EX-VIVO EVIDENCE FOR ASYMMETRIC TYROSINE PHOSPHORYLATION OF ZAP-70 ONDOUBLE-POSITIVE THYMOCYTES IN THE POSITIVE SELECTION PROCESS

Antigen stimulation via TCR in mature T cells provides rapid induction of tyrosine phosphorylation of intracellular substrates including ZAP -70. To study the potential involvement of tyrosine phosphorylation in CD4(+)CD8(+) [double-positive (DP)] thymocytes in the positive select ion process in vivo, we isolated and analyzed them in the presence of phosphatase inhibitor. DP thymocytes were obtained from TCR transgenic mice (TCR-Tg) expressing MHC class I- or class If-restricted TCR in s electing and non-selecting MHC backgrounds respectively. The phosphory lation of ZAP-70 in DP thymocytes of class I-restricted TCR-Tg was sig nificantly higher in the positively selecting background than in the n on-selecting one. However, such a phosphorylation difference between s electing and non-selecting TCR-Tg was found to be considerably less in class Ii-restricted TCR-Tg. A similar bias for ZAP-70 phosphorylation was also observed on selecting DP thymocytes when I-A(beta) deficient - and beta(2)-microglobulin-deficient mice were compared, These ex viv o studies suggest that TCR-mediated signaling on DP thymocytes induces ZAP-70 phosphorylation under a different manner of engagement of TCR to class I and class II molecules in the positive selection process.