Figure 3. Tcf7^+22kb enforces the continuous downregulation of Tcf7 in acute LCMV infection.

(A) Experimental setup (top) and expression of TCF-1 and CD25 in adoptively transferred WT and Tcf7^Δ+22kb P14 cells on 3.5 dpi (bottom).

(B) Histograms (top) and quantification (bottom) of CD25⁺ cells in P14 cells and TCF-1⁺ cells in CD25⁺ and CD25⁻ cells in donor WT (closed circles) and Tcf7^Δ+22kb (open circles) P14 cells on 3.5 dpi. Data were pooled from three experiments (n = 4–5 per experiment), and statistical differences were assessed by paired Wilcoxon tests.

(C) Representative flow plots showing expression of TCF-1 and CD25 by endogenous gp33-specific CD8⁺ T cells in spleens of WT and Tcf7^Δ+22kb mice on 5 dpi. Data from five experiments are shown as mean ± SD (n = 13 per genotype). Unpaired t tests with Welch correction.

(D) Total numbers of gp33- and np396-specific CD8⁺ T cells in spleens of LCMV-Arm infected mice on 5 dpi, pooled from two experiments (n = 5 per genotype), shown with median, and analyzed by the Mann-Whitney test.

(E) Representative flow plots showing expression of TCF-1 and KLRG1 by endogenous gp33-specific CD8⁺ T cells in spleens of WT and Tcf7^Δ+22kb mice on 8 dpi.

(F) Frequencies of TCF-1⁺ cells in total gp33-specific, KLRG1⁺, and KLRG1⁻ cells (left) and KLRG1⁺ cells of gp33-specific CD8⁺ T cells with indicated genotypes, pooled from two experiments (n = 5 WT and 6 Tcf7^Δ+22kb), and shown as mean ± SD with p values by unpaired t tests.

(G) Numbers of gp33- and np396-specific CD8⁺ T cells in WT and Tcf7^Δ+22kb mice on 8 dpi with LCMV-Arm, shown as medians and analyzed by the Mann- Whitney test (n = 5 WT and 6 Tcf7^Δ+22kb).

(H) Representative flow plots showing expression of TCF-1 and CD25 by endogenous gp33-specific CD8⁺ T cells in spleens of WT and Prdm1^−/− mice on 5 dpi with LCMV-Arm. Data represented as the median of data from two experiments (n = 5 WT and 4 Prdm1^−/− mice). Mann-Whitney test.

(I) Schematic of experiment for inhibition of Tcf7^+22kb and Tcf7^+17kb elements by electroporation of Cas9/sgRNA RNPs into WT and Tcf7^Δ+22kb P14 cells.

(J) Representative flow plots of adjacent internal control (WT unelectroporated) and corresponding combinations of P14 genotype and RNP in P14 cells isolated from the spleen on 5 dpi with LCMV-Arm.

(K) Percentage of P14 cells of each genotype and RNP combination retaining TCF-1 expression on 5 dpi with LCMV-Arm (n = 3, representative of three experiments). Data shown as mean ± SD and analyzed by one-way ANOVA and Dunnett’s multiple comparisons test.

See also Figure S2.