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. 2017 Apr 7;2(10):eaah7152.
doi: 10.1126/sciimmunol.aah7152.

Eomesodermin promotes the development of type 1 regulatory T (TR1) cells

Ping Zhang  1 , Jason S Lee  2 , Kate H Gartlan  2 , Iona S Schuster  3   4 , Iain Comerford  5 , Antiopi Varelias  2 , Md Ashik Ullah  2 , Slavica Vuckovic  2 , Motoko Koyama  2 , Rachel D Kuns  2 , Kelly R Locke  2 , Kirrilee J Beckett  2 , Stuart D Olver  2 , Luke D Samson  2 , Marcela Montes de Oca  2 , Fabian de Labastida Rivera  2 , Andrew D Clouston  6 , Gabrielle T Belz  7   8 , Bruce R Blazar  9 , Kelli P MacDonald  2 , Shaun R McColl  5 , Ranjeny Thomas  10 , Christian R Engwerda  2 , Mariapia A Degli-Esposti  3   4 , Axel Kallies  7   8 , Siok-Keen Tey  2   11 , Geoffrey R Hill  1   11
Affiliations

Eomesodermin promotes the development of type 1 regulatory T (TR1) cells

Ping Zhang et al. Sci Immunol. .

V体育平台登录 - Abstract

Type 1 regulatory T (TR1) cells are Foxp3- interleukin-10 (IL-10)-producing CD4+ T cells with potent immunosuppressive properties, but their requirements for lineage development have remained elusive. We show that TR1 cells constitute the most abundant regulatory population after allogeneic bone marrow transplantation (BMT), express the transcription factor Eomesodermin (Eomes), and are critical for the prevention of graft-versus-host disease VSports手机版. We demonstrate that Eomes is required for TR1 cell differentiation, during which it acts in concert with the transcription factor B lymphocyte-induced maturation protein-1 (Blimp-1) by transcriptionally activating IL-10 expression and repressing differentiation into other T helper cell lineages. We further show that Eomes induction in TR1 cells requires T-bet and donor macrophage-derived IL-27. Thus, we define the cellular and transcriptional control of TR1 cell differentiation during BMT, opening new avenues to therapeutic manipulation. .

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V体育官网入口 - Figures

Figure 1
Figure 1. TR1 cells constitute the major regulatory T cell after allogeneic BMT
(A – E) B6 (Syn) and B6D2F1 (Allo) mice were transplanted with B6 CD3+ T (Il10GFP/Foxp3RFP). (A) Gating strategy after BMT for analysis and FACS sorting of TR1 (red), Treg (blue) and Tcon (green) cells. (B) Schema of BMT. (C) Expression of IL-10 and FoxP3 in the spleen at d14 (representative of >3 experiments). (D) Frequencies of TR1 and Treg cells at d 14 (Il10GFP+: solid bar; Il10GFPneg: open bar). (E) CD4+ T cell subsets in spleen after BMT (n = 8 – 9 per group each time point). (F) B6D2F1 mice were transplanted with B6 CD4+ T (Il10GFP and Foxp3RFP) and frequencies of TR1 and Treg cells in the spleen at d14 (n = 14). (G) Suppression of proliferation of CFSE labelled B6 CD4+ and CD8+ responder T cells in vitro by naïve Treg cells versus TR1, Treg and Tcon “suppressors” sorted from 10 transplant recipients at d14 (data combined from 2 experiments). (H) Experimental BMT schema showing adoptive transfer of sorted TR1 cells to treat established acute GVHD and (I) survival of recipients are shown (n = 8 in TCD group, others n = 11 – 12). Data represents mean ± SEM.
Figure 2
Figure 2. TR1 cells express Eomes and display a distinct phenotypic profile
(A – C) B6D2F1 mice were transplanted with Il10GFPFoxp3RFP B6 CD3+ T cells. CD4+ T cells from spleen were FACS sorted into TR1, Treg and Tcon cells at d14 as in Fig 1A. (Data from 3 experiments, ND = not detectable) (A) Cytokine production in culture supernatant of T cell subsets. (B) Expression of transcription factors in T cell subsets (TR1: red, Treg: blue, Tcon: green, isotype: gray). (C) Expression of cytokines and Eomes in T cell subsets. Data represents mean ± SEM.
Figure 3
Figure 3. Eomes is required for TR1 cell differentiation
(A – D) B6D2F1 mice were transplanted with primary or retrovirally transduced (Mock-GFP or Eomes-GFP) CD4+ T cells. (A) Expression of IL-10, IFNγ, FoxP3, Eomes and T-bet (Eomes+IL-10+: open bar; EomesnegIL-10+: solid bar, n = 10 per group) in recipients of WT or Eomes−/− CD4+ T cells at d14 (n = 10 per group). (B) Expression of IL-10, IFNγ, and Eomes in transduced WT or Eomes−/− CD4+ T cells at d7 (n = 8 per group) and (C) transcription of Il10 and related genes (data are from 4–5 pooled animals in triplicate reactions, representative of 2 independent experiments). (D) CD4+ T cells or Foxp3RFPnegIl10GFP+ TR1 cells were FACS sorted from spleen and liver at d14 (representative of 3 experiments). A schematic diagram of the mouse IL-10 promoter indicates Eomes binding sites upstream of the TSS with each sequence shown. Recruitment of Eomes to the Il10 promoter and control regions in CD4+ T cells from TR1 cells (data are from 30 pooled animals in triplicate reactions) and recruitment of RNA Pol II to the Il10 promoter in WT or Eomes−/− CD4+ T cells (data are from 10 pooled animals in triplicate reactions). Data represents mean ± SEM.
Figure 4
Figure 4. Eomes+ TR1 cells are dependent on Blimp-1, IL-27 and IL-10
(A – F) B6D2F1 mice were transplanted with primary or retrovirally transduced (Mock-GFP or Eomes-GFP) CD4+ T cells and spleen examined after BMT. (A) Expression of Eomes, IL-10 and IFNγ in WT or Blimp-1−/− CD4+ T cells at d14 (n = 14 – 15 per group). (B) Expression of Eomes, IL-10 and IFNγ (n = 18, 17 for WT; n = 13, 14 for Blimp-1−/−) in transduced CD4+ T cells at d7–10. (C) Recruitment of Eomes to Il10 promoter in transduced CD4+ T cells (WT or Blimp-1−/−) at d10 (data are from 4 animals in duplicate or triplicate reactions). (D) Expression of T-bet, Eomes and IL-10 in WT or Il27r−/− CD4+ T cells at d14 (n = 10 per group). (E) Expression of Eomes and IFNγ+IL-10+ TR1 cells in WT or Il10r−/− CD4+ T cells at d14 (n = 10 per group). (F) Expression of Eomes and IL-10 (Eomes+IL-10+: open bar; Eomes+IL-10neg: solid bar) in CD4+ T cells in recipients of WT or Il10−/− CD4+CD25neg T cells at d14 (n = 10 – 11 per group). Data represents mean ± SEM.
Figure 5
Figure 5. Attenuation of GVHD by Eomes+ TR1 cells
(A – E) B6D2F1 recipients were transplanted with CD4+ T cells and survival or histopathology examined. (A) Survival of recipients of WT or Blimp-1−/− CD4+ T cells (2×106 per mouse) (n = 11 per T cell group, n = 7 in TCD; 2 experiments). (B) Survival of recipients of WT or Il27r−/− CD4+CD25neg T cells (2×106 per mouse) (n = 12 per T cell group, n = 7 in TCD; 2 experiments). (C and D) Histology in recipients of (C) WT versus Il10−/− or (D) WT versus Il10fl/fl × Lck-cre CD4+CD25neg T cells (1×106 per mouse) at d28 (n = 6 per T cell group, n = 3 in TCD group). (E) Survival of recipients of WT or Eomes−/− CD4+CD25neg T cells (1×106 per mouse) (n = 12 per T cell group, n = 7 in TCD; 2 experiments). Histology represents mean ± SEM.
Figure 6
Figure 6. Eomes and T-bet jointly regulate TR1 cell development
(A and B) B6.WT or B6.Ifngr−/− CD3+ T cells were transplanted into B6D2F1 mice and splenic CD4+ T cells examined at d14. (A) Representative plots show expression of T-bet and Eomes and (B) frequencies of TR1 and Treg cells and expression of IL-10 and Eomes (n = 10 per group). (C) B6.Il10GFPFoxp3RFP CD3+ T cells were transplanted into B6D2F1 mice receiving αIFNγ or control mAb and splenic CD4+ T cells examined at d12 (n = 5 per group). Frequencies of TR1 and Treg cells and expression of Eomes and IL-10 are shown. (D) B6D2F1 mice were transplanted with WT or Tbx21−/− CD4+ T cells and expression of transcription factors and cytokines in splenic CD4+ T cells at d12 shown (n = 10 per group). (E) B6D2F1 mice were transplanted with retrovirally (Mock-GFP or Eomes-GFP) transduced WT or Tbx21−/− CD4+ T cells and expression of IL-10, IFNγ, IL-4 and GATA-3 in splenic CD4+ T cells at d7 shown (n = 8 per group). (F) Co-expression of T-bet and Eomes in CD4+ T cells over time (representative of at least 2 experiments). (G) Splenic CD4+ T cells from naïve mice FACS sorted to 4 subsets based on Il10GFP and Foxp3RFP expression and T-bet and Eomes evaluated (representative of 2 experiments). Data represents mean ± SEM.
Figure 7
Figure 7. Recipient DC and macrophage-derived IL-27 promote the development of TR1 cells
(A – K) B6D2F1 mice were transplanted with TCD BM and CD4+ T cells and spleen examined. (A) Correlation of TR1 cells (Il10GFP+Foxp3RFPneg) with proportions of recipient DC at d14 (n = 26). (B) Frequencies of Treg (Foxp3GFP+) and TR1 (IFNγ+IL-10+) cells at d14 in the presence or absence of CD40L inhibition (n = 8 per group, grafts were CD4+Foxp3GFPneg). (C) WT.B6D2F1 or CD11c-DOG × DBA/2 F1 recipients were treated with DT to deplete recipient cDC and received B6.WT or MHC-II−/− BM respectively. Expression of TR1, Treg cells, Eomes and IL-10 at d14 are shown (n = 10 and 7 respectively). (D) Recipients of WT or CD11c-DOG BM were treated with DT to deplete donor cDC with expression of TR1 and Treg cells at d10 shown (n = 10 per group). (E) Data from (A) and (B) demonstrate correlation between numbers of TR1 cells and IL-27+ cells per spleen at d14 (n = 20). (F) Recipients were treated with IL-6R and spleens analyzed at d5. Phosphorylation of STAT1 and STAT3 in response to IL-6 or IL-27 (n = 10 per group). (G and H) Recipients were treated with IL-6R and spleens analyzed at d10. (G) Expression of Foxp3RFPnegIl10GFP+ TR1, Foxp3RFP+ Treg, Eomes and IL-10 in donor CD4+ T cells and (H) numbers of IL-27+ cells with intensity (MFI) of IL-27 (n = 9 – 10 per group). (I and J) Phenotypes of CD3neg IL-27 secreting cells at d14 are shown. (K) Expression of IL-27 from recipient DC at d+1 after BMT. (L and M) B6.WT or B6.Foxp3GFP-DTR mice were treated with DT for up to 2 weeks and spleens analyzed. (L) Phenotype of IL-27 secreting macrophage in CD3neg splenocytes and (M) expression of Eomes+IL-10+ cells over time with representative plots at d14. Data represents mean ± SEM.
Figure 8
Figure 8. Co-expression of T-bet and Eomes identifies a TR1 cells enriched population in human CD4+ T cells
(A) Representative plots show the correlation of Eomes to CD25, FOXP3 and cytokines in CD4+ T cells in healthy individuals and at d60 after clinical allo-BMT. (B) Frequencies of TR1 cells defined as IFNγ+IL-10+ or Eomes+IL-10+ in CD4+ T cells in healthy donors (n = 27) or d60 after clinical allo-BMT (n = 43). (C – E) Expression of cytokines in the T-betloEomeshi population relative to total CD4+ T cells or subpopulations defined with differential expression of Eomes and T-bet in healthy individuals (HD, n = 27) and at d60 after allo-BMT (BMT, n = 43). Data represents median ± interquartile range.
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