Abstract Early-life microbiota represent an indispensable factor for the proper development and function of host metabolism and the immune system. We have demonstrated that neonatal exposure to antibiotics for the first 3 weeks (NeoATB) leads to obesity in adulthood, characterized by gut microbiota dysbiosis and dysregulated immune responses. Here, we demonstrate that feeding D-mannose suppresses NeoATB-induced obesity, accompanied by improved glucose tolerance and decreased insulin resistance. Mechanistically, D-mannose feeding decreased hypoxia and increased oxygenation and recovery of metabolic activity of adipocytes. D-mannose restored CD4 + Foxp3 + ST2 + Tregs, leading to a reduction of Th1 pro-inflammatory cells in the adipose tissue of NeoATB mice. Significantly, we revealed that D-mannose treatment reversed the dysregulated ratios of phylum Firmicutes to phylum Bacteroidetes in obese NeoATB mice, which was surprisingly attributed to D-mannose-mediated suppression of the growth of Firmicutes rather than an increase in the growth of Bacteroidetes. These findings should have therapeutic implications for the treatment of obesity in human patients.

Abstract Background: Ewing sarcoma (EWS) is a rare, aggressive bone and soft tissue malignancy predominantly affecting children and young adults. Despite advances in multimodal therapy, treatment options remain limited for refractory or relapsed EWS (1). Chimeric antigen receptor (CAR) T-cell therapy has shown marked efficacy in hematologic malignancies and is being developed for solid tumors. AZD0754 is a STEAP2-targeted CAR T-cell therapy incorporating a dominant-negative TGFβRII (dnTGFβRII) armoring strategy and is in Phase 1 clinical development for prostate cancer. Recent published proteomic analyses identified STEAP2 expression in EWS patient samples and cell line-derived xenografts, supporting STEAP2 as a potential candidate immunotherapeutic target (2). Methods: STEAP2 surface expression was evaluated across different human EWS cell lines by flow cytometry. AZD0754-mediated cytotoxicity was assessed in vitro using the xCELLigence real-time cell analysis platform. Based on in vitro results, A673, RD-ES, and SK-NEP-1 xenograft models were selected for in vivo efficacy studies. Mice received varying doses of AZD0754, and tumor growth and overall survival were monitored. Blood was collected at different time points for serum cytokine analysis to assess pharmacodynamic activity. Results: EWS cell lines expressed detectable cell surface expression of STEAP2 at receptor densities much lower than workhorse prostate cancer cell lines. Despite this, AZD0754 was capable of inducing antigen-dependent cytotoxicity in vitro and suppressing tumor growth in EWS xenograft models. AZD0754 exhibited dose-dependent antitumor activity that correlated with serum IFNγ levels, indicating on-target immune engagement. Conclusions: AZD0754 demonstrates robust preclinical anti-tumor activity against EWS in vitro and in vivo, with dose-dependent efficacy and corresponding cytokine levels. These findings support STEAP2 as a potential therapeutic target in EWS. STEAP2 targeting has the potential to address a critical unmet need in EWS by expanding treatment options to biologic and immune-based therapies. References1. National Center for Biotechnology Information (2021). Ewing Sarcoma. NCBI Bookshelf. 2. Mooney B, Negri GL, Shyp T, Delaidelli A, et al. Surface and global proteome analyses identify ENPP1 and other surface proteins as actionable immunotherapeutic targets in Ewing sarcoma. Clin Cancer Res.2024;30(5):1022-1037. doi:10.1158/1078-0432.CCR-23-2187. Citation Format: Peter Zanvit, Brianna Janocha, Shannon Breen, Christine Fazenbaker, Ryan Golden, Jonathan Fitzgerald, Mark Cobbold, Gordon Moody, Emily Bosco. Efficacy of AZD0754, a dominant-negative TGFβRII-armored STEAP2 CAR T-cell therapy, in Ewing sarcoma xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7808.

Abstract Introduction The neonatal dysbiosis of gut microbiota represents a critical environmental factor promoting obesity, but the underlying immunological mechanisms remain unknown. Methods Neonatal mice (from day1) were treated with broad-spectrum antibiotics for the first 3 weeks of life (NeoATB) only . The mice were then hosted under a normal environment for rest of their life. Results We show here that neonatal treatment of mice with broad-spectrum antibiotics for the first 3 weeks of life (NeoATB) resulted in the development of obesity and metabolic abnormalities in adulthood. The NeoATB mice exhibited a permanent dysbiosis of gut microbiota, decreased CD4+Foxp3+ regulatory T cells (Tregs), and increased proinflammatory Th1 cells in visceral adipose tissue (VAT). Mechanistically, neonatal antibiotic treatment resulted in increased intestinal permeability that allowed bacterial translocation into the VAT and liver and an increase in systemic LPS levels. Consequently, VAT dendritic cells were activated via the TLR4 pathway to increase IL-12-triggered Th1-inflammation. Moreover, the decrease in VAT Tregs in NeoATB mice was attributed to reduced infiltration of Tregs from the periphery, decreased expansion of adipose IL-33-mediated ST2+ Tregs, and reduced conversion of local Tregs from VAT CD4+CD25- Foxp3- T cells. Conclusion Thus, we have revealed a previously unrecognized immunological link between neonatal microbiota dysbiosis and the development of obesity. Funding Source NIH, NIDCR IRP Topic Categories Mucosal and Regional Immunology (MUC)

Adipocyte deposition is believed to be a primary characteristic of age-related thymic involution, but the underlying cellular and molecular mechanisms remain unknown. We show here that thymic mesenchymal stromal cells (tMSCs) have a higher tendency to differentiate into adipocytes and melanocortin-2 receptor accessory protein (MRAP) is a potential driver of tMSCs adipogenesis. Furthermore, we discover that thymosin-α1 promotes MRAP expression in tMSCs through FoxO1 signaling pathway. Additionally, the proportion of tMSCs increase in older mice compared to young mice. Importantly, MRAP is also necessary for human thymic MSCs to differentiate into adipocytes when exposed to thymosin-α1. Single-cell RNA-seq analysis of human thymus revealed an accumulation of tMSCs and adipocytes during aging, indicating a strong potential for adipogenic differentiation in age-related thymic involution. Thus, we have revealed MRAP as a key factor in promoting thymic MSCs adipogenesis triggered by thymosin-α1 and FoxO1 pathway, which may serve as potential target to hinder adiposity in age-related thymic involution.

Evaluation of CLDN18.2-targeting CAR-T cells in vivo. A, NSG mice bearing PaTu8988s HS xenografts (CLDN18.2 H-score = 268) were dosed by tail vein with 9e6 CAR+ CLDN18.2 Bz CAR-T cells; total T-cell infusion number was matched across groups. Tumor volume and body weight were measured biweekly (n = 9). Serum levels of IFNγ were measured at 4, 7, and 14 days after infusion (n = 3). B, Schematic representation of a second-generation CAR-T design modified to replace 4-1BB with a CD28 costimulatory domain (28z). The average transduction efficiency (CAR+, day 9) of multiple healthy donors for clone 9 28z is shown. Representative FC plots of CAR surface expression at day 9 after lentivirus transduction were compared with UT control for a single donor. C, NSG mice bearing PaTu8988s HS xenografts were dosed as described in A with clone 9 CD28z or Bz CAR-T (n = 6) at indicated doses. Serum levels of IFNγ were measured at 4, 7, and 14 days after infusion (n = 3). D, Representative images of CLDN18.2 (top row) and CD3 (bottom row) staining in the stomachs of mice dosed with clone 9 CAR-T cells from C at indicated time points. All data represent mean ± SEM of replicate experiments or animals.

Development and evaluation of CLDN18.2-targeting CAR-T cells in vitro. A, Schematic representation of second-generation CAR-T lentivirus design, which includes a 4-1BB costimulatory domain (Bz). The table shows for each CLDN18.2-reactive clone the relative binding affinity (human and mouse), reactivity to mutant CLDN18.2 (M149L), and average transduction efficiency (CAR+, day 9) of multiple healthy donors. Representative FC plots of CAR surface expression at day 9 after lentivirus transduction were compared with UT control for a single donor. B, CLDN18.2 cell surface expression of various cell lines as determined by FC with 5 μg/mL CLDN18.2-reactive clones compared with nonspecific isotype antibody (R347). C, Epitope characterization of CLDN18.2-reactive clones. The AlphaFold structure on the far left (red) represents all sites of point mutation in HEK293 cells that vary between CLDN18.1 and CLDN18.2 in the first extracellular loop; the color-coded diagrams represent sites that influence respective clone binding. D, Percent cytolysis of HEK293 + huCLDN18.1, HEK293 + huCLDN18.2, HEK293 + muCLDN18.1, HEK293 + muCLDN18.2, and PaTu8988s HS cells determined by xCELLigence RTCA assay after 48 hours of co-culture with CLDN18.2 CAR-T cells at a 1:1 E:T ratio. The supernatants from the xCELLigence assay were collected at 24 hours for cytokine assessment (Meso Scale Discovery) assay. All data represent mean ± SEM of replicate experiments.

Abstract CLDN18.2 expression has been observed in pancreatic, gastric and esophageal cancers, whereas normal tissue expression of CLDN18.2 is mostly restricted to differentiated gastric mucosal epithelial cells. This expression profile renders CLDN18.2 an ideal tumor associated antigen (TAA) for CAR-T targeting and an armored targeted CAR-T, AZD6422, is in clinical development. While CAR-T are beginning to show clinical promise in solid tumors, a consistent challenge is to overcome the immunosuppressive tumor microenvironment (TME) where immune and stromal cells secrete high levels of TGFb. Since the majority of pre-clinical CAR-T efficacy data is generated utilizing immunocompromised mouse models, which lack a proper TME, the team utilized syngeneic xenograft models to circumvent this challenge. ​ In vitro studies showed that CLDN18.2 targeted murine CAR-T cells generated by retroviral transduction induced efficient cytolysis of target positive mouse tumor cells accompanied by cytokine release. To model the clinical scenario, tumor-bearing mice were lymphodepleted with total body irradiation (TBI) to enhance CAR-T engraftment. The dosage of TBI used in these studies was predetermined to have caused leukopenia and minimal impact on tumor growth. We observed substantial tumor control and extended tumor efficacy in mice treated with mCAR-T cells compared to those infused with untransduced T cells or in non-target bearing control tumors in the absence of gross toxicities. ​ This syngeneic mouse model platform will additionally allow us to investigate how mCAR-T cells perform within a fully competent TME in combination with other therapeutic agents targeting cancer cells via altered mechanism of action. ​ Citation Format: Shannon Breen, Rosa Carrasco, Kelly McGlinchey, Brianna Janocha, Peter Zanvit, Lorenzo Ortiz, Benjamin Clark, Aleksandra Toloczko, Mark Cobbold, Gordon Moody, Emily Bosco, Allison Barrett. Utilization of CLDN18.2 syngeneic mouse models to study chimeric antigen receptor T cells in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1435.

Optimized manufacturing protocol, STAR-T, for the generation of the CAR-T product. A, Baseline characteristics of donor-matched dnTGFβRII CAR-T cells with traditional manufacture (day 10) vs. AZD6422 (day 4), including CAR+ expression, percent CD4 and CD8 expression, and T-cell phenotypic status as determined by cell surface expression of CCR7 and CD45RO. Results are shown for naïve (CCR7+/CD45RO−, Tn), central memory (CCR7+/CD45RO+, Tcm), effector memory (CCR7−/CD45RO+, Tem), and effector (CCR7−/CD45RO−, Teff) cells. Data are shown as mean ± SEM of representative donors. B, Comparison of bioenergetic profiles of traditionally manufactured dnTGFβRII CAR-T cells vs. AZD6422. Spare respiratory capacity was determined as the differential between basal and maximum respiration. 2-DG, 2-deoxy-D-glucose; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; Rot/AA, rotenone and antimycin A. C, Serial restimulation assay to examine cytotoxicity and persistence of dnTGFβRII CAR-T cells and AZD6422. CAR-T cells were co-cultured at a ratio of 1:2 with BXPC3 + CLDN18.2, tumor lysis was measured every 3 to 4 days, and IFNγ was profiled at 24 hours after each new co-culture. Representative of multiple donors. D, Results of quantitative FC to determine cell surface expression of CLDN18.2 across multiple cancer cell lines. Percent cytolysis was determined by xCELLigence RTCA assay after 48 hours of co-culture with AZD6422 at an E:T ratio of 1:1. Data represent mean ± SEM of replicate experiments.

AbstractPurpose: Claudin 18.2 (CLDN18.2) is a surface membrane protein that is crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers. Thus, CLDN18.2 is suited for exploration as a clinical target for chimeric antigen receptor T-cell (CAR-T) therapy in these indications. Although CAR-T therapies show promise, a challenge faced in their development for solid tumors is the immunosuppressive tumor microenvironment, which is often characterized by the presence of immune and stromal cells secreting high levels of TGFβ. The addition of TGFβ armoring can potentially expand CAR-T activity in solid tumors. We report on the preclinical development of a CLDN18.2-targeting CAR-T therapy showing effectiveness in patient models with CLDN18.2-positive gastric, esophageal, and pancreatic tumors. Experimental Design: The lead lentivirus product contains a unique single-chain variable fragment; CD28 and CD3z costimulatory and signaling domains; and dominant-negative TGF-β receptor armoring, enhancing targeting and safety and counteracting suppression. We developed a shortened cell manufacturing process to enhance the potency of the final product AZD6422. Results: AZD6422 exhibited significant antitumor activity and tolerability in multiple patient-derived tumor xenograft models with various CLDN18.2 and TGF-β levels, as determined by IHC. The efficacy of armored CAR-T cells in tumor models with elevated TGFβ was increased in vitro and in vivo. In vitro restimulation assays established greater persistence and cytolytic function of AZD6422 compared with a traditionally manufactured CAR-T. Conclusions: AZD6422 was safe and efficacious in patient-derived, CLDN18.2-positive murine models of gastrointestinal cancers. Our data support further clinical development of AZD6422 for patients with these cancers.

In vivo antitumor activity of AZD6422 in PDX models of gastric cancer, PDAC, and esophageal adenocarcinoma. Activity and tolerability of AZD6422 are shown in various PDX models of esophageal adenocarcinoma (A, ES11085; D, ES_9500), gastric cancer (E, GA_9275), and PDAC (B, PANC_22; C, PANC_12; F, PANC_24). Each model was selected to represent a range of CLDN18.2 (shown at 10× scan) and TGFβ expression. NSG MHC-DKO mice received a single tail-vein infusion of 1e6 AZD6422, donor-matched UT, or vehicle when the average tumor volume reached 150 mm3. Tumor volumes and body weights were measured biweekly until study completion on day 35, and blood was collected for cytokine analysis on days 7 and 14. Data are shown as mean ± SEM (n = 5).

γδ intestinal intraepithelial lymphocytes (IELs) constitute the majority of IELs with unique CD8αα+ homodimers that are distinct from γδT cells in other tissues. However, it remains largely unclear how those cells develop. Here we show that transforming growth factor beta (TGF-β) signaling controls the development of TCRγδ+CD8αα+ IELs. Deletion of TGF-β receptors or Smad3 and Smad2 in bone marrow stem cells caused a deficiency of TCRγδ+CD8αα+ IELs in mixed bone marrow chimeric mice. Mechanistically, TGF-β is required for the development of TCRγδ+CD8αα+ IELs thymic precursors (CD44–CD25– γδ thymocytes). In addition, TGF-β signaling induced CD8α in thymic γδT cells and maintained CD8α expression and survival in TCRγδ+CD8αα+ IELs. Moreover, TGF-β also indirectly controls TCRγδ+CD8αα+ IELs by modulating the function of intestinal epithelial cells (IECs). Importantly, TGF-β signaling in TCRγδ+CD8αα+ IELs safeguarded the integrity of the intestinal barrier in dextran sulfate sodium (DSS)-induced colitis.

Prostate cancer is generally considered an immunologically “cold” tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to “heat up” the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-β type II receptor, bolstering its activity in the TGF-β–rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-β–rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line–derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

Background Prostate cancer is thought of as immunologically 'cold', rendering tumors insensitive to immunotherapy, and limiting treatment options. Prostate cancer 'coldness' may be due to multiple factors, including a TGFβ rich immunosuppressive tumor microenvironment, dysfunctional T cells, and lack of tumor associated antigens (TAA). We identified STEAP2 as a highly prevalent prostate cancer antigen, displaying high, homogeneous cell surface expression across all stages of disease, with the potential for superior therapeutic targeting. Methods A novel lead generation approach facilitated the development of a potent and specific armored STEAP2 CAR-T therapeutic candidate, AZD0754. This 2nd-generation CAR-T product is armored with dominant-negative transforming growth factor beta (TGFβ) receptor II (dnTGFβRII), bolstering activity in the TGFβ-rich immunosuppressive environment of prostate cancer. In a series of in vitro experiments, we evaluated the effect of enzalutamide (androgen receptor (AR) antagonist) on the viability, gene expression and function of AZD0754 and tumor cells alone or in co-culture of AZD0754 and tumor cells. Furthermore, in vivo activity of AZD0754 was tested across a panel of prostate cancer patient derived xenograft models (PDX) alone and in combination with enzalutamide. Results In vitro experiments demonstrated that the viability and anti-tumor function of AZD0754 was maintained in the presence of enzalutamide. Moreover, AZD0754 CAR-T treatment induced significant anti-tumor efficacy and corresponding IFNγ production in a panel of prostate PDX models that closely mimic the genomic and phenotypic features of human prostate cancer, even in models with low antigen density and stromal derived-TGFβ. Given the correlation between STEAP2 expression and androgen receptor expression, we further investigated the efficacy of AZD0754 in a combination study with the enzalutamide, in prostate cancer PDX models. We found no significant effect of enzalutamide on STEAP2 expression, and our results highlighted the combinatorial activity of enzalutamide and AZD0754 CAR-T therapy. Conclusions Together, these data underscore the potential therapeutic tractability of STEAP2 in prostate cancer and build confidence in the STEAP2 expression threshold required for AZD0754 activity. Moreover, our study explores potential combination strategies involving standard-of-care therapies.

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-β), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-β and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.

Abstract Adipocyte deposition is believed to be a primary characteristic of age-related thymic involution, but the underlying cellular and molecular mechanisms remain unknown. We show here that thymic mesenchymal stromal cells (tMSCs) are differentiated into adipocytes through melanocortin-2 receptor accessory protein (MRAP). Specifically, we found that in contrast to bone-forming MSCs isolated from dental pulp tissues, tMSCs were more susceptible to differentiate into adipocytes under adipogenic condition, but hardly to differentiate into bone tissues in osteogenic medium. We identified MRAP as a potential factor to drive tMSCs adipogenesis by global RNA-seq analysis and confirmed its key role by knockdown of Mrap in tMSCs in vitro and null mutation of Mrap in mice. The aged Mrap-knockout mice had enhanced thymic size and increased thymic weight with less adipocytes accumulation in the thymus. We further uncovered that thymosin-α1 promoted MRAP expression in tMSCs through FoxO1 signaling pathway. Moreover, tMSCs proportion increased in old mice compared to young mice. Importantly, human thymic MSCs also required MRAP to differentiate into adipocytes in response to thymosin-a1. Thus, we have revealed MRAP as a key factor in promoting thymic MSCs adipogenesis triggered by thymosin- α1 and FoxO1 pathway, which may serve as potential target to hinder adiposity in age- related thymic involution.

Adipose-derived mesenchymal stromal cells (ADSCs) play important roles in the alleviation of inflammation and autoimmune diseases. Interleukin-33 (IL-33), a member of the IL-1 family, has been shown to regulate innate and adaptive immunity. However, it is still unknown whether ADSCs regulate immune responses via IL-33. We show here that ADSCs produced IL-33 in response to IL-1β stimulation, which depended on TAK1, ERK, and p38 pathways. ADSCs-derived IL-33 drove the proliferation of CD4+Foxp3+ST2+ regulatory T cells (Tregs) and alleviated experimental autoimmune Sjögren syndrome in mice. Importantly, human ADSCs also produced IL-33 in response to IL-1β. Thus, we have revealed a previously unrecognized immunoregulatory function of ADSCs by IL-33 production in experimental autoimmunity, which may have clinical applications for human immunopathology.

Abstract Immune dysregulation has long been proposed as a component of premature ovarian insufficiency (POI), but the underlying mediators and mechanisms remain largely unknown. Here we showed that patients with POI had augmented T helper 1 (TH1) responses and regulatory T (Treg) cell deficiency in both the periphery and the ovary compared to the control women. The increased ratio of TH1:Treg cells was strongly correlated with the severity of POI. In mouse models of POI, the increased infiltration of TH1 cells in the ovary resulted in follicle atresia and ovarian insufficiency, which could be prevented and reversed by Treg cells. Importantly, interferon (IFN) ‐γ and tumor necrosis factor (TNF) ‐α cooperatively promoted the apoptosis of granulosa cells and suppressed their steroidogenesis by modulating CTGF and CYP19A1. We have thus revealed a previously unrecognized Treg cell deficiency‐mediated TH1 response in the pathogenesis of POI, which should have implications for therapeutic interventions in patients with POI.

The molecular pathways underlying the development of innate lymphoid cells (ILCs) are mostly unknown. Here we show that TGF-β signaling programs the development of ILC2s from their progenitors. Specifically, the deficiency of TGF-β receptor II in bone marrow progenitors results in inefficient development of ILC2s, but not ILC1s or ILC3s. Mechanistically, TGF-β signaling is required for the generation and maintenance of ILC2 progenitors (ILC2p). In addition, TGF-β upregulates the expression of the IL-33 receptor gene Il1rl1 (encoding IL-1 receptor-like 1, also known as ST2) in ILC2p and common helper-like innate lymphoid progenitors (CHILP), at least partially through the MEK-dependent pathway. These findings identify a function of TGF-β in the development of ILC2s from their progenitors.

Cutaneous wound healing is associated with the unpleasant sensation of itching. Here we investigated the mechanisms underlying this type of itch, focusing on the contribution of soluble factors released during healing. We found high amounts of interleukin 31 (IL-31) in skin wound tissue during the peak of itch responses. Il31(-/-) mice lacked wound-induced itch responses. IL-31 was released by dermal conventional type 2 dendritic cells (cDC2s) recruited to wounds and increased itch sensory neuron sensitivity. Transfer of cDC2s isolated from late-stage wounds into healthy skin was sufficient to induce itching in a manner dependent on IL-31 expression. Addition of the cytokine TGF-beta 1, which promotes wound healing, to dermal DCs in vitro was sufficient to induce Il31 expression, and Tgfbr1(f/f) CD11c-Cre mice exhibited reduced scratching and decreased Il31 expression in wounds in vivo. Thus, cDC2s promote itching during skin would healing via a TGF-beta-IL-31 axis with implications for treatment of wound itching.