CD19 CAR T-cell functionality in a 3D stroma/leukemia model

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults, characterized by accumulation of mature, monoclonal B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite advances in targeted therapies, CLL remains incurable for most patients, highlighting the need for novel therapeutic strategies. Although CAR T-cell therapy has markedly improved outcomes in acute lymphoblastic leukemia (ALL) over the past decades, its efficacy in relapsed/refractory (r/r) CLL remains limited, reflecting fundamental differences in disease biology and immune response. The manipulation of the tumor microenvironment (TME) by malignant B cells within protective niches plays a central role in disease progression and therapy resistance, emphasizing the importance to understand microenvironmental contributions to treatment failure. To investigate disease-specific differences in CAR T-cell functionality, a three-dimensional (3D) scaffold-based co-culture model was utilized that incorporates primary CLL or ALL cells, autologous CAR T cells, and bone marrow-derived stromal cells (BMSCs). This model recapitulates key features of the stromal TME. Its spatial heterogeneity allows distinction of cells in the scaffold periphery from those residing deeper within the stromal matrix, facilitating the investigation of CAR T-cell function under physiologically relevant conditions. Comparative analyses revealed pronounced differences in CAR T-cell function between CLL- and ALL-derived CAR T cells after 3D co-culture. Despite strong activation, CLL CAR T cells exhibited limited proliferation and diminished cytolytic activity, accompanied by reduced co-localization with malignant B cells. Additionally, they were enriched in further differentiated and exhausted phenotypes. Notably, defects in cytotoxicity and exhaustion were enhanced in core regions, indicating that intrinsic CLL CAR T-cell dysfunction is amplified by TME-mediated suppression. In the CLL 3D model, IL-10 secretion was notably higher than in ALL co-cultures, reflecting disease-specific immunosuppressive signaling. Additionally, a higher proportion of CXCR4⁺ CLL cells and CXCL12+ BMSCs was localized in core regions, highlighting spatial regulation of stromal support via the CXCR4/CXCL12 axis. Targeting these TME-mediated mechanisms led to an improvement in CLL CAR T-cell function. IL-10 blockade enhanced CLL clearance in a patient- and region-dependent manner and consistently reduced CAR T-cell exhaustion, while CXCR4 blockade resulted in enhanced cytotoxicity and decreased exhaustion of CAR T cells. In summary, this study highlights impairments in CAR T-cell functionality in CLL and demonstrates that combination treatments targeting the TME can partially mitigate these defects. The autologous 3D model is a translationally relevant system to dissect disease-specific immune interactions and evaluate novel therapeutic approaches to improve outcomes in CLL.