On day 3 after the last of 5 anti-CD3 injections (day 8 after plasmid injection), approximately 80% to 90% of CD4+ and CD8+ T cells were depleted in spleen, lymph nodes, and blood of anti-CD3 only and plasmid + anti-CD3Ctreated mice but not in plasmid onlyCtreated animals (Figure 4A,D). to develop safe and effective methods to modulate immune responses against transgene products, vectors, and/or gene-engineered cells to ensure the success of gene therapy. Therapeutic biologic compounds have been used successfully to modulate the immune system. The development of the first humanized anti-CD3 monoclonal antibody (OKT3) spurred a spate of studies with CD3-specific antibodies for the treatment of immune-mediated diseases.10C15 Clinical trials with this monoclonal antibody included treatment of acute renal allograft rejection,16,17 autoimmune insulin-dependent diabetes,18 and psoriatic arthritis,19 and prevention of islet allograft rejection.20 The results of these trials are encouraging, and anti-CD3 treatment did not elicit major side effects.16,17 Anti-CD3 therapy was demonstrated to promote, in both transplantation21,22 and autoimmune settings,23 antigen-specific immune tolerance. In patients with recent onset autoimmune diabetes, reversion of hyperglycemia was achieved by combination therapy of anti-CD3 injections and intranasal application of proinsulin peptide, but not by either WZ8040 agent alone.23 It was suggested that the combination therapy induces large numbers of islet cell-specific regulatory T cells (Tregs), whereas anti-CD3 or proinsulin alone was insufficient to prime Tregs. Experimental evidence supports the hypothesis that there are 2 subsets of Tregs that differ in specificity and effector mechanism.24 Natural Tregs emerge from the thymus as a distinct lineage, whereas adaptive Tregs are induced at the periphery from CD4+CD25? T cells under specific conditions, ie, antigenic stimulation in the presence of a particular cytokine environment or altered T-cell receptor signal transduction. Studies in mice engineered to express a Foxp3 reporter confirmed that Foxp3 is a lineage marker of Tregs and correlates with suppressor activity irrespective of CD25 expression.25C27 In particular, in an autoimmune nonobese diabetic mouse model, anti-CD3 treatment induced adaptive CD4+CD25lowFoxp3+ Tregs in the periphery that suppress T-cell immunity in a transforming growth factor- (TGF-)Cdependent manner.28 Here we report the successful use of the anti-CD3 monoclonal antibody in modulating immune responses against FVIII after gene therapy. Five consecutive injections of anti-CD3 induced tolerance to FVIII in hemophilia A mice, manifested by persistence of FVIII activity and the absence of circulatory FVIII-specific inhibitory antibodies. The treatment with anti-CD3 caused temporary depletion of CD4+ and CD8+ T cells and increased the frequency of CD4+Foxp3+ Tregs. Long-term tolerance was confirmed Rabbit Polyclonal to MLH1 by a second plasmid challenge to anti-CD3 tolerized mice without eliciting FVIII-specific immune responses. In addition, anti-CD3 antibody WZ8040 treatment did not damper immune response toward unrelated antigens after treated mice recovered from transient immunosuppression. Methods Mice All mice were kept in accordance with National WZ8040 Institutes of Health guidelines for animal care and the guideline of Seattle Children’s Research Institute, and maintained at a specific pathogenCfree facility. The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of Seattle Children’s Research Institute. Hemophilia A mice in a 129/SV C57BL/6 mixed genetic background were generated by targeted disruption of exon 16 of the gene29 and bred in our animal facility. Antibodies AntiCmouse-CD3? monoclonal antibodies (145-2C11), antiCmouse-CD25 monoclonal antibodies (PC61), and mouse IgG1 isotype control were purchased from BioXCell. AntiCmouse-Foxp3 (FJK-16s)Cfluorescein isothiocyanate, antimouse-CD25 (PC61)Callophycocyanin, antiCmouse-CD25 (7D4)Callophycocyanin, antiCmouse-CD4 (L3T4)Cphycoerythrin, and antiCmouse-CD8 (Ly3)Callophycocyanin were purchased from eBioscience. Anti-CD4 (L3T4)CAlexaFlour 700 was purchased from BD Biosciences PharMingen. Gene WZ8040 transfer of FVIII into hemophilia A mice with immunomodulation regimen by anti-CD3 antibodies Hemophilia A mice were injected with 50 g of plasmid (pBS-HCRHP-FVIIIA) in 2 mL of phosphate-buffered saline via tail vein in 8 to 10 seconds. For immunomodulation, plasmid-treated mice were given intravenous injections of anti-CD3 antibody at a dose of 40 g at the time of plasmid injection and subsequent daily injections for 4 additional days. Groups of anti-CD3 onlyCtreated mice, plasmid onlyCtreated mice, and naive mice were included as controls. Blood samples were taken from the retro-orbital plexus periodically and assessed for FVIII activity and anti-FVIII antibody levels. Selected immunomodulated mice received a second plasmid challenge at 23 weeks after the first plasmid injection or received 2.5 units ( 500 ng) recombinant human FVIII (Baxter) emulsified in complete Freund adjuvant (CFA; Sigma-Aldrich) at 12.