PD-1 Mediates Lineage Fate Determination and Function of Myeloid Cells in Response to Tumor-Mediated "Emergency" Myelopoiesis

PD-1 is an inhibitory receptor for which blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of how PD-1 compromises anti-tumor function and how such effect is reversed by PD-1 blockade remain poorly understood. The rapid change in hematopoietic output that occurs in response to immunologic stress is known as "emergency" myelopoiesis. This process is co-opted by tumors to evade the immune system by inducing potent immunosuppression. Cancer-driven granulo-monocytopoiesis modifies the composition of the hematopoietic output by stimulating the expansion of tumor promoting immature myeloid populations, defined as myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Monocytic (M-) and granulocytic (PMN-) MDSCs and TAMs differentiate from granulocyte/macrophage progenitors (GMP) in the bone marrow. We analyzed the myeloid compartment of tumor-bearing mice to determine the expression and function of PD-1 in myeloid cells during cancer-driven emergency myelopoiesis. Using three different mouse tumor models (MC38, B16 and MC17), we determined that PD-1 and its natural ligand PD-L1, were expressed at high levels in all subsets of myeloid cells isolated from the spleen of tumor-bearing mice, including CD11b⁺Ly6C⁺ M-MDSC, CD11b⁺Ly6G⁺ PMN-MDSC, CD11b⁺F/480⁺ macrophages and CD11b⁺HLA-DR⁺ dendritic cells (DC). This was in contrast to the significantly lower or undetectable levels of PD-1 expressed in these cellular counterparts isolated from non-tumor bearing mice. High expression of PD-1 and PD-L1 was also identified in these myeloid subsets isolated from the tumor site and at sites of secondary myelopoiesis in the gut. Because these myeloid cell populations differentiate from GMP upon cancer-mediated emergency myelopoiesis, we examined bone marrow myeloid progenitors in tumor bearing and control, non-tumor bearing mice. In non-tumor bearing mice, PD-1 was detected in Lin^-/Sca⁺/Kit⁺ (LSK) myeloid progenitors, whereas PD-L1 was expressed in Lin^-/Sca^-/Kit⁺ (LK), common myeloid progenitors (CMP) and GMP. In tumor-bearing mice, both PD-1 and PD-L1 expression was significantly higher in all myeloid progenitors compared to tumor-free control mice. To determine whether PD-1-mediated signaling in myeloid cells might affect the magnitude and composition of the myeloid output that is altered by cancer-mediated emergency response, we used PD-1 deficient mice. PD-1 ablation, which decreased tumor growth and metastasis, stimulated the differentiation of hematopoietic progenitor cells and resulted in increase of Ly6C^hi monocytes and monocyte-derived DC (mo-DC). Abrogation of PD-1 signaling enhanced the maturation of MDSCs by inducing the lineage commitment transcription factors IRF8, IRF4 and RORC1. IRF8 and IRF4 promote monocyte/macrophage and hinder granulocyte differentiation, whereas RORC1 is required for expansion of Ly6C^hi monocytes and resolution of inflammation. To examine whether the effects of PD-1 on cancer-mediated emergency myelopoiesis and on myeloid lineage commitment and differentiation were mediated by myeloid cell-intrinsic mechanisms, we generated mice with conditional targeting of the PD-1 gene in the myeloid compartment by crossing PD-1^fl/fl with LysM-Cre mice. Myeloid-specific ablation of PD-1 signaling in PD-1^fl/fl/LysM-Cre mice impaired tumor growth to a similar extent as global PD-1 ablation in PD1-KO mice and induced a myeloid cell fate switch from MDSCs to effector-like myeloid cells that could be identified in spleen and tumor. Further, abrogation of PD-1 signaling in the myeloid compartment prevented the expansion of tumor-stress associated GMP in PD-1^fl/fl/LysM-Cre mice and resulted in increased differentiation of committed monocyte progenitors (CMOP) and monocyte progenitors (MP) in the bone marrow. Moreover, PD-1 deletion significantly diminished NO production by M-MDSC and reversed their ability to suppress antigen-specific T cell responses. Our results reveal a previously unidentified role of the PD-1:PD-L1 pathway in the differentiation of lineage-committed myeloid progenitors during tumor-mediated emergency myelopoiesis to promote MDSCs and suppress effector monocytes and mo-DCs. Switch of myeloid progenitor fate commitment might be a key mechanism by which PD-1 blockade mediates its anti-tumor function.