The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITRhiPD-1hiCD25hi (Triplehi) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITRloPD-1loCD25lo (Triplelo) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4+ Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triplehi-like and Triplelo-like CD4+ T cells zsuper> T cells infiltrated the skin, whereas Scurfy TripleloCD4+ T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.
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We thank U. Schneider for animal husbandry, E. Traunecker and T. Krebs for cell sorting, and G. DeLibero, L. Jeker and O. Stepanek for reviewing the manuscript. This study was funded by grants 310030-149972/1 [SNF], Sybilla [EU FP7], and TerraIncognita [ERC] (E.P.); RO1-DK095077, U19 AI109858 and UMass DERC grant DK32520 (E.S.H.); T32 AI 007349 (B.D.S.); Federal Ministry of Education and Research grant (BMBF), German Center for Diabetes Research (grant DZD e.V., FKZ01GI0924) and Center for Regenerative Therapies Dresden, Cluster of Excellence grant FZT 111 (K.K.), Programme Grant from MRC (G.A.); Project IBS-R005-D1 from the Inst. for Basic Science, Korean Ministry of Science (C.D.S.) and Oncosuisse KFS-3169 (L.M.T.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) CD4+ T cells from B6 LNs were analyzed for Foxp3, GITR, PD-1 and CD25 protein expression by flow cytometry. Gates (left plot) show Triplehi (GITRhiPD-1hiCD25hi, red), Tripleint (GITRintPD-1intCD25int, grey) and Triplelo Treg cell (GITRloPD-1loCD25lo, brown) frequencies among total CD4+Foxp3+ cells. Histogram and bar graph (right) shows CD25 expression on Triplehi, Tripleint and Triplelo Treg cells (n=4 mice). (b-d) Expression of homing and activation markers on B6 LN Triplehi (red), Tripleint (grey) and Triplelo Treg cells (brown) analyzed by flow cytometry (n= 4 mice). (e) Triplehi (red), Tripleint (grey) and Triplelo (brown) Treg cells were analyzed for CD5 (n=4 mice) and Nur77-GFP (n=2 mice) expression by flow cytometry. (f) In vivo proliferation of Triplehi (red), Tripleint (grey), Triplelo (brown) Treg cells and CD4+ Tconv cells (blue) isolated from B6 LNs. Percentages of proliferating (BrdU+) cells are shown (n=4 mice). Bar graphs show mean ± s.e.m. Data is taken from 2-3 independent experiments.
Supplementary Figure 2 Nrp-1 and Helios expression in Triplehi and Triplelo Treg cells from SPF, GF and AF mice.
Lymph node cells from SPF, germ free (GF) and antigen free (AF) B6 mice were isolated, and Triplehi (red) and Triplelo (brown) Treg cells were analyzed for Nrp-1 (top) and Helios (bottom) expression by flow cytometry. Bar graphs show geometric mean of fluorescence intensity of Nrp-1 or Helios protein expression in Triplehi and Triplelo Treg cells (n=2 mice) NS= not significant (Kruskal-Wallis Test). Bar graphs display mean ± s.e.m. Data is taken from one experiment.
Supplementary Figure 3 Sorting strategy of CD4+ Tconv, Triplehi and Triplelo Treg cells from YAe62β-tg mice.
Splenocytes from YAe62β-tg TCRα+/KO mice were FACS sorted based upon the expression of Foxp3-GFP, PD-1 and GITR. (a) Gating strategy for FACS sorting of magnetically isolated CD4+ splenic populations from YAe62β-tg TCRα+/KO. (b) Frequency of Triplehi and Triplelo Treg cell populations among Foxp3+ CD4+ T cells in the 3 independent sorted samples (2 pooled mice per sample). Bars are the mean of the 3 samples. (c) Representative flow cytometric analysis of the CD4+ Tconv cell population following FACS sorting. (d) Representative flow cytometric analysis of the Triplehi Treg cell population following FACS sorting. (e) Representative flow cytometric analysis of the Triplelo Treg cell population following FACS sorting.
Supplementary Figure 4 Thymic Treg cell development in the presence of negatively selecting peptides.
Antigen affinity influences TCR downstream signaling. (a) Re-aggregate thymic organ cultures were established from B3K508TCR-tg, MHC II KO thymocytes, which are arrested at the CD4+ CD8+ DP stage and thymic epithelial cells from B6 mice. Peptides were added at the following concentrations: 20 μM P-1A (threshold affinity negative selector), 2 μM P2A (intermediate affinity negative selector), and 0.2 μM 3K (high affinity negative selector). After 7d in culture, thymocytes were stained for CD4 and Foxp3 and analyzed by flow cytometry. (b) Histograms shows flow cytometric analysis for CD25 (left) and Helios (right) protein expression of 3BK508TCR-tg Rag2–/– Foxp3+ CD4SP thymocytes, 48h after stimulation with P-1A (1μM, brown), P2A (1μM, grey) or 3K (1μM,red) peptide presented on mature B6 BMDCs in the presence of IL-2 and TGF-β. (c,d) Phosphorylation of CD3ζ, Erk and cJun in 3BK508TCR-tg CD4SP thymocytes stimulated with P-1A (grey line), P2A (brown line), 3K (red line) or no peptide (light grey filled) was determined after 90min, 24 and 48h by flow cytometry. (c) Representative histograms of fluorescence intensity in thymocytes stained for pCD3ζ, pJun and pErk following 24 and 48h of antigen stimulation and (d) geometric mean fluorescence intensity of pCD3ζ, pJun and pErk after 90min, 24h and 48h of peptide stimulation. (n=2 independent experiments)
Supplementary Figure 5 Sorting strategy to obtain Triplehi and Triplelo Treg cells and design of experimental lympho-proliferation.
(a) Magnetic bead enriched CD4+ T cells isolated from pooled LNs of 6-10 week old Foxp3eGFP mice were sorted for CD4+GFP+GITRhiPD-1hi (TriplehiTreg cells) and CD4+GFP+GITRloPD-1lo (TripleloTreg cells). Purity of each population was > 97%. (b) Lymphoproliferation induction by acute Treg cell ablation and B6 Treg cell treatment. 2.5x105 sorted B6 Treg cells, (unaffected by DTx) from pooled B6 LNs were injected intravenously (i.v.) into 6-10 week old Foxp3DTR mice. Three days later, host Foxp3+ cells were depleted intra peritoneal (i.p.) injection of DTx every other day for 10 days.
Supplementary Figure 6 Colitis-induction protocol and maintenance of Treg cell phenotype following adoptive transfer.
(a) To induce colitis, 6-10 week old T cell deficient CD3ε–/– mice received 3.2x105 sorted naive CD4+ Tconv cells (CD4+CD25–) isolated from B6 Ly5.1+ mice (B6Tconv). In some groups, 0.8 x105 sorted, Ly5.2+ Triplehi or Triplelo Treg cells from pooled B6 LNs were co-transferred along with Tconv cells. In experiments where iTreg cell generation was inhibited during colitis induction, 6-10 week old T cell deficient CD3ε–/– recipients were first injected i.v. with 3.2x105 sorted, naive CD4 Tconv cells isolated from Foxp3DTR, Ly5.1+ mice (Foxp3DTR Tconv cells). In some groups, 0.8 x105 sorted B6 TripleloTreg cells (unaffected by DTx) were co-transferred along with 3.2x105 sorted naive Foxp3DTRTconv cells. To deplete Tconv cell-derived iTreg cells, recipients were injected intraperitoneal every third day with DTx (10μg/kg). Mice were weighed weekly at the same time of day and were sacrificed when they lost > 20% of their initial body weight or at six weeks following adoptive transfer. (b) Flow cytometry analyzing GITR and PD-1 expression in LN cells isolated from mice that received Triplehi or TripleloTreg cells (described in a.The majority of transferred Triplelow Tregs maintain low PD-1 expression, although GITR expression is increased on Triplelow Tregs after six weeks in this lymphopenic environment. Data is from one experiment representative for four independent experiments with similar results.
Supplementary Figure 7 Sorting strategy, experimental setup for adoptive transfer of Scurfy disease, pathology of adoptively transferred mice and α4β7 integrin expression on transferred Scurfy cells homing to mLNs.
(a) Sorting strategy to obtain Scurfy Triplehi and Scurfy Triplelo CD4+ T cells. Magnetic bead enriched CD4+ T cells isolated from pooled LNs from 2 week old, male Foxp3-deficient mice were sorted for CD4+GITRhiCD25hi (Scurfy Triplehi CD4+ T cells) and CD4+GITRloCD25lo (Scurfy TripleloCD4+ T cells) T cells. Purity of each population was > 97%. (b) To transfer scurfy disease, 5x105 sorted Scurfy Triplehi or Scurfy Triplelo CD4+ T cells were injected i.v. into 6-10 week old CD3ε–/– recipient mice. Mice were weighed weekly at the same time of day and were sacrificed when they lost > 20% of their initial body weight or at six weeks following adoptive transfer. Photographs of peripheral and mesenteric LNs (c) and skin (tail) and colons (d) from CD3ε–/– mice six weeks after receiving either Scurfy Triplehi or Scurfy Triplelo CD4+ T cells. (e) Flow cytometric analysis of α4β7 integrin expression on Scurfy CD4+ T cells isolated from mLNs of CD3ε–/– mice six weeks after receiving either Scurfy Triplehi (top panel, orange bar) or Scurfy Triplelo (lower panel, purple bar) CD4+ T cells (n=3 mice each group). Data are from one experiment (d). Images for (c, e) are from one experiment representative of 5 independent experiments with similar results. Mean ± s.e.m., *P<0.05 (unpaired, two-tailed t-test).
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Wyss, L., Stadinski, B., King, C. et al. Affinity for self antigen selects Treg cells with distinct functional properties. Nat Immunol 17, 1093–1101 (2016). https://doi.org/10.1038/ni.3522
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