Precursor frequency analysis of bryostatin activated lymphocytes.

Bryostatin 1 (Bryo) is a potent activator of protein kinase C. When T cells are stimulated with Bryo and the calcium ionophore ionomycin (Io), they expand rapidly in low-dose IL-2 (20 U/ml). We have shown that Bryo/Io-activated T-cells from murine tumor-draining lymph nodes have striking antigen-specific antitumor efficacy in vivo. To account for the specificity despite using a nonspecific T cell activation method, it was postulated that the Bryo/Io combination might preferentially activate antigen-sensitized T cells. To test this hypothesis, an allogeneic response model was used. C57BL/6 mice were primed by intraperitoneal (ip) injection with DBA/2 mouse splenocytes. After 9 days, the C57BL/6 spleens were harvested and H-2d-specific cytolytic T lymphocyte (CTL) precursor frequency (PF) was determined by limiting dilution analysis (LDA). A portion of the same spleen cells was treated for 18 hr with Bryo/Io and expanded for 7 days in IL-2 (20 U/ml); the LDA was then repeated to analyze PF after expansion. The entire experiment was also done with responder and stimulator strains reversed (DBA/2 mice immunized with C57BL/6 cells). The resulting PF values were [table: see text] Normal spleen cells treated with Bryo/Io exhibited < 10% release vs the same targets at any effector:target ratio, ruling out nonspecific cytolysis after Bryo/Io. T lymphocytes or CD8+ T cells were selected from primed splenocytes and the PF values before and after Bryo/Io were analyzed. These data showed that the increase in PF after expansion could not be attributed to T cell or CD8+ cell enrichment alone.(ABSTRACT TRUNCATED AT 250 WORDS)