Generation of The Immune Response

Transcription

1 Generation of The Immune Response

2 Introduction The adaptive immune system recognizes antigens using randomly generated T cell and B cell surface receptors Antigen-specific clonal expansion takes place upon encounter with antigens Adaptive immune responses develop slowly upon first encounter but they develop rapidly in subsequent exposures due to immunological memory

3 T cells play a pivotal role as antigen-specific regulators and sometimes as effector cells The adaptive immune response is largely directed by T cells The nature of the T cell response to antigens is strongly influenced by the nature of the presentation Induction of most adaptive immune responses requires that T cells interact with APCs APCs also provide costimulatory signals for T cells

4 Central Role of Helper T Cells

5 Phases of IR Generation Antigen Processing and Presentation Antigen Recognition and Lymphocyte Activation Recruitment and Stimulation of Effectors

6 Initiation of an Adaptive Immune Response The interface between APC and naïve T cell is called the immunologic synapse The innate immune system is the gateway to the adaptive immune responses Adaptive immune responses are initiated by cells of the innate system Adaptive immune responses require the proteolytic processing of protein antigens that then can be bound by MHC molecules on APCs

7 Antigen Processing and Presentation Whether a peptide fragment is loaded on MHC class I or class II depends largely on the nature of the antigen Exogenous proteins are loaded into MHC class II molecules, whereas those synthesized within the cell s cytoplasm load into class I MHC molecules

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10 MHC Class II and Exogenous Proteins First, they are internalized by phagocytic cells leading to the formation of phagosomes Lysosomes then fuse with the phagosomes forming a phagolysosome that cleaves the protein into small peptides Within the endoplasmic reticulum, MHC class II molecules are synthesized and associated with an invariant (Ii) chain

11 The purpose of the Ii chain is to keep the peptide-binding cleft of class II MHC molecules free of the cell s own peptide, until peptides of exogenous source are available Exocytotic vesicles containing MHC class II-Ii complexes bud off from the endoplasmic reticulum and fuse with peptide-containing phagolysosome

12 Proteolytic enzymes within the vesicle cleave Ii so that only a 24-amino acid peptide known as class II-associated invariant chain peptide (CLIP) remains in the peptide binding cleft Also present within the vesicle is a nonpolymorphic molecule known as HLA-DM which facilitate CLIP removal making the cleft available for binding by exogenous peptides Vesicles containing pmhc class II complexes are transported and displayed on the cell surface

13 The invariant chain Class IIassociated invariantchain peptide (CLIP)

14 The invariant chain prevents MHC class II from acquiring peptides too soon

15 MHC Class I and Endogenous Peptides Cytoplasmic proteins are constantly degraded in all nucleated cells Ubiquitin, a highly conserved 76-amino acid protein, covalently attaches to proteins destined for destruction by the proteasome Postproteasome peptides are transported to the endoplasmic reticulum TAP serves as a gatekeeper to the endoplasmic reticulum

16 Postproteasome peptides of 6-25 amino acids are transported from the cytoplasm into the endoplasmic reticulum by the TAP dimer Newly synthesized MHC class I molecules bind to the calnexin within the endoplasmic reticulum The calnexin-class I polypeptide (Iα) complex allows β2 microglobulin to associate with class Iα On the formation of the Iα:β2 complex, calnexin is replaced with calreticulin and tapasin proteins

17 Tapasin associates the nascent class Iα:β2 complex with the TAP dimer to allow the loading of peptides into the MHC class Iα:β2 complex Peptide loading stabilizes the MHC I:β2 complex; without a peptide, the complex rapidly disintegrates Peptide-class I complexes are transported by specialized vesicles that export molecules to the cell surface for display

18 MHC Class I binds endogenous antigen

19 MHC class I molecules do not leave the endoplasmic reticulum unless they bind peptides

20 Antigen Presentation Pathways

21 The two signal model for lymphocyte activation (antigen alone is insufficient) Memory B cell Antigen TCR T H 2 activation, for example B cell activation, for example (Mature dendritic cell) (Mature naive T cell) (Armed effector T cell) Proliferation and differentiation of the T cell to effector function Proliferation and differentiation of the B cell to effector function Signal 1 comes from recognition of antigen Signal 2 comes from another (activated) cells

22 Antigen Recognition The first step in building the immunologic synapse is TCR recognition of peptide-mhc complex (pmhc) TCR interaction with MHC is stabilized with CD4 or CD8 Responses by CD4+ cells are generally directed to extracellular antigens and those by CD8+ to intracellular antigens Interaction of the pmhc-tcr-cd4 or pmhc-tcr- CD8 complex provides a signal to the T cell

23 Although necessary, this first signal is not sufficient to stimulate naïve T cells to proliferate and differentiate A costimulatory molecule must provide a second signal One of these molecules is CD45 (CLA) which activates kinases such as Fyn Inositol triphosphate kinase (IP3K) in association with CD28 and Fyn activate the guanine-nucleotide exchange factor (GEF) Ras

24 Ras activates the mitogen activated protein (MAP) kinase cascade, ultimately leading to the activation of AP-1, a family of factors that initiate transcription of a number of proteins Mature dendritic cells or activated macrophages express high levels of B7-1 (CD80) and B7-2 (CD86) and may interact with CD28 expressed by naïve T cells to provide a costimulatory signal to the T cell

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26 Other interactions that can provide a costimulatory signal or amplify the immunologic synapse include CD40 and CD154 (CD40 ligand) CD40-CD154 interaction stimulates the expression of CD80/CD86 by APCs In addition, mature dendritic cells and activated macrophages may increase the expression of ICAM-1 that interacts with LFA-1 on T cells

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28 T Cell Signal Transduction The immunologic synapse functions to stabilize noncovalent interactions between APC and T cell and move T cell molecules into close proximity forming a cluster The close proximity of cytoplasmic tails of certain T cell molecules lead to the activation of protein tyrosine kinase and adapter proteins through immunoreceptor tyrosine-based activation motifs (ITAMs)

29 TCR lacks ITAMs and it signals via CD3, where the cytoplasmic portion of its ξ chain contains three ITAMs and each of the other chains contains a single ITAM As the immunologic synapse is formed, CD4 or CD8 engages with class II or class I, which activates a Src family tyrosine kinase, Lck, on the cytoplasmic tail of CD4 or CD8 Activated Lck phosphorylates the ITAMs in the CD3 complex

30 Phosphorylation of multiple ξ chain ITAMs results in binding of the cytoplasmic tyrosine kinase, ZAP-70 ITAM-bound ZAP-70 is phosphorylated by Lck Activated ZAP-70 then phosphorylates the remaining ITAMs on the ξ chain, as well as other molecules Among these are phospholipase C-g (PLC-g) and the linker of activation for T cells (LAT)

31 Phosphatidylinositol 4, 5-biphosphate (PIP2, a membrane phospholipid) is cleaved by PLC-g to form inisitol triphosphate (IP3) and diacylglycerol (DAG) which functions as a secondary messenger PLC-g activation increases intracellular Ca ++ activating calneurin, a calcium dependent phosphatase Calneurin dephosphorylates cytoplasmic nuclear factor of activated T cells (NFAT) required for the activation of cytokine genes such as IL-2 and IL-4

32 DAG in the presence of Ca ++ activates protein kinase C (PKC), that in turn phosphorylates the inhibitor of NF-kB (IkB) that is complexed with nuclear factor kb (NF-kB), a hetrodimeric nuclear transcription factor Once IkB is phosphorylated, NF-kB migrates from the cytoplasm to the nucleus, allowing the activation of a number of cytokine genes ZAP-70 also phosphorylates an adaptor protein, the so called linker of activation for T cells (LAT)

33 LAT activates small guanine-nucleotide exchange factors (GEFs), such as rat sarcoma (Ras) and Ras-related C3 botulinum toxin substrate (Rac) proteins These proteins activate a cascade of intracellular enzymes through either MAP kinase or stress-activated protein (SAP) kinase pathways This ultimately leads to the activation of the activator protein-1 (AP-1) family of transcription factors and the initiation of transcription of specific genes

34 The activation of transcription factors leads to the synthesis of a number of cytokines and receptors, as well as T cell proliferation and differentiation The nature of the resulting immune response depends on a number of factors that are yet poorly understood

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36 Activated or Effector T Cells Initial recognition of antigen by naïve T cells, called priming, is crucial for the proliferation and differentiation of T cells One developmental pathway results in the differentiation of Th1 cells that generally responds to intracellular pathogens by recruiting and activating macrophages and monocytes The second results in the differentiation of Th2 cells that generally respond to extracellular pathogens by stimulating B cells to differentiate into plasma cells and secrete antibody

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38 Antigen Recognition by B Cells Unlike the TCR, BCR recognizes epitopes on soluble molecules without involvement of other cells or molecules BCR is a membrane-bound IgM and IgD that is associated with invariant Igα and Igβ molecules Cytoplasmic tails of Igα and Igβ contain ITAM motifs Antigen-induced cross-linking of BCRs clusters Igα and Igβ molecules and leads to ITAM phosphorylation by sarcoma (Src) kinases (e.g., Lyn, Blk, or Fyn)

39 Cross-linking by BCR requires identical epitopes The phosphorylated ITAMs provide a docking site for the Syk tyrosine kinase, a B cell equivalent of T cell ZAP-70 Syk phosphorylates itself and a number of downstream signaling molecules, including PLC-g and adaptor proteins Plasma membrane PIP2 is cleaved by PLC-g into IP3 and DAG

40 IP3 increases cytoplasmic Ca ++ and DAG induces enzymes such as PKC to activate transcription factors, including NFAT, NK-kB, Myc, and AP-1 In addition, PKC phosphorylates serine - threonine residues BCR cross-linking results in the Ras phosphorylation and leads to the activation of MAP kinase

41 B cells may be activated by the interaction of the BCR and complement receptor 2 (CR2) CD19 has a cytoplasmic tail with ITAM motifs and is associated with CR2 on the B cell surface Engagement of a microbe by BCR and of the microbebound C3b by CR2 results in ITAM phosphorylation of Igα, Igβ, and CD19, and subsequent B cell activation BCR-bound antigens are internalized by endocytosis, enzymatically degraded, and displayed on the B cell surface as pmhc class II molecules

42 Activated B cells enter the cell cycle and increase their expression of CD80/86, IL-2R, IL-4R, and antiapoptotic proteins They now await a T cell signal Communication between B and T cells determines what antibodies will be generated against the appropriate antigen Activated B cells express both pmhc class II and CD80/86 The CD4+ T cell recognizes both pmhc class II by the TCR and CD80/86 by CD28

43 These two signals stimulate the T cell to express CD154 (CD40 ligand) and to secrete cytokines The T cell cytokines induce B cell CD40 expression CD40:CD154 signaling causes B cell proliferation and differentiation The T cell cytokines serve to stimulate B cell proliferation and differentiation into plasma cells and promote isotype switching

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45 T cell help to B cells Signal 2 - T cell help B Th Th Y 1. T cell antigen receptor Signal 1 antigen & antigen receptor 2. Co-receptor (CD4) 3.CD40 Ligand

46 Signal 2 T cell help - Signal 2 Cytokines IL-4 B Th IL-5 IL-6 IFN-g Cytokines TGF-b Signal 1 B cells are inherently prone to die by apoptosis Signal 1 & 2 unregulate Bcl-X L in the B cell and Bcl-X L prevents apoptosis Signal 1 & 2 thus allow the B cell to survive T cells regulate the survival of B cells and thus control the clonal selection of B cells Y

47 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 B Y Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 T cell help - Signal 2 activates hypermutation Day 6 Signal 2 Signal 1 Deleterious mutation Beneficial mutation Neutral mutation Th Receipt of signal 2 by the B cell also activates hypermutation in the CDR - encoding parts of the Ig genes Day 8 Day 12 Day 18 Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection Signal 2, and thus T cells, regulate which B cells are clonally selected. Low affinity Ig takes up and presents Ag to T cells inefficiently. Inefficient presentation to T cells does not induce CD40. With no signal 2 delivered by CD40, low affinity B cells die. Only B cells with high affinity Ig survive - This is affinity maturation

48 Control of Affinity & Affinity Maturation Five B cell antigen receptors - all specific for, but with different affinities due to somatic hypermutation of Ig genes in the germinal centre B B B B B Only this cell, that has a high affinity for antigen can express CD40. Only this cell can receive signal 2 Only this cell is rescued from apoptosis i.e. clonally selected The cells with lower affinity receptors die of apoptosis by neglect

49 Effector Functions of the Adaptive Immune System Cells of the adaptive immune system often use cells and molecules of the innate system to focus and deliver a fatal blow to an invader The adaptive immune system amplify and intensify effector functions of the innate system The terms cellular and humoral adaptive immune responses are actually misnomers, all immune responses have a cellular basis

50 Cell-Mediated Immune Responses Delayed Type Hypersensivity - It is a reaction localized to the immediate area surrounding the site of injection that develops within hours - It is characterized by localized induration and erythema, and may lead to necrosis and ulceration - It develops in response to intracellular antigenic insult being mediated by CD4+ Th1 like contact dermatitis - Macrophages and monocytes are recruited and activate killing of both infected and normal cells

51 Leukocyte Migration Activated Th1 cells, showing increased levels of integrins, namely LFA-1 and VLA-4, are present in circulation Phagocytes within tissues at site of infection secrete TNF which signals nearby epithelial cells to increase the levels of selectins and ligands for LFA-1 and VLA- 4 on their luminal surfaces Interaction between adhesion molecules and their ligands allow activated Th1 cells to localize at the epithelium overlying the site of inflammation followed by their migration to that site

52 Macrophage Activation On arrival at the inflammation site, Th1 cells interact with macrophages Recognition of pmhc by Th1 TCR causes Th1 to express CD154 and secrete IFNg activating the macrophage The activated macrophage upregulates CD80/86 expression, secretes IL-1, IL-12, and TNF, increases MHC class II expression, and increase the production of degradative enzymes and reactive oxygen intermediates (ROI)

53 Functions of Activated Macrophages The activated macrophage is a killing machine It increases the production of lysosomal enzymes and ROIs IL-1 and TNF attract polymorphonuclear cells to the site Activated macrophages and neutrophils remove cellular debris, infected cells, and even some normal cells Activated macrophages stimulate repair by secreting platelet-derived growth factor, TGFβ, and fibroblast growth factor

54 Cytotoxic T Cell Responses Target cell recognition subsequent to pmhc-tcr- CD8 interaction leads to upregulation of Fas ligand Contact between the CTL and target cell lasts for only a few minutes, during which a fatal hit is delivered to the target cell Perforins and granzymes are released upon contact with the target cell Perforin creates osmotic imbalance and granzymes enter through pores to activate caspases leading to apoptotic lysis

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56 Humoral Immunity Antibody Production T-Dependent Antigens: Antibody production requires assistance from T helper cells. Macrophages ingest antigen and present it to T H cell. T H cell stimulates B cells specific for antigen to become plasma cells. Antigens are mainly proteins on viruses, bacteria, foreign red blood cells, and hapten -carrier molecules. T-Independent Antigens: Antibody production does not require assistance from T cells. Antigens are mainly polysaccharides or LPS with repeating subunits (bacterial capsules). Weaker immune response than for T-dependent antigens.

57 Humoral Response to T Dependent Antigens

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59 T Independent Antigens (TI-1) LPS LPS binding protein TLR 4 CD14 Bacterial Lipopolysaccharides, (TI- 1 antigens), bind to host LPS binding protein in plasma LPS/LPSBP is captured by CD14 on the B cell surface Toll - like receptor 4 (TLR4) interacts with the CD14/LPS/LPSBP complex B Cell Activation of B cell

60 T Independent Antigens (TI-1 e.g. LPS) LPS complexes with CD14, LPSBP & TLR4 B B B B B B Y Y Y Six different B cells will require 6 different antigens to activate them At high dose TI-1 antigens (like LPS) will POLYCLONALLY ACTIVATE all of the B cells irrespective of their specificity. TI-1 antigens are called MITOGENS Y Y Y Y YY Y YY YY Y YY YY Y YY YYY YY YY Y YY YY YY

61 Why are babies unresponsive to TI-2 antigens? In adults: TI-2 Antigen Immature B-2 Cell Y Y Mature B-1 Y Adult immature B cells that bind to multivalent self Ag undergo apoptosis Y Y Y IgM Adult non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent epitopes produce IgM WITHOUT T cell help.

62 Why are babies unresponsive to TI-2 antigens? In babies: All B cells, B-1 & B-2, are immature TI-2 Antigen Immature B-1 Cell Y Y Immature B cells that bind to multivalent self Ag undergo apoptosis As with adult B cells, immature B cells that bind multivalent self Ag undergo apoptosis Hence babies do not respond to TI-2 antigens. Babies are, therefore susceptible to pathogens with multivalent antigens such as those on pneumococcus

63 Humoral Immune Responses Once secreted by plasma cells, antibodies can mediate a variety of antigen specific responses: - Formation of immune complexes with soluble or insoluble antigens - Activation of the complement pathway - ADCC (NK cells and Eosinophils) - Sensitization of Mast cells (IgE) - Fetal protection (maternal IgG) - Protection of mucosal surfaces (IgA)

64 Immunologic Memory Second encounter with an antigen generates an immune response rapidly This long-lasting ability to recall an immune response is called memory, a hallmark of the adaptive immune system Antigen specific cells that have been clonally expanded and have undergone some activation previously can be mobilized rapidly The new products, in the case of antibodies, have increased affinity and remove and destroy the antigen quickly

65 To permit these accelerated responses, several of the rules governing the activation of naïve cells have been relaxed for those that are already activated Antigen Presentation: - Unlike antigen-naïve CD4 + T cells, memory T cells do not need to undergo several rounds of division - Memory CD8 + T only need to recognize pmhc class I and do not require costimulation - Essentially, activated CTLs have been given license to kill without any further consultation by another leukocyte

66 Effectiveness of memory More responder cells available: Frequency higher than naïve cells More efficient antigen recognition/activation: May not require costimulatory signals for activation Rapid and effective migration to tissues and lymph nodes: Expresses different homing chemokine receptors than naïve T cells More effective function: Produce qualitatively and quantitatively more cytokines (T cells) or antibodies (B cells) Longer lasting: Naïve cells live for few days/months; memory cells persist for years

67 Secondary T Cell Responses The initial activation of antigen-naïve T cells increases their numbers by more than 1000-fold Many of these cells will undergo apoptosis once the antigenic threat is removed from the body A number of effector and/or memory T cells will persist at levels fold greater than their antigen-naïve predecessors

68 Because effector T cells and long-lived memory cells have very similar, if not identical, surface molecules, it has been very difficult to distinguish one from the other Both are, however, differentiated from antigen-naïve T cells by the type of CD45 isoform expressed on cell surface Antigen-naïve T cells express the greater molecular weight isoform CD45RA, whereas the lower molecular weight isoform, CD45RO is expressed in greater density by effector /memory T cells

69 Secondary B Cell Responses The initial humoral response can be divided into four distinct phases; lag, log, plateau, and decline B cells undergo expansion during the lag phase Antibody production and increase in titer takes place during the log phase to reach a plateau in 10 to 14 days with a lag period of 5 to 7 days in primary responses Low affinity IgM predominates in the primary immune response Duration of the plateau varies depending on many factors

70 Secondary B cell responses have a greatly reduced lag phase, an accelerated log phase, and a plateau phase in which Ab titers can reach 1000-to fold greater than those of primary response The decline phase is prolonged because of the large amounts of antibodies produced Secondary responses are dominated by IgG Depending on the nature of the antigen, IgA and/or IgE may be produced in large amounts

71 Schematic representation of memory response (B cell)

72 In responding to a secondary antigenic insult, B cells undergo hypermutation of their variable region This leads to additional variation and effective fine-tuning the fit of the antibody to a particular antigenic epitope which is referred to as affinity maturation