Tumor-Infiltrating Foxp 3 CD 4 + CD 25 + TCells Predict Poor Survival in Renal Cell Carcinoma

Purpose: RegulatoryTcells (Tregs) have been implicated as inhibitors of antitumoral immunity, and evidence suggests that elimination ofTregs may augment natural and pharmacologic immunity.We tested for the presence of putativeTregs within renal cell carcinoma (RCC) tumors. Experimental Design:We identified170 patientswhounderwent 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 CD4CD25Foxp3 and CD4CD25Foxp3 Tcells with death from RCCwere evaluated using Cox proportional hazards regressionmodels. 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 CD4CD25Foxp3 Tcells.The presence of Foxp3 Tcells was not significantly associatedwith RCC death univariately. One hundred forty-three (84.1%) tumors harbored CD4CD25Foxp3 T cells.The indicator forz10%CD4CD25Foxp3 Tcells was significantly associatedwith 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: Increasedpresence of CD4CD25Foxp3 Tcells wasnot significantly associated with RCC death. In contrast, CD4CD25Foxp3 Tcells, which may represent a unique set of Tregs or activated helperTcells, was significantly associated with outcome. In the United States, an estimated 38,890 new cases of renal cell carcinoma (RCC) are predicted to occur in 2006, with 12,840 deaths (1). Despite the progress in radiographic testing, almost one third of patients present with advanced disease, either locally advanced or metastatic. Surgery remains the mainstay treatment of localized disease with excellent longterm survival. Unfortunately, median survival with metastatic disease is only 13 months with a 5-year survival of <10% (2). Consequently, it is imperative to identify alternative medical and surgical treatment modalities to effectively manage advanced RCC. RCC is considered to be an immunogenic cancer, with pathologic specimens frequently harboring large numbers of tumor-infiltrating lymphocytes (TIL; refs. 3, 4). Reports of spontaneous regression and limited responses to cytokine therapies support this view. Biological modifiers, including interleukin-2 (IL-2) and IFN-a, have, at best, induced modest rates of response against metastatic RCC, ranging from 10% to 20% (2, 5). Evolving therapies with stem cell transplantation (6, 7) and tumor vaccines (8) have also exhibited promise in preliminary investigations. Although these novel immunebased approaches show some clinical activity, it seems that their effectiveness is limited by one or more mechanisms, such as tumor-induced immune suppression in the tumor microenvironment. One mechanism by which cancers evade immune destruction is by recruiting regulatory cells into the tumor microenvironment, including regulatory T cells (Tregs). In the last few years, there has been a resurgence of interest among immunologists about T-cell–mediated immune suppression. T cells were first shown to suppress immune responses in the 1970s (9, 10). Although some work continued through the 1980s (11, 12), the failure to identify antigen-specific factors and unique identifying molecules that caused immune suppression led to stagnation of this field. Interest was rekindled in the mid-1990s when Sakaguchi et al. (13) showed that a small population of Imaging, Diagnosis, Prognosis Authors’Affiliations: Departments of Urology, Immunology, Pathology, and Health Sciences Research, Mayo Clinic, Rochester, Minnesota Received 8/28/06; revised1/8/07; accepted1/23/07. Grant support: The Richard M. Schulze Family Foundation, the Commonwealth Foundation for Cancer Research, and the Helen andMartin Kimmel Foundation. 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: K.L. Knutson and E.D. Kwon contributed equally to this work. Requests for reprints: Eugene D. Kwon, Department of Urology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-284-5365; Fax: 507-284-1637; E-mail: kwon.eugene@mayo.edu. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-2139 www.aacrjournals.org Clin Cancer Res 2007;13(7) April 1, 2007 2075 Research. on August 29, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from CD4 T cells that coexpressed the IL-2 receptor a-chain (CD25) could control autoreactive CD4 T cells in vivo. In vitro murine studies have since confirmed that certain CD4CD25 T cells can function as potent inhibitors of T-cell–mediated immunity (14, 15), and subsequent studies have revealed a similar CD4CD25 T-cell population in humans (16, 17). Our current understanding now suggests that Tregs constitute a family of specialized T cells that can be divided into two broad subsets: natural and adaptive. The natural Treg population develops as a separate T-cell lineage in the thymus (18, 19). These cells are antigen specific and populate in the periphery to guard against autoimmune reactions. The second subset of ‘‘adaptive’’ Treg cells seems to develop from mature, peripheral CD4 T cells in response to tissue-specific or foreign antigens and mediates regulation through the release of soluble cytokines (IL-10; refs. 18, 19). However, one problem with the study of Tregs is the lack of specific cell markers that distinguish them from other lymphocytes. Recently, the ability to characterize Tregs improved with the identification of the transcription factor Foxp3, which has been shown to be a critical component of natural Treg generation and function (20). Murine and human natural CD4CD25 Tregs express high levels of Foxp3 compared with other lymphocytes, permitting its use as a relatively specific marker. Although research into the effect of Tregs on human cancer has been limited, several key studies have emerged in recent years showing their importance. Cancer patients seem to have increased numbers of both peripherally circulating and tumor-associated CD4CD25 Tregs (21, 22). Murine studies have shown that antibodyor immunotoxin-mediated depletion of CD25 cells can enhance tumor immunity and rejection (23, 24). Curiel et al. (25) were the first to show a direct link between Tregs and human tumor antigen-specific T-cell responses and positively correlate with poorer prognosis. In the current study, we evaluated intratumoral CD4CD25Foxp3 Tregs as well as CD4CD25Foxp3 T cells in RCC. Our findings reveal that it is the Foxp3 subset of CD4CD25 T cells and not the Foxp3 subset that correlates with worse pathologic features of RCC and cancer-specific survival. Materials andMethods Patient selection. On approval from the Institutional Review Board, we identified 196 patients treated with radical nephrectomy or nephron-sparing surgery for unilateral, sporadic clear cell RCC between 2000 and 2002 who had fresh-frozen tumor tissue available for study (26). Of these, 170 (86.7%) patients had an adequate tumor specimen available for staining. There was no difference in patient outcome between those with and without tissue available for study (P = 0.372, log-rank test). The cancer-specific survival rates (SE, number still at risk) at 2 years for patients with and without tissue available for study were 83.7% (2.9%, 134) and 88.5% (6.3%, 20), respectively. Pathologic features. The pathologic features examined included histologic subtype, tumor size, the 2002 tumor-node-metastasis (TNM) stage groupings, nuclear grade, coagulative tumor necrosis, the presence of lymphocytic infiltration, and the presence of tumor B7-H1 expression. The microscopic slides from all specimens were reviewed by a urologic pathologist (J.C.C.) without knowledge of patient outcome. Histologic subtype was classified according to the Union Internationale Contre le Cancer, American Joint Committee on Cancer, and Heidelberg guidelines (27, 28). Nuclear grade was assigned using standardized criteria as described previously (29). Histologic tumor necrosis was defined as the presence of any microscopic coagulative tumor necrosis (30). B7-H1 is a costimulatory glycoprotein whose aberrant expression by tumor cells has been shown to increase the risk of RCC-specific death (26). B7-H1 immunohistochemical staining and quantification were done as described previously (26). Immunohistochemistry. Frozen sections of RCC were brought to room temperature and fixed in 20jC acetone for 10 min and air dried at room temperature for 30 min. Sections were rehydrated in PBS and blocked for endogenous avidin/biotin using an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA). Sections were immunostained with mouse anti-human CD25 IgG1 at 1:10 dilution (DAKO, Carpinteria, CA) and visualized with Fluorescein Avidin DCS at a 1:300 dilution (Vector Laboratories) followed by staining with mouse antihuman CD4 IgG1 at a 1:100 dilution (BD PharMingen, San Diego, CA) and visualized with Texas Red Avidin DCS at a 1:400 dilution (Vector Laboratories) using the multiple immunofluorescent staining method of the MOM kit (Vector Laboratories). For Foxp3 immunostaining, sections were blocked for avidin/biotin and incubated for 5 min in 0.1% goat serum in PBS and then incubated with rabbit anti-human Foxp3 IgG at a 1:75 dilution (Abcam, Cambridge, MA) for 30 min. Sections were incubated with biotinylated antirabbit IgG at a 1:200 dilution (Vector Laboratories) for 30 min, washed with PBS, and incubated for 5 min with AMCA