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. 2013 Feb 1;190(3):1076-84.
doi: 10.4049/jimmunol.1202659. Epub 2012 Dec 31.

TNFR2 is critical for the stabilization of the CD4+Foxp3+ regulatory T. cell phenotype in the inflammatory environment

Xin Chen  1 , Xueqiang Wu, Qiong Zhou, O M Zack Howard, Mihai G Netea, Joost J Oppenheim
Affiliations

TNFR2 is critical for the stabilization of the CD4+Foxp3+ regulatory T. cell phenotype in the inflammatory environment

Xin Chen et al. J Immunol. .

Abstract

Several lines of evidence indicate the instability of CD4(+)Foxp3(+) regulatory T cells (Tregs). We have therefore investigated means of promoting the stability of Tregs. In this study, we found that the proportion of Tregs in mouse strains deficient in TNFR2 or its ligands was reduced in the thymus and peripheral lymphoid tissues, suggesting a potential role of TNFR2 in promoting the sustained expression of Foxp3. We observed that upon in vitro activation with plate-bound anti-CD3 Ab and soluble anti-CD28 Ab, Foxp3 expression by highly purified mouse Tregs was markedly downregulated. Importantly, TNF partially abrogated this effect of TCR stimulation and stabilized Foxp3 expression. This effect of TNF was blocked by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Furthermore, TNF was not able to maintain Foxp3 expression by TNFR2-deficient Tregs. In a mouse colitis model induced by transfer of naive CD4 cells into Rag1(-/-) mice, the disease could be inhibited by cotransfer of wild-type Tregs, but not by cotransfer of TNFR2-deficient Tregs. Furthermore, in the lamina propria of the colitis model, most wild-type Tregs maintained Foxp3 expression. In contrast, an increased number of TNFR2-deficient Tregs lost Foxp3 expression. Thus, our data clearly show that TNFR2 is critical for the phenotypic and functional stability of Tregs in the inflammatory environment. This effect of TNF should be taken into account when designing future therapy of autoimmunity and graft-versus-host disease by using TNF inhibitors.

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Figures

Figure 1
Figure 1
Reduced number of Tregs in TNFR2 deficient mice. Cells from thymus, spleen and LNs in WT mice (C57BL/6) and TNFR2−/− mice were stained with CD3, CD4, CD8, TNFR2, and FoxP3. The expression of FoxP3 was analyzed by FACS, gating on CD3+CD4+ cells or CD3+CD4+CD8− cells (CD4 single positive cells, e.g., CD4 SP cells). (A) Proportion of CD4+FoxP3+ cells in the total thymocytes derived from WT or TNFR2−/− mice. (B) Expression of FoxP3 and TNFR2 on CD4 SP thymocytes from WT or TNFR2−/− mice. (C~D) Proportion of CD4+FoxP3+ cells in the total splenic and LN cells from WT or TNFR2−/− mice. In (A and B), left shows the typical FACS plots, and right shows summary (N=3). (C) shows the typical FACS plots and (D) shows the summary (N=3). (E –F) Expression of FoxP3 and TNFR2 on CD4+ T cells in the spleen and LNs from WT or TNFR2−/− mice. (E) Shows the typical FACS plots and (F) shows the summary (N=3). (G) Number of CD4+FoxP3+ Tregs in the spleen from WT or TNFR2−/− mouse. Number in the FACS plot shows the proportion of positive cells in the indicated gating or respective quadrants. Comparison between two indicated groups: * p<0.05; ** p<0.01. Data shown are representatives of at least 3 separate experiments with same results.
Figure 2
Figure 2
Reduced number of Tregs in TNFR2 ligands deficient mice. Cells from thymus, spleen and LNs in WT mice (C57BL/6) or TNF/LTα/LTβ triple KO mice were stained with CD3, CD4, CD8, TNFR2, and FoxP3. The expression of FoxP3 was analyzed by FACS, gating on CD3+CD4+ cells or CD3+CD4+CD8− cells. (A) Proportion of CD4+FoxP3+ cells in total thymocytes from WT or Triple KO mice. (B) Proportion of FoxP3+ cells in the CD4+CD8− thymocytes from WT or Triple KO mouse. In (A and B), left shows the typical FACS plots, and right shows summary (N=3~6). (C) Proportion of CD4+FoxP3+ cells in the spleen from WT or Triple KO mice. (D) Proportion of FoxP3+ cells in CD4+ splenic cells from WT or Triple KO mice. Number in the FACS data shows the proportion of cells in the indicated gating. Comparison between two indicated groups: * p<0.05; ** p<0.01. Data shown are representative of at least 3 separate experiments with same results.
Figure 3
Figure 3
Plasticity of Tregs under proinflammatory stimulation in vitro. Splenic and LN cells from FoxP3/gfp KI mice were stained with CD4. FoxP3/gfp+ or FoxP3/gfp− CD4 cells were flow-sorted. The cells were activated in vitro with plate-bound anti-CD3 and soluble anti-CD28 Abs, in Th0 (no cytokine added) or in Th1 (IL-12 plus anti-IL-4 Ab) or Th17 (IL-6, or IL-6 plus TGFβ) polarizing culture condition. After 5 days, the cells were re-stimulated with BD Leukocyte Activation Cocktail and intracellular IFNγ and IL-17A were analyzed by FACS. Numbers in FACS plots indicate the percentage of cells in the respective quadrant. The data shown are representative of at least three separate experiments with the same results.
Figure 4
Figure 4
TNF stabilizes FoxP3 expression on Tregs in vitro. Flow-sorted CD4+FoxP3/gfp+ cells were stimulated as described in Fig 3, with medium alone or with IL-6, in the presence of TNF or not. After re-activation, intracellular expression of FoxP3 and IL-17A was analyzed with FACS. Numbers indicate the percentage of cells in the respective quadrant. The data shown are representative of at least three separate experiments with the same results.
Figure 5
Figure 5
TNFR2 mediates the effect of TNF in maintaining FoxP3 expression in vitro. (A) Flow-sorted CD4+FoxP3/gfp+ cells were stimulated as in Fig 3, in the presence of TNF (10 ng/mL), and 10 µg/mL of isotype control Ham IgG, or anti-TNFR1 Ab or anti-TNFR2 Ab. FoxP3 expression was analyzed by FACS. Typical histograms and summary (N=3) are shown. (B) Tregs were flow-sorted from WT and TNFR2−/− mice, based on surface expression of CD4+CD25+. The expression of FoxP3 was analyzed by FACS. (C) CD4+ CD25+ cells from TNFR2 KO mice were activated as described in Fig 3, in the presence of TNF (10 ng/mL) or not. The expression of FoxP3 was analyzed by FACS. Typical FACS plot and summary (N=3) are shown. Numbers represent the percentage of cells within the indicated gate. The data shown are representative of at least three separate experiments with the same results. Compared with indicated group, * p<0.05; **p<0.01.
Figure 6
Figure 6
TNFR2 is required for the immunosuppressive function of Tregs in vivo. CD45.1+ CD4+CD25 –CD45RBhi naïve T (nCD4) cells were transferred alone or co-transferred with CD45.2+ WT or TNFR2−/− Treg cells into Rag1−/− mice. A. Cumulative incidence of colitis. Incidence of colitis in mice co-transferred with naïve CD4 cells and WT Tregs was markedly decreased, by comparison with mice transferred with naïve CD4 cells alone or co-transferred with naïve CD4 cells and TNFR2−/− Tregs (p=0.0132 and 0.0163, respectively). (B) After eight weeks, cLP cells were isolated. The intraceullar expression of IFNγ and IL-17A by initial transferred naïve WT CD4 cells was analyzed by FACS, gating on CD45.1+ cells. Typical FACS plot and summary (N=3~5) are shown. Data shown are representative of three separate experiments with similar results. * p<0.05; ** p<0.01.
Figure 7
Figure 7
Critical role of TNFR2 in stabilizing FoxP3 expression in vivo. CD45.1+ CD4+CD25 –CD45RBhi naïve T cells were transferred alone or co-transferred with CD45.2+ WT or TNFR2−/− Treg cells into Rag1−/− mice. After eight weeks, cLP cells were analyzed by FACS. (A) Reduced relative number of co-transferred TNFR2−/− Tregs. Proportion of CD45.2+ initial Treg cells in total transferred CD4 cells was determined. For comparison, pre-transferred cells were shown in the upper panel. (B) Reduced FoxP3 expression by initial Tregs from TNFR2−/− mice in cLP. Expression of IL-17A and FoxP3 was analyzed by FACS, by gating on CD45.2+ initial Tregs. Numbers in the FACS plots represent the percentage of cells in the indicated gate or quadrant. Typical FACS plot and summary (N=3~5) are shown. Data shown are representative of three separate experiments with similar results. * p<0.05; ** p<0.01.
Figure 8
Figure 8
TNFR2 expression is critical for the expansion of Tregs in competitive environment. CD45.1+ Tregs and CD45.2+ Tregs were flow-sorted from WT mice and TNFR2−/− mice respectively and co-transferred into Rag1−/− mice at ratio of 1:1. (A) Profile of pre-transferred Tregs. Left shows expression of TNFR2 and CD45.2 and right shows CD45.2 expression alone. (B) Flow-sorted Tregs from WT mice (solid histogram) and TNFR2−/− mice (grey histogram) express comparable levels of FoxP3. Dashed line: isotype control. (C) Ten weeks after transfer, Tregs present in the cLP were analyzed by FACS, gating on CD45+TCRβ+ cells. Left: expression of TCRβ and CD45.2 on cLP Tregs; right: summary of proportion of WT and TNFR2−/− Tregs in total transferred cells present in cLP (N=5). (D) FoxP3 expression on initial Tregs from WT or TNFR2−/− mice co-transferred into Rag1−/− mice (left) and summary of proportion of FoxP3-expressing cells in total transferred cells (right, N=5). The number shown in the FACS plots represent the proportion of cells in the respective quadrant or gate. Data shown are representatives of three separate experiments with similar results. * p<0.05; *** p<0.001.
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