To better understand immune responses to brain tumors and to develop possible approaches for immunotherapy, we have investigated the leukocyte populations infiltrating the rat 9L gliosarcoma. By immunocytochemical analyses of the cells infiltrating the tumor, we observed a substantial number of cells expressing natural killer cell receptor protein 1 (NKR-P1), a marker expressed only on rat lymphocytes capable of non-MHC-restricted cytotoxicity, Previous investigations have determined the existence of three populations of NKR-P1(+) lymphocytes in normal rats, including NKR-P1(bright)/T-cell receptor (TCR)(-)/CD3(-)/CD5(-) (similar to 5-15%), NKR-P1(dim)/TCR alpha beta(+)/CD3(+)/CD5(+) (similar to 1-5%), and NKR-P1(dim)/TCR gamma delta(+)/CD3(+)/CD5(+) (similar to 0.5-2%). By one-parameter flow cytometry, it was determined that NKR-P1(+) cells constituted 30-60% of the lymphocytes in 9L tumors. Among splenic lymphocytes or peripheral blood leukocytes, NKR-P1(bright) cells are 1.5-4.5 times more numerous than NKR-P1(dim) cells. In striking contrast, NKR-P1(dim) cells were 4-5 times more numerous than NKR-P1(bright) cells among lymphocytes isolated from 9L tumors, Using quantitative analyses of laser confocal microscopic scans, we determined that NKR-P1(dim) cells were similar to 4 times as numerous as NKR-P1(bright) cells in situ, confirming flow cytometric findings, By two-color flow cytometric analyses, it was observed that similar to 5-10% of the cells were NKR-P1(bright)/CD5(-)/TCR-, a phenotype representative of NK cells. Also, similar to 11-25% of the cells were NKR-P1(dim)/CD5(+)/TCR+ cells, corresponding to the T-cell subset with non-MHC-restricted lytic function, In addition, we observed a cell population among 9L-derived lymphocytes with a NKR-P1(dim)/CD5(-)/TCR- phenotype (similar to 15-25%), Cells of this phenotype have not been reported previously, and most likely represent NK cells down-modulated for expression of NKR-P1. Alternatively, they might represent cells of unknown origin or cells down-modulated for expression of T-cell markers in the microenvironment of 9L tumors. We also compared the lytic capacity of NKR-P1(+) populations derived from normal animals and from 9L gliosarcomas, In these experiments, it was determined that, although cells isolated from 9L tumors had some capacity to lyse tumor target cells, they were clearly less efficient than cells isolated from normal splenocytes. Cumulatively, these data suggest that there is selective localization of cells capable of mediating antitumor responses in 9L, but that tumor-associated factors may down-regulate their function and expression of NKR-P1.
