Figure 5. Direct binding of Blimp1 to Tcf7^+22kb restricts Tcf7 in CD8⁺ T cells in acute infection.

(A) Representative flow plots and statistical analysis of total numbers and phenotypic frequencies of gp33-specific specific WT and Tcf7^Δ+22kb CD8⁺ T cells on 60 dpi with LCMV-Arm infection. Data from two experiments (n = 7 WT and 10 Tcf7^Δ+22kb) are shown with medians and analyzed by Mann-Whitney tests.

(B) Representative flow plots (top) and quantitation of total numbers and phenotypes (bottom) of gp33-specific WT and Tcf7^Δ+22kb CD8⁺ T cells in LCMV-Arm immune mice 5 days after rechallenge with LCMV-c13. n = 12 per genotype, pooled from five experiments, shown with medians and p values determined by Mann-Whitney tests.

(C) Ratio of WT and Tcf7^Δ+22kb gp33-specific CD8⁺ T cells following primary LCMV-Arm infection of mixed bone marrow chimeras. n = 23, pooled from three experiments. Data analyzed by the paired Wilcoxon test of the ratio of Tcf7^Δ+22kb:WT gp33-specific cells on 8 and 70 dpi LCMV-Arm.

(D) Ratio of WT and Tcf7^Δ+22kb gp33-specific CD8⁺ T cells in LCMV-Arm immune mixed bone marrow chimeras following LM-GP rechallenge. n = 18, pooled from two experiments. Data analyzed by the paired Wilcoxon test of the ratio of Tcf7^Δ+22kb:WT gp33-specific CD8⁺ cells pre-rechallenge and 30 dpi with LM-GP.

(E) Experimental schematic (top) of competitive adoptive transfer of WT/Cd19 RNP and Tcf7^Δ+22kb/Tcf7^+17kb RNP MPEC and SLEC. Log₂ transformation of the ratio of Tcf7^{Δ+17kb/+22kb} to WT P14 cells (bottom left) in sorted MPEC and SLEC from P14 secondary recipients on 21 days post-transfer. Data shown as median and quartiles and were quantified with one-sample t and Wilcoxon tests with a theoretical mean of 0 (n = 9 SLEC and 5 MPEC from three experiments). Quantitation (bottom middle) and representative histograms (bottom right) of CFSE dilution in MPEC and SLEC on 21 days post-transfer. Data analyzed by two-way ANOVA and uncorrected Fisher’s LSD. (n = 4 SLEC and 4 MPEC from two experiments).

(F) Experimental schematic (top) of adoptive transfer of secondary WT and Tcf7^Δ+22kb KLRG1⁺ and KLRG1⁻ memory cells. Quantitation of fold expansion (bottom left) of transferred MPEC and SLEC cells from WT and Tcf7^Δ+22kb donor mice on 7 dpi LCMV-Arm (n = 10 per group from two experiments). Data are shown as medians and were analyzed by two-way ANOVA and uncorrected Fisher’s LSD.

(G) TCF-1 and KLRG1 expression in co-transferred WT and Tcf7^{ΔBlimp1−22} P14 cells on 8 dpi with LCMV-Arm. n = 3–5, representative of two experiments per genotype. Frequencies of indicated populations in each recipient mouse were analyzed by paired t test.

(H) Analysis of CpG methylation at a Tcf7-DMR in CD25⁺ and CD25⁻ P14 cells from WT and Tcf7^{ΔBlimp1−22} P14 cells on 5 dpi with LCMV-Arm. Data shown are technical triplicates of n = 3 mice and representative of two experiments, analyzed by a Wilcoxon test.

(I) Frequencies of tdTomato labeling in total gp33-specific CD8⁺ and TCF-1⁺ KLRG1⁺ and TCF-1⁺ KLRG1⁻ gp33-specific CD8⁺ T cells in Prdm1^creERT2/+ R26^tdTomato mice after TAM administration on 3 and 4 dpi and analyzed on 6, 10, 21, and 36–40 dpi. Represented as mean ± SEM.

See also Figures S4 and S5.