Fig 2: FACS gating strategy for mouse and human TBR2 sortingFirst P1 is selected to get all nuclei separated from debris, then P2 and P3 population is selected for single nuclei, rejecting doublets, or multiplets. For mouse (A), since the TBR2 population was rather smear like, instead of completely separated population, TBR2+ gating was done conservatively to make sure no TBR2- nuclei mis-sorted. This heavily depends upon sorting efficiency and accuracy of a given FACS machine. (B) Human TBR2 sorting.

Fig 3: IL-12 can enhance CD8+ Tem cells differentiation through T-bet and Eomes influenced by mTOR activated.The peripheral blood mononuclear cells (PBMC) obtained from IPA model mice, IPA treated with IL-12 or rapamycin and control animals were detected using flow cytometry after 7 days Aspergillus fumigatus inhaled by the nose. The side scatter (SSC) and CD8a were applied to gate CD8+T lymphocytes, CD44+CD45+CD62±/- represented effective memory T cell (Tem). And then the TEMs were stained with IFN-?, mTOR, S6K, T-BET, EOMES. The data were presented as mean ± S.D. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Fig 4: EGR2 loss leads to altered phenotypes in exhausted cell subsets.a–e H2-DbGP33-41 tetramer-stained CD8+ T cells were sorted from CD4-depleted, chronic LCMV infected WT or cKO mice at day 20 p.i., and subjected to multiplexed scRNAseq. a tSNE plot showing clusters identified within the pooled WT and cKO cells using published signatures. b Proportional distribution of the clusters identified in a within WT and cKO cells. c tSNE plot from a showing the location of WT (aqua) and cKO (orange) cells. d Volcano plots generated by comparing gene expression within cKO vs WT cells in each cluster from a. Dotted lines indicate p-value (0.05) and log2 fold change (>0.8 or <-0.8) cut-off values. Red dots indicate genes that satisfy both p-value and fold change cut-offs, while blue dots indicate genes that satisfy the p-value but not the fold change cut-off. Genes up in cKO are on the right, while genes up in WT are on the left. e GSEA analysis of the comparisons from d looking at enrichment of genes selectively upregulated in exhaustion (“Exhaustion signature”; upper plots in red), or genes selectively up in effector cells at day 8 of acute vs chronic LCMV (“Effector signature”; lower plots in blue). f, g Differentially expressed markers identified by the scRNAseq analysis in d were confirmed in H2-DbGP33–41 tetramer-stained CD8+ T cells from CD4-depleted, chronic LCMV infected WT or cKO mice at day 20 p.i. Representative plots (f) and pooled data (g) are shown. For the markers in g, WT n = 20 (PD-1, EOMES), 10 (TIGIT, Ly6C, GzmB, FR4, IL-10Ra, CD160), 6 (Ly6A), and KO n = 22 (PD-1, EOMES), 12 (TIGIT, Ly6C, GzmB, FR4, IL-10Ra, CD160), 6 (Ly6A), mice per group from 2–5 independent experiments. p-values were calculated using a two-tailed Mann–Whitney test (TCF1- Ly6C, TCF1- Ly6A, TCF1+ GzmB, TCF1- GzmB, TCF1+ CD160) or a two-tailed unpaired T test (all others). For indicated significant differences, exact p values (left to right graphs) for TCF1+= <0.0001, 0.0466, 0.0014, 0.0015, 0.0056, 0.0070, <0.0001, <0.0001, for TCF1-= <0.0001, <0.0001, 0.0059, 0.0090, 0.0411, <0.0001, 0.0084. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Post sorting validation for sorting efficiencyNotice that in TBR2- nuclei, there is no signal, while all TBR2+ DAPI nuclei have an Alexa 488 (green) signal, confirming 100% pure population. Scale bar, 5 µm.