Fig 2: Regulation of pri-miR-181a/b1 expression by TCF1.a Naive CD4 T cells from young adults were transfected with TCF7 siRNA or control siRNA and assayed for pri-miR-181a/b1 expression after 48 h by q-PCR. Data are shown as mean±SEM (n = 3). b ChIP assay was performed on naïve CD4 T cells with anti-human TCF1 antibodies, and the sequence representing a previously identified pri-miR-181a/b1 enhancer region (chr1:198,904,100-169) was amplified. Precipitation with normal IgG was used as control. Data are shown as mean±SEM (n = 3). c Sequences corresponding to pri-miR-181a/b1 enhancer region (chr1:198,904,065-558) were cloned into a pGL4.27 [luc2P/minP/Hygro] plasmid. Dual-luciferase reporter assays were performed in HEK293T cells transfected with TCF7 siRNA, YY1 siRNA, or control siRNA. Data are shown as mean±SEM (n = 3). d, e Increasing amounts (0 ng, 30 ng or 100 ng) of TCF7 (d) and CTNNB1 (e) –containing plasmids were co-transfected with the pri-miR-181a/b1-enhancer-Luc2p reporter construct into HEK293T cells. Reporter activities after 48 h are shown as mean±SEM (n = 3). Comparisons were done by two-tailed paired t test in a, b; or, by one-way ANOVA with post-hoc Tukey test in c, d, and e. Significance levels are indicated as *P < 0.05, **P < 0.005, ***P < 0.0001.

Fig 3: CXCR3-deficient CD8+ T cells retain a memory phenotype in the RP(A–C) CXCR3 KO TG6-GFP neonatal chimeras were infected with T. gondii and analyzed by confocal microscopy after 6 weeks. Sections were stained with antibodies against TCF1 (A), Ki67 (B), and KLRG1 (C). B220 staining was used to identify B cell zones (B) to map the T cell zone (T) and RP regions. Yellow arrows highlight examples of cells staining positively for the specified marker; white arrows highlight examples of negative staining.(D) Summary data showing the proportion of GFP cells found in the WP or RP that are identified as positive or negative for expression of the indicated marker. Bar graphs display the proportion of WT or CXCR3 KO GFP cells expressing the protein of interest (TCF1, Ki67, or KLRG1) within the WP, BC, or RP.Data for WT cells are combined from at least three mice across two different experiments, and data for KO cells are combined from four mice taken across two experiments.

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: TCF7 knockdown reduces the proliferation and survival of CML K562/G01 cells. (A and B) CCK-8 assays were used to detect the cell viability of the Scramble and TCF7_KD groups at 12, 24, 48, 72, 96 and 120 h. (C and D) Cell cycle distribution of the Scramble and TCF7_KD groups during the logarithmic growth phase. (E) Cell cycle distribution of the Scramble and TCF7_KD groups following serum starvation and release at 0, 8, 16, 24, and 32 h. (F) Flow cytometry cell counts of the Scramble and TCF7_KD groups at 0, 12, 24, 48, 72, and 96 h. One-way ANOVA with Tukey's post hoc test were performed. *P<0.05, **P<0.01, ***P<0.001. TCF7, transcription factor 7; CML, chronic myeloid leukemia; KD, knockdown.