Membrane-engineered Wharton's Jelly derived mesenchymal stem cells with anti-CD2 antibody coating modulate activated CD3+ T-cell responses.

OBJECTIVE

Mesenchymal stem cells (MSCs) regulate immunity through paracrine signaling and direct interactions by secreting cytokines and biological factors, yet effective therapy requires large numbers of MSCs. This study aimed to improve the interaction between human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) and activated CD2⁺/CD3⁺ T lymphocytes by incorporating anti-CD2 antibodies into their cell membranes. This strategy was grounded in the recognition that MSC immunoregulatory efficacy depends on both soluble mediators and receptor-mediated cell-cell contact, and that strengthening this interface enhances their functional engagement with activated T-cells.

METHODS

In this study, hWJ-MSCs were coated with a palmitic acid-conjugated protein G (PPG) system to anchor anti-CD2 antibodies onto the cell membrane. Cytocompatibility of coating was evaluated using WST-8 assay, and 20 µg/mL was identified as optimal concentration. After coating and T-cell isolation, a 48-hour co-culture was established with phytohemagglutinin (PHA)-stimulated CD2⁺/CD3⁺ T lymphocytes and β-cells. Cellular responses were evaluated using WST-8, Live/Dead staining, CFSE proliferation assay, DCFDA-based ROS analysis, and CD3 flow cytometry to assess viability, proliferation, and oxidative activity of PBMCs. β-cell function was assessed by measuring insulin secretion using a single ELISA assay as a functional outcome indicator, without further mechanistic analysis.

RESULTS

The WST-8 assay demonstrated that 20 µg/mL PPG maintained approximately 80 % cell viability, and this concentration was selected as the optimal dose for subsequent experiments. Anti-CD2 and anti-PPG antibodies were purified using the FPLC method. CD25 and CD2 marker expression increased following PHA stimulation. CFSE staining showed higher fluorescence intensity in the MSC-coated group than in the non-coated group, indicating suppressed T-cell proliferation. DCFDA staining revealed reduced ROS levels in the coated group, while the WST-8 test indicated lower viability in MSC-coated groups relative to controls. ELISA analysis showed the highest insulin secretion in the MSC-coated group.

CONCLUSION

Anti-CD2 antibody-conjugated PPG-coated hWJ-MSCs reduced T-cell viability, proliferation, and WST-8-based metabolic activity while also attenuating DCFDA-detected ROS compared with uncoated cells. ROS levels remained elevated in CD2-only group. Coated MSCs therefore produced a more controlled and less inflammatory T-cell response within co-culture system. These findings suggest that surface-engineered hWJ-MSCs may enhance immunoregulatory performance of MSC-based approaches for T-cell-mediated autoimmune disorders, including Type 1 Diabetes Mellitus (T1DM).