Contact Hypersensitivity Model Research Articles

Sialyl Lewis X Defines an Activated and Functional Regulatory T Cell Subpopulation in Mice.

Attempts have been made to elucidate the functional markers of regulatory T cells (Tregs), CD4+Foxp3+ T cells with an immunosuppressive function. Sialyl Lewis X (sLex), a tetrasaccharide Ag, is involved in leukocyte trafficking as selectin ligands and is a marker of highly differentiated Tregs in humans. However, the importance of sLex in murine Tregs remains unknown. In this study, we report that sLex defines the activated and functional subset of murine Tregs. The contact hypersensitivity model showed that murine Tregs strongly express sLex upon activation, accompanied by functional Treg marker elevation, such as Foxp3, CD25, CD103, CD39, and granzyme B. RNA sequencing analysis revealed sLex-positive (sLex+) Tregs expressed genes involved in Treg function at a higher level than sLex-negative (sLex-) Tregs. Using an invitro suppression assay, we found that sLex+ Tregs could more efficiently suppress naive CD4+ T cell proliferation than sLex- Tregs. In the murine contact hypersensitivity elicitation model, the topical sLex+ Treg injection into the ears suppressed ear inflammation more efficiently than that of sLex- Tregs. Our results indicate that sLex could serve as a unique surface marker of activated and functional Tregs with immunosuppressive functions in mice.

An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Immunomodulatory effects of nanoparticles on dendritic cells in a model of allergic contact dermatitis: importance of PD-L2 expression

Nanoparticle (NP) skin exposure is linked to an increased prevalence of allergic contact dermatitis. In our prior studies using the mouse contact hypersensitivity (CHS) model, we reported that silica 20 nm (SiO2) NPs suppressed the allergic response and titanium dioxide NPs doped with manganese (mTiO2) exacerbated it. In this work, we conducted in vitro experiments using bone marrow-derived dendritic cells (BMDCs) to study the combinatorial effect of the potent 2,4-dinitrofluorobenzene (DNFB) hapten sensitizer with SiO2 and mTiO2 NPs on BMDC cytotoxicity, cytokine secretion and phenotype using the B7 family ligands. Results show that DNFB and mTiO2 behave similarly and exhibit proinflammatory characteristics while SiO2 promotes a naive phenotype. We observe that the B7-H3 (CD276) ligand is only expressed on CD80 + (B7-1) BMDCs. Results from adoptive transfer CHS studies, combined with BMDC phenotype analysis, point to the importance of PD-L2 expression in modulating the adaptive immune response. This work identifies metrics that can be used to predict the effects of NPs on contact allergy and to guide efforts to engineer cell-based therapies to induce hapten specific immune tolerance.

The junctional adhesion molecule-like protein (JAML) is important for the inflammatory response during contact hypersensitivity.

The junctional adhesion molecule-like protein (JAML) plays important roles in wound healing and activation of epidermal γδ T cells in mice. Whether JAML plays a role in contact hypersensitivity (CHS), the animal model of allergic contact dermatitis (ACD), is not known. To examine the role of JAML in CHS, we used various mouse models of CHS in JAML knockout (KO) and wild-type (WT) mice. Furthermore, the expression of the JAML ligand coxsackievirus and adenovirus receptor (CXADR) on keratinocytes was accessed in vitro and in vivo. JAML KO mice had a diminished inflammatory response during both the sensitization and elicitation phase of CHS and had reduced numbers of CD8+ and CD4+ T cells in the epidermis. Furthermore, interferon γ (IFNγ), interleukin 1β (IL-1β) and CXCL10 production were significantly reduced in JAML KO mice during the elicitation phase. We found that CD8+ T cells express JAML and that JAML is essential for rapid flare-up responses to contact allergens. Finally, we show that keratinocytes up-regulate the JAML ligand CXADR following exposure to contact allergens. Our study is the first to show a central role of JAML in CHS and reveals a potential new target for the treatment of ACD in humans.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

Inhibition of DYRK1B suppresses inflammation in allergic contact dermatitis model and Th1/Th17 immune response

Allergic contact dermatitis (ACD) is a type IV hypersensitivity mainly mediated by Th1/Th17 immune response. Topical corticosteroid is currently the first-line treatment for allergic contact dermatitis (ACD) and systemic administration of immunosuppressive drugs are used in patients with severe disseminated cases. However, increased risk of adverse effects has limited their use. Thus, the development of a novel immunosuppressant for ACD with low toxicity is a challenging issue. In this study, we began our study by using a murine contact hypersensitivity (CHS) model of ACD to examine the immunosuppressive effects of DYRK1B inhibition. We found that mice treated with a selective DYRK1B inhibitor show reduced ear inflammation. In addition, a significant reduction of Th1 and Th17 cells in the regional lymph node upon DYRK1B inhibition was observed by FACS analysis. Studies in vitro further revealed that DYRK1B inhibitor does not only suppressed Th1 and Th17 differentiation, but also promotes regulatory T cells (Treg) differentiation. Mechanistically, FOXO1 signaling was enhanced due to the suppression of FOXO1Ser329 phosphorylation in the presence of DYRK1B inhibitor. Therefore, these findings suggest that DYRK1B regulates CD4 T cell differentiation through FOXO1 phosphorylation and DYRK1B inhibitor has a potential as a novel agent for treatment of ACD.

Anti-inflammatory effect of hydrogen sulfide donor sodium-sulfide in an experimental mouse model of contact hypersensitivity

Background: Hydrogen sulfide (H2S), synthesized by most immune cells, has been shown to exert anti-inflammatory and antipruritic effects. The effect of H2S on allergic contact dermatitis (ACD), an inflammatory skin disease that negatively affects the quality of life, is unknown. Objectives: We planned to investigate the antipruritic and anti-inflammatory effects of the H2S donor sodium sulfide (Na2S) in the experimental mouse model of contact hypersensitivity (CHS), which is widely used for ACD research. Methods: CHS was created in Balb/c mice using 1-fluoro-2, 4-dinitrobenzene. Na2S was administered systemically (0.2-2-20 mg/kg/i.p.) and locally (1-3-10 nmol/both ear/i.d.) at 3 h and 25 h after the challenge. Ear thickness and the number of scratches were determined at 24 h and 48 h following the challenge. Ear tissue and serum interferon-gamma, interleukin (IL)-2, IL-4, and IL-5 cytokine levels were evaluated by enzyme-linked immunosorbent assay (ELISA). H and E staining was performed for histopathological studies. CD4+ and CD8+ T cells located in the skin were examined by immunohistochemical staining. Results: Locally (1-3-10 nmol/ear/i.d., P < 0.001, P < 0.0001, P < 0.0001, respectively) and systemically (2–20 mg/kg/i.p., P < 0.01, P < 0.0001, respectively), Na2S administration decreased ear thickness dose dependently. Local (1-3-10 nmol/ear/i.d.) Na2S treatment decreased serum IL-2 levels (P < 0.01, P < 0.05, and P < 0.01, respectively). Na2S administered locally (3–10 nmol/ear/i.d., P < 0.05) and systemically (20 mg/kg/i. p., P < 0.05) decreased the number of CD4+ T lymphocytes. Conclusion: Locally and systemically administered Na2S reduces ear thickness, which is one of the symptoms of CHS, probably by preventing CD4+ T lymphocyte infiltration and proliferation and decreasing IL-2 synthesis.

Comparison of adjuvant mechanisms of medium-chain triacylglycerol in a mouse FITC-induced contact hypersensitivity model

The number of allergy sufferers has been increasing with the increase in chemicals to which we are potentially exposed. We have discovered that tributyrin, a short-chain triacylglycerol (TAG), enhanced fluorescein isothiocyanate (FITC)-induced contact hypersensitivity in a mouse model. Medium-chain triacylglycerols (MCTs) are used in cosmetics, with which we come into direct contact frequently, to maintain skin conditions and as a thickening agent for cosmetics. In this study, we examined whether MCTs with different side chain lengths enhanced skin sensitization to FITC in the mouse model. During skin sensitization to FITC, the presence of tributyrin (side chain carbon number, 4; C4) as well as that of each MCT, tricaproin (C6), tricaprylin (C8), or tricaprin (C10), resulted in enhanced skin sensitization, whereas that of trilaurin (C12) did not. As to the mechanism underlying the enhanced sensitization, three MCTs (C6, C8 and C10) facilitated migration of FTIC-presenting CD11c+ dendritic cells to draining lymph nodes. These results indicated that not only tributyrin but also MCTs, up to side chain carbon number 10, have an adjuvant effect on FITC-induced skin hypersensitivity in mice.

A rose flavor compound activating the NRF2 pathway in dendritic cells ameliorates contact hypersensitivity in mice.

Dendritic cells (DCs), which are typical antigen-presenting cells, localize to various sites in the body, particularly the front line of infection as sentinels, and are involved in innate and adaptive immune responses. Although the functions of DCs, such as pathogen-induced cytokine production and antigen-specific T cell activation, are important for host defenses against infection and tumorigenesis, the hyper- and/or extended activation of DCs leads to inflammatory and autoimmune diseases. In the present study, β-damascone, a major ingredient of rose fragrance, was selected from an aroma library as a candidate compound that suppresses antigen-induced immune responses. β-Damascone inhibited the functions of DCs, including the antigen-dependent proliferation of T cells, DC-induced Th1 development, and the TLR ligand-induced production of inflammatory cytokines by DCs. The β-damascone treatment also increased the protein level of the transcription factor NF-E2-related factor 2 (NRF2), which plays key roles in antioxidant responses, and the transcription of Hmox1 and Nqo1, target genes of NRF2, in DCs. Nrf2 -/ - DCs induced Th1-development and produced large amount of IL-12p40 even in the presence of β-damascone, whereas these functions by Nrf2 +/- DCs were inhibited by β-damascone under the same conditions. The intake of β-damascone suppressed ear swelling in contact hypersensitivity (CHS) model mice, but not in CHS-induced Nrf2 -/ - mice. Collectively, the present results indicate the potential of the rose aroma compound β-damascone, which suppresses DC-mediated immune responses by activating the NRF2 pathway in DCs, for the prevention and/or attenuation of immune-mediated diseases.

Hochuekkito exerts the anti-allergic effects via activating regulatory T cells in a murine model of contact hypersensitivity.

Hochuekkito (HET) is a Kampo prescription, used for the clinical treatment of skin diseases such as atopic dermatitis (AD), in Japan. Oral administration of HET exerts anti-allergic effects in an experimental dermatitis mice model and in patients with atopic dermatitis; however, the mechanism underlying the anti-allergic effects of HET is still unclear. Therefore, we investigated the immunopharmacological properties of the anti-allergic actions of HET using a 2,4,6-trinitrochlorobenzene (TNCB)-induced murine contact hypersensitivity (CHS) model and adoptive cell transfer experiments. Oral administration of HET (1.4g/kg) exhibited anti-allergic effects in a TNCB-induced CHS model via activation of Tregs; this activation was observed even without antigen sensitization in donor mice. Activation was dependent on the duration of HET administration and required at least 4days of dosing. In addition, the anti-allergic effects of HET through the activation of Tregs were not antigen specific. Flow cytometry results indicated that the proportion of CD4+CD25+Foxp3+ cells in the splenic lymphocytes increased after oral administration of HET. Therefore, oral administration of HET induced both inducible regulatory T cells (iTregs) and thymus-derived naturally occurring regulatory T cells (nTregs). Ginseng radix and Bupleuri radix were involved in the anti-allergic actions of HET through the induction and/or activation of Tregs; Bupleuri radix participated in the activation of nTregs. In conclusion, our findings suggest that HET exerts the anti-allergic effects through the induction and/or activation of Tregs. These findings elucidate the usefulness of HET as an immunomodulator.

Topical Photodynamic Therapy Generates Bioactive Microvesicle Particles: Evidence for a Pathway Involved in Immunosuppressive Effects

Although effective in treating actinic damage, topical photodynamic therapy (PDT) has been shown to be immunosuppressive through unknown mechanisms, which could potentially limit its effectiveness. Multiple types of environmental stressors, including PDT, can produce the immunosuppressive lipid mediator platelet-activating factor (PAF). Because PAF can produce subcellular microvesicle particles (MVPs), these studies tested whether PDT can generate PAF and MVP release and whether these are involved in PDT-induced immunosuppression. Previously, topical PDT using blue light and 5-aminolevulinic acid was found to be a potent stimulus for PAF production in mice and human skin explants and human patients, and we show that experimental PDT also generates high levels of MVP. PDT-generated MVPs were independent of the PAF receptor but were dependent on the MVP-generating enzyme acid sphingomyelinase. Patients undergoing topical PDT treatment to at least 10% of body surface area showed local and systemic immunosuppression as measured by inhibition of delayed-type hypersensitivity reactions. Finally, using a murine model of contact hypersensitivity, PDT immunosuppression was blocked by genetic and pharmacologic inhibition of acid sphingomyelinase and genetic inhibition of PAF receptor signaling. These studies describe a mechanism involving MVP through which PDT exerts immunomodulatory effects, providing a potential target to improve its effectiveness.

Tolerance to 2,4-Dinitrofluorobenzene‒Induced Contact Hypersensitivity Is Mediated by CD73-Expressing Tissue-Homing Regulatory T Cells

Regulatory T cells (Tregs) express CD73, an ectonucleotidase that converts adenosine (Ado) monophosphate to Ado, which has been shown to suppress immune reactions. To investigate the role(s) of CD73+ Tregs during the induction of tolerance, we used a 2,4-dinitrofluorobenzene‒driven contact hypersensitivity model, in which tolerance can be induced by pretreating wild type mice with 2,4-dinitrothiocyanobenzene. CD73-deficient mice were unable to acquire tolerance. Likewise, transfer of CD73‒/‒ Tregs failed to suppress 2,4-dinitrofluorobenzene‒induced ear swelling in wild type mice, whereas transfer of wild type‒derived Tregs into CD73‒/‒ mice re-established tolerance. This indicates a crucial role of CD73+ Tregs for skin-induced tolerance. Furthermore, we found that 2,4-dinitrothiocyanobenzene induces more activated CD73+ tissue-homing Tregs (marked by Ki-67, CTLA4, CCR4, CD103, CCR6, and CD49b expression) in draining lymph nodes and blood, eventually accumulating in the skin. The application of anti-CD73 antibodies that block CD73-derived Ado production as well as the injection of Ado deaminase, which degrades Ado in tissues, abrogated tolerance induction. Thus, our data indicate that CD73+ Ado-producing Tregs are crucial for the regulation of contact hypersensitivity reactions and tolerance induction in the skin and that manipulating the function(s) of CD73 in tissues may offer a tool to influence autoimmunity and inflammation invivo.

201 Role of keratinocyte proline-rich protein in psoriatic skin inflammation

Keratinocyte proline-rich protein (KPRP), an epidermal marker expressed in stratified squamous epithelia, is known to be involved in skin barrier function. However, the pathological function of KPRP in skin inflammation remains largely unknown. Here we show the role of Kprp during the development of imiquimod-induced psoriatic skin lesions in mice. Epidermal thickness and erythema were significantly changed in Kprp+/- mice compared to Kprp-/- or wild-type mice. Similarly, in the contact hypersensitivity model, Kprp+/- mice showed a significant change in skin inflammation compared to wild-type mice.

006 Conventional dendritic cell 2 as an activator for cytotoxic T lymphocytes in the elicitation phase of murine contact hypersensitivity

Cytotoxic T lymphocyte (CTL) has been found to be activated by cutaneous dendritic cell (DC) in inducible skin-associated lymphoid tissue (iSALT) in a murine contact hypersensitivity (CHS) model. Cutaneous DCs are classified into Langerhans cell (LC), conventional DC1 (cDC1), conventional DC2 (cDC2) and monocyte-derived DC (MoDC); however, which DC subsets activate CTL within iSALT during the elicitation phase of CHS has remained unclear. To clarify this, we used Langerin-diphtheria toxin receptor (DTR), XCR1-DTR and CD301b-DTR mice to deplete LC, cDC1 and cDC2, respectively.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

Skin microbiota of oxazolone-induced contact hypersensitivity mouse model.

Contact allergy is a common skin allergy, which can be studied utilising contact hypersensitivity (CHS) animal model. However, it is not clear, whether CHS is a suitable model to investigate skin microbiota interactions. We characterised the effect of contact dermatitis on the skin microbiota and studied the biological effects of oxazolone (OXA) -induced inflammation on skin thickness, immune cell numbers and changes of the microbiota in CHS mouse model (n = 72) for 28 days. Through 16S rRNA gene sequencing we defined the composition of bacterial communities and associations of bacteria with inflammation. We observed that the vehicle solution of acetone and olive oil induced bacterial community changes on day 1, and OXA-induced changes were observed mainly on day 7. Many of the notably enriched bacteria present in the OXA-challenged positive group represented the genus Faecalibaculum which were most likely derived from the cage environment. Additionally, skin inflammation correlated negatively with Streptococcus, which is considered a native skin bacterium, and positively with Muribacter muris, which is typical in oral environment. Skin inflammation favoured colonisation of cage-derived faecal bacteria, and additionally mouse grooming transferred oral bacteria on the skin. Due to the observed changes, we conclude that CHS model could be used for certain skin microbiome-related research set-ups. However, since vehicle exposure can alter the skin microbiome as such, future studies should include considerations such as careful control sampling and statistical tests to account for potential confounding factors.

Location bias contributes to functionally selective responses of biased CXCR3 agonists

Some G protein-coupled receptor (GPCR) ligands act as “biased agonists” that preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the GPCR CXC chemokine receptor 3 (CXCR3). The signaling profile of CXCR3 changes as it traffics from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling is critical for biased activation of G proteins, β-arrestins, and extracellular-signal-regulated kinase (ERK). In CD8 + T cells, the chemokines promote unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation is dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

Decreased peripheral basophil counts in urticaria and mouse model of oxazolone-induced hypersensitivity, the latter suggesting basopenia reflecting migration to skin.

A decrease in the number of basophils in the peripheral blood, or basopenia, has been noted, reflecting the activity of chronic spontaneous urticaria (CSU). Infiltration of basophils into the skin has also been reported, but the mechanism of basopenia in CSU has not been clarified. The phenomenon of basopenia during the active phase of urticaria was confirmed, and basophil numbers increased following symptom improvement in 15 out of 17 patients treated with omalizumab and in 13 of 15 patients treated with antihistamines. Our examination by immunostaining also revealed basophil infiltration of the CSU lesions, as in previous reports, but since most of our patients were already taking oral steroids, it was not considered appropriate to examine the relationship between basophil numbers in tissue and peripheral blood. Then, we used mouse model of contact hypersensitivity with a single application of oxazolone, which is known to stimulate basophil infiltration, and investigated basophil counts in the skin, peripheral blood, and bone marrow. In this model, a decrease in peripheral blood basophil numbers was observed one day after challenge, but not after 2 days, reflecting supplementation from the bone marrow. Indeed, when cultured basophils expressing GFP were transplanted into the peripheral blood, GFP-positive basophil numbers in the peripheral blood remained low even after 2 days of challenge. Despite differences among species and models, these results suggest that one reason for the decrease of basophils in the peripheral blood in CSU may involve migration of circulating basophils into the skin.

34232 Inhibition of tissue resident memory-T cells as a therapy for contact hypersensitivity

Background: Systemic therapy for eczema targets the immune system during active inflammation. Disease inevitably returns to previously inflamed but healed skin due to the persistence of tissue resident memory T (Trm) cells responding to autoantigens/allergens. The survival of Trm cells is regulated by IL-7 and IL-15. We present a mouse model of recurrent contact hypersensitivity (CHS) and suggest a novel approach to the treatment of inflammatory skin diseases through inhibition of Trm cells thereby establishing long-term disease remission. Methods: Mice were sensitized on the abdomen with the allergen 2,4-dinitrofluorobenzene (DNFB) (day −5). Mice were challenged on day 0 with DNFB, and then rechallenged on days 30 and 60. Ear swelling was measured every 12 hours for 96 hours postchallenge. Ear skin was harvested 2 days (inflamed skin) and 15 days (healed skin) after each DNFB ear challenge (days 32, 45, 62 and 75). Results: Serial DNFB challenges produced progressively stronger peak CHS responses (1-month vs. 2-months re-challenge; 8.8 vs. 12.0 μm, *P < .05). The number of Trm cells and expression of IL-15-receptor increased in healed skin compared with inflamed skin. There were increases in Trm cell numbers, and Trm expression of IL-7- and IL-15-receptor in skin at 2 months compared with 1-month DNFB rechallenge. Conclusions: Repeat CHS inflammation via serial DNFB challenge lead to accumulation of Trm cells expressing IL-7 and IL-15-receptors in healed skin. We suggest that inhibition of IL-7/IL-15-receptors during disease quiescence to reduce Trm cell numbers, may be a novel strategy to prevent dermatitis recurrence and maintain long-term remission.