Search

Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (p50) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that RelA, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any RelA in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients may impair their capacity to respond normally to various stimuli.

Previously we showed that IL2 expanded tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma mediated non-major histocompatibility complex-restricted cytotoxicity. Phenotypic analysis showed that cultured TILs were composed mostly of T-lymphocytes with varying numbers of CD4+, CD8+, and CD56+ (Leu19+) populations. Here we compared the cytolytic activity of the two predominant TIL subsets, CD3+CD4+ and CD3+CD8+, to that of the CD56+ populations. Using magnetic beads coated with antibodies to either CD4 or CD8, CD3+CD4+, and CD3+CD8+ TILs were isolated in a highly enriched form (greater than 92%) and could be expanded for over 40 days in vitro with 1000 units/ml IL2. In a 4-h 51Cr release assay the CD4+ and CD8+ TILs showed minimal lytic activity, whereas unseparated cells exhibited significant levels of non-major histocompatibility complex-restricted cytotoxicity. The lytic activity seen in the 4-h assay with unseparated TILs appeared to be related to the presence of CD56+ populations. With one exception none of the purified CD4+ or CD8+ TILs expressed any significant levels of CD56, while the unseparated TILs contained varying numbers of CD3+CD56+ and CD3-CD56+ populations. Cell-sorting experiments verified that the CD56+ populations were responsible for most of the lytic activity in 4 h even though CD3+CD56- cells represented the predominant cell type. Although CD3+CD56- TILs were minimally lytic in 4 h, we show here that both CD3+CD4+ and CD3+CD8+ subsets displayed substantial cytotoxicity in long-term assays. In the 18-h 51Cr release assay 5 of 6 CD4+ and 2 of 3 CD8+ TILs were lytic for the autologous tumor. In two cases, restimulation with the autologous tumor induced augmented cytolytic activity of TIL subsets and in one case induced lytic activity in 4 h. The cytotoxic activity of TIL subsets was further examined using a 72-h assay in which TILs were cocultured with a confluent layer of tumor cells. The degree of cytotoxicity was quantitated by measuring the amount of crystal violet dye that was incorporated by tumor cells which remained after the incubation period. CD4+ and CD8+ TILs typically caused greater than a 50% reduction of tumor cells in 3 days and the level of reduction was increased when IL2 was added to the cultures. All the CD4+ and CD8+ subset preparations were cytotoxic in the 3-day assay even though some were not lytic for certain targets in the 18-h 51Cr release assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent studies suggest an immune modulator role for C-reactive protein (CRP). We have tested the effect of CRP in a tumor system designed for study of metastases. Fibrosarcoma T241 was implanted on one hind foot of syngeneic C57BL/6 mice. After 17 days, the tumor-bearing feet were amputated, and i.v. therapy of liposomes containing CRP or control reagents was started. Examination of the lungs on Day 35 showed that CRP:liposome-treated animals had significantly fewer and smaller metastases as compared with those in the control groups. Moreover, 38% of the animals in the former groups were completely free of metastases as compared with 0 to 2% of the controls. Significantly, enhanced survival was also noted in the CRP liposome-treated group. CRP may have biological response modifier" function of value in cancer therapy.

Two human renal carcinoma cell lines have been established from the same patient. One cell line (CCF-RC1) was obtained from the primary tumor and the second (CCF-RC2) was established from cells of the renal vein effluent of the perfused tumorous kidney. Although they were established from the same patient, the cell lines differed in certain biological properties. They have been passaged up to 50 times in vitro for about two years. Each has an epithelial morphology and exhibits mutilayering. Cell cycle time of CCF-RC1 and CCF-RC2 was 34 and 36 h, respectively. They exhibited anchorage independent growth, and the plating efficiency of CCF-RC2 in soft agar was higher than that of CCF-RC1. Both lines induced tumors in nude mice at the site of s.c. injection closely resembling the original tumor in histological examination. Electron microscopic features of both tumors in nude mice were consistent with epithelial origin. Doubling time of CCF-RC1 and CCF-RC2 in nude mice was 11 and 12 days, respectively. CCF-RC1 and CCF-RC2 have hypotetraploid karyotype and modal numbers of 83 and 73, presenting two and three marker chromosomes, respectively. Immunocytology with commercial monoclonal antibodies against renal carcinoma (URO-3) and cytokeratin (Mac 6) showed positive reactions with both lines, suggesting that these cell lines derived from renal epithelium. A murine monoclonal antibody (2E11) was generated against CCF-RC2 by the hybridoma technique; 2E11 reacted with CCF-RC2, but not with CCF-RC1. These cell lines may provide a useful model for the study of tumor heterogeneity and its relationship to metastasis.