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Chapter 5

Chapter 5. Cell-Mediated Immune Response. Cell-Mediated Purpose. Combat infection of intracellular microbes virally infected cells have virus replicating inside the cytoplasm phagocytes that pick up microbes but they are resistant to destruction, grow in vesicles or cytoplasm

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Chapter 5

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  1. Chapter 5 Cell-Mediated Immune Response

  2. Cell-Mediated Purpose • Combat infection of intracellular microbes • virally infected cells have virus replicating inside the cytoplasm • phagocytes that pick up microbes but they are resistant to destruction, grow in vesicles or cytoplasm • Removal mediated by T-lymphocytes in adaptive immune system • CD4+ helper T-cells help B-cells make Ab (other arm of adaptive immune response) • must interact with other cells such as phagocytes, infected host cells or B-cells • specific for MHC class for the type of T-cell

  3. Types of Intracellular Microbes

  4. Phases of T-Cell Response • Sequential steps that result in increase in the numbers of Ag-specific T-cells and conversion of naive T-cells to effector cells • Naive T lymphocytes circulate looking for Ag, express the receptor but can’t perform the effector function • must be stimulated to differentiate into effector cell (T-helper/CTL) – initiated by Ag recognition • done in peripheral lymphoid tissue • also gets signals from microbes and/or innate immune system

  5. Phases (cont) • Combination of all signals cause Ag-specific T-cells to secrete cytokines • happens to CD4+ and CD8+ cells • cytokine and Ag and microbe act as 2nd signal that causes proliferation to increase the numbers of Ag-specific T-cells • called CLONAL EXPANSION • fraction undergoes differentiation and switch functions from recognizing Ag to effector T-cells to eliminate microbes • as microbe is eliminated, effector T-cells die and return to basal level of lymphocytes

  6. Phases of Activation

  7. Ag Recognition and Costimulation • 3 categories, each with distinct functions • adhesion • recognition • signaling • Requires multiple receptors on T-cell recognizing APC • TCR – peptide/MHC • CD8 or CD4 – MHC • adhesion molecules on T-cell – APC • receptors for costimulators for 2nd signal from APC • Anything other than TCR are accessory molecules • invariant among T-cells

  8. Ag Recognition and Costimulation • CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) • LFA-1 (Leukocyte Function Associated Antigen-1) • ICAM (Intracellular Adhesion Molecule-1) • VLA-4 (Very Late Antigen) • VCAM-1 (Vascular Cell Adhesion Molecule) • B7-1 (CD80) • B7-2(CD86) Know the molecule and ligand

  9. MHC-Associated Peptide Recognition • Initiation signal – TCR and CD8+ or CD4+ receptor recognize peptide-MHC on APC • cytosolic proteins – MHC I – CD8+ CTL • vesicular proteins – MHC II – CD4+ helper • TCR recognizes peptide and AA residues on MHC around peptide binding cleft • CD4 and CD8 are co-receptors on MHC-restricted T-cell – help to bind TCR to MHC • recognize at sites separate from the peptide binding site that helps to ensure the correct T-cell response • Must get 2 or more TCR and co-receptors to bind MHC-peptides to initiate the signaling pathways • must encounter an array of Ag for a long time or multiple times to begin the activation – threshold needed to initiate response

  10. Biochemical Signals • T-cell activation requires proteins linked to TCR to form TCR complex and to CD4or CD8 coreceptor • 2 set of molecules • Ag receptors with much diversity • those that are conserved signals • TCR recognizes the Ag but can’t pass on the signal so TCR associates with CD3 (complex of 3 proteins) and with a homodimer of  chain • now signal can be passed to the interior of the T-cell • TCR, CD3 and  make up TCR complex

  11. Unusual T-Cell Activation • There are chemicals that can bind TCR of many or all T-cell clones regardless of peptide • polyclonal activators are used to study activation, diagnosis diseases of T-cells function, etc • Ab to TCR or CD3; carbohydrate molecules like phytohemagglutinin; super Ag (microbial protein) • Microbial super Ag can cause a huge T-cell response and excess release of cytokines that can make the host very sick

  12. Adhesion Molecules Functions • Adhesion molecule on T cell recognize ligands on APC and stabilize binding of T-cell to APC • TCR:MHC binding usually has low affinity • probably due to selection process • must maintain contact until signaling threshold is achieved • Most important molecules are the INTEGRINS – heterodimeric proteins • Leukocyte Function-Associated Ag-1 (LFA-1) on T-cell binds Intracellular Adhesion Molecule-1 (ICAM-1) on APC

  13. Integrin Avidity • On naïve T-cell, LFA-1 is in a low affinity state • Exposure to chemokines from the innate immune response to Ag allows for the expression of high affinity state – clusters within minutes of TCR:MHC binding • TCR:MHC binds tightly at infection site and is a positive feedback loop • Integrins also aid in directing migration of effector T-cells (Chapter 6)

  14. Role of Costimulators • Full T-cell activation requires costimulators on the surface of APC – provide 2nd stimuli to T-cell • 2 best defined are B7-1 (CD80) and B7-2 (CD86) • both are on professional APC and their expression is increased when in contact with microbe • B7 molecules recognize CD28 expressed on nearly all T-cells and along with TCR:MHC-peptide/co-receptor – essential for activating T-cells • ensures full activation of naïve T-cells • absence of B7 and CD28 interactions = no activation • CD40L (ligand) on T-cells and CD40 (receptor) on APC – not a direct enhancement of T-cell activation • causes the expression of more B7 molecules and activates APC to secrete cytokines • IL-12 enhances T-cell proliferation and makes APC better at stimulating T-cells

  15. Costimulation of T-Cells • Use adjuvants to get a T-cell dependent immune response in vaccines • microbial proteins, usually mycobacterium, that stimulate macrophages and other APS • Understand T-cell activation may have clinical applications • B7:CD28 and CD40L:CD40 inhibitors are in clinical trials to block graft rejection • may eventually be used in tumor biology

  16. Activation of CD8+ T-Cells • Activation of CD8+:MHC-peptide recognition require costimulation and/or helper T-cells • CD8+ CTL in some viral infections require concomitant activation of CD4+ helper T-cell • cross presentation of Ag on dendritic cells – MHC I and MHC II • both CD4+ and CD8+ cells can be activated by 1 dendritic cell • CD4+ cell may produce cytokines or membrane molecules to help activate CD8+ cells – clonal expansion of T-cell and differentiation into effector/memory CTL • may explain CLT deficiency to virus infection in HIV and patients because of lack of CD4+ (cell death by HIV) • Not all viral infection require CD4+ help to make CTL

  17. Limiting and Terminating Immune Response • Proteins homologous to CD28 are critical for limiting and terminating immune responses • Inhibitory receptor CTLA-4 is like CD28 recognizes B7 on APC and PD-1 recognizes different but related ligands on many cell types • induced in activated T-cells • CTLA-4 involved in inhibitory response to some tumors • PD-1 inhibits responses to some infection and infection becomes chronic • Not sure how activation or inhibition is determined

  18. Biochemical Pathways • T-cells express proteins involved in proliferation, differentiation and effector function • naïve T-cells have low levels of expression of proteins • Ag recognition triggers new transcription and translation within minutes • Pathways link Ag recognition with T-cell responses consist of activation enzymes, recruit adaptor proteins and production of active transcription factors

  19. Signal Transduction • Cross-linking of TCR (bringing together multiple TCR) – at or near TCR complex • orderly redistribution of other proteins on APC/T-cell • bring in adhesion and signaling molecules – important for optimal induction of activating signals in T-cells • Cell:cell link is called the immunological synapse • may function to have effector molecules and cytokines in this region or the enzymes needed to degrade or inhibit signal to inhibit lymphocyte activation

  20. Start of Signal • Cluster of CD4 or CD8 co-receptors activate Tyr protein kinase called Lck that is non-covalently attached to the C’ end of co-receptor • T-cell signaling proteins (CD3 and  chain) have immunoreceptor Tyr-based activation motifs (ITAMs) – critical for signaling • Activated Lck phosphorylates ITAMs and becomes a docking site for a Tyr kinase called ZAP-70 (zeta chain associated protein of 70kD) also phosphorylated by Lck, which then phosphorylates various adapter molecules • Ca-NFATpathways; Ras/Rac-MAP kinase; PKC -NF-B pathways

  21. Signal Transduction

  22. Calcium-NFAT Pathway • Nuclear factor of activated T-cells (NFAT) – transcription factor – dependent on Ca2+ ions • ZAP-70 phosphorylates phospholipase C (PLC) which cleaves plasma membrane phospholipid – inositol-phospholipids – making the by-product inositol 1,4,5-triphosphate (IP3) which stimulates Ca2+ release from ER stores • As this occurs, TCR complex signals cause influx of extracellular Ca2+ • Cytoplasmic Ca2+ binds calmodulin and this complex activates a phosphatase – calcineurin – removes a phosphate from inactive NFAT to make it active, moves to nucleus and then binds to the genes for IL-2 and components of IL-2 receptor • Cyclosporin binds and inhibits calcineurin to inhibit T-cell activation against the organ transplant (Chapter 10)

  23. Ras/Rac – MAP Kinase Pathway • Includes GTP binding proteins Ras and Rac that are biologically active when bound to GTP • requires several adapter proteins and a cascade of enzymes that eventually activates one of a family of mitogen-activated protein (MAP) kinase • Initiated by ZAP-70 dependent phosphorylation and accumulation of adapter proteins at membrane • recruit Ras or Rac and activate by exchange of GDP for GTP • Ras-GTP and Rac-GTP initiate different pathways leading to distinct MAP kinases • extracellular signal regulated kinase (ERK) and c-Jun-amino(N) terminal kinase (JNK) promote cFos expression and phosphorylation of c-Jun that creates the transcription factor AP-1 (activating protein-1) that enhances several T-cells genes

  24. PKC  - NF-B Pathway • Actibation of Ser-Thr kinase activates protein kinase C (PKC) and activation of NFB (transcription factor) • PKC is activated by diacylglycerol (2nd by-product of PLC activity) • PKC (isoform of PKC) linked to activation of NFB • NFB is usually inactive in naive T-cells because it is linked to IB (inhibitor) which is released on activation of TCR; NFB moves to nucleus and activates transcription of several genes • Transcription factors lead to production of cytokines, cytokine receptors, cell cycle inducers and effector molecules (such as CD40L)

  25. Other Biochemical Events • 4th pathway • Signal transduction involves lipid kinase called phosphatidylinositol-3 (PI-3) kinase that phosphorylates PIP2 to PIP3 • Activates ser-thr kinase AKT that has many roles • stimulates expression of anti-apoptotic proteins – survival signal • also trigger CD28 and IL-2 receptors

  26. NFAT, AP-1 and NF-B • All are transcription factors • Stimulate production of cytokines, cytokine receptors, cell cycle inducers and effector molecules like CD40L

  27. B7 and CD28 • B7 binding to CD28 is necessary for full T-cell response but signals from CD28 are not well understood • may amplify TCR signals • may initiate own set of signals

  28. Response • Response of T-lymphocytes to Ag and constimulation are mediated by cytokines that are secreted by the T-cell and acts on T-cells as well as other cells of the immune system

  29. Cytokine Activity • Secretion of cytokines and the expression of cytokine receptors • Response to costimulators and Ag • especially CD4+ cells secrete several different cytokines with many activities • effector CD4+ cells secrete other cytokines • Cytokine function as mediators of immunity and inflammation • innate immunity – macrophage secrete • adaptive immunity – T-cells secrete • some similarities but also distinct and have different roles in the immune response

  30. KNOW THIS!!!!!

  31. KNOW THIS!!!!

  32. IL-2 and T-Cell Activation • 1-2 hours after CD4+ cells IL-2 is secreted which enhances the ability of T-cells to respond to IL-2 by way of regulation of IL-2 receptor expression • IL-2 receptor has 3 protein chains, naïve T-cells express 2 of the 3 proteins (c) which can’t bind IL-2 with high affinity • 3rd chain () gets expressed after activation to complete receptor • now have receptor that can strongly bind IL-2, preferentially on the same T-cell • IL-2 stimulates T-cell proliferation – forces cells into cell cycle • acts as a T-cell “growth hormone” • CD8+ T-cell – doesn’t appear to make large amounts IL-2, may depend on CD4+ helper cells to provide IL-2

  33. IL-2 Interactions

  34. Clonal Expansion • Within 1-2 days after activation, T-cells begin to proliferate and start expansion of Ag specific lymphocytes • CD8+ T-cells • before infection 1 in 105to 106 is specific for Ag • after viral infections, 10-20% of all lymphocytes in lymphoid organs may be specific for that virus, an increase of 100,000 fold increase in Ag-specific for that virus • CD4+ T-cells • see only a 100 to 1000 fold increase • Difference may be due to difference in function • CD8+ are effector cells themselves – kill infected cells • CD4+ effector cells secrete cytokines that activated other effector cells; small amount of cytokine needed to cause action

  35. Clonal Expansion Surprises • Expansion is in specific T-cells for Ag but not other T-cells that do not recognize Ag • Even though microbe may be complex usually <5 immunodominant peptides used for expansion

  36. Differentiation of Naïve Cells • Need to become effector cells to eradicate the infection • Changes in gene expression – cytokines in CD4+ and CD8+ cells or cytolytic proteins in CD8+ CTLs • Appear in 3-4 days and leave lymphoid tissue to move to the site of infection • CD4+ and CD8+ perform different functions so differentiation is also different

  37. CD4+ Effector Cells • Respond to Ag by making surface molecules and cytokines to mainly activate macrophages and B-cells • CD40L (ligand) is the most important – expressed after Ag recognition and costimulation • binds to CD40, mainly on macrophages, B-cells or dendritic cells • binding activates macrophages and B-cells • binding of dendritic cells cause expression of co-stimulators and T-cell activating cytokines • interaction produces T-cells activating cytokine causing positive feedback on APC-induced positive cell activation

  38. CD4 Effector Function

  39. Helper T-Cell Subsets • Different types of CD4+ effector T-cells with distinct function • Subsets of effector cells produce distinct sets of cytokines that perform different functions • TH1 and TH2 cells • recently identified group called TH17 because of signature cytokine – IL-17 • regulatory T-cells is also subset (Chapter 9)

  40. Types of CD4+ Cells and Cytokines • TH1 and TH2 cells are distinguished by cytokines but also by cytokine receptors and adhesion molecules they express • Other CD4+ cells produce various mixtures of cytokines – not readily classified

  41. KNOW * ITEMS!!! * * * * *

  42. TH1 Cells • Most important cytokine produced – IFN, called that because it “interfered” with viral infections • potent activator of macrophages • stimulate Ab isotypes that promote phagocytosis by binding the FC receptor • activate compliment and cause phagocytosis by binding phagocyte complement receptors • Stimulate phagocyte-mediated ingestion and killing of microbes • Stimulates the expression of MHCII and B7 costimulators on APC; especially macrophages which display Ags then amplify T-cells responses

  43. TH1 Cells

  44. TH2 Cells • Produce IL-4 stimulates IgE Ab production • Produce IL-5 which activates eosinophils • Stimulates phagocyte-independent, eosinophil mediated immunity • usually against helminthic infections (parasites) • IL-4, IL-10 or IL-13 inhibit macrophage activation and suppress TH1 cell-mediated response (return to in Chapter 6)

  45. TH17 Cells • Secrete cytokines IL-17 and IL-22 • principal mediators of inflammation in a number of immunologic responses • Experimental models suggest TH17 cells involved in defense against some bacterial and fungal infections • Require IL-6 and IL-1 from macrophage and dendritic cell, IL-23 and TGF • Promote recruitment of PMN/monocytes – principal role

  46. Differentiation of TH1, TH2 and TH17 • Not a random process but is regulated by stimuli that naïve CD4+ T-cell receive when they encounter microbial Ag • Macrophage and dendritic cells make IL-12 in response to many bacteria and viruses; NK cells make IFN • T-cell encounters Ag on APC also get exposed to IL-12 • promotes TH1 proliferation and release of IFN to activate macrophages • innate immune response of IL-12 by APC will drive the formation of TH1helper cells (adaptive immune response) • If there is no IL-12 as in helminth infection, then the T-cell will secrete IL-4 and get a TH2 response • may also be influenced by type of dendritic cell that can dictate the TH response • specialization of immune response • Differentiation of one type will inhibit the development of the other one to make most effective response to microbe

  47. TH1

  48. TH2

  49. CD8+ T-Cell Differentiation • Activated by Ag and co-stimulators • Differentiate into CTLs that are able to kill infected cells expressing the Ag • secrete proteins that make pores in membranes of infected cells and induce DNA fragmentation and apoptotic death (Chapter 6)

  50. Development of Memory • Fraction of Ag-activated T-cells differentiate into long-lived memory T-cells – even after infection is eradicated and innate immune reaction to infection pathogen is over • Subsets of memory – not known how it happens • in mucosal barrier is effector memory = rapid effector functions • in lymphoid tissue is central memory cells = populate lymphoid tissue and responsible for rapid clonal expansion after re-exposure • Memory T-cells do not produce cytokine or kill cells • rapidly do so when encounter Ag again

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