A novel platform for detecting CD28 and CTLA-4 modulation in PBMCs for immunotherapy development 3356

Abstract Description Immune checkpoint proteins such as CD28 and CTLA-4 play critical roles in regulating T-cell activation and immune responses, making them important targets for immunotherapy development. This study explores the application of AlphaLISA™ technology to measure changes in CD28 and CTLA-4 levels in peripheral blood mononuclear cells (PBMCs) under immune-stimulating and immunosuppressive conditions. AlphaLISA kits for CD28 and CTLA-4 were used to generate standard curves in various buffers and media, demonstrating high sensitivity with detection limits in the low pg/mL range and dynamic ranges spanning over four logs. PBMCs were stimulated with CD3/CD28 Dynabeads™ at varying cell densities and time points to assess assay performance. CD28 levels increased 3.5-fold and CTLA-4 increased 4-fold after 48 hours, underscoring the assay’s sensitivity to changes in checkpoint protein levels. Dose-response studies with immune modulators revealed that phytohemagglutinin significantly enhanced CD28 and CTLA-4 expression, demonstrating the potent activation of T-cells under these conditions. In contrast, treatment with the immunosuppressants rapamycin and cyclosporin A reduced expression, highlighting their inhibitory effects on T-cells. Overall, these findings demonstrate the exceptional sensitivity and versatility of AlphaLISA, a homogeneous, no-wash platform, for quantifying checkpoint protein modulation in PBMCs, providing valuable insights for immunotherapy research and drug development. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)