VISTA re-programs macrophage biology through the combined regulation of tolerance and anti-inflammatory pathways

1) BMDMs treated with anti-VISTA under tolerizing and immunizing conditions. 2) Time course analysis of anti-VISTA-induced alterations in human monocytes after anti-VISTA treatment. 3) Time course analysis of anti-VISTA-induced alterations in BMDMs after anti-VISTA treatment. 4) Single cell ATAC-seq of BMDMs tolerized in the presence or IgG or anti-VISTA. Overall design: 1) BMDMs were stimulated with 10 ng/ml LPS and control Ig or anti-VISTA for 15 hours, washed 5 times with 1× PBS, then allowed to rest for 2 hours in LPS-free complete medium. BMDMs were then stimulated with 1 µg/ml LPS for 4 h. For immunization experiments, the same protocol as the tolerization experiment was followed but the initial LPS stimulation was omitted. 2) Human monocytes treated with IgG or Anti-VISTA (10 mg/ml) for indicated time points. 3) BMDMs treated with IgG or Anti-VISTA (10 mg/ml) for indicated time points. 4) BMDMs were stimulated with 10 ng/ml LPS and control Ig or anti-VISTA for 15 hours, washed 5 times with 1× PBS, then allowed to rest for 2 hours in LPS-free complete medium.