Molecular Events in T Cell Activation Flashcards
What is the CD3 complex
• alpha and beta chains recognize antigen but don’t have signaling ability
• When cells were transfected with cDNA encoding the TCRa and TCRb chains, the heterodimers formed were degraded and did not appear on the cell surface → suggests something else is also involved
• TCR needs to form the CD3 complex to signal:
− e/d heterodimer
− e/y heterodimer
− z homodimer
• All CD3 chains contain ITAM motifs
What are ITAMs?
- Signaling from the TCR is initiated by tyrosine phosphorylation within ITAMs
- CD3 y, d and e chains have 1 ITAM each, and z chains have 3, giving the TCR a total of 10 ITAMs
- This motif also present in the signaling chains of BCR and NK cell receptor
- Each ITAM has two tyrosine residues that become phosphorylated when the receptor binds the ligand, providing sites for recruitment of SH2 domains of signaling proteins
Describe the early events in T cell signalling (ITAMS)
Signaling via ITAMS of the TCR:
• TCRs and BCRs must be able to respond to their specific antigen even when present at low levels
• This is especially important for T cells, as the APC will display many different peptides, and so the number of peptide:MHC complexes specific for a particular TCR is likely to be very low
• Some estimates suggest naïve CD4 T cell can become activated by as few as 50 peptide:mHC complexes, CD8 maybe even less.
• It is still unclear precisely how binding of a peptide:MHC to a TCR transmits the signal across the membrane.
There are 3 suggestions:
Recall how ligand induced receptor dimerization is common for many receptors, eg) TLR
- Signalling is initiated by TCR dimerization through formation of psudo-dimeric peptide:MHC complexes containing one antigen peptide:MHC molecule and one self-peptide:MHC on the surface of an APC. This model relies on weak interaction between the TCR and self peptide:MHC, but would explain signaling induced by low densities of antigenic peptides
- Signaling may involve receptor oligomerisation or clustering, as antibodies that bind to and cross-link TCRs can activate T cells. However, antigens are vastly outnumbered on the cell surface, therefore unlikely there would be enough to induce oligomerisation. However, assemblies of small numbers of TCRs called microclusters have been seen in the immunological synapse
- Antigenic peptide:MHC may induce a conformational change in the TCR that generates a signal, but there is no evidence to support this.
Following signal transmission across the membrane, the first intracellular signal generated is phosphorylation of the ITAMs:
1. When TCRs and co-receptors are brought together by binding to peptide:MHC complexes on the surface of an APC, recruitment of the co-receptor assicated kinase Lck leads to phosphorylation of the ITAMs
2. Lck is constitutively associated with the cytoplasmic domains of CD4/CD8
3. The kinase ZAP-70 is recruited to the ITAMs via SH2 domains
4. Recruited ZAP-70 is activated via phosphorylation by Lck
5. Activated ZAP-70 can phosphorylate several different proteins to trigger signaling
6. Signaling is proposed to be enhanced by TCR complex clustering in the immunological synapse.
➢ TCRs cluster in the cSMAC
➢ Lck clusteres in the pSMAC
7. ZAP-70 phosphorylates and recruits scaffold proteins LAT and SLP-76 to the activated TCR complex
8. An adaptor protein Gads holds the phosphor-LAT and SLP-76 together.
9. The Gads:SLP-76-LAT complex recruits PLC-y
− It binds to by its PH domain to PIP3 formed by phosphorylation of PIP2 by PI3-kinase
− An additional signal is needed to activate PI3-kinase → co-stimulation by CD28 engagement.
10. PLC-y is phosphorylated and activated by Itk, which is recruited by interactions with SLP-76.
11. Activated PLC-y catalyses the breakdown of PIP2 to DAG and IP3:
− DAG is confined to the membrane, serves as a molecular target to recruit other signaling molecules to the membrane
− Recruits RasGRP to activate Ras
− Recruits PKC- θ to result in activation of NFkB
− IP3 triggers calcium-mediated signaling events by binding to Ca2+ channels on the ER
− The depletion of Ca2+ stores in the ER causues the aggregation of the ER sensor STIM1
− Aggreated STIM1 stimulates the second phase of Ca2+ release by opening channels in the plasma membrane
Signal 1 in this example:
• T cell binding to MHC leading to recruitment of Lck and phosphorylation of ITAMs
Signal 2:
• CD28 binding to CD80/86 leads to activation if PI3-kinase
− Produces PIP3 which recruits the PLC-y and the Itk needed to activate PLC-y
− PIP3 can also re-inforce signals from the TCR by recruiting PDK, which activates AKT, which enhances cell survival
Describe the Calcineurin pathway of downstream T cell signaling
• Stimulated by Ca2+ influx
• Increased cytosolic Ca2+ leads to activation of a family of TFs called NFAT.
• In resting T cells, NFAT is kept in the cytosol by phosphorylation by GSK3 and CK2
• Cytoplasmic Ca2+ binds calmodulin, which in turn binds and activates calcineurin.
• Calcineurin dephosphorylates NFAT, exposing its NLS and allowing it to translocate to the nucleus
• Inside the nucleus, NFAT functions in turning on genes crucial for T cell activation, eg) IL-2
• The importance of NFAT in T cell activation is illustrated by the effects of calcineurin inhibition
− Cyclosporin A & tacrolimus can inhibit calcineurin
− They can act as immunosuppressants and are widely used to preject organ rejection
Describe the MAPK pathway of downstream TCR signalling
• Ras activation stimulates the MAPK relay and induces expression of the TF AP-1
• The DAG generated by PLC-y activates the RasGRP which activates Ras
• Activated Ras triggers the Raf-MEK-ERK cascade, ending in the activation of MAPK
• Signalling by MAPK cascades Is facilitated by scaffolds that bind all 3 kinases in the relay:
➢ Kinase suppressor of Ras (KSR) scaffold associates with Raf, MEK and ERK during TCR signaling, localizing everything to the membrane
➢ It accelerates the interactions between the components of the cascade
• ERK acts indirectly to generate AP-1
Describe the NFkB pathway of downstream TCR signaling
- PLC can produce DAG, which recruits PKC- θ (isoform of PKC restricted to T cells and muscles)
- Mice lacking PKC- θ develop in the thymus, but have a defect in activation of NFkB and AP-1
- PKC- θ phosphorylates the large membrane-associated scaffold CARMA-1
- CARMA-1 oligomerises and recruits TRAF-6
- TRAF-6 stimulates the degradation of IkB by activating TAK1, which activates IKK
- IKK phosphorylates IkB, resulting in its ubiquitination and degradation
- Active NFkB can then translocate to the nucleus
- PKC- θ can also activate JNK, and may be able to activate AP-1 by this route.
What is the end result of downstream TCR signaling pathways?
• In T cells, one of the major functions of NFAT, AP-1 and NFkB is to act together to stimulate expression of the gene for IL-2
• IL-2 Is essential for promoting T cell proliferation and differentiation into effector cells.
• The IL-2 promoter contains multiple regulatory elements:
− Some are already bound by eg, Oct-1, which is produced constitutively in lymphocytes, but isn’t enough on its own
− Only when AP-1, NFAT and NFkB are activated and bound to the promoter is the gene expressed.
How does TCR signaling lead to cytoskeletal reorganisation?
• Antigen recognition by the T cell stimulates a burst of actin polymerization at the synapse, generating a lamellopodial sheet structure that spreads over the surface of the APC
• The actin related protein Arp2/3 complex, which stimulates the growth of branched actin arrays, has a central role in this process.
• Arp2/3 is coupled to the LAT-Slp76 signalosome
• Actin polymerization is temporarily associated with an increase in integrin-mediated cell adhesion
• The upregulation of the function of integrins is directly affected by PLC-y and other components of the LAT-Slp76 complex
− PLC-y activates integrins via DAG → PKC- θ → Rap GTPase activation
• Integrin activation promotes enhanced adhesion of the T cell to the APC, facilitating establishment of a long-lived T cell APC contact
• TCR signaling also induces the polarisaiton of the MTOC to the synapse
