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Skin tissue resident memory T cells (T RM ) provide superior protection to a second infection. In this study, we evaluated the use of topical CpG oligodeoxynucleotide (ODN) as adjuvant to generate skin T RM in mice. Topical or s.c. CpG ODN adjuvant administration at the time of a s.c. Ag injection led to an accumulation of CD103 - CD8 T cells in the epidermis. However, only mice with CpG ODN administered topically had significant numbers of CD103 + Ag-specific CD8 T cells persisting in the local epidermal skin, enhanced circulating memory cells in the blood, and showed protection from intradermal challenge with melanoma cells. Generation of Ag-specific CD8 T cells was dependent on TLR9 expression on hematopoietic cells and partially dependent on receptor expression on stromal cells. Topical challenge of immunized mice at a distal site led to significant expansion of Ag-specific T cells in the blood and accumulation in the challenged skin. We demonstrate that local and systemic T cell memory can be generated with topical CpG ODN at the time of s.c. immunization, suggesting a new method of enhancing current vaccine formulations to generate tissue T RM ., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Hollow microneedles can overcome the stratum corneum (SC) barrier and deposit a compound directly into the viable epidermis or the dermis, unlike adhesive patches that rely on drug diffusion across the SC. The traditional one-dimensional methods used to study the diffusivity of drugs across the skin layers are not very accurate for hollow microneedles, since the ejection of compounds out of microneedle lumens resembles a point-source spreading in all directions and is highly dependent on injection depth. This paper presents a technique that is useful for studying drug injection using hollow microneedles at various depths below the SC. This technique uses confocal microscopy to image the distribution of a fluorescent compound in the skin after injection. The fluorescence distribution in the skin is observed over time and applied to a spherical Gaussian diffusion model for limited source diffusion to determine the diffusion coefficient of the compound in the skin. Applied to freshly excised pig skin, the diffusion coefficient for the anti-cancer drug doxorubicin was measured as 4.61 × 10(-9) cm(2)/s, while the diffusion coefficient in previously refrigerated or frozen pig skin was 1.31 × 10(-8) cm(2)/s and 4.21 × 10(-8) cm(2)/s, respectively. Our data suggests that skin storage conditions can substantially alter the diffusion of drugs. The use of refrigerated and, even more so, previously frozen skin should be avoided for quantitative transdermal drug delivery studies.

Background: Cutaneous necrotizing vasculitides (CNV) represent a heterogeneous group of inflammatory diseases affecting the skin blood vessels, characterized histologically by transmural inflammation of the blood vessel wall with fibrinoid necrosis and clinically characterized by palpable purpura, leading to ulceration. These syndromes represent a spectrum of disease from limited cutaneous small vessel vasculitis to rapidly progressive systemic vasculitis. Moreover, a number of diseases can mimic vasculitis in the skin, thereby presenting diagnostic difficulties for physicians., Objective: We present an update of CNV and vasculopathies based on recent literature and clinical experience. We provide a dermatologic approach to the patient presenting with purpura and ischemic skin necrosis focusing on the subtle features that may help physicians discern between primary and secondary causes and the differences between vasculitis and vasculopathy.

Background: Vitiligo is characterized by the death of melanocytes in the skin. This is associated with the presence of T cell infiltrates in the lesional borders. However, at present, there is no detailed and systematic characterization on whether additional cellular or molecular changes are present inside vitiligo lesions. Further, it is unknown if the normal appearing non-lesional skin of vitiligo patients is in fact normal. The purpose of this study is to systematically characterize the molecular and cellular characteristics of the lesional and non-lesional skin of vitiligo patients., Methods and Materials: Paired lesional and non-lesional skin biopsies from twenty-three vitiligo patients and normal skin biopsies from sixteen healthy volunteers were obtained with informed consent. The following aspects were analyzed: (1) transcriptome changes present in vitiligo skin using DNA microarrays and qRT-PCR; (2) abnormal cellular infiltrates in vitiligo skin explant cultures using flow cytometry; and (3) distribution of the abnormal cellular infiltrates in vitiligo skin using immunofluorescence microscopy., Results: Compared with normal skin, vitiligo lesional skin contained 17 genes (mostly melanocyte-specific genes) whose expression was decreased or absent. In contrast, the relative expression of 13 genes was up-regulated. The up-regulated genes point to aberrant activity of the innate immune system, especially natural killer cells in vitiligo. Strikingly, the markers of heightened innate immune responses were also found to be up-regulated in the non-lesional skin of vitiligo patients., Conclusions and Clinical Implications: As the first systematic transcriptome characterization of the skin in vitiligo patients, this study revealed previously unknown molecular markers that strongly suggest aberrant innate immune activation in the microenvironment of vitiligo skin. Since these changes involve both lesional and non-lesional skin, our results suggest that therapies targeting the entire skin surface may improve treatment outcomes. Finally, this study revealed novel mediators that may facilitate future development of vitiligo therapies.

1,25-Dihydroxyvitamin D(3) is immunosuppressive both in vivo and in vitro. Topical vitamin D analogs such as calcipotriol alter keratinocyte function, but their effects on cutaneous immune responses are less well understood. We demonstrate that exposure of the skin to calcipotriol before transcutaneous immunization with OVA protein and CpG adjuvant prevents Ag-specific CD8(+) T cell priming coincident with Langerhans cell depletion in the skin. Immunization through calcipotriol-treated skin induces CD4(+)CD25(+) regulatory T cells (Treg) that prevent subsequent Ag-specific CD8(+) T cell proliferation and IFN-gamma production. Treg induced by calcipotriol are able to inhibit the induction and the elicitation of protein contact hypersensitivity. Topical calcipotriol treatment also induces RANKL (receptor activator of NF-kappaB ligand) expression by keratinocytes, a TNF family member involved in modulation of skin dendritic cells. UV light B induces Ag-specific tolerance when it is applied before transcutaneous immunization. We suggest that UV light B-induced tolerance is induced via a vitamin D receptor-dependent mechanism as vitamin D receptor (VDR) knockout mice fail to increase FoxP3(+) Treg in their peripheral draining lymph node following irradiation. Additionally, keratinocytes of VDR(-/-) mice fail to induce RANKL upon UV irradiation or calcipotriol treatment. The in vivo expansion of Ag-specific Treg with the topical application of the vitamin D analog calcipotriol followed by transcutaneous immunization is a simple method to augment functional Ag-specific CD4(+)CD25(+)Foxp3(+) Treg populations and mimics Ag-specific UV-induced tolerance.

Skin manifestations of lupus erythematosus (LE) are common and are a significant source of morbidity. Lupus-specific skin disease includes chronic cutaneous lupus, subacute cutaneous lupus and acute cutaneous lupus. These conditions have in common a lichenoid tissue reaction. The best-characterized mouse model of spontaneous onset lupus-like skin disease is the MRL/lpr mouse. We review features of this mouse model pertinent to lupus skin disease. Transgenically altered mice indicate an important role for keratinocytes and skin dendritic cells in the development of cutaneous LE. Ultraviolet light (UV) is an environmental factor that has been implicated in exacerbation of systemic disease in LE. Two inbred mouse strains, BXSB and NOD, demonstrate UV accelerated, and UV induced, systemic LE-like disease respectively. We discuss mechanisms of UV-mediated acceleration of disease in these models. A better understanding of these mouse models will lead to an improved understanding of clinical cutaneous lupus erythematosus.

Dendritic cells are the coordinators of the adaptive immune response. Chronic activation of skin dendritic cells by keratinocyte expression of CD40 ligand (CD40L; CD154) leads to autoimmunity. In this issue, systemic administration of tacrolimus is shown by Loser et al. to effectively treat autoimmunity in a murine model involving transgenic keratinocyte expression of CD40L.

UV radiation of the skin impairs immune responses to haptens and to tumor Ags. Transcutaneous immunization (TCI) is an effective method of inducing immune responses to protein and peptide Ag. We explore the effect of UV irradiation on TCI. The generation of Ag-specific CTL to OVA protein, but not class I MHC-restricted OVA peptide, is inhibited by TCI through UV-irradiated skin. Consequently, the induction of protein contact hypersensitivity and in vivo Ag-specific CTL activity following OVA protein immunization is prevented. Application of haptens to UV-exposed skin induces hapten-specific tolerance. We demonstrate that application of protein or class II MHC-restricted OVA peptide to UV-irradiated skin induces transferable Ag-specific tolerance. This tolerance is mediated by CD4(+)CD25(+) T regulatory (T(reg)) cells. These Ag-specific T(reg) cells inhibit the priming of CTL following protein immunization in the presence of CpG adjuvant. IL-10 deficiency is known to prevent hapten-specific tolerance induction. In this study, we demonstrate, using IL-10-deficient mice and adoptive T cell transfer, that IL-10 is required for the direct inhibition of CTL priming following immunization through UV-irradiated skin. However, IL-10 is not required for the induction of T(reg) cells through UV-irradiated skin as IL-10-deficient T(reg) cells are able to mediate tolerance. Rather, host-derived IL-10 is required for the function of UV-generated T(reg) cells. These experiments indicate that protein and peptide TCI through UV-irradiated skin may be used to induce robust Ag-specific tolerance to neo-Ags and that UV-induced T(reg) cells mediate their effects in part through the modulation of IL-10.

Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide+cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4(+) and CD8(+) T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG(1), IgG(2a), and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited.

Microneedles are small needle-like structures that are almost invisible to the naked eye. They have an immense potential to serve as a valuable tool in many medical applications, such as painless vaccination. Microneedles work by breaking through the stratum corneum, the outermost barrier layer of the skin, and providing a direct path for drug delivery into the skin. A lot of research has been presented over the past two decades on the applications of microneedles, yet the fundamental mechanism of how they interact, pressure, and penetrate the skin in its native state is worth examining further. As such, a major difficulty with understanding the mechanism of microneedle-skin interaction is the lack of an artificial mechanical human skin model to use as a standardized substrate. In this research news, the development of an artificial mechanical skin model based on a thorough mechanical study of fresh human and porcine skin samples is presented. The artificial mechanical skin model can be used to study the mechanical interactions between microneedles and skin, but not diffusion of molecules across skin. This model can assist in improving the performance of microneedles by enhancing the reproducibility of microneedle depth insertions for optimal drug delivery and biosensing. [ABSTRACT FROM AUTHOR]

[Display omitted] • Separating adjuvant timing and location from antigen exposure. • Prior intradermal adjuvant benefits subcutaneous vaccination outcome. • Toward single-dose vaccination. Adjuvant activity of the Toll receptor 9 agonist CpG 1826 was compared when given subcutaneously (s.c.) together with ovalbumin (s.c.[CpG + Ova]), or when given by either s.c. or intradermally (i.d.) routes two days prior to s.c. ovalbumin. Frequencies of CD8 + effector (T EFF) and central memory (T CM) T cells along with total IgG, IgG2c, and IgG1 titres were measured to ascertain how timing and location of CpG conditioning influenced vaccination outcome. Prior treatment with CpG enhanced T EFF , T CM , as well as total IgG responses. T EFF and T CM responses were greatest when CpG was given intradermally and prior to s.c. ovalbumin, conditions that eliminated the fraction of T CM 'non-responders' observed after s.c.[CpG + Ova] vaccination. IgG responses were polarized toward IgG2c after early s.c. CpG but toward IgG1 after early i.d. CpG. Separating CpG adjuvant and antigen application in time and space can improve vaccination outcome. [ABSTRACT FROM AUTHOR]

Abstract: We explored the topical use of resiquimod (R-848), a Toll-like receptor (TLR) 7/8 agonist, in gel formulation, to enhance cross-priming to subcutaneously administered protein antigen in a murine model. Resiquimod application at the time of subcutaneous administration of ovalbumin generated robust antigen-specific CTL as detected by tetramers, IFN-γ ELISPOT assays and standard cytotoxicity assays. Induced CTL were capable of mediating antigen-specific killing in vivo as measured by in vivo cytotoxicity assays and an ability to protect against B16-OVA tumor challenge. Multiple serial applications of topical resiquimod increased the frequency of antigen-specific CTL when compared to single application. This enhanced frequency was noted despite a marked inhibition of adjuvant mediated pro-inflammatory cytokine release following repeated administration. Topical resiquimod is a potent adjuvant for locally administered subcutaneous vaccines, inducing clinically relevant CTL responses following single application at the time of subcutaneous vaccination. [Copyright &y& Elsevier]

The identification of tumor antigens has spurred the development of efficient adjuvants and novel delivery systems for cancer immunotherapy. To this end, a peptide-based vaccine consisting of the Antennapedia transduction sequence (ANTP) attached to an antigenic peptide was designed to enhance per-cutaneous delivery into cells of the epidermis and dermis. Here we show that the topical application of OVA257–264 linked to ANTP in mice onto tape-stripped skin resulted in enhanced delivery of the antigen through the skin whereas OVA257–264 alone remained distributed uniformly on the skin surface. This delivery correlated with an increase in the CTL response against OVA. When mixed with CpG oligodinucleotides (ODN), the recombinant antigen protected mice from tumor challenge. These data provide the first indication that in vivo use of a translocation sequence can enhance delivery of therapeutic peptides and increase anti-tumor immunity through a simple and safe mechanism involving enhanced penetration of the skin barrier. [Copyright &y& Elsevier]

The immune system of the skin has recently been exploited for the development of non-invasive vaccine technologies. However, one of the limitations of current vaccine protocols is the inefficient priming of cytotoxic T lymphocytes (CTL). In this study, we report that the application of either an immunodominant class I MHC restricted ovalbumin peptide or whole ovalbumin protein, to tape-stripped skin together with the co-application of the bacterial enterotoxin cholera toxin (CT) induces antigen-specific CTL. Tape-stripping (TS) was found to enhance the magnitude of antibody responses to co-administered protein and to promote the generation of antigen-specific IgG2a responses. As well, both cholera toxin and tape-stripping enhanced epidermal dendritic cell (DC) immigration into draining lymph nodes. The adjuvant effect of co-administered cholera toxin and tape-stripping in promoting CTL priming was not dependent on IL-12. Epicutaneous immunization has previously been shown to induce robust antibody responses to administered protein antigen. We now demonstrate the induction of robust and persistent CTL responses to epicutaneously administered protein antigen. Epicutaneous immunization is cheap, simple and effective. These findings suggest the potential use of the skin for the generation of protective immune responses to both viral and tumor challenge. [Copyright &y& Elsevier]