Cellular Basis of the Immune Response

1. Cellular Basis of the Immune Response Robert Fleischmann, Ph.D. Professor Department of Urologic Surgery Office: G144 Diehl Hall Telephone Number: (612) 626-5034 E-Mail Address: rfleisch@umn.edu

2. Objectives • To understand the major players in adaptive immunity • To understand the nature and general functions of cytokines • To understand the identity and functions of the cytokines that are most important for adaptive immunity • To understand the differential regulation for induction of Th1 and Th2 cells • To understand the general features of cytokine/cytokine receptor interactions • To understand the mechanisms by which interferons, as model cytokines, activate their target cells • To understand the positives and negatives of the clinical use of interferons, as model cytokines

3. Jose Jimenez, age 3 months, is brought to the clinic with an extensive fluffy white growth in his mouth. Jose has a history of viral and bacterial infections that never seem to quite go away. Indeed, he has had diarrheal disease for the last two months that has caused him to lose weight. What do you think is happening to Jose? What tests would you wish to run?

4. Jose is diagnosed with Candida albicans. He is evaluated for immunodeficiency disease. His total WBC count is low normal. His differential count shows a preponderance of PMNs with very few lymphocytes. He has a normal level of IgG and an absence of IgM and IgA. Exposure of his lymphocytes to T cell and B cell mitogens fails to give proliferation. What would you wish to do next?

5. Fluorescence cell sorting of his lymphocytes shows a complete absence of CD3 cells and of CD16 cells. All of his lymphocytes appear to be positive for CD20. What do these results mean?

6. The fluorescence cell sorting results indicate that Jose has non-functional B cells but has no T cells nor NK cells. What would you do next?

7. Cells of the Immune System

8. Overview of Major Players of Adaptive Immunity • Dendritic cells, macrophages, and B cells are professional antigen-presenting cells. • CD4+ T cells (Th cells) are exposed to antigen bound to MHC Class II on antigen-presenting cells and are driven to differentiate into Th1 or Th2 cells. • Th1 cells drive the differentiation and proliferation of CD8+ cytotoxic T cells (CTLs). • Th2 cells drive the differentiation and proliferation of B cells to become antibody-producing plasma cells.

9. Activated Lymphocytes Differentiate Into Effector Cells • Note how the lymphocyte enlarges as it differentiates. • Note that T and B lymphocytes cannot be visually distinguished from each other until they are fully differentiated to their respective effector cells.

10. Lymphocyte Differentiation and Proliferation Is Driven by Cytokines • Cytokines are proteins that help the cells of the immune system to communicate with one another. • Cytokines are usually soluble mediator molecules, though some may remain cell-bound.

11. Cytokines Are Often Named for the Type of Cell that Produces Them • Macrophages: monokines • Lymphocytes: lymphokines or interleukins • Generic: cytokines

12. Individual Cytokines Activate Target Cells by Binding to Their Specific Receptors • Each cytokine binds to its unique receptor. • Binding affinities are very strong, with dissociation constants in the range of 10-10 to 10-12 (Abs are in the range of 10-7 to 10-10). • The receptor may be composed of multiple subunits with one specific subunit and one or more common subunits (also shared as subunits of receptors for other cytokines).

13. Cytokine Receptor Expression May Be Regulated • Some cytokine receptors are constitutively present but may be down-regulated by their specific cytokine ligand or by other cytokines (example: IFN receptors). • Some cytokine receptors are induced by treatment of target cells with other cytokines (example: IL-1 induces resting T cells that express the intermediate-affinity IL-2 receptor [composed of and  chains] to express the high-affinity receptor [composed of , , and  chains]).

14. Cytokine Cell Targets • Autocrine action: the target cell of the cytokine is the same as the producing cell. • Paracrine action: the target cell of the cytokine a nearby cell that is different from the producing cell. • Endocrine action: the target cell of the cytokine is a distant cell that is reached through the circulation.

15. Autocrine Action of Cytokines 1. A Th1 cell is stimulated to produce mRNA for IL-2. 2. The IL-2 mRNA is transported to the cytoplasm where it is translated into IL-2 protein. 3. The IL-2 is secreted. 4. The IL-2 binds to a receptor on the producing Th1 cell, activating it. CD4+ Th1 cell

16. Paracrine Action of Cytokines CD8+ T cell 1. A Th1 cell is stimulated to produce mRNA for IFN-. 2. The IFN- mRNA is transported to the cytoplasm where it is translated into IFN- protein. 3. The IFN- is secreted. 4. The IFN- binds to a receptor on the CD8+ T cell, activating it. CD4+ Th1 cell

17. Endocrine Action of Cytokines 1. A macrophage is stimulated to produce mRNA for GM-CSF. 2. The GM-CSF mRNA is transported to the cytoplasm where it is translated into GM- CSF protein. Stem Cell 3. The GM-CSF is secreted. 4. The GM-CSF travels through the blood to the bone marrow where it binds to a receptor on a hematopoietic stem cell, activating it. Macrophage

18. General Properties of Cytokines • Cytokines secretion is generally a brief, self-limiting event. • Secreted cytokines generally have their greatest effects in the immediate area where they are produced. • Cytokines are active at picomolar concentrations.

19. Cytokine Secretion Is a Brief, Self-Limiting Event • Induction of cytokine mRNAs is a transient event. • In general, cytokine mRNAs are unstable and have a short half-life. • After their translation, most cytokines are rapidly glycosylated and secreted.

20. Individual Cytokines May Be Produced by A Single Cell Type or by Many Cells Types • Single Cell Type • IL-2 is produced by Th1 cells. • IL-4 is produced by Th2 cells. • Multiple Cell Types • GM-CSF can be produced by T cells, macrophages, endothelial cells, and a wide range of fibroblasts. • IFN- can be produced by virtually all fibroblasts and epithelial cells.

21. Individual Cytokines May Act on a Single Cell Type or on Many Cell Types • Single Cell Type • IL-10 acts on macrophages to block IL-12, subsequently blocking Th1 cells. • IL-12 acts on Th1 cells. • Multiple Cell Types (Pleiotropism) • IFNs act on multiple cell types. • IL-1 acts on T cells, B cells, macrophages, endothelial cells, fibroblasts, and epithelial cells.

22. An Individual Cytokine May Have Multiple Effects on a Target Cell: Pleiotrophy • IFN- • Induces antiviral proteins • Induces antiproliferative effects • Induces MHC Class I antigens • Stimulates NK cell activation • Stimulates IL-12 production.

23. Different Cytokines Can Share Similar Activities (Redundancy) • IFN-, IFN-, and IFN- share similar activities, while retaining some unique properties. • IL-1, TNF, and IL-6 can all cause fever. • IL-2 and IL-15 share similar abilities to activate T cell proliferation and differentiation, while retaining some unique properties.

24. Individual Cytokines May Influence the Action of Other Cytokines • They may have antagonistic effects. • IL-12 stimulates Th1 cell activation and proliferation, while IL-10 blocks macrophage production of IL-12 and therefore blocks Th1 cell activation and proliferation • They may have additive effects. • IFN- plus IFN-have additive effects. • They may have synergistic effects. • IFN- plus IFN- or IFN- plus IFN- have synergistic effects.

25. Cytokine Attributes

26. Cytokines Belong to Five Families • IL-1 family • Hematopoietin family • Interferon family • Tumor necrosis family • Chemokine family

27. Most Cellular Responses to Cytokines Require mRNA and Protein Synthesis • Cytokine binds to receptor. • A transmembrane alteration occurs allowing the intracellular part of the receptor to be activated. • Second messenger molecules are activated. • Second messenger molecules directly or indirectly activate transcription of specific genes. • Translation of the mRNA for the specific genes results in new cell function.

28. Many Cytokines Act as Growth Regulators • CSFs stimulate bone marrow stem cells to grow. • IL-2 and IL-15 stimulate Th1 cells to grow and IL-2 stimulates Th2 cells to grow. • IL-10 blocks Th1 cell growth by blocking macrophages from making IL-12. • IFN- blocks Th2 cell growth by blocking Th2 from making IL-4.

29. How Do the Actions of Cytokines Affect Immunity?

30. Cytokine Actions • Some cytokines are involved in mediating and regulating innate immunity (Pro-inflammatory cytokines). • Some cytokines are involved in mediating and regulating specific immunity. • Some cytokines are involved in mediating and regulating both innate immunity and specific immunity.

31. Most Important Cytokines

32. IL-2 IL-3 GM-CSF IFN- CD4 Th1 CD8 T cell IL-1 IL-12 M Cytolysis: FAS/FAS ligand TNF Granzyme Perforin IL-4 IL-5 IL-6 IL-9 IL10 IL-13 Antibody CD4 Th2 B cell IL-1 Principle Cytokines in Acquired Immunity

33. Triggers for Th1 Cell Predomination • Naïve helper T cells are strongly stimulated to become Th1 cells and cell-mediated immunity predominates: • When IL12 production by macrophages is high. • IL-23 and IL-27, relatives of IL-12 that share some similarities to IL-12, also play a role to activate Th1 cells. • When IFN- levels are high. • IL-18, a relative of IL-1 that shares some similarities to IL-1, also activates Th1 cells.

34. Triggers for Th2 Cell Predomination • Naïve helper T cells are strongly stimulated to become Th2 cells and humoral immunity predominates: • When IL-4 levels are high. • When IL-10 levels are high.

35. Who Holds the High Trumps? • If IL-4 levels are low and IL-12 and IFN- levels are high, Th1 cells develop. • If IL-4 levels are high, Th2 cells develop. • Thus, IL-4 holds the high trumps.

36. Is There Cross-Regulation? • If naïve T cells are triggered to become Th1 cells, they produce IFN- which shuts down Th2 proliferation. • If naïve T cells are triggered to become Th2 cells, they produce IL-10 which shuts down macrophage production of IL-12, removing the stimulus for naïve helper T cells to become Th1 cells. • Thus, each local environment establishes its own balance of Th1 and Th2 cells.

37. IL-2 IL-3 GM-CSF IFN- CD4 Th1 CD8 T cell IL-1 IL-12 M Cytolysis: FAS/FAS ligand TNF Granzyme Perforin IL-4 IL-5 IL-6 IL-9 IL10 IL-13 Antibody CD4 Th2 B cell IL-1 Principle Cytokines in Acquired Immunity IFN-

38. Treg Cells • Another subset of CD4+ T cells has been identified: Treg cells. • Treg cells produce IL-4, IL-10, and TGF- • Treg cells suppresses T helper cell activity through contact with the T helper cell. • Limits autoimmune T cell activity.

39. Why Is T helper Cell Regulation Important? • Ex., Mycobacterium leprae infection • If Th1 cells predominate, tuberuloid leprosy develops. • Most of the Mycobacteria are walled off in granulomas, with slow tissue destruction and nerve damage caused by a few “free” Mycobacteria. • The patient survives. • If Th2 cells predominate, lepromatous leprosy develops. • The Mycobacteria become widely disseminated in macrophages and the patient has infection of the bone and cartilage, with extensive tissue and nerve damage. • The patient dies.

41. Cytokines Activate Their Target Cells by Binding to Specific Receptors • Cytokines bind to specific receptors on the cell surface. • Cytokines have their signal passed through the cell membrane by changes in receptor configuration. • Cytokines have their signal passed through the cytoplasm by the generation of “second” messengers. • Cytokine activated transcription factors up-regulate and down-regulate transcription.

42. Cytokine Families and the Receptors To Which They Bind • IL-1 family • Ig Domain Receptor Superfamily • Hematopoietin family • Type I Cytokine Receptor Superfamily • Interferon family • Type II Cytokine Receptor Family • TNF family • TNF Receptor Family • Chemokine family • Seven Transmembrane Helix Family

43. Role of the Different Receptor Subunits • Specific subunit ( subunit, low affinity subunit) • Binds specifically to a particular cytokine • Does not transduce a signal through the membrane • Common subunit ( subunit,  subunit, gp130) • Binds to two or more cytokines that are members of the same group, when they are bound to the specific subunit • Transduces a signal through the membrane • Note that the loss of the common subunit eliminates the activity of all cytokines of the family. • Example, loss of common  chain causes loss of IL-2, IL-15, IL-7, IL-9, and IL-4 activities. • Loss of common  chain causes severe combined immunodeficiency disorder (bubble baby)

44. Low Affinity Versus High Affinity Receptors

45. Consequences of Signaling Through a Common Subunit Explains redundancy: (1) IL-3, GM-CSF & IL-5 induce bone marrow proliferation (2) all three induce eosinophil proliferation and basophil degranulation Note, however, that not all cells express  subunits for the three different cytokines, so there can still be some specificity.

46. Consequences of Signaling Through a Common Subunit Because of a limited number of  subunits, IL-3 and GM-CSF exhibit antagonism. This is particularly apparent if IL-3 is added before GM-CSF.

47. How Does a Cytokine Bind to Its Receptor? • Example: The IL-2 receptor is composed of three subunits. •  chain: binds IL-2 •  chain: binds IL-2 •  chain: signal transducing chain (common  chain for IL-2, IL-4, IL-7, IL-15, IL-21)

48. Affinities of IL-2 Receptor Multimers The strength of cytokine binding increases with the addition of the common subunits.

49. Note: (1) IL-2 binds to a pocket formed by the  and  subunits (2) the  subunit binds to the  subunit but not to the  subunit

50. Signal Transduction Across the Cell Membrane • Example: IFN signal transduction.