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Alterations in NF{kappa}B Activation in T Lymphocytes of Patients With Renal Cell Carcinoma

Robert G. Uzzo, Peter E. Clark, Patricia Rayman, Tracy Bloom, Lisa Rybicki, Andrew C. Novick, Ronald M. Bukowski, James H. Finke

Affiliations of authors: R. G. Uzzo, P. E. Clark (Departments of Immunology, Lerner Research Institute, and Urology), P. Rayman, T. Bloom (Department of Immunology, Lerner Research Institute), L. Rybicki (Department of Biostastics), A. C. Novick (Department of Urology), R. M. Bukowski (Departments of Immunology, Lerner Research Institute, and Urology, and Experimental Therapeutics Program), J. H. Finke (Departments of Immunology, Lerner Research Institute, and Urology, and Experimental Therapeutics Program), The Cleveland Clinic Foundation, OH.

Correspondence to: Robert G. Uzzo, M.D., Department of Immunology, NB3-29, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.

T cells represent an important component in the development of an effective antitumor immune response. Although most solid tumors are infiltrated with T lymphocytes, including some clones capable of preferentially recognizing malignant cells, T-cell immunity fails to develop adequately in patients with tumors (1-4). Defects in proliferation and effector functions have been noted in peripheral blood T cells, while more pronounced alterations have been reported for T cells infiltrating the tumor (5,6). Moreover, cytokine gene expression normally associated with the development of an effective antitumor immune response is absent in the tumor (7).

Alterations in expression and activity of intracellular signaling elements have been reported in T cells from patients with tumors (8). These include defective activation of the transcription factor NF{kappa}B in both tumor-bearing mice and patients with renal cell carcinoma (RCC) (9,10). The major problem appears to be an impaired nuclear accumulation of NF{kappa}B complexes, resulting in a loss of binding to {kappa}B sequences. NF{kappa}B plays an important role in the development of T-cell-mediated immune responses through its control of a diverse set of genes that include cytokines (interleukin 2 [IL-2] and tumor necrosis factor-{alpha}) and receptor genes (11-13). The importance of NF{kappa}B activation in T-cell immunity has been documented using knockout mice where deletions of individual NF{kappa}B family members resulted in defective T- and B-cell functions (14,15). Current evidence suggests that NF{kappa}B is also important for cell survival because its activation may induce genes that protect T cells from apoptosis (16,17).

An important question to address is whether the tumor itself is responsible for impaired T-cell signaling. In animal models, tumor progression has been associated with reduced NF{kappa}B activation and {kappa}B-dependent gene expression (9); however, the cause of NF{kappa}B suppression in patients with cancer is not known. To address this issue, we performed several types of experiments. One set determined if removal of the tumor would result in normal NF{kappa}B activation in patients with previously defective T cells. Initial experiments characterized defects in NF{kappa}B signaling in three sets of patients: those with localized (n = 46) or metastatic (n = 42) RCC and patients with advanced disease (i.e., stage III/IV) but with no evidence of disease (NED) after surgical treatment who were then referred to our institution within 10 weeks postoperatively for enrollment into an adjuvant IL-2 protocol (n = 18). Results from these populations were compared with each other and those derived from T cells of healthy volunteers (n = 53). Approval was obtained from the institutional review board of The Cleveland Clinic Foundation and all patients gave informed written consent. NF{kappa}B activation was detected by measuring the binding of nuclear NF{kappa}B complexes to a radiolabeled {kappa}B sequence-specific probe (10). In normal resting T cells, there is variable expression of the NF{kappa}B1 (p50/p50) homodimer (fast-migrating band) but no expression of the RelA (p65)/NF{kappa}B1 (p50) heterodimer (RelA/p50) (slow-migrating band). Activation results primarily in the nuclear translocation of the transactivating RelA/p50 heterodimer and to a lesser extent of the p50/p50 homodimer (Fig. 1).Go While stimulation by phorbol myristate acetate and ionomycin did not result in NF{kappa}B-binding activity in only 6% (three of 53) of healthy volunteers, binding activity in T cells from patients with localized RCC was defective in 63% (29 of 46). In patients with metastatic disease, {kappa}B binding was defective in 69% (29 of 42). Both differed statistically from corresponding controls (Fisher's exact test: two-sided P<.0001) (Table 1).Go The fact that the degree of impaired NF{kappa}B activity was similar for local versus metastatic disease is not surprising because patients with advanced localized disease may have a higher tumor bulk than those with minimal metastatic disease. Although this T-cell defect may be related to tumor burden, this proved difficult to quantitate clinically.



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Fig. 1. Representative data of impaired NF{kappa}B activation and its reversal. Peripheral blood-derived T cells were isolated (>97% CD3+) as previously described using negative antibody selection (18). Cells were then stimulated for 2 hours with phorbol myristate acetate (PMA) (10 ng/mL) plus ionomycin (Iono) (0.75 µg/mL) or cultured in media alone. {kappa}B-binding activity was detected by incubating nuclear extract (10 µg of protein) in 25 µL total reaction volume containing 20 mmol/L HEPES (pH 7.9), 80 mmol/L NaCl, 0.1 mol/L ethylenediaminetetraacetate (EDTA), 1 mmol/L dithiothreitol (DTT), 8% glycerol, and 2 µg of poly (dI-dC) for 15 minutes at 4 °C. The reaction mixture was then incubated with the 32P-labeled double-stranded {kappa}B oligonucleotide corresponding to the interleukin 2R{alpha} gene for 20 minutes prior to electrophoresis of samples in a 6% nondenaturing polyacrylamide gel with 0.25x TBE buffer (22.3 mmol/L Tris, 22.2 mmol/L boric acid, and 0.5 mmol/L EDTA). Gels were dried and analyzed by autoradiography and densitometry. Electrophoretic mobility shift assay (EMSA) shows two {kappa}B-binding complexes in nuclear extracts of normal T cells after activation. The slower migrating band is the RelA/NF{kappa}B1(p50) heterodimer, whereas the faster migrating band is a homodimer of NF{kappa}B1 (p50/p50) (4). A) Analysis of {kappa}B-binding activity in T cells from a normal healthy volunteer (control) and three patients with renal cell carcinoma (RCC) rendered surgically NED (no evidence of disease) prior to enrollment in an adjuvant interleukin 2 (IL-2) trial (patient Nos. 1, 2, and 3). B) {kappa}B-binding activity in two patients (patient Nos. 4 and 5) with localized RCC preoperatively (Pre-op) and postoperatively (Post-op). C) Reversal of NF{kappa}B defect in patient T cells following in vitro culture. After T-cell isolation, a portion of cells was stimulated with PMA/Iono for 2 hours. The remaining T cells were cultured for 48-72 hours in RPMI-1640 medium (BioWhittaker, Inc., Walkersville, MD) supplemented with 2% fetal calf serum prior to stimulation with PMA/Iono. Nuclear extract was isolated and EMSA was performed on samples from freshly isolated and cultured T cells from the same patients. D) Inhibition of NF{kappa}B activation by soluble products from RCCs. Supernatant fluid from RCC was prepared as previously described (18). Briefly, 1 g of 3 x 3-mm explants of tumor was cultured in T-75 flask with 15 mL of Dulbecco's modified Eagle medium without any additional supplements. After 3-4 days of culture at 37 °C with 95% air and 5% CO2, supernatant was harvested, filtered (0.45 µm), and stored at -70 °C. Tissue from uninvolved area of the same kidney served as control supernatant when available. T cells from healthy volunteers were cultured in the presence and absence of RCC or normal kidney supernatant (20%-50% total volume) for 18 hours. Thereafter, T cells were stimulated with PMA/Iono for 2 hours prior to isolating nuclear extracts and performing EMSA.

 

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Table 1. Inhibition and reversal of NF{kappa}B activation in patients with renal cell carcinoma (RCC)

 
In patients with no evidence of disease after surgery, only 17% (three of 18) demonstrated impaired NF{kappa}B activation. There was no statistically significant difference in the frequency of this defect in T cells from healthy donors versus patients with no evidence of disease postoperatively (Fisher's exact test; P = .33) (Table 1Go; Fig. 1Go, A). These results prompted us to compare T cells from patients preoperatively and postoperatively. Follow-up peripheral blood samples were obtained postoperatively from 17 patients with RCC whose NF{kappa}B-binding activity was defective preoperatively. In this group of patients, reversal of the defect was observed in 35% (six of 17; 95% confidence interval [CI] = 14%-62%) within 8 weeks of complete tumor removal (Fig. 1Go, B; Table 1Go). These findings show that, in a subset of patients with RCC, the surgical removal of tumor restores normal NF{kappa}B activation in T cells, suggesting a role for tumor-induced immune suppression.

In a second set of experiments, we determined if the removal of peripheral blood T cells from patients with tumors could restore NF{kappa}B activation. T cells isolated from 12 patients with RCC with defective NF{kappa}B activation were cultured in medium for 48-72 hours and then subjected to activation and electrophoretic mobility shift assays. This in vitro culture resulted in reversal of the {kappa}B-binding defect in 58% (seven of 12; 95% CI = 28%-85%) of the patients (Fig. 1Go, C; Table 1Go). T cells from patients whose NF{kappa}B-binding defect had reversed in vivo or in vitro demonstrated normal binding to {kappa}B sequences when comparing unstimulated to stimulated band intensities (two-sided Wilcoxon signed rank test—in vivo reversal P = .031, n = 6, median difference in band intensity = 87 pixels; in vitro reversal P = .016, n = 7, median difference = 75.3), while those that remained defective had no such induction (two-sided Wilcoxon signed rank test—in vivo reversal P = .12, n = 11, median difference in band intensity = 3.8 pixels; in vitro reversal P = 1, n = 5, median difference = 1.7). The level of induction of NF{kappa}B in T cells whose binding defect was reversed during in vivo and in vitro assays did not differ from their normal corresponding controls. These findings are consistent with suppression of NF{kappa}B mediated by the tumor.

Soluble products derived from cultures of renal tumor explants could suppress NF{kappa}B activation in T cells from healthy volunteers (18) (Fig. 1Go, D). Supernatant fluid from 23 (68%) of 34 RCC tumors suppressed NF{kappa}B activation, whereas only three (30%) of 10 supernatants from uninvolved kidney had any effect. Kolenko et al. (19) have earlier demonstrated that the ability of T cells to proliferate was also diminished, indicating that soluble products can suppress signaling events requisite for T-cell activation.

The data presented here suggest that RCC tumors can induce defects in T-cell signaling events central to the development of an effective antitumor immune response. Our data demonstrate that soluble tumor products induce T-cell {kappa}B-binding defects that are reversible in a subset of patients. The fact that the defect was not reversed in all patients examined may be a function of when the patient T cells were re-examined in relation to their surgery. A greater number of reversals may be seen if T cells from patients with persistent defects are re-evaluated at longer intervals postoperatively. Furthermore, this defect does not appear to be unique to RCC because the NF{kappa}B defect is observed in T cells from patients with other cancers, such as multiple myeloma (Finke JH: unpublished data), as well as solid tumors, such as those of the colon and pancreas (20). Nonetheless, renal tumors represent one mechanism whereby NF{kappa}B can be suppressed in T cells.

Although alterations in signal transduction molecules have been reported in patients with cancer and tumor-bearing animals, few studies (21-23) have examined the mechanisms responsible for these defects. Previous data (19) suggest that soluble products derived from RCC explants but not normal kidney inhibit expression of the IL2R-associated protein tyrosine kinase JAK3 and T-cell proliferation. In addition, soluble products of RCC tumors inhibit stimulus-dependent nuclear translocation of NF{kappa}B by one of two mechanisms. In the first, there is blocking of phosphorylation and subsequent degradation of the inhibitor I{kappa}B{alpha} (18). In the other, there is impaired nuclear accumulation of the Rel dimers without impairment of I{kappa}B{alpha} degradation. The latter phenotype may be induced by tumor-derived glycosphingolipids (gangliosides) (Uzzo RG, Rayman P, Kolenko V, Clark PE, Cathcart MK, Bloom T, et al.: manuscript submitted for publication). Both of these phenotypes have been observed in peripheral blood T cells from patients with RCC (18). The functional consequences of impaired NF{kappa}B are currently under study. Our recent findings suggest that suppression of NF{kappa}B may make peripheral blood T cells from patients with RCC more susceptible to apoptosis upon activation (24).

The defect in NF{kappa}B activation in T cells of patients with RCC may be mediated by tumor or tumor-derived soluble products. A better understanding of the nature of the inhibitory products and their mechanism of suppression may provide additional insight into how tumors can evade destruction by the immune system.

NOTES

Supported by Public Health Service grant CA56937 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by the American Foundation for Urologic Diseases.

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Manuscript received October 1, 1998; revised January 27, 1999; accepted February 2, 1999.


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