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BTN2A1 Targeting Reprograms M2-like Macrophages and TAMs via SYK and MAPK Signaling

ABSTRACT: Tumor-associated macrophages (TAMs), often adopting an immunosuppressive M2-like phenotype, correlate with unfavorable cancer outcomes. Our investigation unveiled elevated expression of the butyrophilin (BTN)2A1 in M2-like TAMs across diverse cancer types. We developed anti-BTN2A1 monoclonal antibodies (mAb) and notably, one clone demonstrated a robust inhibitory effect on M2-like macrophage differentiation, inducing a shift towards an M1-like phenotype both in vitro and ex vivo in TAMs from patients with cancer. Macrophages treated with this anti-BTN2A1 mAb exhibited enhanced support for T-cell proliferation and IFNγ secretion. Indeed, M1-like and M2-like macrophage differentiation involves complex signaling pathways leading to specific gene expression profiles. Typically, stimuli from the TME induce pathways including JAK/STATs, MAPK, PI3K/AKT, NOTCH and NF-κB influencing TAMs polarization. We investigated the signaling pathways activated in monocytes and M-CSF-induced M2-like macrophages upon BTN2A1 engagement using anti-BTN2A mAb5. After 30 min of treatment, we observed phosphorylation of MAPKs (i.e. ERK1/2, P38 and JNK) in CD14+ monocytes and in M2-like macrophages, unlike isotype control-treated cells. In contrast, the PI3K/AKT, JAK/STAT, and NF-kB pathways were not consistently activated after 30 min of anti-BTN2A mAb5 treatment. Since BTN2A1 lacks intrinsic kinase activity, we explored whether BTN2A1 could form complexes with molecular partners in M2-like macrophages that provide kinase activity. We prepared lysates from M2-like macrophages treated with mAb5 or with its isotype control for 30 min and performed immunoprecipitation (IP) using another anti-BTN2A mAb followed by western blotting with anti-phosphoTyrosine (pTyr). A 70 kDa was co-immunoprecipitated with BTN2A1 and revealed by the anti-pTyr in lysates from anti-BTN2A mAb5-treated M2-like macrophages. To identify which Tyrosine kinase is enriched in anti-BTN2A mAb5-treated M2-like macrophages, we performed LC-MS/MS on anti-pTyr immunoprecipitates from treated cells. Several tyrosine kinases, including P38α/δ and JNK, were enriched. Notably, SYK was also identified and matched the 72kDa molecular weight of the pTyr band co-immunoprecipitated with BTN2A1.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Macrophage

DISEASE(S): Cancer

SUBMITTER: AUDEBERT Stephane

LAB HEAD: OLIVE Daniel, PU-PH,

PROVIDER: PXD055262 | Pride | 2024-09-02

REPOSITORIES: Pride

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Project description:Macrophage polarization between the M2 (repair, pro-tumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of CSF-1R that contribute to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain-of-function and loss-of-function with bioinformatic analysis of the macrophage CSF-1R pTyr-721-regulated transcriptome, we uncovered miR-21 as a downstream molecular switch controlling macrophage activation and identified ERK1/2 and NF-M-NM-:B as CSF-1R pTyr-721-regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the proinflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21-regulated mRNAs revealed that 80% of the CSF-1-regulated canonical miR-21 targets are pro-inflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R-mediated activation of ERK1/2 and NF-M-NM-:B. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide (LPS). M-bM-^@M-^C These results identify the macrophage miR-21 network as a novel target for controlling macrophage polarization. We performed microarray-based analysis on four mouse macrophage cell lines, two CSF-1R pTyr-721-expressing cell lines (M-/-.WT and M-/-.3ABY721) and two CSF-1R Tyr-721-deficient lines (M-/-.Y721F and M-/-.3AB). Total RNA (two biological replicates) was extracted from CSF-1-starved cells (UR) or from cells constitutively grown in CSF-1 (CONST). Additionally, CSF-1-starved M-/-.WT and M-/-.Y721F cell lines were stimulated with CSF-1 for 0min, 20 min, 60 min and 180 min and used for total RNA extraction. Total RNA preparations with Ribosomal Integrity Numbers (RIN) > 9.5 were used for microarray analysis. A total of 100 ug/cell line/replicate was used for gene expresion analysis on the Affymetrix Mouse Gene ST1.0 chips at the Genomics Core at Einstein, according to manufacturerM-bM-^@M-^Ys instructions. Differential expression analysis was performed using the M-bM-^@M-^XlimmaM-bM-^@M-^Y package of R/Bioconductor to identify significantly differentially expressed mRNAs over time, in response to CSF-1 treatment and to the genotype. CSF1-regulated genes were identified according to the cutoff with both expression folder change > 1.5 and p-value < 0.05.

			Action	DRS
	Ctrle_Rep1.raw	Raw
	Ctrle_Rep1_20230716024257.raw	Raw
	Ctrle_Rep1_20230717073938.raw	Raw
	Ctrle_Rep2.raw	Raw
	Ctrle_Rep2_20230716122151.raw	Raw

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BTN2A1 Targeting Reprograms M2-like Macrophages and TAMs via SYK and MAPK Signaling

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