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GM-CSF nitration is a new driver of myeloid suppressor cell activity in tumors

ABSTRACT: Reactive oxygen species, including RNS, contribute to the control of multiple immune cell functions within the tumor microenvironment (TME). Tumor-infiltrating myeloid cells (TIMs) represent the archetype of tolerogenic cells that actively contribute to dismantle effective immunity against cancer are among the cells most influenced by these molecules. TIMs inhibit T cell functions and promote tumor progression by several mechanisms including the amplification of the oxidative/nitrosative stress within the TME. In tumors, TIM expansion and differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced by cancer and immune cells. Nevertheless, the role of GM-CSF in tumors has not yet been fully elucidated. In this study, we show that GM-CSF activity is significantly affected by RNS-triggered post-translational modifications. The nitration of a single tryptophan residue in the sequence of GM-CSF nourishes the expansion of highly immunosuppressive myeloid subsets in tumor-bearing hosts. Importantly, tumors from colorectal cancer patients express higher levels of nitrated tryptophan compared to non-neoplastic tissues. Collectively, our data identify a novel and selective target that can be exploited to remodel the TME and foster protective immunity against cancer.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Glycine Max Mus Musculus (mouse)

SUBMITTER: Giorgio Arrigoni

LAB HEAD: Giorgio Arrigoni

PROVIDER: PXD027049 | Pride | 2021-10-04

REPOSITORIES: Pride

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			Action	DRS
	26Sept2013_Molon_CSF2_Mouse_Degraded.RAW	Raw
	26Sept2013_Molon_CSF2_Mouse_Degraded.msf	Msf
	26Sept2013_Molon_CSF2_Mouse_Nitrated.RAW	Raw
	26Sept2013_Molon_CSF2_Mouse_Nitrated.msf	Msf
	26Sept2013_Molon_CSF2_Mouse_NotTreated.RAW	Raw

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Project description:Background: Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a hematopoietic growth factor and adjuvant in cancer immunotherapy via stimulation of dendritic cells/APCs. However, GM-CSF has yielded inconsistent results and its role regarding in vivo modulation of macrophages remains underexplored. We previously demonstrated that 100ng “high-dose” intratumor (IT) GM-CSF ablated tumor blood vessels and worsened tumor hypoxia after 3 weeks through tumor-associated macrophage (TAM) soluble VEGFR-1 production in PyMT murine breast cancer. Here, we investigate a role for “low-dose” IT GM-CSF on tumor oxygen and the impact on immunotherapy response, TAMs/myeloid cells, and TILs relative to “high-dose”. Methods: We performed IT injections of dose-specific GM-CSF or saline controls and then evaluated phenotypic effects after 3 weeks. We used Electron Paramagnetic Resonance Oximetry to measure and image in vivo tumor oxygen in real-time, and fluorescent immunohistochemistry to assess tumor blood vessels. IT GM-CSF doses were tested in priming PyMT tumors for sensitization to PD1. We performed RNA Sequencing of TAM and CD8 TIL to observe transcriptional changes coupled with flow cytometry of peripheral blood monocytes, tumor myeloid, and TIL populations in immunology cold" PyMT tumors response to dose-optimized GM-CSF. Lastly, we assessed and compared effects of IT GM-CSF on TAM and TIL in an immunologically “hot” 4T1 breast cancer model. Results: 5ng IT GM-CSF significantly increased PyMT tumor oxygen without augmenting tumor growth and promoted tumor vessel health via increased pericyte coverage. Priming of PyMT tumors with 5ng IT GM-CSF (“low-dose”, hypoxia reduced) sensitized “cold” PyMT tumors to PD1, but this synergy was not observed with 100ng (“high-dose”, hypoxia exacerbated). Immunologically, 5ng GM-CSF did not increase monocyte mobilization or alter phenotypic marker expression on TAMs, but reduced hypoxic and inflammatory transcriptional programs in macrophages and CD8 TIL isolated from PyMT tumors. 100ng increased infiltration of myeloid cells and TAMs but these TAMs had reduced MHCII expression, suggesting support of immune-suppressive TAM under hypoxia. Some tumors exhibited increased CD8 polyfunctionality either dose suggesting mild CD8 TIL priming. On the other hand, 100ng in “hot” 4T1 tumors resulted in increased TAM MHCII and other immunostimulatory molecules with moderate increases in CD8 TIL polyfunctionality and exhausted PD1hiTIM3+ phenotype, indicating that GM-CSF may have opposing effects on macrophage modulation based on tumor immunological status.

Project description:Tumor growth is associated with a profound alteration of myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Analyzing the cytokines affecting myelo-monocytic differentiation produced by various experimental tumors, we found that GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of IFN- -producing CD8+ T cells upon in vivo adoptive transfer. Moreover, adoptive transfer of syngeneic, GM-CSF+IL-6-conditioned MDSCs to diabetic mice transplanted with allogeneic pancreatic islets resulted in long term acceptance of the allograft and correction of the diabetic status. Cytokines inducing MDSCs acted on a common molecular pathway. Immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on C/EBP transcription factor, a key component of the emergency myelopoiesis triggered by stress and inflammation. Adoptive transfer of tumor antigen-specific CD8+ T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBP in myeloid compartment. These data unveil another link between inflammation and cancer and identify a novel molecular target to control tumor-induced immune suppression. We used gene expression analysis to identify those factors, secreted by tumor-infiltrating MDSC, which could drive emathopoiesis. Moreover we compare gene expression profile of tumor-induced MDSC, obtained from either the spleen and the tumor infiltrate of tumor bearing mice, and in vitro bone marrow-derived MDSC. CD11b+ cells were immunomagnetically enriched from various murine tissue and experimental conditions, and cRNA samples were prepared accordingly to Expression Analysis: Technical Manual. 701021 Rev. 5. Santa Clara, CA, Affymetrix; 2004, and hybridized to the Affymetrix GeneChip MOE430 2.0 array which contains more than 45,000 probe sets, representing more than 34,000 genes. CD11b+ cells obtained from the spleen of healthy BALB/c and C57BL/6 mice were used as reference sample for tumor induced CD11b+ MDSC, enriched from either the spleen and the tumor infiltrate of tumor-bearing mice. Moreover CD11b+ cells enriched from fresh bone marrow were used as reference sample for in vitro bone marrow-differentiated MDSC, obtained with either GM-CSF+IL-6 and GM-CSF+G-CSF 4 days cytokine cocktail treatment.

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GM-CSF nitration is a new driver of myeloid suppressor cell activity in tumors

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