Pioli, P. D. | Search bioRxiv

bioRxiv2022

Thymus Antibody-Secreting Cells Represent a Tissue Specific Population That Possess an Activated Cellular Phenotype

Antibody-secreting cells (ASCs) are key contributors to humoral immunity through immunoglobulin production and the potential to be long-lived. ASCs have been recently identified in the thymus (THY) which is of interest as autoreactive THY B cells regulate T cell tolerance. Here, we showed that the young female THY was skewed towards higher production of ASCs relative to males. However, these differences disappeared with age. Intravenous labeling combined with blockade of CD154(CD40L) suggested that THY ASCs were generated in situ in a T cell dependent fashion. Single cell RNA-sequencing revealed that THY ASCs possessed a transcriptional signature indicative of interferon signaling and activation. Flow cytometry confirmed that THY ASCs had increased levels of Toll-like receptor 7 as well as the activation markers CD69 and major histocompatibility class II. Overall, THY ASCs are a unique population that require further investigation to fully appreciate their biological significance.

immunology↗

bioRxiv2022

Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions

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-1{beta}.

immunology↗