1. Cytokines and Thelper subsets MCB 150, Pr Coscoy

2. I. Characteristics of Cytokines (CKs)  CKs are small proteins (<30 kDa)  CKs are similar to hormones and growth factors.  CKs control the immune system.  Cytokines include subsets called lymphokines, monokines, chemokines, etc.

3. Cytokine Network  CKs are part of complex system that regulates the immune system.  CKs are primarily produced by immune system but many other organs (liver, brain, endocrine glands) make CKs to influence immune response.

4. Cytokine Network

5. T helper and Macrophage Interaction highlighted Effector T cell Th cells control immune system through CKs.

6. Activation of Cells = Production of Cytokines  Production of CKs is usually part of immune cell activation.  Cytokines are produced in response to different stimuli (e.g antigen receptor, cytokine/chemokine receptor).

7. T cell IL-2 IL-2 gene B cell IL-2 Receptor IL-4 Cytokine Network on a Local Level IL-4 Receptor IL-4 gene

8. II. Measuring Cytokines  Protein amount by ELISA. Good for in vitro experiments.  Protein amount by bioactivity assay using CK dependent cell lines.  RNA message by PCR.

9. Antigen Capture ELISA for IL-2

10. III. Actions of Cytokines  Autocrine acts on same cell that produced it. IL-2 for T cell activation  Paracrine acts on nearby cells. T cell help for B cells  Endocrine acts on cell at a distance (through bloodstream). Inflammatory cytokines.

11. Actions of Cytokines IL-2 for T-cell activation T-cell help for B cells Inflammatory cytokines

12. IV. Properties of Cytokines Pleiotropy  Same cytokine has different effects on cells can be activating or inhibiting.  Example: IL-10 can be inhibitory to macrophages and Th1 cells yet activating for Th2 cells and B cells.

13. Synergy Experimental Example Proliferation of CTLLs (CTLLs are cytokine dependent cell line)  Both IL-2 or IL-4 activate CTLLs in vitro.  Maximal proliferation obtained when both IL-2 and IL-4 added.  Both cytokines need to be blocked in order to inhibit growth.

14. Properties of Cytokines Pleiotropy Redundancy Synergy Antagonism

15. V. Functions Of Cytokines

16. VI. CD4 + T helper Subsets Th1/Th2 Cytokine Bias  CD4+ T helper cells can be divided into subsets based on their cytokine production.  T h 1 cells produce IL-2, IFN- , TNF-  CKs which activate cell mediated immunity  T h 2 cells activate IL-4, IL-6, IL-10 CKs that activate humoral immunity These Th subsets were originally identified using mouse T cell clones.

17. Mouse Th Subset Cytokine Th1 Th2 Table 12-4 from Goldsby

18. Th0 ---> Th1 or Th2 Original mouse experiments on Th cells (Mosmann et al (DNAX) 1986 J Immunol) Antigen specific T cells placed in culture with antigen and APCs to make T cell lines.  Spleen cells (T h 0) add IL-12  T h 1 cells  IL-2, IFN- , TNF-   Spleen cells (T h 0) add IL-4  T h 2 cells  IL-4, IL-6,IL-10  (T h 0 --precursor cell that produces IL-2, IL-4, and IFN- .)

19. Th1/Th2 Naïve T h 0 IL-2, IL-4, IFN-  IL-4 IL-12 Effector T h 1 cell IL-2, IFN-  Effector T h 2 cell IL-4, IL-6, IL-10

20. Th1/Th2 Antagonism IL-4 blocks Th1 IFN-  blocks Th2 IL-4 IL-12 T h 1 cell IL-2, IFN-  T h 2 cell IL-4, IL-6, IL-10 IFN-  IL-4

21. Th1/Th2 Regulation T-bet is a transcription factor that is required for Th1 specific genes such as IL-12R  IL-12 T h 1 cell IL-2, IFN-  T h 2 cell IL-4, IL-6, IL-10 IFN-  Enhances T-bet IL-4 blocks T-bet

22. IL-4 vs IFN-   T-bet (Th1 associated) activated by IFN-  and turned off by IL-4.  Conversely in Th2 transcription factor GATA-3 activated by IL- 4 turned off by IFN- .

23. Role for Th1 vs Th2 in Immune Response  Both subsets activated in lymph nodes (LN) immune responses to complex antigens.  Th1 cells leave LN to find activated endothelium tissue to activate macrophages.  Th2 cells can stay in LN to activate B cells.

24. What controls Th1 vs Th2? 1) Amount of antigen. Mouse experiments originally showed high dose for Th1. 2) MHC and TCR affinity. High affinity TCR = Th1. 3) Dendritic cell subsets during activation. APC subsets activate Th1 or Th2 preferentially. 4) Toll-like receptor activation.

25. Influence of APC Subsets on Th1/ Th2 Dendritic cell  Myeloid-like dendritic cells produce abundant IL-12 and drive Th1.  Lymphoid-like dendritic cells produce low levels of IL-12 are permissive for Th2.

26. Toll-like receptors (TLRs) Influence of APCs on Th1/ Th2  Evidence for TLR activation influencing Dendritic cell maturation. –TLR9 binds bacterial CpG DNA –TLR4 binding to bacterial heat shock proteins  TLR activation induces APC expression of IL-12, IL-23, IL-27 Th1

27. TLR vs IL-12 in Th1/ Th2 development  New evidence suggests that TLR activation influencing Th1 outcome through initiation of TLR adapter molecule MyD88.  May be more important than IL-12 for Th1.

28. MyD88-/- mice fail to control acute Toxoplasma infection Control mice ( ) MyD88-/- mice ( ) IL-12p40-/- mice ( ) Survival curve shows MyD88 is just as important as IL-12 for Th1 response after Toxoplasma infection. Scanga et al The Journal of Immunology, 2002, 168: 5997-6001.

29. MyD88-/- mice default to Th2 Role for TLR Activation in Th1/Th2 IFN-  IL-4 IL-5 IL-10 IL-13 A. MyD88-/- mice Response to Th2 pathogen B. MyD88-/- mice Response to Th1 pathogen C. WT mice Response to Th1 pathogen

30. Th Cytokine Bias in Disease Examples Leishmania in mice (Richard Locksley)  C57Bl.6 mice mice have Th1 immune response and resolve infection.  BALB/c mice produce Th2 cytokines unable to control Leishmania lesions. Leprosy in Humans (Robert Modlin)  Tuberculoid form has Th1 response and limits disease (healing).  Lepromatous form has Th2 response and uncontrolled disease (leprosy).

31. Th Cytokine Bias in Disease: Leprosy Skin disease caused by Mycobacterium leprae Lepromatous: has Th2 response and uncontrolled disease (leprosy). Tuberculoid: has Th1 response and limits disease (healing).

32. Cytokine Bias in Leprosy RNA from skin lesions of patients

33. Cytokines as Ligands APC Target T Cell TNF-  or Lymphotoxin Ligand LT  and  Receptors Soluble or membrane bound Receptor

34. VII. Cytokine Receptors  Expression of cytokine receptors controls the ability of a cytokine to act on a cell.  Cell activation increases cytokine receptor expression.

35. Cytokine Receptor Families 5 different families of receptors based on common structural motifs. --> see book for more details

36. IL-2 Receptor Subfamily Shared common  subunit Only IL-2 and IL-15 have unique alpha subunit

37. X-Linked SCID Common  chain Deficiency  Mutation in  chain so unable to signal through IL-2, IL-4, IL-7, IL-9, IL-15.  No T cells abnormal thymus.  Immunocompromised susceptible to infections. SCID Patient with severe Candida in mouth.

38. GM-CSF Receptor Subfamily Hematopoietin Receptors Low affinity receptors compete for common  subunit to get high affinity binding. GM-CSF Granulocyte monocyte Colony stimulating factor

39. GM-CSF Receptor Subfamily Hematopoietin Receptors IL-3, IL-5 GM-CSF activate common  subunit. Cytokine Receptor Signal through JAKs and STATs

40. Cytokine Receptor Signaling JAKs and STATs (Model of Signal) Binding of cytokine ligand brings together receptor subunits JAKs (Janus Associated Kinases) are tyrosine kinases that phosphorylate tyrosines. STAT (Signal Transducers and Activators of Transcription ) dimerize for function.

41. JAKs and STATs Usage by CK Receptors Overlapping and Unique JAK1 is commonly used by CKs from completely different CKR families STAT6 is ONLY used IL-4.

42. The Yin and Yang of Th1 and Th2 Immune Responses