VISTA Is a Novel Regulator of Macrophage Biology

Background:V-domain Ig suppressor of T cell activation (VISTA) is an immune checkpoint receptor with an established role as an inhibitory receptor on T cells. However, its role in myeloid cell biology remains unclear. Owing to the much higher expression of VISTA on monocytes and macrophages, we investigated its role in regulating myeloid biology.

Methods: Mouse bone marrow-derived macrophages (BMDMs) were differentiated and treated with agonistic anti-VISTA mAb under steady-state and conditions of M1 inflammatory polarization (LPS + IFN-𝛾). Human CD14+ monocytes were isolated from peripheral blood and treated with anti-VISTA in vitro. Phosphoarrays were used to assess signaling downstream of VISTA engagement and Luminex analysis was used to evaluate cytokine production. Time-course total RNA-seq was performed on BMDMs (at 1, 2, 4, 8 and 16 hours of anti-VISTA treatment). To evaluate the effect of anti-VISTA in vivo, single-cell RNA-seq (scRNA-seq) was performed on FACS sorted CD11b⁺ myeloid populations 12 hours after mice were treated with intravenous anti-VISTA.

Results:

Anti-VISTA treatment of BMDMs and human monocytes in vitro induced rapid phosphorylation events including robust phosphorylation of Raf-1 kinase at the suppressive serine residue 43.Next, we generated a comprehensive timestamp of VISTA activation in BMDMs in vitro by time-course deep RNA-seq analysis of agonistic anti-VISTA treated cells under rigorous M1 inflammatory polarization. This revealed a unique activation state marked by the upregulation of inhibitory cytokines IL-10 and IL1RA as well as ablation of IL-12 family cytokines, suggestive of an M2-like signature. The presence of an M2-like immunoregulatory signature defined by upregulation of the IL-10 was also seen in macrophages identified post clustering of in vivo scRNA-seq data on myeloid cells.

Conclusion:

Using novel agonistic anti-VISTA antibodies, we reveal that VISTA triggering on myeloid cells can induce an alternative anti-inflammatory cell-state, even under conditions of strong inflammatory polarization. This introduces VISTA as an intrinsic checkpoint of macrophage tolerance.