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

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.