Inhibition of tumor-specific cytotoxic T-lymphocyte responses by transforming growth factor beta 1.

Transforming growth factor beta (TGF-beta) is a potent immunosuppressive cytokine that is produced by neoplastic and normal cells. It has not been demonstrated directly, however, that TGF-beta can inhibit antigen-specific T-cell responses to tumor cells in vitro. We show here that generation of antitumor cytotoxic T-lymphocyte (CTL) activity in mixed-lymphocyte tumor cultures of splenocytes from DBA/2 mice immunized with the syngeneic P815 mastocytoma + Corynebacterium parvum was consistently and profoundly inhibited when 0.675 to 10 ng/ml of TGF-beta were added on Day 0 of culture. TGF-beta added on Day 1 or later had little or no effect. In contrast to the results with P815 immune mice, mixed-lymphocyte tumor cultures established with splenocytes from P815 tumor-bearing hosts showed variable degrees of inhibition by TGF-beta, depending on the stage of the ongoing in vivo immune response. Addition of recombinant murine tumor necrosis factor alpha (1,000 or 10,000 units/ml) partially reversed inhibition of CTL responses by TGF-beta, while recombinant interleukin 2 nearly completely reversed the suppression. These data indicate that one level at which TGF-beta may act to inhibit mixed-lymphocyte tumor cultures is that of cytokine production. To determine whether TGF-beta also has any direct effect on CTL, P815-specific CTL clones derived from tumor-bearing host mice were utilized. We found that proliferation of rested CTL clones in response to tumor cells + interleukin 2 was inhibited by 5 ng/ml of TGF-beta, while the interleukin 2-dependent reactivation of cytolytic activity was not affected by TGF-beta. In contrast to rested CTL, when TGF-beta was added to cultures of previously activated CTL, proliferation was not inhibited. These data demonstrate that TGF-beta has profound inhibitory effects on the in vitro generation of effector CTL from tumor-specific murine splenocytes, and this inhibition may be an indirect result of suppressed cytokine production as well as a direct antiproliferative effect on CTL.

Enhancement of cytotoxic T lymphocyte growth from spleens of P815-tumor-bearing host mice with mafosfamide.

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.

A cytotoxic T-lymphocyte clone derived from mice with progressively growing tumors.

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.

Activation and in vitro expansion of tumor-reactive T lymphocytes from lymph nodes draining human primary breast cancers.

The feasibility of in vitro activation of lymphocytes from the draining lymph nodes (DLN) of breast cancer patients was examined. Lymphocytes isolated from 48 DLN from 12 patients were examined for their proliferative responses to rIL-2, autologous tumor cells, or rIL-2 plus tumor cells. Three general patterns of cellular responses were observed. Cells from some DLN (17%) were unresponsive to any stimuli. Lymphocytes from 52% of the DLN responded moderately to rIL-2 alone. The combination of rIL-2 and tumor antigen had a synergistic effect on the proliferation of cells from 31% of the DLN assayed. Phorbol dibutyrate and ionomycin plus rIL-2 stimulated expansion of DLN lymphocytes by up to 850-fold after 35 days. These expanded cell populations, as well as those stimulated with antigen plus rIL-2, were predominantly CD3+ and CD16- cells, varying in proportions of CD4+ and CD8+ subsets. Both populations were cytotoxic against autologous tumor, MCF-7, and K562 target cells.

Cyclophosphamide and abrogation of tumor-induced suppressor T cell activity.

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.

Activation and expansion of cytotoxic T lymphocytes from tumor-draining lymph nodes.

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.