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Published on: November 28, 2015
Nucleotide receptor P2X7/STAT6 pathway regulates macrophage M2 polarization and its application in CAR-T
1the Inner Mongolia Key Laboratory for Molecular Regulation of the Cell and the State Key Laboratory of Reproductive Regulation, School of Life Sciences, Inner Mongolia University, Hohhot 010010, Inner Mongolia Autonomous Region, P.R. China.
Background:
A key factor underlying the failure of Chimeric Antigen Receptor-T Cell (CAR-T) therapy in ovarian cancer (OC) is the presence of an immunosuppressive tumor microenvironment, which is intricately linked to M2 polarization among tumor-infiltrating macrophages. P2X7 receptor has been previously documented as expressed within these macrophages and its correlation with M2 polarization is evident. This investigation scrutinizes whether silencing of P2X7 receptor within macrophages could lead to augmented anti-tumor potency of CAR-T.
Method:
Human peripheral blood mononuclear cells were artificially differentiated into macrophages or M2 macrophage in vitro. After silencing P2X7 receptor and/or overexpressing STAT6 within macrophages, the M1 and M2 markers were evaluated via flow cytometry, ELISA, and qRT-PCR. Additionally, the phosphorylation level of STAT6 was monitored by western blot. We engineered CAR-T cells targeting the non-functional P2X7 (nfP2X7) receptor, and co-cultured them with macrophages silencing P2X7 receptor along with OC cells. The anti-tumor effect of these CAR-T cells was assessed through evaluating OC cell viability, lactate dehydrogenase release, and IFN-γ levels.
Result:
P2X7 receptor silencing promotes M1 macrophage marker expression (CD86, TNF-α, IL-6, IL-1β), diminishes M2 macrophage marker expression (CD206 and IL-10) and suppresses STAT6 phosphorylation, whereas STAT6 overexpression reverses these phenomena. Furthermore, M2 macrophage suppresses the toxic effect of CAR-T cells on OC cells, while silencing the P2X7 receptor nullifies the immunosuppressive effect of M2 macrophages on CAR-T cells.
Conclusion:
Silencing P2X7 receptor can reverse M2 macrophage polarization by suppressing STAT6 activation, thereby enhancing the anti-tumor efficacy of CAR-T cells targeting nfP2X7 receptor in OC cell lines.
Insights
Silencing the P2X7 receptor in macrophages reverses M2 polarization and enhances Chimeric Antigen Receptor-T Cell (CAR-T) therapy efficacy against ovarian cancer. This approach overcomes the immunosuppressive tumor microenvironment, boosting anti-tumor activity.
Area of Science:
- Immunology
- Oncology
- Cell Biology
Background:
- Ovarian cancer (OC) Chimeric Antigen Receptor-T Cell (CAR-T) therapy often fails due to an immunosuppressive tumor microenvironment.
- Tumor-infiltrating M2-polarized macrophages contribute to this immunosuppression.
- The P2X7 receptor is expressed in M2 macrophages and linked to their polarization.
Purpose of the Study:
- To investigate if silencing the P2X7 receptor in macrophages can enhance the anti-tumor potency of CAR-T cells in ovarian cancer.
- To explore the role of P2X7 receptor and STAT6 in macrophage polarization and CAR-T cell function.
Main Methods:
- Macrophages were differentiated in vitro, and P2X7 receptor was silenced, with or without STAT6 overexpression.
- Macrophage polarization markers (M1/M2) were assessed using flow cytometry, ELISA, and qRT-PCR.
- CAR-T cells targeting non-functional P2X7 (nfP2X7) were co-cultured with modified macrophages and OC cells to evaluate anti-tumor effects.
Main Results:
- P2X7 receptor silencing promoted M1 markers (CD86, TNF-α, IL-6, IL-1β) and reduced M2 markers (CD206, IL-10), suppressing STAT6 phosphorylation.
- STAT6 overexpression reversed these effects.
- M2 macrophages suppressed CAR-T cell anti-tumor activity, but P2X7 receptor silencing nullified this immunosuppression.
Conclusions:
- Silencing the P2X7 receptor reverses M2 macrophage polarization by inhibiting STAT6 activation.
- This strategy enhances the anti-tumor efficacy of CAR-T cells targeting nfP2X7 receptor in ovarian cancer models.
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