7. Specialised Cells: CD4 T Cells

Learning Outcomes: L15-16

What distinguishes the main subsets of CD4 T cell? What is “signal 3”? And what is signal 3 for Th1 + Th2 induction? What intracellular signalling pathways cause Th1 + Th2 induction? What pathogens are Th1 + Th2 cells effective against? How do Th1 + Th2 “help” B cell class switching? How do Th1 cells “help” macrophage activation? How do CD4 T cells “help” CD8 T cell responses? Which pathogens are killed by Th17 cells? What is “Signal 3” for Th17 differentiation and what are the downstream signalling pathways? What are Th17 effector mechanisms? Why do we need regulatory T cells? What are natural and induced Tregs? What is the relationship between Tregs and Th17 cells? How do Tregs suppress immune responses?  

Recap: T Cell Activation

Image (binary/octet-stream)

CD4 T Cell Subsets

Image (binary/octet-stream)

Rubrica: : CD4 T cell subsets make signature cytokines

CD4 T Cell Subsets

Distinct subsets of CD4 T cells coordinate distinct immune responses to target different pathogens, subsets are distinguished by signature cytokines  Cytokines= proteins secreted by immune and non-immune cells that influence immune function: Interleukins, interferons, tumour necrosis factors, transforming growth factor CD4 T cell differentiation events: Production of polarising cytokines - signal 3  Cytokine signalling events in CD4 T cells Effector functions of CD4 T cell subsets

1. Produce Polarising Cytokines: Th1

Signal 3-Th1 differentiation, supported by ILC1-derived IFNϒ Summary: PAMPs stimulate DCs to produce IL12-> main driver of Th1 differentiation (signal 3) DC recognises bacterial PAMP- presents peptide on MHC-II PAMP stimulates DC to produce cytokine IL-12 IL-12 binds IL-12R on naive T cells and elicits signal in naive CD4 T cell ILC1 aLso express IL12R and will bind IL12 and produce IFNϒ IFNϒ binds to IFNϒR on naive CD4 T cell Sum of signals (1 +2 ) + 3= cytokines (IL12 and IFNϒ) = cause naive T cell to be activated to Th1 cell that produces signature cytokine IFNϒ

1. Produce Polarising Cytokines: Th1

Image (binary/octet-stream)

1. Produce Polarising Cytokines: Th2

Signal 3-Th2 differentiation Th2 is response induced by parasitic worms  DC present peptide on MHC-11 (dont know PAMP or PRR)  Parasitic worms cause tissue damage which activates ILC2 to produce IL-4 (signal 3) IL-4 binds IL-4R on naive CD4 T cell Sum of signals (1 +2 ) + 3= cytokines (IL4 = cause naive T cell to be activated to Th2 cell that produces signature cytokines IL4 and IL5

1. Produce Polarising Cytokines: Th2

Image (binary/octet-stream)

2. Cytokine Signalling in CD4 T Cells

How does Il-12 induce Th1 differentiation and how does IL-4 induce Th2 differentiation T cell signalling: JAK-STAT pathway GENERAL JAK is associated with cytokine receptor When cytokine binds receptor, JAKs activated, phosphorylate cytokine receptor Phosphorylation acts as docking site for STAT proteins and recruit them to receptor STATs close to JAKs and get phosphorylated, then translocate to nucleus  STATs bind to promoter of  and transcribe lineage specific genes: cytokines, cytokine receptors, TFs Lineage specific TFs promote the production of signature cytokines by different CD4 T cell subsets

Image (binary/octet-stream)

Image (binary/octet-stream)

2. Cytokine Signalling in CD4 T Cells

Rubrica: : JAK-STAT pathway

Rubrica: : Lineage specific TFs induce signature cytokines

2. Cytokine Signalling in CD4 T Cells: Th1

IL-12 signal cytokine binds IL-12R, phosphorylation through JAKs of intracellular portion of IL12R Recruit STAT4, phosphorylated and translocates to nucleus  Activated STAT4 binds to TH1-specific genes: T-bet= lineage specific TF- promotes more response to IL12 IL12R- signal 3 cytokine receptor- promotes cell to be more responsive to IL12 IFNϒ= hallmark cytokine of Th1 cells T-bet binds to all 3 gene loci, activates, reinforces and stabilises Th1 differentiation, promoting expression of all 3

2. Cytokine Signalling in CD4 T Cells: Th1

Image (binary/octet-stream)

2. Cytokine Signalling in CD4 T Cells: Th2

IL-4 signal cytokine binds IL-4R, phosphorylation through JAKs of intracellular portion of IL12R Recruit STAT6, phosphorylated and translocates to nucleus  Activated STAT6 binds to TH2-specific genes: GATA3= lineage specific TF- promotes more response to IL-4 IL4R- signal 3 cytokine receptor- promotes cell to be more responsive to IL-4 IL4 +IL5= hallmark cytokine of Th2 cells GATA binds to all 3 gene loci, activates, reinforces and stabilises Th1 differentiation, promoting expression of all 3

2. Cytokine Signalling in CD4 T Cells: Th2

Image (binary/octet-stream)

Summary of Differentiation so far

Image (binary/octet-stream)

Overview of Th1 + Th2 Effector Function

Image (binary/octet-stream)

Image (binary/octet-stream)

3. Effector Functions of CD4 T cells

Image (binary/octet-stream)

3. Effector Functions of CD4 T cells

1. Activated CD4 T cells help Ab class switch Ab's have different isotypes-> starts as IgM and IgD but class switch into IgG, IgE, IgA Isotypes have different heavy chain constant regions: Bind different Fc receptors Differing ability to activate complement CD4 T cell signature cytokines direct Ab class switching: Th1 cell produced IFNϒ and B cells express the IFNϒR- Th1 cell IFNϒ induces B cells to class switch to IgG2a Th2 cell IL-4 induces B cell to class switch to IgG1 + IgE

Activated CD4 T cells help Ab class switch

Image (binary/octet-stream)

Activated CD4 T cells help Ab class switch

Eg of Importance of class switching: Parasitic worms induce Th2 cell to produce IL4, induces class switch to IgE  Innate immune cells (granulocytes) express receptor for IgE (FcεR) Immune cells bind IgE, armed with IgE (receptor)  May see pathogen molecules that IgE recognises and bind-> activate immune cells  Th2 also produces IL5 cytokine= survival factor and differentiation factor for eosinophils Produces lots of eosinophils in blood all armed with IgE Mast cells will degranulate- release toxic granules onto parasite to attack ​​​​​​​    

Activated CD4 T cells help Ab class switch

Image (binary/octet-stream)

3. Effector Functions of CD4 T cells

2. Activated CD4 T cells help macrophages  CD4 T cell activated by DC in LN, macrophage far away in peripheral tissue fighting infection Activated T cell needs to migrate from LN to peripheral tissue 

Activation-induced changes in CD4 T cell surface proteins Naive CD4 T cells have lots of L-selectin (to enter HEV) and integrin LFA1 that gets them into LN​​​​​​​ Following activation: ​​​​​​​Loss of L-selectin Increase integrins (more LFA1 and new VLA-4) Induction of CD40L ligand

Image (binary/octet-stream)

Activated CD4 T cells help macrophages 

Activated CD4 T cells help macrophages

T cells migrate from blood into inflamed tissues: Tissue resident macrophage produces inflammatory cytokines recognised by receptor on endothelial cell Endothelium activated, expressed high levels of adhesion molecules: ICAM1 (binds LFA1), VCAM1 (binds VLA4) Binding of adhesion molecules to integrins arrests activated T cell on endothelial cell and migration into tissue controlled by chemokine gradient

Image (binary/octet-stream)

Activated CD4 T cells help macrophages

Image (binary/octet-stream)

Macrophages present antigens, re-stimulate activated CD4 T cell Present antigens on MHC-II T cell with correct antigen specificity TCR signals delivered by MHC-II: Th1 produce IFNγ + express high levels of CD40L CD40L binds CD40 on mac Activate macrophage to kill intracellular pathogens  

3. Effector Functions of CD4 T cells

3. CD4 T cells help CD8 CTL activation: Activated CD4 T cells licence DCs to fully activate CD8 T cells: DC presents peptide on MHC-I and binds TCR on CD8 T cell = weak activation Simultaneous presentation of MHC-II:peptide to CD4 T cell (different peptide derived from same pathogen), activated CD4 T cell produces IL2= further activation CD8 CD40L (induced by cross-talk between CD4 + DC) binds CD40 on DC and causes upregulation of other co-stimulatory molecules on DC for CD8 = strong activation of CD8 ​​​​    

CD4 T cells help CD8 CTL activation

Image (binary/octet-stream)

Type 1 vs Type 2 Immunity

Th1 and Th2 cells are coordinators- directors of broader immune responses (Type 1 and Type 2 immunity) Type 1 immunity: Bacteria activates DC and macrophages to produce IL12 ILC1 produce IFNϒ, supports differentiation into Th1 Lots of production of IFNϒ- Th1 and ILC1 Activation of macrophages to kill intracellular pathogens Type 2 immunity: Worm activates IL4 production ILC2 produces IL4 which supports differentiation of Th2 cells Th2 produce IL4 and IL5 cytokines- lead to activation of B cells to produce IgE class switch Ab When eosinophils and mast cells recognise parasite they degranulate and kill pathogen

Type 1 vs Type 2 Immunity

Image (binary/octet-stream)

Th17 Cells

Make large amounts of cytokine IL-17 Initiate effector immune responses against pathogens not dealt with by Th1 and Th2 cells: Extracellular bacteria and fungi      

How are Th17 Cells Induced?

PAMPs bind DC PRR, uptake antigens present on MHC-II Trigger DCs to produce signal 3= active TGFβ and IL-6 (inflammatory cytokine) Initial Th17 differentiation ​​​​​​​DCs present peptides to CD4 T cell, which binds as well as cytokines to receptors on CD4 T cell triggering initial differentiation into Th17 Th17 characterised by production of IL-17 Need additional signal to keep alive- DCs produce IL-23 cytokine as part of signal 3

How are Th17 Cells Induced?

Image (binary/octet-stream)

Cytokine-induced Signalling in Th17 Cells

IL-6 binds IL-6R on T cell, activates JAKs to phsophorylate intracellular portion of IL-6R Phosphotyrosine acts as docking site for STAT3, phosphorylated by JAKs, translocates to nucleus and bind Th17 specfic genes STAT3 alone not sufficient to induce gene expression TGFβ binds to receptor on T cell and activates SMAD proteins  SMADs and STAT3 bind and induce expression of Th17 specific genes IL23 (not there upon initial activation) binds to its receptor on T cell and signals for survival and expansion RORγT TF protein is expressed and will take over to stabilise expression of all 3 genes (RORγT TF , IL-23R, IL-17)

Image (binary/octet-stream)

Cytokine-induced Signalling in Th17 Cells

Th17 Effector Functions

Recruited to initial sites of inflammation: Infection of extracellular pathogen causes DC to mature and present, DC migrate down afferent lymphatic to draining LN  Interacts with T cell with correct specificity, differentiation into Th17 Some Th17 cells leave via efferent lymphatic, end up in blood and recruited back to inflamed tissue Tissue resident macrophage induced by pathogen to secrete inflammatory cytokines, act on endothelial wall of blood vessel, activate expression of adhesion molecules, chemokine gradient pulls Th17 cells into tissue

Th17 Effector Functions: Neutrophils

Once at site of inflammation, recruit neutrophils: Interaction between mac and specific Th17 cell via MHC-II:peptide- reactivates Th-17 (first in LN),  produces IL-17 IL-17 binds to receptor (broadly expressed by non-immune cells (stromal: fibroblast, eptihelial cells) Triggers non-immune cells to produce lots of chemokine CXCL8 CXCL8 recruits neutrophils from blood to site to kill pathogen

Th17 Effector Functions: Neutrophils

Image (binary/octet-stream)

Summary: Th17

DC produce active TGFβ, IL-6, IL-23 in response to fungi or extracellular bacteria = Signal 3 for Th17 differentiation TGFβ + IL-6 induce initial Th17 differentiation IL-23 promotes expansion + survival of Th17 cells Lineage-specific TF for Th17 cells is RORγT Th17 cells produce IL-17 This induces non-immune cells at sites of infection to produce CXCL8 = neutrophil chemoattractant Recruited neutrophils kill pathogen

Summary of T Cell Subset so far

Image (binary/octet-stream)

Regulatory T Cells

Immune responses are inflammatory and potentially pathogenic... autoimmunity and immunopathology (damages tissue even after clearance) Central tolerance: deletion of self-reactive T cells in thymus Some self-reactive T cells escape this negative selection, so need peripheral tolerance Peripheral tolerance: stop activation of mature self-reactive T cells Via co-stimulation- in absence of infection APCs have low level of CD80/CD86 Not always efficient,  need specialised cells to turn off immune responses.. Regulatory T cells

2 Types of Regulatory T Cell (Treg)

Image (binary/octet-stream)

Still have TCR and express CD4 like normal T cells  Difference=repressive, turn off effector T cells, APCs etc

Natural Tregs

nTregs differentiate in thymus in response to stronger than normal TCR stimulation TCR binds to self-peptide:MHC stronger than normal but not strong enough to be deleted Induces expression of Treg lineage-specific TF: Foxp3 T cells exit thymus and circulate through LN and spleen Function: they recognise self-peptides and have stronger than normal self-reactivity, recognise T cells that escaped negative selection and recirculating->  directly inhibit autoreactive T cell

Natural Tregs

Image (binary/octet-stream)

Image (binary/octet-stream)

Induced Tregs

iTregs differentiate in the periphery  Signal 3 for iTreg is TGFB (in absence of inflammatory signal) DC take up antigen, produce active TGFB which binds receptor on naive CD4 and upregulates foxp3 positive iTreg iTreg potentially specific for any antigen: pathogen, self-antigen, commensal

Differentiation of iTreg vs Th17 Cells

Image (binary/octet-stream)

Pathogens induce production of inflammatory cytokines (eg IL-6) that switch immune suppression to Th17 inflammatory response 

iTregs Supress Immune Response

Via cell surface molecules: Treg express high levels of CTLA-4 (co-inhibitory molecule)  CTLA-4 binds and sequesters DC CD80/86 from naive cells - no signal 2 CTLA-4 can physically rip CD80/86 from APCs= no ability co-stimulate Tregs express high levels of IL-2R Act as sink for IL-2, take it away from activated cells so they cant proliferate Outcompete activated T cells for IL-2

Via cell surface molecules

Image (binary/octet-stream)

Rubrica: : Express IL-2R: Outcompete activated T cells for IL-2

Image (binary/octet-stream)

Rubrica: : Express CTLA-4: no co-stimulation (no signal 2)

iTregs Supress Immune Response

Via secretion of immunosuppressive cytokines: Inhibit Il-12 production by DCs (no Th1 response) Reduce MHC-II + CD80/86 levels on DCs  Directly inhibit T cell proliferation  Generate more Tregs (TGFβ)

Image (binary/octet-stream)

Mutations in Tregs

Mutations to Foxp3 gene of X chromosome = IPEX syndrome Cant make functional Foxp3 protein, lack Tregs, die by autoimmune diseases Fatal early (childhood) onset autoimmune disease in males:  Diabetes, anemia, splenomegaly, skin inflammation, cytokine storm

Tregs Summary

Tregs are essential for peripheral tolerance nTregs differentiate in thymus + turn-off self-reactive T cells In absence of inflammatory cytokines, TGFβ induces iTregs iTregs suppress immune responses through surface + secreted molecules