Conditions for generating and expanding cytotoxic tumor-specific, tumor-infiltrating lymphocytes (TIL) were studied to improve the efficacy of adoptive cancer immunotherapy. Thus, we have examined the growth and cytolytic patterns of bulk culture TIL from human renal cell carcinoma (RCC) cultured in low (20 U/ml) or high (1,000 U/ml) dose interleukin (IL)-2, with or without irradiated autologous tumor stimulation. By 55 days in culture, TIL grown in the presence of IL-2 without tumor stimulation lost their lytic activity, whereas those exposed to tumor stimulation maintained high levels of cytotoxicity against autologous and/or nonautologous tumor targets. Only TIL grown with low dose IL-2 and autologous tumor maintained long-term (over 4 months in culture) specific cytotoxicity against the autologous tumor, even upon cryopreservation and regrowth. These TIL were 88-97% and 80% positive for CD3 and CD8, with a persistent subset exhibiting CD4 + CD8 + double positive staining. Their specific cytotoxic activity was major histocompatibility complex Class I-restricted and inhibited by pretreating the TIL with anti-CD3 monoclonal antibody. TIL exposed to the four types of culture conditions, low or high dose IL-2, with or without irradiated autologous tumors, and exhibiting different lytic specificities, all expressed mRNA for interferon-gamma and tumor necrosis factor (TNF)-alpha, but not for IL-1-beta, IL-4, IL-6, and granulocyte-macrophage colony stimulating factor. The degree of TNF-alpha mRNA expression correlated with the degree of autologous tumor-specific cytotoxicity of these TIL. This initial report demonstrates that antigen-specific cytotoxicity against the autologous tumor does, in fact, exist within the RCC tumors. Furthermore, by modifying culture conditions, cytotoxic T-lymphocytes with anti-tumor activity can be selectively grown from the heterogeneous bulk cultures of TIL. Key Words: Lymphokine expression-Renal cell carcinoma-Specific immunity-Tumor-infiltrating lymphocytes.Lymphocytes in suspensions of the original tumor mass, when cultured in the presence of interleukin (IL)-2, expand and destroy the tumor cells to yield a pure population of tumor-infiltrating lym-

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Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of T cells with potent antitumor activity against a wide variety of tumors. TIL from renal cell cancer (RCC) typically exhibit diminished growth and antitumor activity after four weeks in vitro. We have therefore investigated effects of varying doses of interleukin-6 (IL-6) (0, 25, 100 units/ml.) on in vitro expansion, proliferation, cytotoxicity, and expression of cell surface phenotypes of long term renal TIL cultures from three RCC patients. Among the various conditions tested, three of three TIL cultures displayed a mild increase in cell expansion when grown in IL-2 with the addition of 100 units/ml. of IL-6. Two of three TIL cultures grown in IL-2 and 100 U/ml. of IL-6 demonstrated enhanced proliferation as determined by 3H-thymidine uptake. TIL could not be isolated or maintained in vitro when grown in the presence of IL-6 alone without IL-2. IL-6 was also found to enhance the long term non-specific cytotoxicity against an allogeneic nonrenal tumor target. No consistent effect on autologous tumor-specific cytotoxicity was demonstrated. We conclude that IL-6, when used in combination with IL-2, may modestly enhance the long-term growth of RCC-derived TIL.

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