Immunosuppressive activity of cyclosporin A in vivo via NFAT-independent inhibition of TCR signaling

Abstract

Abstract Calcineurin inhibitors (CNIs) such as cyclosporin A (CsA) and FK506 are widely used in treatment of autoimmune diseases and tissue transplant rejection. It is well known that CNIs inhibit NFAT-dependent gene expression, which is thought to be the major mechanism of their therapeutic effectiveness. We recently reported that CNIs inhibit TCR-proximal signaling and the upregulation of LFA-1/ICAM1 binding by preventing calcineurin’s dephosphorylation of Lck Ser-59 (LckS59), an inhibitory modification. This raises the possibility that CsA’s inhibition of TCR signaling may contribute to immunosuppression in clinical settings. Here we find that LckS59A (which cannot accept a phosphate at Ala59) knock-in T cells are resistant to CsA’s inhibition of TCR-proximal signaling. Correspondingly, whereas CsA prevented LckWT T cells from antigen-induced and LFA-1/ICAM-mediated binding to APCs in vitro, LckS59A T cells were resistant. Moreover, CsA was able to reverse existing LckWT T:APC interactions within minutes whereas LckS59A T:DC binding remained stable. To determine if disruption of T:DC binding occurs in vivo, we performed 2-photon imaging of lymph nodes from mice in which labeled antigen-specific T cells and antigen-pulsed DC had been injected. Antigen-specific T:DC formed as early as 3 hr after T cell injection. Notably, CsA markedly reduced the formation of antigen-specific LckWT T:DC but not LckS59A T:DC clusters in lymph nodes. Given that T:DC adhesion is an early and necessary step in the generation of T cell immunity, these data demonstrate a new and perhaps major immunosuppressive mechanism for CNIs immunosuppression.