Recombinant Adenosine Deaminase Ameliorates Inflammation, Vascular Disease, and Fibrosis in Preclinical Models of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEGylated adenosine deaminase (PEG-ADA) in preclinical models of SSc. METHODS: The effects of PEG-ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra-2 mice and in a B10.D2→BALB/c (H-2d ) model of sclerodermatous chronic graft-versus-host disease (GVHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness skin model. RESULTS: PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) (P = 0.0079; n = 6), dermal fibrosis by 51.8% (P = 0.0006; n = 6), and intestinal fibrosis by 17.7% (P = 0.0228; n = 6) in Fra-2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) (P = 0.0063; n = 8), and in a human full-thickness skin model. PEG-ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) (P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) (P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) (P = 0.0147; n = 6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) (P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) (P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra-2 mice. CONCLUSION: Treatment with PEG-ADA ameliorates the 3 cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications, as PEG-ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA-deficient severe combined immunodeficiency disease.

Mycobacterial Hsp65-IgG-expressing tolerogenic B cells confer protection against adjuvant-induced arthritis in Lewis rats.

OBJECTIVE: Tolerization of T cells directed against a target autoantigen is a desired goal of experimental approaches for the treatment of autoimmune diseases, and novel and improved methods of tolerance induction are continuously being sought. Because most traditional methods of tolerance induction using soluble antigen are effective in the prevention of autoimmunity but fail to control established disease, this study was carried out to explore an innovative tolerogenic approach for the treatment of ongoing disease, using the rat adjuvant-induced arthritis (AIA) model of human rheumatoid arthritis. METHODS: Lewis (RT.1(l)) rats were injected subcutaneously with heat-killed Mycobacterium tuberculosis H37Ra to induce AIA. Before or after AIA induction, Lewis rats were treated intraperitoneally (IP) with tolerogenic B cells expressing a fusion construct of mycobacterial 65-kd heat-shock protein (Hsp65) and IgG heavy-chain. For comparison, control rats were treated IP with ovalbumin (OVA)-IgG-expressing B cells or soluble mycobacterial Hsp65, and the effects on AIA were observed. We also tested the immune response to mycobacterial Hsp65 in B cell-tolerized rats. RESULTS: Administration of tolerogenic mycobacterial Hsp65-expressing B cells as well as soluble mycobacterial Hsp65, but not OVA-expressing B cells, resulted in a significant decrease in the severity of subsequent AIA. However, in rats with established disease, only the B cell regimen of mycobacterial Hsp65, but not the soluble antigen, suppressed ongoing AIA. CONCLUSION: Mycobacterial Hsp65-IgG-expressing B cells can successfully attenuate the progression of AIA. This study introduces a promising approach for the treatment of arthritis that should be further explored.

Treatment of diabetes in NOD mice by gene transfer of Ig-fusion proteins into B cells: role of T regulatory cells.

We previously reported that retrovirally mediated gene expression of Ig fusion proteins leads to specific immunologic tolerance and successful treatment of autoimmune conditions. Thus, a single dose of GAD65-IgG- or (Pro) Insulin-IgG-transduced B cells delays the onset and decreases the incidence of diabetes in young (7-12 weeks old) NOD female mice. Herein, we tested the role of regulatory T cells by in vivo treatment with anti-CD25 before B-cell gene therapy or by in vitro ablation of CD25+ cells from tolerized hosts in an adoptive transfer model. Our results demonstrate that anti-CD25 treatment, like cyclophosphamide, partially blocks the efficacy of gene therapy for tolerance. Moreover, B-cell therapy is effective at preventing diabetes transfer by female T cells (from older diabetic mice) into intact male recipients with normal islets, but failed to do so in NOD-scid recipients. This is due in part to homeostatic proliferation but also to the absence of CD25+ T cells in the latter hosts. Tolerance induced in younger NOD females can be stably transferred to NOD-scid recipients. However, physical removal of CD25+ cells abrogates the transfer of tolerance. Therefore, we conclude that CD4+, CD25+ regulatory T cells are required for the induction as well as maintenance of tolerance in this gene therapy model. The phenotype of these induced regulatory T cells is under investigation.

Mechanisms of gene therapy for tolerance: B7 signaling is required for peptide-IgG gene-transferred tolerance induction.

LPS-activated B cells, transduced with IgG fusion proteins, are highly tolerogenic APCs. To analyze the mechanisms for this B cell-delivered gene therapy, we first followed the fate of CFSE-labeled B cell blasts. These cells primarily localized to the spleen, where a small population persisted for at least 1 mo after injection. By day 7 after injection, approximately 95% of the transduced cells had divided at least once, presumably an effect of the in vitro LPS activation into the cycle, because resting cells did not divide. B cells from gld donors were not tolerogenic, initially suggesting a role for Fas ligand (FasL) in tolerance. Because transduced normal B cells expressed only low levels of FasL and did not kill Fas-expressing Jurkat or A20 B lymphoma cells in vitro, these data suggest that gld B cells are not tolerogenic due to unique characteristics of these B cells rather than the lack of functional FasL expression. The transduced B cell blasts displayed significant up-regulation of both B7 costimulatory molecules, and B7.2 up-regulation was maintained through day 7 in vivo. When B cells from B7 knockout donors were transduced to express Ig fusion proteins, they were not tolerogenic in two different mouse strains and Ag models. Moreover, anti-B7 Ab blocked tolerance induction in this model, a result consistent with a role for B7 in tolerance induction. We propose that tolerance may be induced in this model by B7-driven negative regulatory signaling, but tolerance is maintained by a lack of signal 2, because expression of B7 is eventually lost in vivo.

Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases.

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.