T cell receptor proximal signaling

Division Of Basic Sciences - Nci

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Abstract

Cyclosporin A (CsA) and FK506 are widely used immunosuppressive drugs that inhibit the Ca2+-activated phosphatase calcineurin. Calcineurin's major activity in T cells is thought to be dephosphorylation (and thus activation) of the NFAT family of transcription factors, which are important for cytokine production and cell proliferation. It inhibition of this process that is thought to underlie the therapeutic efficacy of CsA in the treatment of transplant rejection and autoimmune diseases. It has been reported that CsA also inhibits the classic MAP kinase cascade, and therefore the stress-induced activation of p38. Signaling via the TCR results in the activation of the MAP kinase p38 by a MAPK cascade-independent mechanism: phosphorylation of p38 Y323 by ZAP-70. Therefore, we were surprised to observe that CsA also inhibited p38 activation downstream of TCR signaling. This led us to explore the molecular mechanism for this inhibition, and we have made the following observations: 1. Remarkably, CsA and FK506 inhibited a number of key events in proximal T cell signaling. These include phosphorylation of ZAP-70 on Y493, LAT, and SLP76, whereas phosphorylation of ZAP-70 Y319, PLCgamma1, and ERK were intact. 2. Calcineurin itself is recruited to the TCR signalosome within two minutes of activation and then slowly dissociates over the next 30-60 minutes. This was confirmed both by co-immunoprecipitation studies as well as imaging of TCR micro clusters by confocal microscopy. 3. Calcineurin recruitment requires Lck signaling and ZAP-70, but not ZAP-70 signaling. Therefore, we believe that it is binding phosphorylated ZAP-70. 4. siRNA-mediated knockdown of calcineurin recapitulates inhibition of ZAP-70 493, and introduction of ZAP-70 Y493R (which cannot be phosphorylated on residue 493) results in signaling defects similar to those described above (point 1). 5. The calcineurin target in the TCR signalosome is Lck S59, whose phosphorylation has been reported to inhibit TCR signaling. Consistent with this, introduction of Lck mutants (Lck S59A, which can not be phosphorylated, and Lck S59E, a phospho-mimetic) resulted in enhanced and inhibited signaling, respectively. 6. To determine if inhibition of proximal TCR signaling could be a novel mechanism for it's immunosuppressive effects, we looked for important NFAT-independent events downstream of TCR signaling. LFA-1-dependent adhesion to ICAM-1 is a very rapid and essential step in T cell activation and egress from the blood to tissue. We found that CsA inhibited conversion of LFA-1 from the inactive to the active state and blocked cell adhesion to ICAM-1-coated wells. Studies with T cells expressing the Lck-mutants confirmed that the phosphorylation status of S59 was responsible. Followup studies have shown that the addition of CsA to T cells already activated and bound to ICAM-1-coated plates or antigen-pulsed APC results in rapid (minutes) reversal of adhesion. These results have shown that, unexpectedly, calcineurin is an integral member of the TCR signaling complex. We have performed in vivo studies to assess the affect of cyclosporin A on T cell activation. We have B6 mice in which LckS59A (unable to be phosphorylated and thus not a calcineurin target) has been knocked into the WT locus. We used these mice to study acute graft-vs-host disease (aGVHD), in which H-2b T cells are injected into irradiated (H-2b X H-2a)F1 mice. CsA is a standard treatment for this disease, and it is accepted that it works by inhibiting NF-AT-dependent cytokine production. By comparing the effect of CsA on disease caused by WT or LckS59A T cells we can determine if NF-AT, TCR signaling, or a combination of the two is responsible for CsA efficacy. We found that despite effective inhibition of NFAT-dependent cytokine production, aGVHD progression was largely unaffected by CsA when LckS59A T cells were the transferred effectors. There was less CD8 T cell infiltration in the liver, and the cells that were there expressed very little of the cytolytic protein perforin. Moreover, 2-photon imaging of antigen-specific T:DC interactions in lymph nodes in WT or LckS59A mice treated with CsA found that the former, but not the latter, had markedly decreased T:DC interations and fewer T cell clusters, consistent with the effect of CsA on LFA-1/ICAM1 binding. These results demonstrate that the current paradigm that CsA ameliorates aGVHD by inhibiting NFAT activation is incorrect. Rather, it does so by inhibiting TCR signaling that otherwise increases inLFA-1-affinity andp erforin expression, and no doubt other not-yet-characterized responses. In addition to these, we have found that siRNA-mediated knockdown of calcineuring alpha and beta results in a more profound loss of TCR signaling than inhibition of calcineurin enzymatic activity. We have generated CRISPR/Cas9 KO of the calcineurin alpha and beta forms of the A chain in Jurkat T cells and are using confocal microscopy to characterize the ability of these cells to form signaling TCR microclusters and propogate downstream events.