Interleukin-6 (IL-6) is a recently characterized pleiotropic cytokine with antitumor activity. We investigated the production of IL-6 by renal cell cancer (RCC) and the growth effects of IL-6 on RCC. Using immunoperoxidase staining, cytoplasmic IL-6 was detected in four of four renal tumor lines and in tumor cells from freshly nephrectomized RCC. We found that IL-6 mRNA was expressed at basal culture conditions by seven of ten RCC tumor lines tested. Biologically active IL-6, as measured by the B9 assay, was produced by all ten RCC tumor lines. The addition of tumor necrosis factor alpha (TNF alpha) significantly augmented the expression of IL-6 mRNA in five RCC tumor lines (P less than 0.05). The combination of interferon gamma IFN gamma and TNF alpha further enhanced the augmented IL-6 mRNA accumulation seen with TNF alpha alone (P less than 0.05). TNF alpha also significantly stimulated the production of biologically active IL-6 (P less than 0.01). Furthermore, IFN gamma and TNF alpha were found to enhance IL-6 bioactivity synergistically (P less than 0.05). The growth effects of IL-6 on RCC were also investigated in two experimental systems: IL-6 was found to stimulate proliferative responses in six of six RCC tumor lines as measured by thymidine-uptake assays; however, only one of six tumor lines displayed an increase in proliferative response of greater than 21% (113%). The growth effect of IL-6 was further tested in clonogenic assays. One of the tumor lines tested displayed an enhanced growth response of up to 200%. We conclude that IL-6 is produced by RCC; this production is enhanced by TNF alpha with synergistic effects seen with IFN gamma at both mRNA and protein levels. In turn, IL-6 may have a modest stimulatory growth effect on certain RCC tumor lines.

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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