1Cytokines and Thelper subsetsMCB 150, Pr CoscoyI. Characteristics of Cytokines(CKs)CKs are small proteins (<30 kDa)CKs are similar to hormones and growthfactors.CKs control the immune system. Cytokines include subsets called lymphokines, monokines,chemokines, etc.Cytokine NetworkCKs are part of complex system thatregulates the immune system.CKs are primarily produced by immunesystem but many other organs (liver, brain,endocrine glands) make CKs to influenceimmune response.Cytokine Network2T helper and Macrophage Interactionhighlighted Effector T cellTh cells control immunesystem through CKs.Activation of Cells= Production of CytokinesProduction of CKs is usually part of immune cellactivation.Cytokines are produced in response to differentstimuli (e.g antigen receptor, cytokine/chemokinereceptor). T cell IL-2IL-2 geneB cellIL-2 ReceptorIL-4Cytokine Network on a Local LevelIL-4 ReceptorIL-4 geneII. Measuring CytokinesProtein amount by ELISA. Good for in vitro experiments.Protein amount by bioactivity assay usingCK dependent cell lines.RNA message by PCR.3Antigen Capture ELISA for IL-2III. 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 (throughbloodstream). Inflammatory cytokines.Actions of CytokinesIL-2 forT-cell activationT-cell helpfor B cellsInflammatory cytokinesIV. Properties of CytokinesPleiotropySame cytokine has different effects on cellscan be activating or inhibiting.Example: IL-10 can be inhibitory tomacrophages and Th1 cells yet activatingfor Th2 cells and B cells.4SynergyExperimental ExampleProliferation of CTLLs(CTLLs are cytokine dependent cell line)Both IL-2 or IL-4 activate CTLLs in vitro.Maximal proliferation obtained when bothIL-2 and IL-4 added.Both cytokines need to be blocked in orderto inhibit growth.Properties ofCytokinesPleiotropyRedundancySynergyAntagonismV. FunctionsOf CytokinesVI. CD4+ T helper SubsetsTh1/Th2 Cytokine BiasCD4+ Thelper cells can be divided into subsetsbased on their cytokine production.Th1 cells produce IL-2, IFN-γ, TNF-βCKs which activate cell mediated immunityTh2 cells activate IL-4, IL-6, IL-10 CKs that activate humoral immunity These Th subsets were originally identified using mouse T cell clones.5Mouse Th SubsetCytokine Th1 Th2Table 12-4 from GoldsbyTh0 ---> Th1 or Th2Original mouse experiments on Th cells(Mosmann et al (DNAX) 1986 J Immunol)Antigen specific T cells placed in culture withantigen and APCs to make T cell lines.Spleen cells (Th0) add IL-12  Th1 cellsIL-2, IFN-γ, TNF-βSpleen cells (Th0) add IL-4  Th2 cells IL-4, IL-6,IL-10 (Th0 --precursor cell that produces IL-2, IL-4, and IFN-γ.)Th1/Th2Naïve Th0 IL-2, IL-4, IFN-γIL-4IL-12EffectorTh1 cellIL-2, IFN-γEffectorTh2 cellIL-4, IL-6, IL-10Th1/Th2 AntagonismIL-4 blocks Th1 IFN-γ blocks Th2IL-4IL-12 Th1 cellIL-2, IFN-γTh2 cellIL-4, IL-6, IL-10IFN-γIL-46Th1/Th2 RegulationT-bet is a transcription factor that is requiredfor Th1 specific genes such as IL-12RβIL-12 Th1 cellIL-2, IFN-γTh2 cellIL-4, IL-6, IL-10IFN-γEnhances T-betIL-4 blocks T-betIL-4 vs IFN-γT-bet (Th1 associated)activated by IFN-γ andturned off by IL-4.Conversely in Th2transcription factorGATA-3 activated by IL-4 turned off by IFN-γ.Role for Th1 vs Th2 inImmune ResponseBoth subsets activated in lymph nodes (LN)immune responses to complex antigens.Th1 cells leave LN to find activatedendothelium tissue to activate macrophages.Th2 cells can stay in LN to activate B cells.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.7Influence of APC Subsets on Th1/ Th2Dendritic cellMyeloid-like dendritic cells produceabundant IL-12 and drive Th1.Lymphoid-like dendritic cells produce lowlevels of IL-12 are permissive for Th2.Toll-like receptors (TLRs) Influence of APCs on Th1/ Th2Evidence for TLR activation influencingDendritic cell maturation.– TLR9 binds bacterial CpG DNA– TLR4 binding to bacterial heat shock proteinsTLR activation induces APC expression ofIL-12, IL-23, IL-27 Th1TLR vs IL-12 in Th1/ Th2 developmentNew evidence suggests that TLR activationinfluencing Th1 outcome through initiationof TLR adapter molecule MyD88.May be more important than IL-12 for Th1.MyD88-/- mice fail to controlacute Toxoplasma infectionControl mice ( )MyD88-/- mice ( )IL-12p40-/- mice ( ) Survival curve shows MyD88 is just as important as IL-12 forTh1 response after Toxoplasma infection.Scanga et al The Journal of Immunology, 2002, 168: 5997-6001.8MyD88-/- mice default to Th2Role for TLR Activation in Th1/Th2IFN-γ IL-4 IL-5 IL-10 IL-13A. MyD88-/- miceResponse toTh2 pathogenB. MyD88-/- miceResponse to Th1 pathogenC. WT mice Response to Th1 pathogen Th Cytokine Bias in DiseaseExamplesLeishmania in mice (Richard Locksley) C57Bl.6 mice mice have Th1 immune response andresolve infection. BALB/c mice produce Th2 cytokines unable to controlLeishmania lesions.Leprosy in Humans (Robert Modlin) Tuberculoid form has Th1 response and limits disease(healing). Lepromatous form has Th2 response and uncontrolleddisease (leprosy). Th Cytokine Bias in Disease:LeprosySkin disease caused by Mycobacterium leprae Lepromatous: has Th2response and uncontrolleddisease (leprosy).Tuberculoid: has Th1response and limits disease(healing). Cytokine Bias in LeprosyRNA from skin lesions of patients9Cytokines as LigandsAPCTargetT Cell TNF-β or LymphotoxinLigandLT α and β Receptors Soluble or membrane boundReceptorVII. Cytokine ReceptorsExpression of cytokine receptors controlsthe ability of a cytokine to act on a cell.Cell activation increases cytokine receptorexpression.Cytokine ReceptorFamilies5 different families of receptors based on common structural motifs. --> see book for more detailsIL-2 Receptor Subfamily Shared common γ subunitOnly IL-2 and IL-15 have unique alpha subunit10X-Linked SCID Common γ chain DeficiencyMutation in γ chain so unable to signalthrough IL-2, IL-4, IL-7, IL-9, IL-15.No T cells abnormal thymus.Immunocompromisedsusceptible to