013 Heterogeneous CD4 migratory T cells rapidly and constitutively infiltrate skin and co-exist with populations of resident memory T cells (T RM ) C Park 1 , Y Pan 1 , X Fu 2 , T Tian 1 , S Divito 1 , C Lin 2 , R Clark 1 and TS Kupper 1 1 Dermatology, BWH/HMS, Boston, MA and 2 Wellman Laboratories, MGH, Boston, MA Long term resident T RM cells have long been described in skin, but recently populations of migratory T cells have been identified that co-exist in human skin with T RM . While the function of skin CD4 T RM 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 T RM 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 T RM , 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 (fin- gilomod). These migratory CD4 cells expressed CCR7, CD27, were heterogeneous for L- selectin, and were negative for KLRG-1. This phenotypic profile suggests that they are a combination of skin homing T CM cells and the recently described “T migratory memory” (T MM ) cells, rather than T EM cells (which are uniformly KLRG-1+). In contrast, Th17 CD4 T RM 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 T MM cells coexisting with Th17 CD4 T RM cells. These results suggest that heterogeneous populations of CD4 T cells constitutively migrate into skin and provide immunosurveillance that may modify that provided by T RM . 014 Commensal microbes augment expression of the hair follicle chemokine Ccl20 to facilitate accumulation of Tregs in neonatal skin TC Scharschmidt 1 , KS Vasquez 1 , ML Pauli 1 , H Truong 1 , JL Sonnenburg 3 , SE Millar 2 and MD Rosenblum 1 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 hy- pothesized 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. 015 Norepinephrine (NE) biases Langerhans cell (LC) antigen presentation to T cells toward a Th17 response via actions on endothelial cells W Ding, L Xu, L Stohl, JA Wagner and RD Granstein Weill Cornell Medicine, New York, NY We now report that exposure of primary dermal microvascular endothelial cells (pDMECs) to NE induces them to bias LC antigen presentation to CD4 + T cells toward generation of Th17 cells. BALB/c pDMECs (1x10 4 /well) were plated in 96-well round bottom plates in medium containing 10 -8 M to 10 -5 M NE. After 3 h, plates were washed 4 times and 1x10 4 BALB/c LCs and 2x10 5 CD4 + T cells from DO11.10 mice (BALB/c background) were added per well followed by addition of a fragment of chicken ovalbumin (cOVA 323-339 ) to 10 mM in 200 ml of medium. DO11.10 mice express transgenes in their T cell receptors that recognize cOVA 323-339 . After 48 h, supernatants were assayed for cytokine content by ELISA. Addition of NE-exposed pDMECs significantly enhanced IL-6 and IL-17A production while inhibiting interferon-g (IFNg) and IL-22 release compared to wells containing non-treated pDMECs. When T cells were harvested from these cultures and analyzed by FACS for intracellular cytokines, increased numbers of cells containing IL-17A were observed along with a decrease in cells containing IFNg. RNA was extracted from T cells from these cultures. By quantitative RT-PCR the presence of NE-treated pDMECs led to a significant increase in mRNA for IL-17A and RORgT while IFNg, IL-22 and T-bet mRNA were decreased. Similar experiments in which Transwell inserts were used to separate pDMECs from LCs and responding T cells showed that the effect on cytokine release did not depend on cell-cell contact. Thus, NE can influence the outcome of antigen presentation toward a Th17-type immune response through actions on ECs that induce release of a soluble mediator(s) mediating the effects. As dermal blood vessels and lymph nodes are associated with sympathetic nerves, we hypothesize that, when the sympathetic nervous system is activated by stress, NE release biases antigen presentation towards the Th17 pole via actions on ECs. This may exacerbate psoriasis and, perhaps, other inflammatory skin disorders involving Th17 cells. This pathway may prove to be a therapeutic target. 016 Calcitonin gene-related peptide (CGRP)-treated primary dermal microvascular endothelial cells (pDMECs) bias the outcome of antigen (Ag) presentation toward the Th17 pole RD Granstein, L Stohl, L Xu, JA Wagner and W Ding Weill Cornell Medicine, New York, NY CGRP endows ECs with the ability, as bystanders, to bias LC Ag presentation to responsive CD4 + T cells toward generation of Th17 cells. We have now investigated the specificity of the effect, tested whether it is receptor-mediated and examined the role of IL-6 in this phe- nomenon. BALB/c pDMECs (1x10 4 /well) were plated in 96-well plates and treated for 3 hrs with 0.1 to 100 nM CGRP or medium alone. Cells were then washed extensively and 1x10 4 LCs (BALB/c) and 2x10 5 CD4 + T cells from DO11.10 transgenic mice (BALB/c background) were added per well in the presence or absence of 10 mM cOVA 323-339 . (T cells from DO11.10 mice express transgenes such that they respond spontaneously to cOVA 323-339 .) Supernatants were harvested 48 hrs later and analyzed for cytokine content. CGRP exposed- ECs significantly enhanced IL-6 and IL-17A production compared to wells containing medium-treated ECs while significantly inhibiting interferon-g, IL-22 and IL-4 production. Experiments comparing the effects of CGRP in this assay to adrenomedullin and substance P demonstrated the specificity of the CGRP effect. Pre-treatment of pDMECs with the CGRP inhibitor CGRP 8-37 inhibited the CGRP effect. Experiments utilizing Transwell plates to separate pDMECs from LCs and responding T cells showed that the effects did not depend on cell-cell contact. Pretreatment of pDMECs with siRNA to IL-6 indicated that much of the effect observed, although not all, could be inhibited by suppressing IL-6 release. Finally, addition of IL-6 to cultures of LCs presenting Ag to responding T cells demonstrated effects similar to those seen when CGRP-treated pDMECs were added to such culture wells. Thus, the effect of CGRP-treated pDMECs on biasing Ag presentation towards the Th17 pole is specific, depends on action at the CGRP receptor and is at least partially due to induction of IL-6 release by CGRP-treated pDMECs. These results demonstrate a novel pathway for possible manipulation in disorders marked by unwanted or inappropriate Th17 cell activity. 017 Chemo-immunotherapy of melanoma using dissolvable microneedle arrays BE Friedman 1 , C Donahue 1 , G Erdos 1 and L Falo 1,2,3 1 Department of Dermatology, Uni- versity of Pittsburgh School of Medicine, Pittsburgh, PA, 2 Clinical and Translational Science Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA and 3 Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA We have previously demonstrated that dissolvable microneedle arrays (MNAs) can co-deliver cargos directly to the microenvironment of cutaneous tumors while avoiding systemic dis- tribution. As doxorubicin has been shown to induce immunogenic cell death and type I IFN has been implicated in the inflammatory tumor phenotype associated with improved clinical outcomes, we sought to investigate if MNA co-delivery of doxorubicin and a TLR agonist that stimulates type I IFN signaling could induce both immunogenic cell death in tumor cells and proinflammatory changes in the tumor microenvironment implicated in better clinical out- comes. Efficacy of topical chemo-immunotherapy of melanoma was evaluated in mice bearing established B16 tumors that were subsequently treated with MNAs containing Poly(I:C) (MNA-Poly), doxorubicin (MNA-Dox), or Poly(I:C) and doxorubicin (MNA-Dox- Poly). All treatment groups demonstrated improved survival over untreated mice, with the greatest improvement seen in mice treated with MNA-Dox-Poly (80% survival versus 0% survival in untreated mice at 60 days post-inoculation). Both MNA-Dox and MNA-Dox-Poly treatments induced substantial cell death, with associated increased levels of activated caspase-3 in tumor cells as determined by TUNEL and flow-based caspase-3 assays. Further, flow cytometry and histological analyses revealed that mice treated with MNA-Poly, MNA- Dox and MNA-Dox-Poly demonstrated increased intra-tumoral levels of neutrophils and increased CD8:CD4 T-cell ratios, with greatest increases seen in MNA-Dox-Poly treated mice. Together these data suggest the potential of MNA administered doxorubicin and adjuvant to induce immunogenic tumor cell death and provide insight into associated inflammatory changes in the tumor microenvironment associated with improved survival in melanoma patients. 018 Cytokine and chemokine receptor profiles of human interleukin 9 producing T helper cells show close relationship to T H 2 cells C Micosse ´ 1 , A Frei 2 , TS Kupper 3 , R Clark 3 , D Yerly 4 and C Schlapbach 1 1 Department of Dermatology, University Hospital of Bern, Bern, Switzerland, 2 Department of Microbiology and Immunology, Stanford University, Stanford, CA, 3 Department of Dermatology, Harvard Medical School / Brigham and Women’s Hospital, Boston, MA and 4 Division of Allergol- ogy, University Hospital of Bern, Bern, Switzerland Interleukin 9 (IL-9) producing T helper cells have been proposed as a novel T H cell subset (T H 9 cells). In mice, T H 9 cells mediate superior anti-tumor immunity. However, little is known about human T H 9 cells and therefore a bona fide T H 9 lineage has been called into question. We thus extended our previous findings that IL-9 is produced transiently after activation by a distinct population human skin-tropic T H cells. Analysis of the surface phenotype revealed that IL-9 producing T H cells are effector memory T cells and significantly enriched in the CXCR3 - /CCR4 + /CCR6 - /CCR8 + population. Thus, they show a chemokine receptor profile similar to that of T H 2 cells (CXCR3 - /CCR4 + /CCR6 - ). Accordingly, a T cell clone generated from the CXCR3 - /CCR4 + /CCR6 - population showed strong but transient expression of IL-9 followed by upregulation of the T H 2 cytokines IL-13 and IL-5. Close relation of IL-9 producing cells with T H 2 cells was substantiated by mass cytometry using a novel method for multi- plexed simultaneous detection of transcripts in intact cells: In activated T cells from healthy humans, up to 67% of T H cells expressing il9 transcripts co-expressed il13. Unsupervised clustering of mass cytometry data classified IL-9 producing cells as closely related to T H 2 cells. Interestingly, IL-9 expression depends on autocrine/paracrine signals, as inhibition of protein secretion downregulated IL-9 transcription but not that of other T H cell cytokines. Taken together, our data show that human IL-9 producing T H cells are closely related to T H 2 cells and depend on autocrine/paracrine signals. Our findings establish a basis for the study of the transcriptional regulation, lineage specification and plasticity of the T H 9 phenotype and, ultimately, its contribution to tumor immunity in humans. Adaptive Immunity & Vaccination | ABSTRACTS www.jidonline.org S3