Skin thymic stromal lymphopoietin initiates Th2 responses through an orchestrated immune cascade

Leyva-Castillo, Juan Manuel; Hener, Pierre; Michea, Paula; Karasuyama, Hajime; Chan, Susan; Soumelis, Vassili; Li, Mei

doi:10.1038/ncomms3847

Article
Published: 28 November 2013

Skin thymic stromal lymphopoietin initiates Th2 responses through an orchestrated immune cascade

Juan Manuel Leyva-Castillo¹,
Pierre Hener¹,
Paula Michea^2,3,
Hajime Karasuyama^4,5,
Susan Chan¹,
Vassili Soumelis^2,3 &
…
Mei Li^1,6,7

Nature Communications volume 4, Article number: 2847 (2013) Cite this article

7259 Accesses
130 Citations
1 Altmetric
Metrics details

Subjects

Abstract

Thymic stromal lymphopoietin (TSLP) has emerged as a key initiator in Th2 immune responses, but the TSLP-driven immune cascade leading to Th2 initiation remains to be delineated. Here, by dissecting the cellular network triggered by mouse skin TSLP in vivo, we uncover that TSLP-promoted IL-4 induction in CD4⁺ T cells in skin-draining lymph nodes is driven by an orchestrated ‘DC-T-Baso-T’ cascade, which represents a sequential cooperation of dendritic cells (DCs), CD4⁺ T cells and basophils. Moreover, we reveal that TSLP-activated DCs prime naive CD4⁺ T cells to produce IL-3 via OX40L signalling and demonstrate that the OX40L-IL-3 axis has a critical role in mediating basophil recruitment, CD4⁺ T-cell expansion and Th2 priming. These findings thus add novel insights into the cellular network and signal axis underlying the initiation of Th2 immune responses.

You have full access to this article via your institution.

Download PDF

Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity

Article 27 March 2024

Jason B. Ross, Lara M. Myers, … Irving L. Weissman

Immune microniches shape intestinal Treg function

Article Open access 03 April 2024

Yisu Gu, Raquel Bartolomé-Casado, … Fiona Powrie

Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence

Article Open access 10 April 2024

Carlene L. Zindl, C. Garrett Wilson, … Casey T. Weaver

Introduction

Thymic stromal lymphopoietin (TSLP) is a short-chain four α-helical bundle type I cytokine, which is expressed mainly by epithelial cells in the skin, lung and intestine¹. Studies in recent years have implicated TSLP as a master regulator of Th2 inflammatory responses in allergic diseases^2,3. A link between TSLP and human atopic dermatitis (AD) was initially suggested following observations showing an increased expression of TSLP in the epidermis of AD skin, as well as that human TSLP-conditioned dendritic cells (DCs) induced Th2 development⁴. Using mouse models, our previous studies⁵ and those of others⁶ showed that induced expression of TSLP in mouse skin keratinocytes (KCs) triggers an AD-like syndrome, characterized by infiltration of CD4⁺ T cells and eosinophils, increased levels of Th2 cytokines and elevated serum IgE levels. Our studies further revealed that TSLP expression was regulated by nuclear receptors (that is, retinoid X receptors, retinoid acid receptors and vitamin D receptors) and found that topical treatment of mouse skin with the vitamin D3 low-calcemic analogue MC903 (known also as calcipotriol) induced TSLP expression in epidermal KCs, which subsequently generated an AD syndrome^7,8. Furthermore, transgenic mice overexpressing TSLP in lung epithelial cells⁹ were shown to develop a spontaneous asthmatic lung inflammation, and intradermal¹⁰ or intranasal¹¹ delivery of TSLP induced tissue inflammation and Th2 cytokine production.

However, although these studies established that TSLP was a key initiator of Th2 inflammatory responses, the underlying cellular and molecular mechanism remained still elusive. Numerous data (see reviews^3,12 and references therein) suggested that TSLP may act on various immune cells including DCs, T cells, NK cells, NKT cells, B cells, mast cells, eosinophils, basophils, as well as innate lymphoid cells¹³, but these data were based either on in vitro TSLP stimulation of a particular cell type or at a particular snapshot time of analyses, and therefore did not provide information on the immune cascade driven by TSLP within the tissue microenvironment. To achieve a better mechanistic understanding of TSLP-triggered immune responses, it is crucial to delineate the nature of key cellular factors and how they sequentially act and cross-talk in an in vivo context to initiate Th2 responses.

Taking advantage of our established mouse experimental model to induce TSLP expression in epidermal KCs by topical treatment with MC903 (refs 7, 8), we dissect here the immune cascade leading to Th2 initiation. Particularly, we focus on the initiation of IL-4 (the master regulator that promotes Th2 differentiation¹⁴) in CD4⁺ T cells of skin-draining lymph nodes (LNs), where the T-cell priming could take place. We reveal that TSLP drives a ‘DC-T-Baso-T’ cellular cascade representing a sequential and orchestrated action of DCs, CD4⁺ T cells and basophils, which subsequently leads to IL-4 priming in CD4⁺ T cells. Importantly, we uncover that TSLP-activated DCs, through OX40L signalling, prime naive CD4 T cells to produce IL-3 and identify the critical role of TSLP (KCs)-OX40L (DCs)-IL-3 (CD4 T cells) axis in mediating basophil recruitment, CD4⁺ T-cell expansion and Th2 priming.

Results

Skin TSLP induces IL-4 priming in CD4⁺ T cells

TSLP expression was induced in skin keratinocytes of wild-type (WT) Balb/c mice via MC903 (but not vehicle ETOH) treatment on ears every other day starting from day (D) 0 (Fig. 1a). As reported^7,8, the TSLP expression was increased in treated ears (Fig. 1b, left panel) but not in the ear-draining lymph nodes (EDLN) (Fig. 1b, right panel). To investigate TSLP-triggered Th2 priming, we first analysed IL-4 expression in EDLN. MC903-induced IL-4 expression became evident at D5, which was barely detected at D3 (Fig. 1c). In contrast, there was no difference in IFNγ or IL-17 expression in EDLN of MC903- or ETOH-treated mice at D5 (Supplementary Fig. S1), suggesting that MC903 selectively induces Th2 differentiation. Importantly, the IL-4 induction by MC903 was abolished in Tslp^−/− mutant mice (Fig. 1d), demonstrating that TSLP was required for IL-4 expression in draining LNs.

**Figure 1: Skin topical MC903 treatment induces TSLP-dependent Th2 priming in CD4⁺ T cells in the skin-draining lymph nodes.**

We next investigated whether TSLP-triggered IL-4 expression occurs in CD4⁺ T cells. Upon MC903 treatment, the total CD4⁺ T-cell number increased in EDLN of WT but not of Tslp^−/− mice (Fig. 1e), and IL-4 expression was found to be induced in purified CD4⁺ T cells from EDLN of MC903-treated mice, when compared with ETOH-treated mice (Fig. 1f). Accordingly, TSLP-induced IL-4 expression was completely abolished in Rag1^−/− mice that lack mature T and B cells (Fig. 1g). Moreover, intracellular staining showed that IL-4⁺ cells were detected in the CD3⁺CD4⁺ population (Fig. 1h), and their frequency was increased in EDLN from MC903-treated mice (Fig. 1h). Together, these results demonstrated that, upon MC903 topical treatment, skin-derived TSLP induced IL-4 expression in CD4⁺ T cells in the draining LNs.

Dendritic cells are essential for the skin TSLP-induced IL-4 priming

DCs are known to have a pivotal role in T-cell-mediated immunity including Th2 responses. Accumulation of DCs in EDLN was first examined at different time points upon MC903 treatment. An increase in CD11c⁺MHCII⁺ cell number in EDLN became apparent at D3 (Fig. 2a) before IL-4 induction could be detected (see Fig. 1c). To determine whether DCs are required for TSLP-triggered Th2 initiation, we made use of CD11c-DTR transgenic mice¹⁵ in which CD11c^hi DCs can be depleted by the administration of diphtheria toxin (DT). CD11c-DTR^Tg/0 mice and their control littermates (CD11c-DTR^0/0) received an intraperitoneal (i.p.) injection of DT at D1 (thereafter called CD11c^DEP mice and CD11c^CT mice, respectively). When analysed one day later (that is, D0), a significant reduction of the CD11c^hiMHCII⁺ cells was observed in both ear dermis and EDLN of CD11c^DEP mice (Fig. 2b). These mice were then treated with ETOH or MC903 at D0, D2 and D4 as indicated in Fig. 1a. Depletion of DCs did not affect the expression levels of skin TSLP induced by MC903 (Fig. 2c). However, IL-4 induction in EDLN (Fig. 2d), as well as in purified CD4⁺ T cells from EDLN (Fig. 2e), was abolished in MC903-treated CD11c^DEP mice, indicating an essential role of DCs in TSLP-triggered IL-4 priming. Furthermore, we found that the number of CD4⁺ T cells increased in MC903-treated CD11c^CT mice at D5 (as compared with ETOH treatment) but not in CD11c^DEP mice (Fig. 2f).

**Figure 2: CD11c^hi dendritic cells are crucial for skin TSLP-promoted IL-4 priming.**

We also examined whether epidermal Langerhans cells (LCs) could be implicated in TSLP-induced IL-4 priming. LCs were not depleted in the above CD11c^DEP mice¹⁶. To deplete langerin⁺ cells including epidermal LCs, we injected DT to Lang-DTREGFP^+/− mice¹⁷ (called thereafter Lang^DEP mice). Interestingly, similar increases in IL-4 expression and CD4⁺ T-cell number in EDLN were observed in MC903-treated Lang^DEP and Lang^CT (DT-injected Lang-DTREGFP^+/+ control littermates) (Fig. 2g,h), indicating that LCs were not implicated in TSLP-driven IL-4 induction in CD4⁺ T cells at D5.

Basophils are required for TSLP-triggered IL-4 priming

The role of basophils in Th2 cell differentiation in different models of allergy remains highly controversial (see reviews^18,19 and refs therein). To investigate their contribution in TSLP-induced Th2 priming, we first examined whether basophils were recruited into EDLN upon MC903 skin treatment. Flow cytometry analyses showed that basophils (identified as CD3⁻B220⁻CD49b⁺CD45^intFcεRIα⁺CD117⁻Gr1⁻)²⁰ were barely detected in EDLN at D0, whereas their number was increased at D5 upon MC903 treatment (Fig. 3a). Kinetic analyses showed that such an increase started at D3 (Fig. 3b). MC903-induced basophil recruitment in EDLN was driven by TSLP, as it was completely abolished in Tslp^−/− mice (Fig. 3c).

**Figure 3: Basophils are required for skin TSLP-promoted IL-4 priming in CD4⁺ T cells.**

We next depleted basophils in WT mice via i.p. injection of an antibody (Ab) specific to FcεRIα (MAR-1)^21,22, before MC903 treatment. Depletion of basophils was confirmed at D5 in EDLN from MAR-1-injected mice (Fig. 3d). Of note, the number of DCs at D5 was similarly increased in both isotype and MAR-1 Ab-injected mice upon MC903 treatment (Fig. 3e). In addition, the increase in CD4⁺ T-cell number was similar in EDLN from MAR-1- or isotype-injected mice (Fig. 3f), suggesting that, in contrast to DC depletion (Fig. 2f), basophil depletion did not affect CD4⁺ T-cell expansion. However, TSLP-triggered IL-4 expression in CD4⁺ T cells in EDLN was clearly decreased in MAR-1-injected mice (Fig. 3g,h). This was also confirmed by IL-4 intracellular staining (Fig. 3i).

As recent studies have raised concerns about possible deleterious effects of MAR-1 on other cells than basophils¹⁸, such as mast cells²¹ or subset of DCs²³, particularly when high doses of MAR-1 were administrated²³, we further performed experiments using two other depletion strategies, that is, Ba103 (antibody specific to CD200R3)-mediated basophil depletion²⁴ (Supplementary Fig. S2a) and DT-mediated basophil depletion in Mcpt8^DTR mice²² (Fig. 3j). Both of these basophil depletions led to a significantly diminished IL-4 induction in CD4⁺ T cells upon MC903 treatment (Fig. 3k and Supplementary Fig. S2c,d), while the increase in CD4⁺ T-cell number was not altered by basophil depletion (Fig. 3l and Supplementary Fig. S2b). Thus, we concluded that basophils have an important role in priming CD4⁺ T cells to express IL-4, whereas they are dispensable for the expansion of CD4⁺ T cells.

DCs and CD4⁺ T cells are required for basophil recruitment

We then explored the cellular mechanism through which basophils are recruited into EDLN. Upon the depletion of DCs in CD11c^DEP mice, TSLP-triggered basophil recruitment was abolished, as shown by decreases in both the frequency and total number of basophils (Fig. 4a), indicating that DCs were crucial for basophil recruitment into EDLN.

**Figure 4: DCs and CD4⁺ T cells are required for TSLP-promoted basophil recruitment in skin-draining lymph nodes.**

As DC depletion abolished CD4⁺ T-cell expansion (Fig. 2f), we next examined whether CD4⁺ T cells may have a role in recruiting basophils in EDLN. Upon MC903 treatment, basophil recruitment was abolished in Rag1^−/− mice but could be restored in these mice when reconstituted with WT CD4⁺ T cells (Fig. 4b). Together, these results indicated that TSLP-triggered basophil recruitment into the EDLN required both DCs and CD4⁺ T cells.

TSLP-activated DCs trigger IL-3 expression by CD4⁺ T cells

On the basis of the above results, we posited that the basophil recruitment could be mediated by factor(s) derived from DC-primed CD4⁺ T cells. As IL-3 derived from CD4⁺ T cells has been recently implicated in basophil recruitment to LNs in a helminth infection mouse model²⁵, we examined whether IL-3 could be involved in TSLP-triggered basophil recruitment in EDLN. We first performed kinetic analyses of IL-3 expression upon MC903 treatment and found that IL-3 mRNAs were not detected at D0 or D1 but started to increase from D3 (Fig. 5a), which correlated well with basophil recruitment in EDLN (see Fig. 3b). Interestingly, the IL-3 increase was detected earlier (at D3) than that of IL-4 (at D5) (compare Figs 1c and 5a), indicating that IL-3 induction could precede IL-4 priming.

**Figure 5: TSLP-activated DCs induce IL-3 expression in CD4⁺ T cells.**

Next, we showed that MC903-induced IL-3 expression was abolished in Tslp^−/− mice (Fig. 5b). The induction of IL-3 was abrogated in MC903-treated Rag1^−/− mice, but adoptive transfer of WT CD4⁺ T cells fully restored IL-3 expression (Fig. 5c). In addition, intracellular staining showed that the frequency of IL-3⁺CD4⁺ T cells was increased in EDLN upon MC903 treatment (Fig. 5d). These results demonstrated that TSLP triggers the IL-3 expression by CD4⁺ T cells. Furthermore, upon DC depletion, TSLP-triggered IL-3 expression in EDLN was totally abolished; in contrast, it was not affected by basophil depletion, thus indicating that, in TSLP-driven immune cascade, IL-3 expression by CD4⁺ T cells is downstream of DCs but not of basophils (Fig. 5e).

We further sought to verify that TSLP-stimulated DCs trigger an early IL-3 expression in CD4⁺ T cells, by employing an in vitro human DC-naïve CD4⁺ T cell co-culture system^4,26. Previous studies using this system have shown that DCs activated by TSLP could prime allogenic naive CD4⁺ T cells to produce more IL-4 after 6 or 7 days of co-culture^4,26. By performing kinetic analyses, we found with interest that, upon the priming with TSLP-DC, naive CD4⁺ T cells produced an increased amount of IL-3 as early as after 1 day of co-culture (Fig. 5f, left panel; compare TSLP-DC with medium(med)-conditioned DC); in contrast, the increase in IL-4 production began to be seen only after 5 days of co-culture (Fig. 5f, right panel). Such a kinetic fits with the timing observed in vivo in our mouse model, both of which indicate that TSLP-activated DCs prime CD4⁺ T cells to produce IL-3 at a stage that precedes the initiation of IL-4 expression.

IL-3 is crucial for basophil recruitment and IL-4 induction

What could be the possible role of IL-3 in TSLP-triggered Th2 initiation process? We then applied MC903 topical treatment on Il3^−/− mice and found that these mice exhibited a decrease in basophil recruitment in EDLN, compared with MC903-treated WT mice (Fig. 6a). In addition, there was lesser increase in CD4⁺ T-cell number in EDLN (Fig. 6b) These data indicated that IL-3 had an important role in both basophil recruitment and CD4⁺ T-cell expansion in skin-draining LNs. RT–PCR analyses showed that TSLP-driven IL-4 expression in EDLN at D5 was significantly reduced in Il3^−/− mice (Fig. 6c). Moreover, intracellular staining showed a lower frequency of IL-4⁺CD4⁺ T cells in MC903-treated Il3^−/− mice (Fig. 6d), indicating that IL-3 was required for TSLP-driven IL-4 induction in CD4⁺ T cells.

**Figure 6: IL-3 produced by CD4⁺ T cells is required for basophil recruitment and for IL-4 priming in CD4⁺ T cells.**

Furthermore, upon MC903 treatment, Rag1^−/− mice reconstituted with Il3^−/− CD4⁺ T cells showed a decrease in basophil recruitment (Fig. 6e), CD4⁺ T-cell number (Fig. 6f), as well as IL-4 expression (Fig. 6g), when compared with Rag1^−/− mice reconstituted of WT CD4⁺ T cells. These results thus demonstrated that IL-3 produced by CD4⁺ T cells is crucial for recruiting basophils into skin-draining LNs and for inducing IL-4 expression in CD4⁺ T cells.

The OX40L-OX40 signalling promotes IL-3 expression

We then explored which molecules derived from TSLP-targeted DCs could promote IL-3 expression by CD4⁺ T cells. Human TSLP-activated DCs are known to express OX40L, which promotes inflammatory Th2 cell responses²⁶. Indeed, we found that DCs purified from EDLN of MC903-treated WT mice at D3 exhibited a significant induction of OX40L expression (Fig. 7a), together with an increase in the number of CD4⁺ T cells expressing OX40 (the receptor of OX40L) (Supplementary Fig. S3). Expression of the costimulatory molecules CD80 and CD86 also exhibited a tendency to increase, whereas that of CD40, IL-12p35, IL-12/IL-23p40 and IL-23p19 remained unchanged (Fig. 7a).

**Figure 7: A functional axis of OX40L IL-3 in TSLP-promoted Th2 priming.**

To investigate whether OX40L(DCs)-OX40 (T cells) signalling could mediate IL-3 production in CD4⁺ T cells, WT mice were i.p. injected with neutralizing anti-OX40L Ab (RM134L)²⁷ at D0 and D2 during the MC903 treatment. Blockade of OX40L in vivo led to a significant decrease of IL-3 expression in EDLN at both D3 and D5 (Fig. 7b); concomitantly, these mice exhibited a significant decrease in basophil recruitment (Fig. 7c), CD4⁺ T-cell expansion (Fig. 7d) and IL-4 priming in CD4⁺ T cells (Fig. 7e,f) at D5.

To further explore the potential direct effect of OX40L-OX40 signalling on IL-3 expression in naive CD4⁺ T cells, we employed an in vitro culture system, in which mouse naive CD4⁺ T cells (CD3⁺CD4⁺CD62L⁺) were cultured in the presence of polyclonal stimulation of anti-CD3 or anti-CD3/anti-CD28. In both cases, activation of the OX40 signal by an agonist anti-OX40 Ab²⁷ induced IL-3 production, at both mRNA and protein levels (Fig. 7g). Taken together, these in vivo and in vitro data demonstrated that TSLP-triggered OX40L-OX40 signalling promoted IL-3 production by naive CD4⁺ T cells.

Discussion

Employing a topical MC903 treatment to induce TSLP expression in mouse skin KCs, we have dissected the cellular network and signal axis underlying how TSLP drives the initiation of Th2 immune responses in vivo. Our data demonstrate that TSLP-promoted IL-4 initiation in CD4⁺ T cells of skin-draining LNs involves an orchestrated ‘DC-T-Baso-T’ cascade. Moreover, our results reveal that TSLP-activated DCs first prime naive CD4⁺ T cells to produce IL-3 through OX40L-OX40 signalling. Subsequently, IL-3 mediates the recruitment of basophils, and basophils in turn promote IL-4 priming in CD4⁺ T cells. Moreover, IL-3 has an additional role in the optimal expansion of CD4⁺ T cells, in which basophils are not implicated. These are schematically presented in Fig. 8 and further discussed below.

**Figure 8: A schematic representation of how skin TSLP initiates Th2 responses through an orchestrated DC-T-Baso-T cascade.**

Although TSLP appears to act on various immune cell types (see reviews^3,12), for example, TSLP could directly promote IL-4 production in mouse CD4⁺ T cells in vitro²⁸, we demonstrate that, in the in vivo context, it is DCs that have a primary role in the initiation of Th2 response driven by skin TSLP (Fig. 8). Indeed, upon DC depletion, IL-3 induction in CD4⁺ T cells, basophil recruitment in skin-draining LNs and IL-4 priming are all abolished. Of interest, we find that CD11c^hi DCs, instead of epidermal LCs, are involved in TSLP-driven Th2 priming. In keeping with our data, a recent study showed that, ablation of Stat5, a transcriptional factor implicated in TSLP signalling, in CD11c^hi DCs but not in LCs, diminished the induction of Th2 contact hypersensitivity²⁹. It remains yet to be determined whether TSLP produced by KCs activates dermal CD11c^hi DCs and triggers their migration to skin-draining LNs, or TSLP entering the draining LNs (for example, through lymphatic vessel) may activate directly the LN CD11c^hi DCs (see Fig. 8). Further studies are also required to investigate which subset(s) of skin or LN DCs³⁰ could be the major factor(s) in TSLP-triggered immune response.

Although being the primary factor, DCs are not sufficient for optimal IL-4 priming in CD4⁺ T cells, as we find that basophils are also crucial. As mentioned before, the role of basophils in Th2 cell differentiation has been highly controversial^{23,25,31,32,33,34,35,36} (see also reviews^18,19). One argument has been on approaches of basophil depletion, particularly the possible effects of MAR-1 on other cells than basophils^21,23. We have utilized three different strategies (that is, MAR-1, Ba103 or DT) to deplete basophils and found that all of them led to the abrogation of IL-4 induction in CD4⁺ T cells, thus unequivocally demonstrating that basophils are crucial for an optimal IL-4 initiation triggered by TSLP. It is notable that, upon a longer treatment of MC903 (until D10), IL-4 (as well as IL-13) level in LNs was significantly lower in basophil-depleted mice compared with WT control, although the induction of these cytokines was not completely abolished (Supplementary Fig. S4). This suggests that, in the absence of basophils, other mechanisms (for example, expansion of CD4⁺ T cells) may possibly contribute to Th2 differentiation (see discussion below). Nevertheless, the controversial reports on the contribution of basophils in Th2 responses in various mouse models may also reflect different requirement of basophils (and TSLP) in different contexts. One major determinant could be the nature of antigen. For example, studies have reported that certain parasite-associated antigens may by pass the requirement of TSLP³⁷, basophils²⁵ and even IL-4 (refs 14, 38) for generating Th2 responses, whereas we have shown that common protein allergen requires TSLP³⁹ for developing Th2 sensitization.

Our data also add further insights into basophil recruitment and function in draining LNs. First, in contrast to previous reports concluding that either DCs³⁵ or CD4⁺ T cells⁴⁰ alone were sufficient to promote basophil recruitment to draining LNs, we reveal that TSLP-triggered basophil recruitment into skin-draining LNs is mediated by DC-primed naive CD4⁺ T cells, and therefore it requires both DCs and CD4⁺ T cells (Fig. 8). Of great importance, our study uncovers for the first time that TSLP-activated DCs prime CD4⁺ T cells to produce IL-3 at a stage that precedes the induction of IL-4, and IL-3 in turn recruits basophils. Second, our results show that, in contrast to DCs, basophils are dispensable for promoting CD4⁺ T-cell expansion. Therefore, although basophils have a role in IL-4 induction in CD4⁺ T cells, they do not seem to behave like the bona fide APCs (antigen presenting cells) to stimulate T-cell proliferation in the draining LNs.

How could basophils contribute to TSLP-triggered IL-4 priming? It is possible that basophils provide an initial source of IL-4, particularly under the IL-3 stimulation^31,41,42,43, and thereby participate in building up a self-reinforcing IL-4 loop in primed CD4⁺ T cells¹⁸. Indeed, we observed that basophils sorted from naive mouse spleens produced higher amount of IL-4 upon the in vitro stimulation of IL-3 (but not of TSLP) (Supplementary Fig. S5). To further address the role of IL-4 from basophils, it will be necessary to generate mice in which IL-4 can be selectively and inducibly ablated in basophils. Of note, although eosinophils or mast cells could also provide an initial source of IL-4 (refs 44, 45), and both have been suggested as direct targets of TSLP (see review³), we found that neither eosinophils nor mast cells contributed to TSLP-driven IL-4 initiation because the depletion of either eosinophils (Δdbl GATA mice⁴⁶) or mast cells (Kit^wsh/wsh mice⁴⁷) did not affect IL-4 induction (Supplementary Fig. S6). Nevertheless, besides IL-4, whether basophils may provide other factor(s) for priming CD4⁺ T cells and whether such a process implicates a direct contact or a close location between basophils and CD4⁺ T cells remains still to be clarified.

Our analyses identify the OX40L-IL-3 axis in the initiation of Th2 response (Fig. 8). Upon TSLP induction in skin, elevated levels of OX40L and OX40 were seen in LN DCs (Fig. 7a) and CD4⁺ T cells (Supplementary Fig. S3), respectively. Indeed, the OX40L-OX40 signalling has been implicated in T-cell-mediated immunity, for example, through regulating the expansion, survival and effector function of T cells²⁷, and has been suggested to have an important role for TSLP-triggered Th2 polarization²⁶ or TSLP-mediated Th2 lung inflammation¹¹. By in vivo OX40L blocking (Fig. 7b-f) or in vitro OX40 activation (Fig. 7g), we demonstrate for the first time that OX40L-OX40 signalling induces IL-3 expression in naive CD4⁺ T cells during the Th2 initiation process, although a possible link between IL-3 expression to OX40 signal has been suggested^48,49. Interestingly, this is in contrast to the cytokine IL-2, as the expression of IL-2 was not induced upon the OX40 activation (Supplementary Fig. S7), which is in agreement with a previous study showing that OX40 signalling promoted IL-2 expression in effector but not in naive T cells⁵⁰.

The role of IL-3 in Th2 initiation is also of interest. IL-3 has been recognized to be a cytokine produced mainly by activated CD4⁺ T cells, for example, in response to parasite infection⁵¹, but little is known about its role in Th differentiation. Importantly, our data indicate that, in TSLP-triggered immune cascade, the induction of IL-3 is prior to IL-4 in CD4⁺ T cells, and, moreover, IL-3 is critical for IL-4 priming. In addition to its role in mediating basophil recruitment to the skin-draining LNs (see above), IL-3 is also implicated in DC-primed CD4⁺ T-cell expansion (Fig.8). Indeed, the ablation of IL-3 leads to a significant reduction in CD4⁺ T-cell expansion (Fig. 6b,f), as observed by the blocking of OX40L (Fig. 7d), or the depletion of DCs (Fig. 2f). These analyses suggest a previously unrecognized function for IL-3 in CD4⁺ T-cell expansion, besides its better-appreciated role in the development, survival and function of mast cells and basophils^25,52,53. How IL-3 has such a role needs to be further explored. Nevertheless, as the expansion of CD4⁺ T cells does not implicate basophils (Fig. 3f,l and Supplementary Fig. S1b), this suggests that, in the absence of basophils, the expansion of CD4⁺ T cells may contribute to the Th2 cell differentiation probably through autocrine production of IL-4 (ref. 14) (see Supplementary Fig. S4).

In keeping with the mouse in vivo data, human DCs activated by TSLP in vitro also prime naive CD4⁺ T cells to produce IL-3, notably at an early stage (Fig. 5f). Although the in vitro co-culture of TSLP-DCs and CD4⁺ T cells leads to the increased production of IL-4 (refs 4, 26), we speculate that, in the in vivo context, the presence of basophils would contribute to achieve an optimal and/or earlier Th2 initiation. We are currently underway to establish the in vitro DC-T-basophil co-culture system to explore this speculation and to examine whether the TSLP-OX40L-IL-3 axis has a similar role in the induction of Th2 immune responses in humans.

In conclusion, our results in the present study characterize a ‘DC-T-Baso-T’ cellular cascade and reveal OX40L-IL-3 as an important axis in mediating the Th2 priming triggered by skin-derived cytokine TSLP. Further questions are raised. First, do these Th2 initiation mechanisms (Fig. 8) stand in the physiological context(s), and, if yes, in which context(s)? Second, are these Th2 initiation mechanisms specific for TSLP or applied as well to other Th2 promoting cytokines like IL-33 or IL-25; moreover, are they specific for skin or applicable also to other interface barriers including airways or gut epithelia? Notably, it has been recently shown that, in addition to TSLP, IL-25 or IL-33 was capable to promote OX40L in DCs in vitro, and blocking the OX40L signalling diminished Th2 lung inflammation or food allergy⁵⁴. It will be important to functionally validate the implication of OX40L-IL-3 axis in the context of allergy and other diseases related to Th2 immunity^45,55, using pathophysiological animal models and in humans, which may potentially lead to the development of new therapeutic or preventive strategies.

Methods

Mice

Balb/c mice and Rag1−^/− mice (on Balb/c genetic background) were purchased from Charles River Laboratories. Il3^−/− mice⁵⁶ were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Tslp^−/− (on Balb/c genetic background)⁷, MCPT8^DTR (on Balb/c genetic background)²², Lang-DTREGFP^+/− (on Balb/c genetic background)¹⁷ and CD11c-DTR^Tg/0 (on C57B6/J genetic background)¹⁵ mice were previously described. Breeding and maintenance of mice were performed under institutional guidelines, and all of the experimental protocols were approved by the animal care and ethic committee of animal experimentation of the Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) and Institut Clinique de la Souris (ICS).

Antibodies and flow cytometry

The following antibodies were used: purified anti-FcγRIII/II (CD32/16; Fc Block; clone 2.4G2, (1:50)), FITC-conjugated anti-Gr1 (clone RB6-8C5, (1:100)), PE-conjugated anti-siglec-F (clone E50-2440, (1:50)), V500-conjugated anti-CD3 (clone 500-A2, (1:25)), PerCP-Cy5.5-conjugated anti-CD19 (clone1D3, (1:25)), biotin-conjugated anti-CD4 (clone GK1.5, (1:50)), PerCP-Cy5.5-conjugated anti-CD8a (clone 53-6.7, (1:25)), APC-conjugated anti-IL-4 (clone 11B11, (1:25)) and biotin-conjugated anti-CD11c (clone HL3, (1:50)) were from BD Biosciences; biotin-conjugated anti-CD49b (clone DX5, (1:50)), Alexa700-conjugated anti-CD8a (clone 53-6.7, (1:25)) and FITC-conjugated MHCII (clone M5/114.15.2, (1:50)) were from eBioscience; APC-Cy7-conjugated anti-CD117 (c-kit; clone 2B8, (1:25)), PE-Cy7-conjugated anti-FCεRIα (clone MAR-1, (1:25)), PE-conjugated anti-IL-3 (clone MP2-8F8, (1:25)) and PerCP-Cy5.5- or PE-Cy7-conjugated anti-CD45R (B220; clone RA3-6B2, (1:50)) were from Biolegend; Qdot-585-conjugated streptavidin (1:50) was from Invitrogen; and PE-conjugated streptavidin (1:100) was from Jackson ImmunoResearch.

For surface marker staining, ear-draining lymph node (EDLN) cells were incubated with an anti-FcγRII/III Ab (2.4G2) on ice for 10 min to block Fc receptors, washed and incubated with a mix of antibodies on ice for 10 min. Finally, cells were washed and analysed.

For intracellular staining, EDLN cells were stimulated for 5 h at 5 × 10⁶ cells per ml in the presence of PMA (50 ng ml⁻¹; Sigma), ionomycin (500 ng ml⁻¹; Sigma) and BD GolgiStop (BD Biosciences). After washing, cells were incubated with anti-FcγRII/III Ab (2.4G2), followed by surface staining for CD3, CD8 and CD4. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-IL-4 or anti-IL-3 antibodies.

Stained cells were analysed on a LSRII flow cytometer (BD Biosciences). Results were analysed using FlowJo (Treestar).

MC903 topical treatment

MC903 (calcipotriol; Leo Pharma, Denmark) was dissolved in ethanol (ETOH) and topically applied on mouse ears (1 nmol in 25 μl per ear). As vehicle control, the same volume of ETOH was applied.

RNA extraction and quantitative RT–PCR

RNA was extracted using TRI Reagent (Sigma-Aldrich) or the RNeasy micro kit (Qiagen). RNA was reverse transcribed using random oligonucleotide hexamers and amplified by quantitative PCR with a Ligthcycler 480 (Roche Diagnostics) and the QuantiTect SYBR Green kit (Qiagen), according to the manufacturer’s instructions. Relative RNA levels were calculated using hypoxanthine phosphoribosyltransferase (HPRT) as internal control. For the analyses of each set of IL-4 or IL-3 expression, an arbitrary unit of 100 was given to the samples with the highest level, and the remaining samples were plotted relative to this value. For the analyses of OX40L, CD80, CD86, CD40, IL-12p35, IL12/23p40, IL-23p19, levels from ETOH-treated samples were set as 100.

Sequences of PCR primers are provided in Table 1 (as reported³⁹).

Table 1 Sequences of PCR primers.

Full size table

Isolation of DCs and CD4⁺ cells from lymph nodes

For DCs isolation, LNs were chopped and digested in 2 mg ml⁻¹ collagenase D (Roche), 2.5% FCS and 100 U ml⁻¹ DNAse I (Roche) in PBS at 37 °C for 30 min and then filtered through a 70-μm cell strainer (BD). Single-cell suspensions were incubated with Fc block (FcγRII/III m Ab 2.4G2) for 10 min, and DCs were enriched using CD11c Microbeads (Miltenyi Biotec). CD11c⁺MHCII⁺ cells were then sorted with FACS Aria II (BD) (purity was>95%).

For CD4⁺ cell isolation, EDLN were chopped and mechanically disrupted and then filtered through a 70-μm cell strainer. CD4⁺ cells were isolated using CD4⁺ T-cell isolation kit II (Miltenyi Biotec) (purity was >95%).

Depletion of DCs or Langerhans cells in mice

For depletion of CD11c^hi DCs, CD11c-DTR^Tg/0 mice¹⁵ were injected i.p. with diphtheria toxin (DT) (100 ng per 25 g body weight) at D -1 (that is, one day before MC903 or ETOH treatment). DT-injected CD11c-DTR^0/0 littermate mice were used as controls. For depletion of Langerhans cells, Lang-DTREGFP^+/− mice¹⁷ received an i.p. injection of DT (1 μg per 25 g body weight) at D -1. The DT-injected wild-type littermate mice were used as controls. These mice were then treated with MC903 or ETOH at D0, D2 and D4, as indicated.

Basophil depletion in mice

For antibody-mediated depletion of basophils, wild-type Balb/c mice were injected i.p. twice daily for 3 days from D -4 (that is, four days before MC903 or ETOH treatment) to D -2 (that is, two days before MC903 or ETOH treatment) with 5 μg of Armenian hamster-anti-mouse FcεRIα (clone MAR-1, eBioscience) or injected i.v. with 30 μg of rat-anti-mouse CD200R3 (clone Ba103, Hycult Biotech) at D -2. For DT-mediated depletion of basophils, MCPT8^DTR mice (in Balb/c genetic background) received an i.p. injection of DT (750 ng per 20 g body weight) at D -2. These mice were then treated with MC903 or ETOH at D0, D2 and D4, as indicated.

Adoptive transfer of CD4⁺ T cells

CD4⁺ T cells were purified from spleens of wild-type Balb/c or Il3^−/− mice using mouse CD4⁺ T-cell Isolation Kit II (Miltenyi) and were intravenously (i.v.) injected (5 × 10⁶ cells) to Rag1^−/− recipient mice 24 h before MC903 treatment.

Naive T-cell culture

Naive CD4⁺ T cells from spleens of wild-type Balb/c mice were isolated using the CD4⁺CD62L⁺ T-cell isolation kit II (Miltenyi Biotec), following the manufacturer’s instructions. Purified naive T cells (1 × 10⁶ per ml) were cultured in anti-CD3 (2 μg ml⁻¹; clone 145-2C11, eBioscience)-coated wells either in the presence or absence of the anti-CD28 (2 μg ml⁻¹; clone 37–51, eBioscience) antibody in RPMI 1640 medium supplemented with 10% of FCS and 2 mM of L-glutamine. To activate OX40 signalling, agonistic rat-anti-mouse OX40 Ab (5 μg ml⁻¹; clone OX86, Biolegend) was added to the culture. Cells and supernatants were collected for RT–PCR or ELISA analyses. IL-3 protein levels in culture supernatants were measured using the mouse IL-3 Quantikine ELISA kit (R&D) (the detection limit was 7.8 pg ml⁻¹).

In vivo blockade of OX40L

To block OX40L–OX40 interaction, mice were injected i.p. with 300 μg of rat anti-mouse-OX40L neutralizing mAb (clone RM134L, rat IgG2bκ, BioXcell) or isotype control antibody IgG2b, given at D0 and D2 at the same time during MC903 skin treatment (see Fig. 1a).

Human DC-naive CD4⁺ T-cell co-culture

Buffy coats were obtained from healthy donors (Saint-Antoine Crozatier Blood Bank, Paris, France) with informed consent for research use in accordance with the declaration of Helsinki. CD11c⁺ DC were isolated from peripheral blood mononuclear cells by using the Easy Sep human pan-DC pre-enrichment kit (Stem Cell) followed by cell sorting to lineage⁻CD11c⁺ cells by flow cytometry (purity was >99%). Naive CD4⁺ T cells (CD4⁺CD25⁻CD45RA⁺ CD45RO⁻) were purified from healthy donors as previously described^57,58. DCs were seeded in 96-well flat-bottom plates in medium alone or in presence of human TSLP (50 ng ml⁻¹; R&D). Twenty-four hours after, DCs were washed and co-cultured with naive CD4⁺ T cells in complete Yssel medium at a ratio of 1:5. After 1, 2, 3, 4 or 5 days, cells were washed and re-stimulated with anti-human CD3/CD28-coupled microbeads (Invitrogen) for 24 h as described⁵⁸. Supernatants were collected and assayed for IL-3 and IL-4 levels using cytometric bead assay Flex Sets (BD Biosciences; detection limit was 20 pg ml⁻¹ for both).

Statistical analysis

Data were analysed using SigmaStat (Systat Software, Point Richmond, CA, USA) by Student’s t-test or the Mann–Whitney rank sum nonparametric test depending on results from the Kolmogorov–Smirnov test (with Lilliefors’ correction) for normality and Levene Median test for equal variance.

Additional information

How to cite this article: Leyva-Castillo, J.M. et al. Skin thymic stromal lymphopoietin initiates Th2 responses through an orchestrated immune cascade. Nat. Commun. 4:2847 doi: 10.1038/ncomms3847 (2013).

References

Liu, Y. J. et al. TSLP: an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation. Annu. Rev. Immunol. 25, 193–219 (2007).
Article CAS Google Scholar
Liu, Y. J. Thymic stromal lymphopoietin: master switch for allergic inflammation. J. Exp. Med. 203, 269–273 (2006).
Article Google Scholar
Ziegler, S. F. Thymic stromal lymphopoietin and allergic disease. J. Allergy Clin. Immunol. 130, 845–852 (2012).
Article CAS Google Scholar
Soumelis, V. et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat. Immunol. 3, 673–680 (2002).
Article CAS Google Scholar
Li, M. et al. Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin. Proc. Natl Acad. Sci. USA 102, 14795–14800 (2005).
Article ADS CAS Google Scholar
Yoo, J. et al. Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skin. J. Exp. Med. 202, 541–549 (2005).
Article CAS Google Scholar
Li, M. et al. Induction of thymic stromal lymphopoietin expression in keratinocytes is necessary for generating an atopic dermatitis upon application of the active vitamin D3 analogue MC903 on mouse skin. J. Invest. Dermatol. 129, 498–502 (2009).
Article CAS Google Scholar
Li, M. et al. Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis. Proc. Natl Acad. Sci. USA 103, 11736–11741 (2006).
Article ADS CAS Google Scholar
Zhou, B. et al. Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice. Nat. Immunol. 6, 1047–1053 (2005).
Article CAS Google Scholar
Jessup, H. K. et al. Intradermal administration of thymic stromal lymphopoietin induces a T cell- and eosinophil-dependent systemic Th2 inflammatory response. J. Immunol. 181, 4311–4319 (2008).
Article CAS Google Scholar
Seshasayee, D. et al. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation. J. Clin. Invest. 117, 3868–3878 (2007).
Article CAS Google Scholar
Bartemes, K. R. & Kita, H. Dynamic role of epithelium-derived cytokines in asthma. Clin. Immunol. 143, 222–235 (2012).
Article CAS Google Scholar
Kim, B. S. et al. TSLP Elicits IL-33-Independent Innate Lymphoid Cell Responses to Promote Skin Inflammation. Sci. Transl. Med. 5, 170ra116 (2013).
Article Google Scholar
Paul, W. E. & Zhu, J. How are T(H)2-type immune responses initiated and amplified? Nat. Rev. Immunol. 10, 225–235 (2010).
Article CAS Google Scholar
Jung, S. et al. In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens. Immunity 17, 211–220 (2002).
Article CAS Google Scholar
Romani, N., Clausen, B. E. & Stoitzner, P. Langerhans cells and more: langerin-expressing dendritic cell subsets in the skin. Immunol. Rev. 234, 120–141 (2010).
Article CAS 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 Google Scholar
Karasuyama, H., Mukai, K., Obata, K., Tsujimura, Y. & Wada, T. Nonredundant roles of basophils in immunity. Annu. Rev. Immunol. 29, 45–69 (2011).
Article CAS Google Scholar
Siracusa, M. C., Comeau, M. R. & Artis, D. New insights into basophil biology: initiators, regulators, and effectors of type 2 inflammation. Ann. N. Y. Acad. Sci. 1217, 166–177 (2011).
Article ADS CAS Google Scholar
Min, B. Basophils: what they 'can do' versus what they & apos;actually do&apos. Nat. Immunol. 9, 1333–1339 (2008).
Article CAS Google Scholar
Denzel, A. et al. Basophils enhance immunological memory responses. Nat. Immunol. 9, 733–742 (2008).
Article CAS Google Scholar
Wada, T. et al. Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks. J. Clin. Invest. 120, 2867–2875 (2010).
Article CAS Google Scholar
Hammad, H. et al. Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. J. Exp. Med. 207, 2097–2111 (2010).
Article CAS Google Scholar
Obata, K. et al. Basophils are essential initiators of a novel type of chronic allergic inflammation. Blood 110, 913–920 (2007).
Article CAS Google Scholar
Kim, S. et al. Cutting edge: basophils are transiently recruited into the draining lymph nodes during helminth infection via IL-3, but infection-induced Th2 immunity can develop without basophil lymph node recruitment or IL-3. J. Immunol. 184, 1143–1147 (2010).
Article CAS Google Scholar
Ito, T. et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J. Exp. Med. 202, 1213–1223 (2005).
Article CAS Google Scholar
Croft, M. Control of immunity by the TNFR-related molecule OX40 (CD134). Annu. Rev. Immunol. 28, 57–78 (2010).
Article CAS Google Scholar
Omori, M. & Ziegler, S. Induction of IL-4 Expression in CD4+ T Cells by Thymic Stromal Lymphopoietin. J. Immunol. 178, 1396–1404 (2007).
Article CAS Google Scholar
Bell, B. D. et al. The transcription factor STAT5 is critical in dendritic cells for the development of TH2 but not TH1 responses. Nat. Immunol. 14, 364–371 (2013).
Article CAS Google Scholar
Henri, S. et al. Disentangling the complexity of the skin dendritic cell network. Immunol. Cell Biol. 88, 366–375 (2010).
Article Google Scholar
Yoshimoto, T. et al. Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells. Nat. Immunol. 10, 706–712 (2009).
Article CAS Google Scholar
Sokol, C. L. et al. Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response. Nat. Immunol. 10, 713–720 (2009).
Article CAS Google Scholar
Sokol, C. L., Barton, G. M., Farr, A. G. & Medzhitov, R. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat. Immunol. 9, 310–318 (2008).
Article CAS Google Scholar
Perrigoue, J. G. et al. MHC class II-dependent basophil-CD4+ T cell interactions promote T(H)2 cytokine-dependent immunity. Nat. Immunol. 10, 697–705 (2009).
Article CAS Google Scholar
Tang, H. et al. The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling. Nat. Immunol. 11, 608–617 (2010).
Article CAS Google Scholar
Giacomin, P. R. et al. Thymic Stromal lymphopoietin-dependent basophils promote Th2 cytokine responses following intestinal helminth infection. J. Immunol. 189, 4371–4378 (2012).
Article CAS Google Scholar
Massacand, J. C. et al. Helminth products bypass the need for TSLP in Th2 immune responses by directly modulating dendritic cell function. Proc. Natl Acad. Sci. USA 106, 13968–13973 (2009).
Article ADS CAS Google Scholar
Jankovic, D. et al. Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4+ lymphocytes with a Th2 cytokine profile. J. Immunol. 164, 3047–3055 (2000).
Article CAS Google Scholar
Leyva-Castillo, J. M., Hener, P., Jiang, H. & Li, M. TSLP produced by keratinocytes promotes allergen sensitization through skin and thereby triggers atopic march in mice. J. Invest. Dermatol. 133, 154–163 (2013).
Article CAS Google Scholar
Liang, G. et al. Naive T cells sense the cysteine protease allergen papain through protease-activated receptor 2 and propel TH2 immunity. J. Allergy Clin. Immunol. 129, 1377–1386 e1313 (2012).
Article CAS Google Scholar
Sullivan, B. M. et al. Genetic analysis of basophil function in vivo. Nat. Immunol. 12, 527–535 (2011).
Article CAS Google Scholar
Hida, S., Tadachi, M., Saito, T. & Taki, S. Negative control of basophil expansion by IRF-2 critical for the regulation of Th1/Th2 balance. Blood 106, 2011–2017 (2005).
Article CAS Google Scholar
Yoshimoto, T. et al. IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc. Natl Acad. Sci. USA 96, 13962–13966 (1999).
Article ADS CAS Google Scholar
Voehringer, D., Shinkai, K. & Locksley, R. M. Type 2 immunity reflects orchestrated recruitment of cells committed to IL-4 production. Immunity 20, 267–277 (2004).
Article CAS Google Scholar
Pulendran, B. & Artis, D. New paradigms in type 2 immunity. Science 337, 431–435 (2012).
Article ADS CAS Google Scholar
Yu, C. et al. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo. J. Exp. Med. 195, 1387–1395 (2002).
Article CAS Google Scholar
Grimbaldeston, M. A. et al. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo. Am. J. Pathol. 167, 835–848 (2005).
Article CAS Google Scholar
Huddleston, C. A., Weinberg, A. D. & Parker, D. C. OX40 (CD134) engagement drives differentiation of CD4+ T cells to effector cells. Eur. J. Immunol. 36, 1093–1103 (2006).
Article CAS Google Scholar
Mestas, J., Crampton, S. P., Hori, T. & Hughes, C. C. W. Endothelial cell co-stimulation through OX40 augments and prolongs T cell cytokine synthesis by stabilization of cytokine mRNA. Int. Immunol. 17, 737–747 (2005).
Article CAS Google Scholar
Gramaglia, I., Weinberg, A. D., Lemon, M. & Croft, M. Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J. Immunol. 161, 6510–6517 (1998).
CAS PubMed Google Scholar
Shen, T. et al. T cell-derived IL-3 plays key role in parasite infection-induced basophil production but is dispensable for in vivo basophil survival. Int. Immunol. 20, 1201–1209 (2008).
Article CAS Google Scholar
Lantz, C. S. et al. IL-3 is required for increases in blood basophils in nematode infection in mice and can enhance IgE-dependent IL-4 production by basophils in vitro. Lab. Invest. 88, 1134–1142 (2008).
Article CAS Google Scholar
Lantz, C. S. et al. Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 392, 90–93 (1998).
Article ADS CAS Google Scholar
Chu, D. K. et al. IL-33, but not thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization. J. Allergy Clin. Immunol. 131, 187–200 (2013).
Article CAS Google Scholar
Roan, F. et al. The multiple facets of thymic stromal lymphopoietin (TSLP) during allergic inflammation and beyond. J. Leukoc. Biol. 91, 877–886 (2012).
Article CAS Google Scholar
Mach, N. et al. Involvement of interleukin-3 in delayed-type hypersensitivity. Blood 91, 778–783 (1998).
CAS PubMed Google Scholar
Volpe, E. et al. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat. Immunol. 9, 650–657 (2008).
Article CAS Google Scholar
Bogiatzi, S. I. et al. Multiple-checkpoint inhibition of thymic stromal lymphopoietin-induced T(H)2 response by T(H)17-related cytokines. J. Allergy Clin. Immunol. 130, 233–240 (2012).
Article CAS Google Scholar

Download references

Acknowledgements

We thank the staff of the flow cytometry, cell culture and the animal facilities of Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) and Institut Clinique de la Souris (ICS) for excellent technical assistance. We thank C. Ebel (IGBMC) for help with FACS and cell sorting, and L. Paulen (IGBMC) for genotyping. We are grateful for P. Chambon (IGBMC), D. Metzger (IGBMC), and J. Yang (Freiburg) for helpful discussion and critical reading of the manuscript. We thank B. Malissen (Marseille) for Lang-DTREGFP mice; S. Jung (Rehovot) for CD11c-DTR mice; S. Galli and M. Tsai (Stanford) for their help in transferring Mcpt-8^DTR mice, and LEO Pharma (Denmark) for MC903. This work was supported by funds from l’Agence Nationale de la Recherche (ANR) (to M.L.); Fondation Simone et Cino del Duca (Institut de France) (to M.L.); Societe Francaise d’Allergologie (to M.L.); Association pour la Recherche à l’IGBMC (a predoctoral fellowship to J.M.L.-C.); Institut Curie, Fondation pour la Recherche Médicale, and ANR (to V.S. and P.M.); and JST, CREST (to H.K.). We thank also the supports from Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM) and Université de Strasbourg (UDS).

Author information

Authors and Affiliations

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique UMR7104/Institut National de la Santé et de la Recherche Médicale U964/Université de Strasbourg, Illkirch, 67404, France
Juan Manuel Leyva-Castillo, Pierre Hener, Susan Chan & Mei Li
Institut National de la Santé et de la Recherche Médicale U932, Paris, 75248, France
Paula Michea & Vassili Soumelis
Department of Immunology, Institut Curie, Paris, 75248, France
Paula Michea & Vassili Soumelis
Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, Tokyo, 113-85110, Japan
Hajime Karasuyama
JST, CREST, Tokyo Medical and Dental University Graduate School, Tokyo, 102-0076, Japan
Hajime Karasuyama
University of Strasbourg Institute for Advanced Study (USIAS), Strasbourg, 67083, France
Mei Li
Freiburg Institute for Advanced Studies (FRIAS), Freiburg, 79104, Germany
Mei Li

Authors

Juan Manuel Leyva-Castillo
View author publications
You can also search for this author in PubMed Google Scholar
Pierre Hener
View author publications
You can also search for this author in PubMed Google Scholar
Paula Michea
View author publications
You can also search for this author in PubMed Google Scholar
Hajime Karasuyama
View author publications
You can also search for this author in PubMed Google Scholar
Susan Chan
View author publications
You can also search for this author in PubMed Google Scholar
Vassili Soumelis
View author publications
You can also search for this author in PubMed Google Scholar
Mei Li
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

J.M.L.-C. and M.L. designed experiments; J.M.L.-C. and P.H. performed experiments; P.M. and V.S. designed, performed and analysed the human DC-T co-culture study; H.K. provided Mcpt8DTR mice; S. Chan provided ideas and helps for flow cytometry studies; J.M.L.-C. and M.L. analysed and interpreted data, and wrote the manuscript. M.L. directed the study and supervised the work.

Corresponding author

Correspondence to Mei Li.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1-S7 (PDF 5650 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Leyva-Castillo, J., Hener, P., Michea, P. et al. Skin thymic stromal lymphopoietin initiates Th2 responses through an orchestrated immune cascade. Nat Commun 4, 2847 (2013). https://doi.org/10.1038/ncomms3847

Download citation

Received: 22 May 2013
Accepted: 30 October 2013
Published: 28 November 2013
DOI: https://doi.org/10.1038/ncomms3847

This article is cited by

Role of thymic stromal lymphopoietin in allergy and beyond
- Risa Ebina-Shibuya
- Warren J. Leonard
Nature Reviews Immunology (2023)
Context-dependent function of TSLP and IL-1β in skin allergic sensitization and atopic march
- Justine Segaud
- Wenjin Yao
- Mei Li
Nature Communications (2022)
Oral vitamin D modulates the epidermal expression of the vitamin D receptor and cathelicidin in children with atopic dermatitis
- Carolina Cabalín
- Guillermo Pérez-Mateluna
- Arturo Borzutzky
Archives of Dermatological Research (2022)
Obesity alters pathology and treatment response in inflammatory disease
- Sagar P. Bapat
- Caroline Whitty
- Alexander Marson
Nature (2022)
Pruritus in pediatric patients with atopic dermatitis: a multidisciplinary approach - summary document from an Italian expert group
- May El Hachem
- Giuseppe Di Mauro
- Iride Dello Iacono
Italian Journal of Pediatrics (2020)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.