CD19 expression associates with polyfunctionality and altered secretion dynamics in individual activated human B cells.

The concept of polyfunctional secretion, the ability of cells to secrete multiple, functionally distinct proteins, has received increased attention for B cells as they have been described to secrete both antibodies and cytokines. However, the functional analysis of B cells was mostly performed in bulk, and consequently, the dynamics, quantity, and temporality of poly-secreting B cells are not well understood on the level of the individual cell. Furthermore, data on the existence of B-cell polyfunctionality in healthy settings remain scarce. Therefore, in this work, we developed a microfluidic workflow for the quantitative, simultaneous analysis of B-cell polyfunctionality on the single-cell level, focusing on 4 key secreted proteins-IL-10, IL-6, GM-CSF, and IgG-in switched memory B cells after ex vivo stimulation. Our findings revealed that IgG cytokine co-secretion occurred at the single-cell level, and was induced early by specific stimuli. Notably, ex vivo TLR4 activation of human B cells induced a population of highly polyfunctional B cells, wherein IL-6 and GM-CSF exhibited faster secretion dynamics compared to their mono-secreting counterparts. Moreover, these functional differences correlated with CD19 expression: Higher CD19 levels resulted in increased IL-6 and GM-CSF secretion rates and delayed IgG secretion. This suggests distinct functional properties correlating with CD19 expression levels in ex vivo activated antibody-secreting and B cells. Beyond the quantitative and dynamic description of inflammatory and regulatory B cells, these results offer insights into the complexity and temporal patterns of B-cell (poly)functionalities, opening new avenues for studying disease-related immune regulation.