Hannan, R. T. | Search bioRxiv

bioRxiv2024

Novel wheat germ agglutinin-based mass cytometry cell barcoding reagent for heterogeneous, live or fixed sample

Sample multiplexing in flow cytometry is a powerful technique which allows for reduction of error, inclusion of control samples for batch effect correction, and reduction in both time and consumable usage. Current industry standard for barcoding in mass cytometry is an intracellular reagent, which requires fixation and permeabilization of sample prior to barcoding. We developed a barcode using the ubiquitous and well-tolerated membrane labeling lectin, wheat germ agglutinin. This barcode effectively labels all tested cell types, both live and fixed. We determine that barcode yields, or the ratio of debarcoded cells to total input cells, is stable in live pooled sample for at least an hour. This barcode does not show differential performance across major PBMC lineages. Thus, this universal wheat germ agglutinin-based barcode represents an advance in gentle, non-reactive cell surface barcoding for live cells.

cell biology↗

bioRxiv2024

A protective role for B-1 cells and oxidation-specific epitope IgM in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgMOSE). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM (sIgM-/-) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgMOSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.

immunology↗