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).

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Fig. 1

T cell characteristics and PD-1 expression in T_conv 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)

T_conv 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 T_reg cells of ME and TM

We next examined how T_reg cells expressing forkhead box (Fox) p3 are distributed and differ phenotypically in PB, ME, and TMs. T_reg cells showed greater accumulation in TILs than in PBLs and EILs of patients or in PBLs of healthy controls (Fig. 2a). Interestingly, T_reg 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 T_reg cells and T_conv cells clearly reflect the TME, as PD-1 expression increased in the following order: TILs > EILs > PBLs.

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Fig. 2

PD-1 expression in Foxp3⁺ T_reg 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⁺ T_conv (left) and Foxp3⁻ T_reg (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)

We next investigated whether the characteristics of CD4⁺ T cells, CD8⁺ T cells, and T_reg 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 T_reg cells than there were CD4⁺ or CD8⁺ T_conv cells expressing PD-1. Interestingly, the degree of infiltration of PD-1⁺ T_reg cells did not differ among ME samples derived from the different types of cancer, indicating that the presence of PD-1⁺ T_reg cells in ME is a common feature across cancers of distinct histological origin (Additional file 1: Figure S1). Additionally, we compared the phenotype of T_reg between ascites and pleural effusion. As shown in Additional file 2: Figure S2, significant differences of the percentages of Foxp3⁺ T_reg and PD-1⁺ Foxp3⁺ T_reg 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⁺ T_reg and Foxp3⁻ T_conv cells.

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

To clarify the characteristics of T_reg cells in the TME, we compared the frequency of T_reg 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, T_reg cells were more highly enriched in TILs than in pTILs and PBLs (Fig. 3a). Moreover, more T_reg-expressing ICs were found among TILs than among pTILs and PBLs (Fig. 3b).

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Fig. 3

PD-1-expressing tumor-infiltrating T_reg 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⁺ T_reg, Foxp3⁻ T_conv and CD8⁺ T_conv of these patients. d Enhanced suppression of CD8⁺ T cells by PD-1-expressing tumor-infiltrating T_reg from NSCLC patients. T_reg were isolated from the peripheral blood and tumor tissue from NSCLC patients. Peripheral blood T_reg and tumor-infiltrating T_reg 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 T_reg 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)

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⁺T_reg cells (~ 98%) than in Foxp3⁻T_conv cells (~ 82%) or CD8⁺ T_conv cells (78%). Furthermore, the number of PD-1-expressing tumor-infiltrating Foxp3⁺ T_reg cells was greater than the number of tumor-infiltrating Foxp3⁺ T_reg cells expressing other IC-molecules. It is therefore conceivable that PD-1 expression on T_reg cells is a TME marker. Additionally, we performed the in vitro suppressive assay using isolated CD4⁺CD25⁺ T_reg cells from the peripheral blood and tumor tissue of NSCLC patients and isolated CD8⁺ T cells from the peripheral blood. Each tumor-infiltrating T_reg cells or peripheral T_reg 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 T_reg cells and were more potently inhibited by PD-1^hi tumor-infiltrating T_reg cells than by PD-1^lo PBMC T_reg cells (Fig. 3d).

T_reg 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 T_reg cells of patients with NSCLC. We therefore investigated the T_reg 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.

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Fig. 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)

We next examined whether IC-molecules are preferentially upregulated on T_reg cells (compared to T_conv) 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⁺ T_reg cells was observed in TM and the T_reg 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⁺ T_reg cells were more abundant in the TM than in the PB or spleen (Fig. 5c). Foxp3⁺ T_reg cells in TM showed significant increases in PD-1, TIM-3, TIGIT, and CTLA-4, compared to other tissues (Fig. 5d). Moreover, tumor-infiltrating T_reg cells expressed much higher levels of IC-molecules than tumor-infiltrating T_conv (Fig. 5e). Most T_reg cells (~ 80%), but only a low frequency of T_conv (~ 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 T_reg cells before TC-1 injection and its expression progressively increased over time (Fig. 5f). Thus, expression of IC-molecules, especially PD-1, on T_reg 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).

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Fig. 5

Spatial and temporal dynamics of immune checkpoint (IC) molecule expression on T_reg 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 T_reg (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⁺ T_reg in PB, SP, and LG. e Levels of PD-1, TIM-3, TIGIT, and CTLA-4 expression on T_reg and T_conv in different tissues (PB, SP, and LG). f Changes in the levels of PD-1, TIM-3, TIGIT, and CTLA-4 expression on T_reg 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)

Immunosuppressive function of tumor-infiltrating T_reg 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 T_reg cells. To determine the role of PD-1 on tumor-infiltrating T_reg cells, in the regulation of the CD8⁺ T cell response, we compared the suppressive activity of T_reg expressing high- and low-levels of PD-1 (PD-1^hi T_reg cells from lung TM 3 weeks after TC-1 injection vs. PD-1^lo T_reg cells from the spleen of the same TM-bearing mice). CD4⁺CD25⁺ T_reg cells, isolated using a microbead-based Treg isolation kit (CD4⁺CD25⁺ Regulatory T Cell Isolation kit), was confirmed to be ~ 90% purified Foxp3⁺ T_reg 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 T_reg cells and were more potently inhibited by PD-1^hi tumor-infiltrating T_reg cells than by PD-1^lospleen T_reg cells (Fig. 6a). Similarly, interferon (IFN)-γ production was also more strongly suppressed by PD-1^hi tumor-infiltrating T_reg than by PD-1^lo spleen T_reg cells.

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Fig. 6

Enhanced suppressive function of PD-1-expressing tumor-infiltrating T_reg. a Enhanced suppression of CD8⁺ T cells by PD-1-expressing tumor-infiltrating T_reg. At 3 weeks after intravenous injection of TC-1 cells, T_reg were isolated from the spleen (SP) and lung of mice with TC-1 cell-induced tumors. SP T_reg and tumor-infiltrating T_reg 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 T_reg 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 T_reg-mediated suppression of CD8⁺ T proliferation. CTV-labeled CD8⁺ T cells were stimulated in vitro with CD3/CD28 Dynabeads and cocultured with tumor-infiltrating T_reg 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 T_reg isolated from the lungs of tumor-bearing mice 2 weeks after intravenous injection of TC-1 cells. At this time point, T_reg 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 T_reg, 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 T_reg 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)

To investigate the role of PD-1 upregulation, induced by tumor-infiltrating T_reg cells, we examined whether the interaction between PD-1 on tumor-infiltrating T_reg cells and PD-L1 on CD8⁺ T cells is required for immunosuppression in patients with cancer. PD-1 on tumor-infiltrating T_reg 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 T_reg cells_, expressing an intermediate level of PD-1 that were isolated from lung TM in 2- rather than in 3-weeks after injection because T_reg 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 T_reg cells from that of others.

Additionally, to clarify whether the potent suppressive function of PD-1^hi tumor-infiltrating T_reg cells is mediated via cell-to-cell contact between T_reg and CD8⁺ cells or soluble factors produced from T_reg 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⁺ T_reg 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 T_reg requires cell to cell contact between each cell population (Fig. 6b). Next, we performed the in vivo experiment with TIL T_reg and spleen T_reg cellsalong with Ly5.1⁺CD8⁺ T cells. In line with in vitro data, when TIL T_reg cells was injected, CD8⁺ T cell proliferation was significantly inhibited compared with spleen T_reg and no T_reg cells (Fig. 6c).

As shown in Fig. 6d, tumor-infiltrating T_reg 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 T_reg 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⁺ T_reg cells spatially interact in the TME.