Background Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that antibody-secreting cells regulate multiple aspects of biology through the secretion of various cytokines. In this regard, intracellular flow cytometry is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Methods Paraformaldehyde plus saponin or the eBioscience Foxp3/Transcription Factor Staining Buffer Set were used to evaluate the non-specific intracellular retention of phycoerythrin-containing antibody conjugates by antibody-secreting cells. Results We showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of antibody-secreting cell intracellular protein expression compared to other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within antibody-secreting cells. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between antibody-secreting cells and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen antibody-secreting cells expressed Toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β. Conclusion Increasing the number of centrifugation steps performed on antibody-secreting cells post-fixation leads to inappropriate retention of phycoerythrin-containing antibody conjugates during intracellular flow cytometry.

In humans and mice, sex dichotomies exist in regards to physiological immune responses as well as pathological autoimmune responses. However, very little information exists in regards to how sex patterns the production and function of antibody-secreting plasmablast (PB) and plasma cell (PC) populations. Using the Prdm1-enhanced yellow fluorescent (eYFP) reporter mouse strain, we compared the percentages and numbers of PBs and PCs in the bone marrow (BM), spleen (SPL) and thymus (THY) of young (3 months old) female and male mice. While PB/PC generation was equivalent in the BM and SPL of both sexes, the female THY had significantly increased percentages and numbers of both PBs and PCs when compared to males. This correlated with the overall increase in thymopoiesis present in females. Characterization of THY PBs/PCs demonstrated increased expression of canonical B cell activation markers such as CD69 and MHC II when compared to their BM and SPL counterparts. In some aspects, these differences were sex-based in origin. αCD45 intravenous antibody labeling suggested that THY PBs/PCs were locally generated and not a consequence of immigration from the periphery. As such, female THY demonstrated increased numbers of a germinal center B (GCB) cell-like population expressing both GL7 and CD95(Fas). THY B cells have been previously shown to participate in the regulation of T cell selection and we hypothesized that THY PBs/PCs would be generated in a T cell dependent manner. Indeed, administration of αCD40L blocking antibodies ablated THY GCB, PB and to some extent PC production. In summary, female mice are skewed towards increased THY PB/PC generation. The production of these cells happens locally and is dependent on CD40L-based T cell interactions.

Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that antibody-secreting cells regulate multiple aspects of biology through the secretion of various cytokines. In this regard, intracellular flow cytometry is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Here, we showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of antibody-secreting cell intracellular protein expression compared to other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within antibody-secreting cells. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between antibody-secreting cells and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen antibody-secreting cells expressed Toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β.