IL-21 Is an Accomplice of PD-L1 in the Induction of PD-1-Dependent Treg Generation in Head and Neck Cancer

Regulatory T cells (Tregs) are immunosuppressive cells involved in antitumor immunity. However, the regulation of Treg generation by inflammation in the tumor microenvironment has not been carefully investigated. Here, we demonstrated that IL-21-polarized inflammation was enriched in the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC) and that IL-21 could promote PD-L1-induced Treg generation in a PD-1-dependent manner. Moreover, generated Tregs showed a greater ability to suppress the proliferation of tumor-associated antigen (TAA)-specific T cells than naturally occurring Tregs. Importantly, an anti-PD-1 antibody could inhibit only Treg expansion induced by clinical tumor explants with high expression of IL-21/PD-L1. In addition, neutralizing IL-21 could enhance the anti-PD-1 antibody-mediated inhibitory effect on Treg expansion. Furthermore, simultaneous high expression of IL-21 and PD-L1 was associated with more Treg infiltrates and predicted reduced overall and disease-free survival in patients with HNSCC. These findings indicate that IL-21 in the tumor microenvironment may promote PD-L1-induced, Treg-mediated immune escape in a PD-1-dependent manner and that an IL-21 neutralization strategy may enhance PD-1 blockade-based antitumor immunotherapy by targeting Treg-mediated immune evasion in patients with high expression of IL-21 and PD-L1.


INTRODUCTION
The interaction between cancer cells and their surrounding microenvironment could modulate the immunoediting process, leading to tumor immune privilege (1)(2)(3). In this regard, regulatory T cells (Tregs), which infiltrate a variety of solid tumor microenvironments, are considered a key element in the promotion of immune evasion due to their ability to suppress tumor-specific immune responses and hamper cancer immunotherapy (4)(5)(6)(7). In head and neck squamous cell carcinoma (HNSCC), we reported that Treg depletion can repress tumor growth and evoke antitumor immunity and that the accumulation of Tregs predicts poor survival in HNSCC patients (8)(9)(10). However, factors that impact Treg generation in the tumor microenvironment are still poorly studied.
We recently found that tumor-associated inflammation (TAI) was positively related to HNSCC tumor-infiltrating Treg generation, which in turn promoted immunosuppression (10,11). Thus, it would be interesting to further elucidate the potential mechanisms of TAI that are responsible for Treg generation in the tumor microenvironment.
The cytokine Interleukin (IL)-21 is an immune modulator with pleiotropic effects on multiple immune cell type. Driving inflammation by enhancing the generation and functions of cytotoxic T or NK cells is its classical function, importantly, IL-21 also exhibits immunosuppressive properties (12)(13)(14)(15). It has been described to mediate the expression of IL-10 by cytotoxic cells and B cells and polarization of tumor-associated macrophage. Furthermore, the effects of IL-21 on immune cells have been reported depending on additional signals (16). The inhibitory checkpoint programmed death-ligand 1(PD-L1) is widely expressed in human malignancies. It interacts through its receptor modulating local immune contexture and serving an inhibitory signal to attenuate anti-tumor immune response. However, little information is currently available regarding interactions between IL-21, PD-L1 and Tregs in local tissue.
In the present study, we identified increased IL-21-polarized inflammation in HNSCC. Moreover, IL-21 could upregulate PD-1 expression on CD4 + T cells and boost PD-L1-induced Treg generation through upregulating PD-1 expression. Neutralizing IL-21 could enhance the blockade effect of an anti-PD-1 antibody on Treg generation induced by IL-21 high /PD-L1 high clinical tumor explants. High expression of IL-21/PD-L1 significantly predicted reduced survival in 102 patients with HNSCC. Our findings provide evidence that IL-21-associated inflammation might be modulated within the tumor microenvironment and negatively influence the antitumor immune response.

Patients and Healthy Donors
One hundred thirty-seven patients diagnosed with laryngeal squamous cell carcinoma at the First Affiliated Hospital of Sun Yat-sen University were recruited for the present study. In detail, immunohistochemistry (IHC) was performed to evaluate the clinical relevance of tumor microenvironment factors in 102 patients, and 35 patients were selected for other in vitro studies. None of these 137 selected patients received palliative surgery or neoadjuvant chemo-and/or radiotherapy before sampling. Clinical staging was classified according to the criteria of the seventh edition of the Union for International Cancer Control (UICC). The freshly resected tumor and adjacent non-tumor tissues were kept in cold PBS for downstream analysis.
The adjacent non-tumor tissues were confirmed cancer cells infiltration free by pathological examination. Peripheral blood mononuclear cells (PBMCs) were obtained from 14 healthy donors. All samples were collected after receiving informed consent from the patients, and the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University approved this study (Approval No. 2012-349).

IHC and Staining Evaluation
Paraffin-embedded, formalin-fixed, 5-mm-thick tissue sections ( Table 1) were incubated with antibodies against human IL-21 (10 µg/ml, NBP1-02706, Novus), FOXP3 (5 µg/ml, ab20034, Abcam), PD-L1 (1:200, 13684, Cell Signaling Technology), and anti-p16/INK4a (1:250, 10883-1-AP, Proteintech) and then stained using the Dako Envision System (DakoCytomation) according to the manufacturer's instructions. The procedure for immunohistochemical staining evaluation was described in our previous studies (11). Briefly, for the categorization of samples by IL-21 or PD-L1 expression, specimens with a number of positive cells greater than the median were defined as 'high', and those with a number lower than the median were defined as 'low'. The median level of IL-21 + cells was 4.5 cells per field. The expression of PD-L1 was scored semiquantitatively based on the staining intensity and distribution using the immunoreactive score (IRS). The IRS was calculated as staining intensity (SI) × percentage of positive cells (PP) (IRS= SI × PP). The SI was defined as negative (score 0), weak (score 1), moderate (score 2), and strong (score 3). The PP was defined as 0-5% (score 0), 6-25% (score 1), 26-50% (score 2), 51-75% (score 3), and 76-100% (score 4). The cutoff point between low and high PD-L1 expression was 3. P16 was considered positive if there was strong and diffuse nuclear and cytoplasmic staining present in greater than 70% of tumor cells and believed to correlated with Human papilloma virus (HPV). Cells stained with the indicated antibodies were imaged using Zeiss imaging systems (Axio Scan Z1, Carl Zeiss) at 100× and 400× magnification, and at least 5 fields of view per section at 400× magnification were evaluated.

Tissue-Infiltrating Lymphocyte Isolation
Lymphocytes were isolated from tissue as previously described (17). Briefly, fresh surgical specimens (n=17) were minced into small pieces and subsequently digested with Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS), 1 mg/ml collagenase I, 0.5 mg/ml collagenase II, 1 mg/ ml hyaluronidase, and 100 U/ml DNase (all from Sigma-Aldrich) at 37°C for 20 mins. After being filtered through 70 µm cell strainers, the resulting cell suspensions were processed with density gradient centrifugation using a lymphocyte separation medium (MP Biomedical).

Flow Cytometry
Single-cell suspensions were stained with antibodies against CD45, CD3, CD4, PD-1, CD25, interferon (IFN)-g, IL-17, IL-9, IL-4, FOXP3 (eBioscience), and IL-21 (BD Biosciences) according to the manufacturers' instructions. The dose of each of the above antibodies was 5 µl/test. To detect the cytokine profile, cells were stimulated with Leukocyte Activation Cocktail (2 µl/ml, BD Biosciences) for 5 h before flow cytometric analysis. For intracellular staining, cells were stained with the surface markers listed above and fixed in Fix/Perm Buffer (eBioscience), followed by permeabilization in the presence of antibodies specific for intracellular markers. All the reagents were purchased from BioLegend unless otherwise indicated. The stained cells were acquired on a Cytoflex flow cytometer (Beckman Coulter), and the analysis was performed using FlowJo software (TreeStar).

Preparation of Tumor-Associated Antigen (TAA)-Specific T Cells
Autologous TAA-specific T cells were prepared using antigenloaded DCs as we previously described (17). Briefly, soluble SNU899 cell line lysate antigens prepared by four freeze-thaw cycles (-140°C/42°C/60°C) were added to DC cultures at day 6 at a ratio of 3:1 (SNU899 cells: DCs). Lipopolysaccharide (LPS, 1 µg/ml, Sigma-Aldrich) was added to induce DC maturation. The maturation of the DCs was determined by measuring the expression of HLA-DR, CD80, CD86, and CD83 by flow cytometry. Antigen-loaded DCs were added to autologous T cells as stimulators at a ratio of 1:20.

Treg Generation Asay
CD4 + T cells were activated using a tetrameric complex of anti-CD3/CD28 beads (ImmunoCult human T cell activator, STEMCELL) with or without recombinant human IL-21 (25 ng/ml, R&D Systems). Recombinant human PD-L1 (rhPD-L1, R&D Systems) was incubated at a concentration of 100 ng/ml in 96-well plates overnight in the presence of a goat anti-human IgG Fc antibody for dimerization and plate immobilization. Tregs were generated by stimulating CD4 + T cells with anti-CD3/CD28 beads for 6 days with or without the presence of immobilized PD-L1 or IL-21. To study Treg generation, which may be influenced by the tumor microenvironment, tumor slices from clinical specimens (n= 10) were laid on the bottom of the wells of a 96-well plate and cocultured with CD4 + T cells isolated from PBMCs in the presence of anti-CD3/CD28 beads. For neutralization studies, recombinant human IL-21 R Fc Chimera (10 µg/ml, R&D Systems) and an antihuman PD-1 antibody (10 µg/ml, R&D Systems) were added at the start of the coculture. Cells were harvested at the indicated time and then analyzed with a Cytoflex flow cytometer.

Treg-Mediated Suppression of TAA-Specific T Cells
Using the Regulatory T Cell Isolation Kit (Miltenyi Biotec), generated CD4 + CD25 + CD127 low Tregs were isolated from CD4 + T cells stimulated with anti-CD3/CD28 beads and seeded together with autologous TAA-specific T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, 1 mM, eBioscience) at a ratio of 1:2 with a total of 30,000 cells per well.

Statistical Analysis
All statistical analyses were performed using IBM SPSS software version 22.0 (IBM Corporation). The Kaplan-Meier method was used to evaluate survival distributions, and differences between groups were assessed by the log-rank test. The correlation between variables was evaluated using Spearman's correlation test. Two-sided Student's t tests and ANOVA were used to analyze data from IHC and flow cytometry experiments, and the Bonferroni adjustment for multiple comparisons was used for between-group comparisons. Data are presented as the mean ± SD. P-values <0.05 were considered statistically significant.

IL-21 + Cells Predict Poor Survival in Patients With HNSCC
We subsequently evaluated the clinical relevance of the increased frequency of tumor-infiltrating IL-21-producing cells in 102 HNSCC patients using IHC. Our results showed that IL-21 + cells accumulated in the tumor stroma but not in the tumor nest ( Figure 2A). The levels of IL-21 + cell infiltration in patients with stage IV disease were higher than those with stage I, II or III disease (8.9 ± 3.6 vs. 4.0 ± 2.7, P < 0.001, vs. 4.1 ± 2.9, P < 0.001, vs. 5.6 ± 3.1, P < 0.05, counts per field, respectively), Additionally, those with stage III disease was higher than stage I (5.6 ± 3.1 vs. 4.0 ± 2.7, P < 0.05, counts per field) (Figures 2A, B).
To determine whether IL-21 + cell infiltration in HNSCC correlates with disease prognosis, one hundred and two HNSCC patients were divided into two groups according to the median value of their IL-21 + cell density. The patients with a high level of infiltrating IL-21 + cells had worse overall and disease-free survival than the patients with a low level (Figures 2C, D and Table 2). Cox regression analysis revealed that the IL-21 + counts and TNM stage could be independent predictive factors for overall survival in the HNSCC patients ( Table 3). Taken together, our results identified that the accumulation of IL-21 + cells was associated with disease progression and poor prognosis in patients with HNSCC.

Abundance of IL-21 + Cells Is Positively Correlated With Treg Infiltration in HNSCC
Considering that Tregs are known to suppress antitumor immunity and that this suppression results in disease progression, we next determined whether the level of IL-21 + cells was related to Treg infiltration. We first examined the prevalence of tumor-infiltrating Tregs and IL-21 + cells using immunohistochemical staining for FOXP3 and IL-21. The results showed that FOXP3 + and IL-21 + cells collocated in the same area in the tumor stroma ( Figure 3A) and that the increased density of the IL-21 + cells was positively correlated with that of the FOXP3 + cells ( Figure 3B). Next, flow cytometric analysis showed that the frequency of tumor-infiltrating IL-21 + CD4 + T cells positively correlated with that of FOXP3 + CD25 + CD4 + T cells ( Figures 3C, D), which supported our immunohistochemical results. We further assessed IL-21R expression on Treg cells and conventional T cells (CD45 + CD3 + CD4 + CD8 -CD25 -FOXP3 -), showing that Tregs have remarkably more IL-21R expression (Supplementary Figure 2). These results implied that IL-21 + cells may be involved in Treg infiltration in the HNSCC tumor microenvironment.

PD-L1 and IL-21-Induced Tregs Show a Stronger Ability to Suppress the Proliferation of TAA-Specific T Cells Than Naturally Occurring Tregs
For the analysis of the functionality of the generated Tregs, Tregs were isolated from different groups and cocultured with ability (39.7 ± 7.0% vs. 29.0 ± 5.2%, respectively, P<0.05) ( Figures 4I, J). In addition, TAA-specific CD8 + T cells were assessed as well (Supplementary Figure 3). It revealed that PD-L1 and IL-21-induced Tregs show expected suppressive effects as on TAA-specific CD8 + T cells. Taken together, schematic figure ( Figure 4K) illustrates that IL-21 promoted PD-L1-induced Treg generation in a PD-1-dependent manner, and IL-21 and PD-L1induced Tregs could inhibited TAA-specific T cell proliferation at a greater degree than naturally occurring Tregs.
Neutralizing IL-21 Enhances the Blockade Effect of an Anti-PD-1 Antibody on the Treg Generation Induced by IL-21 high /PD-L1 high Tumor Explants but Not on that Induced by IL-21 low /PD-L1 low Tumor Explants To test whether the HNSCC tumor environment is capable of potentiating Treg generation through IL-21 and PD-L1, we cocultured CD4 + T cells with tumor explants from HNSCC patients with or without IL-21 and/or an anti-PD-1 neutralizing antibody. Accordingly, tumor explants from 10 patients with HNSCC were divided evenly into 2 groups according to the cutoff points for PD-L1 and IL-21 described in the methods section. There were 5 samples with high expression of both IL-21 and PD-L1 (IL-21 high /PD-L1 high ) and 5 samples with low expression of both IL-21 and PD-L1 (IL-21 low /PD-L1 low ). Our results showed that the frequency of Tregs in the group of IL-21 high /PD-L1 high tumor explants + CD4 + T cells was higher than that of the group of IL-21 low /PD-L1 low tumor explants + CD4 + T cells (38.8 ± 8.5% vs. 25.8 ± 2.4%, respectively, P < 0.05) (Figures 5A, B). The anti-PD-1 antibody showed an inhibitory effect on Treg generation when the CD4 + T cells were cultured with the IL-21 high /PD-L1 high tumor explants (31.3 ± 5.0% vs. 38.8 ± 8.5%, P < 0.05). In addition, the combination of IL-21 and the anti-PD-1 antibody had a stronger inhibitory effect on Treg generation than the anti-PD-1 antibody alone ( Figures 5A, B). Treg generation induced by IL-21 high /PD-L1 high tumor explant was further validated by cocultured with TAA-specific T cells (both CD4 and CD8) (Supplementary Figures 4, 5). Using intracellular staining, the cytokine-producing ability of responder CD4 + T cells and CD8 + T cells was assessed. Our results showed that the stimulated responder CD4 + and CD8 + T cells exhibited strong proliferation in both, high IFN-g, IL-2 levels in responder CD4 + T cells, high GranzymeB and Perforin levels in responder CD8 + T cells. Adding the induced Treg by tumor explants with or without anti-PD-1 or/and anti-IL-21 treatment strongly inhibited the proliferation of responder T cells (both CD4 and CD8) (Supplementary Figures 4A, 5A), reduced the IFN-g, IL-2 levels of CD4 + T cells (Supplementary Figures 4B,  D), and cytotoxicity, GranzymeB, Perforin levels of CD8 + T cells (Supplementary Figures 5B, E). However, no significant differences were observed between tumor explant with no antibody, with anti-PD-1, with anti-IL-21 group and with the combination anti-PD-1 and anti-IL-21. (Supplementary Figures 4D, 5E).There data suggested the Tregs induced by tumor explant with the treatment of anti-PD-1 or/and anti-IL-21 are expectedly suppressive as the Tregs induced by tumor explant alone.
The above data suggested that IL-21 neutralization may enhance the effect of PD-1-targeted tumor immunotherapy in only HNSCC patients with high expression of both IL-21 and PD-L1.

Simultaneous High Expression of IL-21 and PD-L1 Is Associated With More Treg Infiltrates and Worse Survival
Given that IL-21 plays a positive role in PD-L1-induced Treg generation, we next investigated the relationships between the expression patterns of IL-21, PD-L1, and FOXP3 in tumor specimens. Our results showed that the tumors with high expression of IL-21 or PD-L1 had more FOXP3 + cells than those with low expression of both IL-21 and PD-L1 (6.8 ± 3.6 vs. 3.2 ± 2.7 counts per field, respectively, P < 0.05) (Figures 6A, B). Furthermore, the tumors with high expression of both IL-21 and PD-L1 had more FOXP3 + cells (9.6 ± 4.9 counts per field) than the tumors with low expression of both IL-21 and PD-L1 (3.2 ± 2.7 counts per field, P < 0.05) or the tumors with high expression of either IL-21 or PD-L1 (6.8 ± 3.6 counts per field, P < 0.01) ( Figures 6A, B). Moreover, in the tumor stage analysis, the PD-L1 expression score in patients with stage IV disease was considerably higher than that in those with stage I, II or III disease (5.8 ± 1.9 vs. 2.0 ± 1.6, P <0.001, vs. 3.1 ± 2.2, P < 0.01, vs. 3.2 ± 1.8, P <0.05, respectively), ( Figure 6C). Fifteen cases (47%) with simultaneous high expression of IL-21 and PD-L1 were observed among the stage III or IV samples, and 20 cases (29%) were observed among the stage I or II samples, while simultaneous low expression of IL-21 and PD-L1 was observed in 8 cases (25%) among the stage III or IV samples and in 36 cases (51%) among the stage I or II samples. Moreover, high expression of either PD-L1 or IL-21 was observed in 9 cases (28%) among the stage III or IV samples, and 14 cases (20%) were observed among the stage I or II samples ( Figure 6D). We finally evaluated whether the simultaneous high expression of IL-21 and PD-L1 correlated with the clinical prognosis of HNSCC patients. As expected, the patients with simultaneous high expression of IL-21 and PD-L1 had worse overall and diseasefree survival than those with simultaneous low expression of PD-L1 and IL-21 (OS: P < 0.01, DFS: P < 0.01), worse overall survival than high expression of either PD-L1 or IL-21 (P < 0.05, DFS not significant) (Figures 6E, F). Overall survival was still significantly different between IL-21 high /PD-L1 high and IL-21 low /PD-L1 low group at stages I + II (P < 0.05) and III + IV (P < 0.05), respectively ( Figures 6G, H). Disease-free survival was significantly different between IL-21 high /PD-L1 high and IL-21 low /PD-L1 low group at stages I + II (P < 0.05), but not stages III + IV (P > 0.05) (Figures 6I, J). Cox regression analysis revealed that the simultaneous high expression of PD-L1 and IL-21 was an independent prognostic marker for overall and disease-free survival in HNSCC patients ( Table 3).

DISCUSSION
Substantial evidence shows that immune cells are habitually recruited into the tumor microenvironment, where they create inflammatory responses and play a critical role in the establishment of the immunosuppressive milieu (23,24). The present study demonstrated that the tumor microenvironment inflammatory factor IL-21 may promote PD-L1-induced, Tregmediated tumor immune escape in a PD-1-dependent manner. These findings provide evidence that the tumor microenvironment inflammation represented by IL-21 plays an important role in inhibiting antitumor immune responses via Treg generation.
To date, few studies have shown that IL-21-associated inflammation exists in human solid tumors, and the immune significance of IL-21 in the tumor microenvironment remains controversial. Some studies have revealed that IL-21 may enhance the cytotoxic activity of CD8 + T cells and natural killer (NK) cells, suggesting that IL-21 functions as an antitumor agent (25,26), while other studies have reported the immunosuppressive significance of IL-21 in promoting tumorassociated macrophage polarization and the expression of IL-10 by cytotoxic cells and B cells (12,14,15). We speculated that these inconsistencies could be attributed to the different tumor microenvironment contexts, IL-21 has been proposed acting in a context-dependent manner (27) local presence of IL-21 within tumor microenvironment is likely to interact with a variety of cell types or environmental factors than its systemic occurrence, for PD-1 ligation was observed to skew TCR repertories (28). It is thus possible that IL-21 selectively support a local immunosuppressive environment, whereas systemic application of IL-21 activates cytotoxic T or NK cells with anti-tumor activity at tumor-distant sites. Our hypothesis is supported by the proposal that tumor immune evasion is selectively modulated in local tumor microenvironment (22). Therefore, it likely depends on the status and type of immune infiltrates and the presence of environmental cofactors such as PD-L1 whether anti-tumor immune response is suppressed or supported by IL-21 in tumor microenvironment. Recent studies reported the discrepant transcriptional and functional properties between tumor infiltrating Tregs, tissue resident Tregs and peripheral blood Tregs, indicating that tumor microenvironment may play an essential role in the regulation of the phenotype and immunosuppressive functionality of Tregs (29,30). We recently showed that HNSCC-associated inflammation can augment the tumor microenvironment Treg population, which implied the inflammatory significance of Treg generation (10). However, the specific relationship between TAI and Treg generation is currently unclear. The present study aimed to evaluate the mechanisms by which IL-21-mediated inflammation regulates the generation and function of Tregs in the tumor microenvironment.
To our knowledge, this is the first study to propose significant crosstalk between IL-21 and PD-L1 in the generation of the tumor microenvironment Tregs that are responsible for the enhanced suppression of TAA-specific T cell proliferation. In detail, we first examined the effect of IL-21 alone on Treg generation, and the results showed that IL-21 inhibited Treg generation, which was inconsistent with our immunohistochemical data that showed that Treg infiltrates positively correlated with the counts of IL-21-positive cells. The above results implied that other tumor microenvironment elements may be involved in the regulation of Treg generation. As PD-L1/PD-1 signaling has recently been shown to modulate the Treg homeostasis and facilitate tumor immune tolerance (18)(19)(20)(21), we hypothesized that PD-L1/PD-1 signaling may be involved in the process of Treg generation regulated by IL-21. Hence, we next added PD-L1 to IL-21-treated CD4 + T cells and found that IL-21 significantly promoted Treg generation in the presence of PD-L1. The potential mechanism is that IL-21 can induce the expression of PD-1 for PD-L1/PD-1 signaling, which is responsible for Treg generation. The mechanism of the upregulation of PD-1 expression induced by IL-21 might be a TCR-triggered calcineurin signaling cascade that leads to the activation of the transcription factors NFATc1 and STAT family proteins (31,32). Moreover, although a recent study reported that cancer cells produced TGF-b1 in tumor supernatants and when CD4 + T cells were cultured in tumor supernatants, FOXP3 + Tregs generation was promoted. While IL-21 was able to inhibit cancer cell-mediated FOXP3 induction in the context of tumor supernatants (33), which was contrary to our study. We believe that this inconsistency may be attributed to that study ignoring the involvement of the tumor microenvironment, and IL-21 may exert double sword effects on Tregs at the context of other immunosuppressive component derived from tumor microenvironment such as PD-L1.
To validate the synergistic effect of IL-21 and PD-L1 on Treg generation in the tumor microenvironment, we used tumor explants freshly isolated from cancer patients to induce Treg generation from CD4 + T cells and to test whether neutralizing IL-21 and PD-1 was capable of blocking Treg generation. Our results showed that compared with either treatment alone, IL-21 neutralization combined with an anti-PD-1 neutralizing antibody could effectively inhibit IL-21 high /PD-L1 high tumor explant-induced Treg generation, whereas this effect was not observed with IL-21 low /PD-L1 low tumor explant-induced Treg generation, indicating that other mechanisms might be involved in Treg generation in tumor microenvironments with low expression of IL-21 and PD-L1. The potential mechanisms that may be involved in Treg generation involve other mediators, such as TGF-b, IL-10, and IDO, and interactions with tolerogenic DCs or stromal cells in the tumor milieu (34)(35)(36)(37)(38). The above clinical data suggest that the IL-21 neutralization strategy may represent a promising immunotherapeutic approach that may enhance PD-1 blockade-based tumor immunotherapy by targeting Treg-mediated immune evasion in only patients with high expression of IL-21 and PD-L1.
To expand the understanding of the correlations between IL-21, PD-L1, and Tregs in the tumor microenvironment and their prediction of disease prognosis, we detected the expression of IL-21, PD-L1, and FOXP3 in 102 newly diagnosed HNSCC patients. Our results showed that enriched IL-21 + cells and PD-L1 expression were positively associated with FOXP3 + cell infiltration and that high expression of IL-21 and PD-L1 correlated with advanced tumor stage and poor overall and disease-free survival in patients with HNSCC.
In conclusion, our results suggest that increased IL-21associated inflammation in the tumor milieu may favor Tregmediated immune evasion through the PD-L1/PD-1 pathway and that the IL-21 neutralization strategy may enhance PD-1 blockade-based tumor immunotherapy by targeting Tregmediated immune evasion in patients with high expression of IL-21 and PD-L1. Understanding the mechanisms of the interactions between Tregs and tumor microenvironment inflammation may be beneficial for optimizing Treg-targeted antitumor strategies.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.