CD301b+ dendritic cell-derived IL-2 dictates CD4+ T helper cell differentiation

Tatsumi, Naoya; El-Fenej, Jihad; Davila-Pagan, Alejandro; Kumamoto, Yosuke

doi:10.1038/s41467-025-55916-9

CD301b⁺ dendritic cell-derived IL-2 dictates CD4⁺ T helper cell differentiation

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Published: 26 February 2025

Volume 16, article number 2002 (2025)
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CD301b⁺ dendritic cell-derived IL-2 dictates CD4⁺ T helper cell differentiation

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Abstract

T helper (Th) cell differentiation is fundamental to functional adaptive immunity. Different subsets of dendritic cells (DC) preferentially induce different types of Th cells, but the DC-derived mechanism for Th type 2 (Th2) differentiation is not fully understood. Here, we show that in mice, CD301b⁺ DCs, a major Th2-inducing DC subset, drive Th2 differentiation through cognate interaction by rapidly inducing IL-2 receptor signalling in CD4⁺ T cells. Mechanistically, CD40 engagement prompts IL-2 production selectively from CD301b⁺ DCs to maximize CD25 expression in CD4⁺ T cells, which instructs the Th2 fate decision, while simultaneously skewing CD4⁺ T cells away from the T follicular helper fate. Moreover, CD301b⁺ DCs utilize their own CD25 to facilitate directed action of IL-2 toward cognate CD4⁺ T cells, as genetic deletion of CD25 in CD301b⁺ DCs results in reduced IL-2-mediated signalling in antigen-specific CD4⁺ T cells and hence their Th2 differentiation. These results highlight the critical role of DC-intrinsic CD40–IL-2 axis in Th cell fate decision.

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Introduction

Activation of antigen-specific CD4⁺ T cells by dendritic cells (DCs) is critical for initiating adaptive immune responses. Upon recognition of cognate peptide-major histocompatibility complex class II (MHCII) complex through the T cell receptor (TCR), naive CD4⁺ T cells rapidly proliferate as they differentiate into distinct subsets of effector T helper (Th) cells such as Th1, Th2, and Th17 cells, each of which have unique functions in immunity¹. DCs play a crucial role in both expansion and differentiation of antigen-specific CD4⁺ T cells not only by presenting antigens with costimulation but also by producing cytokines that affect the Th cell fate decision². Notably, different DC subsets have been shown to produce distinct patterns of cytokines upon stimulation and thereby preferentially induce different types of Th cells. For instance, among the three major DC subsets in mouse skin, including the dermal type 1 conventional DCs (cDC1s), the dermal cDC2s, and the epidermal Langerhans cells (LCs), which have a monocytic origin but have DC-like function, the dermal cDC1s preferentially induce Th1 cell differentiation by producing IL-12^3,4, whereas LCs induce Th17 cell differentiation through IL-6 production^3,5. Such division of labor between DC subsets is not unique to the skin, though the capacity to induce specific types of Th cells is not always confined to the same DC subset, as DC subsets other than cDC1s can be a critical source of IL-12 for inducing Th1 cell differentiation when exposed to certain stimuli^6,7. Likewise, in organs outside of the skin where LCs do not exist, the Th17 cell differentiation is induced by a specialized subset of cDC2s that produce high levels of IL-23 and/or IL-6^8,9,10,11. In contrast, the dermal cDC2s and cDC2s elsewhere have been shown to be required for the differentiation of Th2 cells^{12,13,14,15,16}, which play a crucial role in the development of allergic diseases and protection against helminth parasites. However, unlike the Th1 and Th17 fate induced by defined cytokines, the DC-derived factors acting directly on CD4⁺ T cells for instructing the Th2 cell fate have not been clearly identified.

We and others have shown previously that CD301b⁺ DCs, a major, migratory subset of cDC2s, are required specifically for the Th2 cell differentiation of antigen-specific CD4⁺ T cells induced by allergens, adjuvants, or by helminth infection^12,13,17,18. Diphtheria toxin (DT)-induced depletion of CD301b⁺ DCs in mice expressing the DT receptor (DTR) under the regulation of the Mgl2 (encoding CD301b) promoter (Mgl2-DTR mice) resulted not only in delayed priming but also in reduced Th2 cell differentiation with a compensatory increase in Th1, Th17 and T follicular helper (Tfh) cell differentiation^19,20. These findings suggest that CD301b⁺ DCs dictate the bifurcation of effector CD4⁺ T cell fate, but its mechanism remains unknown.

Here, we show that the Th2 cell fate instruction by CD301b⁺ DCs requires MHCII- and CD40-dependent cognate interaction with CD4⁺ T cells. Mechanistically, the CD40 ligation induces IL-2 production specifically from CD301b⁺ DCs, which is required for the maximal CD25 upregulation and the downstream IL-2 receptor (IL-2R) signaling in antigen-specific CD4⁺ T cells. The full expression of CD25 in antigen-specific CD4⁺ T cells is more strictly required for their differentiation into Th2 than for Th1 cells. In addition, the IL-2R signaling in the cognate CD4⁺ T cells early after priming requires CD25 expression in CD301b⁺ DCs, suggesting that the DC-derived CD25 facilitates the directed action of IL-2. Furthermore, the CD301b⁺ DC-intrinsic CD40–IL-2 axis skews CD4⁺ T cells away from differentiation into Tfh cells. These data highlight the critical role of DC-intrinsic CD40–IL-2 axis in the bifurcation of Th cell fate.

Results

Direct antigen presentation by CD301b⁺ DCs is required for Th2 cell differentiation

CD301b⁺ DCs are required for Th2 cell differentiation induced by papain (protease allergen), alum (type 2 adjuvant), and Nippostrongylus brasiliensis (Nb) (helminth parasite)¹³, but whether they instruct the Th2 cell fate through a cognate interaction or via antigen-independent cues (e.g., cytokines) remains unclear. To address this question, we examined Th2 cell differentiation in mice lacking MHCII specifically in CD301b⁺ DCs (Mgl2^+/Cre;H2ab1^fl/fl mice, hereafter CD301b^ΔMHCII mice)²⁰ by adoptively transferring carboxyfluorescein succinimidyl ester (CFSE)-labeled ovalbumin (OVA)-specific TCR transgenic CD4⁺ T (OT-II) cells²¹ and subcutaneously immunizing in the footpad with OVA plus papain (Fig. 1a). As reported previously, the Cre expression in the Mgl2^Cre strain, examined by the inducible tdTomato (iTom) reporter (Mgl2^+/Cre;R26^LSL-iTom) mice, was restricted to CD301b⁺　PD-L2⁺ migratory (MHCII^hi) cDC2 (migDC2) and some CD301b⁻　PD-L2⁺ migDC2s, the latter of which presumably expressed a low level of CD301b that was not detectable due to the reduced CD301b protein expression from the Mgl2^Cre allele²⁰, while the Cre expression was nearly absent from all other cell types including PD-L2⁻ migDC2s, LCs, XCR1⁺ migDC1s, and LN-resident cDCs (Supplementary Fig. 1a, b). Seven days after the immunization, the OT-II cells in the draining lymph node (dLN) of the CD301b^∆MHCII mice showed normal cell divisions with a mild increase in number (Fig. 1b, c), indicating that they were primed by antigen-presenting cells other than CD301b⁺ DCs. However, as in CD301b⁺ DC-depleted mice¹³, the OT-II cells in CD301b^∆MHCII mice failed to develop IL-4-producing Th2 cells, while their differentiation into IFNγ-producing Th1 cells remained unchanged (Fig. 1d, e). Likewise, when mice were immunized with OVA plus alum, the Th2 differentiation of OT-II cells was impaired in CD301b^ΔMHCII mice without affecting cell divisions, numbers, or differentiation into Th1 cells (Fig. 1f–h). In contrast, the expansion and Th1 differentiation of OT-II cells were not affected in the CD301b^ΔMHCII mice when immunized with OVA plus a Th1-plarizing adjuvant CpG oligodeoxynucleotide (Fig. 1i–k). Lastly, as in CD301b⁺ DC-depleted mice¹³, the CD301b^ΔMHCII mice had similar numbers of CD44⁺ activated CD4⁺ T cells but showed impaired IL-4 production from the activated CD4⁺ T cells in the skin- and lung-dLNs upon subcutaneous infection with Nb, a model in which the parasites quickly migrate from the skin to the lung within one day, indicating the requirement of cognate interaction with CD301b⁺ DC for the parasite-induced Th2 cell differentiation in the endogenous polyclonal CD4⁺ T cells (Fig. 1l–n). Of note, the expression of CD301b is not a simple indicator for maturation status but is rather marking a specific cDC2 subset, as the Cre⁺ and Cre⁻ migDC2s in the Mgl2^+/Cre;R26^LSL-iTom mice and CD301b⁺ and CD301b⁻ migDC2s in wild-type (WT) mice expressed CD86 at a comparable level (Supplementary Fig. 1c–g). These results indicate that cognate interaction with CD301b⁺ DC is required specifically for the differentiation of Th2 cells.

CD301b⁺ DCs are required for inducing full CD25 expression in CD4⁺ T cells

DCs send critical activation signals to antigen-specific naive CD4⁺ T cells, leading to cell cycle entry and rapid upregulation of early activation markers such as CD69 and CD25 in T cells. We have shown previously that the depletion of CD301b⁺ DCs results in an 8- to 16-hour delay in CD69 upregulation and cell cycle entry of antigen-specific CD4⁺ T cells²⁰, but the link between the delayed activation and the selective impairment in Th2 differentiation remains unclear. To gain further insight into the potential defect in CD4⁺ T cell activation in these mice, we transferred CFSE-labeled OT-II cells into DT-treated WT (CD301b⁺ DC-intact) or Mgl2-DTR (CD301b⁺ DC-depleted) mice 24 h after immunization with OVA plus papain in the footpad and then blocked further LN entry of T cells with anti-CD62L monoclonal antibody (mAb) 2 h later (Fig. 2a), which allows us to monitor the activation kinetics of OT-II cells in a synchronized manner²⁰. In agreement with our previous report²⁰, the upregulation of CD69 in OT-II cells was delayed in CD301b⁺ DC-depleted mice but was quickly recovered presumably due to antigen presentation by CD301b⁻ DCs (Fig. 2b). In contrast, CD25 expression was induced in a subset of CD69⁺ OT-II cells at a similar timing and peaked at 16 h after the dLN entry in both CD301b⁺ DC-intact and CD301b⁺ DC-depleted mice, but with significantly lower expression levels in the latter (Fig. 2b, c). The requirement of CD301b⁺ DCs for the full CD25 upregulation is subset-specific, as the depletion of CD207⁺ DCs, including LCs and dermal CD103⁺ cDC1s^22,23,24, in CD207-DTR mice²⁵ did not affect CD25 expression (Fig. 2d).

Fig. 2: CD301b+ DCs are required for maximal CD25 expression and STAT5 activation in antigen-specific CD4+ T cells. — **Fig. 2: CD301b⁺ DCs are required for maximal CD25 expression and STAT5 activation in antigen-specific CD4⁺ T cells.**

CD25 (IL-2Rα) is the high-affinity subunit of the IL-2R complex. Intracellular staining of phosphorylated STAT5 (pSTAT5) in OT-II cells expressing the Nur77-GFP reporter (Nur77-GFP;OT-II), whose expression correlates with the TCR signal strength²⁶, showed a reduction of pSTAT5, the major signaling component of IL-2R, in OT-II cells primed in CD301b⁺ DC-depleted mice (Fig. 2e, f). In contrast, like CD69, expression of the Nur77-GFP reporter soon after the dLN entry was lower in Mgl2-DTR than in WT mice (Fig. 2e, 0 h), but the comparable expression at a later time-point indicated a similar amount of TCR stimulation received by OT-II cells over time regardless of the CD301b⁺ DC depletion status (Fig. 2e, 16 h). The pSTAT5 levels were also reduced in CD301b^∆MHCII mice (Fig. 2f), indicating the requirement of cognate interaction with CD301b⁺ DC for optimal activation of STAT5 in OT-II cells. Furthermore, the subset-specific requirement of CD301b⁺ DCs for the full CD25 upregulation and STAT5 phosphorylation in OT-II cells was also observed when the mice were immunized under a non-Th2 condition with Freund’s complete adjuvant (FCA) (Fig. 2g–i). CD301b⁺ DCs, but not CD207⁺ DCs, were also required for the full upregulation of CD25 in OT-II cells in a more potent Th1 condition with CpG that did not induce any Th2 cells (Supplementary Fig. 2a–c), though they were dispensable for the Th1 cell differentiation (Fig. 1k). The expression of GATA-3, the master regulator of Th2 cell differentiation, was induced in a subset of Nur77-GFP⁺ OT-II cells and showed positive correlation with pSTAT5 levels in the dLN of WT mice immunized with papain (Fig. 2j). The depletion of CD301b⁺ DCs resulted in a reduction of GATA-3 in OT-II cells in association with the impaired activation of STAT5 (Fig. 2j, k). While OT-II cells were transferred into pre-immunized mice in the above data, similar results were obtained when the dLN was pre-seeded with naïve OT-II cells or harvested at later time-points (Supplementary Fig. 2d–h), indicating that the impaired Th2 differentiation in the absence of CD301b⁺ DC-mediated priming was not due to a simple delay in IL-4 production. Collectively, these data indicate that CD301b⁺ DCs deliver a qualitatively distinct signal required for the optimal CD25 upregulation and Th2 cell differentiation in antigen-specific CD4⁺ T cells.

Th2 cell differentiation relies more stringently on IL-2R signaling than Th1 cells

IL-2 plays fundamental roles in CD4⁺ T cell biology^27,28. While some studies have suggested the requirement of IL-2R signaling for Th2 cell differentiation in vitro^29,30, its role in Th2-specific fate decision in vivo remains elusive, as abrogating IL-2R signaling often results in a broader defect in proliferation, differentiation, and/or survival of CD4⁺ T cells^31,32. For instance, upon allergen exposure in the lung, CD25-deficient CD4⁺ T cells have been shown to expand normally in the secondary lymphoid organs but migrate to the lung less effectively than WT CD4⁺ T cells, but the impact of CD25 deficiency on their Th2 fate decision per se remains unclear³³. To definitively address the role of IL-2R signaling in Th2 cell differentiation in vivo, we first neutralized IL-2 by intraperitoneally (i.p.) injecting an anti-IL-2 mAb S4B6-1, which blocks the binding of IL-2 to CD25^34,35, in WT mice immunized with OVA plus papain as in Supplementary Fig. 3a. While the IL-2 blockade had no major impact on cell divisions and induced only a modest decrease in numbers of OT-II cells (Supplementary Fig. 3b, c), it inhibited the differentiation of IL-4-producing Th2 cells without affecting their ability to induce IFNγ-producing Th1 cells (Supplementary Fig. 3d), suggesting that IL-2 is specifically required for Th2 differentiation. Notably, the selective requirement of IL-2 for Th2 differentiation is not specific to the type 2 immune environment, since, when treated with the anti-IL-2 mAb, mice transferred with OT-II cells expressing the IL-4-GFP reporter (4get;OT-II) and immunized with OVA plus FCA, which predominantly induces non-type 2 inflammation, also exhibited significantly reduced IL-4-GFP expression in the donor 4get;OT-II cells without major alteration in their proliferation or IFNγ production (Supplementary Fig. 3e–g).

Since the anti-IL-2 mAb S4B6-1 can either neutralize or potentiate IL-2R signaling in a dose-dependent manner^36,37, we next examined if the reduced, but not completely abrogated, CD25 upregulation in CD4⁺ T cells was responsible for the impaired Th2 differentiation in CD301b⁺ DC-depleted mice by transferring Il2ra (CD25)^+/+, Il2ra^+/−, or Il2ra^−/− OT-II cells into WT mice immunized with OVA plus papain (Supplementary Fig. 3h). As expected, CD25 expression in the Il2ra^+/− OT-II cells was reduced by approximately 50% when measured 16 h after the dLN entry, whereas IL-2 production was increased inversely with the Il2ra gene dosage likely due to the lack of the CD25-dependent negative feedback³⁸ (Supplementary Fig. 3i, j). Nevertheless, STAT5 phosphorylation was reduced by roughly 50% and nearly absent in the Il2ra^+/− and Il2ra^−/− OT-II cells, respectively (Supplementary Fig. 3k), suggesting that the CD25 expression level, rather than the IL-2 production, from OT-II cells is the primarily determinant of STAT5 phosphorylation in this model. The reduction of pSTAT5 in the Il2ra^+/− OT-II cells were associated with lower levels of GATA-3 expression 24 h after the priming (Supplementary Fig. 3l, m), indicating that the maximal IL-2R signaling is necessary for the early commitment to the Th2 cell fate. Five days after immunization with OVA plus papain without CD62L blockade (Supplementary Fig. 3n), >80% of Il2ra^+/− and Il2ra^−/− OT-II cells underwent cell divisions, though there were mild defects in the number of division cycles as well as the total numbers of the donor OT-II cells (Supplementary Fig. 3o, p). However, the divided Il2ra^+/− and Il2ra^−/− OT-II cells failed to produce IL-4 without showing a defect in IFNγ production (Supplementary Fig. 3q). Immunization with OVA plus FCA also resulted in a loss of IL-4 production in the Il2ra^+/− OT-II cells, but in this case with a trend for reduction in IFNγ production without significant impact on proliferation (Supplementary Fig. 3r–t).

While the above data suggest Th cell-intrinsic requirement of full CD25 expression for Th2 cell differentiation, the potentially elevated IL-2 levels in the dLN due to the increased production from the Il2ra^+/− and Il2ra^−/− OT-II cells (Supplementary Fig. 3i, j) may have activated bystander T regulatory (Treg) cells and indirectly suppressed Th cell differentiation as previously shown for self-reactive Th cells^39,40. To further elucidate the role of IL-2R signaling in Th2 fate decision in vivo, we next co-transferred equal numbers of Il2ra^+/+ and Il2ra^+/− or Il2ra^−/− OT-II cells into mice immunized with OVA plus papain, FCA, or CpG, so that the IL-2 availability in the dLN is normalized (Fig. 3a, d). The Il2ra gene dosage-dependent decrease and increase in IL-2 production and STAT5 phosphorylation, respectively, were also observed in this setting (Fig. 3b, c), but, unlike the individual transfer approach (Supplementary Fig. 3o), the Il2ra^+/− OT-II cells showed comparable expansion to the co-transferred Il2ra^+/+ counterpart in papain- and FCA-immunized mice while they were outcompeted by the Il2ra^+/+ OT-II cells in CpG-immunized mice, whereas the Il2ra^−/− OT-II cells expanded significantly less than the Il2ra^+/+ OT-II cells regardless of the adjuvant used (Fig. 3e–g). The reduced expansion was not due to poor survival (Fig. 3h), suggesting that it was instead due to impaired survival and/or recruitment of naïve OT-II cells to the dLN. Importantly, the loss of even a single copy of Il2ra in the donor OT-II cells consistently resulted in a dramatic reduction in IL-4 production, whereas its impact on IFNγ production was variable and less significant (Fig. 3i). When normalized to the Il2ra^+/+ counterpart in the same host, a similar reduction of Th2 cells was observed in the Il2ra^+/− OT-II cells in both papain- and FCA-immunized mice (Fig. 3j). Likewise, there was no significant difference in the reduction in Th1 cells in the Il2ra^+/− OT-II cells between papain-, FCA- and CpG-immunized mice when normalized to the Il2ra^+/+ OT-II cells in the same host (Fig. 3j). Furthermore, the impact of the complete loss of CD25 on both Th1 and Th2 cell differentiation of the Il2ra^−/− OT-II cells was also largely similar between different models when normalized to the Il2ra^+/+ counterpart (Fig. 3j). These data indicate that, despite the substantial difference in the magnitude of Th1 and Th2 cell differentiation induced by different adjuvants, the impact of the loss of CD25 on their differentiation was similar between models and does not correlate with the magnitude of differentiation. Most importantly, the loss of CD25 consistently had a greater impact on Th2 cell differentiation than on Th1 cell differentiation regardless of the model (Fig. 3j). Collectively, these data indicate that the Th2 fate decision by antigen-specific CD4⁺ T cells in vivo requires a potent and cell-intrinsic IL-2R signaling and is more sensitive to a partial loss of CD25 than the Th1 cell differentiation.

**Fig. 3: Th2 cell differentiation relies more stringently on IL-2R signaling than Th1 cells.**

CD301b⁺ DC-derived IL-2 is required for the full CD25 upregulation and Th2 differentiation in antigen-specific CD4⁺ T cells

Following TCR ligation, CD25 is induced in antigen-specific CD4⁺ T cells through two consecutive steps: the initial upregulation induced by the TCR signaling and the subsequent TCR-independent amplification of the Il2ra transcription by the IL-2-driven, CD25-dependent positive feedback^41,42. While the activated CD4⁺ T cells themselves are generally considered as the critical source of IL-2 in this feedback loop^42,43,44, DCs can also produce IL-2 upon activation in both mice and humans^{45,46,47,48,49}, though its role in T cell priming in vivo remains elusive. In mice immunized with OVA plus papain and transferred with OT-II cells (Fig. 4a), we found that CD301b⁺ DCs in the dLN expressed significantly higher levels of IL-2 than those in the non-draining lymph node (ndLN), while the increase was not observed in other DC subsets (Fig. 4b and Supplementary Fig. 4a, b). To examine if CD301b⁺ DC-derived IL-2 plays a critical role in CD25 upregulation and Th2 fate decision in antigen-specific CD4⁺ T cells, we generated mice lacking IL-2 specifically in CD301b⁺ DCs (Mgl2^+/cre;Il2^fl/fl, CD301b^ΔIL-2 mice)⁵⁰ (Supplementary Fig. 4c–f) and monitored the activation and differentiation of the donor OT-II cells upon immunization with OVA plus papain (Fig. 4c–k). Notably, blocking IL-2 in WT recipients resulted in reduced CD25 expression in the donor OT-II cells. Likewise, the donor OT-II cells failed to fully upregulate CD25 in CD301b^ΔIL-2 mice even though nearly all the OT-II cells expressed CD69 (Fig. 4d–f), indicating the specific requirement of CD301b⁺ DC-derived IL-2 for fully upregulating CD25 in antigen-specific CD4⁺ T cells. Furthermore, the differentiation of Th2 cells, monitored using the 4get;OT-II reporter cells, was abolished in the CD301b^∆IL-2 recipients without affecting the cell cycle progression, expansion, or their differentiation into IFNγ-producing Th1 cells (Fig. 4g–k). In contrast, CD301b⁺ DC-derived IL-2 was dispensable for the differentiation and homeostasis of DCs and Treg cells (Supplementary Fig. 4f–h). Taken together, these results demonstrate that CD301b⁺ DC-derived IL-2 is critical for inducing maximal CD25 upregulation and Th2 fate decision by antigen-specific CD4⁺ T cells.

Fig. 4: CD301b+ DC-derived IL-2 and CD25 are required for the maximal IL-2R signaling and Th2 cell differentiation of antigen-specific CD4+ T cells. — **Fig. 4: CD301b⁺ DC-derived IL-2 and CD25 are required for the maximal IL-2R signaling and Th2 cell differentiation of antigen-specific CD4⁺ T cells.**

CD301b⁺ DC-derived CD25 is required for Th2 cell differentiation

The above data suggest that CD301b⁺ DCs provide IL-2 specifically to cognate CD4⁺ T cells, but it remains unclear how the directed action of IL-2 is protected from diffusion, which would lead to rapid consumption of IL-2 by bystander Treg cells rather than by cognate CD4⁺ T cells^{39,40,43,44,51}. Previous studies have suggested that the diffusion of IL-2 is limited by the expression of CD25 on non-target cells^48,52,53,54. We found that the expression of CD25 was higher in CD301b⁺ DCs than in CD301b⁻ DCs (Fig. 4l and Supplementary Fig. 4i). CD301b⁺ DCs also expressed higher levels of CD132 (IL-2Rγ) than CD301b⁻ DCs except for XCR1⁺ migDC1, but they express little, if any, CD122 (IL-2Rβ) (Supplementary Fig. 4j, k), suggesting that the IL-2R in CD301b⁺ DCs does not efficiently signal, as CD122 is required for the signaling²⁸. Indeed, when the total LN cells from naïve WT mice were stimulated ex vivo with IL-2, pSTAT5 was detected in CD4⁺ T cells but not in DCs, while stimulation with GM-CSF, another potent STAT5 activator, induced pSTAT5 in DCs but not in CD4⁺ T cells (Supplementary Fig. 4l), further suggesting that CD25 in CD301b⁺ DCs does not promote IL-2 signaling in cis. Importantly, in mice lacking CD25 specifically in CD301b⁺ DCs (Mgl2^+/Cre;Il2ra^fl/fl, CD301b^ΔCD25 mice)^55,56 (Supplementary Fig. 4m, n), differentiation of the donor OT-II cells into Th2 cells was significantly impaired without affecting their expansion or Th1 cell differentiation or the number of each DC subset (Fig. 4m–q, Supplementary Fig. 4o). The lack of CD25 in CD301b⁺ DCs resulted in a reduction of pSTAT5 specifically in the donor OT-II cells but not in the endogenous Treg cells (Fig. 4r–t), suggesting that CD301b⁺ DC-intrinsic CD25 expression facilitates the directed action of IL-2 toward cognate CD4⁺ T cells. Collectively, these results indicate that CD301b⁺ DCs promote Th2 cell differentiation by reinforcing the availability of IL-2 for cognate CD4⁺ T cells by the DC-intrinsic expression of CD25.

CD40 stimulation induces IL-2 production specifically by CD301b⁺ DCs

To further explore the mechanism for the Th2 cell fate instruction by CD301b⁺ DCs, we performed CITEseq, a single-cell RNA sequencing (scRNAseq) technique with DNA-barcoded mAbs for simultaneously analyzing the expression of mRNA and cell surface markers⁵⁷, on MHCII^hi migratory DCs isolated form skin-dLNs of naïve and papain-immunized mice one day after immunization. Unsupervised clustering analysis of the cell surface markers identified 7 distinct clusters composed of 3 major DC subsets, including LC (cluster 4: CD172a^lo CD326^hi XCR1⁻), cDC1s (cluster 5: CD172a⁻ CD326^int XCR1⁺), and cDC2s (clusters 1, 2, 3, 6 and 7: CD172a^hi CD326⁻ XCR1⁻) (Fig. 5a). The cDC2s were divided into three subpopulations, CD301b^lo, CD301b^int, or CD301b^hi DCs, based on CD301b expression levels (Fig. 5b). Alternatively, the cDC2s were further divided into five subclusters based on their mRNA expression profile, with the subclusters 2 and 5 mainly composed of naive cDC2s, while subclusters 1, 3, and 4 predominantly derived from cDC2s in immunized mice (Fig. 5a, c). While the CD301b expression alone did not identify a single cDC2 subcluster in either naive or immunized mice, cells in the cDC2 subcluster 1, 2 and 3 were more enriched in CD301b^int and CD301b^hi DCs, whereas those in the subcluster 4 and 5 were more abundant in CD301b^lo DCs (Fig. 5b, c). Consistent with the intracellular staining data (Supplementary Fig. 4b), Il2 mRNA was detected in all DC subsets including CD301b^hi DCs (Fig. 5d). The Ingenuity Pathway Analysis⁵⁸ of the genes differentially expressed in CD301b^hi DCs compared with all other DC subsets in papain-immunized mice identified several pathways including the CD40 pathway, which was enriched in CD301b^hi DCs in both naive and papain-immunized mice (Fig. 5e, f). Indeed, CD301b⁺ DCs expressed higher levels of CD40 compared to CD301b⁻ DCs in both dLN and ndLN of papain-immunized mice (Fig. 5g, h). Importantly, stimulation of CD40 by i.p. administration of an agonistic anti-CD40 mAb FGK4.5^59,60,61 induced IL-2 production in CD301b⁺ DCs along with CD80, CD86, and CD25 (Fig. 5i, j). Notably, the CD40-induced IL-2 production was specific to CD301b⁺ DCs among DC subsets (Fig. 5i). Thus, CD40 ligation stimulates IL-2 production and upregulation of costimulatory molecules in CD301b⁺ DCs.

Fig. 5: CD40 stimulation induces IL-2 production by CD301b+ DCs. — **Fig. 5: CD40 stimulation induces IL-2 production by CD301b⁺ DCs.**

CD301b⁺ DC-intrinsic CD40 is required for Th2 cell differentiation

Previous studies have shown the requirement of CD40 in Th2 cell differentiation^62,63, but the underlying mechanism is unknown. Indeed, the 4get;OT-II cells primed with OVA plus papain in CD40-deficient mice expressed significantly lower levels of GFP (reporting Il4)⁶⁴ compared to those in WT mice, whereas their expansion, cell division, and differentiation into Th1 cells remained comparable (Supplementary Fig. 5a–c). Moreover, a similar reduction was observed for Th2 cells but not for Th1 cells when the OT-II cells were primed in mixed bone marrow (BM)-chimeric (BMC) mice reconstituted with a 1:1 mixture of MHCII-deficient and CD40-deficient BM cells, in which cells expressing MHCII do not express CD40, while cells expressing CD40 cannot present antigens to CD4⁺ T cells (Fig. 6a–c). Intriguingly, unlike OT-II cells primed in CD40-deficient mice, the cell division and expansion of OT-II cells were also impaired in MHCII^−/−:CD40^−/− BMC mice (Supplementary Fig. 5d, e), suggesting that the partial loss of MHCII-dependent cognate TCR stimulation in the BMC mice had an additional negative impact on priming. These data indicate the requirement of the concurrent CD40 signaling and antigen presentation in the same cells specifically for Th2 fate instruction.

Fig. 6: CD301b+ DCs instruct Th2 cell fate on CD4+ T cells through the DC-intrinsic CD40–IL-2 axis. — **Fig. 6: CD301b⁺ DCs instruct Th2 cell fate on CD4⁺ T cells through the DC-intrinsic CD40–IL-2 axis.**

We next reconstituted lethally irradiated WT mice with a 1:1 mixture of CD40^−/− and Mgl2-DTR BM cells (Fig. 6d). In the resulting mice (CD301b^ΔCD40 BMC), only CD40^−/− cells remained intact in the CD301b⁺ compartment upon DT injection, while all CD301b⁻ compartments maintained the 1:1 chimerism (Fig. 6e). Although the MHCII expression in CD40^−/− CD301b⁺ DCs was comparable to that in WT CD301b⁺ DCs (Fig. 6f), the OT-II cells primed with OVA plus papain in the CD301b^ΔCD40 BMC mice had significantly reduced Th2 cells but not Th1 cells (Fig. 6g). Similarly to the MHCII^−/−:CD40^−/− BMC mice (Supplementary Fig. 5d, e), there was a slight but significant reduction in the cell division and expansion of OT-II cells in the CD301b^ΔCD40 BMC mice (Supplementary Fig. 5f, g), suggesting that the loss of 50% of CD301b⁺ DCs in these mice partially attenuated the priming itself. Collectively, these data indicate that CD301b⁺ DC-intrinsic CD40 is required for Th2 cell fate instruction.

CD301b⁺ DCs are a sufficient DC subset for Th2 cell fate instruction

The data thus far indicate that direct antigen presentation, CD40 expression, and IL-2 production by CD301b⁺ DCs are all required for Th2 cell fate instruction. We previously showed that adoptively transferring CD301b⁺ DCs sorted from papain-immunized mice and pulsed ex vivo with an antigen is not sufficient to induce Th2 cell differentiation in naïve recipients¹³. This may reflect the involvement of multiple DC subsets for Th cell differentiation as suggested by others^65,66 or may be due to the requirement of cells outside of the DC compartment that were not fully recapitulated in the recipients as the recipients had never been directly exposed to the adjuvant in such experiments. To address if CD301b⁺ DCs are sufficient and no other DC subsets are required for Th2 cell fate instruction, we took advantage of mice expressing a Cre-removable DTR cassette in CD11c⁺ cells (CD11c-dlDTR) crossed with the Mgl2-Cre strain (Mgl2-Cre;CD11c-dlDTR mice), in which CD301b⁺ DCs are protected from DT-induced depletion while CD301b⁻ DCs are susceptible to DT²⁰. Although the number of CD301b⁺ cells in these mice is underestimated due to the loss of CD301b protein expression from the Mgl2^cre allele²⁰, the use of the inducible tdTomato reporter concurrently with these mice (Mgl2-Cre;CD11c-dlDTR;R26^LSL-iTom) confirmed subset-specific protection of Cre-expressing Mgl2 (CD301b)⁺ DCs from DT-induced depletion in the skin-dLNs (Supplementary Fig. 5h–m). In line with the tdTomato expression pattern in the Mgl2^+/Cre;R26^LSL-iTom mice (Supplementary Fig 1a, b), the majority of the protected DCs expressed PD-L2, a marker known to be correlated with CD301b expression in CD172a⁺ cDC2s^12,19, whereas all other DCs, including PD-L2⁻ migDC2s, LCs, XCR1⁺ migDC1s, and LN-resident DCs were effectively depleted (Supplementary Fig. 5i–m). Importantly, OT-II cells primed in the Mgl2-Cre;CD11c-dlDTR mice differentiated normally into Th1 and Th2 cells under both Th2 (papain) and non-Th2 (FCA) immunization conditions (Fig. 6h–j), though modest reduction in their expansion was observed in both conditions (Supplementary Fig. 5n–q). These results indicate that, while DCs other than CD301b⁺ DC subset may be required for the maximal OT-II cell expansion, CD301b⁺ DCs are both required and sufficient for Th2 cell fate instruction in these immunization conditions.

CD301b⁺ DCs instruct Th2 cell fate on CD4⁺ T cells through the DC-intrinsic CD40–IL-2 axis

To further elucidate if the sufficiency of CD301b⁺ DCs for Th2 cell fate instruction is based on their cell-intrinsic CD40–IL-2 axis, we next utilized anti-CD154 (CD40L) blocking mAb (MR-1) or anti-CD40 (FGK4.5) agonistic mAb, both of which serve to inhibit physical interaction between CD40 and CD154^60,61,67,68. Blockade of CD154 in WT mice or genetic deficiency of CD40, but not stimulation of CD40, resulted in impaired upregulation of CD25 without affecting the CD69 expression in OT-II cells primed with OVA plus papain (Fig. 6k–m). Accordingly, the blockade of CD154 blunted Th2 cell differentiation of the donor OT-II cells in WT mice, whereas stimulation of CD40 restored Th2 cell differentiation even when CD154 was blocked, indicating that CD40 stimulation is sufficient to drive Th2 cell differentiation even in the absence of CD154 signaling in CD4⁺ T cells (Fig. 6n, o). However, the CD40 stimulation failed to restore Th2 cell differentiation when IL-2 was additionally neutralized, suggesting that IL-2 is responsible for the Th2 fate instruction downstream of CD40 stimulation (Fig. 6o). Furthermore, the CD40 stimulation did not restore Th2 cell differentiation of the donor OT-II cells in CD301b⁺ DC-depleted mice, CD301b^∆IL-2 mice, or in CD301b^ΔMHCII mice (Fig. 6o), indicating that the CD40 stimulation induces Th2 cell differentiation by inducing IL-2 production from the CD301b⁺ DCs that are in cognate interaction with CD4⁺ T cells. We observed no major impact on proliferation of the donor OT-II cells in any of the conditions (Fig. 6p). Notably, the CD40 stimulation significantly increased the frequencies of IFNγ⁺ Th1 OT-II cells in WT mice (Supplementary Fig. 5r). Unlike CD40-induced Th2 cell differentiation, a trend for an increase in Th1 differentiation, though statistically insignificant, was also observed in CD301b⁺ DC-depleted, CD301b^ΔIL-2, or in CD301b^ΔMHCII mice upon stimulation of CD40, suggesting that the stimulation of CD40 in CD301b⁻ antigen-presenting cells can promote Th1 cell differentiation. Collectively, these data indicate that MHCII- and CD40-dependent interaction between CD301b⁺ DCs and antigen-specific CD4⁺ T cells and the resultant production of IL-2 from CD301b⁺ DCs play a decisive role in Th2 cell fate instruction.

CD301b⁺ DC-intrinsic CD40–IL-2 axis skews CD4⁺ T cells toward non-Tfh effector fate

We have shown previously that the depletion of CD301b⁺ DCs in the Mgl2-DTR mice results in the expansion of Tfh cells in addition to impaired Th2 cell differentiation¹⁹. This Tfh expansion is due at least partially to the loss of PD-L1-dependent suppression of Tfh cells by CD301b⁺ DCs¹⁹, but it remains unclear if CD301b⁺ DCs suppress the initial fate decision of antigen-specific CD4⁺ T cells into Tfh cells. During CD4⁺ T cell priming, Tfh cells downregulate PSGL1 prior to upregulating CXCR5⁶⁹. Unlike CD301b⁺ DC-depleted Mgl2-DTR mice¹⁹, there was no significant increase in mature CXCR5⁺ PD-1⁺ Tfh cells within both OT-II and endogenous CD4⁺ T cell compartments in CD301b^∆MHCII mice immunized with OVA plus papain (Fig. 7a, b), suggesting that the expansion of mature CXCR5⁺ PD-1⁺ Tfh cells in CD301b⁺ DC-depleted mice is mainly driven by the loss of antigen-independent suppression mechanism such as PD-L1-mediated suppression of Tfh cells¹⁹. However, the PSGL1^lo PD-1⁺ ‘immature’ Tfh fraction was significantly increased in both OT-II and endogenous CD4⁺ T cells in CD301b^∆MHCII mice, suggesting that more CD4⁺ T cells are poised to the Tfh cell fate in those mice (Fig. 7c, d).

CD4⁺ T cell-intrinsic IL-2R signaling suppresses Tfh cell differentiation while favoring the differentiation of non-Tfh effector Th cells^70,71,72,73, but the role of T cell-extrinsic source of IL-2 in this bifurcation is unknown. In WT mice transferred with OT-II cells, the blockade of IL-2 by anti-IL-2 mAb resulted in a significant increase in the differentiation of PSGL1^lo PD-1⁺ CD4⁺ T cells upon immunization with OVA plus papain (Fig. 7e, f). Notably, the differentiation of the PSGL1^lo PD-1⁺ CD4⁺ T cells was also increased in OT-II cells primed in CD301b^ΔCD40 BMC mice and CD301b^∆IL-2 mice compared with those primed in respective control mice, which was also observed in their endogenous CD4⁺ T cells (Fig. 7g–l). Interestingly, unlike in CD301b^ΔMHCII mice (Fig. 7a, b), the endogenous, but not OT-II, CXCR5⁺ PD-1⁺ mature Tfh cells were also increased in the CD301b^ΔCD40 BMC mice compared with the WT:Mgl2-DTR mixed BMC control mice (Fig. 7i, j), suggesting that the depletion of 50% of CD301b⁺ DCs in the CD301b^ΔCD40 BMC mice, in combination with the lack of CD40 in the remaining CD301b⁺ DCs, had more pronounced impact on Tfh cell suppression than the CD301b⁺ DC-specific MHCII deficiency alone. Lastly, along with the downregulation of PSGL1, the expression of Bcl-6, the master regulator of Tfh cell differentiation⁷⁴, was also increased in the PD-1⁺ fraction in OT-II cells and showed a trend for an increase in endogenous CD4⁺ T cells in CD301b^ΔIL-2 mice (Fig. 7k–n), further suggesting that more CD4⁺ T cells are poised for Tfh differentiation in these mice. Thus, in addition to driving the differentiation toward Th2 cell fate, these data indicate that the CD301b⁺ DC-intrinsic CD40–IL-2 axis also limits the commitment to Tfh cell fate and promotes non-Tfh effector Th cell fate. Taken together, these results demonstrate the role of CD301b⁺ DC-derived IL-2 in priming and effector fate bifurcation of CD4⁺ T cells (Fig. 8).

Fig. 8: Model for Th cell fate instruction by CD301b+ DCs. — **Fig. 8: Model for Th cell fate instruction by CD301b⁺ DCs.**

Discussion

Th2 cells play a fundamental role in type 2 adaptive immunity such as host protection against helminth parasites and pathogenesis of allergic diseases. The transcription factor GATA-3 has long been known as the cell-intrinsic master regulator for the Th2 cell identity, but the mechanism of how the Th2 cell fate is instructed by exogenous cues in vivo remains unclear. Although IL-4 is commonly used to induce Th2 cell differentiation in cultured CD4⁺ T cells, there is no clear evidence that DCs produce IL-4 to instruct the Th2 cell fate⁷⁵, and the expression of IL-4 receptor in CD4⁺ T cells is not strictly required for their Th2 fate decision in vivo⁷⁶. The present study reveals that the DC-intrinsic CD40–IL-2 axis in CD301b⁺ DCs plays a crucial role in Th2 cell fate instruction by quantitatively regulating the IL-2R signaling in CD4⁺ T cells. Upon encountering antigen-specific naive CD4⁺ T cells, CD301b⁺ DCs not only present the antigen to stimulate the TCR for timely activation but also provide IL-2 to T cells upon CD40 ligation to induce full upregulation of CD25, which is required specifically for Th2 cell differentiation, as Th2 cells rely on the IL-2R signaling for their differentiation more stringently than Th1 cells. Furthermore, the CD301b⁺ DC-derived IL-2 also limits the differentiation into Tfh cells, favoring the commitment to non-Tfh effector Th cells altogether. These data demonstrate that CD301b⁺ DC-derived IL-2 plays a decisive role in Th cell fate instruction and highlight the importance of DC-dependent quantitative control of IL-2R signaling in CD4⁺ T cells for the commitment to Th2 cell fate.

Originally identified as the T cell growth factor secreted by activated T cells themselves^77,78, IL-2 plays a multifaceted role in the proliferation, differentiation, and survival of CD4⁺ T cells. Although recombinant IL-2 is often added as a cell culture supplement to activated CD4⁺ T cells to maintain the survival of ‘Th0’ cells without skewing them toward the Th1 or Th2 status, previous studies have shown that CD4⁺ T cell-intrinsic production and sensing of IL-2 inhibits the differentiation of Th17 and Tfh cells^{70,71,72,79,80}, while it is required for the differentiation of Th1 and Th2 cells^{30,31,72,81,82,83,84}. In Th2 cell differentiation, STAT5, the main signaling component of the IL-2R, has been shown to directly bind to the Il4, Il4ra, and Gata3 loci and promote their expression^30,83,84, leading to the activation of the IL-4–IL-4Rα–STAT6 positive feedback loop to accelerate the differentiation of Th2 cells and dictate their identity^85,86. Accordingly, we have shown previously that CD301b⁺ DCs are required for the upregulation of IL-4Rα in antigen-specific CD4⁺ T cells during the priming²⁰. However, STAT5 also promotes the expression of Th1-associated genes such as Il12rb, Tbx21, and Ifng in the context of Th1 cell differentiation^31,87. Our data indicate that, while both Th1 and Th2 cell differentiation ultimately requires CD25 expression as shown in previous studies^29,72,88,89, Th2 cell differentiation is more sensitive to a partial loss of CD25. These observations suggest that quantitative differences in the IL-2R signaling during CD4⁺ T cell priming lead to qualitative differences in Th cell fate decision.

In line with the Th cell fate regulation by quantitative control of IL-2R signaling, the availability of IL-2 is tightly regulated in the LN microenvironment. Studies have shown that IL-2 produced by antigen-specific CD4⁺ T cells is rapidly quenched by Treg cells and/or DCs to prevent aberrant activation of self-reactive clones and to promote Tfh cell differentiation^{39,40,43,44,51,53}. However, since the IL-2 in CD4⁺ T cells functions predominantly in a paracrine manner⁷³, it is unclear how the IL-2R signaling overshoots the threshold in rare antigen-specific CD4⁺ T cells immediately after priming. Our data indicate that CD301b⁺ DCs are a critical source of IL-2 early after CD4⁺ T cell priming, especially when CD4⁺ T cells require the maximal IL-2R signaling for Th2 cell differentiation. This directed action of IL-2 is facilitated by the expression of CD25 by CD301b⁺ DCs. These data suggest that IL-2 may be directly trans-presented to cognate CD4⁺ T cells through the membrane-bound CD25 as previously suggested in other contexts^48,54, though the precise mechanism needs further investigation. Importantly, IL-2 production in CD301b⁺ DCs is induced by CD40, which needs to have coincided with MHCII-dependent cognate interaction for the Th2 cell fate instruction. Thus, CD301b⁺ DCs appear to ‘kick-start’ the IL-2R signaling in CD4⁺ T cells by directly handing IL-2 over to the cognate clones, thereby minimizing the IL-2 quenching effect by surrounding Treg cells. Interestingly, previous studies have shown the production of IL-2 by DCs at the interface between DCs and CD4⁺ T cells in vitro^46,48, suggesting that direct IL-2 delivery by CD301b⁺ DCs to CD4⁺ T cells through the immunological synapse ensures timely and confined consumption of IL-2 by the cognate clones.

While our data in CD301b⁺ DC-enriched Mgl2-cre;CD11c-dlDTR mice suggest that CD301b⁺ DCs are sufficient for inducing Th2 cell differentiation, the relatively normal Th1 cell differentiation in those mice, despite modestly impaired expansion, also indicates that the cognate interaction with CD301b⁺ DCs alone does not constrain the fate of CD4⁺ T cells to the Th2 status. Further, CD301b⁺ DCs are required for the maximal IL-2R signaling even under non-Th2 immunization conditions with FCA or CpG, but, unlike Th2 cell differentiation, it seems to be dispensable for the commitment to Th1 cell fate. Notably, in CD4⁺ T cells stimulated with cognate peptides in vitro, the dose of the peptide has been shown to correlate with the Th1-to-Th2 ratio while inversely correlating with STAT5 activation due to inhibition of STAT5 by ERK, a signal downstream of the TCR, implicating that Th1 cells only require a lower amount of activated STAT5 for their differentiation while Th2 cell differentiation requires more potent STAT5 activation³⁰. Moreover, a recent study found that IL-6 induced by antigen-independent inflammation dampens Th2 and promotes Th17 cell differentiation by prematurely down-regulating CD25 expression in activated CD4⁺ T cells⁹⁰. Thus, the non-Th2 fate appears to be associated with excess TCR signaling and/or inflammation that results in reduced IL-2R signaling. We reported recently that CD301b⁺ DCs in the LNs are located in closer proximity to high endothelial venules and have earlier access to incoming naive CD4⁺ T cells than LCs and cDC1s, which makes them critical for timely priming of antigen-specific CD4⁺ T cells²⁰. Given that LCs and cDC1s encounter CD4⁺ T cells in a delayed timing from CD301b⁺ DCs but are often required for non-Th2 Th cell differentiation^3,4,91,92, under non-Th2 immunization conditions, the Th2-biased status imprinted early after priming by CD301b⁺ DCs may be overridden by the non-Th2 fate through the subsequent interaction with other DCs, as we recently discussed elsewhere⁹³. The nature of such staggered interaction with multiple DC subsets and the role of CD301b⁺ DCs in these conditions need further investigation, as the non-Th2 fate instruction cues may be delivered through antigen-independent interactions^90,91.

Taken together, our data demonstrate that CD301b⁺ DC-intrinsic CD40–IL-2 axis drives Th2 cell fate instruction by quantitatively regulating the IL-2R signaling in antigen-specific CD4⁺ T cells. While the use of a simplistic immunization approach in this study allows precise analysis of the T cell priming kinetics, the mechanism for disease-associated Th2 cell differentiation may need to be further examined in a more complex setting such as allergies and helminth infection, as such condition can potentially induce Il4ra even in naïve CD4⁺ T cells in an antigen-nonspecific manner⁹⁴. Thus, whether the excess amount of type 2 cytokines released from innate immune cells under these conditions overrides the requirement of DC-derived IL-2 at the time of priming needs to be addressed in future studies.

Methods

Mice

Mgl2-DTR (RRID:IMSR_JAX:023822) and Mgl2-Cre (RRID:IMSR_JAX:037286) mice were a gift from Akiko Iwasaki (Yale University). Il2 ^flox/flox mice⁵⁰ were provided by Weizhou Zhang (University of Florida). OT-II mice on the IL-4-GFP background (4get;OT-II) were a gift from Jason Weinstein (Rutgers New Jersey Medical School). Generation of Mgl2-DTR (Mgl2^+/DTR-EGFP)¹³, Mgl2-Cre and CD11c-dlDTR mice²⁰ were previously described and maintained in our colony. C57BL/6N (B6, Strain Code:027) and congenic CD45.1 (B6.SJL-Ptprc^aPepc^b/BoyCrCrl, Strain Code:564) mice on B6 background were purchased from Charles River Laboratory and propagated in our colony. H2-Ab1 (MHCII) ^flox/flox (B6.129×1-H2-Ab1^b-tmKoni/J, RRID:IMSR_JAX:013181), Il2ra^flox/flox (B6(129S4)-Il2ra^{tm1c(EUCOMM)Wtsi}/TrmaJ, RRID:IMSR_JAX:033093), CD40^−/− (B6.129P2-Cd40^tm1Kik/J, RRID:IMSR_JAX:002928), MHCII^−/− (B6.129S2-H2^dlAb1-Ea/J, RRID:IMSR_JAX:003584), OT-II (B6.Cg-Tg(TcraTcrb)425Cbn/J, RRID:IMSR_JAX:004194), CD207-DTR (B6.129S2-Cd207^{tm3.1(HBEGF/EGFP)Mal/}J, RRID:IMSR_JAX:016940), Rag1^−/− (B6.129S7-Rag1^tm1Mom/J, RRID:IMSR_JAX:002216), Il2ra^−/− (B6;129S4-Il2ra^tm1Dw/J, RRID:IMSR_JAX:002462), Rosa26 loxP-stop-loxP (LSL)-tdTomato (Ai14, R26^LSL-iTom) reporter (B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J, RRID:IMSR_JAX:007914), and Nur77-GFP reporter (C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J, RRID:IMSR_JAX:016617) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in-house.

Mgl2^cre/cre mice were crossed with H2-Ab1^flox/flox, Il2^flox/flox, and Il2ra^flox/flox mice to generate Mgl2^+/cre;H2-Ab1^flox/flox (CD301b^ΔMHCII), Mgl2^+/cre;Il2ra^flox/flox (CD301b^ΔIL-2), and Mgl2^+/cre;Il2ra^flox/flox (CD301b^ΔCD25) mice, respectively. The leaky expression of Cre in the Mgl2^+/cre mice was monitored by flow-cytometry or by qPCR and those with global deletion of MHCII, CD25, or IL-2 were excluded from the analysis as previously described²⁰. Mgl2^cre/cre mice were also crossed with R26^LSL-iTom reporter to generate Mgl2^+/Cre;R26^+/LSL-iTom mice and used for evaluating the Mgl2-Cre expression. CD11c (Itgax)-dlDTR mice were crossed with Mgl2-Cre mice to generate Mgl2^+/Cre;Itgax^+/dlDTR mice. The Mgl2^+/Cre;Itgax^+/dlDTR mice were further crossed with R26^LSL-iTom reporter mice to produce Mgl2^+/Cre;Itgax^+/dlDTR;Rosa26^+/LSL-iTom mice. OT-II and 4get;OT-II mice were bred to CD45.1 mice to produce CD45.1;OT-II and CD45.1;4get;OT-II mice, respectively. Nur77-GFP mice were crossed with CD45.1;OT-II mice to generate CD45.1;Nur77-GFP;OT-II mice. Il2ra^−/− mice were crossed with CD45.1;OT-II mice to generate CD45.1;Il2ra^+/−;OT-II and CD45.1;Il2ra^−/−;OT-II mice. The CD45.1;Il2ra^+/−;OT-II and CD45.1;Il2ra^−/−;OT-II mice were maintained on the Rag1^−/− background and further crossed onto the Nur77-GFP background where indicated. All mice were maintained in a specific pathogen-free facility at Rutgers New Jersey Medical School. For all strains, both male and female mice between 6 and 12 weeks old were used. Age and sex-matched mice were allocated randomly in the experimental groups for comparison. Control mice were co-housed or kept separately in the same room. Mice were euthanized with carbon dioxide (CO₂) inhalation followed by cervical dislocation. All animal experiments in this study have been approved by the Institutional Animal Care and Use Committee at Rutgers University (Protocol Number: PROTO999901093).

BM reconstitution

For generating CD40^−/−;Mgl2-DTR (CD301b^ΔCD40 BMC) and CD40^−/−;MHCII^−/− mixed BMC mice, lethally irradiated (1100 cGy) B6 mice were reconstituted with a 50:50 mixture of CD40^−/− and Mgl2-DTR BM cells, or with CD40^−/− and MHCII^−/− BM cells (2 × 10⁶ cells each), respectively. Those reconstituted with WT and CD40^−/− BM cells were used as a control for CD301b^ΔCD40 and CD40^−/−;MHCII^−/− mixed BMC mice. BMC mice were maintained on antibiotics (sulfamethoxazole and trimethoprim, NDC 0121-0854-16, PAI pharma) in their drinking water for 2 weeks after the reconstitution and rested for at least 6–8 weeks before being used for experiments.

Immunization, Nb infection and in vivo treatment

All immunization procedures were conducted in the rear footpad with 20 µL injection volume per footpad. Mice were immunized as indicated in each Figure with 5 µg low-endotoxin OVA (Worthington Biochemical Corporation) mixed with either 50 µg papain (P4762, Sigma), 10 µL FCA (F5881, Sigma), 10 µL alum (77161, Thermo Scientific), or 10 µg CpG2216 (Invivogen) in phosphate-buffered saline (PBS). For Nb infection, mice were injected subcutaneously in the right ear with 600 third-stage (L3) larvae (gift from William Gause, Rutgers New Jersey Medical School) in 0.2 mL of PBS. For DC depletion, mice were injected i.p. with 500 ng DT in PBS (List Biological Laboratories) at indicated time-points. For CD40 stimulation of DCs in vivo, mice were injected i.p. with 100 μg agonistic anti-CD40 mAb (FGK4.5, BioXcell) in PBS. LNs were harvested 24 h later and restimulated with PMA and ionomycin for intracellularly staining IL-2. For mAb treatment of mice immunized with OVA plus papain, mice were injected i.p. with anti-IL-2 (S4B6-1, BioXcell, 100 μg), anti-CD154 (MR1, BioXcell, 100 μg), anti-CD40 (FGK4.5, BioXcell, 100 μg), anti-CD154 plus anti-CD40 (100 μg each), or anti-IL-2 plus anti-CD154 plus anti-CD40 (100 μg each) mAbs at the time of OT-II transfer or immunization as indicated. Rat IgG2a isotype control (2A3, BioXcell) mAb was injected to make the number of mAbs equal across different groups.

T cell priming assays

For adoptively transferring OT-II cells, CD4⁺ T cells were isolated from the spleen and LNs of indicated naïve OT-II strains with Mouse CD4⁺ T Cell Isolation Kit (STEMCELL Technologies 19852 or BioLegend 480033) and labeled with 1.0 µM CFSE (Thermo Fisher) or CTV (C34571, Thermo Fisher) according to the manufacturer’s protocol. The purity of isolated OT-II cells was typically 90–95%. For analyzing the CD4⁺ T cell activation kinetics, CFSE- or CTV-labeled CD45.1;OT-II cells (1–2 × 10⁶) were adoptively transferred into CD45.2/.2 recipient mice that had been immunized in the rear right footpad 24 h prior. Mice with DTR expression were treated with DT as indicated in each Figure. In some experiments, multiple types of donor cells (1 × 10⁶ cells each) were co-transferred into one mouse. Two hours after the OT-II cell transfer, further LN entry of T cells was blocked by retro-orbitally injecting 100 µg anti-CD62L mAb (clone Mel-14, BE0021, BioXCell). Alternatively, in some experiments, the labeled OT-II cells were adoptively transferred into recipient mice 24 h prior to immunization and further LN entry of T cells was blocked by injecting anti-CD62L mAb at the time of immunization. The left (ndLN) and right (dLN) popliteal LNs were harvested at indicated time-points. For analyzing CD4⁺ T cell differentiation, CFSE- or CTV-labeled OT-II cells (1 × 10⁵ cells) were adoptively transferred into indicated CD45.2/.2 recipient mice, followed by immunization with OVA and an indicated adjuvant in the footpad. The dLNs were harvested at indicated time-points for intracellularly staining cytokines or intranuclear staining of Foxp3.

Cell preparations and flow cytometry

For preparing single-cell suspensions, LNs were minced and enzymatically digested with 2.5 mg/mL collagenase D (11088882001, Sigma) in complete RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C for 30 min. The cells were washed with PBS containing 2 mM EDTA and then stained for dead cells with cell viability dye (Zombie Aqua or Zombie UV, BioLegend) in PBS on ice for 20 min. The cells were washed with 2 mM EDTA/PBS, incubated with 10 µg/mL anti-CD16/CD32 (2.4G2, BioLegend) on ice for 10 min to block non-specific antibody binding, and stained with fluorochrome-conjugated mAbs on ice for 20 min. For intracellular cytokine staining, LN single cell suspensions were stimulated in a 96-well round-bottom plate with Cell Stimulation Cocktail containing PMA and ionomycin (eBioscience 00-4970-03, Thermo Fisher or 423302, BioLegend) at 37 °C for 1 h, and then incubated for another 5 h at 37 °C with additional Protein Transport Inhibitor Cocktail containing Brefeldin A and Monensin (eBioscience 00-4980-03, Thermo Fisher). Cells were then fixed and permeabilized with BD Cytofix/Cytoperm Kit (BD Biosciences) and incubated with anti-cytokine mAbs for 30 min on ice. For intranuclear Foxp3 staining, cells were fixed and permeabilized with Foxp3 Transcription Factor Fixation/Permeabilization Buffer (eBioscience 00-5521-00) on ice for 30 min and incubated with anti-Foxp3 mAb on ice for 30 min. For staining phosphorylated STAT5 and GATA-3, LNs were fixed with BD Phosflow^TM Fix Buffer I (BD Biosciences) at 37 °C for 10 min immediately after the harvest or ex vivo stimulation with IL-2 (50 ng/mL) or GM-CSF (50 ng/mL), and then permeabilized with BD Phosflow^TM Perm Buffer III (BD Biosciences) on ice for 30 min. After blocking with anti-CD16/CD32 (2.4G2, BioLegend), cells were stained for cell surface molecules on ice for 20 min, washed with PBS containing 2 mM EDTA, and then incubated with anti-pSTAT5 and anti-GATA-3 mAbs at room temperature (20–25 °C) for 16 h.

The mAbs to CD4 (RM4-5 or GK1.5), CD8α (53-6.7), CD45R/B220 (RA3-6B2), TCRβ (H57-597), Ly6G (1A8), CD69 (H1.2F3), CD25 (PC61), CD44 (IM7), CD279 (PD-1) (RMP1-30), I-A/I-E (MHCII) (M5/114.15.2), CD11b (M1/70), CD11c (N418), CD326 (G8.8), CD301b (URA1), CD172a (P84), CD273 (PD-L2) (TY25), XCR1 (ZET), CD103 (2E7), CD40 (3/23), CD80 (16-10A1), CD86 (GL-1), CD45.1 (A20), CD45.2 (104), IFNγ (XMG1.2), IL-4 (11B11), IL-2 (JES6-5H4), GATA-3 (16E10A23), Bcl-6 (7D1), and Foxp3 (MF-14) were purchased from BioLegend. The mAb to pSTAT5 (Tyr694) (SRBCZX) was purchased from eBioscience. Anti-CXCR5 (2G8) and PSGL1 (2PH1) mAbs were purchased from BD Biosciences. All mAbs used for flow cytometry were prepared in FACS buffer (1% BSA, 2 mM EDTA, 0.05% sodium azide in PBS), except that mAbs to pSTAT5 and GATA-3 were prepared in 1x BD Perm/Wash buffer (BD Biosciences).

All samples were collected on BD LSRII (BD Biosciences) or Attune NxT (Thermo Fisher) flow cytometer and analyzed with FlowJo software (Version 9.3.2 and 10.5.0, BD).

CITEseq data acquisition and analysis

For CITEseq, B6 mice were immunized with 5 µg OVA plus 50 ug papain in both rear footpads in 20 µL PBS. Popliteal lymph nodes were harvested from immunized mice 1 day later and digested with collagenase D (2.5 mg/mL, Roche) in RPMI-1640 medium supplemented with 10% FBS for 30 min at 37 °C, after which EDTA (5 mM final conc.) was added and incubated for another 10 min to allow complete dissociation. Pooled skin-dLNs from naive B6 mice were used as a control. Single-cell suspensions were collected, and cells were stained with viability dye (Zombie Aqua, BioBegend) in PBS for 20 min on ice. Cells were washed and incubated with anti-CD16/32 (10 µg/mL, clone 2.4G2, BioLegend) for 20 min on ice and then stained with fluorochrome-labeled mAbs for B220 and MHCII and DNA-barcoded TotalSeq mAbs for MHCII, CD11c, CD301b, CD8α, CD11b, CD64, CD103, CD172a, Ly6C and XCR1 (BioLegend) in PBS containing 1%BSA and 2 mM EDTA for 20 min on ice. The TotalSeq mAbs for MHCII, CD11c and CD301b (0.25 mg/mL) were pre-mixed with AlexaFluor 488-, AlexaFluor 546, and AlexaFluor 647-labeled antisense oligonucleotide (1 µM) that is specific to each DNA barcode, respectively, for 15 min at room temperature (20–25 °C) in order to detect these antibodies by flow cytometry for sorting. Live MHCII^hi CD11c⁺ B220⁻ cells were sorted by FACS Aria III cell sorter (BD) and used to generate Gel Beads-in-Emulsion (GEMs) using the Chromium Single-Cell 3’ Reagent v3 kit (1000092, 10X Genomics) according to the manufacturer’s protocol. Briefly, cell suspensions with a > 90% viability were mixed with reverse transcription reagents and loaded onto Chromium Chip B (1000074, 10X Genomics) along with Gel beads and Partitioning oil in the recommended order, and then the chip was processed through 10X Chromium Controller for the generation of GEMs, followed by reverse transcription, cleanup, and cDNA amplification. After the cDNA amplification, size selection was used to separate the cDNA molecules for the 3’ Gexp (Gene Expression) library and ADT library construction. Library quantity and quality control were performed on Qubit 4 Fluorometer using an HS reagent kit (Q33231, Invitrogen) and TapeStation using HS DNA D1000 screen tape (5067-5584, Agilent Technologies). ADT and 3’ Gexp libraries were mixed at the ratio of 1:5 and sequenced on NovaSeq 6000 sequencer (Illumina) with a configuration of 28/8/0/91-bp for cell barcode, sample barcode and mRNA reads, respectively, as recommended by 10X Genomics. Cell Ranger and Loupe browsers were used for data analysis. The aligned data were further analyzed and visualized using Partek Flow (Partek Inc.) and Ingenuity Pathway Analysis (QIAGEN) software.

Statistical analysis

P-values were calculated by two-tailed Student’s unpaired t-test or by paired t-test as indicated using Prism software (version 8, GraphPad). Differences were defined as statistically significant when P-values were <0.05. Data are presented as mean ± SEM. The sample sizes were determined based on the magnitude of the effect being observed, the laboratory’s previous experience, and published literature. The sample size for each experiment is provided in the figure legends. No data were excluded from the analyses. The investigators were not blinded during in vivo treatment or data collection.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The CITEseq data used in this study has been deposited in GEO database: accession number GSE281470. Source Data are provided with this paper. Mgl2-DTR (RRID:IMSR_JAX:023822) and Mgl2-Cre (RRID:IMSR_JAX:037286) mice have been deposited in the Jackson Laboratory. CD11-dlDTR mice are available upon request from Y.K. under an MTA from Rutgers University. Source data are provided with this paper.

References

Zhu, J., Yamane, H. & Paul, W. E. Differentiation of effector CD4 T cell populations (*). Annu. Rev. Immunol. 28, 445–489 (2010).
Article CAS PubMed PubMed Central MATH Google Scholar
Yin, X., Chen, S. & Eisenbarth, S. C. Dendritic cell regulation of T helper cells. Annu. Rev. Immunol. 39, 759–790 (2021).
Article CAS PubMed MATH Google Scholar
Igyarto, B. Z. et al. Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific T helper cell responses. Immunity 35, 260–272 (2011).
Article CAS PubMed Google Scholar
Martinez-Lopez, M., Iborra, S., Conde-Garrosa, R. & Sancho, D. Batf3-dependent CD103+ dendritic cells are major producers of IL-12 that drive local Th1 immunity against Leishmania major infection in mice. Eur. J. Immunol. 45, 119–129 (2015).
Article CAS PubMed Google Scholar
Kashem, S. W. et al. Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation. Immunity 42, 356–366 (2015).
Article CAS PubMed PubMed Central Google Scholar
Hilligan, K. L. et al. Dermal IRF4+ dendritic cells and monocytes license CD4+ T helper cells to distinct cytokine profiles. Nat. Commun. 11, 5637 (2020).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Huang, L. Y., Reis e Sousa, C., Itoh, Y., Inman, J. & Scott, D. E. IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10. J. Immunol. 167, 1423–1430 (2001).
Article CAS PubMed MATH Google Scholar
Lewis, K. L. et al. Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine. Immunity 35, 780–791 (2011).
Article CAS PubMed PubMed Central MATH Google Scholar
Persson, E. K. et al. IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity 38, 958–969 (2013).
Article CAS PubMed Google Scholar
Satpathy, A. T. et al. Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens. Nat. Immunol. 14, 937–948 (2013).
Article CAS PubMed PubMed Central Google Scholar
Schlitzer, A. et al. IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 38, 970–983 (2013).
Article CAS PubMed PubMed Central MATH Google Scholar
Gao, Y. et al. Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells. Immunity 39, 722–732 (2013).
Article CAS PubMed PubMed Central MATH Google Scholar
Kumamoto, Y. et al. CD301b(+) dermal dendritic cells drive T helper 2 cell-mediated immunity. Immunity 39, 733–743 (2013).
Article CAS PubMed Google Scholar
Leon, B. et al. Regulation of T(H)2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells. Nat. Immunol. 13, 681–690 (2012).
Article CAS PubMed PubMed Central MATH Google Scholar
Mayer, J. U. et al. Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote T(H)2 and inhibit T(H)17 cell polarization. Nat. Immunol. 22, 1538–1550 (2021).
Article CAS PubMed MATH Google Scholar
Tussiwand, R. et al. Klf4 expression in conventional dendritic cells is required for T helper 2 cell responses. Immunity 42, 916–928 (2015).
Article CAS PubMed PubMed Central MATH Google Scholar
Murakami, R. et al. A unique dermal dendritic cell subset that skews the immune response toward Th2. PLoS ONE 8, e73270 (2013).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Sokol, C. L., Camire, R. B., Jones, M. C. & Luster, A. D. The chemokine receptor CCR8 promotes the migration of dendritic cells into the lymph node parenchyma to initiate the allergic immune response. Immunity 49, 449–463.e446 (2018).
Article CAS PubMed PubMed Central Google Scholar
Kumamoto, Y., Hirai, T., Wong, P. W., Kaplan, D. H. & Iwasaki, A. CD301b+ dendritic cells suppress T follicular helper cells and antibody responses to protein antigens. Elife 5, e17979 (2016).
Tatsumi, N., Codrington, A. L., El-Fenej, J., Phondge, V. & Kumamoto, Y. Effective CD4 T cell priming requires repertoire scanning by CD301b(+) migratory cDC2 cells upon lymph node entry. Sci. Immunol. 6, eabg0336 (2021).
Article CAS PubMed PubMed Central Google Scholar
Barnden, M. J., Allison, J., Heath, W. R. & Carbone, F. R. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40 (1998).
Article CAS PubMed Google Scholar
Bursch, L. S. et al. Identification of a novel population of Langerin+ dendritic cells. J. Exp. Med. 204, 3147–3156 (2007).
Article CAS PubMed PubMed Central MATH Google Scholar
Ginhoux, F. et al. Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state. J. Exp. Med. 204, 3133–3146 (2007).
Article CAS PubMed PubMed Central MATH Google Scholar
Poulin, L. F. et al. The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells. J. Exp. Med. 204, 3119–3131 (2007).
Article CAS PubMed PubMed Central MATH Google Scholar
Kissenpfennig, A. et al. Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity 22, 643–654 (2005).
Article CAS PubMed MATH Google Scholar
Moran, A. E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).
Article CAS PubMed PubMed Central MATH Google Scholar
Osum, K. C. & Jenkins, M. K. Toward a general model of CD4(+) T cell subset specification and memory cell formation. Immunity 56, 475–484 (2023).
Article CAS PubMed PubMed Central MATH Google Scholar
Spolski, R., Li, P. & Leonard, W. J. Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat. Rev. Immunol. 18, 648–659 (2018).
Article CAS PubMed MATH Google Scholar
Hwang, E. S., White, I. A. & Ho, I. C. An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines. Proc. Natl Acad. Sci. USA 99, 13026–13030 (2002).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Yamane, H., Zhu, J. & Paul, W. E. Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment. J. Exp. Med. 202, 793–804 (2005).
Article CAS PubMed PubMed Central Google Scholar
Liao, W., Lin, J. X., Wang, L., Li, P. & Leonard, W. J. Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages. Nat. Immunol. 12, 551–559 (2011).
Article CAS PubMed PubMed Central Google Scholar
Van Parijs, L. et al. Functional responses and apoptosis of CD25 (IL-2R alpha)-deficient T cells expressing a transgenic antigen receptor. J. Immunol. 158, 3738–3745 (1997).
Article PubMed MATH Google Scholar
Hondowicz, B. D. et al. Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma. Immunity 44, 155–166 (2016).
Article CAS PubMed Google Scholar
Le Saout, C. et al. IL-2 mediates CD4+ T cell help in the breakdown of memory-like CD8+ T cell tolerance under lymphopenic conditions. PLoS ONE 5, e12659 (2010).
Article ADS PubMed PubMed Central Google Scholar
Setoguchi, R., Hori, S., Takahashi, T. & Sakaguchi, S. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 201, 723–735 (2005).
Article CAS PubMed PubMed Central MATH Google Scholar
Boyman, O., Kovar, M., Rubinstein, M. P., Surh, C. D. & Sprent, J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311, 1924–1927 (2006).
Article ADS CAS PubMed Google Scholar
Gratz, I. K. et al. Cutting Edge: memory regulatory t cells require IL-7 and not IL-2 for their maintenance in peripheral tissues. J. Immunol. 190, 4483–4487 (2013).
Article CAS PubMed MATH Google Scholar
Villarino, A. V. et al. Helper T cell IL-2 production is limited by negative feedback and STAT-dependent cytokine signals. J. Exp. Med. 204, 65–71 (2007).
Article CAS PubMed PubMed Central MATH Google Scholar
Liu, Z. et al. Immune homeostasis enforced by co-localized effector and regulatory T cells. Nature 528, 225–230 (2015).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Wong, H. S. et al. A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells. Cell 184, 3981–3997.e3922 (2021).
Article CAS PubMed PubMed Central MATH Google Scholar
Pimentel-Muinos, F. X., Munoz-Fernandez, M. A. & Fresno, M. Control of T lymphocyte activation and IL-2 receptor expression by endogenously secreted lymphokines. J. Immunol. 152, 5714–5722 (1994).
Article CAS PubMed MATH Google Scholar
Waysbort, N., Russ, D., Chain, B. M. & Friedman, N. Coupled IL-2-dependent extracellular feedbacks govern two distinct consecutive phases of CD4 T cell activation. J. Immunol. 191, 5822–5830 (2013).
Article CAS PubMed MATH Google Scholar
Busse, D. et al. Competing feedback loops shape IL-2 signaling between helper and regulatory T lymphocytes in cellular microenvironments. Proc. Natl Acad. Sci. USA 107, 3058–3063 (2010).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Feinerman, O. et al. Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response. Mol. Syst. Biol. 6, 437 (2010).
Article CAS PubMed PubMed Central MATH Google Scholar
Granucci, F. et al. Inducible IL-2 production by dendritic cells revealed by global gene expression analysis. Nat. Immunol. 2, 882–888 (2001).
Article CAS PubMed MATH Google Scholar
Granucci, F., Feau, S., Angeli, V., Trottein, F. & Ricciardi-Castagnoli, P. Early IL-2 production by mouse dendritic cells is the result of microbial-induced priming. J. Immunol. 170, 5075–5081 (2003).
Article CAS PubMed Google Scholar
Mencarelli, A. et al. Calcineurin-mediated IL-2 production by CD11c(high)MHCII(+) myeloid cells is crucial for intestinal immune homeostasis. Nat. Commun. 9, 1102 (2018).
Article ADS PubMed PubMed Central MATH Google Scholar
Wuest, S. C. et al. A role for interleukin-2 trans-presentation in dendritic cell-mediated T cell activation in humans, as revealed by daclizumab therapy. Nat. Med. 17, 604–609 (2011).
Article CAS PubMed PubMed Central MATH Google Scholar
Zelante, T. et al. CD103(+) dendritic cells control Th17 cell function in the lung. Cell Rep. 12, 1789–1801 (2015).
Article CAS PubMed MATH Google Scholar
Popmihajlov, Z., Xu, D., Morgan, H., Milligan, Z. & Smith, K. A. Conditional IL-2 gene deletion: consequences for T cell proliferation. Front. Immunol. 3, 102 (2012).
Article CAS PubMed PubMed Central Google Scholar
Leon, B., Bradley, J. E., Lund, F. E., Randall, T. D. & Ballesteros-Tato, A. FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability. Nat. Commun. 5, 3495 (2014).
Article ADS PubMed Google Scholar
Kim, M., Kim, T. J., Kim, H. M., Doh, J. & Lee, K. M. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster. Sci. Rep. 7, 40623 (2017).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Li, J., Lu, E., Yi, T. & Cyster, J. G. EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells. Nature 533, 110–114 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Jones, M. C. et al. CD4 effector TCR avidity for peptide on APC determines the level of memory generated. J. Immunol. 210, 1950–1961 (2023).
Article CAS PubMed MATH Google Scholar
Fan, M. Y. et al. Differential roles of IL-2 signaling in developing versus mature Tregs. Cell Rep. 25, 1204–1213.e1204 (2018).
Article CAS PubMed PubMed Central MATH Google Scholar
Toomer, K. H. et al. Essential and non-overlapping IL-2Ralpha-dependent processes for thymic development and peripheral homeostasis of regulatory T cells. Nat. Commun. 10, 1037 (2019).
Article ADS PubMed PubMed Central MATH Google Scholar
Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865–868 (2017).
Article CAS PubMed PubMed Central MATH Google Scholar
Kramer, A., Green, J., Pollard, J. Jr. & Tugendreich, S. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30, 523–530 (2014).
Article PubMed Google Scholar
Bennett, S. R. et al. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393, 478–480 (1998).
Article ADS CAS PubMed Google Scholar
Rolink, A., Melchers, F. & Andersson, J. The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching. Immunity 5, 319–330 (1996).
Article CAS PubMed Google Scholar
Schoenberger, S. P., Toes, R. E., van der Voort, E. I., Offringa, R. & Melief, C. J. T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 393, 480–483 (1998).
Article ADS CAS PubMed Google Scholar
Jenkins, S. J., Perona-Wright, G. & MacDonald, A. S. Full development of Th2 immunity requires both innate and adaptive sources of CD154. J. Immunol. 180, 8083–8092 (2008).
Article CAS PubMed Google Scholar
MacDonald, A. S., Straw, A. D., Dalton, N. M. & Pearce, E. J. Cutting edge: Th2 response induction by dendritic cells: a role for CD40. J. Immunol. 168, 537–540 (2002).
Article CAS PubMed Google Scholar
Mohrs, M., Shinkai, K., Mohrs, K. & Locksley, R. M. Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity 15, 303–311 (2001).
Article CAS PubMed Google Scholar
Allenspach, E. J., Lemos, M. P., Porrett, P. M., Turka, L. A. & Laufer, T. M. Migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive CD4 T cells. Immunity 29, 795–806 (2008).
Article CAS PubMed PubMed Central Google Scholar
Itano, A. A. et al. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19, 47–57 (2003).
Article CAS PubMed MATH Google Scholar
Foy, T. M. et al. In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J. Exp. Med. 178, 1567–1575 (1993).
Article CAS PubMed MATH Google Scholar
Noelle, R. J. et al. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Natl Acad. Sci. USA 89, 6550–6554 (1992).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Poholek, A. C. et al. In vivo regulation of Bcl6 and T follicular helper cell development. J. Immunol. 185, 313–326 (2010).
Article CAS PubMed Google Scholar
Ballesteros-Tato, A. et al. Interleukin-2 inhibits germinal center formation by limiting T follicular helper cell differentiation. Immunity 36, 847–856 (2012).
Article CAS PubMed PubMed Central Google Scholar
Johnston, R. J., Choi, Y. S., Diamond, J. A., Yang, J. A. & Crotty, S. STAT5 is a potent negative regulator of TFH cell differentiation. J. Exp. Med. 209, 243–250 (2012).
Article CAS PubMed PubMed Central Google Scholar
Ray, J. P. et al. The interleukin-2-mTORc1 kinase axis defines the signaling, differentiation, and metabolism of T helper 1 and follicular B helper T cells. Immunity 43, 690–702 (2015).
Article CAS PubMed PubMed Central MATH Google Scholar
DiToro, D. et al. Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells. Science 361, eaao2933 (2018).
Choi, J. & Crotty, S. Bcl6-mediated transcriptional regulation of follicular helper T cells (T(FH)). Trends Immunol. 42, 336–349 (2021).
Article CAS PubMed PubMed Central Google Scholar
Walker, J. A. & McKenzie, A. N. J. T(H)2 cell development and function. Nat. Rev. Immunol. 18, 121–133 (2018).
Article CAS PubMed MATH Google Scholar
van Panhuys, N. et al. In vivo studies fail to reveal a role for IL-4 or STAT6 signaling in Th2 lymphocyte differentiation. Proc. Natl Acad. Sci. USA 105, 12423–12428 (2008).
Article ADS PubMed PubMed Central Google Scholar
Gillis, S., Ferm, M. M., Ou, W. & Smith, K. A. T cell growth factor: parameters of production and a quantitative microassay for activity. J. Immunol. 120, 2027–2032 (1978).
Article CAS PubMed Google Scholar
Morgan, D. A., Ruscetti, F. W. & Gallo, R. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 193, 1007–1008 (1976).
Article ADS CAS PubMed MATH Google Scholar
Laurence, A. et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 26, 371–381 (2007).
Article CAS PubMed Google Scholar
Yang, X. P. et al. Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5. Nat. Immunol. 12, 247–254 (2011).
Article CAS PubMed PubMed Central MATH Google Scholar
Cote-Sierra, J. et al. Interleukin 2 plays a central role in Th2 differentiation. Proc. Natl Acad. Sci. USA 101, 3880–3885 (2004).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Kagami, S. et al. Stat5a regulates T helper cell differentiation by several distinct mechanisms. Blood 97, 2358–2365 (2001).
Article CAS PubMed MATH Google Scholar
Liao, W. et al. Priming for T helper type 2 differentiation by interleukin 2-mediated induction of interleukin 4 receptor alpha-chain expression. Nat. Immunol. 9, 1288–1296 (2008).
Article CAS PubMed PubMed Central MATH Google Scholar
Zhu, J., Cote-Sierra, J., Guo, L. & Paul, W. E. Stat5 activation plays a critical role in Th2 differentiation. Immunity 19, 739–748 (2003).
Article CAS PubMed Google Scholar
Zheng, W. & Flavell, R. A. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–596 (1997).
Article CAS PubMed MATH Google Scholar
Zhu, J. et al. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat. Immunol. 5, 1157–1165 (2004).
Article CAS PubMed MATH Google Scholar
Shi, M., Lin, T. H., Appell, K. C. & Berg, L. J. Janus-kinase-3-dependent signals induce chromatin remodeling at the Ifng locus during T helper 1 cell differentiation. Immunity 28, 763–773 (2008).
Article CAS PubMed PubMed Central Google Scholar
Hondowicz, B. D., Kim, K. S., Ruterbusch, M. J., Keitany, G. J. & Pepper, M. IL-2 is required for the generation of viral-specific CD4(+) Th1 tissue-resident memory cells and B cells are essential for maintenance in the lung. Eur. J. Immunol. 48, 80–86 (2018).
Article CAS PubMed Google Scholar
Pepper, M., Pagan, A. J., Igyarto, B. Z., Taylor, J. J. & Jenkins, M. K. Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells. Immunity 35, 583–595 (2011).
Article CAS PubMed PubMed Central Google Scholar
Bachus, H. et al. IL-6 prevents Th2 cell polarization by promoting SOCS3-dependent suppression of IL-2 signaling. Cell. Mol. Immunol. 20, 651–665 (2023).
Article CAS PubMed PubMed Central MATH Google Scholar
Ashour, D. et al. IL-12 from endogenous cDC1, and not vaccine DC, is required for Th1 induction. JCI Insight 5, e135143 (2020).
Harpur, C. M. et al. Classical type 1 dendritic cells dominate priming of Th1 responses to herpes simplex virus type 1 skin infection. J. Immunol. 202, 653–663 (2019).
Article CAS PubMed Google Scholar
Tatsumi, N. & Kumamoto, Y. Role of mouse dendritic cell subsets in priming naive CD4 T cells. Curr. Opin. Immunol. 83, 102352 (2023).
Article CAS PubMed PubMed Central MATH Google Scholar
Even, Z. et al. The amalgam of naive CD4(+) T cell transcriptional states is reconfigured by helminth infection to dampen the amplitude of the immune response. Immunity 57, 1893–1907.e1896 (2024).
Article CAS PubMed MATH Google Scholar

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Acknowledgements

We thank Kendall A. Smith for sharing the Il2^flox/flox mice. We thank Weizhou Zhang and Akiko Iwasaki as the original breeders of the Il2^flox/flox mice and Mgl2-Cre and Mgl2-DTR mice, respectively. We thank George Yap for critical reading of the manuscript. We thank the Genomics Center, Comparative Medicine Resources, and the Flow Cytometry Core at Rutgers New Jersey Medical School for technical assistance. This work was supported by NIH grants R01AI132576, R01AI165622, and R21CA259541 to Y.K.

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Center for Immunity and Inflammation, Rutgers New Jersey Medical School, Newark, NJ, USA
Naoya Tatsumi, Jihad El-Fenej, Alejandro Davila-Pagan & Yosuke Kumamoto
Department of Pathology, Immunology and Laboratory Medicine, Rutgers New Jersey Medical School, Newark, NJ, USA
Naoya Tatsumi, Jihad El-Fenej, Alejandro Davila-Pagan & Yosuke Kumamoto

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Naoya Tatsumi
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Jihad El-Fenej
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Alejandro Davila-Pagan
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Yosuke Kumamoto
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N.T. and Y.K. designed experiments. N.T. performed most of the experiments with help from J.E.-F. and A.D.-P. J.E.-F. and Y.K. performed the CITEseq experiments and analyzed the data with assistance from the Flow Cytometry Core and the Genomics Center at Rutgers New Jersey Medical School. A.D.-P. performed the Nb infection experiments and analyzed the data. N.T., J.E.-F., and Y.K. performed statistical and computational analyses and interpreted the data. Y.K. conceptualized and supervised the study and acquired funding. N.T. and Y.K. wrote the paper.

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Correspondence to Yosuke Kumamoto.

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Tatsumi, N., El-Fenej, J., Davila-Pagan, A. et al. CD301b⁺ dendritic cell-derived IL-2 dictates CD4⁺ T helper cell differentiation. Nat Commun 16, 2002 (2025). https://doi.org/10.1038/s41467-025-55916-9

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Received: 17 September 2023
Accepted: 31 December 2024
Published: 26 February 2025
Version of record: 26 February 2025
DOI: https://doi.org/10.1038/s41467-025-55916-9
Springer Nature Limited

CD301b+ dendritic cell-derived IL-2 dictates CD4+ T helper cell differentiation

Abstract

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Introduction

Results

Direct antigen presentation by CD301b+ DCs is required for Th2 cell differentiation

CD301b+ DCs are required for inducing full CD25 expression in CD4+ T cells

Th2 cell differentiation relies more stringently on IL-2R signaling than Th1 cells

CD301b+ DC-derived IL-2 is required for the full CD25 upregulation and Th2 differentiation in antigen-specific CD4+ T cells

CD301b+ DC-derived CD25 is required for Th2 cell differentiation

CD40 stimulation induces IL-2 production specifically by CD301b+ DCs

CD301b+ DC-intrinsic CD40 is required for Th2 cell differentiation

CD301b+ DCs are a sufficient DC subset for Th2 cell fate instruction

CD301b+ DCs instruct Th2 cell fate on CD4+ T cells through the DC-intrinsic CD40–IL-2 axis

CD301b+ DC-intrinsic CD40–IL-2 axis skews CD4+ T cells toward non-Tfh effector fate

Discussion

Methods

Mice

BM reconstitution

Immunization, Nb infection and in vivo treatment

T cell priming assays

Cell preparations and flow cytometry

CITEseq data acquisition and analysis

Statistical analysis

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

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CD301b⁺ dendritic cell-derived IL-2 dictates CD4⁺ T helper cell differentiation

Direct antigen presentation by CD301b⁺ DCs is required for Th2 cell differentiation

CD301b⁺ DCs are required for inducing full CD25 expression in CD4⁺ T cells

CD301b⁺ DC-derived IL-2 is required for the full CD25 upregulation and Th2 differentiation in antigen-specific CD4⁺ T cells

CD301b⁺ DC-derived CD25 is required for Th2 cell differentiation

CD40 stimulation induces IL-2 production specifically by CD301b⁺ DCs

CD301b⁺ DC-intrinsic CD40 is required for Th2 cell differentiation

CD301b⁺ DCs are a sufficient DC subset for Th2 cell fate instruction

CD301b⁺ DCs instruct Th2 cell fate on CD4⁺ T cells through the DC-intrinsic CD40–IL-2 axis

CD301b⁺ DC-intrinsic CD40–IL-2 axis skews CD4⁺ T cells toward non-Tfh effector fate