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Cytokines and Thelper subsets

MCB 150, Pr Coscoy

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.

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 Network

T helper and Macrophage Interactionhighlighted

Effector T cell

Th 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).

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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

II. Measuring Cytokines

Protein amount by ELISA. Good for in vitro experiments.

Protein amount by bioactivity assay usingCK dependent cell lines.

RNA message by PCR.

Antigen Capture ELISA for IL-2 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 (throughbloodstream). Inflammatory cytokines.

Actions of Cytokines

IL-2 forT-cell activation

T-cell helpfor B cells

Inflammatory cytokines

IV. 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.

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SynergyExperimental 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.

Properties ofCytokines

Pleiotropy

Redundancy

Synergy

Antagonism

V. FunctionsOf Cytokines

VI. 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.

Mouse Th Subset

Cytokine Th1 Th2

Table 12-4 from Goldsby

Th0 ---> 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 cellsIL-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.)

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Th1/Th2

Naïve Th0 IL-2, IL-4, IFN-γ

IL-4IL-12

EffectorTh1 cellIL-2, IFN-γ

EffectorTh2 cellIL-4, IL-6, IL-10

Th1/Th2 AntagonismIL-4 blocks Th1 IFN-γ blocks Th2

IL-4IL-12

Th1 cellIL-2, IFN-γ

Th2 cellIL-4, IL-6, IL-10

IFN-γIL-4

Th1/Th2 RegulationT-bet is a transcription factor that is required

for Th1 specific genes such as IL-12Rβ

IL-12

Th1 cellIL-2, IFN-γ

Th2 cellIL-4, IL-6, IL-10

IFN-γEnhances T-betIL-4 blocks T-bet

IL-4 vs IFN-γT-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.

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Influence 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 Th1

TLR 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 infection

Control 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.

MyD88-/- mice default to Th2Role for TLR Activation in Th1/Th2

IFN-γ IL-4 IL-5 IL-10 IL-13

A. MyD88-/- miceResponse toTh2 pathogen

B. MyD88-/- miceResponse to Th1 pathogen

C. WT mice Response to Th1 pathogen

Th Cytokine Bias in DiseaseExamples

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 uncontrolleddisease (leprosy).

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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 Leprosy

RNA from skin lesions of patients

Cytokines as Ligands

APCTarget

T Cell

TNF-β or Lymphotoxin

Ligand

LT α and β Receptors Soluble or membrane bound

Receptor

VII. Cytokine Receptors

Expression of cytokine receptors controlsthe ability of a cytokine to act on a cell.

Cell activation increases cytokine receptorexpression.

Cytokine ReceptorFamilies

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

IL-2 Receptor Subfamily Shared common γ subunit

Only IL-2 and IL-15 have unique alpha subunit

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X-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 infections.

SCID Patient with severe Candida in mouth.

GM-CSF Receptor Subfamily Hematopoietin Receptors

Low affinity receptors compete for common β subunitto get high affinitybinding.

GM-CSFGranulocyte monocyteColony stimulating factor

GM-CSF Receptor Subfamily Hematopoietin Receptors

IL-3, IL-5GM-CSF activate commonβ subunit.

Cytokine Receptor Signal through JAKs and STATs

Cytokine Receptor SignalingJAKs 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 andActivators of Transcription ) dimerize for function.

JAKs and STATsUsage by CK Receptors

Overlapping and Unique

JAK1 is commonly used by CKs from completelydifferent CKR families

STAT6 is ONLY usedIL-4.

The Yin and Yang

of Th1 and Th2

Immune Responses