Basement membrane hydrogels dampen CAR-T cell activation: nanofibrillar cellulose gels as alternative to preserve T cell function in 3D cell cultures.

BackgroundHydrogel-based 3D culture systems are emerging as a valuable tool for preclinical screening of cell-based immunotherapies against solid and hematological malignancies, such as chimeric antigen receptor T (CAR-T) cells. Hydrogels can influence T cell function in a non-desired manner due to their mechanical properties and chemical composition, potentially skewing results in preclinical testing of novel immunotherapeutic compounds. MethodsIn this study, we assess CD4+ T and CAR-T cell activation and proliferation in chemically-undefined matrices (Matrigel and basement membrane extract, BME) and compare them to a synthetic nanofibrillar cellulose (NFC) hydrogel. ResultsRheometric analyses show that NFC is more rigid than Matrigel and BME. Murine CD4+ T cells acquire a regulatory T cell (Treg) phenotype in Matrigel and BME, while this is not observed in NFC. Proliferation and activation of human T cells are higher in NFC than in Matrigel or BME. Similarly, we show that CAR-T cell activation and proliferation is significantly impaired in Matrigel and BME, in contrast to NFC. ConclusionsOur findings highlight the impact of hydrogel choice on (CAR-)T cell behavior, with direct implications for preclinical immunotherapy testing. In contrast to Matrigel and BME, NFC offers a chemically-defined 3D environment where T cell function is preserved. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSIn 3D (preclinical) tumor-killing assays for evaluating engineered T cell cytotoxicity, the surrounding matrix can influence immune cell phenotype and function, potentially skewing T cell activity. Basement membrane hydrogels such as Matrigel and basement membrane extract (BME), widely used as scaffolds for 3D culture, are inherently heterogeneous and contain extracellular matrix components that can influence lymphocyte function. What this study addsHere, we show that (CAR-)T cell function is significantly reduced in Matrigel and BME as compared to standard (2D) culture conditions. In contrast, (CAR-)T cell activity is preserved in synthetic nanofibrillar cellulose (NFC) gels. Importantly, murine T cells spontaneously acquire a Treg phenotype in Matrigel and BME. T cell proliferation and cytokine secretion are >10-fold lower in Matrigel than in NFC. Similarly, CAR-T cell survival and expansion are 10-fold higher in NFC than in Matrigel or BME. How this study might affect research, practice or policyWe report that the intrinsic cytotoxic and proliferative potential of (CAR-)T cells can be underestimated when performing assays in 3D cultures based on Matrigel or BME. As an alternative, we suggest the use of chemically defined synthetic gels, and we show that nanofibrillar cellulose hydrogels are suitable 3D matrices for preserving T cell phenotype and activation.