Jonathan Skupsky

B-Cell-Delivered Gene Therapy Induces Functional T Regulatory Cells and Leads to a Loss of Antigen-Specific Effector Cells

Previous reports have shown that B-cell-mediated gene therapy can induce tolerance in several animal models for autoimmune diseases and inhibitory antibody formation in hemophilia A mice. We know from our previous work that the induction of tolerance following B-cell therapy is dependent upon CD25(+) regulatory T cells (Tregs). To extend these studies and identify the effects of this gene therapy protocol on the target CD4 T cells, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using carboxyfluorescein succinimidyl ester (CFSE) dilution as a measure of T-cell responsiveness to FVIII, we show that CD25(+) Tregs from treated mice are more suppressive than those from control animals. To monitor the induction of antigen-specific Tregs, we repeated these studies in ovalbumin (OVA) peptide-specific DO11.10 T-cell receptor (TCR) transgenic mice. Tregs from DO11.10 mice treated with a tolerogenic OVA-Ig construct are better than polyclonal Tregs at suppressing the proliferation of responder cells stimulated with OVA peptide 323-339 (pOVA). Furthermore, we show that following B-cell therapy, there is an increase in antigen-specific FoxP3(+) Tregs, and there is also a distinct decrease in antigen-specific CD4(+) effector T cells. These changes in the lymphocyte population shift the balance away from effector function toward a tolerogenic phenotype.

Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice

We herein tested the effect of B-cell depletion on tolerance induction to factor VIII (FVIII) in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential depletion effects were used. Thus, IgG1 anti-CD20 selectively depleted follicular B cells and spared marginal zone B cells, whereas IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice, a single dose of either IgG1 or IgG2a anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice by subsequent daily, high-dose FVIII intravenous injection as a model for immune tolerance induction. However, the IgG1, but not the IgG2a, anti-CD20 pretreatment led to a significant increase of regulatory T cells in the spleen. Importantly, 3 months after the partial B-cell depletion with IgG1 anti-CD20, the FVIII-specific hyporesponsive state remained. We suggest a tolerogenic role of the remaining marginal zone B cells as a potential mechanism for anti-CD20 therapy.

Tolerance induction by gene transfer to lymphocytes.

Tolerance must be maintained to prevent deleterious immune responses. Thus, when tolerance is lost, autoimmunity can result. A number of novel approaches to (re-) induce tolerance for potential clinical applications have been developed in the last decade. Our lab has implemented an immunoglobulin-based gene therapy approach, which may have powerful implications for the treatment of human conditions. These include a variety of autoimmune diseases, transplantation, and the immune response to therapeutic proteins (as in the treatment of hemophilia A) or gene therapy per se. We clone the target (immunogenic) protein in frame with an immunoglobulin heavy chain and deliver it via retrovirus to an activated B cell. In our system, we observe tolerance to multiple epitopes of the protein cloned. An important advantage of this regimen is that identification of the precise peptide epitopes of a target protein is not necessary since selection and presentation by the hosts own antigen presenting cells (APCs) eliminates the issue of HLA polymorphism. Additionally, our data indicate that these tolerogenic B cells are stimulating an endogenous population of regulatory T cells, which are effective at suppressing the immune response in both naïve and primed hosts. Thus, this approach has potential for future clinical therapy.

Factor VIII inhibitors: risk factors and methods for prevention and immune modulation.

Patients with hemophilia A are deficient in coagulation Factor VIII. This bleeding disorder can be treated with Factor VIII replacement therapy, but close to a third of patients will be immunized to the treatment and begin to form inhibitory antibodies known as “inhibitors”. These inhibitors will render the treatment ineffective and represent the most severe complication in the treatment of hemophilia A. In this review, we highlight factors involved in inhibitor development and emphasize research being done to modulate the immune response to this life-saving therapy.

Disseminated superficial actinic porokeratosis: a treatment review.

Abstract Disseminated superficial actinic porokeratosis (DSAP) is a chronic disorder of keratinization characterized by numerous papules and plaques distributed over sun-exposed sites. Treatments are poorly standardized and several investigational therapies have demonstrated limited success in treating DSAP. To our knowledge, there have been no systematic reviews of the literature regarding the treatment of this disease. Herein, we review recent studies pertaining to the treatment of DSAP and evaluate the level of evidence for each of these therapeutic modalities.

B-Cell Based Gene Therapy for Inducing Tolerance

The ability of B cells to function as tolerogenic antigen presenting cells (APCs) in vitro and in vivo, makes them ideal targets for gene therapy strategies focused on the induction and re-establishment of tolerance. Current therapy methods employ retroviral vectors for infection of B cells or bone marrow cells and subsequent expression of the target antigen. Moreover, the efficacy of peptide-IgG fusion constructs which take advantage of the tolerogenic properties of IgG has been demonstrated. In this review, we discuss gene therapy approaches mediated by B cells and bone marrow cells for tolerance acquisition in various mouse models for autoimmune disease, hemophilia and transplantation. The results indicate that gene therapy strategies successfully reduce the incidence of disease, or delay disease onset in multiple mouse models for autoimmune disease and hemophilia. Additionally, gene therapy has proven effective in a mouse transplantation model. While these studies show great promise, the mechanisms involved in tolerance, including the role of regulatory T cells, will need to be more clearly defined before the transition to a clinical setting can occur.