{"paper_id":"89b45b96-e290-4fd5-9dea-f94125656b16","body_text":"Jagged-1+ Skin Tregs Modulate the Innate Immune Response to Wound Healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Jagged-1+ Skin Tregs Modulate the Innate Immune Response to Wound Healing Prudence PokWai Lui, Jessie Z. Xu, Hafsah Aziz, Monica Sen, Niwa Ali This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4559523/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Skin-resident regulatory T cells (Tregs) play an irreplaceable role in orchestrating cutaneous immune homeostasis and repair, including the promotion of hair regeneration via the Notch signaling ligand Jagged-1 (Jag1). While skin Tregs are indispensable for facilitating tissue repair post-wounding, it remains unknown if Jag1-expressing skin Tregs impact wound healing. Using a tamoxifen inducible Foxp3creERT2Jag1fl/fl model, we show that loss of functional Jag1 in Tregs significantly delays the rate of full-thickness wound closure. Unlike in hair regeneration, skin Tregs do not utilize Jag1 to impact epithelial stem cells during wound healing. Instead, mice with Treg-specific Jag1 ablation exhibit a significant reduction in Ly6G+ neutrophil accumulation at the wound site. However, during both homeostasis and wound healing, the loss of Jag1 in Tregs does not impact the overall abundance or activation profile of immune cell targets in the skin, such as CD4+ and CD8+ T cells, or pro-inflammatory macrophages. This collectively suggests that skin Tregs may utilize Jag1-Notch signalling to co-ordinate innate cell recruitment under conditions of injury but not homeostasis. Overall, our study demonstrates the importance of Jag1 expression in Tregs to facilitate adequate wound repair in the skin. Biological sciences/Immunology/Lymphocytes/T cells/Cd4 positive t cells/Regulatory t cells Biological sciences/Immunology/Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The skin, the largest mammalian organ, acts as a critical physical barrier from constant environmental traumas. A diverse array of immune and non-immune cell types reside in or migrate to the skin during pre- and post-natal development, influenced by the local inflammatory microenvironment. Over the past decade, skin resident regulatory T cells (Tregs), a major T cell population, have been implicated in various processes, including hair regeneration 1,2 , full-thickness wound healing 3 , epidermal barrier repair 4,5 and fibrosis 6 . These functions are facilitated by the immunosuppressive capacity of skin Tregs, including restriction of pro-inflammatory cytokine production and myeloid cell accumulation 3,4 , as well as their interaction with non-immune tissue cells such as epithelial keratinocytes 5 , fibroblasts 6 and stem cells 1,4 . Multiple single cell studies have illustrated tissue Tregs are transcriptionally distinct from those within secondary lymphoid organs 7–10 . The site where Tregs are seeded determines their phenotype and shapes their functions 7,10 . Previous RNA sequencing analysis identified Jagged-1 (Jag1), one of the five Notch signalling ligands, as a key transcript preferentially expressed in skin Tregs compared to skin-draining lymph node (SDLN) Tregs 1 . Interestingly, intraperitoneal injection of IL-2/anti-IL2 antibody complexes can selectively expand Jag1+Tregs in murine skin but not in SDLNs 11 . In silico data indicate that skin Tregs are also phenotypically distinct from other tissue-resident Tregs 6,9,10 . Ligand-receptor prediction analyses from scRNA-seq datasets further suggest that skin Tregs likely interact with epithelial cells and hair follicle stem cells via Jag1-induced Notch signalling 2,5 , underscoring the potential importance of Jag1 in skin Tregs. Yet, the question of whether Jag1 expression is indeed unique to skin Tregs remains unanswered. Notch signalling is crucial for hair follicle differentiation and postnatal maintenance homeostatically 12 , as well as in regulating wound healing 13 . Conditional deletion of Jag1 in epidermal stem cells (K15 cre Jag1 fl/fl ) delays wound closure, whereas the addition of Jag1 peptide accelerates wound healing 13 , demonstrating the significance of Jag1 in the wound repair process. Although it has been established that Jag1 expressed on non-lymphoid cells can drive T cell fate decisions 14,15 , promote Treg expansion 16–18 , and that Notch signalling regulates Treg immunosuppressive capacity 19 , the function of Treg-derived Jag1 remains largely unknown. Previously, we have shown the perturbation of Jag1 in skin Tregs hinders hair follicle stem cell (HFSC) proliferation, delays the induction of the hair growth phase, and ultimately impedes hair regeneration 1 . Whether the relationship between Jag1+ Tregs and hair regeneration translates to other skin Treg-mediated mechanisms, such as wound healing, remains unexplored. Here, we report that Jag1 is preferentially expressed in skin Tregs compared to Tregs residing in other tissues. Despite Jag1+Tregs displaying higher CTLA4 and CD25 surface expression, the absence of Jag1 in Tregs does not alter skin integrity nor overall cutaneous immune dynamics during homeostasis. However, during full thickness wound healing, mice with Jag1 deficiency in Tregs heal significantly slower than controls. Unlike in hair regeneration, skin Tregs in wounded mice do not utilize Jag1 to alter HFSC activation but rather promote neutrophil accumulation at the wound site. This study sheds light on an alternative function of Jag1 in skin Tregs in facilitating adequate cutaneous wound repair. RESULTS Jagged1 is a Skin Treg Preferential Marker To determine whether Jag1 is uniquely expressed in skin Tregs, we began by re-analysing a bulkRNAseq dataset focusing on Tregs from various tissues 10 . Our analysis confirmed that skin Tregs exhibited the highest levels of Jag1 compared to Tregs from blood, spleen, and other organ tissues (visceral adipose tissue (VAT), lung and colon) (Figure 1A). Differential expression analysis using edgeR and limma packages normalized counts and compared the adjusted p-values (Fig1B) and log fold changes (Fig1C) of Jag1 expression in skin Tregs against other tissue Tregs. We included Cd45 (Ptprc) and common Treg markers (Foxp3, Ctla4, Cd25, and Icos), along with known tissue Treg-associated transcripts (Gata3 and Areg) as controls. Notably, Jag1 expression in skin Tregs was significantly and consistently higher than, not only those in blood or lymphoid organs, but also Tregs residing in lung, colon and VAT (Figure 1B&C). Skin Tregs expressed an average 4.5-fold higher level of Jag1 than other surveyed Tregs, clearly distinguishing Jag1 from other Treg-associated genes. As expected, Cd45 (Ptprc) showed no significant differences (high adjusted p-value and low log fold change) between skin and other tissue Tregs. Skin Tregs expressed similar level of Foxp3 and common Treg markers, as well as tissue Treg-associated genes, compared to Tregs in the lung, colon, and VAT . While Ctla4 and Gata3 expression levels remained similar, Foxp3 and Icos were significantly but mildly upregulated in skin Tregs, when compared to those in blood or spleen. Similar to Jag1, Areg was highly expressed in skin Tregs compared to splenic Tregs, but not when compared to other tissue Tregs. Collectively, this highlights Jag1 as a differentially expressed marker in skin Tregs. To further validate these findings, we performed flow cytometry on Tregs from skin, lung and SDLN. Around 55.59% (±12.61%) of skin Tregs expressed Jag1, in contrast to only 8.18% (±3.20%, p < 0.0001) in lung Tregs and 3.54% (±1.90%, p < 0.0001) in SDLN Tregs (Figure 1D & E, gating strategy in Supplementary Fig1A). The mean fluorescence intensity of Jag1 was significantly higher in skin Tregs (16.72 ± 2.15), in comparison to other CD4+Foxp3- T effector (Teffs) (6.95 ± 6.57, p = 0.0014) and CD8+ T cells (8.10 ± 5.01, p = 0.0041), indicating preferential expression of Jag1 in skin Tregs. We then examined whether these abundant Jag1 pos skin Tregs are phenotypically distinct from non-Jag1-expressing (Jag1 neg ) skin Tregs. In adult mice, Jag1 pos Tregs showed significantly higher proportions and levels of CTLA4 (Supplementary Fig2A-D) and CD25 (Supplementary Fig2E-H), but not ICOS (Supplementary Fig S2I-L). Jag1 pos skin Tregs co-expressed CTLA4 and CD25 more frequently than Jag1 neg Tregs (Supplementary Fig2M&N, 37±4.64% vs 23.5±7.90%, p = 0.0312), suggesting that Jag1 pos Tregs may be phenotypically more active. Jag1 pos Tregs are Dispensable During the Steady State Under homeostatic conditions, skin Tregs suppress long-term CD8 and Teff cell-driven hair follicle-associated inflammation via CD25 Ref. 20 . Given that Jag1 pos skin Tregs express higher levels of CD25 than Jag1 neg skin Tregs, we hypothesised that Jag1 pos skin Tregs may be functionally important during the steady state. To test this, and to avoid influencing early skin development, we created a tamoxifen inducible cell specific model to delete Jag1 in Tregs. This involved crossing mice expressing EGFP-creERT2 gene under the Foxp3 promoter 21 to mice carrying a Jag1 floxed allele with a dysfunctional Delta-Serrate-Lag2 domain of Jag1 22 to generate Foxp3 creERT2 Jag1 fl/fl and Foxp3 creERT2 Jag1 fl/wt , hereafter denoted as “Foxp3 ΔJag1 “ and “Foxp3 Ctrl ”. Following intraperitoneal injection of tamoxifen (Figure 2A), we observed effective downregulation of Jag1 transcript in sorted Tregs from Foxp3 L Jag1 compared to Foxp3 Ctrl mice (Supplementary Figure3A). Expression of Jag1 in Teffs, CD8+ T cells, and Foxp3 expression in Tregs (Supplementary Figure3B) showed no differences between Foxp3 ΔJag1 and Foxp3 Ctrl animals, indicating Treg-specificity of Jag1 deletion in this model. Despite significant systemic loss of Jag1 mRNA in Tregs, the absence of Jag1 did not trigger skin inflammation. H&E staining revealed similar morphology (Figure 2B), epidermal thickness (Figure 2C) and lymphocytic infiltration (Figure 2D) in Foxp3 ΔJag1 and Foxp3 Ctrl animals. Body weight throughout tamoxifen injections (Figure 2E) and SDLN live cell numbers (Figure 2F) remained unchanged between animals with or without Jag1 in Tregs during the steady state. Loss of Jag1 in Tregs did not alter overall CD25 or CTLA4 expression in skin Tregs (Figure 2G&H), nor the abundance and proliferation of skin-resident Tregs, Teff, CD8, gamma-delta T cells (Figure 2I &J) and other myeloid populations (Figure 2K&L, gating strategy in Supplementary Fig1B). Together, this suggests that Jag1 expression in Tregs is dispensable for maintaining skin immune homeostasis. Jag1 pos Tregs are Highly Activated During Early Wound Healing One of the most common skin traumas is cutaneous injury. Treg depletion hinders both epithelial restoration 4 and the full-thickness wound healing process 3 . One known function of Jag1 pos Tregs is their role in facilitating hair follicle stem cell (HFSC) proliferation during hair growth phase transitions 1 . Therefore, we investigated whether Jag1 pos Tregs are involved in wound healing. Wound healing comprises four major overlapping stages: haemostasis, inflammation, proliferation, and remodelling 23 . Skin Treg abundance at full-thickness wound sites peaks at 7 days post wounding (dpw) and returns to homeostatic levels by 14dpw 3 . In Foxp3-DTR mice that permits systemic loss of all Tregs, delayed wound healing is observed only when Tregs are depleted during the inflammation phase, but not later. Jag1 expression in whole skin lysates follows a similar dynamic, being upregulated during the first 7dpw and downregulated from 14dpw 24,25 . Thus, we hypothesised that the first 7 days are likely to be a crucial period in which Jag1 Pos Tregs may have an impact. We first characterised the dynamic expression of Jag1+Tregs by creating two 4mm full- thickness wounds on the dorsum of wildtype adult mice at 0 days post wounding (dpw) and harvesting skin around the wound at inflammatory (2dpw), proliferative (5dpw) and remodelling (12dpw) phases (Figure 3A). Notably, 25%-48% of skin Tregs expressed Jag1 during the first 5 days post-wounding, in contrast to 6.35% (±2.25%, p = 0.0014) at 12dpw (Figure 3B&C). At 5dpw, Jag1 pos +Tregs were significantly more proliferative (Figure 3D&E) and co-expressed higher levels of both CTLA4 and CD25 activation markers (Figure 3F&G), relative to Jag1 neg Tregs. In contrast, no differences in Ki67 or CTLA4+CD25+ proportions were observed between Jag1 pos and Jag1 neg skin Tregs at 2dpw, indicating that 5dpw is a critical period for Jag1+ Tregs’ functional prominence. Jag1+ Tregs Promote Wound Healing We then assessed the role of Jag1 pos Tregs in wound healing by creating two full thickness excisional wounds of the dorsal skin of Foxp3 ΔJag1 and Foxp3 Ctrl mice, and measuring the wound healing rate over time (Figure 4A). Mice lacking Jag1-expressing Tregs showed delayed wound closure compared to wildtype Foxp3 Ctrl mice treated with tamoxifen (Figure 4B & C). The most pronounced difference was observed at 5dpw, with 78.4% (± 9.3%) wound closure in wildtype Foxp3 Ctrl mice versus 66.2% (±15.9%) in Foxp3 ΔJag1 animals (Figure 4D, p = 0.0041). Histologically, Foxp3 ΔJag1 skin showed a trend towards thicker epithelial wound edges (Figure 4E, red arrow), more keratinocyte precipitation at wound edge (Figure 4E,black arrow ) and disorganisation between epithelial layer and pus cells (Fig 4E, white arrow). These results illustrate skin Tregs require Jag1 for effective wound closure particularly during the early (5 dpw) proliferation phase. Jag1+ Tregs Modulate Neutrophil Accumulation During Early Wound Healing We next explored the mechanisms by which skin Tregs use Jag1 to orchestrate wound closure. Upon tissue challenge, skin Tregs primarily perturb inflammation through two mechanisms: either via conventional immunosuppressive capacity 3,4 or enhance tissue repair via cross-talk with epithelial cells and HFSCs 1,4 . Dysregulation of skin Tregs leads to unwanted inflammation, mainly contributed by excessive accumulation and proliferation of neutrophils, pro-inflammatory macrophages, CD4+, and CD8+ T cells during homeostasis 20 , epidermal injury 4 , and full thickness injury 3 . Treg-specific loss of Rbpj, a downstream transcription factor of canonical Notch signalling, has been shown to mediate upregulation of Foxp3, CTLA4 and CD25 expression in splenic Tregs during homeostasis 19 . With Jag1 pos Tregs co-expressing higher level of CTLA4 and CD25 during both homeostasis and wound healing, relative to Jag1 neg Tregs, we questioned whether the absence of Jag1 in Tregs can affect the overall immunosuppressive ability of skin Tregs. We reasoned this may impact the accumulation of T and/or myeloid cells during wound healing and could influence wound closure through regulating local inflammation. Similar to the steady state (Figure 2), the absence of Jag1 in Tregs did not affect overall Treg abundance, as quantified by both flow cytometry and immunofluorescence staining (Supplementary Figure 4A&B). Proliferation and Foxp3 expression in skin Tregs also remained unaffected (Supplementary Figure 4C&D). None of the Treg phenotypic attributes were altered by loss of Jag1, including the proportion of skin Tregs co-expressing CTLA4+CD25+ (Supplementary Figure 4E), CD25 (Supplementary Figure4F) or ICOS (Supplementary Figure4G). IL33 is an alarmin highly induced in keratinocytes in response to cutaneous wounding 26 . IL33 receptor (ST2) is highly expressed in skin Tregs 10 , and recently found crucial for suppressing bleomycin-induced skin fibrosis 27 . However, we found that Tregs from Foxp3 ΔJag1 skin did not alter ST2 expression level either (Supplementary Figure4H). Intriguingly, Jag1 ablation in Tregs resulted in a mild but significant reduction in CTLA4 levels (Supplementary Fig4I). Yet, both the accumulation and proliferation of Teffs and CD8+ T cells remained unchanged between Foxp3 ΔJag1 and Foxp3 Ctrl skin (Supplementary Fig4J&K).Innate cells are first responders to cutaneous wounding. Previously, it has been reported that pro-inflammatory Ly-6C high macrophages accumulate at wound sites at 1 day post full-thickness wounding, contributing up to 60% of skin-resident macrophages, and gradually drop to around 10% at 7dpw 3 . Additionally, Treg depletion leads to a 6-fold increase of Ly-6C high macrophages at 7dpw, suggesting skin Tregs help transit the stages of wound healing from a pro-inflammatory to an anti-inflammatory environment. In contrast, Jag1 loss in Tregs did not lead to the same cellular skewing when compared to deletion of the entire Treg pool 3 . Instead, pro-inflammatory macrophage (defined as CD45+CD11b high F4/80+Ly-6C high Ly-6G low ) accumulation in wounded skin remained indifferent between mice with Jag1-deficient and -sufficient Tregs, at around 6% of total macrophages at 5dpw (Figure 5A&B). Similarly, the accumulation of inflammatory Ly6C+ monocytes was unchanged (Figure 5C&D), suggesting mice with Jag1-deficient Tregs remain capable of transitioning from a pro-inflammatory to an anti-inflammatory state during wound healing. Interestingly, the absence of Jag1 in Tregs led to less neutrophil influx into wounded skin, compared to wildtype controls at 5dpw, quantified by both flow cytometry and immunofluorescence staining (Figure 5E-H). Collectively, Jag1 is unlikely to be a key factor driving the widely appreciated immunosuppressive function of skin Tregs. Rather, Jag1 pos Tregs promote the retention of neutrophils during wound healing. Jag1+ Tregs do not Influence Re-epithelialization Re-epithelialisation is a crucial step for successful wound healing following the inflammatory phase, by preventing excess water loss and further entry of microbial pathogens or debris. Trans-epidermal water loss measurement (TEWL) estimates moisture evaporation externally and is used quantitatively to assess epidermal integrity 28 . Besides suppressing inflammation, skin Tregs drive epidermal barrier repair by promoting bulge HFSC emigration to the epidermis, as well as their proliferation and differentiation 4 . Previous lineage tracing studies have shown that bulge cells repopulate the epidermis at 5 days post full thickness wounding 29 . These studies also indicate that bulge HFSCs are proliferative and can migrate to cutaneous wounds from 4dpw but not earlier 30 , and the derived cells can be detected in the epidermis for up to one year 31 . This indicates that while bulge HFSCs may not contribute to the immediate re-epithelisation of wounds, they play an important role in restoring the skin barrier from 4dpw onwards. Given that Jag1 pos Tregs can drive bulge HFSC (identified as EpCam-Sca1-CD34+CD49f+) proliferation during hair regeneration 1 , we hypothesised Jag1 pos Tregs may also regulate wound closure by mediating bulge HFSC-driven re-epithelisation. However, mice with Jag1 deficiency in Tregs showed a similar TEWL restoration rate to baseline as wildtype controls (Supplementary Fig5A), suggesting that Jag1 pos Tregs do not influence barrier restoration post wounding. In line with this observation, the abundance and proliferation of bulge HFSCs also remain unaffected in Foxp3 D Jag1 mice during wound healing (Supplementary Figure 5C&D, gating strategy in Supplementary Figure 5B), indicating that Jag1 pos Tregs are unlikely to share the same mechanistic interaction with bulge HFSCs as observed in hair regeneration. DISCUSSION Despite understanding the functional importance of skin Tregs, whether skin Tregs carry out their functions through the same molecular mechanisms as Tregs residing in other tissues is incompletely understood. One candidate is TGF- signalling, recently shown to drive both hair regeneration 2 and epithelial barrier repair 5 . Yet, the functional importance of TGF- signalling extends globally to Treg generation and/or maintenance in both lymphoid and non-lymphoid tissues (Reviewed in 32 ). Given that Jag1 is preferentially expressed in skin Tregs, we questioned whether Jag1-mediated signals are uniquely utilized by skin Tregs to drive their skin-related functions. In the current study, we demonstrate that Jag1 is indeed preferentially expressed in skin Tregs. Jag1 expressing Tregs exhibit an activated profile, with upregulation of CD25 and CTLA4, but not ICOS, during both homeostasis and wound healing. Although it remains to be determined whether this upregulation reflects a higher immunosuppressive capacity of Jag1 pos Tregs, our survey of other non-Foxp3 expressing CD4+, CD8+ T cells, pro-inflammatory macrophages, or monocytes, indicates skin Tregs are unlikely to suppress in vivo inflammation through Jag1. Most notably, our study has highlighted the necessity of Jag1 expression in Tregs to facilitate adequate cutaneous wound repair. Consistent with previous findings on skin Tregs, the impact of Jag1 pos skin Tregs is limited to a critical time window (around 5 dpw) in which wound healing transitions from the inflammatory to the proliferation phase. However, rather than suppressing inflammation, Jag1 expression in Tregs seems to drive the retention of the inflammation phase, as evidenced by neutrophil accumulation within the wounded environment. Despite their well-known tissue-damaging properties, neutrophils are crucial facilitators in wound healing. They not only dissipate infiltrating pathogens, damaged cells, and debris, but also play an increasingly appreciated role in resolving inflammation by polarizing anti-inflammatory macrophages, encouraging vascularization, and potentially promoting local cell proliferation to repair tissues (reviewed in 33 ). Pertinently, mice lacking Notch activation in Lyz2+myeloid cells (Lyz2 cre RBPJ fl/fl ) show a milder inflammatory response in the heart, lung and kidney upon lipopolysaccharide exposure 34 . This is also associated with a reduction in neutrophil accumulation, reduced inflammatory cytokine detection in the liver after injury 35 , and pro-inflammatory macrophage reduction in spinal cord lesion sites after compression injury 36 . This suggests that during injury, the activation of Notch signalling in myeloid cells can promote inflammation. Mechanistically, intrinsic Notch signaling plays an important regulatory role in dampening Tregs’ own immunosuppressive functions 19 . Our study also showed that despite high CTLA4 and CD25 expression both homeostatically and during wound healing, Jag1 pos Tregs are unlikely to self-regulate immunosuppressive capacity. While it remains to be elucidated whether Jag1 pos Tregs directly influence Notch signalling in neutrophils, our study has uncovered an unexpected role for Jag1 pos Tregs in modulating the inflammatory phase of cutaneous wound healing. METHODS Animal study design All mouse procedures were approved by local ethical approval at King’s College London (UK) (PP70/8474, establishment license X24D82DFF), and performed under a UK Government Home Office license (PP6051479). All methods were carried out in accordance with relevant guidelines and regulations under the UK animals (Scientific procedures) Act 1986, and were reported in accordance with ARRIVE guidelines. All possible efforts were made to minimize animal suffering. All experiments were performed on animals with no prior procedures. Wildtype C57BL/6 and FoxP3 eGFP-CreERT2 mice (JAX: 016961 ) were purchased from Charles River, and Jag1 fl/fl (JAX: 010618 ) were gift from Prof Rosenblum in UCSF. FoxP3 eGFP-CreERT2 were crossed to Jag1 fl/fl to generate FoxP3 eGFP-CreERT2 Jag1 fl/fl in King’s College London BSU NHH animal unit. Mice were maintained through routine breeding, were fed with a standard chow diet and housed in line with UK regulations. Littermates of the same sex were genotyped and assigned to experimental groups based on genotyping results. Tamoxifen (Sigma, T5648-5G) were sonicated in 37 O C water bath and dissolved in corn oil, at 2.5mg/ml concentration. For conditional Jag1 knockout, 7-10 week old Foxp3 eGFP-Cre-ERT2 Jag1fl/fl and Foxp3 eGFP-Cre-ERT2 Jag1fl/wt or wt/wt were injected with tamoxifen intraperitonially at 75-100mg/kg in the indicated interval before harvesting. All characterisation and steady state experiments were performed on mice of mixed gender, and only females were used for further characterisation in wound healing experiments. Skin wounding assays and analysis Mice were anaesthetised by inhalation of vaporized 1.5% isoflurane, shaved and subcutaneously injected with Vetersgesics. Two full thickness excisional wounds were made on dorsal back of mice under and anaesthesia, using a 4-mm biopsy punch (Stifel Laboratory Research). Wounds were photographed daily till harvest. The same ruler was placed next to wound area for measurement standardization. Wound area was measured using ImageJ 37 (Fiji NIH), and closure ratio of each time point was calculated relative to wound area at 0dpw. Transepidermal water loss (TEWL) of each wound was measured with Tewameter TM 300 probe (Courage + Khazaka electronic GmbH) according to the manufacturer’s protocols. Measurement were made on 0dpw (immediately after excision wound) and every 24 hours thereafter. Each datapoint is an average of four TEWL measurements. Tissue Processing Whole murine dorsal skin was finely minced with scissors and digested in 500ul digestion medium per cm 2 of skin. Digestion medium was prepared with 2mg/ml collagenase (Sigma), 0.1mg/ml DNase (Sigma) and 0.5mg/ml hyaluronidase (Sigma), dissolved in C10 medium [10% FBS, 1% Pen/Strep, 1 mM Na-pyruvate, 1% HEPES, 1% non-essential amino acid, 0.5% 2-mercaptoethanol in RPMI-1640 with L-glutamine medium]. After 45 min incubation at 37 O C 255rpm, single cell suspension was washed with 20ml C10 medium, and filtered through 100um then 40um cell strainer. Lymph nodes were mechanically smashed, washed with FACS buffer [2% Fetal calf serum, 1mM EDTA and 0.1% sodium azaide in PBS], and filtered through 70um cell strainer. Epidermal cells were prepared by floating skin on 0.5%Trypsin-EDTA (Thermofisher) for 1hr at 37 O C, before gently removed from dermal part and washed with C10 medium. Lung was finely minced and digested with 1mg/ml Collagenase A (Sigma) and 0.1mg/ml DNase I (Sigma) in R10 [10% FBS and 1% Pen/Strep in RPMI-1640 medium]. After 1hr at 37 O C 180rpm, single cell suspension is passed through 70um filter, spin for 5 min at 4 O C 1800rpm, and treated with 500ul ACK Lysing Buffer for 30s to 1min before washed with 1xPBS. All single cell suspensions were then centrifuged at 1800rpm at 4 O C for 4 min, and resuspended in 1m FACS buffer. Total live cells were determined using NucleoCounter NC-200 (Chemometec) in 1:20 dilution, before downstream process. For histology, skin tissue was fixed in 10% formalin overnight, followed by PBS washes, stored at 70% Ethanol overnight, and embedded in paraffin. Each sample was cut and levelled to wound area, before manually assembled as tissue microarray blocks using a 7-mm biopsy punch. 5um sections were cut, mounted and sent to Tissueplexia (Scotland) for multiplex-staining using anti-FoxP3 (14-5773-82, Thermofisher) and anti-Ly6G (127602, Biolegend). H&E were performed according to manufacturer’s instruction and imaged using a Nanozoomer (Hamamatsu photonics) with a ×40 objective. Flow cytometry For flow cytometry staining, 1.5-4 million cells per condition were plated in round bottom 96 well plate, and stained with 50ul of stated surface antibodies on ice for 20 min. After washed with FACS buffer, cells were fixed and permeabilised by FoxP3/Transcription Factor Staining buffer set (eBioscience) on ice for another 20 min, before washed with permeabilization buffer and lastly stained with intracellular antibodies (resource table), again on ice for 20 min. Samples were run on Fortessa LSRII (BD Bioscience) in KCL BRC Flow Cytometry Core. For compensation, UltraComp eBeads TM (Thermofisher) were stained with each surface and intracellular antibody following the same cell staining protocol. ArC TM Amine Reactive Compensation Bead Kit (Thermofisher, A10346) were used for GhostDye TM Live/Dead stain. All gating and data analysis were performed using FlowJo v10, while statistics were calculated using Graphpad Prism 10. RNA isolation and quantification PCR Lymph node single cell suspension was resuspended in pre-sort medium [2% FBS, 1% Pen/Strep, 2 mM EDTA, 25mM HEPES in RPMI-1640 without phenol red]. CD45+ CD3+CD4+CD25high (Tregs), CD45+CD3+CD4+CD25neg(Teffs) and CD45+CD3+CD8+ cells were sorted using FACSAria™ Fusion Flow Cytometer (BD) with 100um nozzle in KCL BRC Flow Cytometry Core. Cells were sorted into RPMI-1640 supplemented with 10% heat-inactivated FBS and 1% Pen/Strep, and spun at 300g for 10min at 4 O C. After removal of supernatant, cell pellet were snap-frozen in liquid nitrogen and stored at -80 O C. RNA was extracted using NucloSpin RNA XS, Micro kit for RNA purification (740902, Macherey-Nagel) according manufacturer’s instruction. RNA integrity and concentration were then determined by RNA 6000 Pico Kit on Bioanalyzer (Agilent). RNA were then normalized and synthesied into cDNA using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR were performed using TaqMan TM PreAmp Master Mix Kit (ThermoFisher) on 384-well plate according to manufacturer’s instruction, run with 4 technical replica each condition. The following TaqMan probes were used: Gapdh (Mm99999915_g1, VIC), Foxp3 (Mm00475162_m1, FAM) and Jag1 (Mm00496904_m1, FAM), Plates were then run on CFX384 Touch Real-Time PCR system (BioRad). QUANTIFICATION AND STATISTICAL ANALYSIS Parameters such as sample size, dispersion or precision are reported in Figure Legends. Statistical analyses were performed in Prism 10.1 (GraphPad). Details of the statistics and appropriate test used are also indicated in Figure Legends. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.001, p-values greater than 0.05 was identified as not statistically significant. Bulk RNAseq reanalysis Read-count tables were obtained from NCBI database: GSE182322 (Ref 10 ), and downstream processed according to presented method. Each data point in Figure 1 represents the sum of both gene raw counts from ST2- and ST2+ Tregs per mouse. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse T Cell Panel CD45 Monoclonal Antibody (30-F11), Alexa Fluor 700, eBioscience™ ThermoFisher Cat# 56-0451-82, RRID: AB_891454 CD3 Antibody, anti-mouse, PerCP-Vio® 700, REAfinity™ Miltenyi Biotec Cat# 130-120-826, RRID: AB_2752207 TCRγ/δ Antibody, anti-mouse, PE-Vio® 770, REAfinity™ Miltenyi Biotec Cat# 130-123-290, RRID: AB_2802022 Brilliant Violet 650™ anti-mouse CD4 Antibody BioLegend Cat# 100545, RRID: AB_11126142 CD8a Antibody, anti-mouse, APC-Vio® 770, REAfinity™ Miltenyi Biotec Cat# 130-120-806, RRID: AB_2752203 CD25 Antibody, anti-mouse, PE-Vio® 615, REAfinity™ Miltenyi Biotec Cat# 130-123-028, RRID: AB_2811434 BD Horizon™ BV605 Hamster Anti-Mouse CD27 BD Biosciences Cat# 563365, RRID: AB_2738160 Jagged1 Antibody (E-12) Santa Cruz Biotechnology Cat# Sc-390177, RRID: AB_2892141 Anti-Mouse CD339 (Jagged 1) PE eBioscience Cat# 12-3391-80, RRID: AB_1963616 Brilliant Violet 711™ anti-human/mouse/rat CD278 (ICOS) Antibody BioLegend Cat# 313547, RRID: AB_2734288 BD Horizon™ BV786 Mouse Anti-Ki-67 BD Biosciences Cat# 563756, RRID: AB_2732007 FoxP3 Antibody, anti-mouse, Vio® R667, REAfinity™ Miltenyi Biotec Cat# 130-111-682, RRID: AB_2651771 Brilliant Violet 421™ anti-mouse CD152 Antibody BioLegend Cat# 106311, RRID: AB_10901170 Anti-Mouse/Rat Foxp3 eFluor 450 eBioscience Cat# 48-5773-82 Anti-Mouse CD152 (CTLA4) PE BD Cat# 553720 Anti-Human Ki67 PE-Cy7 BD Cat# 561283 Anti-Mouse TCR gd PerCP-Cy 5.5 Biolegend Cat# 118117 Anti-Mouse CD45 Alexa Fluor 700 eBioscience Cat# 56-0451-82 Anti-Mouse CD25 APC-eFluor 780 eBioscience Cat# 47-0251-82 Brilliant Violet 605 anti-mouse CD8a Antibody Biolegend Cat# 100743 Brilliant Violet 650™ anti-mouse CD4 Antibody Biolegend Cat# 100545 Brilliant Violet 711 anti-mouse CD3 Antibody Biolegend Cat# 100241 Mouse Myeloid Panel BD Horizon™ BUV395 Rat Anti-Mouse Ly-6G BD Biosciences Cat# 565964, RRID: AB_2716852 Brilliant Violet 421™ anti-mouse I-A/I-E Antibody BioLegend Cat# 107632, RRID: AB_2650896 Brilliant Violet 510™ anti-mouse/human CD45R/B220 Antibody BioLegend Cat# 103248, RRID: AB_2650679 Brilliant Violet 510™ anti-mouse CD3 Antibody BioLegend Cat# 100234, RRID: AB_2562555 Brilliant Violet 650™ anti-mouse CD11c Antibody BioLegend Cat# 117339, RRID: AB_2562414 Brilliant Violet 785™ anti-mouse F4/80 Antibody BioLegend Cat# 123141, RRID: AB_2563667 FITC anti-mouse Ly-6C Antibody BioLegend Cat# 128005, RRID: AB_1186134 CD207 (Langerin) Monoclonal Antibody (eBioL31), PE, eBioscience™ ThermoFisher Cat# 12-2075-82, RRID: AB_763452 CD45 Monoclonal Antibody (30-F11), Alexa Fluor 700, eBioscience™ ThermoFisher Cat# 56-0451-82, RRID: AB_891454 APC/Cyanine7 anti-mouse/human CD11b Antibody BioLegend Cat# 101225, RRID: AB_830641 Epidermal panel BD Horizon™ BUV395 Rat Anti-Mouse CD45 BD Biosciences Cat# 564279 CD326 (EpCAM) Antibody, anti-mouse, REAfinity™ Miltenyi Cat# 130-117-871 BD Horizon™ BV786 Mouse Anti-Ki-67 BD Biosciences Cat# 563756 BD Horizon™ BV650 Rat Anti-Human CD49f BD Biosciences Cat# 563707 Alexa Fluor® 700 anti-mouse I-A/I-E Antibody Biolegend Cat# 107622 CD207 (Langerin) Antibody, anti-mouse, PE-Vio® 615, REAfinity™ Miltenyi Cat# 130-112-994 BD Pharmingen™ Alexa Fluor® 647 Rat anti-Mouse CD34 BD Biosciences Cat# 560230 Sca-1 Antibody, anti-mouse, REAfinity™ Miltenyi Cat# 130-106-220 Chemicals, peptides, and recombinant proteins Tamoxifen Sigma-Aldrich Cat# T5648 Collagenase Sigma-Aldrich Cat #C9407 DNase Sigma-Aldrich Cat #DN25 Hyaluronidase Sigma-Aldrich Cat #H3506 Collagenase A Sigma-Aldrich Cat# 10103578001 DNase 1 Sigma-Aldrich Cat# 10104159001 ACK Lysing Buffer ThermoFisher Cat# A1049201 Fetal Bovine Serum ThermoFisher Cat# 10500064 Penicillin-Streptomycin (5,000 U/mL) ThermoFisher Cat# 15070063 Sodium Pyruvate (100 mM) ThermoFisher Cat# 11360070 HEPES (1M) ThermoFisher Cat# 15630056 MEM Non-Essential Amino Acids Solution (100X) ThermoFisher Cat# 11140050 2-mercaptoethanol ThermoFisher Cat# 21985023 RPMI-1640 medium ThermoFisher Cat# 11875093 UltraPure™ 0.5M EDTA, pH 8.0 ThermoFisher Cat# 15575020 PBS, pH 7.4 ThermoFisher Cat# 10010023 FoxP3/Transcription Factor Staining buffer set eBioscience Cat# 00-5523-00 UltraComp eBeads Thermofisher Cat# 01-2222-42 ArCTM Amine Reactive Compensation Bead Kit Thermofisher Cat# A10346 Zombie UV™ Fixable Viability Kit BioLegend Cat# 423107 Deposited data Bulk RNAseq data Spath et al( 13 ) GEO: GSE182322 Experimental Models: Organisms/Strains WT C57BL/6J Charles River RRID: IMSR_CRL:027 Foxp3 eGFP-Cre-ERT2 (Foxp3 tm9(EGFP/cre/ERT2)Ayr /J) C57BL/6 The Jackson Laboratory RRID: IMSR_JAX:016961 Jag1 fl/fl (B6.129S- Jag1 tm2Grid /J) The Jackson Laboratory RRID: IMSR_JAX:010618 Software and Algorithms FlowJo (version 10.8.1) Becton Dickinson https://www.flowjo.com/solutions/flowjo; RRID:SCR_008520 Fuji (Image J) Fuji (Image J) https://fiji.sc NDP.view2 Image viewing software (U12388-01) Hamamatsu https://www.hamamatsu.com/us/en/product/life-science-and-medical-systems/digital-slide-scanner/U12388-01.html RRID:SCR_025177 QuPath (version 0.5.0-arm64) Bankhead et al. https://qupath.github.io/ Prism (version 10.1.0) GraphPad Software http://www.graphpad.com/; RRID:SCR_002798 Inkscape (version 1.3.2) Inkscape https://inkscape.org/; RRID:SCR_014479 R (version 4.3.2) CRAN https://www.r-project.org/ RStudio (version 2021.09.0) RStudio https://www.rstudio.com/ R package: edgeR (version 4.0.16) Robinson et al ( 38 ) http://bioconductor.org/packages/edgeR/ RRID:SCR_012802 R package: limma (version 3.58.1) Ritchie et al http://bioinf.wehi.edu.au/limma/ RRID:SCR_010943 R package: org.Mm.eg.db Carlson M. https://bioconductor.org/packages/org.Mm.eg.db/ (version 3.58.1) RRID:SCR_023488 Other NanoZoomer BD FACSAria II Cell Sorter --- BD LSR-Fortessa analyzer 4-mm biopsy punch Stifel Declarations Acknowledgments We thank the Advanced Cytometry Platform (Flow Core), Research and Development Department at Guy’s and St Thomas' NHS Foundation Trust for assistance with flow cytometry experiments. Funding We acknowledge support by the following grant funding bodies: This work was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society awarded to N.A (Grant Number 213401/Z/18/Z). P.P.L. and J.Z.X are supported by Wellcome Trust PhD fellowships (108874/B/15/Z), and (218452/Z/19/Z). Author contributions P.L.: investigation, data curation, analysis, writing – original draft, writing – Review & Editing; J.Z.X.: investigation and methodology; H.A.: investigation; M.S.: investigation; N.A. conceptualization, supervision, data curation, funding acquisition, writing – original draft, writing – Review & Editing. Declaration of interests The authors declare that they do not have any conflict of interest. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request CONTACT FOR REAGENT AND RESOURCE SHARING Requests for reagents and resources should be directed to the Lead Contact, Niwa Ali ( [email protected] ) References Ali, N. et al. Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation. Cell (2017) doi:10.1016/j.cell.2017.05.002. Liu, Z. et al. Glucocorticoid signaling and regulatory T cells cooperate to maintain the hair follicle stem cell niche. Nat. Immunol. 23 , 1086–1097 (2022). Nosbaum, A. et al. Cutting Edge: Regulatory T Cells Facilitate Cutaneous Wound Healing. J. Immunol. Baltim. Md 1950 196 , 2010–2014 (2016). Mathur, A. N. et al. Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair. Immunity 50 , 655-667.e4 (2019). Moreau, J. M. et al. Regulatory T cells promote innate inflammation following skin barrier breach via TGF-β activation. Sci. Immunol. 6 , eabg2329 (2021). Kalekar, L. A. et al. Regulatory T cells in skin are uniquely poised to suppress profibrotic immune responses. Sci. Immunol. 4 , eaaw2910 (2019). Delacher, M. et al. Genome-wide DNA methylation landscape defines specialization of regulatory T cells in tissues. Nat. Immunol. 18 , 1160–1172 (2017). DiSpirito, J. R. et al. Molecular diversification of regulatory T cells in non-lymphoid tissues. Sci. Immunol. 3 , eaat5861 (2018). Miragaia, R. J. et al. Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of Tissue Adaptation. Immunity 50 , 493-504.e7 (2019). Spath, S., Roan, F., Presnell, S. R., Höllbacher, B. & Ziegler, S. F. Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes. iScience 25 , 104998 (2022). Lee, E., Kim, M. & Lee, Y. J. Selective Expansion of Tregs Using the IL-2 Cytokine Antibody Complex Does Not Reverse Established Alopecia Areata in C3H/HeJ Mice. Front. Immunol. 13 , 874778 (2022). Watt, F. M., Estrach, S. & Ambler, C. A. Epidermal Notch signalling: differentiation, cancer and adhesion. Curr. Opin. Cell Biol. 20 , 171–179 (2008). Chigurupati, S. et al. Involvement of Notch Signaling in Wound Healing. PLOS ONE 2 , e1167 (2007). Lehar, S. M., Dooley, J., Farr, A. G. & Bevan, M. J. Notch ligands Delta 1 and Jagged1 transmit distinct signals to T-cell precursors. Blood 105 , 1440–1447 (2005). Amsen, D., Helbig, C. & Backer, R. A. Notch in T Cell Differentiation: All Things Considered. Trends Immunol. 36 , 802–814 (2015). Yvon, E. S. et al. Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells. Blood 102 , 3815–3821 (2003). Gopisetty, A. et al. OX40L/Jagged1 cosignaling by GM-CSF-induced bone marrow-derived dendritic cells is required for the expansion of functional regulatory T cells. J. Immunol. Baltim. Md 1950 190 , 5516–5525 (2013). Lin, C., Huang, H., Hsieh, C., Fan, C. & Lee, Y. Jagged1‐expressing adenovirus‐infected dendritic cells induce expansion of Foxp3+ regulatory T cells and alleviate T helper type 2‐mediated allergic asthma in mice. Immunology 156 , 199–212 (2019). Charbonnier, L.-M., Wang, S., Georgiev, P., Sefik, E. & Chatila, T. A. Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling. Nat. Immunol. 16 , 1162–1173 (2015). Cohen, J. N. et al. Regulatory T cells in skin mediate immune privilege of the hair follicle stem cell niche. Sci. Immunol. 9 , eadh0152 (2024). Rubtsov, Y. P. et al. Stability of the regulatory T cell lineage in vivo. Science 329 , 1667–1671 (2010). Kiernan, A. E., Xu, J. & Gridley, T. The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet. 2 , e4 (2006). Rodrigues, M., Kosaric, N., Bonham, C. A. & Gurtner, G. C. Wound Healing: A Cellular Perspective. Physiol. Rev. 99 , 665–706 (2019). Chen, X.-D. et al. Effects of porcine acellular dermal matrix treatment on wound healing and scar formation: Role of Jag1 expression in epidermal stem cells. Organogenesis 14 , 25–35 (2018). Shi, Y. et al. Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately. Stem Cell Res. Ther. 6 , 120 (2015). Dai, X. et al. Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB. JID Innov. 3 , 100205 (2023). Cheon, S. Y. et al. IL-33/Regulatory T-Cell Axis Suppresses Skin Fibrosis. J. Invest. Dermatol. 142 , 2668-2676.e4 (2022). Czaika, V. et al. Comparison of Transepidermal Water Loss and Laser Scanning Microscopy Measurements to Assess Their Value in the Characterization of Cutaneous Barrier Defects. Skin Pharmacol. Physiol. 25 , 39–46 (2011). Ito, M. et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat. Med. 11 , 1351–1354 (2005). Huang, S. et al. Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization. Cell Stem Cell 28 , 1582-1596.e6 (2021). Page, M. E., Lombard, P., Ng, F., Göttgens, B. & Jensen, K. B. The Epidermis Comprises Autonomous Compartments Maintained by Distinct Stem Cell Populations. Cell Stem Cell 13 , 471–482 (2013). Moreau, J. M., Velegraki, M., Bolyard, C., Rosenblum, M. D. & Li, Z. Transforming growth factor–β1 in regulatory T cell biology. Sci. Immunol. 7 , eabi4613 (2022). Oliveira-Costa, K. M., Menezes, G. B. & Paula Neto, H. A. Neutrophil accumulation within tissues: A damage x healing dichotomy. Biomed. Pharmacother. 145 , 112422 (2022). Bai, X. et al. Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development. Front. Immunol. 9 , 762 (2018). Yu, H.-C. et al. Blocking Notch signal in myeloid cells alleviates hepatic ischemia reperfusion injury by repressing the activation of NF-κB through CYLD. Sci. Rep. 6 , 32226 (2016). Chen, B.-Y. et al. Myeloid-Specific Blockade of Notch Signaling by RBP-J Knockout Attenuates Spinal Cord Injury Accompanied by Compromised Inflammation Response in Mice. Mol. Neurobiol. 52 , 1378–1390 (2015). Schindelin, J., Rueden, C. T., Hiner, M. C. & Eliceiri, K. W. The ImageJ ecosystem: An open platform for biomedical image analysis. Mol. Reprod. Dev. 82 , 518–529 (2015). Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTARYFIGURES.docx Cite Share Download PDF Status: Published Journal Publication published 09 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 22 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers agreed at journal 24 Jun, 2024 Reviewers invited by journal 24 Jun, 2024 Editor assigned by journal 24 Jun, 2024 Editor invited by journal 13 Jun, 2024 Submission checks completed at journal 12 Jun, 2024 First submitted to journal 10 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4559523\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":319415498,\"identity\":\"275792a9-4f48-4af2-ab81-0bd2e26f4254\",\"order_by\":0,\"name\":\"Prudence PokWai Lui\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"King's College London\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Prudence\",\"middleName\":\"PokWai\",\"lastName\":\"Lui\",\"suffix\":\"\"},{\"id\":319415499,\"identity\":\"1b59d718-69d7-482e-a869-a306ebfd2d04\",\"order_by\":1,\"name\":\"Jessie Z. 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(A)\\u003c/strong\\u003e Jag1 mRNA counts from bulk RNAseq of Tregs from different tissue sites. (n = 5). (\\u003cstrong\\u003eB\\u003c/strong\\u003eand \\u003cstrong\\u003eC\\u003c/strong\\u003e) Heatmap showing the adjusted p-value (B) and log fold change (C) of genes expressed in Tregs from skin compared against those from other tissues. (\\u003cstrong\\u003eD\\u003c/strong\\u003e and \\u003cstrong\\u003eE\\u003c/strong\\u003e) Representative flow cytometry plot and quantification (E) of Jag1+ Tregs in skin, lung and skin-draining lymph nodes (SDLNs) (n = 8-15). (\\u003cstrong\\u003eF\\u003c/strong\\u003e and \\u003cstrong\\u003eG\\u003c/strong\\u003e) Representative histogram (F) and quantification (G) of Jag1 expression in different skin T-cell populations (n= 8). Data in (D,E) or (F,G) were pooled from 3 independent experiments. Individual data points are shown and presented as mean ± SEM. Statistics in (A), (E) and (G) were calculated by one-way ANOVA, ** \\u003cem\\u003ep \\u0026lt;\\u003c/em\\u003e0.01, **** \\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/f1d4121aeb37250b76a3fe19.png\"},{\"id\":59161796,\"identity\":\"105bc489-40bf-4b2e-bd36-064294f315ff\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 05:30:51\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":330735,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eJag1 in \\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003eTregs is dispensable for skin homeostasis. \\u003c/strong\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e) Experimental schematic illustrating intraperitoneal injection of 100mg/kg dose of tamoxifen daily for 5 days, with 5-18 days rests before harvest for downstream analysis. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) Representative H\\u0026amp;E staining of Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eDJag1\\u003c/sup\\u003e skin. Scale bars represent 100mm. (C and D) Quantification of epidermal thickness (C) and lymphocyte infiltration (D) from three regions-of-interest in H\\u0026amp;E staining of Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eDJag1 \\u003c/sup\\u003eskin (n = 2 per group)(E) Average weight of treated animals traced throughout the course of tamoxifen injection and harvest end point (n = 4 per group). (F) Total live cells from Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eDJag1\\u003c/sup\\u003e SDLNs (n = 8 per group). (G and H) Flow cytometric quantification of mean fluorescence intensity (MFI) of CTLA4 (G) and CD25 (H) in Tregs from Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eDJag1\\u003c/sup\\u003e skin (n = 6-7). (I and J) Quantification of skin T cell abundance (I) and proliferation (J) in Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eDJag1\\u003c/sup\\u003e mice (n = 6-7 per group). (K and L) Quantification of\\u0026nbsp; neutrophils (neut), CD11c+ and CD11c- dendritic cells (DCs), macrophages (mac) and monocytes (mono) from wildtype Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eDJag1 \\u003c/sup\\u003emice (n = 4-6 per group). Data in\\u0026nbsp; (E) to (L) were harvested from 4 independent experiments collected between 5 - 18 days post last tamoxifen injection. Data in (B to D) were from 1 and (K and L) from 2 of these 4 experiments.\\u0026nbsp; Each individual data point represent one biological replicate. Results were presented as mean ± SEM. Statistics were calculated by unpaired t-test (C, D, F, G and H) and two-way ANOVA (I, J, K and L), ns = non-significant. \\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/da50d5f13b770f13dafce0a3.png\"},{\"id\":59161792,\"identity\":\"572ba465-317e-4f1a-97a9-caf13d206079\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 05:30:51\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":223599,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eJag1+Tregs are most abundant and activated at 5 days post wounding. (A)\\u003c/strong\\u003eExperimental schematic in which two circular full thickness wounds were generated on the dorsum of wildtype mice using a 4mm punch biopsy. Skin was harvested at 2 days post wound (dpw), 5dpw and 12 dpw. (\\u003cstrong\\u003eB\\u003c/strong\\u003e and \\u003cstrong\\u003eC\\u003c/strong\\u003e) Representative flow plots and quantification of % Tregs expressing Jag1 in wounded skin at 2dpw, 5dpw and 12dpw (n = 4-12 per group). Representative flow plots and quantification of (\\u003cstrong\\u003eD\\u003c/strong\\u003e and \\u003cstrong\\u003eE\\u003c/strong\\u003e) %Ki67 and (\\u003cstrong\\u003eF\\u003c/strong\\u003e and \\u003cstrong\\u003eG\\u003c/strong\\u003e) %CTLA4+CD25+ in Jag1+ Tregs and Jag1- Tregs from 2dpw and 5dpw wounded skin (n = 4-15 per group). Data in (B) to (G) were pooled from 2 independent experiments. Results were presented as individual data points with mean ± SEM in (C) and paired data-points in (E and G). Statistics were calculated by one-way ANOVA (A) and two-way ANOVA (E and G), * \\u003cem\\u003ep \\u0026lt; \\u003c/em\\u003e0.05, **\\u003cem\\u003e p \\u0026lt; \\u003c/em\\u003e0.01, ns = non-significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/75589667e6f2c751c3418e0d.png\"},{\"id\":59161793,\"identity\":\"fbe4d11b-10d1-4637-83e4-58c3ae38d613\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 05:30:51\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":576957,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eJag1+ Tregs are required for wound healing. (A)\\u003c/strong\\u003e Experimental schematic in which Foxp3\\u003csup\\u003eΔJag1\\u003c/sup\\u003e and “Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e mice were injected with 4 doses of tamoxifen, before wounding with 4mm biopsy punches. Two more doses of tamoxifen were given thereafter before wounded skin were harvested at 5dpw. \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Representative clinical images of the healing progression at 0dpw (immediate after wounding), 2dpw, and 5dpw \\u003cstrong\\u003e(C \\u003c/strong\\u003eand\\u003cstrong\\u003e D)\\u003c/strong\\u003e Quantification of wound closure, calculated by fold change against wound area at 0dpw, with (C) showing the kinetics throughout the experiment, and (D) ratio at 5dpw \\u0026nbsp;(n = 20-22 per group). (E) Representative H\\u0026amp;E image of Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eΔJag1 \\u003c/sup\\u003ewounded skin harvested at 5dpw, with labels of scab (S), pus cells (P) and epidermis (Ep). Arrows illustrated region of interest, including epidermal wound edge (red),\\u0026nbsp; keratinocyte precipitation (black) and border between pus cells and epidermis under scab (white). Scale bars represent 250mm. (n = 4 per group). Data were pooled from 4 independent experiments. Results were presented as mean ± SEM in (C) and individual biological replicates as each data point with mean ± SEM in (D). Statistics were calculated by two-way ANOVA in (C), and unpaired t-test in (D).* \\u003cem\\u003ep \\u0026lt; \\u003c/em\\u003e0.05, **\\u003cem\\u003e p \\u0026lt; \\u003c/em\\u003e0.01, *** \\u003cem\\u003ep \\u0026lt; \\u003c/em\\u003e0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/f44453a1f9c259dafc54ca97.png\"},{\"id\":59162426,\"identity\":\"4ea67bb0-6b8c-483d-ba4f-7fbb26c33816\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 05:46:51\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":352746,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eJag1+Tregs impact neutrophil accumulation in wounded skin.\\u003c/strong\\u003e Representative flow plot and quantitation of (\\u003cstrong\\u003eA\\u003c/strong\\u003e and \\u003cstrong\\u003eB\\u003c/strong\\u003e) pro-inflammatory Ly6C+ macrophages, (\\u003cstrong\\u003eC\\u003c/strong\\u003e and \\u003cstrong\\u003eD\\u003c/strong\\u003e) Ly6C+ monocytes and (\\u003cstrong\\u003eE\\u003c/strong\\u003e and \\u003cstrong\\u003eF\\u003c/strong\\u003e) neutrophils in wounded skin at 5dpw (n = 8-10 per group). (G) Representative immunofluorescence staining of Ly6G of wounded skin, with labels of wound site (w) and hair follicle (Hf). Scale bars represent 50mm. (H) Quantification of Ly6G+ neutrophils from (G) within a fixed region-of-interest. (n = 4 per group). Data were pooled from 2 independent experiments. Each individual data point represented a biological replicate, and was collectively presented with mean ± SEM. Statistics were calculated by two-way ANOVA (B, D and F) and unpaired T-test (H). **\\u003cem\\u003e p \\u0026lt; \\u003c/em\\u003e0.01, *** \\u003cem\\u003ep \\u0026lt; \\u003c/em\\u003e0.001, ns = non-significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/f9d25ead12a4c58966a36c03.png\"},{\"id\":64619739,\"identity\":\"ceec4ad7-eae9-4c3f-b4c4-0ab9643760f7\",\"added_by\":\"auto\",\"created_at\":\"2024-09-16 16:17:05\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2663908,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/e13bd9fb-a089-4712-9498-9160fd85e677.pdf\"},{\"id\":59162066,\"identity\":\"0a2087d4-f7f1-46f7-be45-f9c734ff6f37\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 05:38:52\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1573340,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SUPPLEMENTARYFIGURES.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559523/v1/9f340a729c539d0ac158f1f7.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Jagged-1+ Skin Tregs Modulate the Innate Immune Response to Wound Healing\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eThe skin, the largest mammalian organ, acts as a critical physical barrier from constant environmental traumas. A diverse array of immune and non-immune cell types reside in or migrate to the skin during pre- and post-natal development, influenced by the local inflammatory microenvironment. Over the past decade, skin resident regulatory T cells (Tregs), a major T cell population, have been implicated in various processes, including hair regeneration\\u003csup\\u003e1,2\\u003c/sup\\u003e, full-thickness wound healing\\u003csup\\u003e3\\u003c/sup\\u003e, epidermal barrier repair\\u003csup\\u003e4,5\\u003c/sup\\u003e and fibrosis\\u003csup\\u003e6\\u003c/sup\\u003e. These functions are facilitated by the immunosuppressive capacity of skin Tregs, including restriction of pro-inflammatory cytokine production and myeloid cell accumulation\\u003csup\\u003e3,4\\u003c/sup\\u003e, as well as their interaction with non-immune tissue cells such as epithelial keratinocytes\\u003csup\\u003e5\\u003c/sup\\u003e, fibroblasts\\u003csup\\u003e6\\u003c/sup\\u003e and stem cells\\u003csup\\u003e1,4\\u003c/sup\\u003e. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMultiple single cell studies have illustrated tissue Tregs are transcriptionally distinct from those within secondary lymphoid organs\\u003csup\\u003e7\\u0026ndash;10\\u003c/sup\\u003e. The site where Tregs are seeded determines their phenotype and shapes their functions\\u003csup\\u003e7,10\\u003c/sup\\u003e. Previous RNA sequencing analysis identified Jagged-1 (Jag1), one of the five Notch signalling ligands, as a key transcript preferentially expressed in skin Tregs compared to skin-draining lymph node (SDLN) Tregs\\u003csup\\u003e1\\u003c/sup\\u003e. Interestingly, intraperitoneal injection of IL-2/anti-IL2 antibody complexes can selectively expand Jag1+Tregs in murine skin but not in SDLNs\\u003csup\\u003e11\\u003c/sup\\u003e. \\u003cem\\u003eIn silico\\u003c/em\\u003e data indicate that skin Tregs are also phenotypically distinct from other tissue-resident Tregs\\u003csup\\u003e6,9,10\\u003c/sup\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eLigand-receptor prediction analyses from scRNA-seq datasets further suggest that skin Tregs likely interact with epithelial cells and hair follicle stem cells via Jag1-induced Notch signalling\\u003csup\\u003e2,5\\u003c/sup\\u003e, underscoring the potential importance of Jag1 in skin Tregs. Yet, the question of whether Jag1 expression is indeed unique to skin Tregs remains unanswered.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eNotch signalling is crucial for hair follicle differentiation and postnatal maintenance homeostatically\\u003csup\\u003e12\\u003c/sup\\u003e, as well as in regulating wound healing\\u003csup\\u003e13\\u003c/sup\\u003e. \\u0026nbsp;Conditional deletion of Jag1 in epidermal stem cells (K15\\u003csup\\u003ecre\\u003c/sup\\u003eJag1\\u003csup\\u003efl/fl\\u003c/sup\\u003e) delays wound closure, whereas the addition of Jag1 peptide accelerates wound healing\\u003csup\\u003e13\\u003c/sup\\u003e, demonstrating the significance of Jag1 in the wound repair process. Although it has been established that Jag1 expressed on non-lymphoid cells can drive T cell fate decisions\\u003csup\\u003e14,15\\u003c/sup\\u003e, promote Treg expansion\\u003csup\\u003e16\\u0026ndash;18\\u003c/sup\\u003e\\u003cem\\u003e,\\u003c/em\\u003e and that Notch signalling regulates Treg immunosuppressive capacity\\u003csup\\u003e19\\u003c/sup\\u003e, the function of Treg-derived Jag1 remains largely unknown. Previously, we have shown the perturbation of Jag1 in skin Tregs hinders hair follicle stem cell (HFSC) proliferation, delays the induction of the hair growth phase, and ultimately impedes hair regeneration\\u003csup\\u003e1\\u003c/sup\\u003e. Whether the relationship between Jag1+ Tregs and hair regeneration translates to other skin Treg-mediated mechanisms, such as wound healing, remains unexplored. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHere, we report that Jag1 is preferentially expressed in skin Tregs compared to Tregs residing in other tissues. Despite Jag1+Tregs displaying higher CTLA4 and CD25 surface expression, the absence of Jag1 in Tregs does not alter skin integrity nor overall cutaneous immune dynamics during homeostasis. However, during full thickness wound healing, mice with Jag1 deficiency in Tregs heal significantly slower than controls. Unlike in hair regeneration, skin Tregs in wounded mice do not utilize Jag1 to alter HFSC activation but rather promote neutrophil accumulation at the wound site. This study sheds light on an alternative function of Jag1 in skin Tregs in facilitating adequate cutaneous wound repair.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eJagged1 is a Skin Treg Preferential Marker\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo determine whether Jag1 is uniquely expressed in skin Tregs, we began by re-analysing a bulkRNAseq dataset focusing on Tregs from various tissues\\u003csup\\u003e10\\u003c/sup\\u003e. Our analysis confirmed that skin Tregs exhibited the highest levels of \\u003cem\\u003eJag1\\u003c/em\\u003e compared to Tregs from blood, spleen, and other organ tissues (visceral adipose tissue (VAT), lung and colon) (Figure 1A). Differential expression analysis using edgeR and limma packages normalized counts and compared the adjusted p-values (Fig1B) and log fold changes (Fig1C) of Jag1 expression in skin Tregs against other tissue Tregs. \\u0026nbsp;We included Cd45 (Ptprc) and common Treg markers (Foxp3, Ctla4, Cd25, and Icos), along with known tissue Treg-associated transcripts (Gata3 and Areg) as controls. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eNotably, \\u003cem\\u003eJag1\\u003c/em\\u003e expression in skin Tregs was significantly and consistently higher than, not only those in blood or lymphoid organs, but also Tregs residing in lung, colon and VAT (Figure 1B\\u0026amp;C). Skin Tregs expressed an average 4.5-fold higher level of \\u003cem\\u003eJag1\\u0026nbsp;\\u003c/em\\u003ethan other surveyed Tregs, clearly distinguishing Jag1 from other Treg-associated genes. As expected, \\u003cem\\u003eCd45 (Ptprc) showed no significant differences (high adjusted p-value and low log fold change) between skin and other tissue Tregs. Skin Tregs expressed similar level of Foxp3\\u0026nbsp;\\u003c/em\\u003eand common Treg markers, as well as tissue Treg-associated genes, compared to Tregs in the lung, colon, and VAT\\u003cem\\u003e. While Ctla4 and Gata3 expression levels remained similar,\\u0026nbsp;\\u003c/em\\u003e\\u003cem\\u003eFoxp3\\u003c/em\\u003e and \\u003cem\\u003eIcos\\u003c/em\\u003e were significantly but mildly upregulated\\u003cem\\u003e\\u0026nbsp;in skin Tregs, when compared to those in blood or spleen. Similar to Jag1,\\u0026nbsp;\\u003c/em\\u003e\\u003cem\\u003eAreg\\u003c/em\\u003e was highly expressed in skin Tregs compared to splenic Tregs, but not when compared to other tissue Tregs. \\u003cem\\u003eCollectively, this\\u0026nbsp;\\u003c/em\\u003ehighlights Jag1 as a differentially expressed marker in skin Tregs.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTo further validate these findings, we performed flow cytometry on Tregs from skin, lung and SDLN. Around 55.59% (\\u0026plusmn;12.61%) of skin Tregs expressed Jag1, in contrast to only 8.18% (\\u0026plusmn;3.20%, p \\u0026lt; 0.0001) in lung Tregs and 3.54% (\\u0026plusmn;1.90%, p \\u0026lt; 0.0001) in SDLN Tregs (Figure 1D \\u0026amp; E, gating strategy in Supplementary Fig1A). The mean fluorescence intensity of Jag1 was significantly higher in skin Tregs (16.72 \\u0026plusmn; 2.15), in comparison to other CD4+Foxp3- T effector (Teffs) (6.95 \\u0026plusmn; 6.57, p = 0.0014) and CD8+ T cells (8.10 \\u0026plusmn; 5.01, p = 0.0041), indicating preferential expression of Jag1 in skin Tregs.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWe then examined whether these abundant Jag1\\u003csup\\u003epos\\u003c/sup\\u003e skin Tregs are phenotypically distinct from non-Jag1-expressing (Jag1\\u003csup\\u003eneg\\u003c/sup\\u003e) skin Tregs. In adult mice, Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs showed significantly higher proportions and levels of CTLA4 (Supplementary Fig2A-D) and CD25 (Supplementary Fig2E-H), but not ICOS (Supplementary Fig S2I-L). Jag1\\u003csup\\u003epos\\u003c/sup\\u003e skin Tregs co-expressed CTLA4 and CD25 more frequently than Jag1\\u003csup\\u003eneg\\u003c/sup\\u003eTregs (Supplementary Fig2M\\u0026amp;N, 37\\u0026plusmn;4.64% vs 23.5\\u0026plusmn;7.90%, p = 0.0312), suggesting that Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs may be phenotypically more active.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eJag1\\u003csup\\u003epos\\u003c/sup\\u003e Tregs are Dispensable During the Steady State\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eUnder homeostatic conditions, skin Tregs suppress long-term CD8 and Teff cell-driven hair follicle-associated inflammation via CD25\\u003csup\\u003eRef.\\u003c/sup\\u003e\\u003csup\\u003e20\\u003c/sup\\u003e. Given that Jag1\\u003csup\\u003epos\\u003c/sup\\u003e skin Tregs express higher levels of CD25 than Jag1\\u003csup\\u003eneg\\u003c/sup\\u003e skin Tregs, we hypothesised that Jag1\\u003csup\\u003epos\\u003c/sup\\u003e skin Tregs may be functionally important during the steady state. To test this, and to avoid influencing early skin development, we created a tamoxifen inducible cell specific model to delete Jag1 in Tregs. This involved crossing mice expressing EGFP-creERT2 gene under the Foxp3 promoter\\u003csup\\u003e21\\u003c/sup\\u003e to mice carrying a Jag1 floxed allele with a dysfunctional Delta-Serrate-Lag2 domain of Jag1\\u003csup\\u003e22\\u003c/sup\\u003e to generate Foxp3\\u003csup\\u003ecreERT2\\u003c/sup\\u003eJag1\\u003csup\\u003efl/fl\\u003c/sup\\u003e and Foxp3\\u003csup\\u003ecreERT2\\u003c/sup\\u003eJag1\\u003csup\\u003efl/wt\\u003c/sup\\u003e, hereafter denoted as \\u0026ldquo;Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u003c/sup\\u003e\\u0026ldquo; and \\u0026ldquo;Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e\\u0026rdquo;. Following intraperitoneal injection of tamoxifen (Figure 2A), we observed effective downregulation of Jag1 transcript in sorted Tregs from Foxp3\\u003csup\\u003eL\\u003c/sup\\u003e\\u003csup\\u003eJag1\\u003c/sup\\u003e compared to Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e mice (Supplementary Figure3A). Expression of \\u003cem\\u003eJag1\\u003c/em\\u003e in Teffs, CD8+ T cells, and \\u003cem\\u003eFoxp3\\u0026nbsp;\\u003c/em\\u003eexpression\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003ein Tregs (Supplementary Figure3B) showed no differences between Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e animals, indicating Treg-specificity of Jag1 deletion in this model.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eDespite significant systemic loss of Jag1 mRNA in Tregs, the absence of Jag1 did not trigger skin inflammation. H\\u0026amp;E staining revealed similar morphology (Figure 2B), epidermal thickness (Figure 2C) and lymphocytic infiltration (Figure 2D) in Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e animals. Body weight throughout tamoxifen injections (Figure 2E) and SDLN live cell numbers (Figure 2F) remained unchanged between animals with or without Jag1 in Tregs during the steady state. Loss of Jag1 in Tregs did not alter overall CD25 or CTLA4 expression in skin Tregs (Figure 2G\\u0026amp;H), nor the abundance and proliferation of skin-resident Tregs, Teff, CD8, gamma-delta T cells (Figure 2I \\u0026amp;J) and other myeloid populations (Figure 2K\\u0026amp;L, gating strategy in Supplementary Fig1B). Together, this suggests that Jag1 expression in Tregs is dispensable for maintaining skin immune homeostasis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eJag1\\u003csup\\u003epos\\u003c/sup\\u003e Tregs are Highly Activated During Early Wound Healing\\u0026nbsp;\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eOne of the most common skin traumas is cutaneous injury. Treg depletion hinders both epithelial restoration\\u003csup\\u003e4\\u003c/sup\\u003e and the full-thickness wound healing process\\u003csup\\u003e3\\u003c/sup\\u003e. One known function of Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs is their role in facilitating hair follicle stem cell (HFSC) proliferation during hair growth phase transitions\\u003csup\\u003e1\\u003c/sup\\u003e. Therefore, we investigated whether Jag1\\u003csup\\u003epos\\u003c/sup\\u003e Tregs are involved in wound healing. Wound healing comprises four major overlapping stages: haemostasis, inflammation, proliferation, and remodelling\\u003csup\\u003e23\\u003c/sup\\u003e. Skin Treg abundance at full-thickness wound sites peaks at 7 days post wounding (dpw) and returns to homeostatic levels by 14dpw\\u003csup\\u003e3\\u003c/sup\\u003e. In Foxp3-DTR mice that permits systemic loss of all Tregs, delayed wound healing is observed only when Tregs are depleted during the inflammation phase, but not later. Jag1 expression in whole skin lysates follows a similar dynamic, being upregulated during the first 7dpw and downregulated from 14dpw\\u003csup\\u003e24,25\\u003c/sup\\u003e. Thus, we hypothesised that the first 7 days are likely to be a crucial period in which Jag1\\u003csup\\u003ePos\\u003c/sup\\u003e Tregs may have an impact. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWe first characterised the dynamic expression of Jag1+Tregs by creating two 4mm full- thickness wounds on the dorsum of wildtype adult mice at 0 days post wounding (dpw) and harvesting skin around the wound at inflammatory (2dpw), proliferative (5dpw) and remodelling (12dpw) phases (Figure 3A). Notably, 25%-48% of skin Tregs expressed Jag1 during the first 5 days post-wounding, in contrast to 6.35% (\\u0026plusmn;2.25%, p = 0.0014) at 12dpw (Figure 3B\\u0026amp;C). At 5dpw, Jag1\\u003csup\\u003epos\\u003c/sup\\u003e+Tregs were significantly more proliferative (Figure 3D\\u0026amp;E) and co-expressed higher levels of both CTLA4 and CD25 activation markers (Figure 3F\\u0026amp;G), relative to Jag1\\u003csup\\u003eneg\\u003c/sup\\u003eTregs. In contrast, no differences in Ki67 or CTLA4+CD25+ proportions were observed between Jag1\\u003csup\\u003epos\\u003c/sup\\u003e and Jag1\\u003csup\\u003eneg\\u003c/sup\\u003e skin Tregs at 2dpw, indicating that 5dpw is a critical period for Jag1+ Tregs\\u0026rsquo; functional prominence.\\u003cstrong\\u003e\\u003cbr\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eJag1+ Tregs Promote Wound Healing\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe then assessed the role of Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs in wound healing by creating two full thickness excisional wounds of the dorsal skin of Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u003c/sup\\u003e and Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e mice, and measuring the wound healing rate over time (Figure 4A). Mice lacking Jag1-expressing Tregs showed delayed wound closure compared to wildtype Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e mice treated with tamoxifen (Figure 4B \\u0026amp; C). The most pronounced difference was observed at 5dpw, with 78.4% (\\u0026plusmn; 9.3%) wound closure in wildtype Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e mice versus 66.2% (\\u0026plusmn;15.9%) in Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u003c/sup\\u003e animals (Figure 4D, p = 0.0041). Histologically, Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u003c/sup\\u003e skin showed a trend towards thicker epithelial wound edges (Figure 4E, red arrow), more keratinocyte precipitation at wound edge (Figure 4E,black arrow ) and disorganisation between epithelial layer and pus cells (Fig 4E, white arrow). These results illustrate skin Tregs require Jag1 for effective wound closure particularly during the early (5 dpw) proliferation phase.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eJag1+ Tregs\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eModulate Neutrophil Accumulation During Early Wound Healing\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe next explored the mechanisms by which skin Tregs use Jag1 to orchestrate wound closure. Upon tissue challenge, skin Tregs primarily perturb inflammation through two mechanisms: either via conventional immunosuppressive capacity\\u003csup\\u003e3,4\\u003c/sup\\u003e or enhance tissue repair via cross-talk with epithelial cells and HFSCs\\u003csup\\u003e1,4\\u003c/sup\\u003e. Dysregulation of skin Tregs leads to unwanted inflammation, mainly contributed by excessive accumulation and proliferation of neutrophils, pro-inflammatory macrophages, CD4+, and CD8+ T cells during homeostasis \\u003csup\\u003e20\\u003c/sup\\u003e, epidermal injury\\u003csup\\u003e4\\u003c/sup\\u003e, and full thickness injury\\u003csup\\u003e3\\u003c/sup\\u003e. Treg-specific loss of Rbpj, a downstream transcription factor of\\u0026nbsp;canonical Notch signalling,\\u0026nbsp;has been shown to mediate upregulation of Foxp3, CTLA4 and CD25 expression in splenic Tregs during homeostasis\\u003csup\\u003e19\\u003c/sup\\u003e. With Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs co-expressing higher level of CTLA4 and CD25 during both homeostasis and wound healing, relative to Jag1\\u003csup\\u003eneg\\u003c/sup\\u003e Tregs, we questioned whether the absence of Jag1 in Tregs can affect the overall immunosuppressive ability of skin Tregs. We reasoned this may impact the accumulation of T and/or myeloid cells during wound healing and could influence wound closure through regulating local inflammation. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eSimilar to the steady state (Figure 2), the absence of Jag1 in Tregs did not affect overall Treg abundance, as quantified by both flow cytometry and immunofluorescence staining (Supplementary Figure 4A\\u0026amp;B). Proliferation and Foxp3 expression in skin Tregs also remained unaffected (Supplementary Figure 4C\\u0026amp;D). None of the Treg phenotypic attributes were altered by loss of Jag1, including the proportion of skin Tregs co-expressing CTLA4+CD25+ (Supplementary Figure 4E), CD25 (Supplementary Figure4F) or ICOS (Supplementary Figure4G). IL33 is an alarmin highly induced in keratinocytes in response to cutaneous wounding\\u003csup\\u003e26\\u003c/sup\\u003e. \\u0026nbsp;IL33 receptor (ST2) is highly expressed in skin Tregs\\u003csup\\u003e10\\u003c/sup\\u003e, and recently found crucial for suppressing bleomycin-induced skin fibrosis\\u003csup\\u003e27\\u003c/sup\\u003e. However, we found that Tregs from Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u0026nbsp;\\u003c/sup\\u003eskin did not alter ST2 expression level either (Supplementary Figure4H). Intriguingly, Jag1 ablation in Tregs resulted in a mild but significant reduction in CTLA4 levels (Supplementary Fig4I). Yet, both the accumulation and proliferation of Teffs and CD8+ T cells remained unchanged between Foxp3\\u003csup\\u003e\\u0026Delta;Jag1\\u0026nbsp;\\u003c/sup\\u003eand\\u0026nbsp;Foxp3\\u003csup\\u003eCtrl\\u003c/sup\\u003e skin (Supplementary Fig4J\\u0026amp;K).Innate cells are first responders to cutaneous wounding. Previously, it has been reported that pro-inflammatory Ly-6C\\u003csup\\u003ehigh\\u0026nbsp;\\u003c/sup\\u003emacrophages accumulate at wound sites at 1 day post full-thickness wounding, contributing up to 60% of skin-resident macrophages, and gradually drop to around 10% at 7dpw\\u003csup\\u003e3\\u003c/sup\\u003e. \\u0026nbsp;Additionally, Treg depletion leads to a 6-fold increase of Ly-6C\\u003csup\\u003ehigh\\u0026nbsp;\\u003c/sup\\u003emacrophages at 7dpw, suggesting skin Tregs help transit the stages of wound healing from a pro-inflammatory to an anti-inflammatory environment. In contrast, Jag1 loss in Tregs did not lead to the same cellular skewing when compared to deletion of the entire Treg pool\\u003csup\\u003e3\\u003c/sup\\u003e. Instead, pro-inflammatory macrophage (defined as CD45+CD11b\\u003csup\\u003ehigh\\u003c/sup\\u003eF4/80+Ly-6C\\u003csup\\u003ehigh\\u003c/sup\\u003eLy-6G\\u003csup\\u003elow\\u003c/sup\\u003e) accumulation in wounded skin remained indifferent between mice with Jag1-deficient and -sufficient Tregs, at around 6% of total macrophages at 5dpw (Figure 5A\\u0026amp;B). Similarly, the accumulation of inflammatory Ly6C+ monocytes was unchanged (Figure 5C\\u0026amp;D), suggesting mice with Jag1-deficient Tregs remain capable of transitioning from a pro-inflammatory to an anti-inflammatory state during wound healing. Interestingly, the absence of Jag1 in Tregs led to less neutrophil influx into wounded skin, compared to wildtype controls at 5dpw, quantified by both flow cytometry and immunofluorescence staining (Figure 5E-H). Collectively, Jag1 is unlikely to be a key factor driving the widely appreciated immunosuppressive function of skin Tregs. Rather, Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs promote the retention of neutrophils during wound healing.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eJag1+ Tregs do not Influence Re-epithelialization\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRe-epithelialisation is a crucial step for successful wound healing following the inflammatory phase, by preventing excess water loss and further entry of microbial pathogens or debris. Trans-epidermal water loss measurement (TEWL) estimates moisture evaporation externally and is used quantitatively to assess epidermal integrity\\u003csup\\u003e28\\u003c/sup\\u003e. Besides suppressing inflammation, skin Tregs drive epidermal barrier repair by promoting bulge HFSC emigration to the epidermis, as well as their proliferation and differentiation\\u003csup\\u003e4\\u003c/sup\\u003e. Previous lineage tracing studies have shown that bulge cells repopulate the epidermis at 5 days post full thickness wounding\\u003csup\\u003e29\\u003c/sup\\u003e. These studies also indicate that bulge HFSCs are proliferative and can migrate to cutaneous wounds from 4dpw but not earlier\\u003csup\\u003e30\\u003c/sup\\u003e, and the derived cells can be detected in the epidermis for up to one year\\u003csup\\u003e31\\u003c/sup\\u003e. This indicates that while bulge HFSCs may not contribute to the immediate re-epithelisation of wounds, they play an important role in restoring the skin barrier from 4dpw onwards. Given that Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs can drive bulge HFSC (identified as EpCam-Sca1-CD34+CD49f+) proliferation during hair regeneration\\u003csup\\u003e1\\u003c/sup\\u003e, we hypothesised Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs may also regulate wound closure by mediating bulge HFSC-driven re-epithelisation.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHowever, mice with Jag1 deficiency in Tregs showed a similar TEWL restoration rate to baseline as wildtype controls (Supplementary Fig5A), suggesting that Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs do not influence barrier restoration post wounding. In line with this observation, the abundance and proliferation of bulge HFSCs also remain unaffected in Foxp3\\u003csup\\u003eD\\u003c/sup\\u003e\\u003csup\\u003eJag1\\u003c/sup\\u003e mice during wound healing (Supplementary Figure 5C\\u0026amp;D, gating strategy in Supplementary Figure 5B), indicating that Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs are unlikely to share the same mechanistic interaction with bulge HFSCs as observed in hair regeneration.\\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eDespite understanding the functional importance of skin Tregs, whether skin Tregs carry out their functions through the same molecular mechanisms as Tregs residing in other tissues is incompletely understood. One candidate is TGF-\\u0026nbsp;signalling, recently shown to drive both hair regeneration\\u003csup\\u003e2\\u003c/sup\\u003e and epithelial barrier repair \\u003csup\\u003e5\\u003c/sup\\u003e. Yet, the functional importance of TGF- signalling extends globally to Treg generation and/or maintenance in both lymphoid and non-lymphoid tissues (Reviewed in \\u003csup\\u003e32\\u003c/sup\\u003e). Given that Jag1 is preferentially expressed in skin Tregs, we questioned whether Jag1-mediated signals are uniquely utilized by skin Tregs to drive their skin-related functions. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn the current study, we demonstrate that Jag1 is indeed preferentially expressed in skin Tregs. Jag1 expressing Tregs exhibit an activated profile, with upregulation of CD25 and CTLA4, but not ICOS, during both homeostasis and wound healing. Although it remains to be determined whether this upregulation reflects a higher immunosuppressive capacity of Jag1\\u003csup\\u003epos\\u003c/sup\\u003e Tregs, our survey of other non-Foxp3 expressing CD4+, CD8+ T cells, pro-inflammatory macrophages, or monocytes, indicates skin Tregs are unlikely to suppress \\u003cem\\u003ein vivo\\u0026nbsp;\\u003c/em\\u003einflammation through Jag1.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMost notably, our study has highlighted the necessity of Jag1 expression in Tregs to facilitate adequate cutaneous wound repair. Consistent with previous findings on skin Tregs, the impact of Jag1\\u003csup\\u003epos\\u003c/sup\\u003e skin Tregs is limited to a critical time window (around 5 dpw) in which wound healing transitions from the inflammatory to the proliferation phase. However, rather than suppressing inflammation, Jag1 expression in Tregs seems to drive the retention of the inflammation phase, as evidenced by neutrophil accumulation within the wounded environment. Despite their well-known tissue-damaging properties, neutrophils are crucial facilitators in wound healing. They not only dissipate infiltrating pathogens, damaged cells, and debris, but also play an increasingly appreciated role in resolving inflammation by polarizing anti-inflammatory macrophages, encouraging vascularization, and potentially promoting local cell proliferation to repair tissues (reviewed in \\u003csup\\u003e33\\u003c/sup\\u003e). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePertinently, mice lacking Notch activation in Lyz2+myeloid cells (Lyz2\\u003csup\\u003ecre\\u003c/sup\\u003eRBPJ\\u003csup\\u003efl/fl\\u003c/sup\\u003e) show a milder inflammatory response in the heart, lung and kidney upon lipopolysaccharide exposure\\u003csup\\u003e34\\u003c/sup\\u003e. This is also associated with a reduction in neutrophil accumulation, reduced inflammatory cytokine detection in the liver after injury\\u003csup\\u003e35\\u003c/sup\\u003e, and pro-inflammatory macrophage reduction in spinal cord lesion sites after compression injury\\u003csup\\u003e36\\u003c/sup\\u003e. This suggests that during injury, the activation of Notch\\u0026nbsp;signalling\\u0026nbsp;in myeloid cells can promote inflammation. Mechanistically, intrinsic Notch signaling plays an important regulatory role in dampening Tregs\\u0026rsquo; own immunosuppressive functions\\u003csup\\u003e19\\u003c/sup\\u003e. Our study also showed that despite high CTLA4 and CD25 expression both homeostatically and during wound healing, Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs are unlikely to self-regulate immunosuppressive capacity. While it remains to be elucidated whether Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs directly influence Notch\\u0026nbsp;signalling\\u0026nbsp;in neutrophils, our study has uncovered an unexpected role for Jag1\\u003csup\\u003epos\\u003c/sup\\u003eTregs in modulating the inflammatory phase of cutaneous wound healing.\\u003c/p\\u003e\"},{\"header\":\"METHODS\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnimal study design\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll mouse procedures were approved by local ethical approval at King\\u0026rsquo;s College London (UK) (PP70/8474, establishment license X24D82DFF), and performed under a UK Government Home Office license (PP6051479). All methods were carried out in accordance with relevant guidelines and regulations under the UK animals (Scientific procedures) Act 1986, and were reported in accordance with ARRIVE guidelines. All possible efforts were made to minimize animal suffering. All experiments were performed on animals with no prior procedures. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWildtype C57BL/6 and FoxP3\\u003csup\\u003eeGFP-CreERT2\\u0026nbsp;\\u003c/sup\\u003emice (JAX:\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e016961\\u003cstrong\\u003e)\\u003c/strong\\u003e were purchased from Charles River, and Jag1\\u003csup\\u003efl/fl\\u0026nbsp;\\u003c/sup\\u003e(JAX: 010618\\u003cstrong\\u003e)\\u0026nbsp;\\u003c/strong\\u003ewere gift from Prof Rosenblum in UCSF. FoxP3\\u003csup\\u003eeGFP-CreERT2\\u003c/sup\\u003e were crossed to Jag1\\u003csup\\u003efl/fl\\u0026nbsp;\\u003c/sup\\u003eto generate FoxP3\\u003csup\\u003eeGFP-CreERT2\\u003c/sup\\u003e Jag1\\u003csup\\u003efl/fl\\u003c/sup\\u003e in King\\u0026rsquo;s College London BSU NHH animal unit. Mice were maintained through routine breeding, were fed with a standard chow diet and housed in line with UK regulations. Littermates of the same sex were genotyped and assigned to experimental groups based on genotyping results.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTamoxifen (Sigma, T5648-5G) were sonicated in 37\\u003csup\\u003eO\\u003c/sup\\u003eC water bath and dissolved in corn oil, at 2.5mg/ml concentration.\\u0026nbsp;For conditional Jag1 knockout,\\u0026nbsp;7-10 week old\\u0026nbsp;Foxp3\\u003csup\\u003eeGFP-Cre-ERT2\\u003c/sup\\u003eJag1fl/fl and Foxp3\\u003csup\\u003eeGFP-Cre-ERT2\\u003c/sup\\u003eJag1fl/wt or wt/wt were injected with tamoxifen intraperitonially at 75-100mg/kg in the indicated interval before harvesting. All characterisation and steady state experiments were performed on mice of mixed gender, and only females were used for further characterisation in wound healing experiments. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSkin wounding assays and analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMice were anaesthetised by inhalation of vaporized 1.5% isoflurane, shaved and subcutaneously injected with Vetersgesics.\\u0026nbsp;Two full thickness excisional wounds were made on dorsal back of mice under and anaesthesia, using a 4-mm biopsy punch (Stifel Laboratory Research). Wounds were photographed daily till harvest. The same ruler was placed next to wound area for measurement standardization. Wound area was measured using ImageJ\\u003csup\\u003e37\\u003c/sup\\u003e(Fiji NIH), and closure ratio of each time point was calculated relative to wound area at 0dpw. \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTransepidermal water loss (TEWL) of each wound was measured with Tewameter TM 300 probe (Courage + Khazaka electronic GmbH) according to the manufacturer\\u0026rsquo;s protocols. Measurement were made on 0dpw (immediately after excision wound) and every 24 hours thereafter. Each datapoint is an average of four TEWL measurements. \\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTissue Processing\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWhole murine dorsal skin was finely minced with scissors and digested in 500ul digestion medium per cm\\u003csup\\u003e2\\u003c/sup\\u003e of skin. Digestion medium was prepared with 2mg/ml collagenase (Sigma), 0.1mg/ml DNase (Sigma) and 0.5mg/ml hyaluronidase (Sigma), dissolved in C10 medium [10% FBS, 1% Pen/Strep, 1 mM Na-pyruvate, 1% HEPES, 1% non-essential amino acid, 0.5% 2-mercaptoethanol in RPMI-1640 with L-glutamine medium]. After 45 min incubation at 37\\u003csup\\u003eO\\u003c/sup\\u003eC 255rpm, single cell suspension was washed with 20ml C10 medium, and filtered through 100um then 40um cell strainer. Lymph nodes were mechanically smashed, washed with FACS buffer [2% Fetal calf serum, 1mM EDTA and 0.1% sodium azaide in PBS], and filtered through 70um cell strainer. Epidermal cells were prepared by floating skin on 0.5%Trypsin-EDTA (Thermofisher) for 1hr at 37\\u003csup\\u003eO\\u003c/sup\\u003eC, before gently removed from dermal part and washed with C10 medium. Lung was finely minced and digested with 1mg/ml Collagenase A (Sigma) and 0.1mg/ml DNase I (Sigma) in R10 [10% FBS and 1% Pen/Strep in RPMI-1640 medium]. After 1hr at 37\\u003csup\\u003eO\\u003c/sup\\u003eC 180rpm, single cell suspension is passed through 70um filter, spin for 5 min at 4\\u003csup\\u003eO\\u003c/sup\\u003eC 1800rpm, and treated with 500ul ACK Lysing Buffer for 30s to 1min before washed with 1xPBS. All single cell suspensions were then centrifuged at 1800rpm at 4\\u003csup\\u003eO\\u003c/sup\\u003eC for 4 min, and resuspended in 1m FACS buffer. Total live cells were determined using NucleoCounter NC-200 (Chemometec) in 1:20 dilution, before downstream process. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFor histology, skin tissue was fixed in 10% formalin overnight, followed by PBS washes, stored at 70% Ethanol overnight, and embedded in paraffin. Each sample was cut and levelled to wound area, before manually assembled as tissue microarray blocks using a 7-mm biopsy punch. 5um sections were cut, mounted and sent to Tissueplexia (Scotland) for multiplex-staining using anti-FoxP3 (14-5773-82, Thermofisher) and anti-Ly6G (127602, Biolegend).\\u0026nbsp;H\\u0026amp;E were performed according to manufacturer\\u0026rsquo;s instruction and imaged using a Nanozoomer (Hamamatsu photonics) with a \\u0026times;40 objective.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFlow cytometry\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor flow cytometry staining, 1.5-4 million cells per condition were plated in round bottom 96 well plate, and stained with 50ul of stated surface antibodies on ice for 20 min. After washed with FACS buffer, cells were fixed and permeabilised by FoxP3/Transcription Factor Staining buffer set (eBioscience) on ice for another 20 min, before washed with permeabilization buffer and lastly stained with intracellular antibodies (resource table), again on ice for 20 min. Samples were run on Fortessa LSRII (BD Bioscience) in KCL BRC Flow Cytometry Core. For compensation, UltraComp eBeads\\u003csup\\u003eTM\\u003c/sup\\u003e (Thermofisher) were stained with each surface and intracellular antibody following the same cell staining protocol. ArC\\u003csup\\u003eTM\\u003c/sup\\u003e Amine Reactive Compensation Bead Kit (Thermofisher, A10346) were used for GhostDye\\u003csup\\u003eTM\\u003c/sup\\u003e Live/Dead stain. All gating and data analysis were performed using FlowJo v10, while statistics were calculated using Graphpad Prism 10. \\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRNA isolation and quantification PCR\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLymph node single cell suspension was resuspended in pre-sort medium [2% FBS, 1% Pen/Strep, 2 mM EDTA, 25mM HEPES in RPMI-1640 without phenol red]. CD45+ CD3+CD4+CD25high (Tregs), CD45+CD3+CD4+CD25neg(Teffs) and CD45+CD3+CD8+ cells were sorted using FACSAria\\u0026trade; Fusion Flow Cytometer (BD) with 100um nozzle\\u0026nbsp;in KCL BRC Flow Cytometry Core. Cells were sorted into RPMI-1640 supplemented with 10% heat-inactivated FBS and 1%\\u0026nbsp;Pen/Strep, and spun at 300g for 10min at 4\\u003csup\\u003eO\\u003c/sup\\u003eC. After removal of supernatant, cell pellet were snap-frozen in liquid nitrogen and stored at -80\\u003csup\\u003eO\\u003c/sup\\u003eC. RNA was extracted using NucloSpin RNA XS, Micro kit for RNA purification (740902, Macherey-Nagel) according manufacturer\\u0026rsquo;s instruction. RNA integrity and concentration were then determined by RNA 6000 Pico Kit on Bioanalyzer (Agilent). RNA were then normalized and synthesied into cDNA using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR were performed using TaqMan\\u003csup\\u003eTM\\u0026nbsp;\\u003c/sup\\u003ePreAmp Master Mix Kit (ThermoFisher) on 384-well plate according to manufacturer\\u0026rsquo;s instruction, run with 4 technical replica each condition. The following TaqMan probes were used: Gapdh (Mm99999915_g1, VIC), Foxp3 (Mm00475162_m1, FAM) and Jag1 (Mm00496904_m1, FAM), Plates were then run on CFX384 Touch Real-Time PCR system (BioRad). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eQUANTIFICATION AND STATISTICAL ANALYSIS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eParameters such as sample size, dispersion or precision are reported in Figure Legends.\\u0026nbsp;Statistical analyses were performed in Prism 10.1 (GraphPad). Details of the statistics and appropriate test used are also indicated in Figure Legends. *p \\u0026lt; 0.05, **p \\u0026lt; 0.01 ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.001, p-values greater than 0.05 was identified as not statistically significant.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBulk RNAseq reanalysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRead-count tables were obtained from NCBI database: GSE182322 (Ref \\u003csup\\u003e10\\u003c/sup\\u003e), and downstream processed according to presented method. \\u0026nbsp;Each data point in Figure 1 represents the sum of both gene raw counts from ST2- and ST2+ Tregs per mouse. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"601\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eREAGENT or RESOURCE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSOURCE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eIDENTIFIER\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAntibodies\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"97.50415973377704%\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eMouse T Cell Panel\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD45 Monoclonal Antibody (30-F11), Alexa Fluor 700, eBioscience\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 56-0451-82, RRID: AB_891454\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD3 Antibody, anti-mouse, PerCP-Vio\\u0026reg; 700, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eMiltenyi Biotec\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 130-120-826, RRID: AB_2752207\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eTCR\\u0026gamma;/\\u0026delta; Antibody, anti-mouse, PE-Vio\\u0026reg; 770, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eMiltenyi Biotec\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 130-123-290, RRID: AB_2802022\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 650\\u0026trade; anti-mouse CD4 Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 100545, RRID: AB_11126142\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD8a Antibody, anti-mouse, APC-Vio\\u0026reg; 770, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eMiltenyi Biotec\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 130-120-806, RRID: AB_2752203\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD25 Antibody, anti-mouse, PE-Vio\\u0026reg; 615, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eMiltenyi Biotec\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 130-123-028, RRID: AB_2811434\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBD Horizon\\u0026trade; BV605 Hamster Anti-Mouse CD27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 563365, RRID: AB_2738160\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eJagged1 Antibody (E-12)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSanta Cruz Biotechnology\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# Sc-390177, RRID: AB_2892141\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Mouse CD339 (Jagged 1) PE\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eeBioscience\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 12-3391-80, RRID:\\u0026nbsp;AB_1963616\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 711\\u0026trade; anti-human/mouse/rat CD278 (ICOS) Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 313547, RRID: AB_2734288\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBD Horizon\\u0026trade; BV786 Mouse Anti-Ki-67\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 563756, RRID: AB_2732007\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFoxP3 Antibody, anti-mouse, Vio\\u0026reg; R667, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eMiltenyi Biotec\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 130-111-682, RRID: AB_2651771\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 421\\u0026trade; anti-mouse CD152 Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 106311, RRID: AB_10901170\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Mouse/Rat Foxp3 eFluor 450\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eeBioscience\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 48-5773-82\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Mouse CD152 (CTLA4) PE\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBD\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 553720\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Human Ki67 PE-Cy7\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBD\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 561283\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Mouse TCR gd PerCP-Cy 5.5\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBiolegend\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 118117\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Mouse CD45 Alexa Fluor 700\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eeBioscience\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 56-0451-82\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAnti-Mouse CD25 APC-eFluor 780\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eeBioscience\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 47-0251-82\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 605 anti-mouse CD8a Antibody\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBiolegend\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 100743\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 650\\u0026trade; anti-mouse CD4 Antibody\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBiolegend\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 100545\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 711 anti-mouse CD3 Antibody\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBiolegend\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 100241\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"97.50415973377704%\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eMouse Myeloid Panel\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBD Horizon\\u0026trade; BUV395 Rat Anti-Mouse Ly-6G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 565964, RRID: AB_2716852\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 421\\u0026trade; anti-mouse I-A/I-E Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 107632, RRID: AB_2650896\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 510\\u0026trade; anti-mouse/human CD45R/B220 Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 103248, RRID: AB_2650679\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 510\\u0026trade; anti-mouse CD3 Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 100234, RRID: AB_2562555\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 650\\u0026trade; anti-mouse CD11c Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 117339, RRID: AB_2562414\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBrilliant Violet 785\\u0026trade; anti-mouse F4/80 Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 123141, RRID: AB_2563667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFITC anti-mouse Ly-6C Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 128005, RRID: AB_1186134\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD207 (Langerin) Monoclonal Antibody (eBioL31), PE, eBioscience\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 12-2075-82, RRID: AB_763452\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD45 Monoclonal Antibody (30-F11), Alexa Fluor 700, eBioscience\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 56-0451-82, RRID: AB_891454\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eAPC/Cyanine7 anti-mouse/human CD11b Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 101225, RRID: AB_830641\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eEpidermal panel\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Horizon\\u0026trade; BUV395 Rat Anti-Mouse CD45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 564279\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD326 (EpCAM) Antibody, anti-mouse, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eMiltenyi\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 130-117-871\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Horizon\\u0026trade; BV786 Mouse Anti-Ki-67\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 563756\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Horizon\\u0026trade; BV650 Rat Anti-Human CD49f\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 563707\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"43\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eAlexa Fluor\\u0026reg; 700 anti-mouse I-A/I-E Antibody\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBiolegend\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 107622\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCD207 (Langerin) Antibody, anti-mouse, PE-Vio\\u0026reg; 615, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eMiltenyi\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 130-112-994\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003ca href=\\\"https://www.bdbiosciences.com/en-gb/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/alexa-fluor-647-rat-anti-mouse-cd34.560230\\\"\\u003eBD Pharmingen\\u0026trade; Alexa Fluor\\u0026reg; 647 Rat anti-Mouse CD34\\u003c/a\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eBD Biosciences\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 560230\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eSca-1 Antibody, anti-mouse, REAfinity\\u0026trade;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eMiltenyi\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eCat# 130-106-220\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"97.50415973377704%\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eChemicals, peptides, and recombinant proteins\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTamoxifen\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSigma-Aldrich\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# T5648\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCollagenase\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSigma-Aldrich\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat #C9407\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eDNase\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSigma-Aldrich\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat #DN25\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eHyaluronidase\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSigma-Aldrich\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat #H3506\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eCollagenase A\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSigma-Aldrich\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# \\u003cstrong\\u003e10103578001\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eDNase 1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSigma-Aldrich\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCat# 10104159001\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eACK Lysing Buffer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCat#\\u0026nbsp;\\u003c/strong\\u003eA1049201\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFetal Bovine Serum\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 10500064\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003ePenicillin-Streptomycin (5,000 U/mL)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 15070063\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eSodium Pyruvate (100 mM)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 11360070\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eHEPES (1M)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 15630056\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eMEM Non-Essential Amino Acids Solution (100X)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 11140050\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e2-mercaptoethanol\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eCat# 21985023\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"100%\\\" valign=\\\"bottom\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"100%\\\" valign=\\\"bottom\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eRPMI-1640 medium\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 11875093\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eUltraPure\\u0026trade; 0.5M EDTA, pH 8.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 15575020\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003ePBS, pH 7.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermoFisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 10010023\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFoxP3/Transcription Factor Staining buffer set\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eeBioscience\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 00-5523-00\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"43\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eUltraComp eBeads\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermofisher\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 01-2222-42\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eArCTM Amine Reactive Compensation Bead Kit\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThermofisher\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# A10346\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eZombie UV\\u0026trade; Fixable Viability Kit\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBioLegend\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eCat# 423107\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"97.50415973377704%\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eDeposited data\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eBulk RNAseq data\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eSpath et al(\\u003cem\\u003e13\\u003c/em\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eGEO: GSE182322\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"97.50415973377704%\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eExperimental Models: Organisms/Strains\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eWT C57BL/6J\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eCharles River\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eRRID: IMSR_CRL:027\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFoxp3\\u003csup\\u003eeGFP-Cre-ERT2\\u0026nbsp;\\u003c/sup\\u003e\\u003cem\\u003e(Foxp3\\u003csup\\u003etm9(EGFP/cre/ERT2)Ayr\\u003c/sup\\u003e\\u003c/em\\u003e/J) C57BL/6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThe Jackson Laboratory\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eRRID: IMSR_JAX:016961\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"73\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eJag1\\u003csup\\u003efl/fl\\u003c/sup\\u003e (B6.129S-\\u003cem\\u003eJag1\\u003csup\\u003etm2Grid\\u003c/sup\\u003e\\u003c/em\\u003e/J)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eThe Jackson Laboratory\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003eRRID: IMSR_JAX:010618\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"97.50415973377704%\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSoftware and Algorithms\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"21\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFlowJo (version 10.8.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBecton Dickinson\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://www.flowjo.com/solutions/flowjo; RRID:SCR_008520\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"68\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eFuji (Image J)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eFuji (Image J)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://fiji.sc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eNDP.view2 Image viewing software (U12388-01)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eHamamatsu\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://www.hamamatsu.com/us/en/product/life-science-and-medical-systems/digital-slide-scanner/U12388-01.html\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"181\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"94.82758620689656%\\\"\\u003e\\n \\u003cp\\u003eRRID:SCR_025177\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"5.172413793103448%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eQuPath (version 0.5.0-arm64)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eBankhead et al.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://qupath.github.io/\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003ePrism (version 10.1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eGraphPad Software\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttp://www.graphpad.com/;\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"94.82758620689656%\\\"\\u003e\\n \\u003cp\\u003eRRID:SCR_002798\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"5.172413793103448%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eInkscape (version 1.3.2)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eInkscape\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://inkscape.org/;\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"94.82758620689656%\\\"\\u003e\\n \\u003cp\\u003eRRID:SCR_014479\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"5.172413793103448%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eR (version 4.3.2)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eCRAN\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://www.r-project.org/\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\"\\u003e\\n \\u003cp\\u003eRStudio (version 2021.09.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eRStudio\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003ehttps://www.rstudio.com/\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"45\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eR package: edgeR (version 4.0.16)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eRobinson et al (\\u003cem\\u003e38\\u003c/em\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd 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\\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"28.618968386023294%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e4-mm biopsy punch\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"23.12811980033278%\\\"\\u003e\\n \\u003cp\\u003eStifel\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"45.75707154742096%\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"2.4958402662229617%\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd width=\\\"0%\\\" height=\\\"23\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank the Advanced Cytometry Platform (Flow Core), Research and Development Department at Guy\\u0026rsquo;s and St Thomas\\u0026apos; NHS Foundation Trust for assistance with flow cytometry experiments. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe acknowledge support by the following grant funding bodies: This work was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society awarded to N.A (Grant Number 213401/Z/18/Z). P.P.L. and J.Z.X are supported by Wellcome Trust PhD fellowships (108874/B/15/Z), and (218452/Z/19/Z). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eP.L.: investigation, data curation, analysis, writing \\u0026ndash; original draft, writing \\u0026ndash; Review \\u0026amp; Editing; J.Z.X.: investigation and methodology; H.A.: investigation; M.S.: investigation; N.A. conceptualization, supervision, data curation, funding acquisition, writing \\u0026ndash; original draft, writing \\u0026ndash; Review \\u0026amp; Editing.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they do not have any conflict of interest.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCONTACT FOR REAGENT AND RESOURCE SHARING\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRequests for reagents and resources should be directed to the Lead Contact, Niwa Ali (niwa.ali@kcl.ac.uk)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAli, N. \\u003cem\\u003eet al.\\u003c/em\\u003e Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation. \\u003cem\\u003eCell\\u003c/em\\u003e (2017) doi:10.1016/j.cell.2017.05.002.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, Z. \\u003cem\\u003eet al.\\u003c/em\\u003e Glucocorticoid signaling and regulatory T cells cooperate to maintain the hair follicle stem cell niche. \\u003cem\\u003eNat. 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Dev.\\u003c/em\\u003e \\u003cstrong\\u003e82\\u003c/strong\\u003e, 518\\u0026ndash;529 (2015).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4559523/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4559523/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Skin-resident regulatory T cells (Tregs) play an irreplaceable role in orchestrating cutaneous immune homeostasis and repair, including the promotion of hair regeneration via the Notch signaling ligand Jagged-1 (Jag1). While skin Tregs are indispensable for facilitating tissue repair post-wounding, it remains unknown if Jag1-expressing skin Tregs impact wound healing. Using a tamoxifen inducible Foxp3creERT2Jag1fl/fl model, we show that loss of functional Jag1 in Tregs significantly delays the rate of full-thickness wound closure. Unlike in hair regeneration, skin Tregs do not utilize Jag1 to impact epithelial stem cells during wound healing. Instead, mice with Treg-specific Jag1 ablation exhibit a significant reduction in Ly6G+ neutrophil accumulation at the wound site. However, during both homeostasis and wound healing, the loss of Jag1 in Tregs does not impact the overall abundance or activation profile of immune cell targets in the skin, such as CD4+ and CD8+ T cells, or pro-inflammatory macrophages. This collectively suggests that skin Tregs may utilize Jag1-Notch signalling to co-ordinate innate cell recruitment under conditions of injury but not homeostasis. Overall, our study demonstrates the importance of Jag1 expression in Tregs to facilitate adequate wound repair in the skin.\",\"manuscriptTitle\":\"Jagged-1+ Skin Tregs Modulate the Innate Immune Response to Wound Healing\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-27 05:30:47\",\"doi\":\"10.21203/rs.3.rs-4559523/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-07-22T06:03:28+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-18T16:50:57+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-18T07:31:24+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"36815857660746750546346172240836547502\",\"date\":\"2024-06-26T15:41:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"67515929240582186231996494849067712241\",\"date\":\"2024-06-24T13:59:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-06-24T12:58:06+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-06-24T12:54:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-06-13T14:48:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-06-12T09:46:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2024-06-10T17:35:29+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"3b8223ca-276b-4cf3-8352-a6d0c2f0e031\",\"owner\":[],\"postedDate\":\"June 27th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":33773409,\"name\":\"Biological sciences/Immunology/Lymphocytes/T cells/Cd4 positive t cells/Regulatory t cells\"},{\"id\":33773410,\"name\":\"Biological sciences/Immunology/Inflammation\"}],\"tags\":[],\"updatedAt\":\"2024-09-16T16:11:47+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4559523\",\"link\":\"https://doi.org/10.1038/s41598-024-71512-1\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2024-09-09 15:57:44\",\"publishedOnDateReadable\":\"September 9th, 2024\"},\"versionCreatedAt\":\"2024-06-27 05:30:47\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-024-71512-1\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-024-71512-1\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4559523\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4559523\",\"identity\":\"rs-4559523\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}