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Dynamic CD4+ T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection

Nature Immunology volume 22, pages 1524–1537 (2021)Cite this article

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Abstract

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4–TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.

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Fig. 1: PD-L1 blockade specifically amplifies and functionally enhances CD4+ TH1 cells.
Fig. 2: Pretherapy cycling CD4+ SMARTA T cells are expanded upon PD-L1 blockade.
Fig. 3: PD-L1 blockade specifically expands and activates TH1-like Treg cells.
Fig. 4: PD-L1 blockade expands and induces tissue infiltration of Treg cells in nonlymphoid organs.
Fig. 5: PD-L1 blockade enhances TH1 gene programs and terminal differentiation of TH1 cells.
Fig. 6: PD-L1 blockade reorients intracellular interferon and TCR signaling.
Fig. 7: PD-L1 blockade restores virus-specific CD4+ CTL function.

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Data availability

The RNA-seq data generated in this paper have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE163345. Source data are provided with this paper.

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Acknowledgements

We thank past and present members of the Brooks laboratory for technical help and discussion. This work was supported by the Canadian Institutes of Health Research (CIHR) Foundation Grant FDN148386 (D.G.B.), the National Institutes of Health (NIH) grant AI085043 (D.G.B.), the Scotiabank Research Chair to D.G.B, and a training grant from the Fonds de la Recherche en Santé du Québec (L.M.S.).

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Authors and Affiliations

  1. Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada

    Laura M. Snell, Wenxi Xu, Diala Abd-Rabbo, Giselle Boukhaled, Mengdi Guo, Bethany L. Macleod, Heidi J. Elsaesser, Kebria Hezaveh, Nirmin Alsahafi, Sabelo Lukhele, Ramanandan Prabhakaran, Tracy L. McGaha & David G. Brooks

  2. Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA

    Laura M. Snell

  3. Department of Immunology, University of Toronto, Toronto, ON, Canada

    Mengdi Guo, Slava Epelman, Tracy L. McGaha & David G. Brooks

  4. Toronto General Hospital Research Institute, University Health Network (UHN), Toronto, ON, Canada

    Sara Nejat & Slava Epelman

  5. Peter Munk Cardiac Centre, Ted Rogers Centre for Heart Research, Toronto, ON, Canada

    Slava Epelman

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Contributions

L.M.S. and D.G.B. designed the research. L.M.S., W.X., M.G., B.L.M., H.J.E., S.L., K.H. and N.A. performed the experiments. L.M.S., D.A.-R., G.B., S.N., and R.P. analyzed the data. S.E. and T.L.M. contributed technical expertise and discussion. L.M.S. and D.G.B. wrote the paper.

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Correspondence to Laura M. Snell or David G. Brooks.

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Peer review information Nature Immunology thanks Robert Thimme, Santosha Vardhana and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 CyTOF gating scheme.

Plots show the successive gating scheme for (a) SMARTA CD4 + T cells and (b) Treg cells.

Extended Data Fig. 2 Enhancement of virus-specific CD4+ TH1 cells by PD-L1 blockade occurs prior to decreased virus titers.

Virus-specific CD4+ SMARTA T cells were transferred into naïve mice which were subsequently infected with LCMV-Cl13 a day later. Twenty-five days after infection mice were treated with either isotype or anti-PD-L1 antibody and subsequently every 3 days for a total of 3 treatments. Mice were sacrificed 60 h following the first treatment or two days after the third treatment and splenic virus-specific CD4+ SMARTA T cells were analyzed. (a) Longitudinal plasma virus titers pre-treatment and then following one (n = 5) or three (n = 5) isotype or anti-PD-L1 treatments. (b) Log fold change of abundance of CD4+ SMARTA T cell PhenoGraph clusters at 60 h after the first antibody treatment. P-values of cluster abundances are calculated by the edge R test in the diffcyt R package. *, p < 0.05. (c) Total virus-specific CD4+ SMARTA T cell numbers at 60 h after the first isotype (n = 5) or anti-PD-L1 (n = 5) treatment. (d) Proportions and total number of endogenous (that is, non-SMARTA) TH1 cells (CD39 hi) vs TCF-1+ cells after the first isotype (n = 5) or anti-PD-L1 (n = 5) treatment. Foxp3+ Treg cells and SMARTA cells were gated out and plots are gated on the remaining CD4+ PD-1+ (activated) cells. (e) Proportion of virus-specific CD4+ T cells producing IFN-γ, TNF, and IL-10 following ex vivo LCMV-GP61–80 peptide stimulation. Data are from 60 h after the first isotype (n = 5) or anti-PD-L1 (n = 5) treatment. (f) Kinetics of CD4+ SMARTA T cells in each PhenoGraph cluster between 1 and 3 isotype or anti-PD-L1 treatments. (g) Total number of CD4+ SMARTA TH1 (TCF-1-negative) and TCF-1+ virus-specific T cells in liver and lung following 3 anti-PD-L1 treatments. (a–c) n = 5 mice per group examined over four experiments totalling n = 19 isotype and n = 20 anti-PDL1 mice. (d–f) n = 5 mice per group examined over 2 experiments totaling 10 mice per group. Data in (G) is cumulative of 2 experiments with n = 11 for isotype mice and n = 12 for anti-PDL1 treatment groups. *: p < 0.05. unpaired, two-tailed Student’s t-test (a, c–g). Box plots indicate the median, upper and lower quartile, and the whiskers show the high and low value. Line graphs show the average and error bars indicate the standard deviation (SD).

Source data

Extended Data Fig. 3 Pre-therapy cycling CD4+ SMARTA T cell populations are targets of anti-PD-L1 blockade.

(a) Diffusion pseudotime map of CD4+ SMARTA T cells colored by pseudotime starting at Tip (T)3. T1 – T3 designate the algorithm-derived cellular tips and the lines and arrows the pathways derived from T3 as a starting point. (b) Representative flow plots of Ki67+ SMARTA cells following 1 treatment in spleen. (c) UMAPs depict Ki67 expression in CD4+ SMARTA T cells and the graph the proportion of Ki67+ of SMARTA cells in each cluster following the third isotype or anti-PD-L1 treatment. (d) CD4+ SMARTA T cells in the spleen 10 days after LCMV-Cl13 infection mice. (Top) UMAP and heatmap of CyTOF data of CD4 + SMARTA T cells clustered with PhenoGraph. (Bottom) UMAPs illustrating the single cell expression of the indicated protein. The graph shows proportion of SMARTA cells that are Ki67+ in each PhenoGraph cluster. (a) n = 5 mice per group and are representative of total n = 9 mice examined over two experiments. (b, c) n = 5 mice per group and are representative of total n = 10 mice per group over 2 experiments. (d) n = 4 mice per group and representative of n = 9 mice total over 2 experiments. *: p < 0.05. unpaired, two-tailed Student’s t-test between isoytpe and aPD-L1 of each cluster (c), or between clusters 1 (Tfh) and Th1 clusters 4 and 6 (d). Box plots indicate the median, upper and lower quartile, and the whiskers show the high and low value.

Source data

Extended Data Fig. 4 PD-L1 blockade targets cycling, TH1-phenotype Treg cells.

(a) Total number of Foxp3+ Treg cells in each cluster at 60 h after the first isotype or anti-PD-L1 treatment. (b) Representative 2D CyTOF plots of T-bet, Bcl-6 and TCF-1 expression in total Treg cells. (c) To deplete Treg cells, LCMV-Cl13 infected Foxp3DTR mice were treated with DT or as a control PBS on days 23, day 24, day 27 and day 30 after infection. Mice were sacrificed at day 33 and splenocytes isolated for flow cytometric analysis. Representative FACS plots depict SLAM vs GzmB expression in splenic Foxp3-negative effector CD4+ T cells. Box plots depict total numbers of TH1, TFH and GzmB+ Foxp3-negative CD4+ T cells. (d) Total number of Foxp3+ Treg cells in each cluster following 3 treatments. (e) The colors in the heatmap designate the arcsinh ratio of the MSI change for the indicated protein within each PhenoGraph cluster of Treg cells. The graph is comparing protein expression in Foxp3+ Treg cells following the third anti-PD-L1 versus isotype treatment, with (red) increased with anti-PD-L1 treated mice or (blue) increased in isotype-treated mice. P-values are calculated by the limma test in the diffcyt R package. *, p-value<0.05. (f) UMAPs and bar graph depict the expression and proportion of Ki67+ cells in each Treg PhenoGraph cluster following 3 anti-PD-L1 or isotype treatments. (g) Representative protein expression plots gated on Ki67+ Treg cells in spleens of mice following 3 anti-PD-L1 or isotype treatments. (a) n = 5 mice in the isotype group and n = 4 in the anti-PDL1 group. The experiment is representative of n = 18 isotype and n = 16 anti-PDL1 mice examined over three experiments. (c) n = 5 mice in the PBS group and n = 7 mice in the DT group. The experiment is representative of n = 8 mice in the isotype and n = 11 mice in the DT group examined over two experiments. (d–g) n = 5 mice per group.The experiment is representative of n = 15 mice per group examined over three experiments. *: p < 0.05; (unpaired, two-tailed Student’s t-test (a, c, d, f). Box plots indicate the median, upper and lower quartile, and the whiskers show the high and low value.

Source data

Extended Data Fig. 5 Single cell transcriptomic analyses of virus-specific CD4+ T cells following PD-L1 blockade.

(a) Serum Viral Titers in mice prior and post 3 anti-PD-L1 treatments. Line graph indicates average +/-SD of four mice per group. Data are representative of 1 single cell experiment with a total of n = 4 mice per group. Unpaired t-test was used to compare isotype pre and post therapy, and between PDL1 pre and post therapy. (b, c) Data are from one scRNA-seq experiment comprised of pooled cells from four individual mice per group. SMARTA cells were FACS-sorted following the third antibody treatment and analyzed by scRNA-seq. (b) Bar graph depicts differential expression of genes in sorted SLAMF1+ TH1 versus CXCR5+ TFH SMARTA cells from bulk RNA-seq at day 7 after LCMV-Armstrong infection. (c) UMAPs show IFNγ and IL10 gene expression.

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Extended Data Fig. 6 Pathway analysis of CD4+ SMARTA T cells following PD-L1 blockade.

(a) Histograms depict Log10 transformed RNA expression of the ISGs Irf7 and Oas1a, and TH1-related genes Ifngr1 and Ccl5 in total virus-specific SMARTA CD4+ T cells, in the TH1 c2, c3, and in the TFH c5 from the scRNA-seq data from Fig. 5. Numbers in each histogram represent the percent of cells expressing the RNA. Blue histograms are anti-PD-L1 treated and those outlined in black are isotype treated. (b) Box plots indicate the GMFI of Nur77 protein expression in SMARTA cells 60 h after the first isotype (n = 5) or anti-PD-L1 (n = 6) antibody treatment. Data are representative of two experiments totaling 11 isotype and 12 anti-PD-L1 treated mice. *: p < 0.05, unpaired, two-tailed Student’s t-test. (c) The graph shows gene set enrichment analysis (GSEA) of differentially expressed pathways upregulated in CD4+ SMARTA T cells in anti-PD-L1 treatment vs isotype treatment. Pathways are colored by normalized enrichment score (NES). Data are representative of one scRNA-seq experiment comprised of pooled cells from four individual mice per group (Isotype or anti-PD-L1).

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Extended Data Fig. 7 Cytotoxic gene expression in all SMARTA clusters from single cell analysis.

Violin plots depict expression of genes associated with cytotoxic function in all clusters following isotype and anti-PD-L1 treatment. Data are representative of one scRNA-seq experiment comprised of pooled cells from four individual mice per group.

Extended Data Fig. 8 Flow gating scheme for sorting.

Plots show the successive gating scheme for the sorting of SMARTA CD4 + T cells.

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Snell, L.M., Xu, W., Abd-Rabbo, D. et al. Dynamic CD4+ T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection. Nat Immunol 22, 1524–1537 (2021). https://doi.org/10.1038/s41590-021-01060-7

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