receptor (FcRs) expressed by many cell types. Promotes opsonization FcRs present on phagocyte surface recognize antibody coated microbes, bind to them and that leads to enhanced phagocytosis. 2/17/2021 Department of Microbiology 57 Effector Functions of AMI Opsonization of bacteria and phagocytes

58. Transcytosis Poly Ig receptors are expressed on the inner (basolateral) surface of epithelial cells (facing the blood). They bind to dimers of IgA and multimers of IgM antibodies and transfer them through the cell to their apical (outer) surface and into the lumen of an organ (e.g., the intestine). This process – Transcytosis – responsible for accumulation of Abs in lumen of organ. 2/17/2021 Department of Microbiology 58 Effector Functions of AMI Transcytosis of dimeric IgA

59. Mediates mucosal immunity : Transcytosis of IgA to gut lumen provides mucosal immunity by neutralizing the microbes at local mucosal sites. Activates complement mediated inflammation and cytolysis Antigen antibody complex initiates the complement activation via classical pathway. Final complement factors (C5-C9), also called as membrane attack complex has lethal activity by forming pores on the target cells. 2/17/2021 Department of Microbiology 59

60. Promotes Antibody-dependent Cell-mediated Cytotoxicity ADCC : ADCC is principally cell mediated; antibodies direct the cells to reach to the target cells. ADCC is important to provide immunity against- Helminths (eosinophil-IgE mediated), Tumor cells and virus infected cells (NK cell-IgG mediated ). 2/17/2021 Department of Microbiology 60 Effector Functions of AMI (Cont..)

61. Expected Questions Write assay on: Describe in detail about the mechanism of CMI. Describe in detail about the mechanism of AMI. 2/17/2021 Department of Microbiology 61

62. Multiple Choice Questions (MCQ’s) CMI is by virtue of: A. NK cells B. Eosinophil C. Cytotoxic T Cells D. All of the above Ans: D Perforins are produced by: A. Plasma Cells B. Suppressor T cells C. Cytotoxic T Cells D. Memory helper T cells Ans: C 2/17/2021 Department of Microbiology 62

63. MCQ’s Macrophages are major source of: A. IL-1 B. IL-5 C. IL-7 D. IFN- γ Ans: A Professional antigen-presenting cells (APCs) include all, except: A. Dendritic Cells B. Macrophages C. Fibroblasts (Skin) D. B cells Ans: C 2/17/2021 Department of Microbiology 63

64. MCQ’s Cytosolic pathway of antigen presentation – all are true, except: A. Endogenous antigen processed B. Antigen is complexed with MHC I Molecule C. Antigen is presented to TC Cells. D. Occurs only in antigen presenting cells. Ans: D T H 2 cells secrete all the following cytokines, except: A. IL-2 B. IL-4 C. IL-5 D. IL-6 Ans: A 2/17/2021 Department of Microbiology 64