Kuntz, Susan M.

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

Abstract Androgen has been implicated as a negative regulator of host immune function and a factor contributing to the gender dimorphism of autoimmunity. Conversely, androgen deprivation has been suggested to potentiate male host immunity. Studies have shown that removal of androgen in postpubertal male mice produces an increase in size and cellularity of primary and peripheral lymphoid organs, and enhances a variety of immune responses. Yet, few details are known about the effect of androgen removal on T cell-mediated immunity. In this study, we demonstrate two pronounced and independent alterations in T cell immunity that occur in response to androgen deprivation, provided by castration, in postpubertal male mice. First, we show that levels of T cells in peripheral lymphoid tissues of mice are increased by androgen deprivation. Second, T cells from these mice transiently proliferate more vigorously to TCR- and CD28-mediated costimulation as well as to Ag-specific activation. In addition, androgen deprivation accelerates normalization of host T and B cell levels following chemotherapy-induced lymphocyte depletion. Such alterations induced by androgen deprivation may have implications for enhancing immune responses to immunotherapy and for accelerating the recovery of the immune system following chemotherapy.

Abstract Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor Ags via restimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms, which function to limit the recall responses of tumor-specific immunity, remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional antitumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survival. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting in which tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.

Abstract Adoptive γδ T cell immunotherapy has moved briskly into clinical trials prompted by several small studies suggesting abundant accumulation of γδ T cells within renal cell carcinoma (RCC). In this study, we re-examined levels of γδ T cells within RCC tumors and correlated levels of these cells with pathologic features and outcome associated with this form of cancer. Tissues from 248 consecutive clear cell RCC tumors obtained from 2000 to 2003 were stained and quantified for total CD3+ and γδ T cells per mm2. Wilcoxon rank sum and Kruskal-Wallis tests were used to evaluate associations between T cell amounts and prognostic factors (age, gender, tumor size, stage, grade, tumor necrosis). Cox models were used to assess associations with RCC-specific death. Median numbers of total CD3+ and γδ T cells were 281/mm2 (interquartile range (IQR): 149–536) and 2.6/mm2 (IQR: 1.3–4.6), respectively. The median percentage of CD3+ T cells that were γδ T cells was 1.0% (IQR: 0.4–1.9). This low percentage of intratumoral γδ T cells was diluted even further with rising CD3+ T cell infiltration. Percentages of γδ T cells were not associated with even one single clinicopathologic feature examined. Median follow-up for this study was 3.1 years (48 patients died of RCC) and Cox analysis failed to demonstrate that γδ T cells (hazard ratio = 1.02, p = 0.25) were predictive of RCC-specific death. γδ T cells are rare and not recruited nor expanded within RCC tumors. Percentages of γδ T cells fail to correlate with any prognostic features of RCC nor specific death. As such, the role of γδ T cells in RCC immunobiology remains questionable.

Abstract Purpose: Although the prognostic value of B7-H1 and B7-H4 expression by tumor cells in clear cell renal cell carcinoma (ccRCC) has been established, the role of B7-H3 is unknown. As such, we evaluated the association of B7-H3 expression with clinicopathologic outcomes in patients treated for ccRCC. Experimental Design: Nephrectomy specimens from 743 consecutive patients treated for ccRCC at our institution from 1990 to 1999 were evaluated for B7-H3 expression by immunohistochemical staining. Associations of B7-H3 expression with clinical and pathologic features were evaluated using χ2 and Fisher's exact tests. Associations of B7-H3 expression with death from RCC were evaluated using Cox proportional hazards regression models. Results: B7-H3 expression by tumor cells or tumor vasculature was noted in 17% and 95% of specimens, respectively. The presence of either tumor cell or diffuse tumor vasculature expression of B7-H3 was present in 46% of specimens and was associated with multiple adverse clinical and pathologic features. After multivariable adjustment, the presence of either tumor cell or diffuse tumor vasculature B7-H3 expression was significantly associated with an increased risk of death from RCC (risk ratio, 1.38; 95% confidence interval, 1.03-1.84; P = 0.029). Conclusions: Both tumor cell and tumor vasculature B7-H3 expression convey important information to predict ccRCC outcomes. Collectively, our past and present studies pertaining to B7-H ligand expression indicate that ccRCC may use redundant mechanisms to compromise host antitumoral immunity. Future studies will focus on the effect of combined B7-H ligand expression in RCC.

Abstract Purpose: Aberrant expression of T-cell coregulatory molecules has been investigated as a mechanism by which certain cancers may evade host immune surveillance. We evaluated expression of the T-cell coregulators B7-H1, B7-H3, and PD-1 in urothelial cell carcinoma (UCC) of the bladder. Experimental Design: Immunohistochemistry for B7-H1, B7-H3, and PD-1 was done on paraffin-embedded sections from 318 consecutive patients with UCC who underwent radical cystectomy. Expression was correlated with clinicopathologic outcomes and postoperative survival. Results: B7-H3 was widely expressed in UCC, as 222 of 314 (70.7%) tumors showed positive staining. Expression of B7-H3 in UCC was significantly increased compared with adjacent, nontumor urothelium, as a median of 70% of tumor cells expressed B7-H3, compared with 20% of cells in nontumor specimens (P < 0.001). The increase in B7-H3 expression was independent of tumor stage (P = 0.13). Expression of B7-H1 by UCC tumors (P < 0.001) and PD-1 by tumor-infiltrating lymphocytes (P = 0.012) were significantly associated with increased pathologic stage. Patients who had received intravesical bacillus Calmette-Guerin before cystectomy tended to show increased expression of B7-H3 (P = 0.023) and PD-1 (P = 0.071) but were less likely to express B7-H1 (P = 0.027). Moreover, for the subset of patients with organ-confined disease (n = 167), B7-H1 expression independently predicted all-cause mortality after cystectomy (hazard ratio, 3.18; 95% confidence interval, 1.74-5.79; P < 0.001). Conclusions: B7-H3 is highly expressed in UCC across tumor stages, whereas B7-H1 and PD-1 expression are associated with advanced disease. B7-H1 expression predicts mortality after cystectomy for patients with organ-confined tumors. These molecules may represent novel diagnostic or prognostic markers, as well as therapeutic targets, for patients with UCC.

Abstract B7 coregulatory ligands can be aberrantly expressed in human disease. In the context of cancer, these ligands may act as antigen-specific inhibitors of T-cell–mediated antitumoral immunity. We recently reported that B7-H1 expression by carcinomas of the kidney and bladder portends aggressive disease and diminished survival. The expression of these proteins in prostate cancer, however, has not been investigated. We evaluated B7-H3 and B7-H1 protein expression in the pathologic specimens of 338 men treated for clinically localized prostate cancer between 1995 and 1998 with radical retropubic prostatectomy. Expression levels of B7-H3 in prostate cancer were correlated with pathologic indicators of aggressive cancer as well as clinical outcome. We report that B7-H3 is uniformly and aberrantly expressed by adenocarcinomas of the prostate, high-grade prostatic intraepithelial neoplasia, and four prostate cancer cell lines, whereas B7-H1 is rarely expressed. B7-H3 is expressed by benign prostatic epithelia, although at a more reduced level relative to neoplastic tissue. Increasing levels of B7-H3 intensity correlate with worsening clinicopathologic features of prostate cancer. Marked B7-H3 intensity, present in 67 (19.8%) specimens, confers a >4-fold increased risk of cancer progression after surgery (risk ratio, 4.42; P < 0.001). A survey of normal tissues revealed that B7-H3 is expressed within the liver, urothelium, and fetal kidney. In summary, B7-H3 is aberrantly expressed in all prostate cancers and represents an independent predictor of cancer progression following surgery. Moreover, B7-H3 encompasses a novel diagnostic and potential therapeutic target for the clinical management of prostate cancer and, perhaps, other malignancies as well. [Cancer Res 2007;67(16):7893–900]

AbstractPurpose: Regulatory T cells (Tregs) have been implicated as inhibitors of antitumoral immunity, and evidence suggests that elimination of Tregs may augment natural and pharmacologic immunity. We tested for the presence of putative Tregs within renal cell carcinoma (RCC) tumors.Experimental Design: We identified 170 patients who underwent radical or partial nephrectomy for clear cell RCC between 2000 and 2002. Specimens were stained with anti-CD4, anti-CD25, and anti-Foxp3 antibodies and examined using confocal microscopy. Associations of CD4+CD25+Foxp3− and CD4+CD25+Foxp3+ T cells with death from RCC were evaluated using Cox proportional hazards regression models.Results: At last follow-up, 46 of 170 patients had died; of these, 37 died from RCC at a median of 1.4 years following nephrectomy (range, 0-4.4). Among the 124 remaining patients, median follow-up was 3.7 years (range, 0-5.7). Forty-three (25.3%) tumors harbored CD4+CD25+Foxp3+ T cells. The presence of Foxp3+ T cells was not significantly associated with RCC death univariately. One hundred forty-three (84.1%) tumors harbored CD4+CD25+Foxp3− T cells. The indicator for ≥10% CD4+CD25+Foxp3− T cells was significantly associated with RCC death univariately [risk ratio (RR), 2.60; 95% confidence interval (95% CI), 1.35-4.98; P = 0.004], after adjusting for tumor B7-H1 expression (RR, 2.53; 95% CI, 1.32-4.85; P = 0.005) and lymphocytic infiltration (RR, 2.53; 95% CI, 1.32-4.87; P = 0.005).Conclusions: Increased presence of CD4+CD25+Foxp3+ T cells was not significantly associated with RCC death. In contrast, CD4+CD25+Foxp3− T cells, which may represent a unique set of Tregs or activated helper T cells, was significantly associated with outcome.

AbstractPurpose: Clear cell renal cell carcinoma (ccRCC) is an immunogenic tumor that can progress in the presence of an intact host immune system. We previously reported that survivin and B7-H1 are independently associated with disease progression and death when expressed by ccRCC tumors. Herein, we examine the clinical effect of ccRCC combined expression of both survivin and B7-H1.Experimental Design: Specimens from 298 patients who underwent nephrectomy for ccRCC between 1990 and 1994 were immunohistochemically stained for survivin and B7-H1. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations of both markers with ccRCC death were assessed using Cox proportional hazards regression models.Results: At last follow-up, 94 patients died from ccRCC. Among the living patients, the median follow-up was 11.2 years (range, 0-15 years). There were 177 (59.4%) survivinLow/B7-H1−, 51 (17.1%) survivinHi/B7-H1−, 29 (9.7%) survivinLow/B7-H1+, and 41 (13.8%) survivinHi/B7-H1+ tumors. The 5-year cancer-specific survival rates for patients within each group were 89.3%, 59.7%, 70.0%, and 16.2%, respectively. Combined survivinHi/B7-H1+ expression was associated with ccRCC death univariately (risk ratio, 12.82; 95% confidence interval, 7.50-21.92; P < 0.001) and in multivariate analysis (risk ratio, 2.81; 95% confidence interval, 1.56-5.04; P < 0.001). SurvivinHi/B7-H1+ tumors exhibited increased levels of infiltrating mononuclear cells and survivin-specific T cells compared with survivinLow/B7-H1− tumors.Conclusion: Patients with survivinHi/B7-H1+ ccRCC tumors are at increased risk of ccRCC death. SurvivinHi/B7-H1+ tumors also harbor increased amounts of infiltrating mononuclear cells and survivin-specific T cells relative to survivinLow/B7-H1− tumors. Taken together, dual expression of survivin and B7-H1 can be used to predict ccRCC tumor aggressiveness.

B7-H4 is a recently described B7 family coregulatory ligand that has been implicated as an inhibitor of T cell-mediated immunity. Although expression of B7-H4 is typically limited to lymphoid cells, aberrant B7-H4 expression has also been reported in several human malignancies. To date, associations of B7-H4 with clinical outcomes for cancer patients are lacking. Therefore, we examined B7-H4 expression in fresh-frozen tumor specimens from 259 renal cell carcinoma (RCC) patients treated with nephrectomy between 2000 and 2003 and performed correlative outcome analyses. We report that 153 (59.1%) RCC tumor specimens exhibited B7-H4 staining and that tumor cell B7-H4 expression was associated with adverse clinical and pathologic features, including constitutional symptoms, tumor necrosis, and advanced tumor size, stage, and grade. Patients with tumors expressing B7-H4 were also three times more likely to die from RCC compared with patients lacking B7-H4 (risk ratio = 3.05; 95% confidence interval = 1.51–6.14; P = 0.002). Additionally, 211 (81.5%) specimens exhibited tumor vasculature endothelial B7-H4 expression, whereas only 6.5% of normal adjacent renal tissue vessels exhibited endothelial B7-H4 staining. Based on these findings, we conclude that B7-H4 has the potential to be a useful prognostic marker for patients with RCC. In addition, B7-H4 represents a target for attacking tumor cells as well as tumor neovasculature to facilitate immunotherapeutic treatment of RCC tumors. Last, we demonstrate that patients with RCC tumors expressing both B7-H4 and B7-H1 are at an even greater risk of death from RCC.