Deletion of alloreactive T cells by veto cytotoxic T lymphocytes is mediated through extracellular signal-regulated kinase phosphorylation

Background. Anti third-party cytotoxic T lymphocytes (CTLs) were shown to exhibit marked veto activity, thereby inducing transplantation tolerance across major histocompatibility antigens. Elimination of effector cells requires co-expression of CD8 and FasL on the veto cells and is mediated through CD8-major histocompatibility complex (MHC) class I interaction and Fas-Fas ligand signaling. Methods. To further interrogate the signaling events induced in the effector cells on their interaction with veto cell populations, effector cells from 2C transgenic mice were preincubated with different signaling inhibitors and were subject to fluorescence-activated cell sorting and western blot analysis. Results. Screening with inhibitors revealed specific inhibition only with the map kinase (MEK)/extracellular signal regulated kinase (ERK) inhibitor, U0126. Accordingly, fluorescence-activated cell sorting and western blot analysis showed that ERK phosphorylation is induced in the effector cells within 1 hr of incubation with the veto cells. ERK phosphorylation had no effect on the Fas expression level, nor was it reduced when using effector cells from Fas KO mice. Examination of ERK phosphorylation in high and low MHC-I expressing effectors revealed marked differences, suggesting that the interaction between CD8 on the veto CTL, and MHC-I on the effector cells is likely responsible for ERK phosphorylation. Furthermore, XIAP in 2C cells is specifically reduced on binding to the cognate veto cells during the mixed lymphocyte reaction but before the appearance of Annexin V reactivity. Conclusions. These results suggest that the interaction between CD8 on veto CTL and the MHC class I α3 domain on the effector cell, leads to phosphorylation of MEK/ERK in the latter cell, associated with a significant reduction of XIAP levels which, in turn, enables potent triggering of Fas-FasL mediated apoptosis on cognate binding of the veto CTLs.