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317 PD-L1 on radio-resistant cells regulates effector CD8+ T-cell activation during the elicitation phase of contact hypersensitivity
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Universal transcriptomic analysis of host-microbiome interactions in psoriasis T Furnholm1, M Foo1, L Reingold1, K Shedden2 and A Johnston1 1 Dermatology, University of Michigan, Ann Arbor, MI and 2 CSCAR, University of Michigan, Ann Arbor, MI Perturbations in host-microbe interactions underlie many diseases. 16S DNA sequencing has indicated that psoriasis skin and tonsils have markedly altered microbiomes, however, this method identifies bacteria poorly and does not inform about altered microbial function. Thus, we developed new RNA-seq alignment software coupled to a functional and taxonomic annotated gene database, which allows simultaneous analysis of host and microbe gene expression. To identify host-microbe interactions that may drive psoriasis, we performed RNA-seq analysis on lesional skin (LS, n¼11), uninvolved skin (US, n¼10), and tonsils (PT, n¼11) of psoriasis patients and normal controls (NS, NT n¼7). In LS, 799 differentially expressed (>2-fold) human genes mapped to KEGG functions including inflammation (IL1B, IL8, NFkB, TLR4), proliferation (EGF, STAT3, CCND1, MYC), vascularization (VEGF, TEK, ANGPT1), and T cell response (TGFB1, ITGB2, ICAM1, SGK1). This was accompanied by reduced microbial diversity (Shannon Index: LS¼7.2, US¼8.6, NS¼8.3), with decreased Propionibacterium and >4-fold increased S. pyogenes colonization. 9/64 (LS) and 40/90 (PT) of streptococcal species were >2-fold more abundant than in controls, with S. anginosus and S. parasanguinis predominant. Multiple streptococcal species had differentially expressed (>5-fold) virulence factors, including sialidase (NEU1, PT) streptolysin O (Slo, LS), hemolysin (HlyC, PT and LS), exfoliative toxin (EtaA, PT and LS), antimicrobial peptide resistance (DltAD, PT and LS), sec- and TypeIV protein secretion systems and secreted proteases. Psoriasis tonsils yielded 333 other differentially abundant taxa, including Tannerella forsythia, Bulleidia extructa W1219, Dialister invisus, Gemella sanguinis, and Staphylococcus sciuri, all implicated in inflammatory diseases. Our novel RNA-seq pipeline revealed enriched streptococcal superantigens in psoriatic skin and tonsils, together with species-level identification of streptococci and other invasive bacteria that could drive psoriasis and its co-morbidities.
PD-L1 on radio-resistant cells regulates effector CD8D T-cell activation during the elicitation phase of contact hypersensitivity T Hirano1, T Honda1, K Tamada2, L Chen3 and K Kabashima1 1 Department of Dermatology, Kyoto University, Kyoto, Japan, 2 Department of Immunology, Yamaguchi University, Ube, Japan and 3 Department of Immunobiology, Yale University, New Haven, CT The programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway is a negative regulator of effector CD8+ T-cell activation, and is suggested to be involved in the regulation of various inflammatory diseases, including contact dermatitis. However, the role of the PD-1/ PD-L1 pathway in contact dermatitis remains unclear. To address this issue, we subjected PDL1 deficient mice to contact hypersensitivity (CHS), a mouse model of contact dermatitis and a prototype of delayed-type hypersensitivity. PD-L1 deficient mice exhibited significantly exacerbated and prolonged ear swelling responses and increased IFNg production from CD8+ T cells in the skin compared to wild-type (WT) mice. Administration of PD-L1 blocking antibody to WT mice during the sensitization phase did not exacerbate CHS responses, but did during the elicitation phase, indicating that the PD-1/PD-L1 pathway works mainly during the elicitation phase of CHS. Bone marrow (BM) chimera experiments reveled that PD-L1 deficient mice transplanted with BM from WT mice exhibited significantly increased CHS responses compared to the control WT mice transplanted with BM from WT mice, suggesting that radio-resistant cells such as keratinocytes (KCs) or Langerhans cells (LCs) are responsible for the PD-1/PD-L1 dependent regulation. The expression of PD-L1 was significantly increased in KCs during the elicitation phase and depletion of LCs during the elicitation phase did not affect CHS responses, suggesting that KCs, rather than LCs, are involved in PD-1/PDL1-mediated suppression in CHS. Taken together, our data indicate that the PD-1/PD-L1 pathway regulates CD8+ T-cell activation during the elicitation phase in CHS through PD-L1 on radio-resistant cells, possibly on KCs.
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The functional characterization of CARD14 variants in pityriasis rubra pilaris affected skin and keratinocytes A Go¨blo¨s1,2, J Danis1,2, B Ga´l3, K Farkas3, E Varga1, I Korom1, L Keme´ny1,2, Z Bata-Cso¨rgo¨1,2, M Sze´ll3,2 and N Nagy3,2 1 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, University of Szeged, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Pityriasis rubra pilaris (PRP) is a rare papulosquamous skin disorder, which is phenotypically related to psoriasis. The etiology of the disease is unknown. Familial PRP shows autosomal dominant inheritance due to CARD14 mutations, moreover, CARD14 polymorphisms have also been implicated in sporadic PRP. Our genetic screening of the CARD14 gene in a PRP patient with a positive family history of psoriasis revealed three genetic variants (rs117918077, rs2066964, rs28674001). Further in vitro and in situ functional studies were carried out to examine the consequences of the three identified variants, thus providing a link between genetic variants and cellular pathomechanism. Immunofluorescent staining revealed nuclear localization of the NFkB p65 subunit in PRP skin specimen, indicating activated NFkB, in contrast healthy and psoriatic skin sections only showed cytoplasmic staining of inactivated NFkB. NFkBeluciferase reporter assay demonstrated significantly increased NFkB activity in keratinocytes from the PRP patient compared to healthy keratinocytes. Characterization of the cytokine profile of the keratinocytes and PBMCs demonstrated that higher NFkB activation in PRP cells induced higher responses to inflammatory stimuli compared to healthy cells. Our study highlights that functional characterization of rare and common variants of the CARD14 gene can bring us closer to understanding the role of genetic variants in disease pathogenesis.
Resistant residents: Turnover of skin-resident memory T cells J Strobl1, N Bayer1, K Strobl1, B Reininger1, M Bru¨ggen2 and G Stary1 1 Department of Dermatology, Medical University of Vienna, Vienna, Austria and 2 Department of Dermatology, Zurich University Hospital, Zurich, Switzerland Most of our knowledge about the recirculation and distribution of cutaneous tissue-resident memory T cells (TRM) is based on observations from murine studies, while the fate of TRM in human skin is still poorly understood. We followed patients who underwent conditioning regimens (myeloablative therapy, total body irradiation) and subsequent allogeneic hematopoietic stem cell transplantation (HSCT) to track repopulation dynamics of cutaneous T cells over time. Skin biopsies and peripheral blood from 25 patients were taken at four time points before and after conditioning regimens/HSCT. T cells were characterized using molecules for migratory T cells (CCR7, CD62L) and markers defining cutaneous TRM (CD69, CD103). In sex-mismatched transplanted patients, X- and Y-chromosomes on T cells were fluorescently labelled to precisely define their host/donor origin for up to one year after HSCT. All stainings were automatically quantified using imaging analysis software. In concordance with data of peripheral blood, the overall number of T cells in the skin declined upon HSCT. While small populations of central and migratory memory T cells (CD3+CCR7+CD62L+/-) were present in the dermis before HSCT, they disappeared from skin biopsies upon leukocyte depletion by conditioning regimens and repopulated the dermis between week 2 and 14 after HSCT. Notably, a subset of dermal CD69+ T cells with various expression levels of CD103 remained stable throughout all time points. Consistently, XY-chromosome analysis of T cells revealed that host T cells survived in the dermis for at least one year post HSCT. These data present new insights into resilience of skin-resident memory T cells as radiation-resistant and self-contained cells and open a wide range of fundamental questions on T cell biology in peripheral tissues. In addition, host-derived T cells might play a so far unappreciated role in the initiation and propagation of graft-versus-host disease (GVHD), a common and potentially fatal complication of HSCT.
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ADAM10-induced cleavage of semaphorin 4D from eosnophils contributes to antibody production in bullous pemphigoid S Shen, Y Ke, E Dang, T Zhang, H Qiao and G Wang Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Lesional infiltration of eosnophils is a distinguishing histopathologic characteristic for bullous pemphigoid (BP). However, the actual contribution of eosnophils to the pathogenesis of BP is largely unknown. Semaphorin 4D (Sema4D) is constitutively expressed on granulocytes and plays important roles in B cell regulation, but its implication in BP remains obscure. Here, we descried that eosnophils, marked as CD15+ granulocytes in patients with BP presented much lower expression of membrane-bound Sema4D (mSema4D) in both peripheral blood and blister fluid. Consistently, the levels of both lesional and circulating sSema4D were significantly elevated and well correlated with anti-BP180 antibody titer. Moreover, healthy-derived CD15+ granulocytes activated by PMA started spontaneous shedding of mSema4D and generating 120kDa-sized sSema4D into cell supernatants, which can be specifically blocked by ADAM10 inhibitor. Conformably, membrane-bound ADAM10 (mADAM10) was abundantly expressed on activated CD15+ granulocytes despite its absence before activation. Meanwhile, we found that soluble ADAM10 (sADAM10) treatment can directly cause mSema4D cleavage from CD15+ granulocytes. In line with in vitro findings, sADAM10 displayed a prominently high level in the blister fluid and mADMA10 was extensively expressed on CD15+granulocytes from BP patients but nearly absent on those from healthy individuals. Furthermore, in patient-derived PBMCs, by promoting the differentiation of B cell into plasmablasts, sSema4D specifically boosted anti-BP180 antibody production in a time- and dose- dependent manners, which was attributed to CD72-mediated activation of Akt/NF-kB p-65/Erk cascades in B cells. Our findings revealed a novel role of eosnophils in ADAM10dependent Sema4D shedding that ultimately caused augmented pathogenic antibody and aggravated autoimmune responds, thus providing a new insight into the BP pathogenesis.
Human skin-resident innate-like T cells in health and disease R Woolf2,1, O Nussbaumer2 and A Hayday2,3 1 St John’s Institute of Dermatology, King’s College London, UK, London, United Kingdom, 2 Department of Immunobiology, King’s College London, UK, London, United Kingdom and 3 Francis Crick Institute, London, United Kingdom The skin contains many resident immune cells. In the murine epidermis a distinct population of innate-like Vg5Vd1+ T cells has the capacity to directly respond to markers of tissue stress. This lymphoid stress surveillance response (LSSR) contributes to cancer immune-surveillance and also cutaneous atopy. We sought to investigate whether human skin harbours a similar potential for LSSR and if such functional responses are dysregulated in inflammatory skin disease. We adapted an explant protocol to isolate skin-resident lymphocytes from healthy donors and individuals with chronic plaque psoriasis or atopic dermatitis. Cells were characterized by flow cytometry, including functional responses following in vitro activation under different conditions. We found that healthy skin contained a reproducible population of gd T cells: mean 7.6% of lymphocytes. Skin-resident gd T cells predominantly expressed the Vd1 and Vd3 TCR, making them very distinct from those in the blood. Strikingly, these cells were activated by ligands for the NKG2D receptor, independent of their TCR e analogous to innate-like T cell responses in mouse skin. Such responsiveness was not seen in NKG2D+ TCRab+ TRM cells isolated from the same skin samples. Upon activation, skinresident gd T cells invariably showed robust TH1-like/cytotoxic effector responses. gd T cells were also isolated from skin of patients with chronic inflammatory skin disease. Fewer cells were found in atopic dermatitis [AD] lesions (mean 2.1%; p ¼ 0.01). Of note, the activation of such cells in certain AD individuals now led to IL-13/IL-4 production. Likewise, activation of gd T cells from some psoriasis patients uniquely induced IL-17A. These data provide clear evidence of human innate-like gd T cells and for their functional polarisation in discrete disease states. With gd T cells placed in the afferent phase of the immune response, our findings have overt implications for skin immune surveillance and its potentially significant contribution to pathology.
www.jidonline.org S247
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Universal transcriptomic analysis of host-microbiome interactions in psoriasis T Furnholm1, M Foo1, L Reingold1, K Shedden2 and A Johnston1 1 Dermatology, University of Michigan, Ann Arbor, MI and 2 CSCAR, University of Michigan, Ann Arbor, MI Perturbations in host-microbe interactions underlie many diseases. 16S DNA sequencing has indicated that psoriasis skin and tonsils have markedly altered microbiomes, however, this method identifies bacteria poorly and does not inform about altered microbial function. Thus, we developed new RNA-seq alignment software coupled to a functional and taxonomic annotated gene database, which allows simultaneous analysis of host and microbe gene expression. To identify host-microbe interactions that may drive psoriasis, we performed RNA-seq analysis on lesional skin (LS, n¼11), uninvolved skin (US, n¼10), and tonsils (PT, n¼11) of psoriasis patients and normal controls (NS, NT n¼7). In LS, 799 differentially expressed (>2-fold) human genes mapped to KEGG functions including inflammation (IL1B, IL8, NFkB, TLR4), proliferation (EGF, STAT3, CCND1, MYC), vascularization (VEGF, TEK, ANGPT1), and T cell response (TGFB1, ITGB2, ICAM1, SGK1). This was accompanied by reduced microbial diversity (Shannon Index: LS¼7.2, US¼8.6, NS¼8.3), with decreased Propionibacterium and >4-fold increased S. pyogenes colonization. 9/64 (LS) and 40/90 (PT) of streptococcal species were >2-fold more abundant than in controls, with S. anginosus and S. parasanguinis predominant. Multiple streptococcal species had differentially expressed (>5-fold) virulence factors, including sialidase (NEU1, PT) streptolysin O (Slo, LS), hemolysin (HlyC, PT and LS), exfoliative toxin (EtaA, PT and LS), antimicrobial peptide resistance (DltAD, PT and LS), sec- and TypeIV protein secretion systems and secreted proteases. Psoriasis tonsils yielded 333 other differentially abundant taxa, including Tannerella forsythia, Bulleidia extructa W1219, Dialister invisus, Gemella sanguinis, and Staphylococcus sciuri, all implicated in inflammatory diseases. Our novel RNA-seq pipeline revealed enriched streptococcal superantigens in psoriatic skin and tonsils, together with species-level identification of streptococci and other invasive bacteria that could drive psoriasis and its co-morbidities.
PD-L1 on radio-resistant cells regulates effector CD8D T-cell activation during the elicitation phase of contact hypersensitivity T Hirano1, T Honda1, K Tamada2, L Chen3 and K Kabashima1 1 Department of Dermatology, Kyoto University, Kyoto, Japan, 2 Department of Immunology, Yamaguchi University, Ube, Japan and 3 Department of Immunobiology, Yale University, New Haven, CT The programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway is a negative regulator of effector CD8+ T-cell activation, and is suggested to be involved in the regulation of various inflammatory diseases, including contact dermatitis. However, the role of the PD-1/ PD-L1 pathway in contact dermatitis remains unclear. To address this issue, we subjected PDL1 deficient mice to contact hypersensitivity (CHS), a mouse model of contact dermatitis and a prototype of delayed-type hypersensitivity. PD-L1 deficient mice exhibited significantly exacerbated and prolonged ear swelling responses and increased IFNg production from CD8+ T cells in the skin compared to wild-type (WT) mice. Administration of PD-L1 blocking antibody to WT mice during the sensitization phase did not exacerbate CHS responses, but did during the elicitation phase, indicating that the PD-1/PD-L1 pathway works mainly during the elicitation phase of CHS. Bone marrow (BM) chimera experiments reveled that PD-L1 deficient mice transplanted with BM from WT mice exhibited significantly increased CHS responses compared to the control WT mice transplanted with BM from WT mice, suggesting that radio-resistant cells such as keratinocytes (KCs) or Langerhans cells (LCs) are responsible for the PD-1/PD-L1 dependent regulation. The expression of PD-L1 was significantly increased in KCs during the elicitation phase and depletion of LCs during the elicitation phase did not affect CHS responses, suggesting that KCs, rather than LCs, are involved in PD-1/PDL1-mediated suppression in CHS. Taken together, our data indicate that the PD-1/PD-L1 pathway regulates CD8+ T-cell activation during the elicitation phase in CHS through PD-L1 on radio-resistant cells, possibly on KCs.
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The functional characterization of CARD14 variants in pityriasis rubra pilaris affected skin and keratinocytes A Go¨blo¨s1,2, J Danis1,2, B Ga´l3, K Farkas3, E Varga1, I Korom1, L Keme´ny1,2, Z Bata-Cso¨rgo¨1,2, M Sze´ll3,2 and N Nagy3,2 1 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, University of Szeged, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Pityriasis rubra pilaris (PRP) is a rare papulosquamous skin disorder, which is phenotypically related to psoriasis. The etiology of the disease is unknown. Familial PRP shows autosomal dominant inheritance due to CARD14 mutations, moreover, CARD14 polymorphisms have also been implicated in sporadic PRP. Our genetic screening of the CARD14 gene in a PRP patient with a positive family history of psoriasis revealed three genetic variants (rs117918077, rs2066964, rs28674001). Further in vitro and in situ functional studies were carried out to examine the consequences of the three identified variants, thus providing a link between genetic variants and cellular pathomechanism. Immunofluorescent staining revealed nuclear localization of the NFkB p65 subunit in PRP skin specimen, indicating activated NFkB, in contrast healthy and psoriatic skin sections only showed cytoplasmic staining of inactivated NFkB. NFkBeluciferase reporter assay demonstrated significantly increased NFkB activity in keratinocytes from the PRP patient compared to healthy keratinocytes. Characterization of the cytokine profile of the keratinocytes and PBMCs demonstrated that higher NFkB activation in PRP cells induced higher responses to inflammatory stimuli compared to healthy cells. Our study highlights that functional characterization of rare and common variants of the CARD14 gene can bring us closer to understanding the role of genetic variants in disease pathogenesis.
Resistant residents: Turnover of skin-resident memory T cells J Strobl1, N Bayer1, K Strobl1, B Reininger1, M Bru¨ggen2 and G Stary1 1 Department of Dermatology, Medical University of Vienna, Vienna, Austria and 2 Department of Dermatology, Zurich University Hospital, Zurich, Switzerland Most of our knowledge about the recirculation and distribution of cutaneous tissue-resident memory T cells (TRM) is based on observations from murine studies, while the fate of TRM in human skin is still poorly understood. We followed patients who underwent conditioning regimens (myeloablative therapy, total body irradiation) and subsequent allogeneic hematopoietic stem cell transplantation (HSCT) to track repopulation dynamics of cutaneous T cells over time. Skin biopsies and peripheral blood from 25 patients were taken at four time points before and after conditioning regimens/HSCT. T cells were characterized using molecules for migratory T cells (CCR7, CD62L) and markers defining cutaneous TRM (CD69, CD103). In sex-mismatched transplanted patients, X- and Y-chromosomes on T cells were fluorescently labelled to precisely define their host/donor origin for up to one year after HSCT. All stainings were automatically quantified using imaging analysis software. In concordance with data of peripheral blood, the overall number of T cells in the skin declined upon HSCT. While small populations of central and migratory memory T cells (CD3+CCR7+CD62L+/-) were present in the dermis before HSCT, they disappeared from skin biopsies upon leukocyte depletion by conditioning regimens and repopulated the dermis between week 2 and 14 after HSCT. Notably, a subset of dermal CD69+ T cells with various expression levels of CD103 remained stable throughout all time points. Consistently, XY-chromosome analysis of T cells revealed that host T cells survived in the dermis for at least one year post HSCT. These data present new insights into resilience of skin-resident memory T cells as radiation-resistant and self-contained cells and open a wide range of fundamental questions on T cell biology in peripheral tissues. In addition, host-derived T cells might play a so far unappreciated role in the initiation and propagation of graft-versus-host disease (GVHD), a common and potentially fatal complication of HSCT.
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ADAM10-induced cleavage of semaphorin 4D from eosnophils contributes to antibody production in bullous pemphigoid S Shen, Y Ke, E Dang, T Zhang, H Qiao and G Wang Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Lesional infiltration of eosnophils is a distinguishing histopathologic characteristic for bullous pemphigoid (BP). However, the actual contribution of eosnophils to the pathogenesis of BP is largely unknown. Semaphorin 4D (Sema4D) is constitutively expressed on granulocytes and plays important roles in B cell regulation, but its implication in BP remains obscure. Here, we descried that eosnophils, marked as CD15+ granulocytes in patients with BP presented much lower expression of membrane-bound Sema4D (mSema4D) in both peripheral blood and blister fluid. Consistently, the levels of both lesional and circulating sSema4D were significantly elevated and well correlated with anti-BP180 antibody titer. Moreover, healthy-derived CD15+ granulocytes activated by PMA started spontaneous shedding of mSema4D and generating 120kDa-sized sSema4D into cell supernatants, which can be specifically blocked by ADAM10 inhibitor. Conformably, membrane-bound ADAM10 (mADAM10) was abundantly expressed on activated CD15+ granulocytes despite its absence before activation. Meanwhile, we found that soluble ADAM10 (sADAM10) treatment can directly cause mSema4D cleavage from CD15+ granulocytes. In line with in vitro findings, sADAM10 displayed a prominently high level in the blister fluid and mADMA10 was extensively expressed on CD15+granulocytes from BP patients but nearly absent on those from healthy individuals. Furthermore, in patient-derived PBMCs, by promoting the differentiation of B cell into plasmablasts, sSema4D specifically boosted anti-BP180 antibody production in a time- and dose- dependent manners, which was attributed to CD72-mediated activation of Akt/NF-kB p-65/Erk cascades in B cells. Our findings revealed a novel role of eosnophils in ADAM10dependent Sema4D shedding that ultimately caused augmented pathogenic antibody and aggravated autoimmune responds, thus providing a new insight into the BP pathogenesis.
Human skin-resident innate-like T cells in health and disease R Woolf2,1, O Nussbaumer2 and A Hayday2,3 1 St John’s Institute of Dermatology, King’s College London, UK, London, United Kingdom, 2 Department of Immunobiology, King’s College London, UK, London, United Kingdom and 3 Francis Crick Institute, London, United Kingdom The skin contains many resident immune cells. In the murine epidermis a distinct population of innate-like Vg5Vd1+ T cells has the capacity to directly respond to markers of tissue stress. This lymphoid stress surveillance response (LSSR) contributes to cancer immune-surveillance and also cutaneous atopy. We sought to investigate whether human skin harbours a similar potential for LSSR and if such functional responses are dysregulated in inflammatory skin disease. We adapted an explant protocol to isolate skin-resident lymphocytes from healthy donors and individuals with chronic plaque psoriasis or atopic dermatitis. Cells were characterized by flow cytometry, including functional responses following in vitro activation under different conditions. We found that healthy skin contained a reproducible population of gd T cells: mean 7.6% of lymphocytes. Skin-resident gd T cells predominantly expressed the Vd1 and Vd3 TCR, making them very distinct from those in the blood. Strikingly, these cells were activated by ligands for the NKG2D receptor, independent of their TCR e analogous to innate-like T cell responses in mouse skin. Such responsiveness was not seen in NKG2D+ TCRab+ TRM cells isolated from the same skin samples. Upon activation, skinresident gd T cells invariably showed robust TH1-like/cytotoxic effector responses. gd T cells were also isolated from skin of patients with chronic inflammatory skin disease. Fewer cells were found in atopic dermatitis [AD] lesions (mean 2.1%; p ¼ 0.01). Of note, the activation of such cells in certain AD individuals now led to IL-13/IL-4 production. Likewise, activation of gd T cells from some psoriasis patients uniquely induced IL-17A. These data provide clear evidence of human innate-like gd T cells and for their functional polarisation in discrete disease states. With gd T cells placed in the afferent phase of the immune response, our findings have overt implications for skin immune surveillance and its potentially significant contribution to pathology.
www.jidonline.org S247