Cutting edge: in the absence of TGF-β signaling in T cells, fewer CD103+ regulatory T cells develop, but exuberant IFN-γ production renders mice more susceptible to helminth infection

Abstract

Multiple factors control susceptibility of C57BL/6 mice to infection with the helminth Heligmosomoides polygyrus, including TGF-β signaling, which inhibits immunity in vivo. However, mice expressing a T cell-specific dominant-negative TGF-β receptor II (TGF-βRII DN) show dampened Th2 immunity and diminished resistance to infection. Interestingly, H. polygyrus-infected TGF-βRII DN mice show greater frequencies of CD4(+)Foxp3(+)Helios(+) Tregs than infected wild-type mice, but levels of CD103 are greatly reduced on both these cells and on the CD4(+)Foxp3(+)Helios(-) population. Although Th9 and Th17 levels are comparable between infected TGF-βRII DN and wild-type mice, the former develop exaggerated CD4(+) and CD8(+) T cell IFN-γ responses. Increased susceptibility conferred by TGF-βRII DN expression was lost in IFN-γ-deficient mice, although they remained unable to completely clear infection. Hence, overexpression of IFN-γ negatively modulates immunity, and the presence of Helios(+) Tregs may maintain susceptibility on the C57BL/6 background.

Figure 1. Ablation of TGF-β signaling in T cells does not confer resistance in mice to H. polygyrus infection

A. Fecal H. polygyrus egg counts after 14 days of infection in C57BL/6 and TGF-βRII DN mice. Data are pooled from 3 experiments, with mice aged 7-18 wks. Mice were age-matched between groups and no age effect on H. polygyrus susceptibility was seen. B. Adult H. polygyrus counts from the same experiments and time point as (A). C. % IL-13⁺ cells of CD4⁺ lymphocytes in MLNC from naïve (open circles) and 14-day-post-infected (closed circles) C57BL/6 and TGF-βRII DN mice. MLNC were stimulated with PMA/Ionomycin, and stained for flow cytometry. Data are pooled from 2 experiments with 6-9 wk old mice. D. Levels of serum IL-5 in naïve (open circles) and 7-day-post infected (closed circles) mice. Data are pooled from 2 experiments; naïve mice were examined in one of these experiments. Mice were 7-12 wks old and age matched between groups. Abbreviations used in figure: B6= C57BL/6; DN= TGF-βRII DN; H. p.= H. polygyrus-infected. (A-B) were analysed by unpaired T test; and (C-D) by Kruskal-Wallis test.

Figure 2. TGF-βRII DN mice have more T regulatory cells but fewer CD103⁺ Tregs than C57BL/6 animals during H. polygyrus infection

C57BL/6 and TGF-βRII DN mice were left naïve (open circles) or H. polygyrus-infected for 14 days (closed circles), and MLNC were isolated and stained directly ex vivo. Data are representative of the results from 2 experiments each with 2-5 mice per group. Mice were 7-9 wks old. A. % Foxp3⁺ cells among CD4⁺ lymphocytes. B. % Foxp3⁺Helios⁺ cells among CD4⁺ lymphocytes. C. Representative CD103 and Foxp3 staining among CD4⁺ lymphocytes, from 14-day-H. polygyrus-infected C57BL/6 and TGF-βRII DN mice. D. % CD103 among CD4⁺Foxp3⁺ lymphocytes. Abbreviations used as in Figure 1. H. polygyrus-infected groups in (A,B,D) were analysed by unpaired T test.

Figure 3. TGF-βRII DN mice have exaggerated IFN-γ responses yet Th17, Th9 and mast cell responses are not compromised

A-B, E-F. MLNC were isolated from 14-day-H. polygyrus-infected C57BL/6 and TGF-βRII DN mice, stimulated with PMA/Ionomycin, and stained for flow cytometry. A-B. Data are pooled from 3 experiments, with 6-18 wks old mice age-matched between groups. % IL-17A⁺ cells (A) or % IL-9⁺ cells (B) of CD4⁺ lymphocytes. C. Jejunums were sectioned and stained for mast cells with Toluidine Blue. Number of mast cells per μm of villus crypt are shown in naïve (open circles) and 14-day-H. polygyrus infected (closed circles) mice. Data shown are from 7-9 wk old mice and are representative of 2 experiments each with 4-5 mice per group for infected mice; naïve mice were included in one of these experiments. D. Levels of circulating IFN-γ in the sera of naïve (open circles) or 7-day-H. polygyrus-infected C57BL/6 and TGF-βRII DN mice (closed circles). Data are pooled from 2 experiments with 7-12 wk old mice. E-F. % IFN-γ⁺ cells among CD4⁺ (E) or CD8α⁺ (F) TCR-β⁺ lymphocytes. Data shown are from mice 7-9 wk old mice and are representative of 4 experiments each with 2-5 mice per group. Abbreviations used as in Figure 1. (A) was analysed by unpaired T test; (B, D) by Mann Whitney; (C) by Kruskal-Wallis; and (E-F) by unpaired T test between H. polygyrus-infected groups.

Figure 4. Increased susceptibility of TGF-βRII DN mice is reversed in the absence of IFN-γ

A. Fecal egg counts after 14 days of infection in C57BL/6, TGF-βRII DN, TGF-βRII DN IFN-γ^−/− and IFN-γ^−/− mice. Data are pooled from 3 experiments with 6-14 wk old mice age-matched between groups. B. Adult worm counts from the same experiments after 28 days of infection. C-D. Levels of circulating IFN-γ (C) and IL-5 (D) in the same experiments after 7 days of infection. Data are pooled from two experiments with 7-14 wk old mice. E. % Foxp3⁺ T cells among total CD4⁺ lymphocytes. After 28 days of infection, MLNC were stained directly ex vivo. Data are pooled from 2 experiments with 7-14 wk old mice. F. % CD103 among Foxp3⁺ CD4⁺ lymphocytes, in the same experiments as E. Abbreviations used in figure: B6= C57BL/6; DN= TGF-βRII DN; DNγ^−/− = TGF-βRII DN IFN-γ^−/−; γ^−/− = IFN-γ^−/−; H. p.= H. polygyrus-infected. (A,B,E,F) were analysed by ANOVA; (C-D) by Kruskal-Wallis test.