Analysis of different protein kinase C-dependent events in T cells from allogeneic bone marrow transplantation recipients

Abstract

In order to understand the mechanisms underlying the T lymphocyte dysfunction associated to allogeneic bone marrow transplantation (BMT), we assessed two different protein kinase C (PKC) dependent events in T cells from BMT recipients: the PKC-dependent membrane expression and function of the CD69 early activation antigen; and the rapid phorbol ester-induced phosphorylation of PKC protein substrates in lysates from T cells permeabilized with digitonin, in the presence of (gamma-32P)ATP. Most BMT recipient T cells detectably expressed the CD69 surface antigen after 24 h of stimulation with either phorbol 12-myristate 13-acetate (PMA) or anti-CD3 MoAb and PMA, thus indicating that PKC activity is sufficient to induce de novo gene expression. Nevertheless, it is noteworthy that the fluorescent staining intensity with anti-CD69 MoAbs was significantly lower in BMT recipient T cells than in normal T lymphocytes, although no clear-cut correlation was found between the expression of CD69 and the proliferative capacity. However, the pattern of PMA-induced phosphoproteins analysed as early as 1 min after PKC activation in T cells from BMT recipients displaying a low response to mitogenic stimuli, was undistinguishable from that detected in T cells from healthy subjects. In all cases a major 110-kD phosphoprotein was observed, which was inducible with PMA, phorbol 12,13-dibutyrate (PDBu), 1-oleoyl-2-acetylglycerol (OAG) and a phorbol-ester-related activator of PKC (mezerein); moreover, its phosphorylation was blocked by pretreating cells with the PKC inhibitor H-7. Altogether our results suggest that the depressed mitogenic responses, which were also observed in the present study when T cells were stimulated via CD69, cannot be simply attributed to a defective PKC activity.