Cytotoxic T lymphocytes reactive against a syngeneic murine tumor and their specific suppressor T cells are both elicited by in vitro allosensitization.

Abstract

The induction of cytotoxic response against syngeneic or autologous murine or human tumor cells by pool allosensitization in bulk MLC was demonstrated by Bach et al. (1,2). Subsequently, we showed the existence ofmurine cytotoxic cell precursors that can be activated to react with syngeneic tumor cells by single-donor allosensitization in bulk MLC, applying the limiting dilution cultures (LDC) t technique (3, 4). We also showed that a high frequency (1/20) of cytotoxic cells can be elicited in the same manner even against syngeneic, weakly immunogeneic (MT2, mammary carcinoma in C3H mice ; M109, lung carcinoma in BALB/c mice) or nonimmunogenic (PIR-2, x-irradiation-induced lymphoma in 136 mice) tumors (5, 6). We postulated that shared or crossreactive antigenic determinants between the allogeneic leukocytes and the syngeneic tumor cells lead to the elicitation of the antisyngeneic tumor cytotoxic (ASTC) cells. Similar findings were reported by others (7-11) . Recently, we have demonstrated (12) that the ASTC precursors are phenotypically "classical" CTL precursors (i.e., Thy-1' ; Lyt-2+ ; L3T4-; asialo GMl -) rather than classical NK-type lymphokine-activated killer (LAK) cells that might have arisen in the presence of the high concentration of IL-2 (>250 U/ml) in the LDC.In our MLC-LDC system, in which the syngeneic target cells were the syngeneic PIR-2 cells, no ASTC response could be demonstrated by directly testing lymphocytes sensitized in bulk MLC (5, 6). The high ASTC frequency, as well as the antiallotarget response, were demonstrated in LDC wells plated with a low number of MLC-derived responding cells (<200/well). As the number of responding cells plated per well increased beyond a critical number (usually 500/well), a gradual decrease ofASTC response was observed. No anti-PIR-2 cytotoxicity could be detected when >5,000 MLC-derived cells were plated per well in LDC (3-5), whereas the percentage of cytotoxically positive wells against the allotarget, as well as the level of antiallocytotoxicity of the individual wells, remained at maximum .

Chemo-Immunotherapy of murine solid tumors: Enhanced therapeutic effects by interleukin-2 combined with interferon α and the role of specific T cells

“…Limiting-dilution analysis of cytotoxic T lymphocyte (CTL) precursor frequency (see [25]). Splenocytes (500-50 000 cells/well) were incubated (16-24 replicates/group) for 9 days in U-shaped 96-well microtiter plates (in 0.2 ml), in the presence of 1 x 106 irradiated (30 Gy) syngeneic spleen cells (feeder cells) and IL-2 (50 Cetus units/well), with or without 1 x 104 irradiated (100 Gy) stimulating tumor cells.…”

“…Splenocytes [(5-10)x 106/ml] were treated with antiThy-l.2 (1/200), anti-Lyt2.2 (1/100) and anti-L3T4 (1/50) monoclonal antibodies (Cedarlane, Canada), and anti-asialoGM1 antiserum (1/50) (Wako, Japan) together with rabbit complement (1/10) (Cedarlane, Canada) as described previously [25]. For depletion in vivo, a 0.1-ml sample of the above antibodies was administered i.p.…”

“…The cytotoxic activity of individual wells was tested in a 4-h 51Cr-release assay. Frequency analysis was performed as described elsewhere [25,26]. …”

Non-specific anti-proliferative effect of FTY720, a derivative of fungal metabolite from Iscaria sinclarii

“…For allogeneic T cell proliferation (Leshem and Kedar, 1990), splenocytes from BALB/C and C57BL/6 with 1.5:5 ratios were cultured in flat bottom 96-well plates in the presence and absence of various concentrations of FTY720. For splenic T lymphocyte proliferation, splenocytes (5×10 6 cells/ml) from BALB/C were incubated with T cell mitogen [Con A (1 mg/ mL)] with FTY720 for 48 h (Cho, et al, 2000).…”