Chapter 6

1. Chapter 6 Effector Mechanisms of Cell-Mediated Immunity Eradication of Intracellular Microbes

2. Effector Phase of CMI • Carried out by T-cells • NOT antibodies that eradicate intracellular microbes • Phases include • activation • proliferate • differentiate • elimination

3. Types of CMI • 2 types – based on type of intracellular microbes • CD4+ T-cells – activate phagocytes to destroy microbes in vesicles of phagocytes • CD8+ T-cells – kill cells containing microbes or microbial proteins in the cytoplasm • Occasionally overlap in function but fundamentally different mechanism

4. Cell-Mediated Immunity

5. Intracellular Microbes • Many bacteria and some protozoans live in phagocytic vesicles or in the cytoplasm • Viruses that infect phagocytic and nonphagocytic cells and live in the cytoplasm • Naïve T-cell recognizes Ag, differentiate and move to site of infection • Phagocytes ingest microbes – display peptides on appropriate MHC, Ag recognition activates to perform function • MHC II – CD4+; MHC I – CD8+

6. 2 Stages of Ag Recognition • Naïve T cells recognize Ag in lymph node/spleen to become effector cell • Effector T cells recognize same Ag anywhere in the body and eliminate microbe

7. Migration of Effector T-Cells • Movement to the sites of infection • Effector T-cells have many adhesion molecules and cytokine receptors that bind ligands on endothelium on exposure to microbe and chemokine produced at site • Profile of adhesion molecules change in response to differentiation into effector cells • decrease expression of chemokine receptors that hold in T-cell zone and increase expression of sphingosine 1-PO4 so can migrate out of LN

8. Movement of Naïve / Effector T-Cells • Innate immunity responds to infection by responding to secretion of cytokines by macrophages • TNF and IL-1 – act on endothelium cells on small blood vessels adjacent to infection • stimulates endothelium to make more E- and P-selectin and integrins ICAM-1 (intracellular adhesion molecule) and VCAM-1 (vascular cell adhesion molecule) that bind LFA-1 (leukocyte function-associated Ag) and VLA-4 (very-late activation molecules) respectively • Lymphocyte binds weakly and roll along endothelium • comes across ligand and binds tightly and prepare to leave the blood vessel; similar to monocytes ad neutrophils (Chapter 2) • Also lose expression of L-selectin to keep active T-cells out of lymph nodes (L-selectin mediates naïve T-cells to move into lymph node) • Activated T-cells on endothelium – macrophages and endothelial cells produce another set of cytokines called chemokines

10. Glycoproteins on Cell Surface • Attraction in surface molecules between naïve and effector T-cells

11. Chemokines • Attract and stimulate motility of leukocytes • High local concentration – bound to cell surface proteoglycans • Concentration gradient created at extravascular site in response to infectious agents • concentration gradient high at site but lower at blood vessel • draws T-cells through vessel wall to site of infection – follows low to higher concentration

12. Homing Effector T-Cells to Site of Infection • Independent of Ag recognition • dependent on chemokines and adhesions molecules • All T-cells that can recognize different microbial Ag will leave the blood vessel to sites of infection • maximizes ability for specific recognition and elimination • probably will undergo another activation phase to stay in contact and do its job • increase in expression and binding affinity of VLA integrins on T-cells, binds to extracellular matrix and stays close to do job • No recognition – no 2nd activation – go through lymph to lymph node and back to peripheral circulation to home to another site • differentiated T-cells do their job away from the peripheral lymph tissue – not as dependent on co-stimulation as naïve T-cell

13. Migration

14. Effector Function – CD4+ T-Cells • Cell-mediated immunity • Intracellular bacterial infections – found to be able to transfer with T-cells but not serum Ab • specificity due to T-cell but elimination of microbe was by activated macrophages

15. T-Cell Response A – previously exposed T-cells decrease number of bacteria in the spleen, but non-immune do not B – Antibodies to bacteria could not reduce number of bacteria in spleen C – in a test tube, activated macrophages (T-cells turned on) could kill many bacteria but T-cells and resting macrophages could not

16. CD4+ T-Cell – TH1 • Activation of macrophages with phagocytosed microbes to increase the microbiocidal activity to kill the microbe • can actually get by injecting the protein into the person with immunity to that protein • Delayed-Type Hypersensitivity (DTH) happens 24-48 hours after the challenge • delayed because effector T-cell must home to the site of the protein

17. DTH • T-cells and monocytes infiltrate the site • edema, fibrin deposition due to the increased vascular permeability in response to theCD4+ T-cell cytokines and tissue damage by macrophage activation by T-cells • Used diagnostically to determine if previously exposed to antigen • PPD (purified protein derivative) to test for TB

18. Elimination of Phagocytosed Microbes • TH1 subset recognizes macrophage-associated Ag – activate macrophage by CD40L:CD40 interaction • Secrete the macrophage-activating cytokine - IFN

19. Macrophage Activation • Phagosome with the microbes and the lysosome fuse to make the phagolysosome that causes the processing of proteins to be placed in the MHC II • MHC II interacts with CD4+ T-cell causing release of IFN, expresses CD40L which binds to CD40 receptor on macrophage that initiates the biochemical pathways to signal transduction to make transcription factors • interferon can also interact with the macrophage IFN receptor • Gene expression turned on for lysosomal proteases and enzymes – leads to microbicidal reactive oxygen intermediates and NO

20. Macrophage Activation • CD40:CD40L interaction is best because of contact with T-cell; macrophage is also acting as Ag presenting cell • IFN enhances and amplifies response; bidirectional between innate and adaptive immune response

21. macrophage + microbe (innate) activates phagocytes and kills microbe IL-12 INF when encounters Ag on macrophage increase [IFN] stimulate naïve T-cell(CD4+) to TH1 cell (adaptive)

22. CD4+ T-Cell Additional Functions • Ag stimulated CD4+ T-cell secretes TNF – act on vascular endothelium to increase adhesion molecules and production of chemokines • recruits more T-cells, and other WBC to site of infection – enhance phagocytes to help clear infection • TH17 may secrete chemokines to recruit PMNs and monocytes • T-cell stimulated cellular infiltration – inflammation • Helps CD8+ T-cells differentiate to CTLs and B-cells differentiate into Ab-producing • Inflammation = part of DTH sensitivity and innate immune response to enhance T-cell response

23. CD8+ T-Cell • MHC I (cytosolic proteins, may have passed out of the vesicle) can activate macrophage to kill intracellular microbes in the cytoplasm • Similar to CD4+ T-cells • CD40L and IFN- mediated activation • not useful for viruses which replicate in cytoplasm only – mostly because of vesicles is how many macrophages are activated

24. Elimination of Microbes • Done by activated macrophages (Fig 6-7) • Induces expression of enzymes that catalyze production of microbiocidal substances in phagosomes and phagolysosomes • All three activated in the innate immune response with macrophages to microbes • reactive O2 species (ROS) • NO • proteolytic enzyme • TH1 are also potent activators of macrophages mechanism in cell-mediated immunity

25. CMI Importance for Host Defenses • Need CMI when • macrophage not activated by microbes (ineffective innate response) • microbes evolve a mechanism to evade the innate immune response • CMI helps to overcome both of these and change balance back to macrophage/host

26. Macrophage Damage • Macrophage molecules that attack intracellular microbes can also harm normal tissue if released into extracellular milieu • why we see tissue damage in DTH • Can happen when there is a long term CMI response and macrophage activation such as during infections such as with mycobacterium • see granuloma formation • collection of activated lymphocytes and macrophages around microbe with fibrosis and tissue necrosis

27. Other Roles of Activated Macrophages • Secrete cytokines TNF and IL-1 and chemokines • stimulate recruitment of neutrophils, monocytes and effector T-cells to site of infection • Secrete platelet derived growth factor (PDGF) – stimulate fibroblasts and endothelial cells – repairs the tissues after infection • Also increases expression MHC II and costimulators on macrophage which enhance Ag-presentation function and T-cell amplification and CMI

28. Role of TH2 Cells in CMI • Function to enhance eosinophil rich inflammation and to limit the tissue damage caused by macrophages activation • TH2 cells produce IL-4 (IgE Ab) and IL-5 (activates eosinophils) as well as IL-10 which is also produced by other cells • usually helminthic infections • IgE coats helminth, eosinophil recognizes IgE and causes the release of granules that kill the helminth • TH2 cells also secrete IL-4, IL-10 and IL-13 which inhibit macrophage microbiocidal activities – terminated TH1-mediated DTH limiting the tissue injury

29. TH2 and TH1 – relative activation may determine outcome of infection • Activated macrophages are against microbes in vesicles • CTLs are against cytoplasmic and proteins that escape from phagosomes

30. Effector Functions of CTLs • CD8+ CTL recognize MHC class 1-associated peptides and kill these cells • protein Ag synthesized in cytoplasm and those that escape phagocytic vesicles • recognize MHC I peptides on infected cell (target cell) by the TCR on CD8+ T-cell and CD8 co-receptors • cells hold together by integrins on the CTL by binding ligands on the target cell • Ag receptors and co-receptors cluster at site of contact on the target cell – immunological synapse • don’t require co-stimulation or T cell help • can kill any infected cell with appropriate Ag in surface

31. Signal Transduction • Ag recognition by CTL causes signal transduction • causes exocytosis of CTL granule contents which will cause pores to form in the cell • introduce things that can induce DNA fragmentation and apoptosis • Pore forming protein is perforin – inserts into cell membrane and polymerizes by high concentration of Ca2+ ions in extracellular environment to make pore • CTLs secrete granzyme that gets into the cell thru the pores or by receptor mediated endocytosis • granzyme activates caspases that induce apoptosis • caspases are cysteine protease that cleaves at aspartic acid residues

32. Cell Death by 2nd Pathway • Activated CTLs have membrane protein called Fas ligand which binds to death inducing receptor CD95 (Fas) on target cells (Chapter 9) • activates caspases and induces target cell death • DOES NOT require granzyme • minor pathway

33. Net Outcome of Effector Mechanisms of CTLs • Infected cells are killed • Apoptotic cells phagocytosed and eliminated • also kills microbes growing inside of cell • Mechanism inducing DNA fragmentation causes bacterial DNA breakdown • CTLs can detach and go kill another cells • Also secrete cytokine IFN - activates macrophages to destroy phagocytosed microbes and recruit other leukocytes • CD8+ CTLs also help eliminate phagocytosed microbes • like CD4+ T-cells

34. CD4+ and CD8+ Cells • Function together to eradicate intracellular microbes • only in the phagosome – CD4+ T-cell and IFN = macrophage • escape from phagosome – CD8+ CTL

35. Resistance of Microbes to CMI • Different microbes evade the diverse mechanisms to resist T-cell mediated host defense • inhibit the fusion of phagosomes with lysosomes and create pores in phagosomal membranes and escape • many viruses inhibit MHC I expression, block transport of Ag by TAP1 and TAP2 or remove new MHC I molecules from the ER • all lead to reduced peptide loading and viral Ag presentation • some viruses produce inhibitory cytokines or ‘decoy’ receptors to remove IFN to prevent the CMI response • kill cells of the immune system like HIV killing CD4+ cells • viruses can establish chronic nfection by stimulating PD-1 (inhibitory receptor) on CD8+ T-cells – inhibit effector functions

36. !!! !!! !!! !!! !!! Must Know These!!!

37. “Rescue” Mechanisms • NK cells are activated by MHC I deficient cells – help overcome viral immune evasion • Protection from microbes that evade the immune response is through vaccination of host