Figure 6. Tcf7^+22kb mediates TCF-1 downregulation in response to chronic antigen.

(A) Representative flow plots (left) and quantitation (right) of gp33-specific CD8⁺ T cells from WT and Tcf7^Δ+22kb mice on 8 dpi with LCMV-c13. Numbers (represented as median, analyzed by Mann-Whitney test) and frequencies (represented as mean ± SD, analyzed by Welch’s t test) of each population of gp33-specific CD8⁺ T cells. Pooled from two experiments, n = 8.

(B) Representative flow plots (left) and quantitation (right) of gp33-specific CD8⁺ T cells from WT and Tcf7^Δ+22kb mice on 35 dpi with LCMV-c13. n = 21 WT and 12 Tcf7^Δ+22kb mice, pooled from three experiments. Numbers represented as mean ± SD, analyzed by t test. Frequencies represented as median, analyzed by Mann-Whitney tests.

(C) RT-qPCR analysis of plasma LCMV gp transcripts in LCMV-c13 infected WT, heterozygous, or deficient in Tcf7^Δ+22kb mice (n = 20 for WT, n = 6 for Tcf7^{Δ+22kb/Δ+22kb}, and n = 14 for Tcf7^Δ+22kb/+ mice). Data pooled from 4 experiments, shown as medians and analyzed by two-way ANOVA mixed model with Geisser-Greenhouse correction.

(D) Experimental scheme for individual and combined perturbation of Tcf7^+22kb and Tcf7^+17kb in P14 CD8⁺ T cells.

(E) Representative flow plots showing TCF-1 and TIM3 expression in internal control (WT unelectroporated) and P14 cells with indicated RNP/genotype, isolated from the spleen on 8 dpi with LCMV-c13.

(F) Frequencies of TCF-1⁺ P14 cells in each experimental group, shown as mean ± SD, and analyzed by two-way ANOVA and Tukey’s multiple comparisons test. n = 3. Representative of four experiments.

(G) Experimental schematic for adoptive transfer of TIM3⁺ and TIM3⁻ populations of WT/Cd19 RNP and Tcf7^Δ+22kb/Tcf7^+17kb RNP P14 cells from 8 dpi LCMV-c13 infected recipients.

(H) Log₂ transformation of the ratio of Tcf7^{Δ+17kb/+22kb} to WT P14 cells in sorted TIM3⁺ and TIM3⁻ populations on 14 days post-transfer to P14 secondary recipients. Data shown as median and quartiles and quantified with one-sample t and Wilcoxon tests with a theoretical mean of 0 (n = 9 per genotype, pooled from two experiments).

See also Figure S6.