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Tumor microenvironment dictates regulatory T cell phenotype: Upregulated immune checkpoints reinforce suppressive function

Journal for ImmunoTherapy of Cancer volume 7, Article number: 339 (2019) Cite this article

Abstract

Background

Regulatory T (Treg) cells have an immunosuppressive function in cancer, but the underlying mechanism of immunosuppression in the tumor microenvironment (TME) is unclear.

Methods

We compared the phenotypes of T cell subsets, including Treg cells, obtained from peripheral blood, malignant effusion, and tumors of 103 cancer patients. Our primary focus was on the expression of immune checkpoint (IC)-molecules, such as programmed death (PD)-1, T-cell immunoglobulin and mucin-domain containing (TIM)-3, T cell Ig and ITIM domain (TIGIT), and cytotoxic T lymphocyte antigen (CTLA)-4, on Treg cells in paired lymphocytes from blood, peritumoral tissue, and tumors of 12 patients with lung cancer. To identify the immunosuppressive mechanisms acting on tumor-infiltrating Treg cells, we conducted immunosuppressive functional assays in a mouse model.

Results

CD8+, CD4+ T cells, and Treg cells exhibited a gradual upregulation of IC-molecules the closer they were to the tumor. Interestingly, PD-1 expression was more prominent in Treg cells than in conventional T (Tconv) cells. In lung cancer patients, higher levels of IC-molecules were expressed on Treg cells than on Tconv cells, and Treg cells were also more enriched in the tumor than in the peri-tumor and blood. In a mouse lung cancer model, IC-molecules were also preferentially upregulated on Treg cells, compared to Tconv cells. PD-1 showed the greatest increase on most cell types, especially Treg cells, and this increase occurred gradually over time after the cells entered the TME. PD-1 high-expressing tumor-infiltrating Treg cells displayed potent suppressive activity, which could be partially inhibited with a blocking anti-PD-1 antibody.

Conclusions

We demonstrate that the TME confers a suppressive function on Treg cells by upregulating IC-molecule expression. Targeting IC-molecules, including PD-1, on Treg cells may be effective for cancer treatment.

Background

The recent development of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment. ICIs specific for anti-cytotoxic T lymphocyte antigen (CTLA)-4 or anti-programmed death (PD)-1 have improved patient survival and have been approved for the treatment of several cancer types, including non-small cell lung cancer (NSCLC), melanoma, head and neck cancer, bladder cancer, and renal cell cancer [1,2,3].

The tumor microenvironment (TME) and the immune system play critical roles in cancer progression and clinical outcome [4, 5]. Regulatory T (Treg) cells are highly immunosuppressive and contribute to the maintenance of self-tolerance and immune homeostasis in humans [6, 7]. Treg cells infiltrate tumors and promote their progression by suppressing antitumor immunity in the TME. Depleting Treg cells can lead to spontaneous tumor regression due to enhanced antitumor response [7, 8]. Interaction of Treg cells with TME enhances their immunosuppressive function and proliferative capacity. Several studies have shown that tumor-infiltrating Treg cells are phenotypically distinct from those in peripheral blood (PB) and normal tissues [9, 10], suggesting that their immunosuppressive function depends on environmental factors.

Treg cells suppressive functions are associated with the expression of several immune checkpoint molecules (ICs), such as PD-1, CTLA-4, T-cell immunoglobulin and mucin-domain containing-3 (TIM)-3, and T cell Ig and ITIM domain (TIGIT) [3, 6, 11,12,13,14]. CTLA-4 and TIGIT act as tumor suppressors and thus, modulate the immune response in the TME [6, 15, 16]. Although the PD-1/PD ligand (PD-L)1 interaction was shown to promote the conversion of conventional T (Tconv) cells into Treg cells to maintain the latter’s population [17,18,19], it remains controversial whether PD-1 expression by Treg cells suppresses antigen-specific T cell immune responses [20,21,22].

Recent studies have reported that IC-molecules are upregulated on Treg cells within the TME or upon chronic infection and that Treg cells-mediated immunosuppression correlates with the expression of IC-molecules on these cells [6, 12]. The upregulation of these molecules has also been linked to tumor progression, as it likely reinforces the suppressive function of Treg cells in the TME. We previously reported that an increased level of PD-1 on Treg cells during chronic viral infection enhances CD8+ T cell immune suppression via interaction with PD-L1 on CD8+ T cells [12]. On the contrary, high PD-1 expression on Treg cells indicates dysfunctional and exhausted IFN-γ-secreting Treg cells that are enriched in tumor infiltrates and have possibly lost their suppressive function [23]. So far, the precise role of PD-1 in the function of tumor-infiltrating Treg cells in the TME is controversial. Given the significance of PD-1 in modulating immune responses and its paradoxical role as both an activation and exhaustion marker, clarifying the function of PD-1-positive Treg cells and their role in regulating anti-tumor immune responses is important [23].

To evaluate the suppressive function of tumor-infiltrating Treg cells in the TME, we comprehensively compared the phenotypes of T cell subsets, including Treg cells, obtained from PB, malignant effusion (ME), and tumor (TM) samples of patients with cancer. We also characterized Treg cells in paired lymphocyte samples obtained from blood, peri-tumoral tissue, and tumors of patients with lung cancer. Using a lung cancer mouse model, we investigated the suppressive function and mechanism of action of tumor-infiltrating Treg cells in the TME. We found that PD-1 was upregulated in tumor-infiltrating Treg cells and played a role in suppressing CD8+ T cell proliferation through PD-1/PD-L1 interactions. These results suggest that infiltrated PD-1-expressing Treg cells in TME are a potential therapeutic target for anti-cancer treatment.

Methods

Study design

Patients with stage IV cancer with ME and patients with cancer who planned to undergo surgical resection between April 2012 and December 2017 at the Severance Hospital were prospectively enrolled. Inclusion criteria were as follows: 1) over 20 years old; 2) stage IV cancer with malignant pleural effusion or ascites confirmed by cytology, or cancer with scheduled surgery; and 3) written informed consent. We collected 300 cc of effusions and simultaneously obtained 10 cc of PB from patients with stage IV cancer with ME, if available. In patients who underwent surgery, we collected TM-adjacent normal tissue, and 10 cc of PB to isolate peripheral blood lymphocytes (PBLs). The study was approved by the Institutional Review Board of Severance Hospital. We categorized the samples into three groups: 1) PBLs, 2) ME from patients with stage IV cancer, and 3) TM from patients with cancer who underwent surgery. To analyze the characteristics of Treg cells in TME, we also collected paired peritumoral tissue lymphocytes (pTILs), tumor-infiltrating lymphocytes (TILs), and PBLs at the same day from 12 patients with NSCLC who underwent curative resection.

Isolation of PB mononuclear cells and ME lymphocytes

PB mononuclear cells were isolated from 10 cc PB collected into EDTA tubes by separation over a Percoll (Sigma-Aldrich) gradient. Lymphocytes were isolated from 500 cc of ME by discontinuous density gradient centrifugation on Percoll. To isolate TILs, lung TMs were chopped and then incubated with a solution containing 1 mg/mL collagenase type IV (Worthington Biochemical) and 0.01 mg/mL DNaseI (Sigma-Aldrich) at 37 °C for 25 min. TILs were isolated by Percoll gradient after washing dissociated tissues with ice-cold RPMI1640.

Flow cytometry and antibodies

Flow cytometry was performed using a FACS CANTOII (BD Biosciences, Franklin Lakes, NJ, USA) and CytoFLEX (Beckman Coulter, IN, USA). Data were analyzed using FlowJo software (Tree Star, OR, USA).

For immunolabeling of human samples, fluorophore-conjugated monoclonal antibodies against the following proteins were used: CD4 (RPA-T4), CD3 (OKT3), PD-1 (EH12.2H7), and CTLA-4 (BNI3) (all from Biolegend, San Diego, CA, USA); TIM-3 (344823) and TIGIT (741182) (both from R & D Systems, Minneapolis, MN, USA); CD25 (M-A251) (BD Biosciences, Franklin Lakes, NJ, USA); and Foxp3 (PCH101) (eBioscience, San Diego, CA, USA). The LIVE/DEAD Fixable Red Dead Cell Stain kit was from Invitrogen (Carlsbad, CA, USA). Treg cells labeled with various antibodies (except for the antibody against Foxp3) were fixed and permeabilized with Foxp3 fixation/permeabilization solution (eBioscience). Foxp3 antibody was then administered for intracellular labeling of Treg cells. The proportion of CD4+ and CD8+ T cells among total lymphocytes was determined, and the fraction of Foxp3-positive CD4+ T cells was quantified.

For immunolabeling of mouse samples, fluorophore-conjugated monoclonal antibodies against the following proteins were used: CD4 (RM4–5), Ly5.1 (A20), PD-1 (29F.1A12), TIM-3 (RMT3–23), NK1.1 (PK136), and DX5 (DX5) (all from Biolegend); and CD8 (53-6.7), CD25 (PC61.5), CTLA-4 (UC10-4B9), TIGIT (G1GD7), and F4/80 (BM8) (all from eBioscience); and CD11b (M1/79) (BD Biosciences). The LIVE/DEAD Fixable Near-IR Dead Cell Stain kit was from Invitrogen. Treg cells labeled with various antibodies (except for the antibodies against Foxp3 and CTLA-4) were fixed and permeabilized with Foxp3 fixation/permeabilization solution (eBioscience, San Diego, CA, USA). Foxp3 antibody was then administered for intracellular labeling of Treg cells. The proportions of CD4+ and CD8+ T cells among lymphocytes were determined, and the fraction of Foxp3-positive CD4+ T cells was quantified. To prevent myeloid cells from non-specific staining, samples were preincubated with anti-CD16/32 (93) (eBioscience) before immunolabeling with fluorophore-conjugated antibodies.

Mouse TM model and lymphocyte isolation

Female C57BL/6, C57BL/6-Rag2−/−, and C57BL/6-Ly5.1 congenic mice (5–6 weeks) were purchased from Charles River Laboratories (Wilmington, MA, USA) and Jackson Laboratories (Bar Harbor, ME, USA). To generate lung TM bearing mice, 5 × 105 TC-1 cells were intravenously injected into C57BL/6 mice via the tail-vein. Mice were sacrificed on day 21 post-injection. Lymphocytes were isolated from the spleen, normal lung, and lung tumor as previously described [9]. The number of tumor nodules on left upper lobe of the lung was counted at day 12, 16, and 21 post-injection. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Yonsei University Laboratory Animal Research Center (2013–0115).

In vitro suppression assay using mouse lymphocytes

For the Treg cells suppression assay, CD4+CD25+ Treg cells (105/well) were co-cultured with CD8+ T cells (105/well) with Dynabeads mouse T-activator CD3/CD28 (Thermo Fisher Scientific, Waltham, MA, USA) in a 96-well U-bottom plate at 37 °C for 72 h. For the CellTrace Violet dilution assay, CD8+ T cells were isolated from the spleen of naïve mice using a CD8+ T Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and labeled with 5 μM CellTrace Violet (Thermo Fisher Scientific). CD4+CD25+ Treg cells were separately isolated from the spleen and tumor of TM-bearing mice on day 21 post-injection using CD4+CD25+ Regulatory T Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). To inhibit cell migration, Transwell membranes (0.4 mm pore; BD Biosciences) were inserted into 24-well plate. CD4+CD25+ Treg cells (106/well) were co-cultured with CD8+ T cells (106/well) with Dynabeads mouse T-activator CD3/CD28 (Thermo Fisher Scientific) in a 24-well plate at 37 °C for 72 h.

For PD-1 blockade in tumor-infiltrating Treg, CD4+CD25+ Treg cells (2.5 × 104/well) isolated from tumor lymphocytes of TM bearing mice on day 14 post-injection were preincubated with 10 μg/mL anti-PD-1 antibody (RMP1–14) or rat IgG2a isotype control (2A3) (Bio X Cell) at 4 °C for 1 h, washed twice, and then co-cultured with CD8+ T cells (105/well) in the presence of mouse T-activator CD3/CD28 Dynabeads for 68 h.

Adoptive cell transfer for in vivo suppression assay

To examine the functionality of TIL Treg (PD-1hi) and spleen Treg (PD-1lo) cells, CD4+CD25+ Treg cells were isolated from the tumor and spleen of TM bearing mice at day 21 post-injection using CD4+CD25+ Regulatory T Cell Isolation kit (Miltenyi Biotec). Ly5.1+ CD8+ T cells were isolated from naïve C57BL/6-Ly5.1 congenic mice. Ly5.1+ CD8+ T cells (2 × 106) were injected i.v. into recipient Rag2−/− mice alone or with Ly5.2+ TIL Treg or spleen Treg (1 × 106). At day 7 after cell transfer, splenocytes isolated from Rag2−/− mice were analyzed for homeostatic expansion of the Ly5.1+ CD8+ T cell population using FACS.

In vitro suppression assay using human lymphocytes

CD4+CD25+ Treg cells were isolated from the tumor tissue and peripheral blood of NSCLC patients, using human CD4+CD25+CD127dim/− Regulatory T Cells Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ T cells were isolated from the paired peripheral blood of NSCLC patients using human CD8+ T Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequently labeled with 5 μM CellTrace Violet. The CD8+ T cells (105/well) were co-cultured with CD4+CD25+ Treg cells (5 × 104/well) isolated from either tumor tissue or peripheral blood in the presence of 2.5 μl/well of Dynabeads human T-activator CD3/28 (Thermo Fisher Scientific) at 37 °C for 72 h.

Multi-color immunofluorescence analysis

For multicolor immunofluorescence analysis, lungs were isolated, fixed with 2% paraformaldehyde/phosphate buffered saline overnight at 4 °C, and then embedded in OCT compound (Sakura). Tissue blocks were frozen in 2-methyl butane and cooled on dry ice. Frozen blocks were cut to a thickness of 8 μm and mounted on the silane-coated slide. Sections were stained with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen) and with antibodies for anti-CD8α (Clone 53–6.7), anti-CD4 (clone RM4–5), anti-CD279 (clone RMP1–30), and anti-GFP (clone 1GFP63) for amplification of Foxp3-GFP signals (Biolegend). Streptavidin-conjugated horseradish peroxidase was used for staining of biotin-conjugated antibodies, and TSA Cyanine 3 Tyramidetetramethylrhodamine reagent (SAT704A001EA; PerkinElmer) was subsequently added for amplification. Images was acquired using a microscope (Carl Zeiss Co. Ltd) and analyzed with ImageJ 1.50b software.

Statistical analysis

Data were analyzed using Prism 5.0 software (GraphPad Inc., CA, USA). The Student’s t-test, one-way analysis of variance, and the least significant difference test were used, where appropriate, to evaluate the significance of differences among groups. All statistical analyses were conducted with a significance level of α = 0.05 (P < 0.05).

Results

Patient characteristics

We enrolled 103 patients: 72 were stage IV cancer patients with ME, and 31 were patients with operable disease (not stage IV) who underwent surgical resection. Detailed information of the patients from which PB, ME, or TM were obtained is described in Additional file 6: Table S1. The total number of tumor specimens was divided into three groups based on the specimen type: PB, 20.7% (28/135); ME, 56.2% (76/135); and TM, 23.1% (31/135). Detailed analyses of immune subsets as well as the levels of their immune checkpoints were performed in PBLs (PB group), effusion-infiltrating lymphocytes (EILs) (ME group), and TILs (TM group). Primary cancer types in the ME group were NSCLC, 43.1% (31/72); gastric cancer, 22.2% (16/72); colon cancer, 5.6% (4/72), and breast cancer, 5.6% (4/72). The types of ME were ascites, 59.7% (43/76) and pleural effusion, 45.8% (33/76), with four patients having both (Additional file 6: Table S1). The presence of malignant cancer cells and TILs in TM or ME was pathologically or cytologically confirmed (Fig. 1a).

Fig. 1
figure 1

T cell characteristics and PD-1 expression in Tconv of patients with cancer. Malignant effusions, such as ascites, and pleural effusions were extracted from patients with stage IV cancer. Tumor-infiltrating lymphocytes were obtained from the tumors of patients with non-small cell lung cancer (NSCLC) and colon cancer. a Left, computed tomography images showing malignant ascites (upper), pleural effusion (middle), and lung cancer lesions in left lower lobe (bottom) of a patient with NSCLC. Right, cytological analysis of malignant effusion and histological analysis of lung cancer tissue. Red arrows indicate malignant effusion and cancer (left column) and tumor cells (right column). b Proportions of CD4+ and CD8+ T cells among CD3+ T cells. Representative plots (upper) and statistics (bottom) are shown. c, d PD-1 and TIM-3 expression on CD4+ and CD8+ T cells. Representative plots of PD-1 and TIM-3 expression (upper) and percentages of total PD-1+ (bottom left) and TIM-3+ (bottom right) cells among CD4+ and CD8+ T cells are shown. Peripheral blood lymphocytes (PBLs), effusion-infiltrating lymphocytes (EILs), and tumor-infiltrating lymphocytes (TILs) were isolated from healthy control donors (HC) and patients with cancer (PBLs of HC, n = 16; PBLs, n = 28; EILs, n = 76; TILs, n = 31). Lines in the scatterplot represent the mean values. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney test)

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Tconv with exhausted phenotypes are abundant in TM and ME

To investigate T cell subsets in three different tumor specimens, we compared the ratio of CD4+ and CD8+ T cells in PBLs, EILs, and TILs isolated from PB, ME, and TM, respectively. The percentage of CD4+ T cells was higher among TILs than among PBLs or EILs. In contrast, the percentage of CD8+ T cells was markedly lower among TILs than among PBLs or EILs (Fig. 1b), suggesting that the migration of cytotoxic lymphocytes (CTLs) into the TM was inhibited.

The phenotypes of PBLs, EILs, and TILs were compared by quantifying CD4+ and CD8+ T cells expressing PD-1 and TIM-3. The percentage of PD-1- or TIM-3-expressing CD4+ or CD8+ T cells was the highest among TILs, with lower percentages in EILs and PBLs (Fig. 1c, d), suggesting that T cells derived from TM and ME show more pronounced T cell exhaustion than those derived from PBLs.

High expression of PD-1 in Treg cells of ME and TM

We next examined how Treg cells expressing forkhead box (Fox) p3 are distributed and differ phenotypically in PB, ME, and TMs. Treg cells showed greater accumulation in TILs than in PBLs and EILs of patients or in PBLs of healthy controls (Fig. 2a). Interestingly, Treg cells in TILs expressed a higher level of PD-1 than those in PBLs and EILs; moreover, the PD-1-expressing Foxp3+ population among CD4+ T cells was also larger in TILs than in EILs, which, in turn, had a larger population than PBLs (Fig. 2b). To further characterize CD4+ T cells in different tissues, we compared PD-1 in Foxp3+ and Foxp3− CD4+ T cells (Fig. 2c). The proportion of PD-1-expressing cells in both CD4+ cells was larger in EILs and TILs than in PBLs. These results indicate that PD-1 expression by Treg cells and Tconv cells clearly reflect the TME, as PD-1 expression increased in the following order: TILs > EILs > PBLs.

Fig. 2
figure 2

PD-1 expression in Foxp3+ Treg in different tissue types of patients with cancer. a Representative plots of CD25 and Foxp3 expression (left) and proportion of Foxp3+ T cells (right) among CD4+ T cells. b Representative plots of PD-1 and Foxp3 expression (left) and proportion of PD-1 and Foxp3 co-expressing cells among total CD4+ T cells (right). c Summary of PD-1-positive fraction of Foxp3+ Tconv (left) and Foxp3− Treg (right) cell populations among CD4+ T cells. Peripheral blood lymphocytes (PBLs), effusion-infiltrating lymphocytes (EILs), and tumor-infiltrating lymphocytes (TILs) were isolated from healthy control donors (PBL, n = 16) and patients with cancer (PBL, n = 28; EIL, n = 76; TIL, n = 31). Lines in the scatterplot represent the mean values. ns, not significant; **P < 0.01, ***P < 0.001 (Mann-Whitney test)

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We next investigated whether the characteristics of CD4+ T cells, CD8+ T cells, and Treg cells were altered in MEs depending on the cancer type. As shown in Additional file 1: Figure S1, the abundance of these cells and their expression of PD-1 in MEs were comparable in different types of cancer, although it is worth noting that there were more Treg cells than there were CD4+ or CD8+ Tconv cells expressing PD-1. Interestingly, the degree of infiltration of PD-1+ Treg cells did not differ among ME samples derived from the different types of cancer, indicating that the presence of PD-1+ Treg cells in ME is a common feature across cancers of distinct histological origin (Additional file 1: Figure S1). Additionally, we compared the phenotype of Treg between ascites and pleural effusion. As shown in Additional file 2: Figure S2, significant differences of the percentages of Foxp3+ Treg and PD-1+ Foxp3+ Treg cells were not observed between ascites and pleural effusion. Moreover, the ascites and pleural effusions had a similar expression rate of PD-1 in Foxp3+ Treg and Foxp3− Tconv cells.

Tumor-infiltrating Treg are abundant in patients with lung cancer and express multiple IC-molecules

To clarify the characteristics of Treg cells in the TME, we compared the frequency of Treg cells and the expression of IC-molecules such as PD-1, TIM-3, TIGIT, and CTLA-4 in paired sets of tissue-derived lymphocytes, such as PBLs, pTILs, and TILs collected from 12 patients with NSCLC. As expected, Treg cells were more highly enriched in TILs than in pTILs and PBLs (Fig. 3a). Moreover, more Treg-expressing ICs were found among TILs than among pTILs and PBLs (Fig. 3b).

Fig. 3
figure 3

PD-1-expressing tumor-infiltrating Treg and their activated phenotype in patients with non-small cell lung cancer (NSCLC). a CD25 and Foxp3 expression in CD4+ T cells (upper) and proportion of Foxp3+ cells among total CD4+ T cells (lower) in peripheral blood lymphocytes (PBLs), peritumoral infiltrating lymphocytes (pTILs), and tumor-infiltrating lymphocytes (TILs) derived from patients with NSCLC. b Representative plots of PD-1, TIM-3, TIGIT, CTLA-4, and Foxp3 expression in CD4+ T cells (left) and percentage of CD4+ T cells co-expressing PD-1, TIM-3, TIGIT, CTLA-4, and Foxp3 (right). c PD-1, TIM-3, TIGIT, and CTLA-4 expression in Foxp3+ Treg, Foxp3− Tconv and CD8+ Tconv of these patients. d Enhanced suppression of CD8+ T cells by PD-1-expressing tumor-infiltrating Treg from NSCLC patients. Treg were isolated from the peripheral blood and tumor tissue from NSCLC patients. Peripheral blood Treg and tumor-infiltrating Treg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8+ T cells were stimulated in vitro with CD3/CD28 Dynabeads for 96 h in the absence or presence of each Treg population. CTV dilution in proliferating CD8+ T cells is indicated in each histogram. Histograms represent the percentages of proliferating cells. Lines in the bar graph represent the mean and mean ± SEM, respectively. ns, not significant; **P < 0.01, ***P < 0.001 (Mann-Whitney test)

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We also compared IC-molecule expression on different tumor-infiltrating T cell subsets. Among four different IC-molecules, PD-1 most clearly distinguished TME in all T cell subsets, because a significant increase in the PD-1+ population was observed in the following order TIL > pTIL > PBL (Fig. 3c). Notably, PD-1 was higher in tumor-infiltrating Foxp3+Treg cells (~ 98%) than in Foxp3−Tconv cells (~ 82%) or CD8+ Tconv cells (78%). Furthermore, the number of PD-1-expressing tumor-infiltrating Foxp3+ Treg cells was greater than the number of tumor-infiltrating Foxp3+ Treg cells expressing other IC-molecules. It is therefore conceivable that PD-1 expression on Treg cells is a TME marker. Additionally, we performed the in vitro suppressive assay using isolated CD4+CD25+ Treg cells from the peripheral blood and tumor tissue of NSCLC patients and isolated CD8+ T cells from the peripheral blood. Each tumor-infiltrating Treg cells or peripheral Treg cells was co-cultured with peripheral CD8+ T cells with αCD3/CD28 stimulation. CD8+ T cells proliferated at a high rate in the absence of Treg cells and were more potently inhibited by PD-1hi tumor-infiltrating Treg cells than by PD-1lo PBMC Treg cells (Fig. 3d).

Treg numbers and the expression of IC-molecules are altered during cancer progression in a mouse model

We previously showed that immune-exhaustion markers were highly expressed in tumor-infiltrating Treg cells of patients with NSCLC. We therefore investigated the Treg phenotype in greater detail in different tissues, using a mouse lung cancer model. We compared the expression levels of IC-molecules, such as PD-1, TIM-3, and TIGIT, on CD4+ and CD8+ T cells in different tissues from naïve and TM-bearing mice. As in patients with cancer tissue, the expression of IC-molecules in CD4+ and CD8+ T cells was much higher in lung TM than in PB or spleen (Fig. 4a, b). Among the populations expressing IC-molecules, PD-1-expressing CD4+ and CD8+ T cells were more abundant in the TM.

Fig. 4
figure 4

Differential expression of immune checkpoint (IC) molecules on CD4+ and CD8+ T cells in mice with lung cancer. To induce lung adenocarcinoma, TC-1 cells were intravenously injected into syngeneic mice. a, b Tumor-bearing mice at 3 weeks after TC-1 cell injection and naïve control mice were sacrificed, and lymphocytes were isolated from peripheral blood (PB), spleen (SP), and lung (LG). (Left) Expression levels of PD-1, TIM-3, and TIGIT on CD4+ and CD8+ T cells were assessed. (Right) Summary of the proportions of IC molecules expressed on populations of CD4+ and CD8+ T cells in PB, SP, and LG at the tumor site. Numbers in the plot indicate percentages of the corresponding population. Data are representative of three independent experiments (n = 5 mice per group in each experiment). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). The symbol above each column is the P value obtained when SP samples were compared to the corresponding samples from naïve mice (control)

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We next examined whether IC-molecules are preferentially upregulated on Treg cells (compared to Tconv) in TM, as was observed in patient tissues. PB, spleen, and lung lymphocytes were isolated at different time points after TC-1 injection (Fig. 5a). Starting at 12 days after TC-1 injection, an increase in the number of Foxp3+ Treg cells was observed in TM and the Treg cells fraction reached 20% of total CD4+ T cells, a nearly 3-fold increase compared to that in the non-TM lung (Fig. 5b). At 3 weeks after TC-1 injection, Foxp3+ Treg cells were more abundant in the TM than in the PB or spleen (Fig. 5c). Foxp3+ Treg cells in TM showed significant increases in PD-1, TIM-3, TIGIT, and CTLA-4, compared to other tissues (Fig. 5d). Moreover, tumor-infiltrating Treg cells expressed much higher levels of IC-molecules than tumor-infiltrating Tconv (Fig. 5e). Most Treg cells (~ 80%), but only a low frequency of Tconv (~ 20%) expressed PD-1 in TM. PD-1 was markedly upregulated 21 days after TC-1 injection, and the same trend was observed for TIM-3 and TIGIT, although the increases in the levels of these molecules were less prominent (Fig. 5f). Unlike PD-1, TIM-3, and TIGIT, CTLA-4 was already upregulated in Treg cells before TC-1 injection and its expression progressively increased over time (Fig. 5f). Thus, expression of IC-molecules, especially PD-1, on Treg cells increases with TM progression. As tumor numbers increased, immune checkpoints including PD-1, TIM-3, TIGIT, and CTLA-4 increased (Additional file 3: Figure S3).

Fig. 5
figure 5

Spatial and temporal dynamics of immune checkpoint (IC) molecule expression on Treg during cancer progression. a Schedule for establishing the TC-1 lung adenocarcinoma model and tumor formation at each time point. b Representative plots showing CD25 and Foxp3 expression in CD4+ T cells (left) and changes at different time points after TC-1 TM tumor cell injection (right). c Representative plots of Treg (left) and summary of the proportion of Foxp3+ cells among CD4+ T cells (right) in peripheral blood (PB), spleen (SP), and lung (LG). d Levels of PD-1, TIM-3, TIGIT, and CTLA-4 expression on Foxp3+CD4+ Treg in PB, SP, and LG. e Levels of PD-1, TIM-3, TIGIT, and CTLA-4 expression on Treg and Tconv in different tissues (PB, SP, and LG). f Changes in the levels of PD-1, TIM-3, TIGIT, and CTLA-4 expression on Treg at different time points. Data are representative of three independent experiments (n = 5 mice per group in each experiment). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)

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Immunosuppressive function of tumor-infiltrating Treg in CD8+ T cell response is mediated by PD-1/PD-L1 interaction

Among all IC-molecules examined, PD-1 was most highly upregulated in tumor-infiltrating Treg cells. To determine the role of PD-1 on tumor-infiltrating Treg cells, in the regulation of the CD8+ T cell response, we compared the suppressive activity of Treg expressing high- and low-levels of PD-1 (PD-1hi Treg cells from lung TM 3 weeks after TC-1 injection vs. PD-1lo Treg cells from the spleen of the same TM-bearing mice). CD4+CD25+ Treg cells, isolated using a microbead-based Treg isolation kit (CD4+CD25+ Regulatory T Cell Isolation kit), was confirmed to be ~ 90% purified Foxp3+ Treg cells (Additional file 4: Figure S4). Each population was co-cultured with naïve CD8+ cells with or without stimulation by αCD3/CD28. CD8+ T cells proliferated at a high rate in the absence of Treg cells and were more potently inhibited by PD-1hi tumor-infiltrating Treg cells than by PD-1lospleen Treg cells (Fig. 6a). Similarly, interferon (IFN)-γ production was also more strongly suppressed by PD-1hi tumor-infiltrating Treg than by PD-1lo spleen Treg cells.

Fig. 6
figure 6

Enhanced suppressive function of PD-1-expressing tumor-infiltrating Treg. a Enhanced suppression of CD8+ T cells by PD-1-expressing tumor-infiltrating Treg. At 3 weeks after intravenous injection of TC-1 cells, Treg were isolated from the spleen (SP) and lung of mice with TC-1 cell-induced tumors. SP Treg and tumor-infiltrating Treg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8+ T cells were stimulated in vitro with CD3/CD28 Dynabeads for 72 h in the absence or presence of each Treg population. CTV dilution in proliferating CD8+ T cells is indicated in each histogram. Histograms represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. b Contact-dependent Treg-mediated suppression of CD8+ T proliferation. CTV-labeled CD8+ T cells were stimulated in vitro with CD3/CD28 Dynabeads and cocultured with tumor-infiltrating Treg for 72 h in the absence or presence of a transwell membrane. c Homeostatic proliferation of donor Ly5.1+CD8+ T cells in the spleen isolated from Rag2−/− mice at 7 d after adoptive cell transfer. Representative plot (left) and absolute number (right) of donor Ly5.1+CD8+ T cells in the spleen. d PD-1-mediated suppressive activity of tumor-infiltrating Treg isolated from the lungs of tumor-bearing mice 2 weeks after intravenous injection of TC-1 cells. At this time point, Treg expressed intermediate levels of PD-1. CTV-labeled CD8+ T cells were stimulated as shown in (a). Before co-culture of CD8+ T cells with tumor-infiltrating Treg, the latter were pre-incubated with an anti-PD-1 antibody or its isotype as control. CTV dilution in proliferating CD8+ T cells is shown in the histograms, which represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. (e) Representative immunofluorescence images of mouse lung tumor samples. Antibodies against Foxp3, CD8, and PD-1 were used to label and examine the interaction between Treg and CD8+ T cells expressing PD-1. Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)

Full size image

To investigate the role of PD-1 upregulation, induced by tumor-infiltrating Treg cells, we examined whether the interaction between PD-1 on tumor-infiltrating Treg cells and PD-L1 on CD8+ T cells is required for immunosuppression in patients with cancer. PD-1 on tumor-infiltrating Treg cells was blocked by incubation with an anti-PD-1 antibody. Unbound antibody was subsequently removed and the cells were co-cultured with CD8+ T cells. We prepared Treg cells, expressing an intermediate level of PD-1 that were isolated from lung TM in 2- rather than in 3-weeks after injection because Treg cells, highly expressing PD-1, isolated at later time-points, also co-expressed other IC-molecules (Fig. 5), making it difficult to differentiate the role of PD-1 in the suppressive function of Treg cells from that of others.

Additionally, to clarify whether the potent suppressive function of PD-1hi tumor-infiltrating Treg cells is mediated via cell-to-cell contact between Treg and CD8+ cells or soluble factors produced from Treg cells, we conducted the experiments with transwell membrane system to block cell migration (Fig. 6b). Transwell membranes were inserted into 24-well plate. CTV labeled CD8+ T cells and CD4+CD25+ Treg were placed into lower and upper wells, respectively, and αCD3/CD28 was added into both wells for stimulation. Suppression of T cell proliferation was not observed in the presence of the Transwell membrane. This data demonstrated that the suppression of CD8+ T cell proliferation by Treg requires cell to cell contact between each cell population (Fig. 6b). Next, we performed the in vivo experiment with TIL Treg and spleen Treg cellsalong with Ly5.1+CD8+ T cells. In line with in vitro data, when TIL Treg cells was injected, CD8+ T cell proliferation was significantly inhibited compared with spleen Treg and no Treg cells (Fig. 6c).

As shown in Fig. 6d, tumor-infiltrating Treg cells that had been blocked with anti-PD-1 antibody, were significantly impaired in their ability to suppress the proliferation of CD8+ T cells and IFN-γ production as compared to isotype antibody-treated tumor-infiltrating Treg cells. Given that both mouse and human CD8+ T cells can upregulate low affinity Fc receptors following activation [24, 25], we tested whether CD8+ T cells upregulate Fc receptors in our system. We obtained splenocytes of TC-1 tumor bearing mice. We stained splenocytes with CD8, CD44, CD16/32 (FcγRIII/II), and CellTrace Violet and compared the expression of Fc receptor between with and without stimulation. Significant differences of CD16/32 were not observed between groups with and without stimulation (Additional file 5: Figure S5A). To validate the CD16/32 antibody, we analyzed the expression of CD16/32 on NK cells and macrophages. This antibody can specifically detect CD16/32 on these cells, so we excluded the possibility that no detection of CD16/32 on CD8+ T cells after stimulation could be a problem of CD16/32 antibody (Additional file 5: Figure S5B). Taken together, our data demonstrated that the effect of anti-PD-1 is direct effect by blocking of PD-1 pathway signaling rather than the effect of anti-PD-1 antibody mediated by ADCC.

A multi-color immunofluorescence analysis revealed that CD8, PD-1, and Foxp3 were co-localized in mouse tumor tissues (Fig. 6e), implying that CD8+ cells and Foxp3+ Treg cells spatially interact in the TME.

Discussion

In this study, we examined the phenotype and function of Treg cells as well as CD4+ and CD8+ Tconv cells that infiltrated into the TME, including the ME and TM from patients with cancer. We also investigated the mechanism by which Treg cells induce immunosuppression using a mouse lung cancer model. Most tumor-infiltrating Treg cells showed higher PD-1 expression than Tconv cells, implying that PD-1-expressing Treg cells are a biological marker of the TME. Indeed, in Treg cells derived from TMs of patients with NSCLC, PD-1 was the most clearly upregulated IC-molecule. As previously reported, these cells exhibited an enhanced immunosuppressive function that was correlated with the extent of PD-1 upregulation [12]. We speculate that PD-1-expressing tumor-infiltrating Treg cells induce immunosuppression through the interaction of PD-1 and PD-L1, which may contribute to immune escape in TME. Clarifying the link between this phenotype and enhanced suppressive function of tumor-infiltrating Treg cells can provide insight into their suppressive mechanism in patients with cancer.

The predominant function of PD-1 in Treg cells seems to be similar to that of CTLA-4; both proteins contribute to the maintenance of Treg immunosuppressive function [15]. However, PD-1 expression on Treg cells differed by cell location. For instance, PD-1 was expressed by Treg cells in TMs but not in normal tissue or PBLs as depicted in Fig. 3. In contrast, Treg cells had high basal CTLA-4 levels irrespective of the tissue of origin. This supports our assertion that PD-1 on Treg cells is a more useful marker for characterizing the TME. We also examined whether the upregulation of PD-1 on tumor-infiltrating Treg cells can reinforce their basal immune-suppressive function. High PD-1 expression in Treg cells was associated with the suppression of CD8+ T cells and PD-1 blockade abrogated the immune-suppressive function of Treg cells, suggesting that an interaction between PD-1 on Treg cells and PD-L1 expressed by another cell type—likely CD8+ T cells [26]—is necessary for immunosuppression. Thus, elevated PD-1 expression on Treg cells is a potential marker for immune escape in patients with cancer. These findings were consistent with our previously reported data that PD-1 upregulation in Treg cells and the interaction between PD-1 on Treg cells and PD-L1 expressed by effector T cells enhanced T cell-mediated immune suppression during chronic viral infection [12]. Thus, an immunotherapy targeting Treg and PD-1 could be highly effective in patients with cancer.

We also investigated tumor-infiltrating Treg and Tconv cells obtained from ME of patients with stage IV cancer. Most of the earlier studies of TME Treg cells were performed in mice and focused on Treg cells phenotype. Studies in patients with stage IV cancer have been hampered by the difficulty of obtaining sufficient TMs for analysis of T cell populations. To overcome this limitation, we developed an experimental model using ME from human patients with stage IV cancer as representative TME of stage IV cancer. This model will allow future examinations of various mechanistic aspects of human cancer through functional assays.

Several studies have reported IC expression on intra-tumoral Treg cells and suggested potential roles of these ICs in the regulation of the immune response in mice [6, 15, 27]. We also showed here that ICs other than PD-1 were upregulated in Treg cells. Studies on the relative contributions of these IC-molecules to immunosuppression in the TME may lead to the development more effective immunotherapies.

Regarding other PD-1-expressing immune cells than CD8+ T cells and Treg cells in TME and their role, Irving et al. reported that tumor-associated macrophages (TAMs) expressed PD-1 and PD-1-expressing TAMs increased over time in mouse model and progressive disease in human cancers [28]. PD-1 expressed on TAMs reduced their phagocytic potency against tumor cells and blockade of PD-1 pathway restored the macrophage phagocytosis, resulting in enhancing anti-tumor activity of TAMs. This data suggests that PD-1 expressed by TAMs is one of the mechanism for immune evasion. PD-1 expression was also described on NK cells in many different types of human and mouse cancers, where the PD-1 expressed by NK cells negatively regulated NK cell function even though its molecular mechanisms are not clearly demonstrated to date [29,30,31,32,33,34]. In addition, PD-1 has been reported to be expressed on innate lymphoid cells (ILCs), prevalently ILCs type 3 (ILC3s), as well as NK cells in pleural effusion of primary and metastatic tumors, albeit the role of PD-1 on ILC3s was not addressed [35].

Based on these reports, it is plausible that PD-1 expressed by different types of immune cells including CD8+ T cells, Treg cells, NK cells, and ILCs in the TME probably contributes to immune evasion, leading to promotion of tumor cells. However, it has not been addressed yet which types of PD-1-expressing immune cells are most effectively involved in the PD-1-mediated immunosuppression. In addition, to compare the immunosuppressive activity of each immune cell subset, the level of PD-1 expression on each type of cells should be examined. In this regard, further study is needed to determine whether other PD-1-expressing immune cells than Treg cells in the TME compensate for a lack of Treg cells and which types of PD-1-expressing immune cells mostly impact on immune suppression in the TME.

Treg cells expansion in the TME is widely recognized as an obstacle to successful immunotherapy in patients with cancer [5]. Previously, we demonstrated that Treg cells depletion using an anti-CD25 antibody increased the abundance of functional antigen-specific CD8+ T cells during chronic viral infection [12]. Furthermore, treatment with a neutralizing antibody also caused the elimination of non-Treg and rapid replenishment of Treg cells [36]. Thus, functional inactivation of Treg cells and rejuvenation of exhausted T cells by targeting overexpressed PD-1 combined with temporal depletion of Treg cells expressing IC-molecules may be a promising strategy to limit cancer progression.

Conclusions

In conclusion, our study provides insights into Treg cells function and their suppressive mechanism in patients with cancer. We showed that the suppressive function of tumor-infiltrating Treg cells was enhanced by the increase in their relative proportion and by the upregulation of the expression of inhibitory receptors, such as PD-1, TIM-3, and CTLA-4.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

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Acknowledgements

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Declarations

This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review board. We have read and understood your journal’s policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare. The author contributions are described below.

Funding

This work was supported by grants from the National Research Foundation of the Republic of Korea (2018R1A2A1A05076997, 2017R1A5A1014560, 2018M3A9H3024850, and 2019M3A9B6065221 to S-.J.H.; 2017M3A9E9072669, 2017M3A9E8029717, 2017R1D1A1B03029874, and 2019M3A9B6065231 to H.R.K).

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  1. Hye Ryun Kim, Hyo Jin Park, Jimin Son contributed equally to this work and are joint first authors.

Authors and Affiliations

  1. Yonsei Cancer Center, Division of Medical Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Gu, Seoul, 120-752, South Korea

    Hye Ryun Kim, Gamin Kim, Byoung Chul Cho & Sun Young Rha

  2. Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea

    Hyo Jin Park, Jimin Son, Seyeon Park & Sang-Jun Ha

  3. Department of Thoracic and Cardiovascular Surgery, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Gu, Seoul, 120-752, South Korea

    Jin Gu Lee, Kyung Young Chung & Seong Yong Park

  4. Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea

    Nam Hoon Cho & Hyo Sup Shim

  5. Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, South Korea

    Hong In Yoon

  6. Department of Pharmacy, Korea University, Sejong, South Korea

    Hyun Gyung Kim & Yong Woo Jung

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Contributions

Conception and design: HRK, SYP, SYR, S-JH. Financial support: HRK, S-JH. Administrative support: HRK, SYP, SYR, S-JH. Provision of study materials or patients: HRK, HJP, JS, JGL, KYC, NHC, HSS, SP, GMK, HIY, HGK, YWJ, BCC, SYP, SYR, S-JH. Collection and assembly of data: HRK, HJP, JS, JGL, KYC, GMK, HIY, HGK, YWJ, BCC, SYP, SYR, S-JH. Data analysis and interpretation: HRK, HJP, SYP, SYR, S-JH. Manuscript writing: All authors. Final approval of manuscript: All authors.

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Correspondence to Seong Yong Park, Sun Young Rha or Sang-Jun Ha.

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The study was approved by the Institutional Review Board of Severance Hospital. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Yonsei University Laboratory Animal Research Center (2013–0115).

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Supplementary information

Additional file 1: Figure S1

. PD-1 expression on CD4+, CD8+, or Treg in malignant effusion according to the different types of cancer patients including lung cancer, gastric cancer, breast cancer, and others.

Additional file 2: Figure S2

. Comparison of Treg phenotype between ascites and pleural effusion. (A) Percentage of Foxp3 in total CD4+ T cells between ascites and pleural effusion (B) Percentage of PD-1+Foxp3+ in total CD4+ T cells between ascites and pleural effusion (C) Percentage of PD-1+ in Foxp3+CD4 Treg between ascites and pleural effusion (Left), percentage of PD-1+in Foxp3− CD4 T cells between ascites and pleural effusion.

Additional file 3: Figure S3

. Correlation of the immune checkpoint including PD-1, TIM-3, TIGIT and CTLA4 expressed on Treg as tumor nodule number increased. (A) PD-1, (B) TIM-3, (C) TIGIT, and (D) CTLA-4 expression on Treg according to the increased tumor nodules. The number of tumor nodules was measured at day 12, 16, and 21 post-injection (n = 4–7 mice per group). Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

Additional file 4: Figure S4

. Purification of tumor-infiltrating Treg using microbead-based Treg isolation kit. Treg were separately isolated from the spleen and from TM-bearing mice using a CD4+CD25+ Regulatory T Cell Isolation kit for suppressive function analysis. Treg, isolated using a microbead-based Treg isolation kit, demonstrated ~ 90% purified Foxp3+ Treg compared with the 16% prior to isolation.

Additional file 5: Figure S5

. Expression of CD16/32 on CD8+ T cells after in vitro TCR activation and NK cells and macrophages. (A) The expression of CD16/32 on purified CD8+ T cells activated by CD3/28 Dynabeads for 3 d. (B) The expression of CD16/32 on Dx5+NK1.1+ NK cells and CD11b+F4/80+ macrophages isolated from the spleen of naïve mouse.

Additional file 6: Table S1.

Baseline characteristics of patients and specimens.

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Kim, H.R., Park, H.J., Son, J. et al. Tumor microenvironment dictates regulatory T cell phenotype: Upregulated immune checkpoints reinforce suppressive function. j. immunotherapy cancer 7, 339 (2019). https://doi.org/10.1186/s40425-019-0785-8

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