Previously we have demonstrated that the in vitro generation of P815-specific anti-tumor cytotoxic T lymphocytes (CTL) was suppressed by splenic suppressor T cells from late tumor-bearing hosts (TBH). Suppression is not caused by in vitro growth of P815 from splenic metastases, since suppression was also seen with spleen cells from late TBH mice bearing a hypoxanthine/aminopterin/thymidine-sensitive subline (PHS-5) of P815 in the presence of HAT. Cyclophosphamide has been shown to inhibit the induction of suppressor cells selectively in a number of immune responses, but evidence that it can inhibit active tumor-induced suppressor T cells is limited. We have found that suppressor T cells already induced by P815 in syngeneic late TBH are sensitive to low doses of cyclophosphamide (50 mg/kg) given 1 day before spleen harvest, but the in vitro CTL response of late TBH spleen cells could not be restored by pretreating the mice with cyclophosphamide, even when exogenous interleukin-2 was added to the cultures. Although 50 mg/kg cyclophosphamide did not inhibit the CTL response of spleen cells from mice immunized with P815 + Corynebacterium parvum, the same dose of cyclophosphamide eliminated the CTL response of spleen cells from early TBH. Interleukin-2 (IL-2) did not overcome this effect of cyclophosphamide, suggesting a direct effect on CTL. "Ultra-low" -dose cyclophosphamide (10 mg/kg) did not adversely effect early TBH CTL but was still able to eliminate suppressor T cell activity from late TBH. Nevertheless, late TBH CTL remained unresponsive after pretreatment of mice with ultra-low-dose cyclophosphamide, even when exogenous IL-2 was added in vitro. CTL precursor frequency analyses demonstrated that cyclophosphamide pretreatment had little or no effect on the numbers of CTL precursors from early TBH. Late TBH CTL precursor cells were not detectable in these studies, with or without suppressor T cell inhibition by cyclophosphamide pretreatment. Thus, it appears that most CTL precursor cells may be lost or irretrievably inactivated in the spleens of late TBH mice.

Mafosfamide (Mafo) is an analog of cyclophosphamide that does not require hepatic activation and therefore has in vitro activity. The present study was conducted to determine the effects of in vitro treatment with Mafo on the generation and growth of cytotoxic T lymphocytes (CTL) from tumor-bearing host mice (TBH). In contrast to early (day-11) TBH splenocytes, splenocytes from late (days 18-20) P815 TBH mice suppress the in vitro generation of CTL. Treatment of late TBH splenocytes in vitro with 5-15 microM Mafo resulted in a reduced ability of these cells to suppress in vitro CTL generation. Treatment of late TBH splenocytes with 10 microM Mafo also inhibited their ability to suppress adoptive immunotherapy of intradermal tumors with immune splenocytes. These doses of Mafo were selectively toxic to the suppressive effects of late TBH splenocytes, since treatment of early TBH splenocytes with 1-10 microM Mafo did not significantly inhibit CTL generation. Spleen cells from early (days 10-12) TBH mice, carried in long-term in vitro sensitization cultures in the presence of tumor cells and 20 U/ml human recombinant interleukin-2, did not increase in cell number over time. However, when pretreated with 3 microM Mafo, this population of tumor-sensitized lymphocytes demonstrated 450-fold growth over 6 weeks as compared to the static cell numbers for the untreated controls. High levels of tumor-specific cytolytic activity were maintained in these expanded cells. These results suggest that Mafo pretreatment markedly and selectively inhibits suppressor cells that limit long-term expansion of splenic CTL in culture and inhibit adoptive immunotherapy of solid tumors.

Large numbers of cytotoxic T lymphocytes (CTL) could be generated from tumor-draining lymph nodes (DLN) from mice bearing PHS-5 tumor by culturing at low density with autologous tumor cell stimulators and 20 U/ml recombinant interleukin-2 (IL-2). Outgrowth of metastatic tumor cells in culture was prevented by use of this hypoxanthine/aminopterin/thymidine-sensitive mutant of P815, PHS-5. After 9 days in culture, lymphoid cells demonstrated specific cytotoxicity against autologous tumor target cells. Lymph node cells could be expanded continuously in culture with repeated tumor stimulation with up to 7500-fold increase in cell number by 6 weeks; although CTL could be activated from tumor-bearing host spleen cells in short-term culture, they showed no significant growth in long-term cultures. Phenotypically, DLN cells were a mixture of CD8+ and CD4+ cells immediately after harvest but after 2 weeks in culture they were predominantly CD8+ CD4-. CTL could be generated from tumor-bearing mice 10-14 days after i.d. tumor inoculation into the abdominal wall, but the immune response declined both in spleen and DLN by 21 days. Much greater CTL activity could be generated from axillary DLN that contained metastases than from non-draining popliteal nodes that were free of metastatic tumor cells. Some CTL activity could be generated from DLN with the addition of IL-2 alone but was further increased by the addition of more tumor cells as stimulators. When adoptively transferred to a host with 3-day P815 liver metastases, lymphocytes from DLN activated in vitro were able to reduce or eliminate metastases with very little or no IL-2 administered concomitantly. As few as 10(6) cells were therapeutically effective, and in vivo efficacy was tumor-specific, since L5178Y liver metastases were not affected.

Tumor-specific T-cell clones were derived from spleen cells of mice bearing a syngeneic PHS-5 tumor (a P815 mastocytoma mutant). Cells were expanded in vitro and characterized and assayed for activity against the relevant tumor in vivo. Clone cells were CD4-, CD8+ T lymphocytes, as determined by fluorescence activated cell sorting analysis and were specifically cytotoxic against P815 tumor cells in vitro, as shown in chromium 51 release assays. These cells require both antigen and interleukin 2 to proliferate; neither alone is sufficient, even with the addition of interleukin 1. In an experimental P815 liver metastasis model, the adoptive transfer of GD11 or GD11.17 clone cells and injection of recombinant interleukin 2 (7500 U intraperitoneally) 3 days after infusion of tumor cells reduced the number of tumor nodules, while the adoptive transfer of lymphokine-activated killer cells was ineffective.