Fig. 6. TRM precursors identified within the siIEL CD8+ T cell pool early in infection.
(A) Expression of selected genes within single siIEL CD8+ T cells at day 4 post-infection, relative to the mean expression among all cells, demonstrating differential gene expression between cells in Clusters 16 and 20. (B) Cell cycle status of siIEL CD8+ T cells at day 4 post-infection, inferred from transcriptional profiles. (C and D) P14 CD8+ T cells were adoptively transferred into congenic hosts one day prior to infection with LCMV. Splenic and siIEL CD8+ T cells were analyzed by flow cytometry at day 4 post-infection. Data are representative of two independent experiments, with n=2–3 mice per experiment. (C) Representative flow cytometry plots displaying distribution of IL-2Rα expression among total siIEL P14 CD8+ T cells at day 4 post-infection (left), or among total H-2Db GP33–41 tetramer+ endogenous siIEL CD8+ T cells at day 4 post-infection in mice that had not received adoptive transfer of P14 CD8+ T cells (right). (D) Quantification of Ezh2 expression among IL-2Rαlo (gray) and IL-2Rαhi (blue) P14 siIEL CD8+ T cells, as gated in (C), with representative flow cytometry plot (left) and quantification (right). Numbers represent Mean Fluorescence Intensity (MFI) of Ezh2 within the indicated population. Data are representative of (plot to left) or compiled from (graph to right) 2 independent experiments, with n=4 total mice, where each dot represents an individual mouse. (E) Schematic of experimental setup. CD45.1+ P14 CD8+ T cells were adoptively transferred into CD45.2+ congenic hosts one day prior to infection with LCMV. CD45.1+ P14 CD8+ T cells were collected from the siIEL compartment at day 4 post-infection and sorted based on expression of IL-2Rα as shown in (C). IL-2Rαhi and IL-2Rαlo populations were adoptively transferred into secondary hosts that had been infected with LCMV one day prior to transfer. 7 or 30 days later, siIEL P14 CD8+ T cells were analyzed by flow cytometry. (F) Quantification of the proportion of KLRGhi P14 CD8+ T cells present within the siIEL compartment at day 7 post-infection. Data were pooled from 2 independent experiments, with mean and SEM of n=7 (IL-2Rαlo) or 5 (IL-2Rαhi), where each dot represents an individual mouse. (G) Quantification of the percent of siIEL CD45.1+ P14 CD8+ CD69+CD103+ T cells derived from IL-2Rαlo (gray) or IL-2Rαhi (blue) day 4 siIEL cells at day 7 or day 30 post-transfer, with representative flow cytometry plots shown to the left. Day 7 data were pooled from 2 independent experiments, with mean and SEM of n=5 (IL-2Rαhi) or 6 (IL-2Rαlo) hosts, where each dot represents an individual mouse. Day 30 data were pooled from 3 independent experiments, with mean and SEM of n=4 (IL-2Rαhi) or 12 (IL-2Rαlo) hosts, where each dot represents an individual mouse. *P<0.05, **P<0.01 (Paired t-test, D, or Student’s two-tailed t-test, F and G).