Thymic regulatory T cells arise via two distinct developmental programs

Abstract

The developmental programs that generate a broad repertoire of regulatory T cells (T_reg cells) able to respond to both self antigens and non–self antigens remain unclear. Here we found that mature T_reg cells were generated through two distinct developmental programs involving CD25⁺ T_reg cell progenitors (CD25⁺ T_regP) and Foxp3^lo T_reg cell progenitors (Foxp3^lo T_regP). The CD25⁺ T_regP had higher rates of apoptosis and interacted with thymic self-antigens with higher affinity than Foxp3^lo T_regP, and had a T cell antigen receptor (TCR) repertoire and transcriptome distinct from that of Foxp3^lo T_regP. The development of CD25⁺ T_regP and Foxp3^lo T_regP was controlled by distinct signaling pathways and enhancers. Transcriptomic and histocytometric data suggested that CD25⁺ T_regP and Foxp3^lo T_regP arose by coopting negative and positive selection programs, respectively. T_reg cells derived from CD25⁺ T_regP, but not Foxp3^lo T_regP, prevented experimental autoimmune encephalitis. Our findings indicate that T_reg cells arise through two distinct developmental programs that are both required for a comprehensive T_reg cell repertoire capable of establishing immune tolerance.

Regulatory T cells (T_reg cells) play key roles in protecting against autoimmune responses to tissues, preventing inappropriate responses to commensal organisms and dampening effector T cell responses following clearance of pathogens. However, the mechanisms that lead to the development of a population of Treg cells that can mediate such diverse functions remain unclear. T_reg cells were shown to develop through a two-step process in the thymus ^{1, 2}. The first step is driven by strong signals sent through the T cell antigen receptor (TCR), which leads to upregulation of CD25, the key component of the high-affinity receptor for IL-2, as well as the TNF receptor superfamily members GITR, OX40 and TNFR2, but not the upregulation of the transcription factor Foxp3 ^{1, 3}. A second, TCR-independent, step involves the conversion of the CD25⁺ T_regP cells into mature CD25⁺Foxp3⁺ T_reg cells in a manner dependent on the cytokine IL-2 and the transcription factor STAT5 ^{1, 2, 4, 5}. A distinct T_reg cell progenitor population, characterized by low expression of Foxp3 and lacking detectable expression of CD25, was also described in the thymus ⁶. This Foxp3^lo T_regP cell has high expression of GITR and OX40 ³, and can differentiate into mature CD25⁺Foxp3⁺ T_reg cells following stimulation with IL-2 ⁶. The relative contributions of these T_regP cell populations to the mature T_reg cell pool remains controversial.

Here we demonstrate that CD25⁺Foxp3⁻ T_reg cell progenitors (CD25⁺ T_regP hereafter) and CD25⁻Foxp3^lo T_reg progenitors (Foxp3^lo T_regP hereafter) generated mature T_reg cells with relatively comparable efficiency in vitro and in vivo. The two developmental pathways for T_reg cell generation differed in many aspects, including distinct transcriptomes and TCR repertoires. The CD25⁺ T_regP exhibited increased apoptosis, developed into mature T_reg cells with faster kinetics and exhibited greater reactivity with self-antigens in the thymus than Foxp3^lo T_regP. Development of the two T_reg cell progenitor subsets was controlled by different cytokines, signaling pathways, gene enhancers and stromal cells in the thymus. Finally, T_reg cells derived from CD25⁺ T_regP, but not Foxp3^lo T_regP, protected against experimental autoimmune encephalomyelitis. Our data suggest a model in which two distinct T_reg cell progenitor subsets both contribute to generate a broad T_reg cell repertoire able to protect against immune responses to self-antigen, limit immune responses to commensal organisms and resolve immune responses to foreign pathogens.

Results

CD25⁺ and Foxp3^lo T_regP cells differentiate into T_reg cells

To determine if CD25⁺ and Foxp3^lo T_regP are both bona-fide thymic T_reg progenitors we compared their ability to convert into mature T_reg cells in response to low doses of IL-2 for 3 days in vitro. Sorted CD25⁺ T_regP and Foxp3^lo T_regP responded to very low amounts of IL-2 (0.2–1.0 U/mL) by converting to mature CD25⁺Foxp3⁺ T_reg cells (Supplementary Fig. 1a) indicating that although they lack CD25 expression, Foxp3^lo T_regP, which express the low affinity IL-2R, consisting of the IL-2Rβ and IL-2Rγ chains, were responsive to IL-2. Mature CD25⁺Foxp3⁺ T_reg cells exhibited even greater sensitivity to IL-2, as they maintained their phenotype and viability at concentrations of IL-2 (0.04 U/mL) to which CD25⁺ T_regP and Foxp3^lo T_regP cells did not respond (Supplementary Fig. 1a). To confirm these findings in vivo, we used ultrasound-guided intrathymic injection to co-transfer sorted congenically distinct (CD90.2⁺CD45.2⁺ or CD90.1⁺CD45.2⁺) CD25⁺ or Foxp3^lo T_regP cells into the thymi of CD45.1⁺ mice (Fig. 1b, Supplementary Fig. 1b). Six days post-injection, total congenically labelled cells, derived from CD25⁺ or Foxp3^lo T_regP, were recovered from the host thymi with similar efficiency (data not shown). CD25⁺ T_regP and Foxp3^lo T_regP cells converted into mature CD25⁺Foxp3⁺ T_reg cells with approximately the same frequency, although CD25⁺ T_regP did so slightly better (~45±16.9% to 37±23.5%, respectively; Fig. 1b). Thus, both CD25⁺ T_regP and Foxp3^lo T_regP contributed to the generation of mature T_reg cells with high efficiency in vitro and in vivo.

Figure 1.

Two thymic T_reg progenitor cell populations exist. a) Gating scheme used to quantify or isolate CD25⁺Foxp3⁻ and CD25⁻Foxp3^lo T_regP cell populations throughout the manuscript. b) Quantification of the proportion of sorted congenically distinct (CD90.2⁺CD45.2⁺ or CD90.1⁺CD45.2⁺) CD25⁺ and Foxp3^lo T_regP cells co-injected into the thymi of CD45.1⁺ mice that differentiated into CD25⁺Foxp3⁺ T_reg cells 6 post-injection. Each dot represents a single recipient mouse; pairing represents data within the same recipient thymi. Data represents 3 independent experiments, n=9 mice. Data was analyzed by a two-sided paired t test. * P<0.05.

CD25⁺ T_regP and Foxp3^lo T_regP cells have distinct TCR repertoires

To address whether the CD25⁺ T_regP and Foxp3^lo T_regP cells represent distinct subsets of cells with different TCR repertoires, or whether the T_reg cell developmental pathway chosen only reflects the stochastic expression of CD25 or Foxp3, we used mice expressing a fixed TCliβ TCRb transgene on a TCRa^+/− heterozygous background expressing a Foxp3-RFP reporter ^{7, 8, 9}. Using these mice we carried out high throughput sequencing of the TCR Trav14 genes in conventional CD25⁻Foxp3⁻ thymocytes, CD25⁺ T_regP, Foxp3^lo T_regP and mature CD25⁺Foxp3⁺ T_reg cells. Consistent with previously published results ^{7, 10, 11}, we found little overlap between the Trav14 repertoire of conventional CD25⁻Foxp3⁻ thymocytes and mature thymic CD25⁺Foxp3⁺ T_reg cells (Fig. 2a). As reported ¹, we found substantial overlap between the Trav14 repertoire of CD25⁺ T_regP cells and mature CD25⁺Foxp3⁺ T_reg cells (Fig. 2a,b, Table 1). Importantly, the TCR repertoire of the Foxp3^lo T_regP also overlapped substantially with that of mature CD25⁺Foxp3⁺ T_reg cells (Fig. 2a). However, the TCR repertoires of the CD25⁺ T_regP and Foxp3^lo T_regP cell populations showed very little overlap, despite their significant individual overlap with the repertoire of mature T_reg cells (Fig. 2a,b). To narrow our analysis to TCRs that are known to be represented in the mature T_reg cell pool, we reanalyzed our TCR repertoire data but this time excluding all the TCRs not detected in the thymic mature T_reg cell pool. The ratios of Morisita-Horn indices (T_regP to T_regP versus T_regP to T_reg cell) calculated using TCRs detected only in mature CD25⁺Foxp3⁺ T_reg cells in the thymus were not significantly different than the ratios obtained when using all TCRs(Supplementary Table 1). Thus, CD25⁺ T_regP and Foxp3^lo T_regP cells had distinct TCR repertoires and contributed unique TCR clones to the mature T_reg cell repertoire.

Figure 2.

CD25⁺ and Foxp3^lo T_regP are distinct thymic T_reg cell lineages. a) Morisita-Horn indices comparing similarity of Vα2 CDR3 repertoires generated by TCR sequencing of CD4⁺CD25⁻Foxp3⁻, CD4⁺CD25⁺Foxp3⁻ T_regP, CD4⁺CD25⁻Foxp3^lo T_regP and CD4⁺CD25⁺Foxp3^lo mature T_reg cells isolated from Tcliβ⁺TCRa^+/− mice. b) Plot of the ratio of Morisita-Horn similarity indices comparing TCRs in CD25⁺ and Foxp3^lo T_regP cell populations, and TCRs in CD25⁺T_regP and mature T_reg cells or Foxp3^lo T_regP and mature T_reg cells. CD25⁺ T_regP cell comparisons are shown in green and Foxp3^lo T_regP cell comparisons are shown in orange. Data represents 2 independent experiments, n=4 mice. Data was analyzed by a two-sided paired t test. c,d) Flow cytomyerty analysis of Nur77-GFP MFI in CD25⁺Foxp3⁻ T_regP, CD25⁻Foxp3^lo TregP and CD25⁺Foxp3⁺ mature T_reg cells obtained from thymi of Nur77-GFP reporter mice. Dots represent individual mice. Data are displayed as mean ± SD and represents 7 independent experiments, n=19 mice. Data was analyzed via a two-sided paired Friedman test with Dunn’s multiple comparisons test. e) Left- Three-dimensional tSNE plots from 10X Genomics single-cell RNA-Seq data set for sorted CD25⁺ T_regP, Foxp3_lo T_regP and CD25⁺Foxp3⁺ T_reg cells displaying relationships between individual cells with color-coding based on flow cytometry sort origin. Right- Three-dimensional tSNE plots of identical data to left panel but color-coded based on gene expression profiles. f) Heatmap for the top 10 differentially regulated genes from each cluster derived from subfigure e. Each column represents gene expression for an individual cell; yellow is up and purple is down. Data from e,f is representative of 3 independent experiments, n=3 mice. *P<0.05, **P<0.0005, ***P<0.0001.

Table 1:

List of representative TCR Vα2 CDR3 sequences and the relative read distribution in each sorted population (conventional defined as CD4⁺CD8⁻CD25⁻Foxp3⁻).

Amino acid sequence	Reads Conventional	Read CD25+ TregP cells	Reads Foxp3lo TregP cells	Reads Treg cells
CAASGSAGNKLTF	0	0	2737	7714
CAAKSGSFNKLTF	0	1888	0	3587
CAAKHSGTYQRF	0	0	1176	3093
CAAPSSGSWQLIF	0	2028	659	1697
CAASYYNQGKLIF	0	349	0	1801
CAASKGSNYQLIW	0	569	0	1729
CAASAPYNQGKLIF	0	0	4221	1428
CAASSSGSFNKLTF	0	0	1392	1399
CAASLDYSNNRLTL	0	1015	863	1097
CAARASGSWQLIF	0	224	1044	2080
CAAASSGSWQLIF	0	432	268	1177
CAARNYNQGKLIF	1092	5278	2532	7041
CAASGTGGYKVVF	199	317	2870	1128

CD25⁺ T_regP and Foxp3^lo T_regP cells have distinct affinity for self-antigen

Next we examined the types of antigens that CD25⁺ T_regP and Foxp3^lo T_regP interacted with. The relative abundance of either T_regP cell subset was similar in germ-free mice compared to specific pathogen-free mice (Supplementary Fig. 2a,b) or in C57Bl/6 mice co-housed with pet store mice, which have a normalized microbial experience ¹², compared to specific pathogen-free mice (Supplementary Fig. 2c), suggesting that interactions with self antigens were the major driver of T_reg cell selection in the thymus. We used expression of the transcription factor Nur77, whose abundance is directly proportional to TCR signal strength, to assess the strength of the interaction of CD25⁺ T_regP and Foxp3^lo T_regP cells with self-antigens in the thymus. In Nur77-GFP mice, in which GFP expression correlates with the strength of TCR stimulation ^{13, 14}, mature CD25⁺Foxp3⁺ T_reg cells have higher expression of Nur77-GFP than conventional CD4⁺Foxp3⁻ T cells ^{3, 13, 14}, corresponding with the higher degree of self-reactivity attributed to T_reg cells ⁷. In this system, expression of Nur77-GFP in CD25⁺ T_regP cells was significantly higher compared to that recorded in mature T_reg cells (Fig. 2c,d), while the expression of Nur77-GFP in Foxp3^lo T_regP cells was significantly lower than that of CD25⁺ T_regP cells (Fig. 2c,d). These findings indicate that CD25⁺ T_regP and Foxp3^lo T_regP cells have distinct affinities for self-antigens in the thymus.

We next assessed the transcriptomic differences between the T_regP subsets using single-cell RNA-Seq. CD25⁺ T_regP cells, Foxp3^lo T_regP cells and mature, CD25⁺Foxp3⁺ T_reg cells sorted from thymi of Foxp3-GFP mice, in which an IRES-GFP construct was knocked into the3’ UTR of the Foxp3 gene, and were used to create individual single cell RNA-Seq libraries that were subjected to high-throughput sequencing. The transcriptomic data from these individual libraries was then combined for joint analysis. Individual cells were color-coded based on sort origin and a combined dimensional reduction with graph-based clustering approach followed by a shared nearest-neighbor modularity optimization-based clustering algorithm (Seurat R package) was applied to this combined data set to identify cell groups with distinct gene expression. This analysis generated clusters of cells that closely confirmed the original sorted populations (Fig. 2e), based on expression of Il2ra and Foxp3 (Supplementary Fig. 3a). The analysis also identified a small subset of contaminating thymocytes that expressed Rag1, Cd8b1 and Dntt and therefore likely represent CD4⁺CD8⁺ double positive thymocytes (Fig. 2f). Heat maps based on the top ten most differentially expressed genes for each cell subset discriminated all four cell clusters (Fig. 2f). Analysis of differentially expressed genes indicated that there were ~180 reproducibly differentially expressed genes between CD25⁺ and Foxp3^lo T_regP cell subsets (Fig. 2f and Supplementary Table 2). Similar results were obtained in an independent single-cell RNA-Seq study with individually sorted CD25⁺ T_regP cell, Foxp3^lo T_regP cell and mature T_reg cell libraries (Supplementary Fig. 3b) or when CD25⁺ T_regP, Foxp3^lo T_regP cells and mature T_reg cell subsets were combined into one library (data not shown). CD25⁺ T_regP were enriched in pro-apoptotic genes and genes involved in negative selection (Nr4a1 and Bcl2l11) (Supplementary Fig. 3c, Supplementary Table 2), consistent with stronger TCR signaling in this subset. Foxp3^lo T_regP had increased expression of Ms4a4b and Ms4a6b (Fig. 2f), which encode MS4A4B and MS4A6B, which bind to GITR and enhance signaling via TCR and GITR ¹⁵ and may facilitate differentiation of lower affinity CD4⁺ thymocytes into Foxp3^lo T_regP and enhance sensitivity of Foxp3^lo T_regP to IL-2 ³. Thus, CD25⁺ T_regP cells and Foxp3^lo T_regP cells had distinct interactions with self-antigens presented in the thymus and unique transcriptomes indicative of distinct modes of selection and differentiation.

CD25⁺ T_regP and Foxp3^lo T_regP cells are at distinct developmental stages

The cell surface markers CD24 and Qa2, or CD69 and MHCI, can be used as surrogates to analyze thymocyte age ¹⁶. We found that CD25⁺ T_regP cells were largely CD24^hiQa2^lo (87%) and CD69^hi MHC1⁺, and thus representative of immature CD4⁺ thymocytes. In contrast, only 38% of Foxp3^lo T_regP cells were found in the CD24^hiQa2^lo gate with the remainder being CD24^loQa2^hi, indicative of a more mature stage of thymocyte development (Supplementary Fig. 4a,b). To examine this issue more precisely, we used Rag2-GFP transgenic mice, in which GFP expression is controlled by the Rag2 gene regulatory elements, to determine the kinetics of differentiation for CD25⁺ T_regP and Foxp3^lo T_regP cells. The Rag2-GFP transgene turns off after positive selection in CD4⁺CD8⁺ thymocytes, after which the GFP protein decays with a relatively slow half-life, allowing GFP⁺ recent thymic emigrants to be distinguished from older GFP⁻ T cells in peripheral blood and lymphoid organs ¹⁷. To determine whether within the GFP⁺ thymocyte fraction a gradient in GFP expression could distinguish thymocytes at different stages of development, we gated on CD4⁺RAG2-GFP⁺ thymocytes and examined CD25 and Foxp3 expression in different bins determined by expression of RAG2-GFP. RAG2-GFP^brightest cells (bin 1) were all CD25⁻Foxp3⁻ CD4SP thymocytes (Fig. 3a). CD25⁺ T_regP, but no Foxp3^lo T_regP cells or mature CD25⁺Foxp3⁺ T_reg cells were detected in bin 2, while Foxp3^lo T_regP and mature CD25⁺Foxp3⁺ T_reg cells appeared in bin 3. CD25⁺ T_regP, Foxp3^lo T_regP and mature CD25⁺Foxp3⁺ T_reg cells were all detected in bins 4–6, while CD25⁺ T_regP cells were no longer detected in bin 7, as these cells differentiate, die or leave the thymus (Fig. 3a and Supplementary Video 1). Bin 8 contained GFP^– cells (Fig. 3a), representing fully mature recirculating T cells. This analysis indicated that the CD25⁺ T_regP cells differentiated rapidly, while Foxp3^lo T_regP cells took longer to develop.

Figure 3.

CD25⁺ and Foxp3^lo T_regP cells are in discrete selection stages. a) Representative histograms of RAG2-GFP expression in CD4SP thymocytes obtained from thymi of RAG2-GFP mice. Bins are displayed from high (Bin 1) to low (Bin 8) RAG-GFP expression and cells in each bin are plotted for CD25 vs Foxp3 expression. Data shown are concatenated results from 3 mice and are representative of 7 independent experiments, n=9 mice. b) Representative flow cytometry plots of CD73 staining in CD4SP thymocytes and RAG2-GFP expression in CD73⁻ and CD73⁺ compartments. Data is representative of 5 experiments, n=5 mice. c) Representative flow cytometry plots (top) and quantification of the percent of CD4⁺CD73⁻ thymocytes differentiating into each T_regP cell population in WT versus Itgal^−/− thymi. Data represents 3 independent experiments, n= 16 WT and Itgal^+/− mice, n=12 Itgal^−/− mice. Data was analyzed by two-sided Mann-Whitney test. d) Left panels, Representative example of Annexin V expression on CD4⁺CD73⁻CD25⁺Foxp3⁻ T_regP (top green histogram) and CD4⁺CD73⁻CD25⁻Foxp3^lo T_regP (bottom orange histogram) thymocytes from Foxp3-GFP mice. Right panel, quantification of Annexin V staining on CD25⁺ T_regP and Foxp3_lo T_regP. Data represents 3 independent experiments, n=9 mice. Data were analyzed by two-sided Wilcoxon matched-pairs signed rank test. e) Left and middle panels, represenative examples of staining for CD25 and Foxp3 on CD4⁺CD73⁻ gated thymocytes from wild-type mice either negative (left panel) or positive (middle panel) for cleaved caspase-3. Right panel, quantification of the ratio of cleaved caspase-3-positive to cleaved caspase-3 negative CD25⁺ T_regP (green circles) or Foxp3lo T_regP; data represents 4 independent experiments, n=10 mice. Data were analyzed by two-sided Wilcoxon matched-pairs signed rank test. All data are displayed as mean ± SD. *P<0.05, **P<0.005, ns-not significant.

About half of CD25⁺Foxp3⁺ T_reg cells in the thymus represent mature recirculating cells ^{18, 19}. Because 18% of Foxp3^lo T_regP cells consisted of recirculating Rag2-GFP⁻ T cells (Supplementary Fig. 5), we looked for cell surface markers that could distinguish newly developed RAG2-GFP⁺ thymocytes from recirculating RAG2-GFP⁻ T cells. Over 99% of CD73⁻ cells were RAG2-GFP⁺, while >85% of CD73⁺ cells were RAG2-GFP⁻ (Fig. 3b), suggesting expression of CD73 could distinguish between developing thymocytes and recirculating mature T cells in the thymus in the absence of the Rag2-GFP transgenic reporter. To assess whether Foxp3^lo T_regP cells, which arose with slower kinetics and exhibited reduced TCR signal strength, were more dependent on adhesion molecules like LFA-1, which help prolong T cell-APC contacts and enhance TCR signaling ²⁰, we examined T_reg cell development in Itgal^−/− mice, which are LFA-1 deficient. CD73⁻ Foxp3^lo T_regP, but not CD73⁻ CD25⁺ T_regP cells were decreased in Itgal^−/− thymi compared to wild-type thymi (Fig. 3c). Thus, development of Foxp3^lo T_regP cells was more dependent on stable T cell-APC interactions or LFA-1-dependent co-stimulation compared to CD25⁺ T_regP cells.

Based on Foxp3 overexpression studies, Foxp3^lo T_regP cells were proposed to be very susceptible to apoptosis ⁶. Because our single cell RNA-Seq, based on Bcl2l11 and Nr4a1 expression, suggested that CD25⁺ T_regP cells might be more apoptotic than Foxp3^lo T_regP cells (Supplementary Fig. 3c, Supplementary Table 2) we used annexin V staining to identify apoptotic cells in the thymus. Annexin V⁺ cells were detected in both T_regP cell subsets, although there were substantially more annexin V⁺ cells in the CD73⁻ CD25⁺ T_regP (12%) than in the CD73⁻ Foxp3^lo T_regP cells (3.3%; Fig. 3d). We also stained thymocytes for cleaved caspase-3 (casp-3). CD25⁺ T_regP were enriched four-fold for casp-3⁺ cells compared to Foxp3^lo T_regP cells (Fig. 3e). Thus, CD25⁺ T_regP cells interacted most strongly with self-antigen in the thymus and contained a higher fraction of apoptotic cells, suggesting they were undergoing negative selection.

We next examined where Foxp3^lo TregP were located in the thymus. Histocytometry ²¹ experiments in C57Bl/6 mice thymi indicated that mature CD25⁺Foxp3⁺ T_reg cells were largely restricted to the thymic medulla (Fig.4a,b), consistent with agonist-driven T_reg selection occurring in the thymic medulla. In contrast, Foxp3^lo T_regP cells were found both in the thymic medulla and some (20%) in the thymic cortex (Fig. 4 a,b), suggesting that at least some Foxp3^lo T_regP cells were selected on cortical antigens. Thus, CD25⁺ and Foxp3^lo T_regP subsets differentiate with distinct kinetics and exhibit different rates of apoptosis; moreover, a fraction of the Foxp3^lo T_regP subset share features with conventional (i.e., non-Treg) T cells undergoing positive selection, which also occurs in the thymic cortex.

Figure 4.

T_reg cells and Foxp3^lo T_regP cells show different localization in the thymus. a) Top left panels, representative histocytometry dot plots of thymocytes stained with antibodies to CD25, Foxp3 and TCRβ used to denote Foxp3^lo T_regP cells (CD25⁻Foxp3⁺TCRβ⁺) and T_reg cells (CD25⁺Foxp3⁺TCRβ⁺) within stained thymic sections. Bottom left panels, representative images of Foxp3^lo T_regP cells and T_reg cells. Right panels, thymic sections stained with antibodies to TCRb and RORgt used to delineate the thymic medulla (M) and cortex (C). Distribution of CD25⁺Foxp3⁺TCRβ⁺ T_reg cells (blue) and CD25⁻Foxp3⁺TCRβ⁺ Foxp3^lo T_regP cells (orange) within the thymic medulla and cortex (right). Scale bar represents 200μm. b) Distribution of Foxp3^lo T_regP cells and T_reg cells between thymic cortex and medulla, p value determined by Fisher’s exact test. Data represents 1 experiment, n=2 mice. ***P<0.0001.

NF-kB is critical for the development of Foxp3^lo T_regP cells

We next assessed the effect of co-stimulation and downstream signaling pathways on the development of CD25⁺ T_regP and Foxp3^lo T_regP. Cd28^−/− thymi exhibited a 1.7-fold decrease in abundance of CD25⁺ T_regP, and a 6.7-fold decrease in abundance of Foxp3^lo T_regP compared to thymi from wild-type mice (Fig. 5a). Similar results were found in CD28-AYAA mice, which express a mutant CD28 lacking the PYAP motif that links CD28 to activation of the transcription factor NF-κB (data not shown), but not in CD28-Y170F mice, which express a mutant CD28 lacking Y170, which links CD28 to signaling through PI3K (data not shown). Conversely, IKK-CA mice, which express a constitutively active from of IKK that leads to constitutive NF-κB activation, had a selective increase in Foxp3^lo T_regP cells (data not shown). A caveat of the analysis in CD28-AYAA, CD28-Y170F and IKK-CA mice is the lack of CD73 staining to exclude recirculating mature effector T cells and T_reg cells from the CD25⁺Foxp3⁻ T_regP and CD25⁻FOXP3^lo T_regP gates, respectively. Finally, we examined the effect of the downstream transcription factor NF-κB1 on T_reg cell development. Nfkb1^−/− mice had a 3.6 fold reduction in the abundance of CD73⁻Foxp3^lo T_regP cells compared to thymi from wild-type mice, while CD73⁻CD25⁺ T_regP cell abundance was unaffected (Fig. 5b). These results suggest that co-stimulation, and especially activation of NF-κB1, were selectively required for the formation of Foxp3^lo T_regP.

Figure 5.

Foxp3^lo T_regP cells are dependent on NFκB1 activation and the Foxp3 regulatory element Cns3. a), Left panels, shown are representative flow cytometry plots for CD4⁺CD8⁻CD73⁻ gated thymocytes antibodies from wild-type and Cd28^−/− mice stained with antibodies to CD25 and Foxp3 (1 experiment, n=4 wild-type and 5 Cd28^−/− mice). Right panels show cumulative data for all mice and depict the relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type and Cd28^−/− mice. b), Shown are representative flow cytometry plots for CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type and Nfkb1^−/− mice stained with antibodies to CD25 and Foxp3 (3 independent experiments, n=19 wild-type and 11 Nfkb1^−/− mice). Right panels show cumulative data for all mice and depict the relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type and Nfkb1^−/− mice c) Top panels, shown are representative flow cytometry plots for CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type, FOXP3-GFP^KIN, and Cns3^−/− mice stained with antibodies to CD25 and Foxp3 (4 independent experiments, n=8 wild-type, 14 Foxp3-GFP^KIN, and 14 Cns3^−/− mice). Right panels show quantification of cumulative data for all mice and depict relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type (black circles), Foxp3-GFP^KIN (black squares), and Cns3^−/− (black triangles) mice. d) Shown are the percentages of CD25⁺Foxp3⁺ mature T_reg cells generated after stimulating sorted CD4⁺CD8⁻CD73⁻CD25⁺ T_regP cells from thymi of Foxp3-GFP^KIN and Cns3^−/− mice for 3 days with 0 U/ml IL2 (black circles, n=5 Foxp3-GFP^KIN and 5 Cns3^−/− mice), 0.2 U/ml IL2 (black squares, n=4 Foxp3-GFP^KIN and 5 Cns3^−/− mice), 1 U/ml IL2 (black triangles, n=5 Foxp3-GFP^KIN and 6 Cns3^−/− mice) and 100 U/ml (open circles, n= Foxp3-GFP^KIN and 5 Cns3^−/− mice) and come from 2 independent experiments. e) Quantification of cumulative data from 2 independent experiments showing the relative percentage of CD25⁺ T_regP, Foxp3^lo T_regP and mature CD25⁺Foxp3⁺ T_reg cells from CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type (black circles) or EDEL mice, which lack the CaRE4 enhancer (black squares). Data represents 2 independent experiments, n=10 wild-type and 10 EDEL mice. a,b) Data was analyzed by two-sided unpaired t test. c) CD25⁺ T_regP cells (%) and Foxp3^lo T_regP cells (%) were analyzed by one-way ANOVA with Tukey’s multiple comparison test and T_reg cells (%) was analyzed by Kruskal-Wallis test with Dunn’s multiple comparison test. d) Data was analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test. e) Data was analyzed by two-way ANOVA with Sidak multiple comparisons test. All data are displayed as mean ± standard deviation. **P<0.005, ***P<0.0001, ns- not significant.

CD25⁺ T_regP and Foxp3^lo T_regP development is regulated by distinct enhancers

To examine whether Foxp3 was required for the development of T_reg cells from both CD25⁺ T_regP and Foxp3^lo T_regP we used Foxp3-GFP^KIN mice, in which a GFP reporter construct is knocked-into the Foxp3 locus and generates a GFP-Foxp3 fusion protein ²². These mice express normal amounts of GFP-Foxp3 protein, but have been described as functional Foxp3 hypomorphs ^{23, 24}. Following gating on CD4⁺CD73⁻ thymocytes, we found that Foxp3-GFP^KIN mice had a significant reduction in the frequency Foxp3^lo T_regP compared to thymi from wild-type mice, while the abundance CD25⁺ T_regP was unaffected (Fig. 5c). To examine this in more detail we analyzed Foxp3-GFP^KIN mice lacking the Foxp3 regulatory element Cns3 (called Cns3^−/− mice hereafter). Cns3^−/− mice, which are known to have a ~40% reduction in the frequency of T_reg cells in the thymus ²⁵, have selective defects in immune tolerance and a T_reg cell bias towards higher self-reactivity ²⁶. Cns3^−/− mice lacked Foxp3^lo T_regP cells (Fig. 5c). Consistent with previous reports ²⁵, mature CD25⁺Foxp3⁺ T_reg cells were also substantially reduced in Cns3^−/− mice compared to wild-type mice, and the defect was about twice as large (~80% reduction) when gating on CD73⁻ cells to eliminate mature recirculating T_reg cells (Fig. 5c). Importantly, mature CD25⁺Foxp3⁺ CD73⁺ T_reg cells in Cns3^−/− and wild-type mice expressed comparable amounts of Foxp3 (Supplementary Fig. 6a), indicating that Cns3 was not required for Foxp3 expression. CD25⁺ T_regP cells isolated from wild-type, Foxp3-GFP^KIN or Cns3^−/− mice cultured in vitro upregulated Foxp3 and differentiated into mature CD25⁺Foxp3⁺ T_reg cells with comparable efficiency when stimulated with IL-2 (Fig. 5d), suggesting Cns3 was not required for the expression of Foxp3 in mature T_reg cells. These results indicated that the development of T_reg cells from Foxp3^lo T_regP was blocked in the absence of Cns3 and mature T_reg cells developed only from CD25⁺ T_regP cells in Cns3^−/− mice.

A non-coding SNP that contributes to an increased risk for autoimmunity in humans was previously described in the Il2ra locus ^{27, 28, 29}, specifically in enhancer CaRE4, which is required for rapid induction of Il2ra following TCR activation ³⁰. Next, we examined the role of this autoimmunity-associated Il2ra enhancer in T_reg cell development. Deletion of the CaRE4 Il2ra enhancer in the NOD mice background led to a significant reduction in the percentages of thymic CD25⁺ T_regP cells and mature T_reg cells compared to wild-type NOD mice (Fig. 5e). In contrast, the percentage of Foxp3^lo T_regP cells was slightly increased, perhaps as a compensatory mechanism. This decrease in CD25⁺ T_regP cells was not due to a lack of CD25 expression in general, as the expression of CD25 on mature T_reg cells, or on the remaining CD25⁺ T_regP, was not reduced compared to wild-type controls (Supplementary Fig. 6b and data not shown). Collectively these data suggest that the development of T_reg cells from CD25⁺ T_regP and Foxp3^lo T_regP cells was controlled by distinct regulatory circuits.

Inhibition of negative selection pathways expand CD25⁺ T_regP cells

To further probe if CD25⁺ T_regP cells were undergoing negative selection, we examined the development of T_reg cells in Itk^−/− mice, as mice lacking the tyrosine kinase ITK have defects in negative selection ³¹. Itk^−/− thymi had higher frequencies of mature thymic CD25⁺Foxp3⁺ T_reg cells ³², as well as CD25⁺ T_regP and Foxp3^lo T_regP cells (Fig. 6a). The adaptor protein ADAP, which is downstream of ITK, is also required for efficient negative selection³³. Thymi from Adap^−/− mice showed an increase in the abundance CD25⁺ T_regP, but no change in Foxp3^lo T_regP cells compared to wild-type (Fig. 6b). The frequency of mature CD25⁺Foxp3⁺ T_reg cells in the thymus was also significantly increased in Adap^−/− mice compared to wild-type (Fig. 6b). Thus, Adap^−/− mice have a selective increase in CD25⁺ T_regP cells compared to wild-type mice. A potential explanation for the discrepancy between Itk^−/− and Adap^−/− mice is that ITK deficiency is known to induce increased production of IL-4 in iNKT cells in the thymus ^{34, 35}, while ADAP deficiency does not. To examine if the different phenotypes in terms of T_reg cell development in Adap^−/− and Itk^−/− mice were linked to IL-4 production we examined T_reg cell development in Itk^−/− and Itk^−/− x Il4ra^−/− mice. Compared to Itk^−/− mice, the increase in abundance of Foxp3^lo T_regP cells was lost in the thymi of Itk^−/− x Il4ra^−/− mice (Fig. 6a,c), suggesting that the increase in Foxp3^lo T_regP cells in Itk^−/− mice was due to the increased amounts of IL-4 present in the thymi of these mice. Thus CD25⁺ TregP cells are selectively pruned by the ITK-ADAP pathway required for negative selection.

Figure 6.

Itk^−/− mice have increased T_reg cell production from both T_regP cell pathways via distinct molecular mechanisms. a) Left panels, representative flow cytometry plots of CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type versus Itk^−/− mice stained with antibodies to CD25 and FOXP3. Right panels show cumulative data for all mice and depict the relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type, Itk^−/−, and Itk^−/− x Il4ra^−/−mice (3 independent experiments, n=15 wild-type, 17 Itk^−/− mice, and 11 Itk^−/− x Il4ra^−/−). b) Left panels, representative flow cytometry plots of CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type versus Adap^−/− mice stained with antibodies to CD25 and FOXP3. Right panels show cumulative data for all mice and depict the relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type, and Adap^−/−mice (3 independent experiments, n=12 wild-type and 12 Adap^−/− mice). c) .representative flow cytometry plots of CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type, Itk^−/− and Itk^−/− x Il4ra^−/−mice stained with antibodies to CD25 and FOXP3. a) CD25⁺ T_regP cell (%) was analyzed by one-way ANOVA with Tukey’s multiple comparisons test; Foxp3^lo T_regP cells (%) was analyzed by Kruskal-Wallis with Dunn’s multiple comparisons test. b) Data was analyzed by two-sided Mann-Whitney test. All data are displayed as mean ± SD. *P<0.05, ***P<0.0001, ns-not significant.

CD25⁺ T_regP and Foxp3^lo T_regP cells have distinct cytokine responsiveness

IL-2, and to a lesser degree IL-15, are the predominant cytokines that drive the STAT5-dependent differentiation of CD25⁺ T_regP cells into mature T_reg cells ^{1, 2, 4, 36, 37}. Because the ability of Foxp3^lo T_regP cells to differentiate into mature T_reg cells in response to IL-4 has not been evaluated, we asked if IL-4 could affect the differentiation of CD25⁺ T_regP or Foxp3^lo T_regP cell subsets into mature T_reg cells. As reported previously ^{1, 37}, IL-4 did not cause robust conversion of thymically derived CD25⁺ T_regP cells into mature T_reg cells, but supported substantial conversion of thymically-derived Foxp3^lo T_regP into mature CD25⁺Foxp3⁺ T_reg cells in an in vitro assay (Fig. 7a). Foxp3^lo T_regP had slightly higher expression of the IL-4 receptor (IL-4Ra) compared to CD25⁺ T_regP (Fig. 7b), which we considered unlikely to account for the difference in the differentiation of CD25⁺ T_regP and Foxp3^lo T_regP cells in response to IL-4. In addition, although IL-4 converted Foxp3^lo T_regP cells into mature T_reg cells, these cells expressed less CD25 and Foxp3 than those stimulated with IL-2 alone (Supplementary Fig. 7a–d).

Figure 7.

CD25⁺ and Foxp3^lo T_regP cells have distinct cytokine responsiveness. a) Percent of T_reg cells generated from sorted CD4⁺CD8⁻CD73⁻CD25⁺FOXP3⁻ and CD4⁺CD8⁻CD73⁻CD25⁺FOXP3⁻ T_regP cell subsets stimulated for 3 days with no cytokine (black circles, n=9 CD25⁺ T_regP and 7 Foxp3^lo T_regP cultures), 0.5 ng/ml IL4 (black squares, n= 3 CD25⁺ T_regP and 2 Foxp3^lo T_regP cultures), 1 ng/ml IL4 (black triangles, n= 9 CD25⁺ T_regP and 7 Foxp3^lo T_regP cultures), and 100 ng/ml IL4 (open circles, n= 9 CD25⁺ T_regP and 7 Foxp3^lo T_regP cultures) . Data is representative of 3 independent experiments (excluding 0.5 ng/mL IL4 stimulation which is from 1 experiment) and was analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test. b) IL4Ra gMFI in CD25⁺ (black circles) and Foxp3^lo (black squares) T_regP cells from thymi of wild-type mice. Data is representative of 2 independent experiments, n=10 mice. Data analyzed by two-sided Wilcoxon matched-pairs signed rank test. c) Percent of CD4⁺CD8⁻CD73⁻CD25⁺Foxp3⁻ T_regP, CD4⁺CD8⁻CD73⁻CD25⁻Foxp3^lo T_regP, and CD4⁺CD8⁻CD73⁻CD25⁺Foxp3⁺ T_reg cells in thymi from wild-type (black circles, n=11 mice) and Pou2f3^−/− mice (black squares, n= 11 mice). Data is representative of 2 independent experiments, and was analyzed by a two-sided unpaired t test. d) Left panels, representative flow cytometry plots of CD4⁺CD8⁻CD73⁻ gated thymocytes from wildtype BALB/c and Cd1d^−/− BALB/c mice stained with antibodies to CD25 and Foxp3. Right panels, percent of CD4⁺CD8⁻CD73⁻CD25⁺Foxp3⁻ T_regP (left) and CD4⁺CD8⁻CD73⁻CD25⁻Foxp3^lo T_regP (right) in wild-type Balb/c (back circles) and Cd1d−/− Balb/c (black squares) mice. Data is representative of 2 independent experiments, n=6 mice-WT and 11 mice-Cd1d^−/− and was analyzed by two-sided unpaired t-test. All data are displayed as mean ± SD. *P<0.05, **P<0.005, ***P<0.0001, ns-not significant.

What cells drive production of IL-4 in the thymus remains unclear. In humans, the Hassall’s corpuscles, a distinct anatomical feature of the thymus containing cells that resemble Tuft cells, are important for T_reg cell development ³⁸. Tuft cells were also reported in the murine thymi ^{39, 40} and are major producers of IL-25, which induces IL-4 production in other cell types ⁴¹. To test whether thymic tuft cells influenced T_regP cell differentiation, we examined T_reg cell development in Pou2f3^−/− mice, which lack the transcription factor POU2F3, which is required for the development of tuft cells. We observed an ~30% decrease in frequency of Foxp3^lo T_regP, but not CD25⁺ T_regP cell development in Pou2f3^−/− thymi compared to wild-type (Fig. 7c). To determine if iNKT cells, the canonical producers of IL-4 in the thymus, selectively affected the development of Foxp3^lo T_regP cells we examined the frequency of T_regP in Cd1d^−/− mice, which lack NKT cells. There was an ~20% reduction in the abundance of Foxp3^lo T_regP cells in the Cd1d^−/− thymi compared to wild-type Balb/c controls (Fig. 7d). This effect was only observed in Balb/c mice, which produce abundant NKT-derived IL-4, and not in C57Bl/6 mice, in which NKT cells make very little IL-4 (data not shown). Thus, CD25⁺ T_regP and Foxp3^lo T_regP cells development have distinct dependence on thymic tuft cells, and this is likely partially mediated via tuft cell induction of IL-4 production in iNKT cells.

CD25⁺ and Foxp3^lo TRP exhibit distinct functions

Next we tested whether the mature T_reg cells derived from CD25⁺ T_regP or Foxp3^lo T_regP cells had different abilities to prevent distinct types of autoimmune diseases, driven by different auto-antigens. To test whether mature T_reg cells generated from CD25⁺ T_regP cells differed in their ability to suppress autoimmunity in the central nervous system when compared to Foxp3^lo T_regP-generated T_reg cells, we isolated thymic CD25⁺ T_regP or Foxp3^lo T_regP cells, transferred them separately into individual C57Bl/6 host mice 1 day before immunization with MOG_35–55 peptide and monitored the development of EAE symptoms. Transfer of mature T_reg cells is known to prevent or ameliorate symptoms in this EAE model ⁴². Mice receiving Foxp3^lo T_regP showed similar disease scores throughout the study as mice without T_regP transfer (Fig. 8a). In contrast, disease progression and severity in mice that received CD25⁺ T_regP were significantly blunted from day 14 through 18 after disease induction compared to mice receiving no T_regP or Foxp3^lo T_regP cells (Fig. 8a,b). The number of congenically-marked donor Foxp3⁺ T_reg cells observed following transfer of CD25⁺ or Foxp3^lo T_regP cells was similar in the spleen at the endpoint of the experiment, 20 days after T_regP transfer (Fig. 8c), suggesting the conversion of CD25⁺ T_regP and Foxp3^lo T_regP into mature T_reg cells was similar. However, transferred Foxp3^lo T_regP cells prevented weight loss in a T cell transfer model of colitis (Supplementary Fig. 8), demonstrating the suppressor activity of the Foxp3^lo T_regP-derived T_reg cells was normal. Finally, we used MOG:I-A^b tetramers in combination with magnetic bead enrichment approaches to identify MOG:I-A^b-specific T_reg cells and T_regP in the thymus of wild-type mice. We found MOG:I-A^b-specific T cells in the CD25⁺ T_regP and mature T_reg cell subsets, but only observed one MOG:I-A^b-specific T cell in the Foxp3^lo T_regP in 15 mice examined (Fig. 8d,e), suggesting MOG:I-A^b-specific T_reg cells were almost always generated from the CD25⁺ T_regP and were specifically required for protection against EAE.

Figure 8.

CD25⁺ and Foxp3^lo T_regP cells are functionally distinct. a) Clinical score over 20-day time course of MOG_35–55 peptide-induced experimental autoimmune encephalomyelitis in wild-type mice treated with no T_reg cells (red line and circles), Foxp3^lo T_regP (orange line and circles) and CD25+ T_regP (green lines and circles). Data was analyzed by two-sided unpaired t-tests with Holm-Sidak multiple comparisons correction. Asterisks mark values with adjusted p-values less than 0.05. Bars represent mean ± SEM. b) Cumulative EAE scores for control mice (no treatment, red circles), or mice injected with CD25+ T_regP (green squares) or Foxp3^lo T_regP (yellow triangles). Data was analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test. Bar represents median. c) Number of donor T_reg cells recovered from the spleen of EAE-induced mice receiving CD25⁺ T_regP (black squares) and Foxp3^lo T_regP (black triangles). Data was analyzed by a two-sided unpaired t test. Bars represent mean ± SD. a-c) Data represent 2 independent experiments, n=6 mice per group. d) Representative gating strategy for thymic tetramer pulldowns. Dual tetramer gates are drawn on CD8⁺ thymocytes such that ~0% of CD8⁺ thymocytes appear in the double tetramer positive gate. This gate is applied to CD4⁺CD73⁻ thymocytes to identify bona fide MOG_38–48:I-A^b specific thymocytes. e) Representative flow cytometry plots of MOG tetramer pulldowns, concatenated from 6 thymi from wild-type mice. Left plot, number of MOG:I-A^b dual-tetramer⁺ T cells within the CD4⁺CD73⁻ thymocyte cell gate. Middle plot, percentage of MOG:I-A^b dual tetramer⁺ cells in CD25⁺T_regP cell, Foxp3^lo T_regP cell and T_reg cell gates. Right plot, cumulative data showing percentage of percentage of CD25⁺ T_regP cells (black squares), Foxp3^lo T_regP cells (black triangles), and Treg cells (open circles) in thymi from 15 wild-type mice. Data was analyzed by two-sided Wilcoxon matched-pairs signed rank test and is pooled from 3 independent experiments. Bars represent mean ± SD. *P<0.05, ns-not significant.

Discussion

Here we found that both CD25⁺ T_regP and Foxp3^lo T_regP contributed to mature T_reg development in the thymus, and that in our hands they did so relatively equivalently. However, these two distinct TregP cell subsets differed in many important ways. They utilized different signaling pathways and enhancers for their differentiation, were affected in distinct ways by different stromal cells and cytokines and expressed distinct TCR repertoires and RNA transcriptomes. Most importantly, T_reg cells derived from CD25⁺T_regP versus Foxp3^lo T_regP subsets had distinct roles in protecting against autoimmunity. Thus, there are at least two different developmental pathways in the thymus that contribute substantially to the generation of the mature T_reg cell repertoire.

Several pieces of evidence support the notion that mature T_reg cells derived from both CD25⁺ T_regP and Foxp3^lo T_regP cells are required for full maintenance of immune tolerance. Both the Foxp3-GFP^KIN hypomorph and Cns3^−/− mice had defects in generating Foxp3^lo T_regP and specific defects in immune tolerance. The Foxp3-GFP^KIN mice have a relatively mild defect in immune tolerance that is only revealed on distinct genetic backgrounds^{23, 24}. In contrast, Cns3^−/− mice have unique defects in immune tolerance, such as greater inflammation in the lung than wild-type mice²⁶. In addition, Cns3^−/− mice have increased titers of specific autoantibodies ²⁶. Because Cns3^−/− mice selectively lacked Foxp3^lo but not CD25⁺ T_regP, this suggests a unique role for Foxp3^lo T_regP-derived mature T_reg cells in preventing autoimmunity.

Additional studies suggest a unique role for CD25⁺ T_regP in promoting immune tolerance. The CaRE4 enhancer in the Il2ra locus harbors an autoimmunity risk variant that promotes susceptibility to inflammatory bowel disease, but protection against diabetes ^{27, 28, 29}. Deletion of this enhancer resulted in a selective decrease in CD25⁺ T_regP, suggesting that CD25⁺ T_regP-derived T_reg cells could have an important role in protecting against inflammatory bowel disease. Likewise, Cns3^−/− mice exhibit increased protection against EAE ²⁶. Because T_reg cells in Cns3^−/− mice are almost exclusively derived from CD25⁺ T_regP, this suggests CD25⁺ T_regP -derived T_reg cells protect against EAE. We directly confirmed this idea by showing that CD25⁺ T_regP-derived T_reg cells provided robust protection from EAE while Foxp3^lo T_regP-derived T_reg cells did not. Thus, modulating the frequency of CD25⁺ T_regP results in differential protection against autoimmunity. This observation has important translational implications, as it indicates that T_reg cells with select TCRs can be identified that have uniquely potent efficacy versus specific types of autoimmune disease.

The presence of more than one developmental pathway leading to mature T_reg cells raises the question of why such a system has evolved. T_reg cells are required to prevent responses to self-antigens and commensal antigens, as well as dampen anti-pathogen immune responses once these agents have been cleared. Establishing such a diverse repertoire requires the generation of T_reg cells able to recognize high-affinity self-antigens. Such a population could be generated via a process of agonist selection in the thymus. However, agonist selection is unlikely to generate the broad repertoire of T_reg cells needed to prevent immune responses to commensal organisms or to limit responses to foreign pathogens. In many ways this resembles the problem faced by conventional thymocytes, which must generate a repertoire able to recognize a vast array of antigens that they never see in the thymus. Thus, establishing a broader non-self-focused repertoire for T_reg cells may require a process that resembles positive selection for conventional thymocytes. We show that T_reg cells developed through two distinct developmental programs that exhibit such characteristics. CD25⁺ T_regP developed through a process of agonist selection that shared many similarities with the underlying process of negative selection and would result in mature T_reg cells focused on TCRs with high-affinity for thymic self-antigens. Foxp3^lo T_regP developed through a mechanism that could be akin to positive selection of T_reg cells, resulting in a broader repertoire able to react with both self- and non-self antigens. As such, thymic T_reg cells derived from Foxp3^lo T_regP could complement the function of induced peripheral T_reg cells. Our model also suggests that the Cns3 regulatory element evolved to promote the development of mature T_reg cells through the Foxp3^lo T_regP pathway. Finally, the genetic variability that altered the relative balance of these two developmental pathways also altered the T_reg cell repertoire and correlated with susceptibility to distinct forms of autoimmunity. Given the differences in signaling pathways and cytokines that controlled these two developmental pathways, specific targeting of each T_reg cell population might help patients with various autoimmune defects.

Online Methods

Mice.

Mice were housed in specific pathogen–free facilities at the University of Minnesota, Cornell University, Salk Institute or University of California San Francisco, and experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committees of these respective institutions. Exceptions are germ-free mice housed in germ-free facilities at the University of Chicago and mice with a normalized microbial experience which were housed in the University of Minnesota’s mouse vivarium. Pet store mice were purchased from various pet stores in the greater Minneapolis-St. Paul metropolitan area. Information about the age of the pet store mice was not available from the vendor. Co-housing of SPF mice with sex-matched pet store partner was performed as described¹² within the University of Minnesota BSL-3 facility. Conversion efficiency was confirmed by assessing the conversion of naïve CD8⁺ T cells into CD8⁺ memory T cells; effective conversion correlated with ~30–60% CD8⁺CD44^hi T cells. All relevant ethical guidelines have been followed. Foxp3-GFP mice (006772), Foxp3-RFP mice (008374) were from the Jackson Laboratory. CD45.1⁺ (B6.SJL) mice were from the US National Cancer Institute. Nur77-GFP BAC reporter mice, Rag2-GFP reporter mice, Cns3^−/− and Foxp3-GFP^KI, CD28^−/−, Nfkb1^−/−, Itk^−/−, Itk^−/− x Il4Ra^−/−, Adap^−/−, Il2ra EDEL, Itgal^−/−, Rag2^−/−, Cd1d^−/− and Tcliβ x TCRα^+/− have been described previously ^{7, 14, 17, 22, 25, 30, 35, 43, 44, 45, 46, 47, 48}. Mice were generally six to eight weeks old but ranged from four to sixteen weeks old. Mice were randomly selected for experiments, in age-matched cohorts. The investigators were not ‘blinded’ to genotype during data acquisition.

Tissue preparation and cell isolation.

For analysis of thymocyte and Treg development thymi were mechanically dissociated into 1x PBS with 2% FBS and 2 mM EDTA, pH 7.4, using frosted glass slides. Cells suspensions were passed through 70 µm filters and washed prior to staining.

Flow cytometry and antibodies.

All flow cytometry analysis was conducted in the University of Minnesota Flow Cytometry Core Facility using BD LSR II and Fortessa cytometers (BD, San Jose, CA). For surface staining, cells were stained for 20 minutes with fluorochrome-conjugated antibodies prior to washing and analysis or intracellular staining. Intracellular detection of Foxp3, Cleaved Caspase-3 and GFP was performed as previously described ² using the eBioscience Transcription Factor staining kit. When staining for GFP, Rag2-GFP thymi were fixed for 10 minutes at room temperature in 1.6% PFA prior to intracellular staining of GFP and Foxp3 using the eBioscience Transcription Factor staining kit. For apoptosis assays, thymi were harvested and mechanically dissociated into 1x PBS on ice. Following surface staining cells were washed into Annexin V binding buffer (eBioscience San Diego, CA) and stained with Annexin V. Specific antibodies used are listed below. For tetramer staining, single cell thymocyte suspensions were treated with Dasatinib (Axon Medchem) at 50nM in complete RPMI for 20 minutes at 37°C followed by dual (APC and PE conjugated) MOG_38–48 tetramer ⁴⁹ staining at 10nM for an additional 45 minutes in complete RPMI (10% FBS, 1% glutamine, 1% penicillin-streptomycin, 1% NEAA, 10mM HEPES, 50uM BME) at 37°C. Tetramer stained cells were washed and incubated with anti-APC and anti-PE microbeads (Miltenyi Biotec) for 30 minutes on ice. Cells were washed and run over a LS column and unbound and bound fractions were collected, stained and analyzed as described above. A complete list of staining reagents is available as a supplemental reagent table.

Ultrasound guided intrathymic injection

Injections were performed largely as described previously ⁵⁰. Briefly, mice were anesthetized with 2–4% isofluorane in medical gas (21% oxygen, 79% nitrogen) in an acrylic chamber then transferred to the warmed ultrasound platform face up with the snout fastened in a facemask delivering 2–4% isofluorane and medical gas. Depilatory cream was used to remove hair from the mid-upper ventral thoracic region prior to applying ultrasound gel. Using the Vevo 2100 ultrasound, the MS550D probe was lowered parallel to the left or right side of the sternum. The ultrasound image generated was used to guide an insulin syringe (27G) into the thymus and visualize the injection of the 10–20uL cell suspension.

Immunofluorescence and histo-cytometry

Immunofluorescence analysis of thymi was described previously ⁵¹. Thymi were washed, fixed with 4% paraformaldehyde (PFA) for 1 h and snap frozen. 5-μm sections were blocked with PBS containing 5% bovine serum albumin (BSA) and goat serum (Jackson Laboratory) before staining. The sections were then covered with Prolong anti-fade mounting medium (Life Technologies) and images were obtained 1 to 3 d later with a Leica DM6000B Epi-Fluorescent microscope.

Histo-cytometry analysis was performed as described previously ²¹.

TCR sequencing

CD4SP thymocytes were enriched by magnetic depletion using biotinylated anti-CD8/Ter119 antibodies followed by secondary labelling with streptavidin–conjugated microbeads (Miltenyi Biotec). Enriched cells are labelled with fluorchrome-conjugated anti-CD4, CD25 and streptavidin, while Foxp3 is marked with the RFP reporter, prior to sorting on the BD Aria sorter (BD Biosciences). TCR sequencing was performed as described previously ⁵². Briefly, CD4⁺CD25⁻Foxp3⁻, CD4⁺CD25⁺Foxp3⁻, CD4⁺CD25⁻Foxp3^lo and CD4⁺CD25⁺Foxp3⁺ populations are sorted into lysis buffer (Buffer RLT+BME). cDNA was generated from these samples using the RNeasy kit (QIAGEN Valencia, CA). CDR3 Vα2 TCRs were sequenced using an Ion Torrent as previously described. TCRs representing >20% of the reads in the T_reg compartment, which influence distributions disproportionally, were excluded from analysis as described ⁵³.

T_reg progenitor conversion assays.

T_reg progenitors were isolated as previously described ². Briefly, Foxp3-GFP thymi were dissected, dissociated, and pooled CD4SP cells were enriched by magnetically depleting with biotinylated anti-CD8 and Ter119 antibodies (eBioscience) followed by secondary labeling with streptavidin-conjugated microbeads (Miltenyi Biotec). Enriched CD4SP cells were stained with fluorchrome-conjugated anti-CD4, CD25, CD73 and streptavidin prior to sorting CD4⁺CD73⁻CD25⁺GFP⁻, CD4⁺CD73⁻CD25⁻GFP⁺, or CD4⁺CD73⁻CD25⁺GFP⁺ cells using a BD Facs Aria sorter (BD Biosciences). Purified T_reg cell progenitors or T_reg cells were incubated in complete RPMI and supplemented with human IL2 from R&D Systems (Minneapolis, MN) or mouse IL4 from Tonbo Biosciences (San Diego, CA). After 72 hours, cells were harvested, stained with anti-CD4, CD8 and CD25 antibodies and analyzed by flow cytometry for the percentage or number of cells that express CD25 and GFP after incubation.

Mapping and analysis of single cell sequencing

CD4⁺CD73⁻CD25⁺Foxp3⁻, CD4⁺CD73⁻CD25⁻Foxp3^lo and CD4⁺CD73⁻CD25⁺Foxp3⁺ cells were isolated from a single Foxp3-GFP thymus using a BD Facs Aria sorter. Cells were resuspended at 10⁶ cells/mL in 50%FBS in 1xPBS before being counted and captured using the 10X Genomics (San Francisco, CA) Single Cell 3’ Solution.

A custom genome was created by adding the sequence for the FoxP3-GFP construct ⁵⁴ as a new chromosome to the Ensembl GRCm38 reference and gtf file (version 89). The gene annotation file (gtf) was then filtered further to contain only protein coding genes. The 10x Genomic Cellranger pipeline (version 2.2.0 https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome) was used to align reads and generate counts for each sorted population (or library). Sorted populations were then combined using the depth normalization mode.

The Seurat R Package (version 1.4.0.12) was used to analyze the mapped single cell reads. The data was filtered to include cells that contained 100–2500 unique gene counts and expressed more than 5 genes. Global-scaling normalization was applied to the filtered data as described in the default settings in the Seurat package. The data was then scaled to regress out sequencing depth. Linear dimensional reduction (PCA) was performed on the most variable genes (2010 genes). Heatmap analysis of different PCs and an elbow plot were created to decide on proceeding with 9 PCs for cluster analysis using the Seurat function ‘FindCluster’. To visualize the clusters, non-linear dimensional reduction (tSNE) was performed using the 9 PCs. Clusters were assigned to a specific population by comparing cells labeled for the original sorted population and cellular markers. The R packages plot3D (version 1.1.1) and threejs (version 0.3.1) were used to create 3D visualization of the tSNE data. Differential expression (“bimod” McDavid et al., 2013) was calculated for each cluster against all other clusters to identify potential markers for each individual cluster. Differential expression was also calculated between individual clusters using the same method.

Experimental Autoimmune Encephalomyelitis Induction

EAE was induced in mice as described previously ⁵⁵. Briefly, on Day −1 sorted T_regP populations (4×10⁵-5×10⁵) were transferred i.v. into CD45.1 congenic mice. On Day 0 mice were immunized with 200ug of MOG_35–55 emulsified in CFA with 4mg/mL heat killed Tb. Immunization was performed with 2, 50uL s.c. injections of emulsion on left/right flank of the lower back. Mice were treated with 200ng Pertussis Toxin on day 0 and day 2 via i.p. injection in 1xPBS. Mice were monitored for disease progression and treated with s.c. normal saline or given wet food on the cage floor, as described previously ⁵⁵.

Transfer colitis induction

Colitis was induced as previously described ⁵⁶. Briefly, 5×10⁵ CD4⁺CD45RB^hi cells were transferred via i.v. or i.p. injection into Rag^−/− recipient mice. Sorted T_regP were transferred via i.p. injection in a split dose (2.5×10⁵ on days 1 and 7) or a single dose (5×10⁵ on day 21). Mice were weighed weekly to monitor disease progress.

Statistical analysis

Statistical tests used to analyze data are included within the figure legends. Briefly, comparisons of 2 groups were done by either, paired t-test (paired, normal data), Wilcoxon matched-pairs test (paired, non-normal data), t-test (non-paired, normal data) or Mann-Whitney (non-paired, non-normal data); tests were always two-sided. Comparisons of 3 or more groups was done by one-way ANOVA (non-paired, normal data), Kruskal-Wallis (non-paired, non-normal data), or Friedman test (paired, non-normal data). P-values <0.05 were considered significant. Statistics were calculated using Prism (GraphPad Software, LaJolla, CA). All data, except those specifically mentioned in figure legends, are displayed as mean ± standard deviation.