IL‐3 is essential for ICOS‐L stabilization on mast cells, and sustains the IL‐33‐induced RORγt+ Treg generation via enhanced IL‐6 induction

Summary IL‐33 is a member of the IL‐1 family. By binding to its receptor ST2 (IL‐33R) on mast cells, IL‐33 induces the MyD88‐dependent activation of the TAK1‐IKK2 signalling module resulting in activation of the MAP kinases p38, JNK1/2 and ERK1/2, and of NFκB. Depending on the kinases activated in these pathways, the IL‐33‐induced signalling is essential for production of IL‐6 or IL‐2. This was shown to control the dichotomy between RORγt+ and Helios+ Tregs, respectively. SCF, the ligand of c‐Kit (CD117), can enhance these effects. Here, we show that IL‐3, another growth factor for mast cells, is essential for the expression of ICOS‐L on BMMCs, and costimulation with IL‐3 potentiated the IL‐33‐induced IL‐6 production similar to SCF. In contrast to the enhanced IL‐2 production by SCF‐induced modulation of the IL‐33 signalling, IL‐3 blocked the production of IL‐2. Consequently, IL‐3 shifted the IL‐33‐induced Treg dichotomy towards RORγt+ Tregs at the expense of RORγt− Helios+ Tregs. However, ICOS‐L expression was downregulated by IL‐33. In line with that, ICOS‐L did not play any important role in the Treg modulation by IL‐3/IL‐33‐activated mast cells. These findings demonstrate that different from the mast cell growth factor SCF, IL‐3 can alter the IL‐33‐induced and mast cell‐dependent regulation of Treg subpopulations by modulating mast cell‐derived cytokine profiles.


INTRODUCTION
Mast cells are critical modulators of the innate and adaptive immune responses by recruitment of leucocytes [1][2][3] and by support of the maturation of DCs and T lymphocytes. 4,5 Mast cells can induce tissue inflammation 6 and therefore, contribute to immune responses against pathogens 4 or to the development of allergic diseases 7 and autoimmunity. [8][9][10] Therefore, understanding of mast cell activities is fundamental to influence the balance of tissue-protecting and tissue-destructing processes. Hallmarks of mast cells are the strong expression of the highaffinity Fce-receptor I (FceRI), c-Kit (CD117) and the IL-3 receptor (CD123). FceRI represents the best-characterized mechanism of mast cell activation, which results in the production and the release of inflammatory mediators by degranulation. Besides CD117 activation by the stem cell factor (SCF), IL-3 activation of CD123 signalling is essential for mast cell differentiation and survival in vivo and in vitro. 11 Activation of IL-3R induces the activation of the IKK2 signalling, 12 and MAP kinases such as ERK1/ 2, JNK and p38. 12 All of these signals are involved in mast cell differentiation, survival and proliferation. 12,13 Besides FceRI, the Toll-like/IL-1 receptor (TIR) family member IL-33R is also highly expressed on mast cells. 14 IL-33, the ligand for the IL-33R, is an IL-1 family member, which induces the activation of the MyD88-TAK1-IKK2 signalling module. This results in activation of the p38-MAPKAPK2/ MAPKAPK3 (p38-MK2/3) signalling module, ERK1/2, JNK1/2 and NFjB. 1,15,16 Both the IL-33-induced activation of NFjB and of the p38-MK2/3 signalling module result in the transcriptional and translational regulation of the cytokine production. 1 IL-33 is released upon damage of endothelial and epithelial cells, 17 and thus, IL-33 is termed an alarmin, which signals the loss of integrity of cellular layers with barrier functions. Mast cells represent a major cellular sensor for such damage-released IL-33 in peripheral tissues and are a component of the first line of response to cell injury. 18 Although such mechanisms result in allergic inflammation, 2,18 IL-33-activated mast cells also downregulate inflammatory responses in peripheral tissues by inducing the formation of regulatory T cells (T regs ). 19 T regs are dominant immune-suppressive T cells characterized by the transcription factor FoxP3. One stable subpopulation of FoxP3 + T regs is characterized by the expression of the transcription factor RORct 20 and can predominantly be found in gut-associated lymphoid tissues (GALT) or on mucosal surface. 20 Recently, we found that IL-33-activated mast cells control the dichotomy between RORct + and HELIOS + T regs via the production of IL-6. 21 Growth factors such as IL-3 and SCF modulate IL-33-induced cytokine responses in mast cells. 1,12,22,23 ICOS is a coactivating receptor expressed on Treg cells and is essential for the maintenance of FoxP3. 24 On the other hand, the ligand of ICOS, ICOS-L, can be induced on mast cells. 25 Stimulation of Tregs by ICOS-L promotes Treg induction and stability. 26 General blockade of the ICOS-L/ICOS costimulatory axis is of immunosuppressive character in vivo as blocking ICOS-L reduces the severity of symptoms in G6PI-induced arthritis 27 or collagen-induced arthritis 28 resulting in inhibited Th effector responses. 29 However, the lack of ICOS signalling specifically in Tregs worsens the symptoms of diabetes and releases the suppression of IBD. 24,30 In the presented work, we aimed to investigate the influence of IL-3 on the IL-33-induced generation of RORct + T regs . We show that IL-3 is a main factor to stabilize the expression of ICOS-L on mast cells. Besides that, IL-3 potentiated the IL-33-induced IL-6 response. In summary, IL-3 exhibits potential immunoregulatory capacities via ICOS-L stabilization, but more importantly dramatically enhanced the IL-33-induced production of IL-6, and thus promotes the shift of T reg subset characteristic towards RORct + T regs .

Mice
We used sex-and age-matched wt and mk2 À/À /mk3 À/À double-deficient (mk2/3 ko ) C57BL/6. 31 Mice were maintained according to the guidelines of the institutional and governmental committees for animal welfare of the Animal Research Facility of the Jena University Hospital and Medizinische Hochschule Hannover, Institut f€ ur Zellbiochemie. We isolated organs from sacrificed wt C57BL/6 and mk2 À/À /3 À/À double-deficient (mk2/3 ko ) mice. Organ isolations are approved by the Th€ uringer Landesamt f€ ur Lebensmittelsicherheit und Verbraucherschutz; Bad Langensalza. All mice were used under the license twz-36-2017 for the Institute of Immunology, Jena.

Flow cytometry
BMMCs were harvested and washed with PBA-E (PBS, 5 mg/ml, BSA, 10 mM NaN3, 2 mM EDTA). To block unspecific binding prior to antibody staining, BMMCs were preincubated in the presence of rat IgG (Jackson) at 4°C for 5 min. For the determination of the purity of the BMMC cultures, cells were stained with PE-anti-CD117 (Biolegend) and FITC-anti-FceRI (Biolegend). Furthermore, we used FITC-anti-CD117 (Biolegend), APC-Cy7anti-CD117 (Biolegend) and PE-anti-CD275/ICOS-L (Biolegend) as indicated in the figures and figure legends. All antibodies were diluted in PBA-E and were incubated at 4°C. After 20 min, BMMCs were washed with PBA-E and analysed by using LSR II flow cytometer (BD). For the analysis of T reg /BMMC cocultures, we used the FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher). According to the manufacturer's protocol, cells were washed and fixed and were intracellularly stained with APC-Cy7-anti-CD4 (Thermo Fisher), PacBlue-anti-FoxP3 (Thermo Fisher), PE-anti-RORct (Thermo Fisher) and PerCP-eFluor 710-anti-HELIOS (Thermo Fisher). Subsequently, cells were analysed with the LSR II flow cytometer (BD) and data were analysed by using FlowJo 10 (Treestar Inc., Ashland, OR).

ELISA
BMMCs were seeded (10 6 cells/ml) in IL-3-free medium. After 1 h, BMMCs were preincubated with DMSO (vehicle) or inhibitors (all from Merck) for 30 min. The inhibitors were used as indicated in the figures and the figure legends. Afterwards, cells were prestimulated with IL-3 (50 ng/ml) for 30 min and then stimulated with IL-33 (50 ng/ml) (both from Peprotech) for 24 h. Supernatants were collected and analysed for IL-6 or IL-2 by using matched paired antibodies (eBioscience). For ELISA experiments, we used biological replicates separated into at least three technical replicates.

RT-PCR
Upon mast cell stimulation, total RNA was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's instruction. 500 ng RNA was reversely transcribed using Oligo(dt)18 primer and SuperScript TM IV Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer's instruction. RT-qPCR was performed using 2 ll of template cDNA, primers for Icosl (Mouse ICOS ligand qPCR primer pair, Sino Biological, MP200220) or glyceraldehyde-3-phosphate-dehydrogenase (Gapdh, forward 5 0 TTGGCCGTATTGGGCGCCTG 3 0 , reverse 5 0 CACCCTTCAAGTGGGCCCCG 3 0 ) as housekeeping gene. Reaction volume was calculated to 20 ll, and PowerUp SYBR Green Master Mix (Applied Biosystems) was used for detection. Samples were analysed in duplicates in the AB 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Expression levels of Icosl transcripts were normalized to Gapdh.

IL-3 induced an ERK1/2-dependent maintenance of the surface expression of ICOS-L on mast cells
Similar to dendritic cells, mast cells have been reported to express ICOS-L. 32 To characterize the regulation of ICOS-L on mast cells in vitro, we monitored the expression of ICOS-L during BMMC generation. A representative gating strategy is shown in Figure S1A. ICOS-L is upregulated after the 3 rd and 4 th week of BMMC differentiation and remains stably expressed on CD117 + BMMCs ( Figure 1A-C). To exclude a secondary effect of the IL-3conditioned supernatant used for mast cell generation, we blocked IL-3 in these cultures by the addition of a monoclonal anti-IL-3 antibody. The downregulation of ICOS-L upon blockade of IL-3 indicated that on fully differentiated mast cells, the ICOS-L expression is maintained particularly by IL-3 ( Figure 1D). Upon removal of the IL-3-conditioned supernatant from the mast cell culture by washing the cells, ICOS-L expression was downregulated on mast cells upon reculture in medium without IL-3 supplementation after 24 h ( Figure 1E). In the absence of IL-3, IL-33 or SCF was insufficient to maintain the expression of ICOS-L on mast cells ( Figure 1E). Next, we determined the signalling pathways, which maintain ICOS-L on mast cells. IL-3 stimulation of mast cells induced activation of MAP kinases ERK, p38 and JNK ( Figure 1F-I). To investigate the role of these kinases in the IL-3-mediated stabilization of ICOS-L on the surface, we inhibited the kinase activities with the help of well-described inhibitors, namely SP600125 (JNK inhibitor 33 ), SB203580 (p38 inhibitor 34 ) and UO126 (ERK inhibitor 35 ). However, only ERK inhibition resulted in a significant decrease in ICOS-L surface expression (Figure 1J). Depletion of IL-3 signals did similarly result in reduced ERK activation, indicating that ERK is a major signalling node important for the IL-3-induced ICOS-L expression on the surface of mast cells (Figure 1K).

IL-3 promotes the IL-33-induced and mast celldependent formation of RORct + T regs independently from ICOS-L
We recently showed that IL-33-stimulated BMMCs induce RORct + T regs from RORct À T regs . 21 We analysed Tregs in the cocultures with mast cells as represented by the gating strategy shown in Figure S1B. When we added IL-3 to these cultures, we did not observe any effect on the overall T reg frequencies among CD4 + Th cells (Figure 2A). However, we observed a strong increase in the capacities of BMMCs to induce RORct + T regs in the presence of IL-3 ( Figure 2B). While the presence of IL-3 did slightly improve the survival of the mast cells in these cocultures ( Figure S2A,B), the overall T reg frequencies remained comparable in the presence and absence of IL-3 ( Figure 2A). Therefore, we conclude that the increase in RORct + T regs was not mediated by an improved mast cell survival.
Recently, ICOS-L on dendritic cells has been linked to the formation of RORct + T regs . 36 On the other hand, T regs have been shown to express ICOS, but not ICOS-L. 37 Because IL-3 stabilizes ICOS-L and addition of IL-3 to the IL-33 stimulated cocultures induced more RORct + T regs , we stimulated T regs and BMMCs with IL-33 or with IL-3 and IL-33 in the presence of a blocking ICOS-L antibody. Unexpectedly, anti-ICOS-L did not reduce the formation of RORct + T regs ( Figure 2C). In line with IL-33 being not able to stabilize ICOS-L expression, we observed a dominant destabilization of ICOS-L on IL-3stimulated mast cells within 24 h of IL-33 stimulation ( Figure 2D,E). Upon IL-33 costimulation, the IL-3-mediated upregulation of Icosl transcripts was blocked after 24 h and remained at levels comparable to the absence of IL-3 ( Figure 2F). Collectively, we could demonstrate an increase in RORct + T reg generation by IL-33-activated mast cells in the presence of additional IL-3, which was unexpectedly independent from the IL-3-maintained ICOS-L expression.

IL-3 potentiates the IL-33-induced IL-6 and GM-CSF production
Given that IL-3 enhanced the IL-33-induced and mast cell-dependent formation of RORct + T regs , we speculated that IL-3 modulates the IL-33-induced cytokine response in mast cells. Confirming and extending our recently published data, IL-3 potentiated the IL-33-induced IL-6 [ Figure 3 and 12 ]. This was mediated by IKK2, because inhibition of the kinase with the inhibitor IKK-16 (IKKiVII 38 ) resulted in a complete block of the enhanced IL-6 response induced by IL-33 in the presence of IL-3 ( Figure 3A). Different from its role in ICOS-L surface stabilization ( Figure 1J), inhibition of ERK did not show any effect on the IL-3-mediated increase in the IL-33-induced IL-6 production (Figure 3B). While inhibition of JNK only showed a very mild reduction of the IL-3-mediated enhancement of IL-6 production ( Figure 3C), inhibition of p38 did abolish any IL-33-induced effect ( Figure 3D). In line with the essential role of the p38-MK2/3 signalling module for the IL-33-induced IL-6 production in mast cells, 1,21 , the potentiating effect of IL-3 on the IL-33-induced IL-6 was completely absent in MK2/3 doubleknockout mast cells ( Figure 3E). To exclude that this effect is mediated by a destabilization of p38 in MK2/3 double-knockout mast cells, 39 we blocked MK2/3 functions with the inhibitor PF3644022 40 and could reproduce the abolished IL-6 production of the mutant mast cells ( Figure 3F). This demonstrates that the p38/MK2/3 signalling module is essential for mast cells costimulated with IL-3 and IL-33.

IL-3/IL-33-activated mast cells favour the formation RORct + T regs in dependency of IL-6
The finding that IL-3 potentiated the IL-33-induced formation of RORct + T regs by mast cells (Figure 2A,B) and the IL-33-induced IL-6 production ( Figure 3A-E) prompted us to investigate whether the enhancive effect of IL-3 was dependent on IL-6. In line with our previous work, 21 blockade of IL-6 did neither alter the overall T reg levels in the presence of IL-33-stimulated BMMCs nor in the presence of IL-3-IL-33-costimulated BMMCs (Figure 3G,H). Comparable to the essential role of IL-6 for the generation of RORct + T regs by IL-33-stimulated mast cells, even that enhanced generation of RORct + T regs was completely blocked by removal of IL-6 from the culture ( Figure 3I,J). Interestingly, the increase in IL-33-induced RORct + Tregs in the presence of IL-3 happened at the expense of RORct À Helios + T regs (Figure 3I,J). Collectively, IL-6 is also essential for an IL-3-mediated increase in RORct + T regs by IL-33-stimulated mast cells.
IL-3 blocks the IL-33-induced IL-2 production and reduces the frequency of RORct À Helios + T regs A maintenance factor for Helios + T regs is IL-2. 21 To investigate whether the reduction of RORct À Helios + T regs can be rescued by the addition of IL-2, we added IL-2 to the coculture of IL-3-IL-33-stimulated BMMCs and T regs . Interestingly, the addition of IL-2 recovered the maintenance of RORct À Helios + T regs in the presence of IL-3 and IL-33 ( Figure 4A,B). This suggested that the reduction in RORct À Helios + T regs resulted from a deficiency of IL-2 in the culture, which is normally produced by IL-33stimulated BMMCs. 21 In contrast to conditions with SCF costimulation, 21 IL-3 strongly reduced the IL-33-induced production of IL-2 by mast cells ( Figure 4C). In line with its lacking suppressive effect on the IL-33-induced IL-2 secretion by mast cells, 21 SCF costimulation did not reduce the RORct À Helios + T regs in the presence of IL-33stimulated BMMCs ( Figure 4D). Collectively, these data demonstrate that different from SCF, the presence of IL-3 alters the cytokine profile induced by IL-33-activated mast cells and therefore enhanced the shift of the balanced formation of RORct + T regs and Helios + T regs towards RORct + T regs .

DISCUSSION
Here, we demonstrate that IL-3 strongly impacts the IL-33-induced cytokine profile in mast cells. Costimulation with IL-3 and IL-33 promotes p38-MK2/3-mediated effector functions. This results in a potentiated IL-6, but an inhibited IL-2 response.
The IL-33-induced IL-6 production is mediated by the TAK1-IKK2-IjB-p65 signalling pathway. 22 While IL-3   *** IL-6 (relative production) IL-6 (relative production) IL-6 (relative production) IL-6 (relative production) IL-6 (relative production) IL-6 (relative production)  alone does not activate NFjB per s e, 12 the enhanced production of IL-6 by costimulation with IL-3 and IL-33 suggests that IL-3 modulated the NFjB activity. In earlier studies, we could show that SCF enhances the IL-33-induced activation of the TAK/IKK complex, which results in an increase in the activities of NFjB, and ultimately enhanced the production of IL-6. 22,23 Therefore, we suggest that IL-3 also enhances the IL-33-induced activation of TAK1/IKK2 complex, which leads to an increased destabilization of IjB 12 and therefore primes NFjB for enhanced activation. While the IL-33-induced production of IL-6 in the presence of IL-3 was potentiated independently from ERK1/2 and JNK1/2, it was mediated via the p38-MK2/3 signalling module similar to stimulation with IL-33 alone. 21 It is widely accepted that IL-3 activates p38 in mast cells, 41 but it is not entirely clear whether p38 is activated by TAK-1 via MKKs 42 or whether it is subjected to interaction with a TAK-IKK-TPL2 complex. 43 In contrast to the enhanced IL-6 production, IL-3-mediated mechanism of costimulation did not result in a potentiated IL-33-induced IL-2 production. Notably, IL-3 even inhibited the IL-33-induced IL-2 production. The mechanism how IL-3 counter-regulated the IL-33-induced ERK1/2-dependent IL-2 production remains unknown. An autocrine feedback loop via IL-2-IL2R activation is highly unlikely, because mast cells express the IL-2Ra and the IL-2Rc chain, but not the IL-2Rb chain, which is essential for IL-2 signalling. 44 However, IL-33 induces expression of the IL-33-induced ERK1/2-specific dualspecificity phosphatase 5 (DUSP5) in eosinophils. 45 DUSP5 has been demonstrated to negatively regulate the ERK activity, 46 which is essential for the IL-33-mediated induction of IL-2 in mast cells. 21 Therefore, as a potential mechanism we suggest an enhanced expression of DUSP5 in IL-3/IL-33-stimulated BMMCs, which in turn controls the production of IL-2 via deactivation of ERK1/2.
We recently showed that among mast cell produced cytokines, SCF potentiates both IL-33-induced IL-6 and IL-2. 21 According to this, SCF increased the formation of RORct + T regs , whereas the maintenance of Helios + T regs is not affected. 21 In the absence of IL-3, IL-33-activated mast cells mediate a balanced production of IL-6 and IL-2 and thus regulate the dichotomy between RORct + T regs and Helios + T regs . 21 Therefore, mast cell-derived IL-6 promotes the formation and stability of RORct + T regs , whereas the produced IL-2 maintains HELIOS + T regs . 21 In the presence of IL-3, the IL-33-induced IL-6 response is potentiated and the production of IL-2 is blocked. This altered cytokine profile correlated with an enhanced shift of the dichotomy between RORct + T regs and HELIOS + T regs towards RORct + T regs ( Figure S3).
Recently, Kim et al. showed that ICOS-L on DCs is essential to maintain the formation of RORct + T regs . 36 The expression of ICOS-L on mast cells is dependent on IL-3 and is mediated by MAPK signalling via ERK, but not by p38 or JNK ( Figure S3A). However, when we tested the role of ICOS-L by using a blocking ICOS-L antibody, we did not detect any influence on the formation of RORct + T regs or maintenance of HELIOS + T regs . Concomitantly, we detected a strong downregulation of ICOS-L on BMMCs in the presence of IL-33, thereby explaining the absence of a blocking effect of ICOS-L. This indicated that ICOS-L is not involved in the IL-33induced mast cell-mediated dichotomy between RORct + and Helios + T regs . Surprisingly, we did not observe any strong change in FoxP3 expression within the T reg compartment depending on the presence of IL-3. Although IL-3 is considered as a survival factor for mast cells, 47 the cell viability was hardly reduced during most of the coculture period, and therefore, increased mast cell viability would not account for the effect of IL-3 on the RORct + Treg. These observations suggest that IL-33 stimulation maintains T regs mainly via factors different from ICOS-L or IL-2. Considering the alarmin function of IL-33, one could speculate that ICOS-L-or IL-2-driven regulatory mechanisms are blocked in situations of tissue destruction, but by generation of RORct + T regs , the shutdown of the damage-induced immune response would be initiated.
Being effective in protection from colitis, RORct + T regs have been established to reflect a key T reg subpopulation. 48,49 However, the formation of RORct + T regs in vivo has been shown to essentially depend on IL-6, for example in the gastrointestinal tract. 50,51 As a modulator of intestinal inflammatory diseases, IL-33 is released during inflammation and controls the severity of the immune response via the activation of mast cells. 52,53 In such a context, SCF might be secreted, which not only facilitates the differentiation and survival of mast cells, but also might control their responses during the recovery phase upon inflammation. 53 In the presented work, we addressed another important factor in mast cell biology different from SCF: IL-3. By a yet unknown selective impact on the cytokine production, IL-3 did enhance IL-6, but decreased IL-2 production induced by IL-33 (Figure S3B,C). That appeared to be different from SCF costimulation that not only enhanced IL-33-induced formation of RORct + T regs , but also did not reduce IL-2 production by mast cells 21 and therefore maintained Helios + T regs . Taken together, these observations indicate that IL-3 facilitates the IL-33-dependent mast cell activation different from SCF resulting in altered mast cell-dependent modulation of RORct + T regs or HELIOS + T reg responses to tissue injury ( Figure S3C). IL-3 is mainly produced by T cells, 13 which preferentially locate in skin or mucosal tissues and can be activated by the alarmin IL-33. 54 Therefore, tissue-prevalent mast cells stimulated by a combination of IL-3 and IL-33 would secrete high levels of IL-6, but not IL-2, and represent potential mediators of the negative regulatory role of IL-3 in colitis-mediated immune responses by induction of RORct + Tregs. 48 Given the specific modulatory effects of IL-3 on mast cell function, the manipulation of IL-3 levels in mast cell-mediated responses should be considered for treatment of inflammatory diseases.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Gating strategy for the flow cytometrical analysis of BMMCs and T regs . Cellular events were defined in the FSC-A/SSC-A plot. Subsequently, among these gated cells, singlets were identified via FSC-H/FSC-W and SSC-H/SSC-W analysis. (A) Mast cells were identified among living cells (DAPI À ) via the expression of CD117. (B) FoxP3 + CD4 + Tregs were identified among CD4 high SSC-A low culture cells via the expression of FoxP3. Figure S2. BMMC survival starts to decrease after 48 h of IL-3 deprivation. BMMCs were washed and cultured in medium alone or with 50 ng/ml recombinant IL-33 for 72 h. If indicated, 50 ng/ml recombinant IL-3 was added to the cultures. After 24 h, 48 h and 72 h the frequencies of DAPI + cells among the CD117 + cells were analysed by flowcytometry. FACS plot show representative results for the indicated conditions (A). Data from 3 independent biological replicates are shown in (B). Figure S3. Model of the interaction of IL-3 and IL-33 as presented in the manuscript. (A) IL-3 is essential for the surface expression of ICOS-L via ERK1/2. (B) IL-33 induces the production of IL-6 and IL-2 via TAK1-IKK2 signalling and via TAK1-p38-MK2/3 signalling. While IL-6 is important for the induction of RORct + Tregs, IL-2 supports the stability of Helios + Tregs. (C) We presented that IL-33 inhibits the IL-3-induced ICOS-L expression.
Via a yet unknown mechanism, IL-3 did almost completely block the IL-2 production induced by IL-33 but potentiated the production of IL-6. Consequently, IL-3 shifted the IL-33-induced T reg dichotomy towards RORct + T regs at the expense of RORct -Helios + T regs .