We have described a method for the generation, from fresh human renal cell cancers, of lymphoid cells that are capable of exhibiting significant antitumor reactivity when tested in short term 51Cr release assays. Tumor cell suspensions obtained from 37 consecutive fresh human renal cell cancer specimens (35 patients) could be separated by using enzymatic techniques and culturing in medium containing recombinant interleukin 2 (IL-2). The total cell recovery was 1.5 × 109 ± 2.2 (SE) per tumor with a range of 1 × 108 to 5 × 109 cells. The percentage of tumor cells in the suspension ranged from 6 to 75% with a mean of 39.1 ± 3.3%. The remaining cells were predominantly lymphocytes. Viability of mononuclear cells was greater than 90%. Activated tumor-infiltrating lymphocytes (TIL) within these tumors expand and by 10 to 14 days after initiation of culture a 5- to 15-fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells. Lymphocytes were recultured in fresh medium containing IL-2 and continued to expand between 2- and 10-fold every 4 to 6 days for an average of 33.7 ± 4.5 days, resulting in greater than 50,000-fold increase in the total number of lymphocytes. The average number of splits was 4.9 ± 0.8, with a range of 0 to 21. In 11 of 11 cases tested, TIL exhibited a far better expansion capability in vitro compared to that of peripheral blood lymphocytes obtained from the same patient and grown under identical conditions. The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+). With continued in vitro expansion (up to 50 days) there was a concomitant increase in the helper T (Leu 3+) and pan T populations (Leu 4+) and decrease in Leu 2+ and HLA-DR+ cells. Compared with expanded peripheral blood lymphocytes, these cells demonstrated higher levels of IL-2+ receptors and HLA-DR+ antigens.
Renal TIL effectors expanded in IL-2 could lyse almost all autologous tumor targets in 4-h chromium release assays. Allogeneic renal as well as nonrenal targets were equally lysed. TIL lysis of cultured tumor targets K562 and Daudi was significantly better than lysis of autologous, allogeneic-renal, and nonrenal targets. No statistically significant difference in the cytotoxic activity of renal TIL or peripheral blood lymphocyte effectors in killing autologous or allogeneic targets could be demonstrated. The best expansion and cytotoxicity of TIL were achieved in the first 3 weeks in culture, followed by a gradual decrease in both growth rate and lytic potential of the cells. Cryopreserved tumor preparations maintained good autologous cytotoxic activity as well as stable surface markers and could be expanded in about two-thirds of the tumors. This opens the possibility of thawing, activating with interleukin 2, expanding, and reinfusing the patient with his own cells once distant metastases appear.
Murine studies have demonstrated that the adoptive transfer of TIL is from 50 to 100 times more potent than therapy with LAK cells in mediating the destruction of established tumor in a variety of tumor models. The studies described in this paper provide a basis for testing human TIL in the treatment of patients with metastatic renal cell cancer.