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

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells capable of generating all hematopoietic lineages, representing a powerful platform for regenerative immunotherapy. In parallel, chimeric antigen receptor (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-optimized CARs across immune cell types using a human HSPC-derived multilineage CAR-immune cell platform. HSPC-derived T, natural killer (NK), and myeloid cells demonstrated immune activation across different CAR designs, whereas B cells showed CAR design dependency for activation. Hallmark pathway analysis revealed that immune lineage was the dominant determinant of activation-induced transcriptional states, while CAR design modulated signaling programs within lineage-constrained circuits, notably proliferation, inflammation, and cytokine responses. Single-cell InTraSeq highlighted differential activation of signaling pathways depending on CAR design and lineage identity with links to downstream transcriptional programs 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.