Production of stable cytolytic T-cell clones directed against autologous human melanoma.

We have attempted to optimize the production of stable human cytolytic T lymphocyte clones directed against autologous melanoma cell lines. MLTC were restimulated every week with irradiated melanoma cells in medium containing human serum and IL-2. After 21 to 35 days, in 5 out of 6 patients, these cultures expressed a preferential cytolytic activity against the autologous melanoma cells, as compared to autologous EBV-B cells or NK target K562. Limiting dilution of MLTC responder cells was performed at times varying from days 7 to 28, in medium containing IL-2 and allogeneic EBV-B cells as feeders. Approximately 1% of these responder cells gave rise to CTL clones that lysed the autologous melanoma cells, but did not lyse K562 or autologous B cells. It was possible to maintain in culture for several months a large number of CTL clones that retained this specificity with high activity, and multiplied more than 5-fold every week. Some of these CTL clones were dependent on the presence of the autologous melanoma cells for their growth. With one melanoma, the use of autologous CTL clones made it possible to identify 3 different antigens on the tumor cells.

Protection against two spontaneous mouse leukemias conferred by immunogenic variants obtained by mutagenesis.

Two spontaneous mouse leukemias were adapted to culture. In agreement with most reported observations on spontaneous tumors, injection of irradiated cells of the malignant culture cell lines failed to protect mice against these leukemias. These cell lines were treated in vitro with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and stable immunogenic variants (tum-) were obtained, that failed to form progressive tumors in syngeneic CBA/Ht mice. Mice that had rejected tum- variants showed a significant degree of resistance to challenge not only with the original malignant cell line but also with the original transplantable tumor. No protection was observed against syngeneic tumor cells other than those of the parental tumor. These results indicate that these two spontaneous leukemias carry a specific transplantation antigen that can be the target of a rejection response by syngeneic mice. In confirmation of this, we found that lymphocytes of CBA/Ht mice that had rejected tum- variants could be restimulated in vitro so as to develop a cytolytic activity directed against an antigen that was specific for the original tumor cell line.

Immunogenic variants obtained by mutagenesis of mouse Lewis lung carcinoma. Recognition of variant-specific antigens by cytolytic T lymphocytes.

By mutagenesis of a cell line derived from Lewis lung carcinoma (3LL), it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic mice. The possibility of obtaining a cytolytic T cell (CTL) response directed specifically against these tum- variants was examined. With the four variants that were analysed, a significant cytolytic activity was obtained with peritoneal cells from immune mice collected shortly after an intraperitoneal boost and also with spleen cells after a secondary stimulation in vitro. The CTL populations preferentially lysed the immunizing tum- variant, while also showing a cross-reactive lysis against the other variants and the original 3LL cells. Highly active CTL clones could be isolated from limiting dilution microcultures of these CTL populations. The clonal analysis clearly showed the existence of two distinct CTL populations, one directed exclusively against the immunizing variant and another that lysed all 3LL targets equally. This CTL specificity analysis therefore demonstrates directly the presence of new antigens on the 3LL tum- cell variants.