Figure 4

Restoration of IL-2 production and proliferation of tumor-infiltrating lymphocytes in the absence of new T cell migration. B16.SIY-bearing mice were either treated with FTY720 or control vehicle prior to initiation of therapy, to prevent migration of new lymphocytes into the tumor. A. Peripheral blood T cell numbers following FTY720 treatment on the day of tumor harvest for analysis. Open bars depict the number of CD45⁺CD3⁺ T cells detected in 200ul peripheral blood of vehicle treated mice set to 100%. Filled bars represent the number found in FTY720-treated mice, relative to the vehicle-treated group. B &C. Single cell suspensions from tumor were labeled with cell trace and stimulated with plate-bound anti-CD3 antibody for 48 h prior then with anti-CD3 and anti-CD28 in the presence of Brefeldin A. Cells were then analyzed for proliferation by cell trace dilution and production of IL-2 via intracellular staining. Depicted are the percentages of proliferating cells (B) or proliferating and IL-2 producing cells (C) comparing vehicle-treated groups (open bar) to FTY720-treated groups (filled bar). Results are shown as the mean +/− SEM combining two experiments with each having 2 pools of 3 mice. Significance was tested using Mann–Whitney-U test but no significant change between FTY720 and vehicle control could be detected except for increased IL-2 production in the αPD-L1 + IDOi treatment group (p = 0.02).