Background/Purpose: Rheumatoid arthritis (RA) affects approximately 1.3 million adults in the United States, causing significant morbidity and economic burden. Current treatments reduce inflammation and slow disease progression but often have considerable side effects. Regulatory T cells (Tregs) are crucial for maintaining immune tolerance. Folate receptor β (FRβ) is selectively expressed on activated macrophages in inflamed synovial tissues of RA patients, making it an attractive target for immunotherapy. This study investigates the potential of FRβ chimeric antigen receptor Tregs (FRβ CAR-Tregs) to induce monocyte apoptosis and polarization towards an immunosuppressive phenotype, offering a novel therapeutic strategy for RA.

Methods: Human CD4+CD25+CD127^low Tregs were transduced with a CAR construct targeting FRβ. The functionality of FRβ CAR-Tregs was assessed in vitro by co-culturing with human monocytes. Monocyte apoptosis was measured using Annexin V and 7-AAD staining, and immune polarization was evaluated by flow cytometry for markers of M2 macrophages (CD206^hiCD163^+) and dendritic cell regulatory phenotype (DCreg). The role of CD39 expression and IL-10 in the suppressive activity of FRβ CAR-Tregs was also examined. In vivo efficacy was tested using islet transplantation, skin transplantation, and colitis murine models.

Results: FRβ CAR-Tregs significantly reduced the percentage of live CD11b+ monocytes through apoptosis and promoted the polarization of monocytes to M2 macrophages and DCregs. CD39(+) FRβ CAR-Tregs showed greater suppressive potency compared to CD39(-) counterparts, likely due to higher expression of immunosuppressive molecules such as granzyme B (Gzmb). Gzmb knockout reduced the cytotoxic effect of CD39(-) FRβ CAR-Tregs, underscoring its critical role in their suppressive function. Neutralization of IL-10 did not significantly reduce the suppressive activity of FRβ CAR-Tregs, indicating additional mechanisms such as adenosine production through CD39 enzymatic activity. In vivo, FRβ CAR-Tregs enhanced graft survival and reduced rejection in islet and skin transplantation models. In the colitis model, FRβ CAR-Tregs helped maintain body weight, reduced colon inflammation, and decreased systemic inflammation, evidenced by reduced spleen cell counts.

Conclusion: FRβ CAR-Tregs effectively induce monocyte apoptosis and polarization to an immunosuppressive phenotype, suggesting their potential as a novel therapeutic strategy for RA. The study highlights the enhanced suppressive function of CD39(+) FRβ CAR-Tregs, mediated through mechanisms involving granzyme B and IL-10. However, the current study is limited by the lack of RA patient data and the use of non-RA murine models. Future research should focus on validating these findings in RA-specific models and clinical settings, potentially providing a targeted and effective treatment for RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/folate-receptor-%ce%b2-car-tregs-induce-monocyte-apoptosis-and-immune-polarization-potential-for-therapeutic-application-in-rheumatoid-arthritis/