Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes

We have shown that tumor vaccine-sensitized draining lymph node (vDLN) cells activated ex vivo with bryostatin and ionomycin (B/I) were capable of inducing antigen-specific regression of a murine mammary tumor, 4T07. vDLN cells not activated with B/I were ineffective. We hypothesized that B/I selectively activates tumor-sensitized (CD62L(low)) lymphocytes, to account for the highly potent and tumor-specific activity. We hypothesized that CD8+ CD62L(low) cells may be preferentially activated by B/I treatment, infiltrate the tumors and mediate tumor regression in mice. 4T07-IL2 tumor cells were injected into one hind footpad of BALB/c mice. Ten days later, vDLN were harvested and separated based on CD62L expression. After separation, cells were activated with B/I, expanded with IL2 (40 IU/ml) for 10 days, and adoptively transferred to 4TO7 tumor bearing mice. Naive mice were also treated with different subsets of T cells and later were challenged with 4TO7 tumor cells. To test in vitro responses to antigen, expanded lymphocytes were cultured either alone or with irradiated 4T07 tumor cells. Supernatants were harvested after 24 h and tested by ELISA for IFN-gamma. The importance of the host immune response was tested by AIT into 4T07-bearing nude athymic mice. Host mice were depleted in vivo of CD4 or CD8 T cells after vDLN AIT to ascertain the mediators of tumor regression. In order to track B/I activated vDLN cells, they were prestained with CFSE prior to adoptive transfer into tumor-bearing hosts. At various time points, tumors, spleens and lymph nodes of host mice were harvested, dual stained for activation marker expression and analyzed by flow cytometry. CD62L(low) cells expanded 12-fold more than CD62L(high) lymphocytes during the 10 day culture period. Supernatant from CD62L(low) cells + 4T07 cultures contained 33-fold more IFN-gamma than supernatant from CD62L(high) cells + 4T07 cultures (843.9 pg/ml +/- 135.8 vs 25.89 pg/ml +/- 0.01). Adoptive transfer of CD62L(low) lymphocytes induced complete tumor regressions in all mice, while tumors regressed in only 17% of mice treated with CD62L high lymphocytes. Naive mice that received B/I-activated CD62L(low) cells were protected from future tumor challenges, while mice given CD62L high Cells did not exhibit the same resistance to tumor growth. Tumors in nude host mice regressed after AIT treatment. In vivo depletion of CD4 T cells after AIT did not inhibit tumor regression, but CD8 T cell depletion abrogated tumor regression. vDLN cells tracked preferentially to tumor draining lymph nodes and proliferated in vivo, persisting for at least 21 days, and were 95% CD44+ and 39% CD69+. Bryostatin 1 and ionomycin, by increasing PKC activity and intracellular calcium, respectively, mimic intracellular signals that result in T cell activation. CD62L(low) cells are preferentially activated by B/I, leading to a highly effective anti-tumor T cell population.