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In vitro differentiation of M1, M2, and TAM-like macrophages from healthy donor PBMC

ABSTRACT: To understand the immunological role of different macrophage populations in the tumor, we generated transcriptional profiles of macrophages differentiated from PBMC-derived monocytes in vitro under three conditions to compare with: M1-like macrophages were developed in vitro through polarization of human monocytes by IFNg and TLR4 stimulation. This population is considered to have anti-tumor activities; releasing proinflammatory cytokines, performing phagocytosis, and possessing antigen cross presentation abilities. Second, M2-like macrophages were developed in vitro through intensive IL4 stimulation of monocytes. M2 macrophages are known to promote many pro-tumorigenic processes such as angiogenesis, immune suppression, hypoxia induction, tumor cell proliferation, metastasis. Lastly, we deployed a third population that represents a better in vitro model for tumor associated macrophages (TAM), developed under conditions better resembling the TME.Monoctyes derived from two individual patient donors were cultured separately under four (4) different conditions in vitro, for a total of eight (8) samples to be processed for bulk RNA sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE243175 | GEO | 2023/09/18

REPOSITORIES: GEO

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Project description: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.

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In vitro differentiation of M1, M2, and TAM-like macrophages from healthy donor PBMC

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