Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy

Abstract

Chronic viral infections are characterized by a state of CD8⁺ T-cell dysfunction that is associated with expression of the programmed cell death 1 (PD-1) inhibitory receptor. A better understanding of the mechanisms that regulate CD8⁺ T-cell responses during chronic infection is required to improve immunotherapies that restore function in exhausted CD8⁺ T cells. Here we identify a population of virus-specific CD8⁺ T cells that proliferate after blockade of the PD-1 inhibitory pathway in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). These LCMV-specific CD8⁺ T cells expressed the PD-1 inhibitory receptor, but also expressed several costimulatory molecules such as ICOS and CD28. This CD8⁺ T-cell subset was characterized by a unique gene signature that was related to that of CD4⁺ T follicular helper (T_FH) cells, CD8⁺ T cell memory precursors and haematopoietic stem cell progenitors, but that was distinct from that of CD4⁺ T_H1 cells and CD8⁺ terminal effectors. This CD8⁺ T-cell population was found only in lymphoid tissues and resided predominantly in the T-cell zones along with naive CD8⁺ T cells. These PD-1⁺CD8⁺ T cells resembled stem cells during chronic LCMV infection, undergoing self-renewal and also differentiating into the terminally exhausted CD8⁺ T cells that were present in both lymphoid and non-lymphoid tissues. The proliferative burst after PD-1 blockade came almost exclusively from this CD8⁺ T-cell subset. Notably, the transcription factor TCF1 had a cell-intrinsic and essential role in the generation of this CD8⁺ T-cell subset. These findings provide a better understanding of T-cell exhaustion and have implications in the optimization of PD-1-directed immunotherapy in chronic infections and cancer.

Extended Data Figure 1. LCMV GP276-specific CD8⁺ T cells also consist of CXCR5⁺ and CXCR5⁻ CD8⁺ T cell subsets during chronic infection

a, Phenotypic analysis of GP276-specific CD8⁺ T cells in the spleens of immune mice that had cleared an acute LCMV Armstrong infection or mice that were chronically infected with LCMV clone 13 (day 30 after infection (p.i.)). FACS plots showing CXCR5 expression in combination with the indicated markers are gated on GP276 tetramer⁺ CD8⁺ T cells. b, Longitudinal analysis of the numbers of GP276-specific CXCR5⁺Tim-3⁻ and CXCR5⁻Tim-3⁺ CD8⁺ T cells in the spleen at the indicated time after infection. LCMV titers in the serum are shown as the shaded yellow area. Graph shows the mean ± s.e.m. Data are the average of 8 mice from two experiments per time point (total n=48). c, Phenotypic characterization of CXCR5⁺ and CXCR5⁻ GP276-specific CD8⁺ T cells in the spleen of chronically infected mice (>45 days p.i.).

Extended Data Figure 2. Low dose challenge with LCMV clone 13 results in acute infection and does not generate CXCR5⁺ CD8⁺ T cells

Mice were infected with either low dose (2×10² PFU) or high dose (2×10⁶ PFU) of LCMV clone 13, and the generation of CXCR5⁺ CD8⁺ T cells was examined at day 8 and day 35 p.i. a, Serum virus titers at day 8 after infection. b, Representative flow plots of CXCR5 and Tim-3 expression on GP33-specific CD8⁺ T cells in the spleen at day 8 and 35 p.i. Data are obtained from a total of 16 mice with 4 mice per group at each time point. Student’s t-test, where ** P < 0.01.

Extended Data Figure 3. Distinct transcriptional profiles of CXCR5⁺ and CXCR5⁻ CD8⁺ T cells from spleens of mice chronically infected with LCMV

a, Principal component analysis of naïve (CD44^lo) CD8⁺ T cells isolated from uninfected mice and CXCR5⁺Tim-3⁻PD-1⁺ and CXCR5⁻Tim-3⁺PD-1⁺ CD8⁺ T cells isolated from chronically infected mice (>45 days p.i.). Each square represents an individual biological replicate. b, Relative expression of selected genes as determined by Affymetrix microarray analysis. Data are shown as fold change relative to naïve (CD44^lo) CD8⁺ T cells. Graphs show the mean ± s.e.m.

Extended Data Figure 4. LCMV-specific CXCR5⁺ CD8⁺ T cells are more polyfunctional than CXCR5⁻ CD8⁺ T cells

Splenocytes from chronically infected mice (>45 days p.i.) were stimulated with GP33–41 peptide for 5 h followed by phenotypic marker staining and intracellular staining. a, Gating strategy for IFNγ⁺PD-1⁺CXCR5⁺Tim-3⁻ and IFNγ⁺PD-1⁺CXCR5⁻Tim-3⁺ CD8⁺ T cells. b, Representative FACS plots and graph showing the frequency of TNFα- and IL-2-producing cells among IFN-γ⁺ CD8⁺ T cells. Data are representative of 2 experiments (n=4 per experiment). Student’s paired t-test, where ** P < 0.01.

Extended Data Figure 5. Analysis of reactome pathways, mTOR signalling and fatty acid metabolism in CXCR5⁺ and CXCR5⁻ CD8⁺ T cells from LCMV chronically infected mice

a. Reactome pathways in CXCR5⁺Tim-3⁻PD-1⁺ and CXCR5⁻Tim-3⁺PD-1⁺ CD8⁺ T cells isolated from the spleens of mice chronically infected with LCMV (>45 days p.i.). GSEA (nominal P<0.01; 1,000 permutations) was used to identify positive (red, maximum normalized enrichment score (NES) = 3.2) or negative (blue, min NES = −3.7) enrichment of Reactome pathways (http://www.reactome.org/) in CXCR5⁺Tim-3⁻PD-1⁺ and CXCR5⁻Tim-3⁺PD-1⁺ CD8⁺ T cells using meta-analysis. The size of the circles (nodes) represents the number of genes on each pathway. The links between circles (edges) represent the number of genes shared by two given pathways. The networks were generated using Cytoscape. b, GSEA on mTOR signalling and fatty acid metabolism. Bars represent pathways with nominal P-value < 0.01. c,d, Splenocytes from chronically infected mice (>45 days p.i.) were stimulated with medium or GP33–41 peptide for 1 h followed by phenotypic marker staining and phosphorylated S6 ribosomal protein (pS6) staining. Flow cytometry analysis (c) and MFI (d) of pS6 expression in CXCR5⁺ and CXCR5⁻ CD8⁺ T cell subsets after ex vivo stimulation. Data are representative of 2 experiments (n=4 per experiment). Student’s paired t-test, where * P < 0.05.

Extended Data Figure 6. Comparison of gene signatures of CXCR5⁺ and CXCR5⁻ CD8⁺ T cell subsets from chronically infected mice with Id2^−/−Id3^−/− innate T_FH-like CD4⁺ T cells

a, GSEA was performed using genes pre-ranked by the mean Z-score values of each CD8⁺ subset (naive, CXCR5⁺ or CXCR5⁻) calculated across all samples. Splenic CD4⁺ T_FH gene signatures from wild-type mice and thymic innate variant T_FH gene signatures from Id2^−/−Id3^−/− mice (GSE64779) were used as gene sets in our GSEA. Genes were considered up- or downregulated in cell subsets compared to control (sorted CD4⁺TCRβ⁺CD8^– cells) if there was a fold-change > 2 and P < 0.05. b, Heat map illustrating the relative expression of the indicated genes of Id2^−/−Id3^−/− CD4⁺ T cells defined in ref. compared to those of naïve, CXCR5⁺ and CXCR5⁻ CD8⁺ T cells. GSEA analysis revealed some interesting similarities and differences between the CXCR5⁺ CD8⁺ T cells from chronically infected mice and the Id2^−/−Id3^−/− T_FH-like CD4⁺ T cells. These two cell populations are distinct, but share certain biological properties such as increased self-renewal activity. For example, some of the interesting inhibitory and costimulatory molecules such as Pdcd1 (PD-1), Tnfsf14 (LIGHT), Cd28, and Icos were commonly upregulated in both CXCR5⁺ CD8⁺ and innate T_FH-like CD4⁺ T cells, whereas molecules like Cd244 (2B4), Prf1, Fasl and Gzmb were downregulated in both cell types. However, there were also many differences perhaps the most notable being the low expression of Tcf7 (TCF1) in the innate CD4⁺ T cells compared to the high expression of Tcf7 in the CXCR5⁺ CD8⁺ T cells and the critical role of this transcription factor in the generation of these cells. Notably, the CD4⁺ T cell population defined in ref. is genetically deficient in both Id2 and Id3, whereas the CXCR5⁺ CD8⁺ T cells express high levels of Id3. Thus, many aspects of the transcriptional program of these two cell types will be distinct.

Extended Data Figure 7. Tissue distribution of LCMV-specific CXCR5⁺ and CXCR5⁻ CD8⁺ T cells in chronically infected mice

a,b, Frequency (a) and numbers (b) of LCMV-specific CXCR5⁺ and CXCR5⁻ CD8⁺ T cells in the indicated tissues at >45 days p.i. in chronically infected mice (SP, spleen; BM, bone marrow; LN, mesenteric lymph nodes; IEL, intestinal epithelial lymphocytes). c,d, Representative FACS plots (c) and graph (d) showing the frequency of CXCR5⁺Tim-3⁻ GP33-specific CD8⁺ T cells in the blood and spleen at day 8 and day 30 p.i. FACS plots are gated on D^bGP33 tetramer⁺ CD8⁺ T cells. Data are obtained from 4 or 8 mice. Graphs show the mean ± s.e.m. Student’s t-test, where **P < 0.01; *P < 0.05.

Extended Data Figure 8. Anatomic localization of CXCR5⁺ and CXCR5⁻ CD8⁺ T cells in the spleen and in vitro migration of the two CD8⁺ T cell subsets to CXCL13

a, Analysis of TCF-1 and PD-1 expression in CD8⁺ T cell subsets in the spleen of chronically infected mice (>45 days p.i.). Left and middle FACS plots are gated on total CD8⁺ T cells and PD-1⁺ CD8⁺ T cells, respectively. Right FACS plot displays the overlay of TCF-1 and PD-1 expression in gated PD-1⁻, PD-1⁺CXCR5⁺Tim-3⁻ and PD-1⁺CXCR5⁻Tim-3⁺ CD8⁺ T cells. b,c, Nuclear histocytometry analysis of the spleen (b) and frequency (c) of the CD8⁺ T cell subsets within the respective zones of the spleen 10 days after LCMV clone 13 infection (n=3). d, Representative immunofluorescence staining of LCMV clone 13 infection in the spleen (>45 days p.i.). The spleen was stained for LCMV antigen (red), IgD (blue) and CD3 (white) and examined via microscopy (n=4). Note, LCMV infection is mostly in the red pulp. e, CXCR5 expression on splenic naïve (CD44^lo) CD8⁺ T cells, B cells and CXCR5⁺ GP33-specific CD8⁺ T cells from chronically infected mice (>45 days p.i.). f, Relative migration of sorted B cells and CXCR5⁺Tim-3⁻PD-1⁺ and CXCR5⁻Tim-3⁺PD-1⁺ CD8⁺ T cell subsets in response to CXCL13, which is a ligand for CXCR5. Data are combined from two experiments performed in duplicate wells for each sample. Graphs show the mean and s.e.m. Student’s t-test, where **P < 0.01; *P < 0.05.

Extended Data Figure 9. CXCR5⁺ CD8⁺ T cells selectively undergo proliferation after LCMV clone 13 challenge

a, Sorted PD-1⁺CXCR5⁺Tim-3⁻ and PD-1⁺CXCR5⁻Tim-3⁺ CD8⁺ T cells isolated from CD45.2⁺ chronically infected mice (>45 days p.i.) were adoptively transferred into naive CD45.1⁺ recipient mice, followed by LCMV clone 13 challenge. Two sets of adoptive-transfer experiments were performed; one using a low dose of donor cells (2,500 cells) and another with a large dose of donor cells (90,000 cells). The data shown in Fig. 3d, e are from the high-dose transfer experiment. Data in b–d are from the high-dose transfer; and in e–h are from the low-dose transfer. b, Expansion of CXCR5⁺ CD8⁺ T cells in the blood after LCMV clone 13 infection. c, Number of cells in the spleen and liver 14 days after infection. d, Phenotypic analysis of transferred donor CXCR5⁺Tim-3⁻ cells in the spleen 14 days after challenge showing proliferation and differentiation of this subset. Data are representative of 3 experiments (n=4 or 6 mice per experiment). e, Expansion of CXCR5⁺ CD8⁺ T cells (low dose transfer) in the blood after infection with LCMV clone 13. f, Number of cells in the spleen and liver 14 days after challenge. g, Phenotypic analysis of transferred donor CXCR5⁺Tim-3⁻ and CXCR5⁻Tim-3⁺ cells in the spleen and liver at 14 days after infection showing differentiation of CXCR5⁺ CD8⁺ T cells. h, Number of CXCR5⁺Tim-3⁻ and CXCR5⁻Tim-3⁺ CD8⁺ T cells derived from donor CXCR5⁺ or CXCR5⁻ CD8⁺ T cells in the spleen and liver after clone 13 challenge. Dashed line indicates the limit of detection. Data are combined from two experiments (n=8 or 10 per group, total n=18). Graph shows the mean and s.e.m. Student’s t-test, where ** P < 0.01.

Extended Data Figure 10. Enhanced conversion of CXCR5⁺Tim-3⁻ CD8⁺ T cells to Tim-3⁺ CD8⁺ T cells after PD-1 blockade

a, Sorted PD-1⁺CXCR5⁺Tim-3⁻ and PD-1⁺CXCR5⁻Tim-3⁺ CD8⁺ T cells isolated from CD45.2⁺ chronically infected mice (>45 days p.i.) were adoptively transferred into infection-matched CD45.1⁺ recipient mice, followed by treatment with anti-PD-L1 antibody. b, Phenotypic analysis of sorted donor CD8⁺ T cell subsets before transfer and 14 days after the transfer followed by PD-1 blockade. Data are representative of 2 experiments (total n=5, 7, or 9 mice per group).

Figure. 1. Identification of a population of PD-1⁺ CD8⁺ T cells during chronic LCMV infection that has a unique gene signature that resembles both CD4⁺ T_FH cells and CD8⁺ memory precursor T cells

a, Phenotypic analysis of GP33-specific CD8⁺ T cells in the spleens of mice infected with LCMV Armstrong (acute) or clone 13 (chronic) at day 30 post infection (p.i.). b, Longitudinal analysis of the numbers of GP33-specific CXCR5⁺ and CXCR5⁻ CD8⁺ T cells in the spleens of chronically infected mice (n=8 from two experiments per time point). Graph shows the mean and s.e.m.. c, Heat map illustrating the relative expression of genes in naïve CD8⁺ T cells from uninfected mice and CXCR5⁺Tim-3⁻PD-1⁺ and CXCR5⁻Tim-3⁺PD-1⁺ CD8⁺ T cells from chronically infected mice (>45 days p.i.). d, Phenotypic characterization of CXCR5⁺ and CXCR5⁻ GP33-specific CD8⁺ T cells in the spleens of chronically infected mice (>45 days p.i.). e, GSEA for identifying specific gene signatures of the two CD8⁺ T cell subsets from chronically infected mice compared to gene signatures of different subsets of CD4⁺ and CD8⁺ T cells and haematopoietic stem cells. HSC, haematopoietic stem cell; NES, normalized enrichment score.

Figure 2. CXCR5⁺ PD-1⁺ CD8⁺ T cells are found in lymphoid tissues of chronically infected mice and reside predominantly in T cell zones

a, Frequency of PD-1⁺CXCR5⁺Tim-3⁻ and PD-1⁺CXCR5⁻Tim-3⁺ LCMV-specific (GP33+GP276) CD8⁺ T cells in the indicated tissues (45 days p.i.). b, Representative histology of the spleen and nuclear histocytometry analysis to identify the anatomic location of the two CD8⁺ T cell subsets in chronically infected mice. c, Zoomed-in panels for the T cell zone and the red pulp. T, T-cell zone; R, red pulp; B, B-cell zone. d, Frequency of the CD8⁺ T cell subsets within the respective zones. Data are representative of 3 experiments (n=3 per experiment). e, In vivo CD45.2 labelling of CXCR5⁺ and CXCR5⁻ GP33-specific CD8⁺ T cells in spleen, 3 min after injection (n=4). f, Relative gene expression of Ccr7 mRNA in sorted CD8⁺ T cell subsets. g, Migration of sorted CD8⁺ T cell subsets in response to CCL19 and CCL21. Data are combined from 2 experiments done in duplicate wells. h, CXCR5 and CD69 expression on GP33-specific CD8⁺ T cells. Graph shows the mean and s.e.m.. Student’s t-test, where **P< 0.01; *P< 0.05. BM, bone marrow; mLN, mesenteric lymph nodes; IELs, intestinal epithelial lymphocytes; AU, arbitrary units.

Figure 3. CXCR5⁺ CD8⁺ T cells act as stem cells during chronic LCMV infection and respond to PD-1 blockade

a, Experimental set-up showing transfer of congenically marked CTV-labelled PD-1⁺CXCR5⁺Tim-3⁻ and PD-1⁺CXCR5⁻Tim-3⁺ CD8⁺ T cells from chronically infected mice into infection-matched recipient mice. b, Proliferation and differentiation of CXCR5⁺ CD8⁺ T cells in spleen and liver after adoptive transfer. c, FACS plots of donor CD8⁺ T cells in the spleen. Data are representative of 3 experiments (total n=7 or 14 mice per group). d, Representative FACS plots showing expansion of donor CXCR5⁺ CD8⁺ T cells after transfer into naïve mice followed by LCMV clone 13 infection (see Extended Data Fig. 9a for experimental setup). e, Phenotypic analysis of donor CD8⁺ T cell subsets pre-transfer and 14 days after transfer and clone 13 infection. Data are representative of 3 experiments (n=4 or 6 per experiment). f, FACS plots and total number of donor CD8⁺ T cells recovered after adoptive transfer of CXCR5⁺ and CXCR5⁻ CD8⁺ T cells into infection-matched recipient mice followed by PD-1 blockade (see Extended Data Fig. 10a for experimental setup) g, Expansion and differentiation of CXCR5⁺ CD8⁺ T cells after PD-1 blockade. Dashed line indicates the limit of detection. Data are combined results of 2 experiments and are mean ± s.e.m. Student’s t-test, where *P < 0.05.

Figure 4. TCF1 is required for the generation of CXCR5⁺ CD8⁺ T cells during chronic LCMV infection

a, Congenically marked wild-type or Tcf7^−/− LCMV GP33-specific P14 transgenic CD8⁺ T cells were transferred into naïve mice, followed by LCMV clone 13 infection. b,c, CXCR5 and Tim-3 expression on wild-type and Tcf7^−/− P14 cells in the spleen (b) and in tissues (c) at the indicated time points. d, CXCR5 and Tim-3 expression on wild-type or Tcf7^−/− donor P14 cells and on endogenous GP33-specific CD8⁺ T cells in the spleen 20 days p.i. e, Phenotypic analysis of wild-type and Tcf7^−/− P14 cells in the spleen 8 days p.i. f,g, Longitudinal analysis of the numbers (f) and FACS plots (g) of wild-type and Tcf7^−/− P14 cells in the spleen after clone 13 infection. h,i, FACS plots (h) and total number (i) of wild-type and Tcf7^−/− P14 cells in the tissues 30 days p.i. Data are representative of 2 experiments (n=7 or 8 mice per time point). Graph shows the mean and s.e.m. Student’s t-test, where **P < 0.01; *P < 0.05.