CAR design equips diverse immune cells with unique antigen-specific activation identities

Haematopoietic stem and progenitor cells (HSPCs) are multipotent cells, capable of generating all haematopoietic lineages, representing a powerful platform for regenerative immunotherapy. In parallel, CAR-engineering has extended beyond T cells, which have achieved success in treating B cell malignancies, to broader immune cell types alongside cell type-specific CAR design. However, how CAR architecture modulates activation phenotypes across immune lineages remains poorly understood. Here, we systematically compared previously validated, immune cell-optimised CARs across immune cell types using a human HSPC-derived multilineage CAR-immune cell platform. HSPC-derived T, NK and myeloid cells demonstrated immune activation across different CAR designs whereas cell-specific activation showed CAR design dependency in B cells. Hallmark pathway analysis revealed that immune lineage was the dominant determinant of activation-induced transcriptional states while CAR design modulated signalling programmes within lineage constrained programmes, notably proliferation, inflammation and cytokine responses. Single cell InTraSeq highlights differential activation of signalling pathways depending on CAR design and lineage identity with links to downstream transcriptional programmes across T, B and myeloid cells. Although exploratory and performed with limited sample sizes, this demonstrates the activated phenotypes and pathways mediated by CAR design and lineage identity that may inform the development of next-generation CAR-immune cells with tailored activation-induced characteristics.