The Frequency and Suppressor Function of CD4CD25Foxp3 TCells in the Circulation of Patients with Squamous Cell Carcinoma of the Head and Neck

Objective: Immune escape is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). RegulatoryTcells (Treg) might contribute to HNSCC progression by suppressing antitumor immunity, and their attributes in patients are of special interest. Methods:Multicolor flow cytometry was used to study the frequency and phenotype ofTreg in peripheral blood lymphocytes of 35 patients with HNSCC and 15 normal controls (NC). CD4CD25Tcells were purified by fluorescence-activated cell sorting and tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidylester ^ labeled autologous CD4CD25 responder cells. Results: The percentages of circulating CD4CD25 Tcells were increased in HNSCC patients (5 F 3%) versus NC (2 F 1.5%). In patients, this cell subset largely contained CD4CD25 Foxp3 T cells and only few CD25 cells. In addition, the frequency of Treg positive for CD62L, CTLA-4, Fas, FasL, and Foxp3 was greater in the circulation of patients than in NC (P < 0.0001). In HNSCC patients,Tregmediated significantly higher suppression (78F 7%) comparedwithTreg inNC (12F 4%)withP < 0.0001. Surprisingly, higherTreg frequency (P < 0.0059) and levels of suppression (P < 0.0001) were observed in patients with no evident disease (NED) than in untreated patients with active disease (AD). Conclusions: The frequency of T cells with suppressor phenotype and function (Treg) was significantly greater in HNSCC patients who were NED after oncologic therapy relative to those with AD.This finding suggests that oncologic therapy favors expansion ofTreg. Patients with head and neck squamous cell carcinoma (HNSCC) have benefited from recent advances in radiation therapy, chemotherapy, and surgical techniques. However, despite new treatment modalities and their success in terms of organ preservation and overall quality of life, survival rates for this disease have not improved in many years (1). More recent studies have examined the role of host immune responses in HNSCC progression, suggesting that T lymphocytes may play a role in control of tumor growth. Several naturally processed and presented HNSCC-associated antigens have been identified and are known to be recognized by specific CD4 and CD8 T lymphocytes (2). Most immunologic studies have focused on the analysis of CD8 T cells, which have been shown to mediate antitumor immunity (3). The role of CD4 T cells is more complex, as subsets of CD4 T cells are involved in initiating and maintaining anticancer immune responses (4, 5), as well as down-regulating these responses. In HNSCC subjects, antitumor functions of CD8 T lymphocytes are often compromised (6). In recent years, the concept has emerged that peripheral tolerance to tumors is maintained and enhanced by T cells with immunoregulatory function (Treg). In cancer, Treg frequency is increased in the peripheral circulation, and their accumulations in the tumor may be predictive of significantly reduced patient survival (7). We have previously described an enrichment of CD4CD25 T cells among tumor-infiltrating or circulating lymphocytes in HNSCC patients (8). Such increases in Treg could potentially present a significant problem, as these cells could be interfering with antitumor immune responses and inhibit responses to immunotherapies. However, phenotypic characteristics of Treg in HNSCC patients are incompletely understood, and their functional characteristics have not been evaluated. To date, three types of CD4 Treg cells have been partly characterized in humans: (a) CD4CD25Foxp3 type 1 T regulatory (Tr1) cells, which arise in the periphery upon encountering antigen in a tolerogenic environment via a process that is interleukin-10 (IL-10) dependent (9–11); (b) naturally occurring CD4CD25Foxp3 T cells (nTreg), which arise directly in the thymus and have the ability to suppress responses of both CD4CD25 and CD8CD25 T cells Human Cancer Biology Authors’Affiliation: Suite 1.27, Research Pavilion at the Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania Received 6/7/07; revised 7/18/07; accepted 7/24/07. Grant support: NIH grants PO1-CA109688, PO1-DE12321, and RO1-DE13918 to T.L.Whiteside. 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. Requests for reprints: Theresa L.Whiteside, Research Pavilion at the Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 CentreAvenue, Suite 1.27, Pittsburgh, PA 15213-1863. Phone: 412-624-0096; Fax: 412-624-0264; E-mail: [email protected]. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-1403 www.aacrjournals.org Clin Cancer Res 2007;13(21) November1, 2007 6301 Research. on April 8, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from in a contact-dependent, cytokine-independent, and antigen nonspecific manner (12–14); and (c) Th3 cells, which are dependent on IL-4 for functional differentiation (15). Currently, isolation and expansion of human Treg cells subsets into functionally active, disease-specific T cells is, however, difficult due to (a) the paucity of Treg cells in the peripheral blood and (b) the lack of specific identity markers for Treg cells. In humans, CD4CD25 T cells are mixed populations, including suppressor CD4CD25 T cells as well as CD4CD25 T cells, which are nonsuppressive, activated CD4 T cells. Furthermore, expression of Treg markers such as CTLA-4 or GITR can vary depending on cell activation, and these markers have not been useful for discriminating nTreg from effector T-cell populations. Similarly, Foxp3 expression, although more specific for Treg, may also be up-regulated on effector cells following activation (16). Also, due to its intracellular localization, Foxp3 cannot be used for isolation of living Treg cells. Recently, two groups have independently shown that expression of CD127, the a chain of the IL-7 receptor, discriminates CD127 Treg cells from CD127 conventional T cells within the CD25 CD45RO/RA effector/memory and the CD45RARO naive compartments in the human peripheral blood and lymph nodes (17). Nevertheless, T effector cells, which fail to differentiate into CD127 memory T cells after activation, down-regulate CD127 (18). These CD127 pseudoeffector cells persist in the peripheral circulation, displaying the hallmarks of activated effector cells but are unable to mediate effector functions (19). In our previous studies, we have developed flow cytometry– based methods for the characterization of phenotypic and functional attributes of human circulating CD4 Treg (20). Here, we use these methods to evaluate Treg in the peripheral blood of HNSCC patients with active disease (AD) as well as no evident disease (NED) after oncologic therapy. We show that the Treg subset within CD3CD4 is expanded and strongly suppressive in the peripheral circulation of HNSCC patients. Furthermore, in HNSCC patients who are NED after oncologic therapy, the frequency and suppressor function of Treg are higher than those in patients with AD. This finding suggests that oncologic therapy might contribute to the survival and expansion of highly active Treg in patients with cancer. Materials andMethods HNSCC patients and healthy volunteers. Blood samples were obtained from 35 HNSCC patients and 15 age-matched healthy volunteers as controls (NC). All subjects signed an informed consent approved by the Institutional Review Board of the University of Pittsburgh. All patients were seen at the Outpatient Otolaryngology Clinic at the University of Pittsburgh Cancer Institute (UPCI) between January 2006 and April 2007. The patient cohort included males and females with a mean age of 60 years (range, 23–82 years). The NC group included 10 males and 5 females with a mean age of 60 years. A total of 15 patients had AD. All NED patients (n = 20) underwent surgical resection of their tumor with a curative intent, and 14 of these received radiotherapy and/or chemotherapy. Of the 15 AD patients, 11 had untreated primary tumors, and 4 had a recurrent disease. The therapy, if given, was terminated from 3 weeks to 12 months before the time of phlebotomy for this study. The age, sex, and clinicopathologic characteristics of the patients are listed in Table 1. Collection of peripheral blood mononuclear cells. Peripheral venous blood (20–30 mL) was drawn into heparinized tubes. The samples were hand-carried to the laboratory and immediately centrifuged on Ficoll-Hypaque. Peripheral blood mononuclear cells (PBMC) were recovered, washed in AIM-V medium (Invitrogen), counted in a trypan blue dye, and immediately used for experiments. Antibodies. The following anti-human monoclonal antibodies (mAb) were used for flow cytometry: anti–CD3-ECD, anti–CD4PC5, anti–CD8-PC5, anti–GITR-FITC, anti–CD25-FITC, anti–CD62LFITC, anti–HLA-DR-FITC, anti–Foxp3-FITC, anti–CD122-FITC (IL-2R h), anti–CD45RA-FITC, anti–CD45RO-FITC, anti–Fas-FITC, anti– CD127-FITC, anti –CCR7-FITC, anti – TGFh1-FITC, anti – FasL-PE (NOK-1,42 kDa), anti–CD132-PE (IL-2Rg), anti–CD25-PE, anti– CD152-PE (CTLA-4), anti–CCR4-PE, and anti– IL-10-PE. Antibodies and their respective isotypes, used as negative controls for surface and intracellular staining, were all purchased from Beckman Coulter, except for anti–IL-10-PE, anti–CD45RA-FITC, anti–FasL-PE (BD PharMingen), anti–h/TNFRSF18GITR-FITC (clone FAB689F), anti–CCR4-PE, anti– CCR7-FITC (R&D Systems, Inc.), anti–TGFh1-FITC (Antigenix America Inc.), anti–CD127-FITC, and anti–Foxp3-FITC (eBioscience). Before use, all mAbs were titrated using normal resting or activated PBMC to establish optimal staining dilutions. Table 1. Clinicopathologic characteristics of patients with HNSCC who donated PBMC for this study Age (y) Mean range 23–82 Sex Male 27 Female 8 Total 35 Tumor site Nasal cavity 3 Oral cavity 18 Oropharynx 3 Hypopharynx 2 Larynx 6 Not determined 3 Tumor differentiation Poor 5 Moderate 20 Well 4 Not determined 6 Tumor stage T1 7 T2 6 T3 5 T4 11 Unstaged 6 Nodal status N0 20 N1 2 N2 7 N3 0 Unstaged 6 Status at blood draw Active disease (AD) 15 No previous therapy 11 Previous therapy 4 No evident disease (NED) 20 Primary disease 31 Recurrent disease 4 Therapy before blood draw Surgery 20 Radiotherapy 11 Radiochemotherapy 6 Human Cancer Biology www.aacrjournals.org Clin Cancer Res 2007;13(21) November1, 2007 6302 Research. on April 8, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Surface and intracellular staining. To determine the frequency of CD4CD25 and CD8CD25 T cells and the expression of nTreg markers, including intracellular and surface expression of transforming growth factor h1 (TGFh1) and IL-10, PBMCs (at least 2 10 cells per tube) were stained with mAbs included in the above-described panel for 15 min at 4jC. Appropriate isotype Ab controls were used in all experiments. Cells were washed and examined by four-color flow cytometry, as previously described (21). Intracellular staining for Foxp3, CD152 (CTLA-4), and IL-10 was done as previously described (21). Intracytoplasmic expression of TGFh1 and IL-10 was assessed before and after stimulation of PBMC for 4 h with phorbol 12-myristate 13-acetate (20 Ag/mL) and ionomycin (1 Ag/mL). Briefly, samples were first incubated with mAbs against surface markers CD4, CD3, and CD25. After extensive washing, cells were fixed with 4% (v/v) formaldehyde in PBS for 20 min at RT, washed once with PBS containing 0.5% (v/v) bovine serum albumin (BSA; w/v) and 2 nmol/L EDTA, permeabilized with PBS containing 0.5% BSA and 0.1% (v/v) saponin and stained with pre-titrated anti–CTLA-4-PE, anti–Foxp3-FITC, anti–TGFh1-FITC, or anti–IL-10-PE mAb for 30 min at RT. Cells were further washed twice with PBS containing 0.5% BSA Fig. 1. A ^ E, percentages of CD4CD25 and CD4CD25Tcells in PBMC of HNSCC patients and normal controls.The gating strategy used is illustrated in (A ^ C). In (D), percentages (meansF SD) of CD4CD25 (left) and CD4CD25 (right) T-cell subsets within the CD3CD4 T-cell subset in PBMC of HNSCC patients or NC. In (E), flow dot plots for one representative NC and one HNSCC patient and the isotype control for CD25-PE (mouse immunoglobulin G2a (IgG2a)-PE) are shown. CD4CD25Foxp3 TCells in HNSCCPatients www.aacrjournals.org Clin Cancer Res 2007;13(21) November1, 2007 6303 Research. on April 8, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from and 0.2% (v/v) saponin, resuspended in fluorescence-activated cell sorting flow solution and immediately analyzed by flow cytometry. Appropriate isotype controls were included for each sample. Flow cytometry. Flow cytometry was done using a FACScan flow cytometer (Beckman Coulter) equipped with Expo32 software (Beckman Coulter). The acquisition and analysis gates were restricted to the lymphocyte gate as determined by their characteristic forward (FSC) and side-scatter (SSC) properties. FSC an SSC were set in a linear scale. For analysis, 1 10 lymphocytes were acquired. Furthermore, analysis gates were restricted to the CD3CD4, CD4CD25, CD8CD3, CD8CD25, and CD4CD25 T-cell subsets, as appropriate. Cells expressing Treg markers were acquired and analyzed in the FL1 or FL2 logarithmic scale using the set gates. Suppression experiments. Single cell – sorted, fresh CD4CD25 T-cell populations were tested for regulatory function by coculture analysis with at least 0.5 10 carboxyfluorescein diacetate succinimidylester (CFSE)-labeled autologous CD4CD25 responder cells per well at the suppressor/responder (S/R) ratios of 1:1, 1:5, and 1:10. Soluble OKT3 (1 Ag/mL; American Type Culture Collection) and soluble anti-CD28 mAb (1 Ag/mL) were used for stimulation in the presence of 150 IU IL-2/mL for 5 days. CFSE-labeling of R cells was done as previously described (21, 22). Briefly, CD4CD25 T cells separated by single cell sorting were stained with 1.5 Amol/L CFSE (Molecular Probes/Invitrogen) for 10 min at room temperature. The CFSE label was quenched by the addition of an equal volume of FCS (Invitrogen), and then cells were washed extensively with PBS. T-cell populations were classified as suppressive, if they inhibited proliferation of the CD4CD25 R cells in the coculture assay and if decreasing the number of CD4CD25 T cells relative to the number of CD4CD25 R cells in coculture restored proliferation. CD4CD25 T-cell populations that satisfied both of these criteria were classified as suppressor T cells. These criteria were applied to all populations, so that they could be tested regardless of levels of expansion. All CFSE data were analyzed using the ModFit software provided by Verity Software House (Topsham). The percentages of suppression were calculated based on the proliferation index (PI) of responder cells alone compared with the PI of cultures containing responders and Treg. The program determines the percent of cells within each peak, and the sum of all peaks in the control culture is taken as 100% of proliferation and