Cutting Edge: Tissue Antigen Expression Levels Fine-Tune T Cell Differentiation Decisions In Vivo.

Immune homeostasis in peripheral tissues is, to a large degree, maintained by the differentiation and action of regulatory T cells (Treg) specific for tissue Ags. Using a novel mouse model, we have studied the differentiation of naive CD4+ T cells into Foxp3+ Treg in response to a cutaneous Ag (OVA). We found that expression of OVA resulted in fatal autoimmunity and in prevention of peripheral Treg generation. Inhibiting mTOR activity with rapamycin rescued the generation of Foxp3+ T cells. When we varied the level of Ag expression to modulate TCR signaling, we found that low Ag concentrations promoted the generation of Foxp3+ T cells, whereas high levels expanded effector T cells and caused severe autoimmunity. Our findings indicate that the expression level of tissue Ag is a key determinant of the balance between tissue-reactive effector and peripheral Foxp3+ T cells, which determines the choice between tolerance and autoimmunity.

Leveraging immune memory against measles virus as an antitumor strategy in a preclinical model of aggressive squamous cell carcinoma.

Viral antigens are among the strongest elicitors of immune responses. A significant proportion of the human population already carries pre-existing immunity against several childhood viruses, which could potentially be leveraged to fight cancer. We sought to provide proof of concept in mouse models that a pre-existing measles virus (MeV) immunity can be redirected to inhibit tumor growth by directly forcing expression of cognate antigens in the tumor. To this end, we designed DNA vaccines against known MeV cytotoxic and helper T epitopes, and administered these intradermally to mice that were subsequently challenged with syngeneic squamous cancer cells engineered to either express the cognate antigens or not. Alternatively, established wild-type tumors in vaccinated animals were treated intratumorally with in vitro transcribed mRNA encoding the cognate epitopes. Vaccination generated MeV cytotoxic T lymphocyte (CTL) immunity in mice as demonstrated by enhanced interferon gamma production, antigen-specific T cell proliferation, and CTL-mediated specific killing of antigen-pulsed target cells. When challenged with syngeneic tumor cells engineered to express the cognate antigens, 77% of MeV-vaccinated mice rejected the tumor versus 21% in control cohorts. Antitumor responses were largely dependent on the presence of CD8+ cells. Significant protection was observed even when only 25% of the tumor bulk expressed cognate antigens. We therefore tested the strategy therapeutically, allowing tumors to develop in vaccinated mice before intratumoral injection with Viromer nanoparticles complexed with mRNA encoding the cognate antigens. Treatment significantly enhanced overall survival compared with controls, including complete tumor regression in 25% of mice. Our results indicate that redirecting pre-existing viral immunity to fight cancer is a viable alternative that could meaningfully complement current cancer immune therapies such as personalized cancer vaccines and checkpoint inhibitor blockade.

Profiling trial burden and patients' attitudes to improve clinical research in epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) comprises inherited mechanobullous dermatoses with considerable morbidity and mortality. While current treatments are symptomatic, a growing number of innovative therapeutic compounds are evaluated in clinical trials. Clinical research in rare diseases like EB, however, faces many challenges, including sample size requirements and recruitment failures. The objective of this study was to determine attitudes of EB patients towards clinical research and trial participation as well as the assessment of contextual motivating and discouraging factors in an effort to support patient-centered RD trial designing. METHODS: A 53-items questionnaire was handed over to EB patients (of all types and ages) in contact with the EB House Austria, a designated national center of expertise for EB care. Main categories included level of interest in and personal knowledge about clinical studies, pros/cons for participation and extent of individual expenses considered acceptable for participation in a clinical study. Descriptive subgroup analysis was calculated with SPSS 20.0 and Microsoft Excel. RESULTS: Thirty-six individuals (mean age 25.7 years), diagnosed for recessive dystrophic EB (36.1%), EB simplex (33.4%), junctional EB (8.3%), dominant dystrophic EB (2.8%) and acral peeling syndrome (2.8%) participated. Motivation for participation in and the desire to increase personal knowledge about clinical trials were (outmost) high in 57.2 and 66.7%, respectively. Altruism was the major motivating factor, followed by hope that alleviation of the own symptoms can be achieved. The greatest hurdle was travel distance, followed by concerns about possible adverse reactions. Patients diagnosed for severe subgroups (RDEB, JEB) were more impaired by the extent of scheduled invasive investigations and possible adverse reactions of the study medication. Patients with generally milder EB forms and older patients were accepting more frequent outpatient study visits, blood takes, skin biopsies and inpatient admissions in association with trial participation. CONCLUSIONS: This study provides additional indications to better determine and address attitudes towards clinical research among EB patients as well as guidance to improve clinical trial protocols for patient centricity.

A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair.

RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

Topical diacerein for epidermolysis bullosa: a randomized controlled pilot study.

Blistering in epidermolysis bullosa simplex type Dowling-Meara (EBS-DM) is associated with an inflammatory phenotype, which can be disrupted by diacerein in vitro. In this pilot study we hypothesized, that a topical formulation of diacerein 1% reduces blistering. Five patients initially applied diacerein underneath both armpits. Then, each participant received 1% diacerein-cream for one armpit, and placebo for the other (randomized withdrawal). The number of blisters was reduced significantly (left: -78%; right: -66% of baseline) within two weeks and remained significantly below the initial level even during withdrawal in four patients. These findings point to a relevant effect of diacerein and provide important information for a confirmative study.