Background Mononuclear and polymorphonuclear myeloid-derived suppressor cells (M-MDSC and PMN-MDSC) accumulate in many cancer types and impair anti-tumor immune response and reduce cancer immunotherapy efficacy.^{1 2} The role of immune checkpoint protein V-domain Suppressor of T cell Activation (VISTA) in the differentiation and function of MDSC remains incompletely understood.^3–10

Methods Flow analysis was applied for cell surface makers analysis. Gene expression was determined by RT-qPCR. Intracellular proteins and signaling molecules were detected by Western Blotting. Multiplex immunohistochemistry was used to stain the tissue slides. Mitochondrial functions were detected by seahorse metabolic flux analysis.

Results Here, by studying BM-derived MDSCs, we identified that ablation of VISTA significantly reduces the expression of arginase1 (Arg1), iNOS and diminishes the inhibitory effects of MDSC on T cell proliferation. In bone marrow (BM) chimera mouse tumor model, depletion of VISTA reduces the frequency of MDSC and expression of Arg1 and iNOS. Transcriptomic analysis of BM-MDSC indicates that oxidative phosphorylation, Myc-targets, mTORC1, UPR pathways are down-regulated in VISTA-deficient MSDC cells. The deregulation of oxidative and phosphorylation pathway in VISTA knockout MDSC cells correlates the diminished mitochondrial function with significant lower basal oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and lower spare respiratory capacity (SRC). Myeloid specific deletion of VISTA mice demonstrated a durable better tumor control through reduced MDSC differentiation and enhanced T-cell mediated cytotoxicity. Mechanistically, VISTA directly regulates the signaling of MDSC in response to inflammatory stimuli including GM-CSF and IL-6 by augmenting the activation of STAT3. Phosphorylated STAT3 positively regulates the expression of Arg1 and iNOS and modulates the mitochondrial function. Correlated expression of VISTA and Arginase-1 (ARG1), a key enzyme supporting polyamine biosynthesis, was observed in multiple human cancer types. In human endometrial cancer, co-expression of VISTA and ARG1 on tumor-associated myeloid cells is associated with poor survival.

Conclusions These studies unveil the role of VISTA as a central regulator of MDSC differentiation and warrant therapeutically targeting this axis for cancer immunotherapy.

Acknowledgements National Institute of Health/National Cancer Institute R01CA164225 (L.L.W),

National Institute of Health/National Cancer Institute R01CA223804 (L.L.W),

National Institute of Health/National Cancer Institute R21CA258618 (L.L.W),

Department of Defense CDMRP W81XWH-21-MRP-MCAA ME210229 (L.L.W),

Department of Defense CDMRP W81XWH-21-LCRP-IITRA LC210336 (L.L.W),

American Cancer Society RSG-18–045-01-LIB (L.L.W),

The Norma C. and Albert I. Geller Professorship in Ovarian Cancer Research (S.A.),

Case Comprehensive Cancer Center, Genomics Pilot Grant (S.A.),

The V Foundation Scholar award V2020–011 (A.A.C),

Department of Defense Early Career Investigator grant KCRP AKCI-ECI, W81XWH-20–1-0804 (A.A.C),

NCCN Young Investigator Award (A.A.C),

American Cancer Society Research Scholar Grant (RSG-22–067-01-TBE) (A.A.C),

American Cancer Society RSG-22–135-01-IBCD (S.C.-C.H)

Melanoma Research Foundation Career Development Award (S.C.-C.H)

Andrew McDonough B+ Foundation Grant Award (S.C.-C.H)

Case GI SPORE DRP grant 5P50CA150964–08 (S.C.-C.H)

Cancer Research Institute CLIP Investigator Award (S.C.-C.H

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