Programmed Death-1 Homolog (PD-1H/VISTA) Blockade Confers a Synergistic Anti-Leukemia Effect with PD-1 Blockade

Abstract

Monoclonal antibodies (mAbs) to block the interaction of PD-1/PD-L1 (herein anti-PD therapy) have been at the forefront of cancer therapy. Despite its success in many cancers, anti-PD therapy as a single agent has shown modest response rates in acute myeloid leukemia (AML), indicating different mechanisms of immune evasion in human AML. Programmed Death-1 Homolog (PD-1H, also known as VISTA) is a co-inhibitory immunoglobulin broadly found in hematopoietic cells. We previously found that PD-1H surface protein was expressed on AML blasts; AML blast PD-1H inhibited T cell immunity, leading to immune evasion in AML (Kim et al, ASH 2017). Now, we demonstrate that PD-1H is highly expressed on normal myeloid cells and on T cells in AML bone marrow. To determine the role of host immune cell PD-1H in immune evasion of AML, AML cells (C1498) were transplanted into B6 mice with wild-type (WT) or PD-1H knockout (KO) immune cells to assess in vivo proliferation using bioluminescence. In vivo AML growth was delayed in PD-1H KO mice compared with WT mice (mean radiance in PD-1H WT vs. PD-1H KO mice on day 24: 4.4x108 vs. 5.1x105, N=5 per group, p=0.04). To elucidate which immune cell subsets expressing PD-1H contribute to immune evasion in AML, we generated lineage-specific conditional KO mice that lacked expression of PD-1H in either T cells (Cre-Lck) or myeloid cells (Cre-LysM) (macrophages, granulocytes). Following transplantation with AML cells, we assessed in vivo proliferation in both conditional KO mice and littermate controls using bioluminescence. We found that in vivo proliferation of AML was significantly inhibited by myeloid cell-specific KO of PD-1H, compared with littermate controls (mean radiance in Cre-LysM-PD-1Hfl/fl vs. Cre-LysM+PD-1Hfl/fl on day 23: 1.7x109 vs. 1.7x106, N=9 (combined from two independent experiments), p=0.03). T cell-specific KO of PD-1H showed a trend toward anti-leukemia effects, but the effect was not statistically significant (mean radiance in Cre-Lck-PD-1Hfl/fl vs. Cre-Lck+PD-1Hfl/fl on day 34: 2.5x108 vs. 2.2x107, N=6, p=0.1). Taken together, these results show that PD-1H on host myeloid cells rather than on T cells contributes to immune evasion in AML. Consistent with reduced in vivo AML growth in PD-1H KO mice, anti-mouse PD-1H (mPD-1H) mAb treatment significantly inhibited in vivo AML growth compared with isotype control in a syngeneic AML model, while anti-mouse PD-1 (mPD-1) mAb modestly reduced in vivo AML growth. However, treatment with a combination of anti-mPD-1H mAb and anti-mPD-1 mAb synergistically suppressed in vivo growth of AML in WT B6 mice, compared with mice treated with either mAb alone (mean radiance with anti-mPD-1H mAb vs. the combination of anti-mPD-1 mAb and anti-mPD-1H mAb on day 21: 2x106 vs 2.4x105, N=5 per group, p=0.017; anti-mPD-1 mAb vs. the combination: p=0.00053). Finally, we determined the effect of human PD-1H (hPD-1H) in a humanized AML model. NSG-SGM3 mice were first reconstituted with purified human T cells and then subcutaneously injected with human leukemia cells (THP1) expressing or not expressing hPD-1H. Two weeks later, PD-1H+ AML tumors were significantly bigger than PD-1H- AML tumors (PD-1H+ AML tumors: 78.8 mm3 vs. PD-1H- AML tumors: 8.5 mm3, N=5, p<0.0001). Interestingly, treatment with anti-hPD-1H mAb significantly reduced the size of PD-1H+ AML tumors, accompanied by increased T cell infiltration, while anti-hPD-1 mAb did not significantly inhibit tumor growth (anti-hPD-1H mAb: 7.2 mm3 vs. anti-hPD-1 mAb: 51.4 mm3, N=5, p=0.0032). More importantly, the combination treatment of anti-hPD-1H mAb with anti-hPD-1 mAb synergistically reduced the size of PD-1H+ AML tumors (0 mm3, N=5, p=0.0035 vs. anti-hPD-1 alone). These findings extend and validate the results in syngeneic mouse leukemia models by showing that hPD-1H mAb can reverse immune evasion induced by leukemia-associated PD-1H in a humanized AML model. Our studies reveal that host immune cell PD-1H in addition to AML blast PD-1H induces immune evasion in syngeneic and humanized AML mouse models. More importantly, PD-1H blockade by a specific mAb reverses this immune evasion. Finally, PD-1H blockade is potentially synergistic with anti-PD therapy. Our studies implicate PD-1H as a potential target for AML immunotherapy.