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. 2019 Dec 4;7(1):339.
doi: 10.1186/s40425-019-0785-8.

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

Hye Ryun Kim  1 , Hyo Jin Park  2 , Jimin Son  2 , Jin Gu Lee  3 , Kyung Young Chung  3 , Nam Hoon Cho  4 , Hyo Sup Shim  4 , Seyeon Park  2 , Gamin Kim  1 , Hong In Yoon  5 , Hyun Gyung Kim  6 , Yong Woo Jung  6 , Byoung Chul Cho  1 , Seong Yong Park  7 , Sun Young Rha  8 , Sang-Jun Ha  9
Affiliations

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

Hye Ryun Kim et al. J Immunother Cancer. .

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.

Keywords: Immune checkpoints; Programmed cell death 1 receptor; Regulatory T cells; Tumor microenvironment.

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Conflict of interest statement

The authors declare that they have no competing interest that are relevant to this study.

Figures

Fig. 1
Fig. 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)
Fig. 2
Fig. 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)
Fig. 3
Fig. 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)
Fig. 4
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)
Fig. 5
Fig. 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)
Fig. 6
Fig. 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)
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