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bioRxiv · 10.64898/2026.10.22.760934

Stimulation strategy matters: A B-cell focused single-cell transcriptomic study on in vitro stimulated immune cells

D'hamers, T.·Neirinck, J.·Buysse, M.·De Vriendt, C.·Kerre, T.·Haerynck, F.·Hofmans, M.·Bonroy, C.·Everaert, C.
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Abstract

Distinct in vitro B-cell stimulation strategies, defined by stimulation condition and incubation duration, have been described in diverse studies. Often, these approaches are considered interchangeable due to comparable flow-cytometric outputs concerning fluorescence intensities and B-cell subset distributions. However, whether these phenotypic similarities also reflect comparable underlying transcriptional states have not been systematically evaluated yet. In this study, we provide an unbiased comparison of B-cell activation strategies on healthy individuals, resulting in a single-cell transcriptomic atlas (4 donors; ~2,029 cells per sample; 81,160 cells in total with 56.3% annotated as B cells). Two T-cell independent stimulators targeting toll-like receptors (ODN2006 and IFN-{gamma}/R-848), were assessed next to two T-cell dependent stimulators (anti-CD40 and anti-CD3/CD28 antibodies), all incubated for eight and 30 hours. Observed overlap for differential abundance, differential gene expression and cell-cell interactions could directly be linked to T-cell (in)dependent working mechanism. Strategy-specific results uncovered time-point specific transcriptional changes, revealed the largest set of differentially expressed genes for IFN-{gamma}/R-848 and ODN2006, and showed that IFN-{gamma}/R-848 and anti-CD3/CD28 induced the majority of the cell-cell interactions. Ultimately, our reference atlas emphasizes the importance and facilitates the rational selection of optimal B-cell stimulation strategies, particularly for studies aiming to interrogate B-cell dysregulation in immune-mediated diseases.

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D'hamers, T., Neirinck, J., Buysse, M., De Vriendt, C., Kerre, T., Haerynck, F., Hofmans, M., Bonroy, C., Everaert, C.. 2026-10-27. Stimulation strategy matters: A B-cell focused single-cell transcriptomic study on in vitro stimulated immune cells. https://doi.org/10.64898/2026.10.22.760934

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