Fig 1.
scRNA-seq analyses of CD8+ T cells responding to acute vs. chronic infection.
(A) Experimental setup. CD8+CD45.1+ P14 T cells were adoptively transferred into separate CD45.2+ hosts 1 day prior to infection with either LCMV-Arm or LCMV-Cl13. To identify cells that had undergone their first division, some cells were labeled with CFSE prior to adoptive transfer. Splenocytes were harvested at the indicated time points after infection. Donor P14 CD8+ T cells were FACS-isolated and processed for scRNA-seq using the 10x Genomics Chromium platform. (B) UMAP clustering of all CD8+ cells, colored by cluster identity (left), infection type (middle), or time point (right); the three Division 1 clusters are circled for emphasis. (C) Bar graphs indicating the infection type and time point from which cells derived from each cluster are derived; clusters are grouped according to similarity in gene expression based on (D). (D) Hierarchical clustering of clusters based on gene expression profiles. The raw data for the panels in this figure are located in S1 Data file. Fig 1A created with BioRender.com. CFSE, carboxyfluorescein succinimidyl ester; LCMV-Arm, LCMV-Armstrong; LCMV-Cl13, LCMV-Clone 13; scRNA-seq, single-cell RNA-sequencing; UMAP, Uniform Manifold Approximation and Projection.
Fig 2.
CD8+ T cells that have undergone their first division in response to LCMV-Arm vs. LCMV-Cl13 exhibit phenotypic and transcriptional heterogeneity.
(A) Heatmaps representing relative gene expression of the three Division 1 clusters, Div1ARM-EFF, Div1ARM-MEM, and Div1CL13, divided by category; rows represent selected genes and columns represent each of three Division 1 clusters. (B) Enriched pathways among the three Division 1 clusters. (C) Representative flow cytometry plots (left) displaying expression of Ezh2, CD25 (IL-2Rα), and T-bet protein among gated Division 1 (second CFSE peak) P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of P14 T cells responding to LCMV-Arm (blue) or LCMV-Cl13 (red) expressing each molecule. Data are shown as mean ± SEM. (D, E) RNA velocities of Div1ARM (D, top) or Div1Cl13 (E, top) subclusters derived from scVelo projected onto a UMAP-based embedding. Putative driver genes derived from scVelo that may regulate the CD8+ Division 1 T cell response to LMCV-Arm (D, bottom) vs. LCMV-Cl13 (E, bottom), represented as heatmaps. Individual lines above each heatmap represent single cells; colors correspond to subcluster identities among Div1ARM (D, top) or Div1Cl13 (E, top) cells. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001 (Student’s t test). Data are representative of 2 to 3 independent experiments. The raw data for the panels in this figure are located in S1 Data file. CFSE, carboxyfluorescein succinimidyl ester; LCMV-Arm, LCMV-Armstrong; LCMV-Cl13, LCMV-Clone 13; MFI, mean fluorescence intensity; UMAP, Uniform Manifold Approximation and Projection.
Fig 3.
Ezh2-mediated epigenetic repression may partially influence exhaustion.
(A) Experimental setup. Control CD45.1+ (wild-type, “WT”) and Ezh2-deficient CD45.1.2+(Ezh2fl/flCd4Cre+, “Ezh2 KO”) CD8+ P14 T cells were cotransferred into congenically distinct CD45.2+ recipient mice prior to infection with LCMV-Cl13; recipient mice were killed at 5–7 days post-infection and splenocytes analyzed by flow cytometry. (B) Representative flow cytometry plots (left) displaying expression of PD1, TCF1, TOX, Granzyme A, IL-2, or TNF protein among gated donor WT or Ezh2 KO P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of WT (blue) or Ezh2 KO (red) P14 T cells responding to LCMV-Cl13. (C) Experimental setup. CD8+ P14 T cells were transduced with an empty vector control (EV, CD45.1+) or Ezh2 overexpression (Ezh2-OE, CD45.1.2+) construct prior to adoptive cotransfer into CD45.2+ recipient mice prior to infection with LCMV-Cl13; recipient mice were killed at 5–7 days post-infection and splenocytes analyzed by flow cytometry. (D) Representative flow cytometry plots (left) displaying expression of PD1, TCF1, TOX, and Granzyme A among gated donor EV or Ezh2-OE P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of EV (blue) or Ezh2-OE (red) P14 T cells expressing each molecule. (E, F) Control CD45.1+ (wild-type, “WT”) and IFNAR1-deficient CD45.1.2+(Ifnar1-/-, “IFNAR1 KO”) CD8+ P14 T cells were labeled with CFSE (E only) and cotransferred into congenically distinct CD45.2+ recipient mice prior to infection with LCMV-Cl13; recipient mice were killed at 2 (E) or 5 days (F) post-infection and splenocytes analyzed by flow cytometry. Representative flow cytometry plots (left) displaying expression of Ezh2 protein among gated Division 1 (second CFSE peak) WT vs. IFNAR1 KO P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of WT (blue) or IFNAR1 KO (red) P14 T cells expressing each molecule. (F) Representative flow cytometry plots (left) displaying expression of Ezh2, PD1, TOX, and TIM3 protein among gated WT vs. IFNAR1 KO P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of WT (blue) or IFNAR1 KO (red) P14 T cells expressing each molecule. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001 (paired t test). Data are representative of 2 to 3 independent experiments. The raw data for the panels in this figure are located in S1 Data file. Fig 3A, 3C and 3E created with BioRender.com. CFSE, carboxyfluorescein succinimidyl ester; EV, empty vector; IL-2, interleukin 2; LCMV-Cl13, LCMV-Clone 13; MFI, mean fluorescence intensity; TNF, tumor necrosis factor; WT, wild-type.