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.

Immunotherapy has long represented a mainstay treatment for patients with advanced renal cell carcinoma (RCC)3 (1, 2). Despite the clinical investigation of various agents for anticancer treatment, immunotherapy with high-dose IL-2 persists as the only therapeutic modality that can produce durable and even complete responses in minor subsets of patients with metastatic RCC (3). It is therefore understandable that many groups remain actively engaged in seeking out novel approaches to manipulate the patient’s immune system to evoke more efficacious immunotherapeutic responses to manage advanced RCC.

The vast majority of human T cell-mediated immunity is provided by T lymphocytes, of which >95% express the αβ TCR. In contrast, <5% of circulating T cells express the γδ TCR and the role of such γδ T cells remains hotly debated and enigmatic. One recent experimental antitumoral approach that has been proposed as a treatment for advanced RCC is adoptive γδ T cell immunotherapy (4, 5, 6). The rationale for this approach has been primarily predicated on: 1) a reported relative abundance of γδ T cells in the mononuclear cell infiltrates of RCC tumors (7), 2) associations of γδ T cells with some pathologic features of RCC and (8), and 3) demonstrations that γδ T cells stimulated with pyrophosphate derivatives exhibit cytolytic activity against RCC target cells ex vivo (4, 7). Of concern, however, is that much of the impetus for clinical trial testing of γδ T cell adoptive immunotherapy has been provided by way of indirect immunologic studies (5, 6), typically involving very small numbers of patient tumor specimens and often without consideration of RCC tumor subtype (4, 7, 8, 9, 10).

In response to these concerns, we quantified levels of γδ T cells present in 248 consecutive clear cell RCC (ccRCC) tumor specimens and correlated these values with widely accepted clinicopathologic prognostic features of ccRCC aggressiveness as well as cancer-specific outcomes. In this study, we report that percentages of intratumoral γδ T cells are consistently low in nearly all tumor specimens examined and failed to correlate with any prognostic feature of ccRCC or with cancer-specific survival. Percentages of T cells bearing the γδ TCR were no higher in the tumor than in the peripheral blood and, in fact, declined even further with rising CD3+ T cells infiltration of ccRCC tumors. Taken together, these data suggest that γδ T cells do not accumulate within ccRCC tumors and argue strongly against γδ T cell clonal expansion or recruitment into ccRCC tumors. Moreover, we did not observe a single association between the low level of γδ T cells within renal tumors and any of the established prognostic clinicopathologic features described for ccRCC. Thus, a role for γδ T cells in altering the biology of ccRCC tumors, either in the normal in situ setting or in the context of immunotherapy, should be carefully reconsidered.

After obtaining institutional review board approval, we identified a consecutive series of patients who were treated surgically with partial or radical nephrectomy for ccRCC from 2000 to 2003 and for whom fresh-frozen tissue was available for evaluation. We restricted our analysis to noncystic clear cell cases because this is the predominant histologic variant of RCC (∼80% of cases) and carries a prognosis that is different from the papillary and chromophobe variants of RCC (11). A total of 248 patients met these inclusion criteria and all were followed postoperatively for the development of important clinical outcomes such as metastasis and death. Symptomatic patients were defined by their presentation with a palpable renal mass, abdominal discomfort, gross hematuria, acute onset varicocele, or constitutional symptoms including rash, sweats, weight loss, fatigue, early satiety, and anorexia. Death was attributed to RCC if it was listed as the immediate or contributing cause of death on the death certificate. In cases where the cause of death was ambiguous or unknown, the entire medical record was reviewed by a urologist and the death was attributed to RCC if the patient had metastatic RCC within 6 mo of the date of death.

PBMCs were isolated from 13 Leukotrap WB leukoreduction filters (Pall) used in the routine processing of normal human donor blood. The filters were backflushed with 50 ml of 5 mM EDTA in PBS and eluted cells were layered onto a Lymphoprep sucrose density gradient (Accurate Chemical & Scientific) that was then centrifuged at 2000 rpm for 30 min. The mononuclear interface layer was collected and washed twice, resuspended in ACK buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.2), and then washed twice in 0.9% NaCl. The remaining cell population was counted using trypan blue exclusion and then cryopreserved at −80°C in Recovery cell freezing media (Invitrogen Life Technologies).

The primary Abs used in this study included polyclonal rabbit anti-human CD3 (A0452; DakoCytomation), Immu510 murine anti-human γδ TCR mAb (Beckman Coulter), and TCR-γδ-1 clone 11F2 murine anti-human γδ TCR mAb (BD Biosciences). For flow cytometry experiments, PE-labeled TCR-γδ-1 (BD Biosciences) and Immu510 labeled with Alexa488 by a Zenon labeling kit (Invitrogen Life Technologies) were used. Isotype control Abs used in various experiments were purchased from BD Biosciences.

Before staining, frozen PBMCs were thawed at 37°C for 2 min, washed with FACS buffer (PBS, 3% FBS, and 1 mM EDTA), and counted using trypan blue exclusion. In brief, 106 cells were used for each experimental condition. Fc receptors were blocked with 2% human serum (Sigma-Aldrich) for 20 min and then the appropriate Abs were added for 30 min, both steps occurring in the dark at 4°C. After two washes in FACS buffer, cell surface expression of CD3 and γδ TCR was quantified by flow cytometry using a FACSCalibur FACS (BD Bisociences) and analyzed with FlowJo software (Tree Star). Gating was done on forward vs side scatter plots for live lymphocytes and ∼2 × 104 cells were counted and included for each analysis. Matched isotype controls for R-PE and Alexa Fluor 488 were used to determine thresholds for positive expression.

ccRCC cryosections were mounted on Superfrost Plus slides, air dried, and fixed in −20°C acetone. Serial sections were stained on a DakoCytomation Autostainer for CD3 and for the γδ TCR using the Biocare Mach 4 HRP-polymer (Biocare Medical) and DakoCytomation EnVision+ System-HRP (diaminobenzidine) kits, respectively. Slides were blocked with H2O2 for 5 min, followed by incubation with primary Ab for 30 min at room temperature. Labeled polymer was then applied at room temperature for 30 min followed by incubation with chromogen substrate for 10 min. Finally, sections were counterstained with Hematoxylin 7211 (Richard-Allan Scientific). Positive controls for CD3 and γδ TCR staining consisted of human tonsillar tissue resected for chronic tonsillitis (12). Irrelevant isotype-matched Abs were used to control for nonspecific staining.

Each tumor specimen was centrally reviewed by a urologic pathologist blinded to clinical outcome to obtain a consistent set of histopathologic prognostic features for analysis, including: International Union Against Cancer histologic subtype (13), nuclear grade (14), tumor-node-metastasis (TNM) 2002 tumor stage (15), tumor size, the presence of histologic coagulative tumor necrosis, and sarcomatoid differentiation (11). To quantify T lymphocytes, numbers of CD3+ and γδ T cells in each of five representative high-powered fields were systematically counted using a Leica DMR microscope (Leica Microsystems). Using a 10/25 eyepiece and a ×40 objective, the Leica DMR has an object field diameter of 0.625 mm resulting in a high-powered field area of 0.307 mm2. We therefore present all CD3+ T cell data as number of cells per mm2 of tumor tissue. γδ T cell counts are also expressed as the number of cells per mm2 of tumor tissue as well as a percentage of overall CD3+ T cells to indicate the contribution of γδ T cells relative to the intratumoral pool of infiltrating CD3+ T cells.

The associations of CD3+ and γδ T cells with clinicopathologic features of the ccRCC cohort were evaluated using Wilcoxon rank sum and Kruskal-Wallis tests. Survival outcomes of the study cohort were estimated using the Kaplan-Meier method. Greenwood’s estimate of the SE was used on the complementary log-log scale to construct 95% confidence intervals (CI) for these estimates. Cox proportional hazards models were used to assess the magnitude of associations between CD3+ and γδ T cells with patient outcome, both univariately and after multivariate adjustment for the SSIGN score. The SSIGN score is a validated prognostic tool for surgically treated ccRCC that incorporates tumor stage, tumor size, tumor grade and the presence of histologic coagulative tumor necrosis to predict cancer-specific survival (16). Graphical methods used to evaluate the functional form of the association of CD3+ and γδ T cells with death from ccRCC did not indicate that these features should be transformed or categorized (even after deleting one γδ T cell outlier).

The agreement of the two anti-γδ TCR Abs in detecting γδ T cells by flow cytometry was evaluated using the cell as the unit of measure and each patient as an independent sample. κ statistics were therefore generated for each of the 13 patients whose PBMCs were studied and these 13 κ values were then tested for pooling using the technique of Donner and Klar for multiple samples (17). Due to the large number of cells counted in FACS experiments and the resulting high level of statistical power, the test for heterogeneity among the 13 independent samples was highly significant (p < 0.001), indicating that pooling of the κ statistics would not be valid. We therefore report the individual κ statistics instead of one overall measure of Ab agreement.

The clinicopathologic characteristics of the study cohort are given in Table I. Median patient age was 64 years (interquartile range (IQR): 56–72) and the median duration of patient follow up was 3.1 years. To date, 68 patients (27%) have died, 48 of whom died of ccRCC at a median of 1.3 years following nephrectomy. The estimated cancer-specific survival rates at 1, 3 and 5 years following surgery were 92% (95% CI, 89–96%; at risk = 222), 81% (95% CI, 76–86%; at risk = 107) and 75% (95% CI, 68–83; at risk = 21), respectively.

Table I.

Clinicopathologic features of 248 patients with ccRCC

FeatureNumber (%)
Age at surgery (years)  
 <65 131 (53) 
 ≥65 117 (47) 
Gender  
 Female 80 (32) 
 Male 168 (68) 
Symptoms at presentation  
 Absent 118 (48) 
 Present 130 (52) 
Constitutional symptoms  
 Absent 207 (84) 
 Present 41 (17) 
Tumor size (cm)  
 <5 98 (40) 
 5–<7 48 (19) 
 7–<10 46 (19) 
 ≥10 56 (23) 
TNM 2002 T stage  
 pT1a 78 (32) 
 pT1b 58 (23) 
 pT2 37 (15) 
 pT3a 28 (11) 
 pT3b 40 (16) 
 pT3c 5 (2) 
 pT4 2 (1) 
TNM 2002 N stage  
 pNx/pN0 233 (94) 
 pN1/pN2 15 (6) 
TNM 2002 M stage  
 pM0 208 (84) 
 pM1 40 (16) 
TNM 2002 stage grouping  
 I 131 (53) 
 II 28 (11) 
 III 48 (19) 
 IV 41 (17) 
Nuclear grade  
 1 13 (5) 
 2 80 (32) 
 3 126 (51) 
 4 29 (12) 
Coagulative tumor necrosis  
 Absent 177 (71) 
 Present 71 (29) 
Sarcomatoid differentiation  
 Absent 240 (97) 
 Present 8 (3) 
FeatureNumber (%)
Age at surgery (years)  
 <65 131 (53) 
 ≥65 117 (47) 
Gender  
 Female 80 (32) 
 Male 168 (68) 
Symptoms at presentation  
 Absent 118 (48) 
 Present 130 (52) 
Constitutional symptoms  
 Absent 207 (84) 
 Present 41 (17) 
Tumor size (cm)  
 <5 98 (40) 
 5–<7 48 (19) 
 7–<10 46 (19) 
 ≥10 56 (23) 
TNM 2002 T stage  
 pT1a 78 (32) 
 pT1b 58 (23) 
 pT2 37 (15) 
 pT3a 28 (11) 
 pT3b 40 (16) 
 pT3c 5 (2) 
 pT4 2 (1) 
TNM 2002 N stage  
 pNx/pN0 233 (94) 
 pN1/pN2 15 (6) 
TNM 2002 M stage  
 pM0 208 (84) 
 pM1 40 (16) 
TNM 2002 stage grouping  
 I 131 (53) 
 II 28 (11) 
 III 48 (19) 
 IV 41 (17) 
Nuclear grade  
 1 13 (5) 
 2 80 (32) 
 3 126 (51) 
 4 29 (12) 
Coagulative tumor necrosis  
 Absent 177 (71) 
 Present 71 (29) 
Sarcomatoid differentiation  
 Absent 240 (97) 
 Present 8 (3) 

We initially optimized our immunohistochemistry (IHC) protocol using the Immu510 Ab because this Ab is a published pan-γδ TCR-recognizing Ab that can be used for IHC study of fresh-frozen human tissues (12, 18). To confirm that Immu510 was capable of accurately identifying γδ T cells, we compared it to a second mAb, TCR-γδ-1 (19), in flow cytometry experiments. Thirteen independent peripheral blood samples were tested for Ab concordance by three double-staining sequences: Immu510 followed 30 min later by TCR-γδ-1, TCR-γδ-1 followed 30 min later by Immu510, and both Abs given concurrently. These experiments revealed that the sequence of Ab staining had no impact on γδ T cell detection by either Ab and that the two Abs recognized similar cells bearing the γδ TCR (Fig. 1). Specifically, the κ statistics for agreement between the two Abs in the 13 patient PBMC samples studied were 0.643, 0.734, 0.790, 0.794, 0.828, 0.861, 0.864, 0.870, 0.904, 0.909, 0.929, 0.939, and 0.973, respectively.

FIGURE 1.

Zebra plots comparing γδ T cell staining characteristics using anti-γδ TCR Immu510 and TCR-γδ-1 in PBMCs from four representative donors (A–D). Figure demonstrates significant concordance in identification of γδ T cells using both Abs.

FIGURE 1.

Zebra plots comparing γδ T cell staining characteristics using anti-γδ TCR Immu510 and TCR-γδ-1 in PBMCs from four representative donors (A–D). Figure demonstrates significant concordance in identification of γδ T cells using both Abs.

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Several small reports have suggested that γδ T cells might be important in the pathogenesis of RCC (Table II and Refs. 4 and 7, 8, 9, 10), and clinical trials testing γδ T cell immunotherapy in RCC patients are already underway (5, 6). We performed IHC for CD3 and the γδ TCR on fresh-frozen tumor specimens obtained from a consecutive series of 248 ccRCC cases. Due to its known enrichment for γδ T cells, human palatine tonsillar tissue, surgically removed for chronic tonsillitis, was used as a positive control (Fig. 2, A and B, and Ref. 12). We observed that the median number of CD3+ T cells in the ccRCC specimens was 281/mm2 (IQR: 149–536) while the median number of γδ T cells was 2.6/mm2 (IQR: 1.3–4.6). When γδ T cells were expressed as a percentage of CD3+ T cells, the median percentage of CD3+ T cells bearing γδ TCR was 1.0% (IQR: 0.4–1.9). However, 3 of 248 tumors contained CD3+ T cell infiltrates comprised of ≥20% γδ T cells (Fig. 2,F). We also observed that the median percentage of γδ T cells decreased (even below peripheral blood values) as intratumoral CD3+ T cell counts increased, suggesting that intratumoral γδ T cells are diluted by increasing T cell infiltration (Fig. 3 A). This observation does not support that γδ T cells accumulate within ccRCC tumors, whether through active recruitment or intratumoral clonal expansion.

Table II.

Study results for unmanipulated γδ T lymphocytes in RCC patients

StudyStudy PopulationSample SizeTumor-Infiltrating LymphocytesPBLs
TechniquesKey FindingsTechniquesKey Findings
Choudhary et al. (9Regional ccRCC IHC γδ T cells present (Vδ1 predominant)   
Kobayashi et al. (8Localized RCC 41 IHC (n= 21) 5% of TILsa have γδ TCR—↑ HLA-DR in RCC γδ T cells FCM TCR cloning 5% of circulating T cells have γδ TCR—↑ γδ T cells with tumor stage—↓ γδ T cells following nephrectomy (n= 9) 
Kowalczyk et al. (10Localized RCC 30 FCM 4% of TILs have γδ TCR—↑ HLA-DR in RCC γδ T cells (n= 19) FCM 5% of circulating T cells have γδ TCR 
Viey et al. (7Metastatic ccRCC 15 IHC (n= 7) Dense T cells in 3 cases γδ T cells present (Vδ2 predominant) FCM 2% of circulating T cells have γδ TCR 
Viey et al. (4 )b Metastatic and localized ccRCC 32 (mets) and 31 (local)   FCM Local: 2% of circulating T cells have γVδ2TCR—Mets: 2% of circulating T cells have γVδ2TCR 
StudyStudy PopulationSample SizeTumor-Infiltrating LymphocytesPBLs
TechniquesKey FindingsTechniquesKey Findings
Choudhary et al. (9Regional ccRCC IHC γδ T cells present (Vδ1 predominant)   
Kobayashi et al. (8Localized RCC 41 IHC (n= 21) 5% of TILsa have γδ TCR—↑ HLA-DR in RCC γδ T cells FCM TCR cloning 5% of circulating T cells have γδ TCR—↑ γδ T cells with tumor stage—↓ γδ T cells following nephrectomy (n= 9) 
Kowalczyk et al. (10Localized RCC 30 FCM 4% of TILs have γδ TCR—↑ HLA-DR in RCC γδ T cells (n= 19) FCM 5% of circulating T cells have γδ TCR 
Viey et al. (7Metastatic ccRCC 15 IHC (n= 7) Dense T cells in 3 cases γδ T cells present (Vδ2 predominant) FCM 2% of circulating T cells have γδ TCR 
Viey et al. (4 )b Metastatic and localized ccRCC 32 (mets) and 31 (local)   FCM Local: 2% of circulating T cells have γVδ2TCR—Mets: 2% of circulating T cells have γVδ2TCR 
a

TIL, Tumor-infiltrating lymphocyte; FCM, flow cytometry, mets/Mets, metastasis.

b

Many of the metastatic patients in study 5 are reported in study 4.

FIGURE 2.

IHC staining of human tissues. Positive tonsillar controls are shown for CD3 (A) and γδ TCR (B). ccRCC tumors with moderate amounts of CD3+ tumor-infiltrating lymphocytes (C and E) and low (D) or high (F) levels of γδ T cells. The tumor exhibiting high levels of infiltrating γδ T cells (F) is representative of one of three ccRCC tumors harboring ≥20% γδ T cells. In contrast, the tumor exhibiting low levels of γδ T cells (D) is representative of the remaining 245 ccRCC tumors studied.

FIGURE 2.

IHC staining of human tissues. Positive tonsillar controls are shown for CD3 (A) and γδ TCR (B). ccRCC tumors with moderate amounts of CD3+ tumor-infiltrating lymphocytes (C and E) and low (D) or high (F) levels of γδ T cells. The tumor exhibiting high levels of infiltrating γδ T cells (F) is representative of one of three ccRCC tumors harboring ≥20% γδ T cells. In contrast, the tumor exhibiting low levels of γδ T cells (D) is representative of the remaining 245 ccRCC tumors studied.

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FIGURE 3.

A, Scatter plot demonstrating intratumoral γδ T cell dilution with increasing CD3+ T cell infiltration. The black line represents the median percentage of γδ T cells predicted by a quantile regression model. The shaded area indicates the normal percentage of γδ T cells present in peripheral blood (1–5%), demonstrating that with increasing CD3+ infiltration intratumoral γδT cell levels drop below peripheral blood levels. B, Jittered scatter plots demonstrating no relationship between absolute γδ T cell counts and tumor SSIGN scores. The bars indicate the median γδT cell counts for each SSIGN score group.

FIGURE 3.

A, Scatter plot demonstrating intratumoral γδ T cell dilution with increasing CD3+ T cell infiltration. The black line represents the median percentage of γδ T cells predicted by a quantile regression model. The shaded area indicates the normal percentage of γδ T cells present in peripheral blood (1–5%), demonstrating that with increasing CD3+ infiltration intratumoral γδT cell levels drop below peripheral blood levels. B, Jittered scatter plots demonstrating no relationship between absolute γδ T cell counts and tumor SSIGN scores. The bars indicate the median γδT cell counts for each SSIGN score group.

Close modal

Associations of intratumoral CD3+ and γδ T cell counts with conventional ccRCC prognostic features were also explored and we observed no associations between the percentage of intratumoral T cells that expressed the γδ TCR and any of the well-established prognostic factors for ccRCC (Table III). We also found no association between absolute γδT cell counts and the SSIGN score (Fig. 3,B), indicating that γδ T cell counts remained low as the severity of disease increased, bolstering the argument that clonal expansion and specific recruitment of γδ T cells does not occur in ccRCC. Univariate Cox models showed that percentages of γδ T cells (hazard ratio = 1.02; 95% CI, 0.99–1.06; p = 0.250) failed to be predictive of cancer-specific survival (Fig. 4). Likewise, after adjusting in multivariate Cox models for the SSIGN score, we still found that percentages of ccRCC-infiltrating γδ T cells (hazard ratio = 0.98; 95% CI. 0.94–1.02; p = 0.392) did not predict cancer-specific survival.

Table III.

Associations between CD3+ and γδ T cells and prognostic features for RCC

FeatureCD3+ T Cells/mm2Percent γδ T Cells
MedianIQRpMedianIQRp
Age at surgery (years)   0.503a    
 <65 277 136–550  0.9 0.4–1.9 0.922a 
 ≥65 282 168–530  1.1 0.4–1.8  
Gender   0.402a    
 Female 270 141–538  1.1 0.5–2.3 0.389a 
 Male 304 172–539  0.9 0.4–1.8  
Symptoms at presentation       
 Absent 264 136–524 0.309a 0.9 0.4–1.7 0.671a 
 Present 293 163–582  1.1 0.4–1.9  
Constitutional symptoms       
 Absent 277 142–530 0.271a 1.0 0.4–1.8 0.534b 
 Present 301 177–697  1.1 0.4–2.6  
Tumor size (cm)       
 <5 264 142–528 0.233b 0.8 0.4–1.6 0.511b 
 5 to <7 249 141–455  1.1 0.4–1.7  
 7 to <10 400 216–616  1.0 0.4–1.8  
 ≥10 295 149–667  1.2 0.5–2.3  
TNM 2002 T stage       
 pT1 248 137–473 0.063b 0.9 0.4–1.7 0.518b 
 pT2 371 207–624  1.1 0.5–1.8  
 pT3 301 170–550  1.1 0.4–2.0  
 pT4 768 588–949  3.9 1.2–6.5  
TNM 2002 N stage       
 pNx/N0 279 147–525 0.358a 1.0 0.4–1.8 0.223a 
 pN1/N2 306 173–706  1.2 0.6–2.7  
TNM 2002 M stage       
 pM0 299 158–521 0.720a 1.0 0.4–1.8 0.478a 
 pM1 231 129–700  1.1 0.5–2.1  
TNM 2002 stage grouping       
 I 248 137–470 0.065b 0.8 0.4–1.7 0.796b 
 II 380 269–672  1.1 0.5–1.9  
 III 347 190–542  1.1 0.4–2.0  
 IV 247 135–689  1.1 0.5–2.1  
Nuclear grade       
 1 164 103–231 <0.001b 1.6 1.2–3.5 0.069b 
 2 235 141–379  0.9 0.4–2.1  
 3 392 185–689  0.9 0.4–1.5  
 4 275 173–504  1.1 0.6–1.8  
Coagulative tumor necrosis       
 Absent 265 141–530 0.139a 1.1 0.4–2.1 0.741a 
 Present 337 170–616  0.9 0.4–1.5  
Sarcomatoid differentiation       
 Absent 285 150–543 0.225a 1.0 0.4–1.9 0.267a 
 Present 210 127–305  1.4 0.8–2.1  
FeatureCD3+ T Cells/mm2Percent γδ T Cells
MedianIQRpMedianIQRp
Age at surgery (years)   0.503a    
 <65 277 136–550  0.9 0.4–1.9 0.922a 
 ≥65 282 168–530  1.1 0.4–1.8  
Gender   0.402a    
 Female 270 141–538  1.1 0.5–2.3 0.389a 
 Male 304 172–539  0.9 0.4–1.8  
Symptoms at presentation       
 Absent 264 136–524 0.309a 0.9 0.4–1.7 0.671a 
 Present 293 163–582  1.1 0.4–1.9  
Constitutional symptoms       
 Absent 277 142–530 0.271a 1.0 0.4–1.8 0.534b 
 Present 301 177–697  1.1 0.4–2.6  
Tumor size (cm)       
 <5 264 142–528 0.233b 0.8 0.4–1.6 0.511b 
 5 to <7 249 141–455  1.1 0.4–1.7  
 7 to <10 400 216–616  1.0 0.4–1.8  
 ≥10 295 149–667  1.2 0.5–2.3  
TNM 2002 T stage       
 pT1 248 137–473 0.063b 0.9 0.4–1.7 0.518b 
 pT2 371 207–624  1.1 0.5–1.8  
 pT3 301 170–550  1.1 0.4–2.0  
 pT4 768 588–949  3.9 1.2–6.5  
TNM 2002 N stage       
 pNx/N0 279 147–525 0.358a 1.0 0.4–1.8 0.223a 
 pN1/N2 306 173–706  1.2 0.6–2.7  
TNM 2002 M stage       
 pM0 299 158–521 0.720a 1.0 0.4–1.8 0.478a 
 pM1 231 129–700  1.1 0.5–2.1  
TNM 2002 stage grouping       
 I 248 137–470 0.065b 0.8 0.4–1.7 0.796b 
 II 380 269–672  1.1 0.5–1.9  
 III 347 190–542  1.1 0.4–2.0  
 IV 247 135–689  1.1 0.5–2.1  
Nuclear grade       
 1 164 103–231 <0.001b 1.6 1.2–3.5 0.069b 
 2 235 141–379  0.9 0.4–2.1  
 3 392 185–689  0.9 0.4–1.5  
 4 275 173–504  1.1 0.6–1.8  
Coagulative tumor necrosis       
 Absent 265 141–530 0.139a 1.1 0.4–2.1 0.741a 
 Present 337 170–616  0.9 0.4–1.5  
Sarcomatoid differentiation       
 Absent 285 150–543 0.225a 1.0 0.4–1.9 0.267a 
 Present 210 127–305  1.4 0.8–2.1  
a

Wilcoxon rank sum test.

b

Kruskal-Wallis test.

FIGURE 4.

Kaplan-Meier plot for RCC-specific survival. The patient cohort is split into four groups based on quartiles of the percentage of CD3+ tumor-infiltrating cells that were γδ T cells: 1st quartile = 0–0.42%, 2nd quartile = 0.42–1.00%, 3rd quartile = 1.00–1.90%, and 4th quartile = >1.90%. A comparison among the curves was evaluated using the log-rank statistic.

FIGURE 4.

Kaplan-Meier plot for RCC-specific survival. The patient cohort is split into four groups based on quartiles of the percentage of CD3+ tumor-infiltrating cells that were γδ T cells: 1st quartile = 0–0.42%, 2nd quartile = 0.42–1.00%, 3rd quartile = 1.00–1.90%, and 4th quartile = >1.90%. A comparison among the curves was evaluated using the log-rank statistic.

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In our present study, we failed to confirm the previous literature suggesting that γδ T cells accumulate to abundant levels within ccRCCs of the kidney. As such, our study raises a fundamental question as to whether or not γδ T cells play a significant role in the biology of ccRCC (4, 7, 8, 9, 10). Limitations of our study include its relatively short 3-year median follow up which is accompanied by a mortality rate of 27%. However, given that the estimated 10-year ccRCC-specific death rate is roughly 35% (11), it is clear that our current study encompasses more than half of all anticipated events and, thus, portrays a representative experience of expected outcomes for patients with ccRCC. In addition, it is unlikely that other patient selection factors significantly influenced the key findings of our study given that the clinicopathologic features of our study cohort (detailed in Table I) closely parallel features of ccRCC patients encountered in most western medical centers.

In several small studies in which histologic subtype of RCC tumors has been largely ignored, it has been reported that levels of γδ T cells within RCC tumors exceed levels of γδ T cells observed in the periphery. In addition, levels of tumor-infiltrating γδ T cells have been reported to correlate with a few prognostic features of RCC (summarized in Table II). It has further been suggested that γδ T cells, including TCR Vγ9Vδ2 T cells, constitute the predominant lymphocyte population infiltrating RCC tumors and that bromohydrin pyrophosphate-stimulated TCR Vγ9Vδ2 γδ T cells exhibit an ability to lyse RCC target cells in vitro (7). Lastly, a considerable body of literature has reported that γδ T cells play a specialized role in mediating inflammatory responses within normal epithelia injured by trauma, infection, or encroaching malignant processes (20). Emboldened by these collective observations, clinical trials have been pursued with the expectation that adoptive γδ T cell therapy may prove useful as an antitumoral immunotherapeutic approach to treat patients with advanced RCC as well as other forms of malignancy (5, 6).

Our present data fail to support the publication of Viey et al. (7), in which it was reported that γδ T cells (as well as Vδ2 TCR-bearing T cells) comprise “the major component” of infiltrating T cells within renal tumors, observations that were based on detailed investigations of only three patient RCC tumor specimens. Specifically, our examination of 248 consecutive specimens clearly demonstrates that only ∼1% of all CD3+ T cells within ccRCC specimens are of the γδ T cell subtype. Thus, on average, percentages of γδ T cells within ccRCC tumors remain comparable to values observed in normal peripheral blood, a finding that is consistent with two other studies: one conducted using IHC (8) and the other using flow cytometry (10), both demonstrating very low levels of intratumoral γδ T cells within RCC tumors. This observation alone raises concerns regarding the relevance of γδ T cells in altering the biology of RCC tumors. It is possible that γδ T cells die soon after interacting with tumor cells, thus making their detection by IHC impossible. However, such an occurrence would support our argument that γδ T cells are not effective tumoricidal cells. If γδ T cells die each time they try to infiltrate and attack a tumor, a phenomenon similar to peripheral clonal deletion would be occurring, which would imply that the tumor has gained the capacity of targeted immunosuppression. Why would we want to treat a tumor with a therapy that it so easily bypasses?

The assertion that adoptive transfer of γδ T cells (stimulated and expanded ex vivo) might produce favorable responses in patients with advanced ccRCC is impossible to rule out with the current study data. It is possible that manipulated γδ T cells might acquire functional capacities that they did not possess naturally and therefore become more tumoricidal. However, our study raises substantive concerns about the likelihood of eliciting such favorable responses using γδ T cell therapy. Specifically, we failed to observe a single correlation between percentages of intratumoral γδ T cells and any of the conventional predictors of ccRCC outcome. This lack of association between percentages of intratumoral γδ T cells and conventional predictors of ccRCC outcome argues against a significant role for γδ T cells in regulating ccRCC biology. This particular conclusion is supported by consensus observations by others that robust predictive markers for cancer typically exhibit significant associations with other established features of disease, often because such markers contribute (either directly or indirectly) to key mechanisms involved with malignant inception or progression (21). Thus, if γδ T cells were indeed mediating antitumoral responses against ccRCC tumors, we would have expected to observe at least some correlation between percentages of γδ T cells within ccRCC tumors and other established clinicopathologic predictors of ccRCC behavior. No such relationships were observed in our present study. Adding to our concerns, we further demonstrate that as levels of infiltrating CD3+ T cells rise within ccRCC tumors, percentages of γδ T cells actually decline, implying a dilution effect that argues against either active recruitment or clonal expansion of γδ T cells within ccRCC tumors. Given that our study of 248 consecutive patient specimens is several times larger than the aggregate of all previous studies published pertaining to γδ T cells within ccRCC, it is highly unlikely that the values we report are artifactual (due to insufficient sampling of tumors). Also, determinations of γδ T cell levels within renal tumors without regard to histologic subtype (i.e., by including papillary or chromophobe RCC tumors) could lead to estimations of γδ T cell levels that are not at all relevant to most patients with RCC. This is important because RCC tumors of the clear cell subtype represent the most common (80%) of all renal malignancies and, in contrast to papillary and chromophobe RCC tumors, ccRCC is notably more aggressive and is more amenable to immunotherapeutic intervention. It is for these critical reasons that we restricted our present study to an examination of ccRCC tumors.

Nevertheless, proponents of adoptive γδ T cell immunotherapy for the treatment of RCC could suggest that our findings do not necessarily preclude unforeseen therapeutic benefits arising from a γδ T cell-based treatment approach and would likely cite two further observations reported by Viey et al. (4, 7): enrichment of TCR Vγ9Vδ2 T cells within RCC tumors and apparent specific lysis of RCC tumor cells by bromohydrin pyrophosphate expanded TCR Vγ9Vδ2 T cells. In response to the these observations, the finding of enrichment for specific TCR VγVδ-paired T cell subsets within RCC tissues is not entirely unexpected since it is well established that γδ T cells (bearing paired VγVδ TCR and exhibiting limited VDJ diversity) distribute themselves throughout the host in an organ-specific fashion following thymic emigration, presumably to provide a first line of immunologic defense against pathogenic insults specific to each organ type or to down-regulate organ-specific TCR αβ T cell-mediated autoimmunity (22, 23). With regard to lysis of RCC tumor cells by γδ T cells, the published literature simply fails to support specific lysis of RCC tumors by Phosphostim-expanded (Innate Pharma) TCR Vγ9Vδ2 T cells. In fact, the study by Viey et al. (7) reported roughly 65% lysis of Daudi (Burkitt’s lymphoma) cells, 40% lysis of autologous RCC cells (as well as allogeneic RCC6 tumor cells), and nearly 20% lysis of normal (uncharacterized) renal parenchymal cells in response to patient-matched, Phosphostim-expanded TCR Vγ9Vδ2 T cells (4-h 51Cr release assay; E:T ratio of 30:1). That Phosphostim-expanded TCR Vγ9Vδ2 T cells exhibit an ability to lyse unpurified normal renal cells in bulk culture (as well as Daudi cells) raises the distinct possibility of autoimmune-type injury to one or more of the 20+ odd cell types that comprise the kidney as well as other tissue types during aggressive antitumoral immunotherapeutic treatment provided by adoptive γδ T cell transfer. Additionally, reports of exaggerated TCR αβ T cell-mediated inflammation in pathogen-challenged (or untreated) γδ T cell-deficient mice also raises questions. In fact, it has recently been suggested that a primary physiologic role of organ-specific γδ T cells may be to down-regulate TCR αβ T cell-mediated pathogenic or autoimmune inflammatory responses in a manner similar to that described for T regulatory cells (24). γδ T cells may therefore be immunosuppressive, not immunoprotective.

Regardless, the physiologic and pathophysiologic function of γδ T cells remains highly enigmatic and may prove to be dependent on organ-, host-, and disease-specific factors. Our study demonstrates that γδ T cells constitute an extremely minor population of cells within ccRCC tumors; a population that is further diluted with increasing tumor infiltration by CD3+ T cells. In addition, we show that levels of γδ T cells fail to correlate with even one single established clinicopathologic feature of ccRCC, including patient outcome. Based on these observations and the other studies cited above, we conclude that γδ T cells may prove to be unlikely arbiters of antitumoral immunity for the treatment of ccRCC. Our study further suggests that adoptive γδ T cell immunotherapy as a potential treatment for advanced ccRCC (currently in phase II trials) should be closely scrutinized with guarded expectations.

We thank Dr. James P. Allison (Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY) for critical review and helpful comments during the preparation of this manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by gifts from The Richard M. Schulze Family Foundation and The Commonwealth Foundation for Cancer Research. Donations were also provided by the Helen and Martin Kimmel Foundation.

3

Abbreviations used in this paper: RCC, renal cell carcinoma; ccRCC, clear cell RCC; CI, confidence interval; IQR, interquartile range; IHC, immunohistochemistry.

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