Markian R. Bochan

Target cell-directed rapid degradation of TNF-α messenger RNA in human cytotoxic T cells

Abstract Previously we have shown that upon exposure to sensitive target cells (TC), human cytotoxic T cells (CTL) and natural killer cells (NK) undergo functional inactivation and specifically degrade mRNA-encoding lytic proteins stored in cytoplasmic granules. These lytic proteins include perforin (PFP) and the serine proteases (granzymes). In this study we show that tumor necrosis factor-alpha (TNF-α) mRNA undergoes rapid down-regulation after interaction with TC, in a manner identical to PFP and serine protease mRNA. Both PFP and TNF-α messages are down-regulated and maintained at low baseline levels while effector cells (EC) are in contact with TC. Moreover, treatment of CTL with antibodies against CD2, CD3, CD8, and class I could not reproduce TC-directed mRNA down-regulation of TNF-α and PFP even though lytic ability was inhibited. Anti-CD2 and anti-CD3, however, markedly induced the expression of TNF-α mRNA within 15 min. The increase of TNF-α mRNA by anti-CD2 and anti-CD3 was offset by the presence of TC, suggesting that TC supply a negative signal to the CTL resulting in degradation of the TNF-α mRNA. Furthermore, treatment of CTL with cycloheximide (CHX) did not prevent PFP message loss and did not allow its recovery, suggesting that de novo protein synthesis is not required for mRNA degradation. In contrast, whereas CHX did not prevent TNF-α message loss, CHX allowed recovery of TNF-α mRNA within 120 min after TC-directed degradation. Taken as a whole, these data suggest that TC provides a negative regulatory signal that triggers the degradation of TNF-α message.

Target cell-directed degradation of perforin mRNA in CTL: Lack of correlation with loss of protein and lytic ability

We have previously shown that CTL and NK cells rapidly down regulate perforin mRNA and become functionally inactive within 4-6 hr after exposure to sensitive target cells (TC). We report here for the first time that CTL also down regulate perforin mRNA upon exposure to resistant, but binding, TC. When three separate human MHC-restricted CTL lines were exposed to resistant TC, perforin mRNA was rapidly degraded. Removal of both extracellular Ca++ and Mg++ prevented perforin message down regulation, whereas removal of Ca++ alone did not, indicating that CTL:TC binding was required. Unlike the response of CTL exposed to sensitive TC, resistant TC did not trigger serine esterase (SE) release, suggesting distinct signalling pathways for perforin mRNA down regulation and granule exocytosis. Moreover, using western analysis, we showed that there was limited (< 10%) perforin protein release after CTL:TC interaction, suggesting that CTL loss of lytic activity after exposure to sensitive TC is not due to massive depletion of perforin. Treatment of CTL with mAb to CD2, CD3, CD2 + CD3, CD8, Class I and LFA-1 did not induce perforin mRNA down regulation. Furthermore, mAb to CD2, CD3, CD8, Class I, Class II, CD54 and LFA-1 did not block TC-mediated perforin mRNA down regulation although lysis of TC was inhibited.