Fig. 5

Signal-regulatory protein alpha (SIRPα) expression on PD-1⁺ CD8⁺ T cells identifies cells with enhanced proliferation and cytolytic ability. CD8⁺ FV-D^b gagL dextramer⁺ splenocytes from 14 dpi (a) or chronically infected mice (b) were analyzed by flow cytometry for programmed cell death protein 1 (PD-1) and SIRPα expression and the gated SIRPα-positive and -negative subsets were analyzed for surface CD107a and intracellular granzyme B. Representative fluorescence-activated cell sorting (FACS) plots with gating strategy are shown in c–f. Data are from two independent experiments (n = 8 total mice) where each dot represents the percentage of (g) intracellular granzyme B, (h) surface CD107a, and (i) intracellular Ki-67 from each cell subset. Mice (j) chronically or (k) acutely infected with Friend virus (FV) were adoptively transferred with 1 × 10⁶ naive FV-specific T cell receptor transgenic CD8⁺ T cells and then at 13–15 days post-transfer the cells were recovered and separated into PD-1⁺ SIRPα⁻ and PD-1⁺ SIRPα⁺ subpopulations by FACS cell sorting. A 2-h in vitro cytotoxicity assay was then performed with the sorted effector cells, as described in Methods. Each dot represents the background-corrected value of substrate fluorescence for an individual sample at target to effector ratios, 1:4 and 1:10. The bar is the mean. The negative control is bead purified CD8⁺ T cells from a naive Y10 mouse and is represented by the horizontal dashed line. Data from chronic infection are from three independent experiments. Subsets of splenic CD8⁺ T cells from mice chronically infected with FV were analyzed by intracellular flow cytometry for T cell factor-1 (TCF-1). l A representative histogram overlay is displayed, depicting the mean fluorescence intensity (MFI) of each labeled subset. m Each dot represents an MFI value of intracellular TCF-1 for a given subset. Data are from 1 of 2 independent experiments; a total of n = 11 mice were analyzed. All bars in the figure represent the mean. ns = p > 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired, two-way t tests). The flow cytometric gating strategy is shown in supplementary Fig. 6h–m