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Lymphocytes Infiltrating Human Ovarian Tumors: Synergy between Tumor Necrosis Factor and Interleukin 2 in the Generation of CD8⁺ Effectors from Tumor-infiltrating Lymphocytes¹

Departments of Pathology [W.Y.L., S. L., A. K., R. B. H., T. L. W.] and Medicine [R. B. H.], University of Pittsburgh School of Medicine, Pittsburgh 15261, and Pittsburgh Cancer Institute [R. B. H., T. L. W.], Pittsburgh, Pennsylvania 15213

Tumor-infiltrating lymphocytes (TIL) were isolated by enzymatic digestion and gradient centrifugation from 18 human ovarian carcinomas. These cells were cultured in a complete medium supplemented with recombinant interleukin 2 (IL2) alone or recombinant IL2 plus recombinant tumor necrosis factor (TNF-), and their growth and antitumor cytotoxicity were determined. TIL cultured in the presence of IL2 plus TNF- (1000 units/ml each) for 6 days showed significantly higher cytotoxicity against fresh autologous tumor targets than did TIL cultured with IL2 alone (e.g., mean lytic units/10⁷ cells for 8 TIL preparations were 290 versus 74; P < 0.05). No differences in [³H]thymidine uptake or natural killer cell activity were observed among these TIL cultures. In titration experiments, optimal synergistic concentrations of IL2 and TNF- were determined as 10² and 10³ units/ml, respectively. Using these concentrations for culturing the TIL, effector cells developed which preferentially lysed autologous tumor and displayed a CD8⁺ phenotype (up to 75% positive). However, the autologous tumor cytotoxicity mediated by these cultured TIL on day 6 was short lived. By day 12, it was replaced by non-major histocompatibility complex-restricted, lymphokine-actived killer cell-like activity mediated by CD3^-CD56⁺ effector cells. Simultaneously, the production of -interferon and interleukin 1 decreased in these cultures. In contrast to TNF-, anti-CD3 antibody synergized with IL2 to increase 2–3-fold TIL proliferation but not their cytotoxic activity against autologous tumor cell targets. These data suggest that TNF- and IL2 synergize early in culture to induce tumor-reactive CD8⁺ effectors, some of which may be specific for autologous ovarian tumor cells. However, the conditions needed to sustain the specific autologous tumor responses in long-term cultures of human TIL remain to be determined.

¹ Supported by American Cancer Society Grant IM27077 to TLW. This is the second article in a series; see Ref. 7.

² To whom requests for reprints should be addressed, at One Children's Place, Room 5725, Pittsburgh, PA 15213-2583.

Received 5/ 5/89. Revised 7/21/89. Accepted 8/ 3/89.