Removal of CD25+ cells from precursor alloreactive cytotoxic T lymphocyte preparations does not prevent T regulatory cell generation after the one-way mixed lymphocyte reaction

265 An effector cell population, alloreactive cytotoxic T lymphocytes (aCTL), sensitized to human leukocyte antigens (HLA) of the tumor host, has shown promise when intratumorally instilled into the resected tumor beds of recurrent gliomas. Half of the six patients treated in a pilot study responded. We hypothesized that the removal of T regulatory (Treg) cells from the precursor aCTL preparations may further optimize the therapy, or perhaps help to explain the nonresponse observed in 3 of the 6 patients. Treg cells are a subset of CD4+ T cells expressing the activation marker CD25 (IL-2 receptor α-chain); they also can express FoxP3, a member of the Forkhead box/winged-helix family of transcriptional regulators. Treg cells normally constitute a low percentage (0.5-6.0%) of peripheral blood mononuclear cells (PBMC) and therefore are a small entity within the normal donor PBMC used as precursor aCTL. A simple magnetic bead removal of the Treg cell compartment from the PBMC could be easily implemented during clinical generation of aCTL. Therefore, we tested to see if the batch removal of CD25+ cells from the PBMC (precursor aCTL) would improve the growth kinetics and cytolytic function of aCTL. Our preliminary data show that the frequency of Treg cells present in PBMC pools increases during culture of the aCTL after performing the one-way mixed lymphocyte reaction (MLR). Dual positive CD4+FoxP3+ Treg cells were found to comprise approximately 1% of the CD4+ T cell population in the PBMC used as precursor aCTL, and by day 14 post-MLR, the frequency of Treg cells increased up to 5-fold. aCTL preparations made from PBMC lacking CD25+ T cells (aCTL -CD25 ) were mixed with inactivated stimulator lymphoblasts at a 10:1 responder to stimulator ratio, and compared to parallel aCTL cultures that were not depleted of CD25+ cells. Growth rate, measured by daily lactate production, was similar throughout a 14 day culture period. Surprisingly, the cytotoxic capability of aCTL -CD25 was significantly inhibited ( p -CD25 cultures were consistently higher on day 14 post-MLR compared to the percentages seen in the undepleted cultures. The data indicate that despite batch removal of CD25+ cells from precursor aCTL, the cytolytic function of aCTL was not improved, and in fact, resulted in an increased population of CD4+FoxP3+ Treg cells.