Laura Strauss

A Unique Subset of CD4+CD25highFoxp3+ T Cells Secreting Interleukin-10 and Transforming Growth Factor-β1 Mediates Suppression in the Tumor Microenvironment

Purpose: Immunosuppression, including that mediated by CD4+CD25highFoxp3+ regulatory T cells (Treg), is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). Tregs with a distinct phenotype in tumor-infiltrating lymphocytes (TIL) contribute to local immune suppression. Experimental Design: The frequency and phenotype of Treg in TIL and/or peripheral blood mononuclear cells (PBMC) in 15 HNSCC patients and PBMC in 15 normal controls were compared. Single-cell sorted CD4+CD25high T cells were tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidyl ester–labeled and activated autologous CD4+CD25− responder T cells. Transwell inserts separating Treg from responders and neutralizing interleukin-10 (IL-10) or transforming growth factor-β1 (TGF-β1) antibodies were used to evaluate the mechanisms used by Treg to suppress responder cell proliferation. Results: In TIL, CD25+ cells were enriched in the CD3+CD4+ subset (13 ± 3%) relative to circulating CD3+CD4+ T cells (3 ± 0.7%) in HNSCC patients (P ≤ 0.01) or normal controls (2 ± 1.5%; P ≤ 0.001). Among the CD3+CD4+ subset, CD25high Treg represented 3 ± 0.5% in TIL, 1 ± 0.3% in PBMC, and 0.4 ± 0.2% in normal controls. Tregs in TIL were GITR+, IL-10+, and TGF-β1+, although circulating Treg up-regulated CD62L and CCR7 but not GITR, IL-10, or TGF-β1. Treg in TIL mediated stronger suppression (P ≤ 0.001) than Treg in PBMC of HNSCC patients. The addition of neutralizing IL-10 and TGF-β antibodies almost completely abrogated suppression (5 ± 2.51%). Transwell inserts partly prevented suppression (60 ± 5% versus 95 ± 5%). Conclusions: Suppression in the tumor microenvironment is mediated by a unique subset of Treg, which produce IL-10 and TGF-β1 and do not require cell-to-cell contact between Treg and responder cells for inhibition.

Selective Survival of Naturally Occurring Human CD4+CD25+Foxp3+ Regulatory T Cells Cultured with Rapamycin

Naturally occurring CD4+CD25+ regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4+CD25+ non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4+CD25high T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4+CD25low or CD4+CD25− T cells did not. The expanding pure populations of CD4+CD25high T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4+CD25− T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4+CD25high T cell populations retained a broad TCR repertoire and, like CD4+ CD25+ T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4+ and CD8+ T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.

Human Circulating CD4+CD25highFoxp3+Regulatory T Cells Kill Autologous CD8+but Not CD4+Responder Cells by Fas-Mediated Apoptosis

Mechanisms utilized by human regulatory T cells (Treg) for elimination of effector cells may vary. We investigated the possibility that the mechanism of Treg suppression depends on Fas/FasL-mediated apoptosis of responder cells (RC). CD4+CD25highFoxp3+ Treg and autologous CD4+CD25− and CD8+CD25− subsets of RC were isolated from blood of 25 cancer patients and 15 normal controls and cocultured in the presence of OKT3 and IL-2 (150 or 1000 IU/ml). Suppression of RC proliferation was measured in CFSE assays. RC and Treg apoptosis was monitored by 7-aminoactinomycin D staining in flow-based cytotoxicity assays. Treg from all subjects expressed CD95+, but only Treg from cancer patients expressed CD95L. These Treg, when activated via TCR plus IL-2, up-regulated CD95 and CD95L expression (p < 0.001) and suppressed CD8+ RC proliferation (p < 0.001) by inducing Fas-mediated apoptosis. However, Treg cocultured with CD4+ RC suppressed proliferation independently of Fas/FasL. In cocultures, Treg were found to be resistant to apoptosis in the presence of 1000 IU/ml IL-2, but at lower IL-2 concentrations (150 IU/ml) they became susceptible to RC-induced death. Thus, Treg and RC can reciprocally regulate Treg survival, depending on IL-2 concentrations present in cocultures. This divergent IL-2-dependent resistance or sensitivity of Treg and RC to apoptosis is amplified in patients with cancer.

Expansion of Human T Regulatory Type 1 Cells in the Microenvironment of Cyclooxygenase 2 Overexpressing Head and Neck Squamous Cell Carcinoma

Cyclooxygenase 2 (COX-2) overexpression and production of prostaglandin E(2) (PGE(2)) by head and neck squamous cell carcinomas (HNSCC) induce type 1 regulatory T (Tr1) cells and contribute to carcinogenesis by creating a tolerogenic milieu. To test this hypothesis, CD4(+)CD25(-) T cells obtained from the peripheral blood of 10 normal donors were cocultured with autologous dendritic cells, irradiated HNSCC cells and cytokines, interleukin 2 (IL-2), IL-10, and IL-15. HNSCC cells were either COX-2 negative, constitutively expressed COX-2, were transfected with COX-2, or had COX-2 expression knocked down by small interfering RNA. Other modifications included coculture plus or minus the COX-inhibitor, Diclofenac, or synthetic PGE(2) in the absence of HNSCC. Lymphocytes proliferating in 10-day cocultures were phenotyped by flow cytometry, studied for cytokine production by ELISA and for suppressor function in CFSE inhibition assays plus or minus anti-IL-10 or anti-transforming growth factor-beta(1) (TGF-beta(1)) monoclonal antibodies (mAb). COX-2(+) HNSCC or exogenous PGE(2) induced outgrowth of Tr1 cells with the CD3(+)CD4(+)CD25(-)IL2Rbeta(+)IL2Rgamma(+)FoxP3(+)CTLA-4(+)IL-10(+)TGF-beta(1)(+)IL-4(-) phenotype and high suppressor functions (range, 46-68%). Small interfering RNA knockout of COX-2 gene in HNSCC led to outgrowth of lymphocytes with decreased IL2Rgamma (P = 0.0001), FoxP3 (P = 0.05), and IL-10 (P = 0.035) expression and low suppressor activity (range, 26-34%). Whereas COX-2(+) cocultures contained IL-10 and TGF-beta(1) (medians, 615 and 824 pg/mL), cytokine levels were decreased (P < 0.0001) in COX-2(-) cocultures. Inhibition of COX-2 enzymatic activity in HNSCC abrogated outgrowth of Tr1 cells. Neutralizing mAbs to IL-10 and/or TGF-beta(1) abolished Tr1-mediated suppression. COX-2 overexpression in HNSCC plays a major role in the induction of Tr1 cells in the tumor microenvironment.

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. Methodology/Principal Findings CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway. Conclusions/Significance RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.

Expression of ICOS on Human Melanoma-Infiltrating CD4+CD25highFoxp3+ T Regulatory Cells: Implications and Impact on Tumor-Mediated Immune Suppression

Objective: Interaction of ICOS with its ligand (ICOSL, B7-H2) promotes T cell responses. As CD4+CD25highFoxp3+ naturally occurring T regulatory cells in melanoma patients express ICOS, we investigated the impact of ICOS on naturally occurring T regulatory cell function. Methods: Expression of ICOS and T regulatory (Treg) cell markers was determined on CD4+CD25high T cells in PBMC and tumor-infiltrating lymphocytes from melanoma patients (n = 10) and PBMC of normal controls (n = 10) by multicolor flow cytometry. Suppression mediated by sorted ICOShigh and ICOSlow Treg was assessed in CFSE-based suppression assays with autologous CD4+CD25− responder cells (RC). Transwell inserts separating Treg from RC were used to evaluate suppression mechanisms used by Treg. ICOShigh or ICOSlow Treg were coincubated with RC ± TCR and IL-2 stimulation. ICOShigh and ICOS− Treg were also expanded under conditions previously shown to induce Tr1 from RC. Results: Treg in tumor-infiltrating lymphocytes expressed ICOS (mean fluorescence intensity = 70 ± 10), while Treg in PBMC had low ICOS expression (mean fluorescence intensity = 3.5 ± 2.5, p ≤ 0.001). ICOShigh Treg up-regulated Treg markers (p ≤ 0.0016) and mediated stronger suppression (p ≤ 0.001) relative to ICOSlow Treg. ICOShigh Treg induced Tr1 cells in nonactivated RC and Th2 cells in preactivated RC. ICOShigh Treg exposed to Tr1 cytokines expressed IL-10 and suppressed RC (92 ± 12%) in contrast to ICOSlow Treg, which mediated low suppression (21 ± 15%; p ≤ 0.0028). Conclusion: ICOShigh Treg can induce diverse immune responses in RC, depending on activation signals and cytokines present. ICOShigh Treg induce Tr1 or Th2 cells depending on the activation state of RC. In a “Tr1” cytokine milieu, ICOShigh Treg transit to Tr1.

Tumor-derived microvesicles in sera of patients with head and neck cancer and their role in tumor progression.

Tumor‐derived membranous vesicles (MV) isolated from sera of the patients with squamous cell carcinomas of the head and neck (HNSCC) induce apoptosis of activated CD8+ T cells. We tested if MV molecular profile and activity correlate with disease progression.

Functional and phenotypic characteristics of CD4+CD25highFoxp3+ Treg clones obtained from peripheral blood of patients with cancer

Circulating human CD4+CD25highFoxp3+ T cell populations (Treg) may contain activated CD4+CD25+ T cells interfering with Treg evaluation. To gain insights into the phenotypic and functional characteristics of Treg in patients with cancer, we have analyzed CD4+CD25high populations at the clonal level. Single‐cell sorted (SCS) CD4+CD25high T cells obtained from PBMC of normal controls (NC) or patients with squamous cell carcinoma of the head and neck (HNSCC) were plated at 1 cell/well in 96 well plates and expanded with anti‐CD3/anti‐CD28 Abs and 1,000 IU IL‐2/mL in the presence or absence of rapamycin (1 nM). All generated clones were evaluated for the phenotype by flow cyometry and suppressor function in CFSE‐based proliferation assays. Clones had heterogeneous CD25 expression levels. Cloning efficiency of CD4+CD25high T cells was low. CD25high clones expressed CTLA‐4, Foxp3, CD62L, but little GITR and suppressed proliferation of autologous CD4+CD25− responder cells. Clones of activated CD4+CD25interm./low cells expressed intermediate to high levels of GITR and HLA‐DR and did not suppress proliferation of responder cells. The number, suppressor phenotype and function of CD25high Treg clones were significantly enhanced in HNSCC patients relative to NC (p ≤ 0.001). CD4+CD25+ populations comprise phenotypically and functionally distinct subsets of CD25+ cells. Only a small fraction of these activated CD4+ T cells are potent suppressor cells characterized by high expression levels of CD25, Foxp3, CTLA‐4 and CD62L. The number of expandable Treg is increased in HNSCC patients.

Effects of Adjuvant Chemoradiotherapy on the Frequency and Function of Regulatory T Cells in Patients with Head and Neck Cancer

Purpose: Regulatory T cells (Treg) accumulate in tumor tissues and the peripheral blood of cancer patients and may persist after therapies. This cross-sectional study examines effects of adjuvant chemoradiotherapy (CRT) on Treg numbers and function in head and neck squamous cell carcinoma (HNSCC) patients. Experimental Design: The frequency and absolute numbers of CD4+, ATP-hydrolyzing CD4+CD39+ and CD8+ T cells, and expression levels of CD39, CD25, TGF-β–associated LAP and GARP on Treg were measured by flow cytometry in 40 healthy donors (NC) and 71 HNSCC patients [29 untreated with active disease (AD); 22 treated with surgery; 20 treated with CRT]. All treated subjects had no evident disease (NED) at the time of phlebotomy. In an additional cohort of 40 subjects with AD (n = 15), NED (n = 10), and NC (n = 15), in vitro sensitivity of CD4+ T-cell subsets to cisplatin and activation-induced cell death (AICD) was tested in Annexin V–binding assays. Results: CRT decreased the frequency of circulating CD4+ T cells (P < 0.002) but increased that of CD4+CD39+ Treg (P ≤ 0.001) compared with untreated or surgery-only patients. Treg frequency remained elevated for >3 years. CRT increased surface expression of LAP, GARP, and CD39 on Treg. In vitro Treg were resistant to AICD or cisplatin but conventional CD4+ T cells (Tconv) were not. CRT-induced Treg from AD or NC subjects upregulated prosurvival proteins whereas Tconv upregulated proapoptotic Bax. Conclusions: Highly suppressive, cisplatin-resistant Treg increase in frequency and persist after CRT and could be responsible for suppression of antitumor immune responses and recurrence in HNSCC. Clin Cancer Res; 19(23); 6585–96. ©2013 AACR.