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. 2018 May 31;7:e33057. doi: 10.7554/eLife.33057

Figure 7. ZFP36 regulates anti-viral immunity.

(A) Virus-specific CD4 + or (B) CD8 +T cells were tracked in peripheral blood using MHC-tetramers after LCMV Armstrong infection (n = 8–9 mice per genotype). (C) Virus-specific CD4 +T cells and CD69 expression on CD4 +T cells in peripheral blood at early time points post-infection (p.i.) (n = 7–8 mice per genotype). (D) Virus-specific CD8 +T cells and CD69 expression on CD8 +T cells in peripheral blood at early time points p.i. (n = 7–8 mice per genotype). (E) Virus-specific CD4 +and (F) CD8 +T cells in spleen after LCMV infection (n = 5–8 mice per genotype). (G) Fraction of CD4 +T cells producing IFN-γ and TNF-α in splenic CD4 +T cells 6 days p.i., after ex vivo stimulation with GP66-77 peptide (n = 7–8 mice per genotype). (H) Levels of IFN-γ and TNF-α(gated on cytokine-producing CD4 +cells) 6 days p.i. after ex vivo stimulation with GP66-77 (n = 7–8 mice per genotype). (I) Raw percentage of bifunctional IFN-γ+TNF-α+CD4+cells in spleen 6 days p.i. after ex vivo stimulation with GP66-77 (left), or normalized to percentage of GP66-77 tetramer +cells (n = 7–8 mice per genotype). (J) Levels of LCMV genomic RNA in spleen measured by RT-qPCR (n = 9–14 per group). For (A–J), mean values ± S.E.M. are shown, with circles as individual mice. Results of two-tailed t-tests: *=p < 0.05; **=p < 0.01; ***=p < 0.001; ****=p < 0.0001. In each panel, one representative experiment of two is shown.

Figure 7.

Figure 7—figure supplement 1. The T cell compartment in naïve Zfp36 KO mice is largely normal.

Figure 7—figure supplement 1.

(A) Counts of CD4 +and CD8+T cells in peripheral blood of WT and Zfp36 KO mice (n = 9–10 per genotype). Mean values ± S.E.M are shown; circles are individual mice (n = 9–10 per genotype) (B) Counts of thymocytes and distribution among T cell development stages in WT and Zfp36 KO mice (n = 11 per genotype; DN = double negative; DP = double positive; CD4-SP = CD4 single positive; CD8-SP = CD8 single positive) (C) Counts of CD4 + and CD8+T cells in spleen or (D) as a proportion of splenocytes in WT and Zfp36 KO mice (n = 5 mice per genotype). (E) Percentages of naïve CD4 +and CD8+T cells in spleen, defined as CD25-CD62L-hiCD44-lo (n = 11 per genotype). For (A–E), mean values ± S.E.M are shown with circles as individual mice. (F) Percentages of CD4 +CD25 hi cells in spleen (n = 11 per genotype). For (A–F), mean values ± S.E.M are shown with circles as individual mice. (G) Percentages of FoxP3 +iTreg cells from WT and Zfp36 KO FoxP3-GFP transgenic mice indicated skewing conditions (mean ±S.E.M. is shown for n = 2 mice per genotype). (H) Percentages of CD4 + and CD8+cells producing the indicated cytokines by intracellular flow cytometry, following 5 hr of PMA/ionomycin stimulation of splenocytes directly ex vivo (mean ± S.E.M. is shown for n = 6 per genotype). (I) Percentages of CD4 +T cells producing the indicated lineage-specific effector cytokines is shown under various skewing conditions (mean ± S.E.M. is shown for n = 3 mice per genotype). For (A–I) results of two-tailed t-tests are shown beneath relevant panels when significant differences were observed: *=p < 0.05. Otherwise, differences were not significant.
Figure 7—figure supplement 2. ZFP36 regulates anti-viral immunity.

Figure 7—figure supplement 2.

(A) The fraction of CD8 +T cells producing IFN-γ and IFN-γ proteins levels were measured splenic CD8 +T cells by ICS 6 days post-infection, after ex vivo stimulation with the LCMV antigenic peptide GP33-41 (n = 5–7 per genotype). (B) The fraction of CD8 +T cells producing TNF-α and TNF-α proteins levels were measured in splenic CD8 +T cells by ICS 6 days post-infection, after ex vivo stimulation with the LCMV antigenic peptide GP33-41 (n = 5–7 per genotype). (C) The raw percentage of bifunctional IFN-γ+TNF-α+CD8+cells in spleen 6 days post-infection after ex vivo stimulation with GP33-41 (left), or normalized to percentage of GP33-41 tetramer +cells (right) (n = 5–7 per genotype). (D) Plots depicting the relationship between LCMV load and levels of tetramer +virus specific T cells in spleens of WT and Zfp36 KO animals, 6 days post-infection. R-squared and p-values are shown for linear regression analysis. For (A–D), mean values ± S.E.M are shown with circles as individual mice. Results of two-tailed t-tests are shown beneath relevant panels when significant differences were observed: *=p < 0.05; **=p < 0.01; ***=p < 0.001, ****=p < 0.0001. Data from one representative experiment of two are shown.
Figure 7—figure supplement 3. LCMV infection in mixed bone marrow chimeras.

Figure 7—figure supplement 3.

(A) Schematic of mixed chimeric experiments. (B) Analysis of T cell numbers in peripheral blood in mixed bone marrow chimeras 10 weeks after constitution. Values were normalized to levels of Thy1.1 WT T cells, and the mean ± S.D. are shown (n = 15 mice). (C) Levels of LCMV-specific CD4 + and (D) CD8 +T cells were determined in peripheral blood by MHC-tetramer staining over a time course of LCMV Armstrong infection. Values are mean ± S.D (n = 15 mice). Data are shown for one representative experiment. A second experiment analyzing d0-d10 showed similar results.