Novel Phenotype Associated within VivoActivated CTL Precursors

A previously undefined phenotype of CD8+cells that appears to representin vivoactivated CTL precursors (CTLP*) has been identified in the spleens of C57Bl/6 mice responding to a P815 tumor allograft. This population was first evident by the transient expression of very high levels of CD28 and CD44 on day 5 of the allograft response and reached maximal levels on days 7 and 8 before declining on day 9. A transient increase in CD69 expression was also observed on these cells on day 5. In contrast, CTL effectors (CTLE), identified by their CD8+CD44hiCD62LloCD45RBlophenotype, were not appreciably detected in the spleen until day 8 and reached maximal levels on day 10. Further characterization of CTLP* on day 7 revealed that they represented blasting cells by increased light scatter and also expressed very high levels of CD54 but not CD122, CD152, or CD154. In addition, the cells had already up-regulated CD49d, asialo GM1, CD11a, and CD95L, and down-regulated their expression of CD62L. A small percentage of these cells also expressed CD25. Day 7 CTLP* sorted on the basis of their CD44xhiand CD54xhiphenotype did not exhibit cytolytic activity in a standard chromium release assay but became cytotoxic when they were cultured in the presence of exogenous murine IL-2 for 5 days. Granzyme B activity, however, was detected in CTLP* on day 7 at levels equivalent to CTLEon day 10. In order to establish a potential precursor relationship between CTLP* and CTLE, mice were treated with various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical that has been shown to dose-dependently suppress thein vivogeneration of CTLEto P815 tumor cells by altering an early stage of CTLPactivation. Results indicated that CTLP* were suppressed by TCDD on day 7 to the same degree that CTLEwere suppressed on day 10. Importantly, for controls and for all doses of TCDD, there were approximately 12.5 CTLEon day 10 for every CTLP* detected on day 7. These results suggested that TCDD acted identically across all doses to inhibit the early stages of activation of CTLPbut did not affect the final stages of differentiation and expansion to CTLE. This interpretation supports the previous observation that TCDD exposure had to occur within the first 3 days of the allograft response in order to induce suppression of CTLEactivity. Taken together, these results support the conclusion thatin vivoactivated CTLPcan be identified by their unique expression of very high levels of CD44, CD28, and/or CD54 prior to their full maturation and clonal expansion to functional CTLE.