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Purpose of review To provide updates on recent advances in the diagnosis and management of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome. Recent findings The number of identified HLA allele associations with DRESS continues to grow. There is increasing evidence indicating viral infection, reactivation, and cross-reactivity may play key roles in disease. Translational work illuminated JAK/STAT activation in recalcitrant disease. There is expanding recognition of rapid-onset DRESS resulting from specific drugs. Summary DRESS is a severe form of adverse drug reaction with potential for significant morbidity and mortality. Recent research advances may improve clinical care. HLA screening can now be performed to prevent disease in susceptible patients and may help identify culprit drugs in the near future. Viral testing should be performed on every patient, and if positive, patients potentially treated with antiviral therapy. JAK inhibitors may be an effective treatment option for DRESS. Early onset of disease relative to drug exposure should not exclude the diagnosis of DRESS.

Delayed-type drug hypersensitivity reactions (dtDHR) are immune-mediated reactions with skin and visceral manifestations ranging from mild to severe. Clinical care is negatively impacted by a limited understanding of disease pathogenesis. Though T cells are believed to orchestrate disease, the type of T cell and the location and mechanism of T cell activation remain unknown. Resident memory T cells (TRM) are a unique T cell population potentially well situated to act as key mediators in disease pathogenesis, but significant obstacles to defining, identifying, and testing TRMin dtDHR preclude definitive conclusions at this time. Deeper mechanistic interrogation to address these unanswered questions is necessary, as involvement of TRMin disease has significant implications for prediction, diagnosis, and treatment of disease.

The authors regret not all contributing authors correctly acknowledged funding. Jonathan Peter’s IMARI-Africa project (AFRISCAR) is part of the EDCTP2 programme supported by the European Union (grant number TMA2017SF-1981 ). The authors would like to apologise for any inconvenience caused.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67(+)) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are potentially life-threatening, immune-mediated adverse reactions characterized by widespread erythema, epidermal necrosis, and detachment of skin and mucosa. Efforts to grow and develop functional international collaborations and a multidisciplinary interactive network focusing on SJS/TEN as an uncommon but high burden disease will be necessary to improve efforts in prevention, early diagnosis and improved acute and long-term management. SJS/TEN 2019: From Science to Translation was a 1.5-day scientific program held April 26-27, 2019, in Vancouver, Canada. The meeting successfully engaged clinicians, researchers, and patients and conducted many productive discussions on research and patient care needs.

Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens. However, the biological pathways that enable the long-term survival of TRM cells are obscure. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM cells, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA β-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity.

During that past two decades, advances in techniques for generating in vitro immune-suppressive dendritic cells (DCs) have heralded the use of these pro-tolerogenic DCs as therapeutics against transplant rejection and autoimmune diseases. In transplantation, previous dogma assumed that systemically administered therapeutic DCs bearing donor antigens (Ags) control the anti-donor response by directly interacting with anti-donor T cells in vivo. However, recent evidence indicates that the exogenously-administered therapeutic DCs instead function as Ag-transporting cells that transfer donor Ags to recipient's Ag-presenting cells (APCs) for presentation to T cells. In secondary lymphoid organs, presentation of acquired donor Ags by recipient's quiescent DCs triggers deficient activation and eventual apoptosis of donor-specific effector T cells, leading to a relative increase in the percentage of donor-specific regulatory T cells. This chapter describes the methodology to generate in vitro immune-suppressive DCs that are resistant to maturation, and to assess in vivo both their survival and their ability to regulate donor-specific T cells in a mouse model.

During that past two decades, advances in techniques for generating in vitro immune-suppressive dendritic cells (DCs) have heralded the use of these pro-tolerogenic DCs as therapeutics against transplant rejection and autoimmune diseases. In transplantation, previous dogma assumed that systemically administered therapeutic DCs bearing donor antigens (Ags) control the anti-donor response by directly interacting with anti-donor T cells in vivo. However, recent evidence indicates that the exogenously-administered therapeutic DCs instead function as Ag-transporting cells that transfer donor Ags to recipient's Ag-presenting cells (APCs) for presentation to T cells. In secondary lymphoid organs, presentation of acquired donor Ags by recipient's quiescent DCs triggers deficient activation and eventual apoptosis of donor-specific effector T cells, leading to a relative increase in the percentage of donor-specific regulatory T cells. This chapter describes the methodology to generate in vitro immune-suppressive DCs that are resistant to maturation, and to assess in vivo both their survival and their ability to regulate donor-specific T cells in a mouse model.

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor β gene (TCRB) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

Increasing evidence indicates the existence of a complex cross-regulation between the most important biosensors of the human body: The immune and nervous systems. Cytokines control body temperature and trigger autoimmune disorders in the central nervous system, whereas neuropeptides released in peripheral tissues and lymphoid organs modulate inflammatory (innate) and adaptive immune responses. Surprisingly, the effects of nerve fibers and the antidromic release of its pro-inflammatory neuropeptides on the leukocytes of the immune system that mediate graft rejection are practically unknown. In the transplantation field, such area of research remains practically unexplored. A recent study by Riol-Blanco et al has revealed new details on how nociceptive nerves regulate the pro-inflammatory function of leukocytes in peripheral tissues. Although the mechanism(s) by which neuroinflammation affects the immune response against the allograft remains unknown, recent data suggest that this new area of research is worth exploring for potential development of novel complementary therapies for prevention/treatment of graft rejection.

Although rare, Stevens-Johnson syndrome and toxic epidermal necrolysis remain among the most devastating of acute conditions involving the skin. In the past 30 years, tremendous progress has been made in understanding the causes and pathobiology of this often life-threatening condition. Su et al demonstrate associations between IL 15 serum levels and the outcome of patients with Stevens-Johnson syndrome and toxic epidermal necrolysis. Their findings provide ideas for further investigations that may help us better understand the role of cytokines in this T-cell mediate disease and provides clues to possible new therapies.

The adaptive immune system is historically divided into cell-mediated immunity and humoral immunity, which were considered to provide defense against intracellular and extracellular pathogens, respectively. As the complexity of each element of the adaptive immune system is revealed, it is now clear that extracellular pathogens can also be targeted by cell-mediated immunity, and intracellular viruses by humoral immunity. The two main effector cell types mediating adaptive immunity are T and B lymphocytes (more commonly called T and B cells). Both cell types are characterized by a nearly infinite variety of unique antigen receptors, so that at the genomic level no two naive T cells or naive B cells are alike. There are two hallmarks of the adaptive immune system, specificity and memory, and lymphocytes are the only immune cells capable of both. Naive lymphocytes are antigen inexperienced, meaning they have not previously recognized and responded to antigen. Their response upon first exposure is termed the primary response. The primary response is characterized by rapid but transient clonal expansion, and this ultimately generates a limited number of long-lived antigen specific lymphocytes, termed memory cells, which upon re-exposure to their cognate antigen produce a more rapid and effective immune response, termed the secondary response. Engineered generation of memory T and B cells to specific pathogens is the basis for vaccination. Specificity to structurally distinct antigen is produced by a combination of genetic events and selection process that allows lymphocytes to recognize and react predominantly to foreign, and much less frequently to self, antigen. The loss of inhibition of recognition of self results in autoimmune disease, and there are a number of mechanisms in place to prevent this from occurring.

Focal dermal hypoplasia (FDH) is an X-linked dominant disease characterized by dermal thinning and fat herniation with other ectodermal and mesodermal abnormalities. There is limited literature regarding the symptomatology and progression of skin, hair, and nail disease. The risk of bone fragility has not been explored either. This cross-sectional survey-based study explored these gaps in knowledge and provides direction for future avenues of research in FDH.

The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.

A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the alloresponse and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.

Problem statement: Organ transplantation is a life-saving and increasi ngly common procedure, as it often serves as the only treatment available for end-stage organ disease. Although th e constant development of new and more effective immunosuppressive drugs has revolutionized the prevention and treatment of acute graft rejection, these drugs have significant toxicity, greatly incr ease patient susceptibility to neoplasms and infection a nd exert little impact on chronic rejection. Approach: The literature was reviewed to illuminate the mech anisms by which the anti-donor immune response is initiated and how cellular therapies im pact this response. Results: Data show that Donor Specific Transfusion, Apoptotic Cell therapies and Dendritic Cell therapies all function as a source o f alloantigen to suppress the anti-donor T cell respo nse. Conclusion: Cellular therapies hold promise in the prevention of solid organ allograft rejection, but require optimization and study in large animal mode ls before clinical implementation.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Introduction La plupart des patients atteints de lymphome T cutane primitif presentent une atteinte limitee a la peau et une evolution indolente. Une minorite de patients developpera une forme agressive pour laquelle il n’existe pas de traitement curatif. Notre capacite a predire l’evolution au stade precoce est actuellement limitee. Materiel et methodes Nous avons sequence le gene TCRB par sequencage haut debit en peau lesionnelle de 208 patients atteints de lymphome T cutane primitif, inclus sur une periode de plus de 15 ans et correle ces donnees a l’evolution clinique par modele de Cox uni- et multivariee. Cette technique de sequencage permet de mesurer precisement la frequence de chacun des clones T dans la peau. Les resultats ont ete valides sur une cohorte independante de 102 patients. L’expression de 78 biomarqueurs potentiels dans la peau a ete mesuree concomitamment par Nanostring. Observations L’analyse transcriptionnelle non supervisee revele un groupe de patients surexprimant des genes exprimes par les lymphocytes T et ayant un pronostic sombre. Ces patients ont une frequence du clone tumoral significativement plus elevee dans la peau lesionnelle. La frequence du clone tumoral en peau lesionnelle est un facteur pronostique de survie sans progression et survie globale chez ces patients ( p p Discussion Cette etude est limitee par son caractere unicentrique mais le nombre de patients etudies, la force de l’association statistique et sa reproductibilite sont des forces. Conclusion La mesure precise du clone tumoral T dans la peau lesionnelle des patients atteints de lymphome T cutane au stade precoce est le premier indice pronostique independant, robuste et reproductible de survie et progression.

To fulfill its immunologic functions, the skin is richly populated with dendritic cells (DCs), the most potent professional Ag-processing and Ag-presenting cells of the immune system. The immune-stimulatory and tolerogenic functions of skin DCs are regulated by the immune and neuroendocrine systems. Pro-inflammatory neuropeptides like substance P and calcitonin gene-related peptide secreted by cutaneous Aδ and C nerve fibers are the main initiators of neuro-inflammatory responses in the skin. Conversely, anti-inflammatory neuropeptides like the products of cleavage of proopiomelanocortin (α-melanocyte-stimulating hormone and adrenocorticotropic hormone) negatively regulate immune responses in the skin. Likewise, the control of immune responses against stimuli applied to the skin depends on the balance between the release and neutralization of pro-inflammatory and anti-inflammatory neuropeptides, a matter that has been somehow overlooked by the immunology field until recently.

Cell proliferation is commonly assayed in the laboratory for research purposes, but is increasingly used clinically to gauge tumor aggressiveness and potentially guide care. Therefore, both researchers and clinicians should have a basic understanding of techniques used to assess cell proliferation. Multiple cell proliferation assays exist, and the choice of method depends on the laboratory resources available, the types of cells/tissues to be studied, and the specific experimental goals. In this article, we identify four overarching categories of cell proliferation assays that signify various stages of the cell cycle: nucleoside-analog incorporation, cell cycle-associated protein detection, use of cytoplasmic proliferation dyes, and indirect measures of cell proliferation. Each method has strengths and limitations that should guide the dermatology investigator's choice of assay.

INTRODUCTION The term “humanized mice” refers to immunodeficient mice containing human cells or tissues or to mice (immunodeficient or not) that have been genetically modified to express human genes. Humanized mouse models are increasingly utilized in many areas of research, such as infectious disease, autoimmune disease, cancer biology, and drug development. Because humanized mice recapitulate human physiology and pathology better than traditional mouse models do, they are employed both in disease modeling and in preclinical investigations of novel therapies. As these models are increasingly utilized in dermatology research, it is important for dermatology researchers and clinicians to have a rudimentary understanding of humanized mice. In this article, we review the basic biology of humanized mice and provide examples of their use in dermatology research.

Graft-versus-host-disease (GVHD) is a major cause of morbidity and mortality in stem cell transplantation (SCT). Acute GVHD is thought to be mediated by donor T cells that target and destroy host tissues. Studies into the host immune system are limited, as it is assumed that host T cells in peripheral tissues such as skin are depleted by conditioning regimens in parallel to T cells in lymphoid organs and blood. However, it is recently appreciated that a high number of memory T cells reside long-term within the organs most affected by GVHD. We investigated whether host skin T cells survive SCT conditioning and are present during acute GVHD. To do so, excess skin biopsy tissue was obtained from patients with acute skin GVHD who had undergone myeloablative or non-myeloablative conditioning and allogeneic SCT with sex-mismatched donor cells. Tissue was stained for T cell markers via immunofluorescence concurrently with in situ hybridization for the X and Y chromosomes. All seventeen patients studied demonstrated persistence of host T cells in skin, often in equal or greater numbers than donor T cells. Host skin T cells survived regardless of myeloablative or non-myeloablative conditioning, of patient age, and of day post-SCT (samples ranged from 12–213 days post-SCT). Comparatively, blood T cell chimerism was 100% donor in nearly all samples, indicating diverging effects of conditioning on T cell compartments. Skin host T cells demonstrated pro-inflammatory cytokine production. These results provide a novel avenue of research into GVHD pathobiology and suggest that alternative conditioning regimens should be employed if host skin T cells are to be targeted.

Pemphigus vulgaris is an uncommon intraepidermal blistering disorder that typically presents with flaccid bullae or erosions. We report a patient with pemphigus vulgaris who presented with several unusual clinical features: tense bullae with dependently layered pus, true target lesions coalescing into annular configurations, and diffuse desquamation that initially raised concern for toxic epidermal necrolysis. We discuss the differential diagnosis and implications of these morphological findings.

013 Heterogeneous CD4 migratory T cells rapidly and constitutively infiltrate skin and co-exist with populations of resident memory T cells (TRM) C Park, Y Pan, X Fu, T Tian, S Divito, C Lin, R Clark and TS Kupper 1 Dermatology, BWH/HMS, Boston, MA and 2 Wellman Laboratories, MGH, Boston, MA Long term resident TRM cells have long been described in skin, but recently populations of migratory T cells have been identified that co-exist in human skin with TRM. While the function of skin CD4 TRM cells is well described in murine models, re-circulating migratory CD4 T cells have not been well characterized in this setting. Thus, to explore the function of putative skin migratory CD4 T cells, we used a model of C. albicans skin infection that we have previously shown generates a TRM population of sessile CD4 Th17 T cells. Months after the infection had resolved, a faster moving population of CD4 T cells could be observed in the reticular dermis by intravital (IV) microscopy; unlike TRM, these cells expressed little CD69 or CD103, produced negligible IL-17, and interacted infrequently with CD11c+ DC. By IV microscopy, these migratory deep dermal cells could actually be observed entering the previously infected skin from blood in real time with rapid kinetics, a process that could be blocked completely by the sphingosine 1 phosphate receptor (S1P1) inhibitor FTY720 (fingilomod). These migratory CD4 cells expressed CCR7, CD27, were heterogeneous for Lselectin, and were negative for KLRG-1. This phenotypic profile suggests that they are a combination of skin homing TCM cells and the recently described “T migratory memory” (TMM) cells, rather than TEM cells (which are uniformly KLRG-1+). In contrast, Th17 CD4 TRM cells in the same field are all negative for these markers. Later in the time course, CD4 FoxP3+ Treg cells also infiltrated the skin and accumulated as a major population of TMM cells coexisting with Th17 CD4 TRM cells. These results suggest that heterogeneous populations of CD4 T cells constitutively migrate into skin and provide immunosurveillance that may modify that provided by TRM. 014 Commensal microbes augment expression of the hair follicle chemokine Ccl20 to facilitate accumulation of Tregs in neonatal skin TC Scharschmidt, KS Vasquez, ML Pauli, H Truong, JL Sonnenburg, SE Millar and MD Rosenblum 1 Dermatology, University of California, San Francisco, San Francisco, CA, 2 Dermatology, University of Pennsylvania, Philadelphia, PA and 3 Microbiology and Immunology, Stanford University, Stanford, CA We have previously reported that a wave of regulatory T cells (Tregs) into neonatal skin is responsible for establishing tolerance to skin commensal microbes. However, mechanisms mediating the abrupt accumulation of this population remain undefined. Tregs localize to hair follicles (HFs) in both mouse and human skin. Migration of Tregs into neonatal skin coincides with initial colonization of the skin by commensal microbes and HF neogenesis. We hypothesized that both commensal microbes and HF development play a role in facilitating Treg migration to neonatal skin. Germ-free neonates as well as K5-Dkk1 mice, which fail to develop HFs, demonstrate reduced numbers of neonatal skin Tregs. Using RNA-seq and flow cytometry we identified Ccr6 as a key receptor preferentially expressed by neonatal skin Tregs. The Ccr6 ligand, Ccl20, is a chemokine known to be produced by HF keratinocytes. We found that expression of Ccl20 increased in neonatal skin during the period of peak Treg influx and was significantly reduced under germ-free conditions. Accordingly, Ccl20 was exquisitely capable of driving migration of neonatal Tregs in vitro. Taken together, these data suggest that HFs and commensal bacteria jointly coordinate migration of Tregs into neonatal skin during this critical developmental window by a Ccl20-dependent mechanism.