Fig. 4.

Ablation of Tpl2 results in the reduction of Tregs in the intestinal mucosa and peripheral lymphoid organs. (A–C) Single-cell suspensions of intestinal mononuclear cells from Apc^+/+/Tpl2^+/+, Apc^+/+/Tpl2^−/−, Apc^min/+/Tpl2^+/+, and Apc^min/+/Tpl2^−/− mice were stained with anti-CD4, anti-CD25, and anti-Foxp3 antibodies, and they were analyzed by flow cytometry for the abundance of CD4⁺/CD25⁺/Foxp3⁺ Treg cells. Five-week-old mice (A; n = 5 per genotype), 7-wk-old mice (B; n = 5 per genotype), and 3-mo-old mice (C; n = 7 per genotype) were included in the analysis. Single-cell suspensions of splenocytes and lymph node lymphocytes (D) and thymocytes (E) from 3-mo-old mice (n = 7 per genotype) were analyzed for the abundance of CD4⁺/CD25⁺/Foxp3⁺ Treg cells as in A–C. Ablation of Tpl2 resulted in the reduction of Tregs in the spleen, lymph nodes, and intestine (iTregs) but not in the thymus (nTregs). The histograms show the mean value for each group. *P < 0.05. (F) Whereas Tpl2^+/+ Tregs partially protect Apc^min/+/Tpl2^+/+ mice from the development of intestinal polyps, Tpl2^−/− Tregs do not. Seven 9-wk-old Apc^min/+/Tpl2^+/+ mice were inoculated with 4.5 × 10⁵ CD4⁺/CD45RB^low/CD25⁺ Tpl2^+/+ Tregs, and eight age-matched Apc^min/+/Tpl2^+/+ mice were inoculated with 4.5 × 10⁵ CD4⁺/CD45RB^low/CD25⁺ Tpl2^−/− Tregs. Eight age-matched Apc^min/+/Tpl2^+/+ mice were used as controls. All mice were killed at the age of 13 wk. The difference in the number of polyps between the control mice and the mice inoculated with the Tpl2^+/+ Tregs was statistically significant (P = 0.01534). NT, not transplanted. (G) Six 9-wk-old Apc^min/+/Tpl2^−/− mice were inoculated with Tpl2^+/+ Tregs as in F. Five age-matched Apc^min/+/Tpl2^−/− mice were used as controls.