Elevated Levels of PD-L1 on MDSCs in Patients with Ph(-) Myeloproliferative Neoplasm

Introduction. We previously reported that PD-1 and PD-L1 were increased in patients with Ph(-) myeloproliferative neoplasm (MPN) ( Wang et al., Leuk Res 2019). The PD-1 inhibitor therapy or immune checkpoint inhibitor therapy (ICI) trial in MPN by Hobbs et al. reported a negative result (Blood, 2020 (supplement )). Resistance to ICI therapy has been reported to be related to myeloid-derived suppressor cells (MDSCs) in melanoma and breast carcinoma. We also previously reported that MDSCs were increased in patients with Ph(-) MPN (Wang et al., Leu Res 2016). Regulation of immune suppression by MDSCs has been reported to be related to PD-1and PD-L1 expression on the MDSC. Therefore, the current study measured PD-1 and PD-L1 expression in MDSCs in patients with Ph(-) MPN.

Materials and Methods. Twenty-six patients, including 11 essential thrombocythemias (ETs), six polycythemia vera (PV), and nine myelofibrosis (six primary MF, one post-ET-MF, two post-PVMF) were compared with ten normal volunteer controls. MDSCs were measured as follows: the pelleted PBMNCs were used for Magnetic-assisted cell separation (MACS, Miltenyi Biotech, Inc.). PBMNCs are sequentially selected for HLA-DR ^- cells, from which we further selected for CD14 ⁺ and CD14 ^- cells using Miltenyic magnetic microbeads kits, respectively, according to the manufacturer's protocol. The resulting HLA-DR ^-CD14 ⁺ and HLA-DR ^-CD14 ^- cells were stored at -80°C until use. Flow cytometric assay:HLA-DR ^-CD14 ⁺ and HLA-DR ^-CD14 ^- cells were stained with both anti-human CD274 (PD-L1) FITC and human CD279 (PD-1) PE (BD Biosceinces, Inc.), together with anti-human CD14 APC or anti-human CD33 APC (BD Biosceinces, Inc.), respectively. Flow cytometric assessment was done using the BD Accuri C6 flow cytometer. While HLA-DR ^-CD14 ⁺Monocytic MDCS cells were gated and assayed for surface expression of PD-1 (CD274) and PDL-1 (CD279), G-MDSC (granulocytic MDSC), and M-NDSC (Monocytic MDSC) were assayed as HLA-DR ^-CD14 ^- , CD33 ⁺ and HLA-DR ^-CD14 ⁺, respectively. These MDSCs were then assayed for surface expression of PD-1 (CD274) and PDL-1 (CD279). The flow data were analyzed using FlowJo V8 (Flowjo, LLC), and the mean fluorescent intensity (MFI) was calculated.

Results. PD-L1 on the m-MDSCs was significantly elevated in patients with MPN (grouping patients with ET, PV, and MF) and MF than controls. PD-1 on the M-MDSCs was not different between ET, PV, MF, or MPN compared with normal controls. Compared to controls, PD-L1 on the G-MDSC was significantly elevated in patients when ET, PV, and MF were grouped as MPN. PD-1 on the G-MDSC were not different between ET, PV, MF, or MPN and controls.

Conclusion. Ph(-) MPN was found to have significantly elevated PD-L1 on the M-MDSCs and G-MDSCs, but PD-1 levels were not significantly elevated. Further studies to employ drugs, such as a combination of IMiDs (e.g., lenalidomide) and anti-MDSC drugs in combination with ICI in vitro and clinical trials, may be warranted.

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