Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade

Abstract

Blocking Programmed Death-1 (PD-1) can reinvigorate exhausted CD8 T cells (T_EX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram T_EX into durable memory T cells (T_MEM) is unclear. We found that reinvigoration of T_EX in mice by PD-L1 blockade caused minimal memory development. After blockade, reinvigorated T_EX became reexhausted if antigen concentration remained high and failed to become T_MEM upon antigen clearance. T_EX acquired an epigenetic profile distinct from that of effector T cells (T_EFF) and T_MEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that T_EX are a distinct lineage of CD8 T cells. Nevertheless, PD-1 pathway blockade resulted in transcriptional rewiring and reengagement of effector circuitry in the T_EX epigenetic landscape. These data indicate that epigenetic fate inflexibility may limit current immunotherapies.

Fig. 1. Anti-PD-L1 induces an effector-like transcriptional program in T_EX cells that is not sustained after cessation of treatment

(A) Consensus hierarchical clustering by 1-Pearson correlation from the microarray on control- or anti-PD-L1–treated T_EX. (B) GSEA of representative Gene Ontology (GO) terms. (C) GSEA of effector genes. (D) Row-normalized heat map of effector-associated genes. (E) Circos plots showing overlap in metagenes identified in anti-PD-L1–-treated T_EX compared to metagenes in T_EFF (left) and T_MEM cells (right). Ribbons connecting areas of the Circos plots indicate shared genes between groups. The microarray includes four independent experiments with 10 to 12 mice per group per experiment. (F) Frequency of LCMV GP33-specific Tcell receptor transgenic “P14” cells among CD8 T cells and (G) Ki67⁺ P14 cells in the peripheral blood. Gray bar indicates antibody treatment period. Error bars represent SEM. (H) Quantification of IFNγ⁺ TNFα⁺ P14 cells. (I) Viral load in the kidney. Data in (F) and (G) are one representative experiment. In (H) and (I), the ⁺1 day time point is combined from two representative experiments, and the ⁺20 week time point is from one representative experiment. Data in (F) to (I) are representative of at least two independent experiments with at least four mice per group per experiment. (J) Principle-component analysis of RNA sequencing (RNA-seq), percentage of variance (% of var.) indicated. The RNA-seq was performed on two to four independent experiments with 5 to 13 mice per group as indicated in the supplementary methods. Each dot represents an independent replicate. Asterisks indicate statistical significance determined by unpaired t tests between groups (*P < 0.05, **P < 0.01, and ***P < 0.001). ns, not significant.

Fig. 2. PD-1 pathway blockade moderately improves antigen-independent persistence and IL-7 signaling in T_EX

(A) Number of D^bGP33⁺ donor CD8 Tcells per million peripheral blood mononuclear cells (PBMCs) at day 27 (compared to day 1) after transfer and (B) number recovered from the spleen. (C) Histograms of CD127 and CD122 expression on T_EX P14 cells (day 35 after clone 13) compared to T_MEM P14 cells or bulk CD44^lo CD62L⁺ T_N cells [day 167 after LCMV strain Armstrong (Arm)]. Values indicate average geometric mean fluorescence intensity (MFI) and SEM. (D) Contour plots of PD-1 versus CD127 from mice in (C). (E) Quantification of (D). Data in (A) to (E) are representative of at least two independent experiments with at least four mice per group. (F) Quantification of phospho-STAT5 induction by P14 cells at day 39 after infection after ex vivo stimulation with IL-7 or IL-15 for 30 min. Values indicate fold change over unstimulated controls. (G) Frequency (of CD8⁺, left) and number (right) of P14 cells in the spleen after 2 weeks of treatment. (H) Plots (left) and quantification (right) of IFNγ⁺ TNFα⁺ P14 cells from (G) after ex vivo peptide stimulation. Data in (F) to (H) are combined from two independent experiments with at least four mice per group. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001) determined as described in the supplementary methods. Blue asterisks indicate analysis of variance (ANOVA) P values; black asterisks indicate post-test P values.

Fig. 3. PD-1 pathway blockade fails to restore memory-like recall capacity or reprogram the epigenetic landscape of T_EX into T_EFF or T_MEM cells

(A to D) The experimental design outlined in fig. S7A was used except that recipient mice were rechallenged with Listeria-GP33 3.5 weeks after transfer. (A) Flow cytometry plots of responding T_MEM, T_EX, or anti-PD-L1–treated T_EX at 6 days after rechallenge with Listeria-GP33. (B) Concatenated flow cytometry plots gated on P14 cells from mice in (A) after ex vivo peptide stimulation. (C) Quantification of donor (Ly5.2⁺) D^bGP33⁺ CD8 Tcells in the spleens shown in (A). (D) Quantification of IFNγ⁺ P14 cells shown in (B). (E) Histograms of PD-1 on donor D^bGP33⁺ cells from mice shown in fig. 2B. Values indicate average geometric MFI and SEM. Data are representative of two independent experiments with at least four mice per group. Asterisks indicate statistical significance (***P < 0.01, and ***P < 0.001) determined as described in the supplementary methods. Blue asterisks indicate ANOVA P values; black asterisks indicate post-test P values. (F) Venn diagrams of ATAC-seq OCRs compared to T_N cells (LFC ≥ 2). Data from the two replicates are combined. (G) Representative ATAC-seq tracks from one independent replicate per group shown at the Ifng and Pcdc1 loci. (H) Cocluster analysis of variance showing enrichment of OCRs in ATAC-seq data set. Solid lines separate cell types, and replicates are shown side by side. (I) Box and whisker plots showing ATAC-seq enrichment from (H). Whiskers represent the interquartile range. (J) Principle-component analysis of all OCRs. For (I) and (J), each replicate is shown. ATAC seq data are from two independent experiments with 2 to 15 mice per group as described in the supplementary methods.

Fig. 4. Differential transcription factor binding after PD-1 pathway block-ade contributes to an altered transcriptional network during T_EX reinvigoration

(A) Enrichment of TF binding motifs in OCRs lost or gained after anti-PD-L1 treatment. (B) Wellington bootstrap analysis of TF binding in pairwise comparisons for each cell type; the top 10 TFs (in boxes) enriched in all OCRs are shown. Full list in table S10. (C) TF footprint for NFATc1 in T_EX and NFκB-p65 in anti-PD-L1–treated T_EX. (D) Integrated network analysis of the transcriptional and epigenetic changes after anti-PD-L1 treatment. Lines connect TFs predicted to have altered activity to corresponding genes regulated. Details in table S11. (E) LFC of genes significantly changed by anti-PD-L1 treatment compared to genes significantly induced by the “partnerless” NFAT construct CA-RIT-NFAT1 (30). (F) Venn diagram showing genes near OCRs containing given TF motifs in T_EFF, T_EX, or both (overlap) (top left). Percentage difference in TF target genes changed (P < 0.05, LFC ≥ 0.3) with anti-PD-L1 in overlap compared to T_EX only (bottom left). Sum of the absolute value of the LFC in expression in TF target genes after anti-PD-L1 treatment (right). ATAC-seq data shown are combined replicates for each condition.