Monocytic Myeloid-Derived Suppressor Cells (Mo-MDSC) Are Increased but Not M1 or M2 TAM Macrophages in the Bone Marrow of Patients with BCR-ABL Negative Myeloid Neoplasm (MPN)

Introduction. Evidences indicate that megakaryocyte/platelet lineage is crucial in BCR-ABL negative MPN, especially in myelofibrosis (MF) induction. Monocyte/Macrophage lineage were also reported to play a significant role . These Monocyte /Macrophage studies were on the blood monocytes such as have increased the intermediate forms of monocytes (CD14⁺ CD16⁺) and Substance P in MF , but in the bone marrow these cell lineages were not studied. Tumor-associated macrophages (TAM) have been classified into two subsets. as the M1 subset (CD86⁺ ) which produces Th1 cytokines such as interferon (IFN)-γ and pro-inflammatory cytokines. They are involved in the host defense against different pathogens and play a role in the anti-tumor immunity. The M2 subset, that secretes IL-10 and other iimmunosuppresive cytokines and are identified as CD206⁺ (in vitro induced by IL4) or CD163⁺(IL10). These M2 macrophages are associated with tumor progression and metastasis. Myeloid suppressor cells (MDSC ) also were found abundantly in the tumor matrix. We previously reported an increase in MDSC in the peripheral blood of BCR-ABL(-) MPN, therefore, these cells were also studied. Material and Methods: Patient and sample preparation: Peripheral blood, bone marrow (BM) aspirate and biopsy were obtained and used in isolation for mononuclear cells (MNCs). 11 MPN patients consisting of 5 ET, 1 PV , 2 PMF, 1 ET-MF, and 2 PV-MF were studied along with 5 controls ,including 4 questionable spike on protein electrophoresis with normal bone marrow exminations and one with leukemoid reaction. Flow cytometric quantification of monocytes, TAMs and MDSCs MNCs from peripheral blood or BM (aspirates and biopsy) were incubated (1X10⁶/ml) with fluorophore-conjugated monoclonal antibodies against the following markers (CD14, HLA-DR, CD86, CD163, and CD206; BD Bioscience, Inc.) The raw flow data were analyzed using FlowJo (v 7.6.2).Thecell populations were gated and identified with the following immunophenotypes: CD14⁺HLA-DR^-/low as the Mo-MDSCs , CD14⁺ CD86⁺as M1 macrophage, and CD14⁺ CD206⁺ or CD14⁺CD163⁺ as M2 TAMs . TAM or Mo-MDSC cells were presented in MFIs, as well as their percentages of total gated MNCs. Results: 1) Mo-MDSC were significantly elevated in MPN compared to that of controls both in the bone marrow aspirates and in the biopsy specimens (Fig1); 2) No difference was identified in M1 macrophages between MPN and controls in the percentage of total gated cells or by the MFI (data,not shown); 3) No difference was noted in M2 macrophages between MPN and controls in the percentage of total gated cells (Fig 2a) or by the MFI (Fig 2b). Conclusions: Mo-MDSC are increased in the bone marrow aspirates and biopsy specimens of patients with BCR-ABL(-) MPN. No difference was noted in the M1 and M2 TAM macrophages between the MPN and control.

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