Transcription factor expression for identifying B cell subsets in cattle by flow cytometry

With their capacity for antibody production, B cells underpin many protective immune responses, but they can also contribute to immunopathology and autoimmunity. Studying their differentiation following antigen encounter is therefore a key aspect of fundamental immunological research. However, in many livestock species, including cattle, the study of B cell subsets is limited by the restricted availability of immunological tools. Here, we demonstrate that cross-reacting anti-human and anti-murine antibodies used in combination with established anti-bovine reagents can be used to identify three major cattle B cell subsets by flow cytometry: a) total mature B cells, b) plasma cells and plasmablasts, and c) germinal centre (GC) B cells. For this, we employed antibodies targeting CD79α, Pax5, Blimp1, IRF4, and Bcl6. CD79α/Pax5 co-expressing total B cells largely overlapped with the previously described CD14^-CD40⁺ phenotype. Putative plasma cells, defined by Blimp1/IRF4 co-expression, were detected in blood and lymph nodes and exhibited reduced Pax5 expression, consistent with a plasma cell phenotype. In contrast, putative GC B cells, identified by Bcl6 expression, were only observed in lymph nodes, as expected for this population. Using foot-and-mouth disease virus-like particles (VLPs) as probes to detect antigen-specific B cells in blood and lymph nodes from VLP-immunised cattle, we show that our newly established phenotypes facilitate the dissection of antigen-driven B cell responses. Notably, VLP-binding B cell subsets were predominantly detected in lymph node cell preparations draining the site of vaccination. Thus, this work significantly expands the toolbox available for studying B cell biology in cattle.