Development and Characterization of a Preclinical Model for the Evaluation of CD205-Mediated Antigen Delivery Therapeutics in Type 1 Diabetes.

Dendritic cells (DCs) are crucial for the production of adaptive immune responses to disease-causing microbes. However, in the steady state (i.e., in the absence of an infection or when Ags are experimentally delivered without a DC-activating adjuvant), DCs present Ags to T cells in a tolerogenic manner and are important for the establishment of peripheral tolerance. Delivery of islet Ags to DCs using Ag-linked Abs to the DC endocytic receptor CD205 has shown promise in the NOD mouse model of type 1 diabetes (T1D). It is important to note, however, that all myeloid DCs express CD205 in humans, whereas in mice, only one of the classical DC subsets does (classical DC1; CD8α+ in spleen). Thus, the evaluation of CD205-targeted treatments in mice will likely not accurately predict the results observed in humans. To overcome this challenge, we have developed and characterized a novel NOD mouse model in which all myeloid DCs transgenically express human CD205 (hCD205). This NOD.hCD205 strain displays a similar T1D incidence profile to standard NOD mice. The presence of the transgene does not alter DC development, phenotype, or function. Importantly, the DCs are able to process and present Ags delivered via hCD205. Because Ags taken up via hCD205 can be presented on both class I and class II MHC, both CD4+ and CD8+ T cells can be modulated. As both T cell subsets are important for T1D pathogenesis, NOD.hCD205 mice represent a unique, patient-relevant tool for the development and optimization of DC-directed T1D therapies.

Genetically modified human CD4(+) T cells can be evaluated in vivo without lethal graft-versus-host disease.

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD-scid IL2rγ(null) (NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4(+) T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA-DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA-DR4 and to be deficient for murine class II MHC molecules. CD4(+) T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3 months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.

An HLA-Transgenic Mouse Model of Type 1 Diabetes That Incorporates the Reduced but Not Abolished Thymic Insulin Expression Seen in Patients.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. Multiple genetic loci contribute to disease susceptibility in humans, with the most responsible locus being the major histocompatibility complex (MHC). Certain MHC alleles are predisposing, including the common HLA-A(∗)02:01. After the MHC, the locus conferring the strongest susceptibility to T1D is the regulatory region of the insulin gene, and alleles associated with reduced thymic insulin expression are predisposing. Mice express two insulin genes, Ins1 and Ins2. While both are expressed in beta cells, only Ins2 is expressed in the thymus. We have developed an HLA-A(∗)02:01-transgenic NOD-based T1D model that is heterozygous for a functional Ins2 gene. These mice exhibit reduced thymic insulin expression and accelerated disease in both genders. Immune cell populations are not grossly altered, and the mice exhibit typical signs of islet autoimmunity, including CD8 T cell responses to beta cell peptides also targeted in HLA-A(∗)02:01-positive type 1 diabetes patients. This model should find utility as a tool to uncover the mechanisms underlying the association between reduced thymic insulin expression and T1D in humans and aid in preclinical studies to evaluate insulin-targeted immunotherapies for the disease.

DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of diabetogenic CD8⁺ T cells independently of PD-1 and PD-L1.

CD8⁺ T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) have been implicated in type 1 diabetes in both humans and non-obese diabetic (NOD) mice, in which T cells specific for IGRP206₋214 are highly prevalent. We sought to manipulate these pathogenic T cells by exploiting the ability of steady-state dendritic cells (DCs) to present antigens in a tolerogenic manner. The endocytic receptor DEC-205 was utilized to deliver an IGRP206₋214 mimotope to DCs in NOD mice, and the impact of this delivery on a polyclonal population of endogenous islet-reactive cognate T cells was determined. Assessment of islet-infiltrating CD8⁺ T cells showed a decrease in the percentage, and the absolute number, of endogenous IGRP206₋214-specific T cells when the mimotope was delivered to DCs, compared with delivery of a specificity control. Employing an adoptive transfer system, deletion of CD8⁺ T cells as a result of DEC-205-mediated antigen targeting was found to occur independently of programmed death-1 (PD-1) and its ligand (PD-L1), both often implicated in the regulation of peripheral T-cell tolerance. Given its promise for the manipulation of self-reactive polyclonal T cells demonstrated here, the distinctive characteristics of this antigen delivery system will be important to appreciate as its potential as an intervention for autoimmune diseases continues to be investigated.

Beyond HLA-A*0201: new HLA-transgenic nonobese diabetic mouse models of type 1 diabetes identify the insulin C-peptide as a rich source of CD8+ T cell epitopes.

Type 1 diabetes is an autoimmune disease characterized by T cell responses to ß cell Ags, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for ß cell-specific CD8(+) T cells in the pathogenic process. As CD8(+) T cells specific for ß cell Ags are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human ß(2)-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify ß cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8(+) T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified in this study have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.

T-cell autoantigens in the non-obese diabetic mouse model of autoimmune diabetes.

The non-obese diabetic (NOD) mouse model of autoimmune (type 1) diabetes has contributed greatly to our understanding of disease pathogenesis and has facilitated the development and testing of therapeutic strategies to combat the disease. Although the model is a valuable immunological tool in its own right, it reaches its fullest potential in areas where its findings translate to the human disease. Perhaps the foremost example of this is the field of T-cell antigen discovery, from which diverse benefits can be derived, including the development of antigen-specific disease interventions. The majority of NOD T-cell antigens are also targets of T-cell autoimmunity in patients with type 1 diabetes, and several of these are currently being evaluated in clinical trials. Here we review the journeys of these antigens from bench to bedside. We also discuss several recently identified NOD T-cell autoantigens whose translational potential warrants further investigation.

Anti-inflammatory activity of sertaconazole nitrate is mediated via activation of a p38-COX-2-PGE2 pathway.

Sertaconazole nitrate is an antifungal agent that exhibits anti-inflammatory activity; however, the mechanism for this action was unknown. We investigated the cellular mechanisms by which sertaconazole exerts its anti-inflammatory activity in keratinocytes and human peripheral blood mononuclear cells (PBMCs). Paradoxically, sertaconazole was found to activate the proinflammatory p38 mitogen-activated protein kinase. Treatment with sertaconazole also resulted in the induction of cyclooxygenase-2 (COX-2) and the subsequent release of prostaglandin E2 (PGE2). Knocking down p38 in keratinocytes using small interfering RNA resulted in an inhibition of sertaconazole-induced PGE2 release confirming that activation of p38 was required for PGE2 production. Additionally, in stimulated keratinocytes and human PBMCs, sertaconazole was found to suppress the release of cytokines. Treatment with anti-PGE2 antiserum or the COX-2 inhibitor NS398 reversed the inhibitory effects of sertaconazole on the release of proinflammatory cytokines, linking endogenous PGE2 with the anti-inflammatory effects. Finally, in an in vivo mouse model of tetradecanoyl phorbol acetate (TPA)-induced dermatitis, the sertaconazole-mediated inhibition of TPA-induced ear edema was reversed by NS398. Biochemical analysis of tissue biopsies revealed increase in PGE2 levels in sertaconazole-treated mice. Thus, activation of the p38-COX-2-PGE2 pathway by agents such as sertaconazole provides anti-inflammatory therapeutic benefits.

Anti-inflammatory and anti-itch activity of sertaconazole nitrate.

Cutaneous fungal infections are frequently associated with an inflammatory component including irritated skin, itching and stinging/burning. Therapeutic anti-fungal agents that have anti-inflammatory activity have the potential to provide clinical benefit beyond fungus eradication. Recently, certain anti-fungal agents have been shown to have intrinsic anti-inflammatory activity, therefore we sought to determine the extent of the anti-inflammatory activity of these compounds. The anti-inflammatory activities of eight anti-fungal agents (butoconazole, ciclopirox olamine, fluconazole, miconazole nitrate, sertaconazole nitrate, terconazole, tioconazole and ketoconazole) were compared in a number of preclinical models of dermal inflammation and pruritus. While butoconazole, ciclopirox olamine, fluconazole, and miconazole nitrate were all found to have anti-inflammatory activity, only sertaconazole nitrate reduced the release of cytokines from activated lymphocytes and mitigated inflammation in animal models of irritant contact dermatitis and neurogenic inflammation. In addition, sertaconazole nitrate inhibited contact hypersensitivity and scratching responses in a murine model of pruritus. Furthermore, the in vitro and in vivo anti-inflammatory activity of sertaconazole nitrate was found to be greater than other topical anti-fungal agents examined. These studies demonstrate that topical administration of clinically relevant concentrations of sertaconazole nitrate resulted in an efficacious anti-inflammatory activity against a broad spectrum of dermal inflammation models and itch. The anti-inflammatory properties of sertaconazole may contribute to the efficacy of the drug in the treatment of cutaneous fungal conditions and provide greater anti-inflammatory activity compared with other anti-fungal agents.