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Microenvironment and Immunology

CTLA-4+ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis

Hyun-Bae Jie, Patrick J. Schuler, Steve C. Lee, Raghvendra M. Srivastava, Athanassios Argiris, Soldano Ferrone, Theresa L. Whiteside and Robert L. Ferris
DOI: 10.1158/0008-5472.CAN-14-2788 Published June 2015

Abstract

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4+FOXP3+ intratumoral Treg expressing CTLA-4, CD39, and TGFβ. These Treg suppressed cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4+FOXP3+ Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFβ and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA-4+ Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Treg-mediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity. Cancer Res; 75(11); 2200–10. ©2015 AACR.

Introduction

EGF receptor (EGFR)-specific therapy is moderately effective in the treatment of head and neck squamous cell cancer (HNSCC; refs. 1, 2). EGFR-specific monoclonal antibodies (Ab) such as cetuximab enhance survival of patients with HNSCC, whereas EGFR-specific tyrosine kinase inhibitors do not (3, 4). However, the response rate for cetuximab therapy in patients with HNSCC is low (15%∼20%). It has been proposed that cetuximab resistance is related to the activity of downstream EGFR-independent pathways (5), decreased EGFR internalization (6), genetic alterations of EGFR (7), or genotype of Fcγ receptor IIIa (CD16) on NK cells (8). This latter mechanism is based on accumulating evidence that cetuximab mediates antibody-dependent cellular cytotoxicity (ADCC) of NK cells (9), which can be augmented by cytokines (10–12). Furthermore, cetuximab can facilitate cross-talk between NK cells and dendritic cells (DC), which induces EGFR-specific cytotoxic T lymphocytes (CTL; refs. 10, 12) and/or activation of the complement system (13). Despite the various potential mechanisms of cetuximab activity, little is known about the effects of anti-EGFR mAb therapy on the cellular immune system in the tumor microenvironment (TME; ref. 2). In addition, the cellular and molecular escape mechanisms negatively regulating ADCC or EGFR-specific CTL activity (12, 14) have yet to be delineated.

The frequency of regulatory T cells (Treg) is increased at tumor sites and among the peripheral blood lymphocytes (PBL) of patients with cancer (15–17), including those with HNSCC (18). The enrichment of Treg in tumor-infiltrating lymphocytes (TIL) may promote cancer cell growth and the metastatic potential by suppressing antitumor immunity. Treg could also suppress NK cell responses through expression of suppressive cytokines and other tumor growth–promoting effects (19). It is known that Treg can inhibit NKG2D expression, impairing NK cell cytotoxicity and DC maturation (20, 21). However, whether Treg inhibit cetuximab-induced, NK cell–mediated ADCC or antitumor T cells in treated patients with cancer is unknown, and the mechanism(s) and clinical implications of such Treg-mediated suppressive effects remain to be elucidated.

In this study, we investigated the effects of cetuximab therapy on the frequency of Treg in the peripheral circulation and in the TME of patients with HNSCC, using freshly isolated TIL from a prospective clinical trial of single-agent, cetuximab-treated patients. We found that Treg suppress NK cell–mediated ADCC in a TGFβ1-dependent manner. The frequency of Treg was inversely associated with NK cell activation at the tumor sites and with the clinical course of the disease in cetuximab-treated patients. Interestingly, NK cells selectively eliminated intratumoral FOXP3+ Treg in the presence of ipilimumab ex vivo and thus decreased the Treg suppression of NK cells mediating cetuximab-driven ADCC. These results indicate that depletion of Treg by targeting CTLA-4 promotes antitumor immunity in the TME and enhances the efficacy of cetuximab therapy.

Patients and Methods

Patients and specimens

All patients were seen in the Outpatient Clinic of the Department of Otolaryngology at the University of Pittsburgh Medical Center (Pittsburgh, PA), and all subjects signed an informed consent approved by the Institutional Review Board of the University of Pittsburgh (IRB #99-06). Peripheral venous blood samples were obtained from cetuximab-treated patients with previously untreated stage III/IV HNSCC, including 22 patients treated with cetuximab plus cisplatin/paclitaxel/radiotherapy followed by 6 months of maintenance single-agent cetuximab (UPCI-05-003, NCT 00226239; ref. 1) and 18 patients receiving single-agent cetuximab on a separate prospective phase II clinical trial (UPCI #08-013, NCT 01218048; refs. 12, 22), as described in Table 1. All analyses were conducted on protocol patients who were receiving single-agent cetuximab. Blood samples were obtained 1 to 7 days before cetuximab therapy and again after the conclusion of therapy (∼1 month). The comparison (cetuximab-naïve) HNSCC cohorts were gender and age-matched, previously cetuximab-untreated patients with HNSCC. No patients were excluded as a result of prior treatments or performance status. Blood from cetuximab-naïve patients with HNSCC was drawn within the same period after completing therapy without cetuximab.

View this table:
Table 1.

Demographics of the cetuximab-treated patients with HNSCC in this study

Collection and processing of PBMC and TIL

Blood samples from patients with cancer and healthy donors (40 mL) were drawn into heparinized tubes and centrifuged on Ficoll-Hypaque gradients (GE Healthcare Bioscience). PBL were recovered, washed in RPMI-1640 or AIM-V medium (Invitrogen), and immediately used for experiments. For TIL isolation, tumor tissues obtained from patients with HNSCC undergoing surgery were collected, placed in sterile medium, and delivered to the laboratory for processing. Tumor tissues were sliced into small pieces, which then were transferred to a cell strainer (70-μm nylon) and mechanically minced using a syringe plunger. No enzymes were used for TIL recovery. The cells that passed through the cell strainer were collected and subjected to Ficoll-Hypaque gradient centrifugation. Mononuclear cells were recovered and used for ADCC assays or were stored at −80°C for l FACS analysis. For pre- and post-cetuximab treatment, peripheral bold mononuclear cells (PBMC) and TIL were isolated from patients with HNSCC before and after therapy, according to the protocol for the UPCI #08-013 trial.

Cell isolation

CD4+CD25+ T cells and CD3−CD56+ NK cells were isolated from freshly obtained PBMC by immunomagnetic sorting (Miltenyi) or by using a human NK cell Enrichment Kit (STEM Cell Technology), respectively, following the manufacturers' instructions. Cell separation was performed using an AutoMACS (for Treg isolation) and EasySep magnet (for NK cell isolation) according to the manufacturer's protocol. Flow-bases cell sorting was used to isolate NK cells from TIL. Viability of separated cells was measured using trypan blue dye.

Monoclonal antibodies and flow cytometry

The mAb used to stain human cells included CD39-FITC/PC7, FOXP3-FITC, FOXP3 PerCP-Cy5.5 (clone PCH101, eBioscience); LAP-PE, PD-1-APC (eBioscience); CD14-ECD, CD4-PC5 (all Beckman Coulter); CD73-PE, CTLA-4-PE, TIM-3-Brillian violet 421, CD107a-PE, CD137-Percp cy5.5, CD25-PE-Cy7, Granzyme B-FITC, Perforin-APC, CD39-APC, CD16-PE-Cy7 (Biolegend); CD25-PE (Miltenyi), LAG-3-ATTO647N conjugate (Enzo Life Sciences); CD56-PE-Texas Red, CD3-Alexa Fluor 405 conjugate (Invitrogen); and CD4-AF700 (BD Biosciences) and their respective isotype controls. All mAb preparations were pretitrated using activated as well as nonactivated PBMC to determine the optimal staining dilution for each. Intracellular staining for Foxp3, granzyme B, and perforin was performed as follows: PBMC or TIL were stained with mAb for surface markers and subsequently fixed and permeabilized (eBioscience). After washing, cells were subjected to intracellular staining for Foxp3, granzyme B, and perforin. Flow cytometry was performed using an EPICS XL-MCL flow cytometer equipped with Expo32 software, a CyAn flow cytometer (Dako) or Fortesa (Becton Dickinson) machine; data were analyzed using Summit V4.3 software or the FlowJo software (TreeStar, Inc.). The acquisition and analysis gates were restricted to the lymphocyte gate based on characteristic properties of the cells in the forward and side scatter. At least 1 × 105 events were acquired for analysis and, where applicable, gates were restricted to CD3+CD4+ for Treg analysis. Ipilimumab was obtained from Bristol Myers Squibb.

Transwell culture

A head and neck squamous cell carcinoma (HNSCC) cell line (JHU029) and NK cells (1:1 ratio, 0.2 × 106 cells/well for each cell type) were cocultured in the presence of cetuximab (10 μg/mL) or human IgG1 in a the upper chamber of a Transwell plate (0.4-μm polyester membrane, 6.5 mm insert, 24-well plate; Costar). In the lower chamber, purified monocytes and CD4+ T cells [1:2 ratio, 0.2 × 106 cells/well for monocytes and 0.4 × 106 cells/well for CD4+ T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE)] were incubated in the presence or absence of TGFβ1 (2.5 ng/mL) and/or anti-CD3 antibody (5 μg/mL). These conditions were previously established by us as optimal for Treg generation (18). Four days later, CD4+ T cells in the lower chamber were harvested for analysis of expansion of CTLA-4+Foxp3+ Treg, and their proliferation was assessed by CFSE dilution using flow cytometry. Four days after incubation, the frequency of CTLA-4+Foxp3+ Treg was assessed by flow cytometry using the PerCP-Cy5.5 dye. To confirm DC maturation, tumor cells, NK cell, and cetuximab in the upper chamber were incubated with monocytes alone in the lower chamber for 48 hours. Monocytes were harvested for analysis of their status by flow cytometry.

ADCC assay

Cetuximab-mediated NK cell cytotoxicity (ADCC) was measured as previously described (22) with the following modifications. Briefly, K562 or PCI-13 target cells were incubated with 2 μmol/L CFSE (Invitrogen) for 10 minutes at 37°C in the dark. Staining with CFSE was quenched with RPMI complete medium, and after 2 washes, the CFSE-labeled target cells were incubated with PBMC or NK in the absence or presence of Treg at various NK cell to target ratios ranging from 1:1 to 10:1 for 3 or 18 hours at 37°C in a 5% CO2 atmosphere. The ratio of NK cells to Treg was 2:1 in this system. After incubation, 7-aminoactinomycin D (7-AAD, Invitrogen; 1 μg/mL) was used to measure cell death, and cells were acquired on a Beckman Coulter XL or Fortesa (Becton Dickinson) cytometer. Target cell death was determined by measuring percentage of 7-AAD+ cells by gating only on CFSE-positive target cells. Target cells in the absence of PBMC or NK cells were used as a negative control, and target cells were incubated for 10 minutes at 56°C as a positive control for cell death. To measure accurate cytotoxicity on a per cell basis, lytic units (LU) were defined as the number of NK cells required to kill 20% of 5 × 104 target cells and then the reciprocal number of this value per 105 NK cells was calculated. The percentage of cytotoxic activity was calculated using the following formula: % specific lysis = (7-AAD+ targets − spontaneous 7-AAD+ targets)/(Maximum lysis − spontaneous 7-AAD+ targets) × 100.

Statistical analysis

Averages were calculated as means. For nonparametric distribution of samples, P values were calculated by Kruskal–Wallis and 2-tailed exact Wilcoxon–Mann–Whitney tests or nonparametric paired test (Wilcoxon matched paired test). Disease-specific survival of patients with HNSCC was defined as the time elapsed from the first treatment with cetuximab until death from HNSCC. Patients were censored if they were alive at last follow-up or had died but were cancer-free at the time of death. For prognostic correlations, we used the Treg frequency of 6%, which was the mean frequency previously found in untreated patients with HNSCC (23), and analyzed the prognosis of those patients with Treg frequencies above or below this level. Treg subclasses were tested for association with disease-free survival with the log-rank test. P < 0.05 was considered to be significant.

Results

Cetuximab-induced increase in circulating Treg correlates with clinical outcome in cetuximab-treated HNSCC patients

To investigate the effects of treatment with cetuximab on Treg in the circulation of patients with HNSCC, the frequency of CD4+CD39+CD25hi Treg in PBL was analyzed before and after cetuximab therapy. Treg measurements were performed in previously untreated, stage III/IV HNSCC patients before receiving cetuximab-based chemoradiotherapy (CRT, UPCI #05-003), and compared with posttreatment Treg frequencies measured during single-agent cetuximab maintenance therapy. As shown in Fig. 1A, the frequency of CD4+CD39+CD25hiFoxp3+ Treg was found to be increased after cetuximab therapy (P = 0.01). For prognostic correlations, we used Treg frequency of 6%, which was the mean frequency previously found in untreated patients with HNSCC (23), and analyzed the prognosis of those patients with Treg frequencies above or below this level.

Figure 1.
Figure 1.

Cetuximab-based therapy increases CD4+CD25hi Treg in HNSCC patients' peripheral blood. A, in 22 patients with HNSCC, the frequency of CD4+CD39+CD25+ Treg in PBL was compared before and after treatment (n = 22). B, correlation of peripheral blood Treg frequencies in patients with HNSCC (n = 20) with clinical outcome. For prognostic correlations, we used the Treg frequency of 6%, which was the mean frequency previously found in untreated patients with HNSCC (23), and analyzed the prognosis of those patients with Treg frequencies above or below this level. The patients were divided into two groups (10 patients per group), one containing patients who had the Treg frequency < 6% and the other with the Treg frequency > 6%. The survival rates of these two groups were compared (median follow-up = 36 months).

Given that Treg suppress NK-mediated ADCC, we hypothesized that the Treg frequency might predict clinical response to cetuximab therapy. Indeed, in a cohort of patients with HNSCC (n = 22) treated with cetuximab, significantly longer survival was observed in patients with lower Treg frequencies. We have previously established a mean ± SD value of 5.3% ± 3.2% for Treg in untreated patients with HNSCC and have now used the value of 6% to separate the patients into two groups. Patients with a Treg frequency lower than 6% fared significantly better than those with the Treg frequency higher than 6% (P = 0.015, Fig. 1B). Those cetuximab-treated patients with the elevated Treg frequency after cetuximab therapy appeared to have worse outcome. This result strongly supports our finding that the frequency of CD4+CD39+CD25hi Treg is a critical factor modulating the efficacy of cetuximab treatment in patients with HNSCC. Although these patients received CRT in conjunction with cetuximab, Treg measurements were performed at baseline (untreated) and during single-agent cetuximab treatment, suggesting that the effect was cetuximab-specific, a finding that was validated in a separate patient cohort below.

Frequency of intratumoral Treg and expression levels of CTLA-4, CD39, and TGFβ are increased after cetuximab monotherapy in HNSCC patients

To further document the cetuximab-specific effects on Treg, a second cohort of patients (n = 18), who received only single-agent neoadjuvant cetuximab in a second prospective phase II clinical trial (UPCI #08-013), was examined. As shown in Fig. 2A, a highly significant and consistent enrichment of CD4+CD25hiFoxp3+ Treg was observed in TIL (P < 0.0001) compared with PBL. The treatment of patients with HNSCC with single-agent cetuximab for 4 weeks significantly increased the number of CD4+CD25hiFoxp3+ intratumoral Treg in these patients with HNSCC as compared with baseline (pre-cetuximab) Treg levels. This result suggests that cetuximab monotherapy promotes Treg expansion in the TME, potentially contributing to immunosuppression and impairing cetuximab-induced immunity (14).

Figure 2.
Figure 2.

Cetuximab monotherapy increases CD25+, CTLA-4+, CD39+, and LAP+ (membrane bound TGFβ+) cells mainly on intratumoral Foxp3+ Treg isolated from 18 patients with HNSCC. Flow cytometric analysis of circulating and intratumoral CD4+CD25hiFoxp3+ Treg isolated from a representative HNSCC patient treated with single-agent cetuximab therapy on a prospective phase II clinical trial (UPCI #08-013). PBL and TIL were respectively isolated from blood and tumors of patients with HNSCC pre- and post-single-agent cetuximab therapy. Percentages of CD25+ (A), CTLA-4+ (C), CD39+ (E), and LAP+ (G) cells in CD4+Foxp3+ Treg were compared pre- and post-single-agent cetuximab therapy for a series of patients with HNSCC. Representative flow cytometric analysis of CD25 (B), CTLA-4 (D), CD39 (F), and LAP (H) on Foxp3+ Treg of CD4+ PBL and TIL isolated from a HNSCC patient (pre- and post-cetuximab treatment). The numbers represent the percentages of CD25+, CTLA-4+, CD39+, LAP+, and Foxp3+ cells in CD4+ T cells.

To characterize the immunosuppressive phenotype of Treg induced during cetuximab therapy, we analyzed expression of immunosuppressive molecules, including the immune checkpoint receptor, CTLA-4, as well as LAP (membrane-bound TGFβ) and CD39 (an ectonucleotidase), present on the Treg surface. Effects of cetuximab monotherapy on expression of CTLA-4, TGFβ, and CD39 were assessed on paired PBL and TIL isolated prospectively from 18 patients with HNSCC treated before and after 4 weeks of single-agent cetuximab therapy. As shown in Fig. 2, cetuximab monotherapy significantly increased the frequency of CD4+Foxp3+ intratumoral Treg expressing CTLA-4, CD39, and TGFβ (P = 0.002, P = 0.005, and P = 0.0001 respectively; Fig. 2C, E and H). Interestingly, a significant increase was only observed in circulating Treg expressing CTLA-4 (P = 0.02; Fig. 2C). We also observed that cetuximab monotherapy significantly increased the frequency of intratumoral CD4+Foxp3+ Treg expressing TIM-3 (P = 0.0015). However, the frequency of CD4+Foxp3+ intratumoral Treg expressing PD-1 was not enhanced by cetuximab monotherapy. Although TIM-3+, PD-1+, and CD39+ cells were also increased in Foxp3−CD4+ TIL by cetuximab monotherapy, CTLA-4+/CD39+ cells were significantly increased among the large fraction of CD4+Foxp3+ Treg from patients after cetuximab treatment, indicating that CTLA-4 and CD39 define a distinct subset of CD4+CD25hiFoxp3+ Treg enriched in the TME of cetuximab-treated patients.

Cetuximab-induced DC maturation contributes to expansion of CTLA-4+ Treg in combination with T-cell receptor triggering

Several studies showed that in addition to cytokines such as TGFβ, signaling mediated by T-cell receptor (TCR) triggering and costimulation may be critical events that expand Treg in the TME (24–26). On the basis of these observations, and the previously reported capacity of cetuximab to promote DC maturation and CD8(+) T-cell priming through “NK-DC cross-talk” (12), we examined whether cetuximab therapy affects the TCR triggering and costimulation by analyzing the expression levels of HLA-DR, CD80, and CD86 on both intratumoral and peripheral blood HLA-DR+CD11c+ cells from these patients with HNSCC. Indeed, DC activation/maturation markers were significantly upregulated on intratumoral HLA-DR+CD11c+ cells after cetuximab (not shown). Given that cetuximab-activated NK cells promote DC maturation (12) and that TGFB is highly expressed in the TME, cetuximab therapy may promote the expansion of intratumoral Treg.

To recapitulate the intratumoral Treg expansion observed in cetuximab-treated patients, we investigated whether the presence of cetuximab increased the frequency of CTLA-4+ Treg using a Transwell plate–based ex vivo system. As shown in Fig. 3A and B, the frequency of CTLA-4+ Treg was increased in the setting of TCR stimulation using agonistic, plate-bound anti-CD3 mAb, compared with isotype control control mAb (P < 0.05, P < 0.005, and P < 0.001, respectively) in the presence or absence of TGFβ. This result was only observed in the presence of cetuximab but not human IgG1 mAb. Under anti-CD3 stimulatory condition, cetuximab treatment significantly increases the frequency of CTLA-4+ Treg in the presence of TGFβ, compared with the absence of it (P < 0.001). Taken together, these results indicate that the treatment with cetuximab can significantly increase the frequency of CTLA-4+ Treg, which is further expanded in the presence of TCR triggering.

Figure 3.
Figure 3.

Treatment with cetuximab combined with TCR triggering induces CTLA-4+ Treg expansion. JHU029 cell line and NK cells (1:1 ratio) were cocultured in the presence of cetuximab or human IgG1 at the upper chamber of transwell plate, whereas at the lower chamber, purified monocytes and CFSE-labeled CD4+ T cells (1:2 ratio) were cultured with TGFβ1 in the presence or absence of anti-CD3 antibody. Similar results were seen of lower magnitude when TGFβ1 was omitted from the cultures. Four days after incubation, the frequency (A and B) and proliferation (C and D) of CTLA-4+Foxp3+ Treg were assessed by flow cytometry using the PerCP-Cy5.5 dye. Representative flow cytometric analysis of CTLA-4+Foxp3+ Treg (A) and their proliferation by CFSE dilution (C) are shown for each condition and their frequency was statistically compared, respectively (B and D).

We also investigated whether incubation with cetuximab induce proliferation of Foxp3+ Treg in the presence of TGFβ1 and/or anti-CD3 antibody by using a CFSE dilution–based assay and flow cytometry to measure the frequency of CTLA-4+FOXP3+ Treg. The presence of cetuximab significantly enhanced proliferation of Foxp3+ Treg under anti-CD3 Ab stimulatory conditions, compared with human IgG1 control mAb in the presence and absence of TGFβ (Fig. 3C and D, P < 0.005). Under stimulation with anti-CD3 Ab, cetuximab treatment significantly expanded Foxp3+ Treg by TGFβ, compared with absence of it (P < 0.001). This result is consistent with our previous report showing that cetuximab-activated NK cells induce DC maturation (12), which may provide antigen stimulation and costimulatory signals to preferentially expand Treg in the TME, where other suppressive signals exist (i.e., TGFβ1).

Cetuximab monotherapy differentially activates both circulating and intratumoral NK cells in HNSCC patients and their functional phenotypes are associated with the frequency of Treg

Previously, we showed that cetuximab-mediated ADCC activity of NK cells was dependent on CD16 (FcγRIIIa; ref. 8). However, this in vitro result has not yet been validated real-time in vivo in cetuximab-treated patients with HNSCC, particularly in TIL freshly isolated from single-agent cetuximab-treated patients. To examine whether cetuximab therapy activated NK cells in these patients, we examined granzyme B and perforin expression reflecting cytotoxic activity, and CD107a, CD137, and CD16 expression as measures of activation status, in both circulating and intratumoral NK cells. As shown in Fig. 4A, although the frequency of NK cells did not change in the PBL or TIL compartment during the therapy, the frequency of circulating NK cells was significantly higher than that of intratumoral NK cells. Similarly, the frequency of CD16+ NK cells also did not change during the cetuximab therapy on either circulating or intratumoral NK cells, albeit the frequency of circulating NK cells was notably higher than that of intratumoral NK cells (Fig. 4B, P < 0.0001). Interestingly, cetuximab therapy significantly upregulated granzyme B+ and perforin+ cells in circulating NK cells (P = 0.03 and P = 0.04, respectively; Fig. 4C and D) while notably upregulating CD107a+ and CD137+ cells on intratumoral NK cells (P = 0.002 and P = 0.005, respectively; Fig. 4E and F). The frequency of CD107a and CD137 positivity was substantially higher on intratumoral NK cells compared with those of circulating NK cells (P < 0.0001 and P = 0.0002, respectively). The frequency of granzyme B+/perforin+ NK cells was significantly higher in PBL compared with tumor-infiltrating NK cells (P < 0.0001), suggesting that a suppressive mechanism may inhibit full cetuximab-mediated NK cell activation in the TME.

Figure 4.
Figure 4.

Cetuximab monotherapy differentially activates circulating and intratumoral NK cells. PBL and TIL were isolated from patients with HNSCC before and after single-agent cetuximab therapy to analyze CD3−CD56+ NK cells for their cytolytic activity–related molecules (granzyme B, perforin, and CD16), degranulation marker (CD107a), and activation molecule (CD137). Percentages of CD3−CD56+ NK cells (A), CD16+(B), granzyme B+(C), perforin+(D), CD107a+(E), CD137+ (F) cells of circulating and intratumoral CD3−CD56+ NK cells were compared in pre- and post-single-agent cetuximab therapy for patients with HNSCC (n = 17).

To determine whether Treg inhibit cetuximab-induced, NK cell–mediated ADCC in vivo, potentially facilitating tumor progression, we correlated the frequency of Treg with functional and phenotypic markers of activated NK cells in the TME. Indeed, CD4+CD25hiFoxp3+ Treg were 7.4 times more enriched in TIL than in PBL. In the presence of these enriched Treg, intratumoral NK cells manifested significantly impaired expression of ADCC-related functional molecules, including granzyme B, perforin, and CD16 compared with those of circulating NK cells. Indeed, significantly lower expression of granzyme B, perforin, and CD16 on intratumoral NK cells was observed. In contrast, in PBL, which had a low frequency of Treg, NK cells maintained intact expression of these cytolytic molecules (not shown). Thus, a strong inverse correlation exists between the frequency of Treg and functional impairment of NK cells in the TME.

On the basis of the association of a high Treg frequency with the impaired NK cell functional phenotype, we tested whether HNSCC patient Treg could directly suppress cetuximab-mediated, NK cell–induced ADCC. To this end, we investigated the effect of CD4+CD25hiFoxp3+ Treg on cetuximab-mediated ADCC ex vivo, finding that, indeed, Treg significantly suppressed cetuximab-mediated cytotoxicity (P = 0.002). This effect was mainly mediated by Treg-derived TGFβ1, as the suppression was significantly abrogated using a neutralizing Ab specific for TGF-β1 (P = 0.01). IL10 appeared to play a minor role, as little inhibition of cetuximab-mediated ADCC mediated by Treg was observed after blockade of IL10 alone (27). Taken together, this result indicates that Treg inhibit cetuximab-induced TIL NK cell–mediated cytotoxicity through TGFβ1 secretion.

Treg are associated with clinical response to monotherapy with cetuximab

To further assess the clinical impact of Treg in predicting response in an independent clinical trial of patients after cetuximab therapy, we compared the extent of Treg induction in objective clinical responders (using posttreatment CT scan) with clinical nonresponders. During cetuximab therapy, we found that clinical responders maintain stable levels of circulating and intratumoral CTLA-4+ Treg (Fig. 5). On the contrary, the frequency of CTLA-4+ Treg was significantly increased among the nonresponder patients' PBL (P = 0.02) and TIL (P = 0.006), indicating that the induction of Treg negatively impacts on the efficacy of cetuximab therapy and that selected targets, such as CTLA-4, which deplete Treg would potentially provide additional therapeutic benefit.

Figure 5.
Figure 5.

Cetuximab monotherapy increases the frequency of CTLA-4+ Treg only in nonresponder group. The frequency of CTLA-4+ Treg in CD4+ PBL (A) and TIL (B) was measured using flow cytometry and compared in pre- and post-single-agent cetuximab monotherapy for 5 responders and 13 nonresponders respectively.

Treatment with ipilimumab enhances cetuximab-mediated ADCC by eliminating intratumoral Treg

We postulated that selective elimination of CTLA-4+ Treg, particularly at the tumor site, might improve the antitumor immunity followed by cetuximab therapy. Recent preclinical studies also showed that anti-CTLA-4 mAb induced rapid elimination of intratumoral Treg in murine tumor models, which improve antitumor immunity (28, 29). However, similar results have not been reported using ipilimumab (anti-CTLA-4 mAb) in humans. Because ipilimumab is a human IgG1 isotype mAb, which like cetuximab has been shown to induce NK cell–mediated ADCC (8, 30), we investigated whether ipilimumab could eliminate intratumoral CTLA-4+ Treg in the presence of NK cells. To test this possibility, we freshly isolated TIL containing Treg from a series of HNSCC tumors (n = 6) and incubated them with ipilimumab in the absence or presence of NK cells. After 72-hour incubation, the frequency of Foxp3+ intratumoral Treg in CD4+ TIL was evaluated using a flow cytometry–based cytotoxicity assay. As shown in Fig. 6A and B, the addition of ipilimumab together with exogenous NK cells significantly reduced (∼40%) the proportion of Foxp3+ Treg in CD4+ TIL. Exogenous NK cells slightly reduced the frequency of TIL Treg even in the absence of ipilimumab compared with TIL control conditions without NK cells, indicating that circulating NK cells can also deplete TIL Treg in an ipilimumab-independent manner (P = 0.04; Fig. 6B). However, NK cells plus ipilimumab did not deplete effector CD4+TIL (CD25+Foxp3− cells) in the presence or absence of ipilimumab, confirming that NK cells selectively deplete Foxp3+ TIL Treg whereas they preserve effector T cells in TIL (Fig. 6C). We also tested whether myeloid cells can eliminate intratumoral Treg in the presence of ipilimumab. In contrast to NK cells, however, myeloid cells did not deplete either intratumoral Treg or effector cells (not shown), indicating that the presence of myeloid cells may be dispensable for Treg elimination mediated by ipilimumab.

Figure 6.
Figure 6.

Treatment with ipilimumab enhances cetuximab-mediated ADCC by eliminating Treg. Ipilimumab was added into TIL cultures in the absence or presence of NK cells (ratio of NK to TIL was 2:1). Three days after incubation, gates were set to include CD4+ TIL to analyze the frequency of Foxp3+ Treg. A, representative FACS dot plot for the frequency of Foxp3+ Treg in CD4+ TIL. The frequency of Foxp3+ TIL Treg (B) or CD25+Foxp3− TIL effector (C) in each condition was compared by analyzing CD4+ TIL of 9 patients with HNSCC. Ipi, ipilimumab; Iso, isotype. D, representative flow cytometric analysis assessing the effect of ipilimumab on cetuximab-mediated ADCC in the presence of intratumoral Treg. Target HNSCC cells (JHU029) labeled with CFSE were incubated with peripheral NK cells in the absence or presence of intratumoral Treg (NK, JHU029 target:Treg ratio is 10:1:5). Cetuximab and/or ipilimumab were added into the coculture as indicated. After 18 hours, gates were set to include only CFSE-labeled target cells to analyze cell death assessed by 7-AAD staining. E, the effect of ipilimumab on cetuximab-/NK cell–mediated ADCC was tested in the presence of intratumoral Treg isolated from patients with HNSCC (n = 6). Specific lysis was calculated as described in Materials and Methods.

Next, we postulated that Treg depletion using ipilimumab-mediated NK cell cytotoxicity could overcome the suppressive activity of Treg against NK cell–mediated HNSCC cell lysis induced by cetuximab. To test this hypothesis, we added ipilimumab or irrelevant isotype control mAb to the cetuximab-mediated ADCC assay system, with cocultured NK and Treg (as described in Fig. 5). As shown in Fig. 6D and E, the cytolytic activity of NK cells previously suppressed by intratumoral Treg was recovered after incubation with ipilimumab (P = 0.03). This result indicates that ipilimumab could reverse the suppressive activity and eliminate intratumoral CTLA-4+ Treg in the presence of NK cells, thus potentially enhancing cetuximab-mediated ADCC activity.

Discussion

In this report, we describe the unexpected finding that cetuximab-based therapy is associated with an increase in CTLA-4+Foxp3+ Treg in the circulation and in the microenvironment in treated patients with HNSCC from two independent clinical trial cohorts. Indeed, higher levels of Treg correlated with worse prognosis in cetuximab-treated patients with HNSCC, as recently seen in lung cancer after neoadjuvant chemotherapy (31). For the first time, we also show that intratumoral NK cell activation and cytotoxicity occurs during cetuximab therapy, which is primarily inhibited by Treg-derived TGFβ1, providing a mechanism for their suppressive effect and impact on clinical course of the disease. Furthermore, we demonstrated that NK cells can selectively eliminate intratumoral Treg in the presence of ipilimumab, which induces recovery of cetuximab ADCC activity from Treg suppression. Importantly, the suppressive effect of these Treg could be substantially abrogated by ipilimumab-mediated NK cell elimination of Treg, a strategy that has the potential to enhance cetuximab-treated patient outcomes.

Although Treg are known to be enriched in the TME compared with peripheral blood, the mechanism underlying Treg enrichment at the tumor site has not been well-elucidated. A recent study showed that a few dominant Treg clones recruited into tumor sites were dramatically expanded through TCR responses in the microenvironment (32). However, little information is available regarding their biologic and clinical impact on mAb-induced immunity and tolerance in TME after mAb therapy. We previously proposed that cetuximab induces not only ADCC of NK cells (8, 30) but also cross-talk between NK cells and DC, leading to generation of tumor antigen (TA)-specific TCR (14). Recently, we reported that cetuximab stimulated IFNγ production from NK cells in vitro and subsequently induced DC maturation and enhanced the generation of TA-specific CD8+ T cells (12). Because TGFβ1, a critical factor for Treg generation, is abundant in the TME, this unique environment in conjunction with TCR triggering for Treg expansion could be promoted by cetuximab therapy in cancer patients' tumor sites.

Treg are reported to express immune checkpoint receptors and suppressive molecules, which are involved in generation and function of Treg. In this study, we demonstrated that single-agent cetuximab therapy significantly increased not only the frequency of intratumoral Foxp3+CD25hi Treg but also their expression of CTLA-4, TIM-3, CD39, and TGFβ/LAP. Of particular interest is the expression of CTLA-4 and CD39 that are detected on the majority of intratumoral Treg, which is further upregulated by cetuximab treatment. Many studies define CTLA-4 as an effector receptor in mediating Treg-suppressive functions (33). For example, Foxp3+ Treg-specific loss of CTLA-4 is sufficient to induce fatal lymphoproliferative disease, which is reminiscent of findings observed in CTLA-4–deficient mice (34). It has been reported that Treg can induce indoleamine 2,3–dioxygenase (IDO) activity in antigen-presenting cells through CTLA-4 and CD80/CD86 interaction. IDO catabolizes tryptophan, which locally depletes tryptophan and subsequently inhibits proliferation of T lymphocytes. In addition, IDO is involved in inhibiting NK cell function by inhibiting activating receptors and cytolytic activity (35). Similarly, it has been suggested that antibody triggering of CTLA-4 can induce the release of TGFβ1 from Treg (36). However, the link between CTLA-4 and IDO/TGFβ1 currently remains controversial, albeit their critical roles in Treg-mediated immunosuppressive activities (33). On the basis of the observation showing that CD39-specific blocking mAb attenuated immunosuppressive function of Treg and subsequently restored CD4 and CD8 T cells (37), it was proposed that blockade of CD39 could also restore NK cell–mediated antitumor immunity (38). Taken together, these results suggest that blockade of CTLA-4 or CD39 enzymatic activity is an attractive target to inhibit immunosuppressive functions of Treg in the TME, thus enhancing the efficacy of cetuximab immunotherapy against HNSCC. Treg from patients with HNSCC potently suppress cetuximab-mediated ADCC in vitro and correlate directly with phenotypic and functional impairment in tumor-infiltrating NK cells from cetuximab-treated patients, pointing to infiltrating Treg as the primary suppressive culprit responsible for impairment of cetuximab-mediated NK cell activation and tumor cell lysis (39). This finding was consistently reported from patients with non–small cell lung cancer, showing that Treg potently inhibit NK-mediated and cetuximab-induced ADCC in vitro (31). TGFβ1 is a critical factor not only to induce Treg but also to suppress NK cell functions, such as ADCC and cytokine production by downregulating FcγRIIIa, CD16. It has been well-established that cetuximab induces ADCC against different cancer cell lines, including HNSCC (9, 40, 41). In addition, several lines of evidence have consistently shown that intratumoral NK cells possess more severe dysfunctional phenotypes than circulating NK cells; this difference may be mediated via several mechanisms (42–44). Another study showed that TGFβ1 impaired CD16-mediated human NK cell ADCC by inhibiting granzyme A and B expression and IFNγ production via SMAD3 (45). Furthermore, it has been reported that TGFβ1 downregulated CD16 expression on peripheral blood NK cells (46), which parallels the impairment of cetuximab-mediated ADCC in the patients with esophageal squamous cell carcinoma (47). CD107a and CD137 on intratumoral NK cells were significantly upregulated compared with those on circulating NK cells, indicating that NK cells were actively stimulated, presumably due to the presence of the targets at the tumor sites. The low level of granzyme B and perforin in intratumoral NK cells may also reflect on theses NK cell phenotypes following their activation at the tumor site. In particular, CD107a and CD137 on intratumoral NK cells were further upregulated during cetuximab therapy, strongly suggesting that cetuximab-mediated ADCC take place at the tumor site. These results collectively support our observation showing that Treg suppress cetuximab-mediated ADCC of NK cells through TGFβ1 and might serve as a potential novel biomarker to predict the efficacy of cetuximab therapy in treating patients with head and neck cancer.

Recently, two preclinical studies using mouse models showed that anti-CTLA-4 mAb eliminated intratumoral Treg in a FcγR-dependent manner (28, 29). Despite the existence of NK cells, macrophages, and DC in TIL, addition of ipilimumab alone into the TIL culture did not reduce the frequency of intratumoral Treg. This result indicates that these intratumoral NK cells are not sufficiently numerous or functionally capable of eliminating TIL Treg in the presence of ipilimumab, which may be due to their impaired cytolytic molecules such as granzyme B, perforin, and/or CD16 expression. On the basis of our observation that peripheral NK cells are relatively more cytolytic than intratumoral NK cells and able to eliminate TIL Treg in the presence of ipilimumab, their directed migration into tumor sites may be essential to eliminate intratumoral Treg, thus enhancing the efficacy of cetuximab in the treatment of patients with HNSCC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: H.-B. Jie, T.L. Whiteside, R.L. Ferris

Development of methodology: H.-B. Jie, P.J. Schuler, S.C. Lee, R.M. Srivastava, T.L. Whiteside, R.L. Ferris

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.-B. Jie, P.J. Schuler, S.C. Lee, R.M. Srivastava, A. Argiris, R.L. Ferris

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H.-B. Jie, P.J. Schuler, S.C. Lee, R.M. Srivastava, T.L. Whiteside, R.L. Ferris

Writing, review, and/or revision of the manuscript: H.-B. Jie, P.J. Schuler, A. Argiris, S. Ferrone, T.L. Whiteside, R.L. Ferris

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H.-B. Jie, R.L. Ferris

Study supervision: R.L. Ferris

Grant Support

This work was supported by NIH grants R01 DE19727, P50 CA097190, CA110249, and University of Pittsburgh Cancer Institute grant P30CA047904.

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.

Acknowledgments

The authors acknowledge excellent technical assistance from Clayton Mathis, Michael Meyer, and Bratislav Janjic (University of Pittsburgh Cancer Institute). They also thank Ferris laboratory members for helpful suggestions.

  • Received September 24, 2014.
  • Revision received January 26, 2015.
  • Accepted February 19, 2015.

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Cancer Research: 75 (11)
June 2015
Volume 75, Issue 11

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CTLA-4+ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis
Hyun-Bae Jie, Patrick J. Schuler, Steve C. Lee, Raghvendra M. Srivastava, Athanassios Argiris, Soldano Ferrone, Theresa L. Whiteside and Robert L. Ferris
Cancer Res June 1 2015 (75) (11) 2200-2210; DOI: 10.1158/0008-5472.CAN-14-2788
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