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987 Glioma-derived factors induce an immune suppressive phenotype in bone marrow-derived CCR2+ myeloid cells
  1. Gregory Takacs,
  2. Julia Garcia,
  3. Alexandra Sherman,
  4. Christian Kreiger,
  5. Defang Luo and
  6. Jeffrey Harrison
  1. University of Florida, Gainesville, FL, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.

Abstract

Background Glioblastoma (GBM) is an aggressive primary brain tumor that is highly resistant to immune checkpoint inhibitors (ICIs). Bone marrow-derived myeloid cells comprise a large proportion of glioma-infiltrating leukocytes and are associated with an immune-suppressive phenotype. We previously characterized monocytic-myeloid derived suppressor cells (M-MDSCs) based on expression of the chemokine receptors CCR2 and CX3CR1. Targeting CCR2, in combination with anti-PD-1, reduced M-MDSCs in the tumor microenvironment and prolonged survival in ICI resistant murine gliomas, KR158B and 005GSC. We also established that M-MDSCs migrate to CCR2 ligands, CCL2 and CCL7, in a redundant manner. Dual targeting of CCL2 and CCL7 limited the presence of CCR2+/CX3CR1+ M-MDSCs in the glioma microenvironment. While knowledge of the mechanisms that recruit myeloid cells to the glioma microenvironment is better understood, it remains unclear what factors drive the myeloid-derived immune suppressive phenotype in GBM. The objective of this study is to evaluate the effect of glioma-derived factors on immature myeloid cells.

Methods C57BL/6 mice were implanted with KR158B glioma cells. Brain tumor tissue was subjected to Luminex cytokine analysis and compared to healthy brain. Bone marrow from Ccr2RFP/WT/Cx3cr1GFP/WT mice was cultured in the presence of KR158B glioma conditioned media followed by fluorescent microscopy and flow cytometry analysis to assess M-MDSCs. T cell suppression assay was conducted using Ly6G-/GR1+ cells (M-MDSCs) isolated from bone marrow cultures supplemented with KR158B conditioned media and freshly isolated T-cells to examine their immune-suppressive phenotype. In vivo iNOS was examined by flow cytometry and immunohistochemical analyses. NOS inhibitor, L-NMMA, was used to evaluate the role of nitric oxide in M-MDSC-mediated T cell suppression.

Results KR158B gliomas differentially upregulate a number of cytokines/growth factors, including CCL2, IL6, G-CSF, GM-CSF, as compared to healthy naive brains. KR158B conditioned media increased the percentage of bone marrow-derived CCR2+/CX3CR1+ cells that express M-MDSC markers. Bone marrow-derived CCR2+/CX3CR1+ cells, expanded in KR158B condition media, suppress both CD4+ and CD8+ T cell proliferation. M-MDSCs in KR158B glioma microenvironment regulate iNOS compared to M-MDSCs present in the bone marrow. NOS inhibitor, L-NMMA, prevented M-MDSC suppression of CD8 T cell proliferation, but had no effect on suppression of CD4 T cells.

Conclusions Glioma-derived factors recruit and induce CCR2+/CX3CR1+ myeloid cells to a CD4/8+ T cell suppressive state that can be partially ameliorated by iNOS inhibition. While additional M-MDSC-mediated immune suppressive mechanisms need to be identified, targeting M-MDSCs holds promise as an effective approach to improve immune-directed interventions in GBM.

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