Cutting Edge: ICOS-Deficient Regulatory T Cells Display Normal Induction of Il10 but Readily Downregulate Expression of Foxp3

Abstract

The ICOS pathway has been implicated in the development and functions of regulatory T (Treg) cells, including those producing IL-10. Treg cell-derived IL-10 is indispensable for the establishment and maintenance of intestinal immune homeostasis. We examined the possible involvement of the ICOS pathway in the accumulation of murine colonic Foxp3- and/or IL-10-expressing cells. We show that ICOS deficiency does not impair induction of IL-10 by intestinal CD4 T cells but, instead, triggers substantial reductions in gut-resident and peripherally derived Foxp3⁺ Treg cells. ICOS deficiency is associated with reduced demethylation of Foxp3 CNS2 and enhanced loss of Foxp3. This instability significantly limits the ability of ICOS-deficient Treg cells to reverse ongoing inflammation. Collectively, our results identify a novel role for ICOS costimulation in imprinting the functional stability of Foxp3 that is required for the retention of full Treg cell function in the periphery.

Figure 1:. Altered distribution of colonic Foxp3⁺ subsets in absence of ICOS.

(A) Total CD4 single-positive T cells from the thymus, spleen (Spl), and large intestine (LI) of co-housed wild type (grey fill) and Icos^−/− (black line) mice were examined for expression of Foxp3. (B) Graph summarizing the frequencies of Foxp3⁺ cells in wild type and Icos^−/− mice analyzed as in A. (C) Graph displaying actual numbers of LI CD4⁺ and Foxp3⁺ cells. (D) Helios expression by Foxp3-gated cells. (E) Graph summarizing the frequencies of Helios⁺ cells among Foxp3⁺ cells. (F) TREC counts among purified splenic CD4 T cells from Icos^+/+ and Icos^−/− mice. (G) Analysis of thymic and lamina propria CD4 T cells from 6-week-old CBir1 transgenic mice. Graphs summarize frequencies of LI Foxp3⁺ cells (H) and numbers of colonic CD4⁺ and Foxp3⁺ cells (I) from Icos^+/+ and Icos^−/− CBir1.Rag^−/− mice analyzed as in G. Graphs represent data from 2 (C, E) or 3 (G) similar experiments each with 3–5 mice per group. Graphs show mean +/− SEM. *p<0.05, ** p<0.01, ***p<0.001, NS=not significant.

Figure 2:. Induction of Il10 in intestinal CD4 T cells independent of ICOS.

(A) LI lamina propria CD4 T cells from 10BiT and 10BiT.Icos^−/− mice were examined for co-expression of Thy1.1 and Foxp3. (B-C) Graphs summarizing frequencies and MFI respectively of Thy1.1⁺ cells among Foxp3⁻, Foxp3⁺, and total CD4 T cells as shown in A. (D) LI lamina propria Foxp3-gated CD4 T cells from 10BiT and 10BiT.Icos^−/− mice were examined for co-expression of Helios and Thy1.1. (E-F) Graphs summarizing frequencies and MFI of Thy1.1⁺ cells among Helios⁻, Helios⁺, and total Foxp3⁺ T cells. (G) Schematic overview of thymocyte transfer experiment. CD4 single-positive Foxp3⁻ thymocytes were FACS-sorted from congenically-marked WT (CD45.1) and Icos^−/− (CD45.2) 10BiT.Foxp3 mice and transferred to Tcrβδ^−/− recipients. After 3 weeks, donor CD4⁺TCRβ⁺ cells from the LI lamina propria were analyzed by FACS. (H) Analysis of Foxp3, IL-17 and Thy1.1 expression by donor T cells 3 weeks after transfer. (I) Graphs summarizing frequencies of the various cell populations from all mice analyzed as in H. (J) Analysis of Foxp3 and Thy1.1 expression by wild type and Icos^−/− T cells recovered from the LI lamina propria. (K) Graphs summarizing frequencies of the various cell populations from mice analyzed as in J. Graphs represent data pooled from 1 of 2 similar experiments with 3–5 mice per group and display mean + SEM. *p<0.05, ***p<0.001, NS=not significant.

Figure 3:. ICOS-deficient Treg cells display robust methylation of Foxp3 CNS2 and readily downregulate Foxp3 ex vivo and in vivo.

(A). Graph shows average methylation of 9 CpG sites of Foxp3 CNS2 as determined by pyrosequencing of purified CD4⁺GFP⁺ cells from Icos^+/+ and Icos^−/− Foxp3^gfp mice. (B) Purified CD4⁺GFP⁺ cells from the WT (CD45.1) and Icos^−/− (CD45.2) Foxp3^gfp mice were co-cultured with anti-CD3 and anti-CD28 plus IL-2 and IL-7. IL-1β, IL-6, IL-12, and IL-23 (stim + cytokines), or anti-IL-6R, anti-IL-12/23p40, and anti-IL-21R (stim + blockade) were added to select wells. Expression of Foxp3 was examined on Day 3. (C) Rag1^−/− mice received naïve CD4⁺ CD45RB^hi T cells purified from B6.CD45.1 mice. Mice in each cage were then randomly assigned to 1 of 3 groups and received either vehicle (PBS), or identical doses of wild type or Icos^−/− Treg cells, each on the CD45.2 background. Mice were weighed weekly until they were euthanized at week 8. (D-E) Histology scores and representative photomicrographs of H&E-stained colonic tissues from mice in the 3 recipient groups, 10X magnification. (F) FACS analysis of LI CD4⁺ cells (left panel) and of Foxp3+ cells among CD45.2⁺ cells (right panel). Graphs summarize relative frequencies and numbers of LI CD45.2⁺ cells (G) and frequencies of Foxp3+ among CD45.2+ analyzed as in F (H). Bar graphs display mean + SEM and represent data from 1 of 2 (A) or 1 of 3 (D, G, H) similar experiments. **p<0.01, ***p<0.001, NS, not significant.

Figure 4:. Downregulation of Foxp3 correlates with the inability of ICOS-deficient Treg cells to reverse ongoing inflammation.

(A) Rag1^−/− mice were injected with CD45.1⁺ CD45RB^hi T cells as in Figure 3 and monitored for 4 weeks. At week 4, mice were randomly assigned to 1 of 3 groups that received PBS, or identical numbers of CD45.2⁺ of wild type or Icos^−/− Treg cells. Mice were weighed weekly until week 14. (B and C) Histology scores and representative photomicrographs of H&E-stained colonic tissues from mice in the 3 recipient groups, 10X magnification. (D) Representative plots depicting relative frequencies of CD4⁺ cells of Treg origin (CD45.2⁺) remaining at the end of the experiment (left panel) and the percentage of CD45.2⁺ cells that still expressed Foxp3 (right panel). (E) Bar graphs displaying relative Foxp3+ cell frequencies and numbers of Foxp3+ cells from all mice analyzed as in D. (F) Graph shows numbers of cells of CD45.2 origin recovered from the LI or recipient mice. All data are from 1 of 2 replicate experiments each with 5 recipients per group. Graphs show mean +/− SEM. ** p<0.01, ***p<0.001, NS=not significant.