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 NFB 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
B complexes, resulting in a loss of binding to
B
sequences. NF
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-
) and receptor genes (11-13). The importance of NF
B activation in T-cell immunity has been documented
using knockout mice where deletions of individual NF
B family members resulted in
defective T- and B-cell functions (14,15). Current evidence suggests that
NF
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
NFB activation and
B-dependent gene expression (9);
however, the cause of NF
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
B activation in patients with previously defective T cells.
Initial experiments characterized defects in NF
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
B activation was detected by measuring the binding of
nuclear NF
B complexes to a radiolabeled
B sequence-specific probe (10). In normal resting T cells, there is variable expression of the NF
B1 (p50/p50)
homodimer (fast-migrating band) but no expression of the RelA (p65)/NF
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).
While stimulation by phorbol myristate acetate and
ionomycin did not result in NF
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,
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).
The fact that the
degree of impaired NF
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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In a second set of experiments, we determined if the removal of peripheral blood T cells from
patients with tumors could restore NFB activation. T cells isolated from 12 patients with
RCC with defective NF
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
B-binding defect in 58% (seven of 12; 95% CI
= 28%-85%) of the patients (Fig. 1
, C; Table 1
). T cells from patients whose NF
B-binding defect had reversed in vivo or in vitro demonstrated normal binding to
B sequences when
comparing unstimulated to stimulated band intensities (two-sided Wilcoxon signed rank
testin 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 testin 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
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
B mediated by the tumor.
Soluble products derived from cultures of renal tumor explants could suppress NFB
activation in T cells from healthy volunteers (18) (Fig. 1
, D). Supernatant fluid from 23 (68%) of 34 RCC tumors suppressed
NF
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 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
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
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 NFB by one of
two mechanisms. In the first, there is blocking of phosphorylation and subsequent degradation of
the inhibitor I
B
(18). In the other, there is impaired nuclear
accumulation of the Rel dimers without impairment of I
B
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
B are currently under study. Our recent findings suggest that suppression of NF
B
may make peripheral blood T cells from patients with RCC more susceptible to apoptosis upon
activation (24).
The defect in NFB 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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