This model has been shown to be dependent on both B7 and CD28, and administration of CTLA4Ig completely abrogates OVA-induced airway inflammation, although it may be less effective when exposed to a more intense regimen of inhaled challenges (10, 31C35). of CTLA4Ig to treat chronic inflammatory disease. Introduction Approaches to augment or interfere with immune cell function may be of benefit in many diseases. Members of the CD28 receptor family both activate and inhibit T cell responses, making them attractive therapeutic targets. CD28 is one of the best studied and was the first Losartan to be targeted with the development Icam1 of CTLA4Ig. CTLA4Ig has been shown to be effective both as well as in numerous animal models of disease (reviewed in (1)). These studies led to the development of the humanized version, abatacept, and the related protein, belatacept, which are approved for use in humans to treat rheumatoid arthritis and prevent renal transplant rejection, respectively (2, 3). Biologics directed against additional members of the CD28 family have also been developed including anti-CTLA-4 antibodies (ipilimumab) to treat malignant melanoma, and promising results have been reported with anti-PD-1 therapy in early cancer trials (4C6). CTLA4Ig is a fusion protein of the extracellular domain of CTLA-4 and IgG1 that binds to both CD80 and CD86 (also referred to as B7-1 and B7-2, or collectively as B7-proteins) and prevents interaction of B7-proteins with their counter-receptors CD28 and CTLA-4 expressed on T cells (7). In addition, CD80 has been shown to bind PD-L1 and inhibit T cell activation and proliferation through this interaction (8). The primary mechanism of action for CTLA4Ig has been thought to be blockade of CD28 and therefore prevention of initial T cell activation. However, we previously demonstrated that CTLA4Ig was effective if administered after initial antigen activation of T cells and that this was independent of CD28 (9). In this current study, we report the mechanism for this novel mode of action for CTLA4Ig. We demonstrate that the effects of CTLA4Ig are mediated by regulatory T cells (Tregs) and TGF and require macrophage derived nitric oxide (NO). These data provide an entirely new insight into how treatment with CTLAA4Ig suppresses inflammation, and may provide information relevant to how endogenous CTLA-4:B7 interactions inhibit Losartan T cell responses. Materials and Methods Mice C57Bl/6J and NOS2-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). STAT1-deficient mice were gifts of Dr M. Holtzman and Dr H. Virgin (Washington University School of Medicine, St Louis, MO). CD80/86-deficient mice and FoxP3-DTR mice were provided by Alexander Rudensky (Memorial Sloan Kettering Cancer Center, NY, NY). SMAD-3 deficient mice were provided by Dr. David Beebe (Washington University School of Medicine, St Louis MO). IDO-deficient mice were provided by Dr. Matthew Ciorba (Washington University Losartan School of Medicine, St Louis MO). FoxP3-IRES-GFP (B6. Cg-FoxP3tm2Tch/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and crossed to OT-II OVA transgenic mice on a RAG 1-deficient background to generate OT-II/FoxP3-GFP/Rag1KO mice. All mice were bred and housed in specific pathogen-free facilities at Washington University School of Medicine. All animal studies have been approved by the Washington University Animal Studies Committee. Antibodies -IFN (clone H22, provided by R. Schreiber, Washington University, St Louis, MO) and -CD4 were purchased from Biolegend (San Diego, CA). -TGF (clone1D11) was purchased from R&D Systems (Minneapolis, MN). Murine CTLA4Ig was provided by Bristol-Myers Squibb (Princeton, NJ.). Experimental allergic airway inflammation Mice were immunized and challenged with OVA (Sigma, St Louis, MO) as previously described (10). When indicated, clodronate liposomes.