Identification of Cellular Sources of IL-2 Needed for Regulatory T Cell Development and Homeostasis

David L. Owen, Shawn A. Mahmud, Kieng B. Vang, Ryan M. Kelly, Bruce R. Blazar, Kendall A. Smith and Michael A. Farrar
J Immunol June 15, 2018, 200 (12) 3926-3933; DOI: https://doi.org/10.4049/jimmunol.1800097

Abstract

The cytokine IL-2 is critical for promoting the development, homeostasis, and function of regulatory T (Treg) cells. The cellular sources of IL-2 that promote these processes remain unclear. T cells, B cells, and dendritic cells (DCs) are known to make IL-2 in peripheral tissues. We found that T cells and DCs in the thymus also make IL-2. To identify cellular sources of IL-2 in Treg cell development and homeostasis, we used Il2FL/FL mice to selectively delete Il2 in T cells, B cells, and DCs. Because IL-15 can partially substitute for IL-2 in Treg cell development, we carried out the majority of these studies on an Il15−/− background. Deletion of Il2 in B cells, DCs, or both these subsets had no effect on Treg cell development, either in wild-type (WT) or Il15−/− mice. Deletion of Il2 in T cells had minimal effects in WT mice but virtually eliminated developing Treg cells in Il15−/− mice. In the spleen and most peripheral lymphoid organs, deletion of Il2 in B cells, DCs, or both subsets had no effect on Treg cell homeostasis. In contrast, deletion of Il2 in T cells led to a significant decrease in Treg cells in either WT or Il15−/− mice. The one exception was the mesenteric lymph nodes where significantly fewer Treg cells were observed when Il2 was deleted in both T cells and DCs. Thus, T cells are the sole source of IL-2 needed for Treg cell development, but DCs can contribute to Treg cell homeostasis in select organs.

Introduction

Regulatory T (Treg) cells play a critical role in preventing self-reactivity, limiting responses to commensal organisms, and dampening responses to pathogens following clearance of the infectious agents. Previous studies have shown that the cytokine IL-2 is critical for the development of Treg cells in the thymus and for their subsequent homeostasis in peripheral lymphoid tissues. Mice lacking either IL-2 or the IL-2R α- or β-chains all exhibit profound autoimmunity, although the reason was initially unclear (1–3). Following the discovery of CD25+ Treg cells by Sakaguchi and colleagues (4), several groups reported that IL-2 is essential in CD4+CD25+FOXP3+ Treg cell development or function (5–12). More recent studies have shown that IL-2 also plays a critical role in the conversion of thymic CD25+FOXP3− and CD25−FOXP3lo Treg progenitor cells into mature Treg cells (13–16). Thus, substantial evidence implicates IL-2 as a key cytokine for the development and homeostasis of FOXP3+ Treg cells.

A fundamental question is “What cells produce the IL-2 needed for Treg cell development and homeostasis?” One obvious candidate is T cells themselves. Initial studies by Yang-Snyder and Rothenberg (17, 18) detected IL-2 production in the thymus and found that this required the presence of T cells. However, developing thymocytes produce much less IL-2 than activated mature T cells. Moreover, the studies by Yang-Snyder and Rothenberg demonstrated that T cells were necessary to detect IL-2 in the thymus but did not demonstrate that the T cells produced the IL-2 themselves. Thus, it is plausible that other cellular sources of IL-2 contribute to Treg cell development. Consistent with this possibility, IL-2 is produced by both B cells and dendritic cells (DCs) under specific circumstances (19, 20). Because both of these cell subsets are found within the thymic medulla where Treg cell development takes place, each could also be a potential source of IL-2 needed for Treg cell development. Supporting this possibility, Robey and colleagues (21) found that DC-dependent development of Treg cells in thymic slices was reduced by 50% when the DC were derived from Il2−/− mice. Thus, there are multiple potential sources of IL-2 that could play an important role in either Treg cell development in the thymus or homeostasis in peripheral lymphoid tissues.

To definitively address what sources of IL-2 are required for Treg cell development and homeostasis, we used mice in which the Il2 gene is flanked by loxP sites (22). We crossed Il2FL/FL mice with Cd4-Cre, Cd79a-Cre, and Cd11c-Cre mice (23–25) to selectively delete IL-2 in T cells, B cells, and DCs, respectively. Furthermore, because IL-15 can partially substitute for IL-2 in Treg cell development, we also crossed these mice onto the Il15−/− background. These studies revealed that the only critical source of IL-2 required for Treg cell development in the thymus was T cells. In contrast, although T cell–derived IL-2 was necessary and sufficient to maintain Treg cells in most peripheral lymphoid tissues, both T cell– and DC-derived IL-2 contributed to Treg cell homeostasis in mesenteric lymph nodes. Thus, multiple cellular sources of IL-2 contribute to mature Treg cell homeostasis.

Materials and Methods

Mice

All mice were housed in specific pathogen–free facilities at the University of Minnesota, and experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee. Cd4-Cre, Cd79a-Cre, Cd11c-Cre, Il2FL/FL, IL-15−/−, and Rag2−/− mice have been described previously (22–27). Mice from mouse facilities of the University of Minnesota 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 and peripheral lymphoid organs were mechanically dissociated into 1× PBS with 2% FBS and 2 mM EDTA (pH 7.4) using frosted glass slides. Cell suspensions were passed through 70-μm filters and washed prior to staining. DCs and medullary thymic epithelial cells (mTECs) were isolated via cutting thymi or spleen into pieces, followed by collagenase D digestion at 37°C and mechanical dissociation.

Flow cytometry and Abs

All flow cytometry analysis was conducted in the University of Minnesota Flow Cytometry Core Facility using BD LSR II and Fortessa cytometers (BD Biosciences, San Jose, CA). For surface staining, cells were stained for 20 min with 1:100 dilutions of fluorochrome-conjugated Abs prior to washing and analysis or intracellular staining. Anti-mouse CD4 (GK1.5), CD8α (53-6.7), CD25 (PC61.5), FOXP3 (FJK-16s), Thy1.2 (30-H12 or 53.21), CD73 (TY/11.8), CD45.2 (104), CD45.1 (A20), Thy1.1 (HIS51), CD44 (IM7), IL-2 (JES6-5H4), CD11c (N418), CD19 (1D3), NK1.1 (PK136), EpCAM (G8.8), CD172α (P84), and CD62L (MEL14) were from eBioscience (San Diego, CA). CD4 (GK1.5, BV786), CD45RA (14.8), and CD172α (P84) were from BD Biosciences. CD3 (17A2 or 145-2C11) and Rat IgG1 (HPRN) were from Tonbo Biosciences (San Diego, CA). UEA1 was from Vector Laboratories (Burlingham, CA). Intracellular detection of FOXP3 was performed as previously described (14) using the eBioscience Transcription Factor staining kit. Intracellular detection of IL-2 was performed with BD Cytofix/Cytoperm kit as described by the manufacturer.

Bone marrow chimeras

Il2−/− × Il15−/− × Rag2−/− recipients were sublethally irradiated with 500 rad and rested before i.v. injection of a 50:50 mixture of bone marrow of the indicated origin. Chimeric mice were analyzed 8–10 wk following donor cell transfer.

In vitro stimulations

Isolated T cells or DCs were stimulated with 100 ng/ml PMA and 1 μM ionomycin for 1 h followed by GolgiPlug treatment for 7 h.

Statistical analysis

Statistical tests used to analyze data are included within the figure legends. Comparisons of three or more groups were done by one-way ANOVA (nonpaired, normal data) using a Tukey’s post hoc test or by Kruskal–Wallis test (nonpaired, nonnormal data) using a Dunn’s multiple-comparison post hoc test. The p values >0.05 were considered significant. Statistics were calculated using Prism (GraphPad Software, LaJolla, CA).

Results

Thymic T cells and DCs can produce IL-2

To determine if developing T cells or thymic DCs produce IL-2, we carried out flow cytometry with intracellular staining for IL-2. Upon stimulation with PMA and ionomycin, both thymocytes and thymic DCs were able to produce IL-2 (Fig 1A, 1B), although a much smaller fraction of DCs produced IL-2 compared with thymocytes. Staining for IL-2 was specific, as staining was lost in Cd4-Cre × Il2FL/FL and Cd11c-Cre × Il2FL/FL mice, respectively (data not shown). Similar results were found when we sorted thymocytes or DCs and assayed for Il2 mRNA levels by quantitative real-time PCR. In contrast, we failed to detect IL-2 production by mTECs (data not shown). These results suggest that either T cells or DCs could be responsible for producing the IL-2 needed for thymic Treg cell development.

FIGURE 1.
FIGURE 1.

Thymocytes and thymic DCs produce IL-2. (A) Thymocytes were stimulated with PMA/ionomycin and stained for IL-2 production. Left panel, A representative flow plot showing IL-2 production in thymocytes pregated on CD3. Right panel, Quantification of percentage of IL-2+ cells; data represent seven experiments, n = 18 mice. (B) Thymic DCs were stimulated with PMA/ionomycin and stained for IL-2 production. Left panel, A representative flow plot showing IL-2 production from thymic DCs. Right panel, Quantification of percentage of IL-2+; data represent three experiments, n = 3 mice. Bars represent mean plus SD. (C) CD25 expression on mTECs (dark gray) compared with isotype staining control (light gray). (D) Expression of CD25 in thymic (dark gray) or splenic (light gray) SIRPα+ DCs.

A previous study has suggested that IL-2 can be trans-presented by CD25 expressed on DCs (28). To examine whether such trans presentation of IL-2 could occur in the thymus, we stained thymic DCs, mTECs, and splenic DCs for CD25. A large fraction of mTECs as well as thymic, but not splenic, conventional SIRPα+ DCs expressed CD25 (Fig. 1C, 1D). Importantly, neither mTECs nor thymic DCs expressed the IL-2R β-chain (data not shown). Thus, CD25 expression by mTECs and DCs is not involved in signaling cell-intrinsic responses to IL-2. Rather, this finding suggests that these APCs in the thymus may use CD25 to increase the local concentration of IL-2 available to Treg progenitor cells and thereby facilitate Treg cell development.

T cell–derived IL-2 is important for Treg cell homeostasis in IL-15–sufficient mice

Given that DCs, B cells, and T cells are the most likely candidates to produce the IL-2 required for Treg cell development and homeostasis, we analyzed Cd4-Cre, Cd11c-Cre, and Cd79a-Cre mice crossed to Il2FL/FL mice. None of these mice exhibited statistically significant reductions in thymic Treg cell production, although Cd4-Cre mice trended toward lower thymic Treg cell abundance (2.5-fold mean reduction from controls, p = 0.39) (Fig. 2A). This is in agreement with previous reports, which showed that IL-15 could rescue IL-2 deficiency in Treg cell development (10, 11). Although thymic Treg cell development was not grossly inhibited, Cd4-Cre × Il2FL/FL mice, but not Cd11c-Cre × Il2FL/FL or Cd79a-Cre × Il2FL/FL mice, exhibited significant reductions in splenic Treg (CD4+CD25+FOXP3+) cells (Fig. 2B). Moreover, when we stained for CD44 and CD62L in CD4 and CD8 effector T cells, we observed significant increases in activated CD4 and CD8 T cells in Cd4-Cre × Il2FL/FL mice but no differences in Cd11c-Cre × Il2FL/FL or Cd79a-Cre × Il2FL/FL mice (Fig. 2C). This result suggests that the Treg cell compartment in Cd4-Cre × Il2FL/FL mice is failing to maintain immune homeostasis, likely because of the defect in Treg cell abundance in the periphery of these mice.

FIGURE 2.
FIGURE 2.

T cell–derived IL-2 is important for Treg cell homeostasis in IL-15–sufficient mice. (A) Quantification of the percentage of (CD25+FOXP3+) Treg cells within CD4+ thymocytes of the indicated CRE+ mice on an Il2FL/FL background. (B) Quantification of the percentage of Treg cells (CD25+FOXP3+) within splenic CD4+ T cells of the indicated mice (C). Quantification of the percentage of activated/effector CD4 (left panel) and CD8 (right panel) T cells in the spleen of the indicated mice. Data are representative of six experiments, n = 13 mice (WT); three experiments, n = 3 mice (Cd4-Cre × Il2FL/FL); four experiments, n = 12 mice (Cd11c-Cre × Il2FL/FL); and one experiment, n = 2 mice (Cd79a-Cre × Il2FL/FL). Data were analyzed by one-way ANOVA.

DC- and B cell–derived IL-2 are dispensable for Treg cell development and homeostasis

Previous studies have reported that IL-15 can substitute for IL-2 during thymic Treg cell development (10, 11). Thus, to understand the role of DC- and B cell–derived IL-2 in Treg cell differentiation, we crossed Cd11c-Cre and Cd79a-Cre mice to Il2FL/FL × Il15−/− mice. We observed that Il2 deletion in DCs, B cells, or both subsets, had no effect on Treg cell development in the thymus (Fig. 3A, 3B). Furthermore, the spleens of these mice also had no differences in Treg cell abundance (Fig. 3C). There was also no increase in the percentage of CD44hiCD62Llo activated CD8+ or CD4+ effector T cells (Fig. 3D). This suggests that Treg cells in mice lacking IL-2 from DCs or B cells can maintain immune tolerance and Treg cell homeostasis in the spleen, even in the absence of compensatory IL-15.

FIGURE 3.
FIGURE 3.

DC- and B cell–derived IL-2 is dispensable for Treg cell development and homeostasis. (A) Representative flow plots of CD25 and FOXP3 expression in CD4SP thymocytes of the indicated CRE+ mice on an Il2FL/FL × Il15−/− background. (B and C) Quantification of the percentage of (CD25+FOXP3+) Treg cells within CD4+ T cells in the thymus (B) and spleen (C). (D) Quantification of the percentage of activated/effector CD4 (left panel) and CD8 (right panel) T cells in the spleen of the indicated mice. Data are representative of nine experiments, n = 17 mice (WT); four experiments, n = 7 mice (Cd11c-Cre); six experiments, n = 11 mice (Cd79a-Cre); four experiments, n = 5 mice (Cd11c-Cre × CDd79a-Cre); and two experiments, n = 7 mice (Cd4-Cre). Data were analyzed by one-way ANOVA (B and D) or Kruskal–Wallis tests (C). WT and Cd4-Cre × Il2FL/FL × Il15−/− data points are identical for (A) and (B) and Fig. 4A, 4B. WT and Cd4-Cre × Il2FL/FL × Il15−/− data points are identical for (C) and (D) and Fig. 6A, 6B.

T cell–derived IL-2 is critical for Treg development in the thymus

The most likely sources of IL-2 that could be important for Treg cell development are thymocytes themselves. To test this possibility, we generated Cd4-Cre × Il2FL/FL × Il15−/− mice and analyzed their thymi for Treg cell development. We observed a striking loss of almost all Treg cells in the thymi of mice lacking IL-2 in T cells in the Il15−/− background (Figs. 3A, 3B, 4). This finding demonstrates that T cell–derived IL-2 is necessary for Treg cell development in the thymus. We also wanted to see if deletion of Il2 in T cells, B cells, and DCs combined would exacerbate the loss of Treg cells observed in Cd4-Cre × Il2FL/FL × Il15−/− mice. We observed no significant differences in Treg cell development when comparing Cd4-Cre × Il2FL/FL × Il15−/− mice with Cd11c-Cre × Cd4-Cre × Il2FL/FL × Il15−/−, Cd79a-Cre × Cd4-Cre × Il2FL/FL × Il15−/−, or Cd79a-Cre × Cd11c-Cre × Cd4-Cre × Il2FL/FL × Il15−/− mice (Fig. 4). These results demonstrate that T cell–derived IL-2 is both necessary and sufficient for Treg cell development in the thymus.

FIGURE 4.
FIGURE 4.

T cell–derived IL-2 is critical for thymic Treg cell differentiation. (A) Representative flow plots of CD25 and FOXP3 expression in CD4SP thymocytes of the indicated CRE+ mice on an Il2FL/FL × ILl5−/− background. (B) Quantification of the percentage of (CD25+FOXP3+) Treg cells within CD4SP thymocytes in the indicated mice strains. Data are representative of nine experiments, n = 17 mice (WT); two experiments, n = 7 mice (Cd4-Cre); six experiments, n = 13 mice (Cd4-Cre × Cd11c-Cre); two experiments, n = 5 mice (Cd4-Cre × Cd79a-Cre); and four experiments, n = 5 mice (Cd4-Cre × Cd11c-Cre × Cd79a-Cre). Data were analyzed by one-way ANOVA. WT and Cd4-Cre × Il2FL/FL × Il15−/− data points are identical for Fig. 3A, 3B and (A) and (B).

Autocrine production of IL-2 by T cells is not required for Treg cell development

One model for Treg cell development could be that developing CD4SP thymocytes that receive a strong TCR stimulus would upregulate IL-2 in an autocrine fashion, which would then bind to the IL-2 receptor on that cell and drive Treg cell differentiation. To test this hypothesis, we created bone marrow chimeras in Rag2−/− × Il2−/− × Il15−/− recipients from a 1:1 mixture of wild-type (WT) bone marrow with either congenically marked WT or Cd4-Cre × IL-2FL/FL × Il15−/− bone marrow. In this study, we observed identical development of Treg cells from IL-2–deficient and –sufficient T cells (Fig. 5). This demonstrates that autocrine signaling of IL-2 by T cells is dispensable for Treg cell development and that bystander T cell production of IL-2 is sufficient to drive this developmental process.

FIGURE 5.
FIGURE 5.

Cell-intrinsic IL-2 is not required for thymic Treg cell differentiation. (A) Representative flow plots of CD25 and FOXP3 expression in the CD4SP thymocytes of the indicated donor origin in chimeric mice. (B) Quantification of the percentage of (CD25+FOXP3+) Treg cells within CD4SP thymocytes from each donor origin. Data are representative of two experiments, n = 13 mice (WT, CD45.1+), 7 mice, T cell–IL-2−/− (Cd4-Cre × Il2FL/FL × Il15−/−), 6 mice, T cell–IL-2+ (Cd4-Cre × Il2FL/+ × Il15−/− or Il2FL/FL × Il15−/−). Data were analyzed by one-way ANOVA.

Production of IL-2 by T cells and DCs is required for Treg cell homeostasis in mesenteric lymph nodes

Given that DC- and B cell–derived IL-2 is dispensable for Treg cell homeostasis in the spleen (Figs. 3C, 3D, 6A, 6B), we wanted to know if T cell–derived IL-2 was responsible for this function. To answer this question, we looked at Treg cell abundance in the spleen and inguinal and mesenteric lymph nodes of Cd4-Cre × Il2FL/FL × Il15−/−, Cd11c-Cre × Cd4-Cre × Il2FL/FL × Il15−/−, Cd79a-Cre × Cd4-Cre × Il2FL/FL × Il15−/−, or Cd79a-Cre × Cd11c-Cre × Cd4-Cre × Il2FL/FL × Il15−/− mice. Deletion of Il2 in T cells resulted in an ∼100-fold reduction in Treg cell proportions in the spleen. No further decrease in Treg cell proportions was observed when combining deletion of Il2 in T cells with deletion of Il2 in DCs, B cells, or all three cell subsets (Fig. 6A). Consistent with these findings, we observed a significant increase in the percentage of activated CD4+ and CD8+ T cells in the spleen of mice with a selective defect in T cell–derived IL-2 (Fig. 6B). Similar results were observed in inguinal lymph nodes (data not shown). Deletion of Il2 in T cells, but not DCs or B cells, also led to a significant decrease in Treg cells in mesenteric lymph nodes. However, deletion of Il2 in both T cells and DCs led to a further ∼10-fold drop in Treg cell abundance in mesenteric lymph nodes (Fig. 6C). Deletion of Il2 in B cells provided no additional decrease in Treg cell abundance under any experimental conditions. Finally, Il2 deletion observed in Cd4-Cre × Il2FL/FL × Il15−/−, Cd11c-Cre × Cd4-Cre × Il2FL/FL × Il15−/−, Cd79a-Cre × Cd4-Cre × Il2FL/FL × Il15−/−, or Cd79a-Cre × Cd11c-Cre × Cd4-Cre × Il2FL/FL × Il15−/− mice led to increased T cell activation in the mesenteric lymph node, consistent with reduced percentages of Treg cells in all of these mouse strains (Fig. 6D). These observations demonstrate the importance of T cell–derived IL-2 in maintaining Treg cell abundance and function in peripheral lymphoid organs but also highlight a role for DCs in contributing to Treg cell homeostasis in mesenteric lymph nodes.

FIGURE 6.
FIGURE 6.

T cell–derived IL-2 is critical for Treg cell homeostasis. (A and B) Quantification of the percentage of (CD25+FOXP3+) Treg cells in the spleen (A) and percentage of activated/effector CD4 (left panel) or CD8 (right panel) T cells in the spleen (B). (C and D) Quantification of the percentage (CD25+FOXP3+) Treg cells in mesenteric lymph nodes, log10 transformed data (C), and percentage of activated/effector CD4 (left panel) or CD8 (right panel) (D). Data are representative of nine experiments, n = 17 mice (WT); four experiments, n = 7 mice (Cd11c-Cre); six experiments, n = 11 mice (Cd79a-Cre); four experiments, n = 5 mice (Cd11c-Cre × CDd79a-Cre); two experiments, n = 7 mice (Cd4-Cre); six experiments, n = 13 mice (Cd4-Cre × Cd11c-Cre); two experiments, n = 5 mice (Cd4-Cre × Cd79a-Cre); and four experiments, n = 6 mice (Cd4-Cre × Cd11c-Cre × Cd79a-Cre). Data were analyzed by Kruskal–Wallis (A) and one-way ANOVA (B–D). WT and Cd4-Cre × Il2FL/FL × Il15−/− data points are identical for Fig. 3C, 3D and (A) and (B).

T cell–derived IL-2 is important for maintaining resting Treg cells

It has been previously observed that resting or central Treg (cTreg) cells are specifically dependent on IL-2 for maintenance (29). Given this dependence, we quantified the proportion of Treg cells that were phenotypically effector (effector Treg [eTreg]; CD44hiCD62Llo) or central (cTreg; CD44loCD62Lhi) Treg cells in mice lacking IL-2 in DCs, B cells, or T cells. In Cd11c-Cre × Il2FL/FL × Il15−/−, Cd79a-Cre × Il2FL/FL × IL-15−/−, or Cd11c × Cd79a-Cre × Il2FL/FL × Il15−/− mice, which lack IL-2 in B cells, DCs, or both these cell subsets, there was no change in the relative percentages of splenic or mesenteric eTreg and cTreg cells (Fig. 7A). In contrast, when IL-2 was lost in the T cell compartment in Cd4-Cre × Il2FL/FL × Il15−/− mice, the proportion of Treg cells that were eTreg cells versus cTreg cells was significantly altered, with increased percentages of eTreg cells and decreased percentages of cTreg cells in the spleen and mesenteric lymph node (Fig. 7B). These results again highlight an important role for T cell–derived IL-2 in Treg cell phenotype and directly demonstrate that cTreg cells are dependent on T cell–derived IL-2.

FIGURE 7.
FIGURE 7.

T cell–derived IL-2 is important in maintaining resting Treg cells. (A and B) Quantification of the percentage of (CD25+FOXP3+) Treg cells in the indicated CRE+ mice, which are the eTreg (left) or cTreg (right) phenotype in the spleen (top) or mesenteric lymph node (bottom). Data are representative of nine experiments, n = 17 mice (WT); four experiments, n = 7 mice (Cd11c-Cre); six experiments, n = 11 mice (Cd79a-Cre); four experiments, n = 5 mice (Cd11c-Cre × CDd79a-Cre); two experiments, n = 7 mice (Cd4-Cre); six experiments, n = 13 mice (Cd4-Cre × Cd11c-Cre); two experiments, n = 5 mice (Cd4-Cre × Cd79a-Cre); and four experiments, n = 6 mice (Cd4-Cre × Cd11c-Cre × Cd79a-Cre). Data were analyzed by one-way ANOVA [(A) cTreg spleen, eTreg mesenteric lymph node, cTreg mesenteric lymph node; (B) eTreg spleen, eTreg mesenteric lymph node) or Kruskal–Wallis [(A) eTreg spleen; (B) cTreg spleen, cTreg mesenteric lymph node]. WT data points are the same for individual conditions in (A) and (B).

Discussion

The cellular sources of IL-2 important for Treg cell development and homeostasis have been a controversial issue for some time. For example, work from the Germain laboratory using intravital imaging observed clustering of pSTAT5+ Treg cells around activated effector T cells producing IL-2, supporting a role for T cell–derived IL-2 in Treg cell homeostasis or function in peripheral lymphoid organs (30). Likewise, work by Sakaguchi and colleagues (12) also supported a role for T cell–derived IL-2 in Treg cell homeostasis. Conversely, work from the Robey laboratory showed a dependence on DC-derived IL-2 for optimal Treg cell differentiation in thymic tissue (21). In this study, we provide direct evidence that production of IL-2 from T cells is critical for Treg cell development, homeostasis, and function in vivo. In the absence of T cell–derived IL-2, there is a significant defect in Treg cell development in the thymus (when IL-15 is absent) and a dramatic reduction in Treg cells in peripheral lymphoid tissues. As Treg cell development in the thymus requires relatively strong signals (16, 31, 32), it is possible that these signals induce autocrine production of IL-2 that subsequently drives IL-2–dependent Treg cell development. However, our studies with mixed bone marrow chimeras demonstrate that bystander T cell production of IL-2 is sufficient to drive Treg cell differentiation in the thymus. This suggests that nearby activated T cells produce the IL-2 needed for Treg cell development. An intriguing possibility is that this process may be enhanced by the selective expression of CD25 on thymic DCs and mTECs, which may allow for trans presentation of IL-2 to developing Treg cells. Such mechanisms have been proposed for peripheral DCs in promoting T cell responses but have not yet been described in thymic Treg cell selection (28). Finally, although DCs and B cells in the thymus are potential sources of IL-2 that could contribute to Treg cell development, we found that they are neither necessary (as Treg cells develop fine in the absence of DC- and B cell–derived IL-2) nor sufficient (as the Treg cell defect is just as severe when IL-2 is deleted in T cells as when it is deleted in all cells). This is consistent with the observation that CD4+ and CD8+ thymocytes appeared to produce much more IL-2 than stimulated thymic DCs. Thus, the only critical cellular source of IL-2 needed for Treg cell development in the thymus appears to be T cells.

Our work also points to a key role for T cell–derived IL-2 on Treg cell homeostasis and function in the spleen and inguinal lymph nodes. In these organs, T cell–derived IL-2 appears to be both necessary and sufficient to maintain normal proportions of Treg cells and their function, as effector T cell activation was only seen in these organs when Il2 was selectively deleted from T cells. Additional deletion of Il2 in DCs and/or B cells did not result in a further reduction in Treg cells in these peripheral lymphoid tissues or affect effector T cell activation, demonstrating that DC- and B cell–derived IL-2 does not influence Treg cell homeostasis or function in these organs. In contrast, we saw a somewhat different effect when examining mesenteric lymph nodes. Loss of T cell–derived IL-2 led to a clear reduction in Treg cells and a concomitant induction of effector T cell activation. Consistent with previous reports (29), loss of T cell–derived IL-2 led to an almost total loss of the cTreg cell phenotype, as the majority of remaining Treg cells were eTreg cells. This again supports the importance of IL-2 in maintaining cTreg cells, whereas eTreg cells can likely be supported by factors other than IL-2 such as ICOS. Interestingly, in the mesenteric lymph nodes, there was a further decrease in Treg cell abundance when IL-2 was deleted from T cells and DCs but not T cells and B cells. This suggests that DC production of IL-2 contributes to Treg cell maintenance in this mucosal-associated tissue. Our findings point to a more general role for DC-derived IL-2 at mucosal interfaces in promoting Treg cell homeostasis. Alternatively, our results are also consistent with the possibility that DC-derived IL-2 is specifically required to maintain a unique subset of Treg cells only found at these locations.

Although our work demonstrated a critical dependence on T cell–derived IL-2 for Treg cell development and homeostasis, it is important to note that these studies focused on the thymus and secondary lymphoid organs. Further investigation will be required to understand if distinct paradigms of IL-2 cellular production and importance exist in unique lymphoid environments, such as the mesenteric lymph node, or in nonlymphoid tissues, like the large and small intestine, where T cells are not as abundant as in lymphoid tissues. Our observations that DC-derived IL-2 contributes to Treg cell homeostasis in the mesenteric lymph node, but not in the spleen or inguinal lymph nodes, is one such example in which different cell types are required to produce IL-2 needed for optimal Treg cell homeostasis in distinct microenvironments. Other cell type–specific knockouts of IL-2 could reveal novel insights into the cellular partners of Treg cells required for maintaining immune tolerance.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Gregory Hubbard, Alyssa Kne, Christopher Reis, Amy Mack, Emilea Sykes, Hannah Wiesolek, Dylan Duerre, Asima Meskic, and Abby Rost for assistance with mouse husbandry and Paul Champoux and the University of Minnesota Flow Cytometry Core for assistance with sorting experiments.

Footnotes

  • This work was supported by National Institutes of Health (NIH) Grant P30CA77598 to the University of Minnesota Flow Cytometry Resource. D.L.O., S.A.M., and K.B.V. were supported by an NIH T32 grant (T32 AI07313). S.A.M. was also supported by NIH Grant F30 DK096844. B.R.B. was supported by NIH Grant R01 HL11879. M.A.F. is supported by NIH Grants R56AI113138 and R01 AI124512 and by the University of Minnesota Masonic Cancer Center.

  • Abbreviations used in this article:

    cTreg
    central Treg
    DC
    dendritic cell
    eTreg
    effector Treg
    mTEC
    medullary thymic epithelial cell
    Treg
    regulatory T
    WT
    wild-type.

  • Received January 23, 2018.
  • Accepted April 15, 2018.

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