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Deletion of PD-1 destabilizes the lineage identity and metabolic fitness of tumor-infiltrating regulatory T cells

Nature Immunology volume 24, pages 148–161 (2023)Cite this article

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Abstract

Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP−Cre-ERT2(+/−) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.

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Fig. 1: Expression of immune checkpoint receptors on CD3+ T cells in various murine tumor models.
Fig. 2: Mice with a conditional deletion of PD-1 in Treg cells show delayed tumor growth because of a reduced TI Treg cell pool.
Fig. 3: TI PD-1cKO Treg cells are more unstable than TI PD-1Ctr Treg cells.
Fig. 4: PD-1KO Treg cells exhibit an unstable phenotype compared to PD-1WT Treg cells in the same TME.
Fig. 5: PD-1KO Treg cells are less stable and suppressive than PD-1WT Treg cells in the same TME.
Fig. 6: PD-1 therapy reduces the TI Treg pool and promotes antitumor immunity.
Fig. 7: Single-cell analysis reveals that PD-1 regulates TI Treg cell stability via the transcriptional programming of lipid metabolism.

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Data availability

Data generated in the study are included within the paper (and its extended data) or are available from the corresponding author upon reasonable request. scRNA-seq and scVDJ-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE164033. Hallmark gene sets related to oxidative phosphorylation, fatty acid metabolism are available from the MSigDB (https://www.gsea-msigdb.org/gsea/msigdb/). Source data are provided with this paper.

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Acknowledgements

This study was supported by National Research Foundation of Korea grants funded by the Korean government (2017R1A5A1014560, 2019M3A9B6065221 to S-.J.H.; 2018R1A5A2025079, 2019M3A9B6065192 to I.L.). This study was also supported by the Korean Health Technology R&D Project (HV20C0144, HN21C1410 to S-.J.H.) through the Korean Health Industry Development Institute funded by the Ministry of Health & Welfare. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Myeong Joon Kim, Kyungsoo Kim.

Authors and Affiliations

  1. Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea

    Myeong Joon Kim, Hyo Jin Park, Kyeong Hee Hong, Ji Hoon Oh, Jimin Son, Dong Jin Park, Dahae Kim & Sang-Jun Ha

  2. Brain Korea 21 (BK21) FOUR Program, Yonsei Education & Research Center for Biosystems, Yonsei University, Seoul, Republic of Korea

    Myeong Joon Kim, Kyungsoo Kim, Kyeong Hee Hong, Ji Hoon Oh, Jimin Son, Dong Jin Park, Dahae Kim, Insuk Lee & Sang-Jun Ha

  3. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea

    Kyungsoo Kim & Insuk Lee

  4. Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea

    Gil-Ran Kim & Je-Min Choi

  5. Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea

    Gil-Ran Kim & Je-Min Choi

  6. Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Republic of Korea

    Je-Min Choi

  7. Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul, Republic of Korea

    Je-Min Choi

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Contributions

M.J.K. conceived the project, designed the experiments, developed the experimental models, performed the experiments, analyzed the FACS data, wrote the manuscript and edited the manuscript. K.K. analyzed the scRNA-seq and scVDJ-seq data and edited the manuscript. H.J.P. developed the experimental models. G.-R.K. performed the EAE experiments and provided advice about the EAE experiments. K.H.H. performed the experiments. J.H.O. and J.S. provided advice for this project. D.J.P. and D.K. assisted with experiments. J.-M.C. supervised the EAE experiments and provided advice for this project. I.L. supervised the single-cell data analysis and edited the manuscript. S.-J.H. conceived the project, designed the experiments, developed the experimental models, supervised the project, wrote the manuscript and edited the manuscript.

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Correspondence to Insuk Lee or Sang-Jun Ha.

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Extended data

Extended Data Fig. 1 Establishment of mice with a conditional deletion of PD-1 in Treg cells.

a. Genetic constructs of Pdcd1fl/fl mice. b. Experimental scheme. c. Representative flow cytometry plots of CD8+ and CD4+ T cells in the PBMCs of PD-1Ctr and PD-1cKO mice treated intraperitoneally with vehicle and TAM at day 1, 3, 7, 11 and 24 post-TAM treatment. d. Kinetics of PD-1, TIM3, TIGIT, LAG3 and CTLA4 expression in T cells in the PBMCs of PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in c. e. Experimental scheme. f. Quantification of the percentages and absolute numbers of T cells in the spleen (S) and lung (L) of PD-1Ctr (n = 6) and PD-1cKO (n = 6) mice at day 12 after vehicle and TAM treatment. g. Quantification of the percentages of PD-1, TIM3, TIGIT, and LAG3 expression in CD8+ T cells, CD4+ Tconv cells, and Treg cells in the spleen and lung of PD-1Ctr (n = 6) and PD-1cKO (n = 6) mice as in f. The data were analyzed by the two-tailed unpaired Student’s t-test (f, g). All bar graphs show the mean ± s.e.m. The data are representative of two independent experiments (b-g).

Source data

Extended Data Fig. 2 Comparison of the phenotype of Treg cells between Foxp3cKO and PD-1Ctr mice.

a. Representative histograms of T cells in the spleen and lung of PD-1Ctr and PD-1cKO mice at day 12 after vehicle and TAM treatment. b. Experimental scheme. c. Quantification of the tumor nodules in the lungs of TAM-treated Foxp3cKO (n = 15) mice and vehicle-treated PD-1Ctr (n = 11) mice injected intravenously with TC-1 cells at day 20 after injection. d. Representative flow cytometry plot of CD4+ T cells in the tumor of TC-1 tumor-bearing Foxp3cKO mice and PD-1Ctr mice as in c. Quantification of the percentages of Treg cells in the tumor of TC-1 tumor-bearing Foxp3cKO (n = 15) mice and PD-1Ctr (n = 11) mice as in c. e. Representative histograms of Ki67 expression in Treg cells in the tumor of TC-1 tumor-bearing Foxp3cKO mice and PD-1Ctr mice as in c. Quantification of the percentages of Ki67+ cells in Treg cells in the tumor of TC-1 tumor-bearing Foxp3cKO (n = 15) mice and PD-1Ctr (n = 6) mice as in c. The data were analyzed by the two-tailed unpaired Student’s t-test (c, d, e). All bar graphs show the mean ± s.e.m. The data are representative of two independent experiments (a). The data were pooled from two independent experiments (c, d, e).

Source data

Extended Data Fig. 3 Conditional deletion of PD-1 in Treg cells enhanced viral control during LCMV-Clone13 infection.

a. Experimental scheme. b. Quantification of viral titer of serum of the LCMV-Cl13-infected PD-1Ctr (n = 10) and PD-1cKO (n = 10) mice as in a at day 8 and 15 after infection. Quantification of viral titer of spleen, lung, and kidney of the LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in a at day 8 and 15 after infection. The dashed line indicates the viral detection limit. c. Representative flow cytometry plots of CD8+ T cells in the PBMC, spleen, and lung of LCMV-Cl13-infected PD-1Ctr and PD-1cKO mice as in a at the indicated time point. d. Kinetics of the percentage of H-2DbGP33+CD8+ T cells and the MFIs of PD-1 expression in H-2DbGP33+CD8+ T cells in the PBMCs of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in a. Quantification of the percentage of H-2DbGP33+CD8+ T cells and the MFIs of PD-1 expression in H-2DbGP33+CD8+ T cells in the spleen, and lung of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in a at day 21 after infection. e. Representative flow cytometry plots of CD4+ T cells in the PBMC, spleen, and lung of LCMV-Cl13-infected PD-1Ctr and PD-1cKO mice as in a at the indicated time point. f. Kinetics of the percentages of Treg cells and the MFIs of Foxp3 expression in Treg cells in the PBMCs of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in a. Quantification of the percentages of Treg cells and the MFIs of Foxp3 expression in Treg cells in the spleen, and lung of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in a at day 21 after infection. g. Representative histograms of Ki67+ and annexinV+ cells in Treg cells in the spleen and lung of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in a at day 21 after infection. h. Quantification of the percentages of Ki67+ and annexinV+ cells in Treg cells in the spleen and lung of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in g. i. Representative flow cytometry plots of CD8+ T cells in the spleen and lung of LCMV-Cl13-infected PD-1Ctr and PD-1cKO mice as in a at day 15 after infection. j. Quantification of the percentages of IFN-γ+TNF+ cells in CD8+ T cells and the ratio of IFN-γ+TNF+CD8+ T cells/IFN-γ+CD8+ T cells in the spleen and lung of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in i. k. Representative flow cytometry plots of CD8+ T cells in the spleen and lung of LCMV-Cl13-infected PD-1Ctr and PD-1cKO mice as in a at day 21 after infection. l. Quantification of the percentages of IFN-γ+TNF+ cells in CD8+ T cells and the ratio of IFN-γ+TNF+CD8+ T cells/IFN-γ+CD8+ T cells in the spleen and lung of LCMV-Cl13-infected PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in k. The numbers in the plots and histograms indicate the percentage of the population (c, e, g, i, k). The data were analyzed by the two-tailed unpaired Student’s t test (b, d, f, h, j, l) and two-way ANOVA with Sidak’s post hoc test (d, f). All bar graphs show the mean ± s.e.m. The data are representative of two independent experiments. The data were pooled from two independent experiments (b, left).

Source data

Extended Data Fig. 4 TI CD8+ T cells and CD4+ Tconv cells in PD-1cKO mice are less exhausted than those in PD-1Ctr mice.

a. Experimental scheme. b. Representative histograms of PD-1 expression in CD3- cells and Treg cells and Foxp3 expression of CD4+ Tconv cells and Treg cells in the spleen and tumor of PD-1Ctr and PD-1cKO mice injected intravenously with TC-1 cells at day 20 after injection. c. Representative histograms of Treg cells in the spleen and tumor of TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. d. Representative flow cytometry plots of TI CD8+ T cells and CD4+ Tconv cells of TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. e. Quantification of the percentages of annexin V+7-AAD+ and Ki67+ cells in TI CD8+ T cells and CD4+ Tconv cells of TC-1 tumor-bearing PD-1Ctr (n = 9) and PD-1cKO (n = 9) mice as in a. f. Representative flow cytometry plots of TI CD8+ T cells of TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. Quantification of the percentages of PD-1+CD44+ and PD-1+Eomes+ cells in TI CD8+ T cells of TC-1 tumor-bearing PD-1Ctr (n = 9) and PD-1cKO (n = 9) mice as in a. g. Representative flow cytometry plots of TI CD4+ Tconv cells of TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. Quantification of the percentages of PD-1+CD44+ and PD-1+Eomes+ cells in TI CD4+ Tconv cells of TC-1 tumor-bearing PD-1Ctr (n = 9) and PD-1cKO (n = 9) mice as in a. The numbers in the plots indicate the percentage of the population (d, f, g). The numbers in the histograms indicate the percentage of positive populations or the MFIs (b, c, d). The data were analyzed by the two-tailed unpaired Student’s t-test (e, f, g). All bar graphs show the mean ± s.e.m. The data are representative of three independent experiments (b, c). The data were pooled from two independent experiments (e, f, g).

Source data

Extended Data Fig. 5 Graphical description of PD-1Cre+/− and PD-1cKORosa26tdTomato mice.

a. Graphical description of PD-1Cre+/− mice. b. Graphical description of PD-1cKORosa26tdTomato mice.

Extended Data Fig. 6 TAM-titrated TC-1 tumor-bearing Pdcd1fl/flFoxp3eGFP−Cre-ERT2 mice for ex vivo suppression assay.

a. Experimental scheme. b. Representative flow cytometry plots of CD4+ T cells in the spleen and tumor of naïve and TC-1 tumor-bearing (as in a.) Pdcd1fl/flFoxp3eGFP−Cre-ERT2 mice at day 20 post-TC-1 intravenous injection on day 8 post-1mg of TAM treatment. c. The purity of sorted CD25+PD-1+ and CD25+PD-1- Treg cells in the tumor of TC-1 tumor-bearing Pdcd1fl/flFoxp3eGFP−Cre-ERT2 mice as in a.

Extended Data Fig. 7 PD-1 expression before tumor inoculation is crucial for maintaining the proliferation of TI Treg cells.

a. Experimental scheme. b. Quantification of tumor nodules and the ratio of CD8+ T cells/Treg cells in the tumor of the indicated TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a at day 20 after injection. c. Representative flow cytometry plots of CD4+ T cells in the spleen and tumor of the indicated TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. The numbers in the plots indicate the percentage of the population. Quantification of the percentages of Treg cells in the spleen and tumor of the indicated TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. d. Representative histograms of Ki67 expression in Treg cells in the spleen and tumor of the indicated TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. The numbers in the histograms indicate the percentage of positive populations. Quantification of the percentages of Ki67+ cells in Treg cells in the spleen and tumor of the indicated TC-1 tumor-bearing PD-1Ctr and PD-1cKO mice as in a. Tx → TC-1 group; PD-1Ctr mice; n = 5, PD-1cKO mice; n = 5 ((b, left), d). Tx → TC-1 group; PD-1Ctr mice; n = 9, PD-1cKO mice; n = 9 ((b, right), c). TC-1 → Tx D2 ~ D6 group; PD-1Ctr mice; n = 6, PD-1cKO mice; n = 3, TC-1 → Tx D9 ~ D13 group; PD-1Ctr mice; n = 5, PD-1cKO mice; n = 5 (b, c, d). The data were analyzed by the two-tailed unpaired Student’s t-test (b, c, d). All bar graphs show the mean ± s.e.m. The data of Tx → TC-1 were pooled from two independent experiments (b, c). The data are representative of three (a-d) independent experiments.

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Extended Data Fig. 8 PD-1 expression is important for naïve Treg cells activation and proliferation.

a. Representative flow cytometry plots and histograms of Treg cells in the spleen of naïve TAM-treated PD-1Cre+/− mice at day 12 after treatment cultured with CD3 antibodies in the presence of dendritic cells for 3 days. b. Quantification of the percentage of CTV- and Ki67+ cells in PD-1WT and PD-1KO Treg cells (n = 3) cultured as in a. c. Representative histograms of Treg cells cultured as in a. d. Quantification of the percentage of TIM3, TIGIT, LAG3 (n = 3), CD103, and CD69 (n = 4) expression and the MFIs of Foxp3, CD25, CD44 (n = 3), CTLA4, and ICOS (n = 4) expression in cultured Treg cells. e. Representative histograms of Treg cells cultured as in a. f. Quantification of the MFIs of Blimp1 and GATA3 expression and the percentage of T-bet and TCF1 expression in cultured Treg cells (n = 4). g. Representative flow cytometry plots of Treg cells cultured as in a with isotype and PD-1 antibody. Quantification of the percentage of CTV- cells in the indicated Treg cells. The numbers in the plots and histograms indicate the percentage of the population or the MFIs. The data were analyzed by the two-tailed unpaired Student’s t-test (b, d, f, g). All bar graphs show the mean ± s.e.m. The data are representative of two independent experiments.

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Extended Data Fig. 9 Loss of PD-1 in Treg cells delays EAE progression by inducing excessive lung inflammation.

a, b. The disease score (a) assessed by ref. 24 and ref. 31. and body weight (b) of PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice immunized with MOG peptide in CFA emulsion at day 12 post-TAM treatment. c. Representative flow cytometry plots of CD4+ T cells and quantification of the percentages of CD4+ T cells and Treg cells and absolute number of CD4+ T cells, CD4+ Tconv cells, and Treg cells in the spinal cord of PD-1Ctr and PD-1cKO mice as in a at day 19 after immunization. PD-1Ctr (n = 12), PD-1cKO (n = 14) mice. d. Representative histograms of Foxp3 expression in Treg cells quantification of the MFI of Foxp3 expression in Treg cells in the spinal cord of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. The numbers in the histogram indicate the MFI. e. Representative flow cytometry plots of CD4+ Tconv cells and quantification of the percentages and absolute number of Tetramer+CD4+ Tconv cells in the spinal cord of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. f. Representative flow cytometry plots of CD4+ Tconv cells and quantification of the percentage of IFN-γ+ and/or IL-17+ cells in CD4+ Tconv cells in the spinal cord of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. g. Images of H&E staining of the lung of PD-1Ctr (n = 5) and PD-1cKO (n = 5) mice as in c. Scale bars: 200 μm. h. Representative flow cytometry plots of CD8+ and CD4+ T cells and quantification of the percentages of CD8+ T cells, CD4+ Tconv cells, and Treg cells in the lung of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. i. Representative flow cytometry plots of CD62L and CD44 expression in CD4+ Tconv cells and quantification of the percentages of CD44+ cells in CD4+ Tconv cells in the lung of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. j. Representative flow cytometry plots of CD4+ Tconv cells and quantification of the percentages and absolute number of Tetramer+CD4+ Tconv cells in the lung of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. k. Representative flow cytometry plots of CD4+ Tconv cells and quantification of the percentage of IFN-γ+ and/or IL-17+ cells in CD4+ Tconv cells in the lung of PD-1Ctr and PD-1cKO mice as in c. PD-1Ctr (n = 5), PD-1cKO (n = 5) mice. The numbers in the plots indicate the percentage of the population (c, e, f, h, i, j, k). The data were analyzed by the two-tailed unpaired Student’s t-test (c, d, e, f, h, i, j, k) and two-way ANOVA with Sidak’s post hoc test (a, b). All bar graphs show the mean ± s.e.m. The data were pooled from two independent experiments (c). The data are representative of two independent experiments (a, b, d–k).

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Extended Data Fig. 10 Deletion of PD-1 in TI Treg cells decreases lipid uptake and mitochondrial mass.

a. Experimental scheme of single-cell analysis. b. UMAP visualization for TI Treg cells of PD-1Cre+/− mice at day 14 post-intravenous injection of TC-1 cells on day 12 post-TAM treatment from the replicate scRNAseq experiments. Different subsets of TI Treg cells in PD-1Cre+/− mice as in b are indicated by color codes. c. UMAP visualization and Contour plot for TI Treg cells in PD-1Cre+/− mice as in b from the replicate scRNAseq experiments. Different subsets of TI Treg cells in PD-1Cre+/− mice as in b are indicated by color codes. d. UMAP visualization of TI Treg cells in PD-1Cre+/− mice as in b from the replicate scRNAseq experiments with an indication of a unique or expanded clonotype. e. The proportion of expanded clonotypes in each TI Treg cell subset in PD-1Cre+/− mice as in b from the replicate scRNAseq experiments. f. Volcano plot of Gene set enrichment analysis (GSEA) between isotype PD-1WT and isotype PD-1KO Treg cells in PD-1Cre+/− mice as in b using MSigDB hallmark gene sets. g. The expression of the indicated gene sets in isotype PD-1WT and isotype PD-1KO Treg cells h. Representative flow cytometry plots of Treg cells in the tumors of PD-1Cre+/− mice injected intravenously with TC-1 cells at day 20 injection. The numbers in the histograms indicate the MFIs. Quantification of the MFIs of lipid uptake and mitochondrial mass in Treg cells in the TC-1 tumor-bearing PD-1Cre+/− mice (n = 6). The data were analyzed by two-tailed unpaired Student’s t-test (h). All bar graphs show the mean ± s.e.m. The data are representative of three independent experiments.

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Supplementary information

Reporting Summary

Supplementary Table

The list of differentially expressed genes between PD-1WT and PD-1KO Treg cells in the same tumor of isotype-treated TC-1 tumor-bearing (as in Extended Data Fig. 10a) PD-1Cre+/− mice at day 14 after injection. Significance of difference in this table was determined by two-sided Wilcoxon rank-sum test.

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Kim, M.J., Kim, K., Park, H.J. et al. Deletion of PD-1 destabilizes the lineage identity and metabolic fitness of tumor-infiltrating regulatory T cells. Nat Immunol 24, 148–161 (2023). https://doi.org/10.1038/s41590-022-01373-1

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