Andreone, S. | bioRxiv Science

bioRxiv2024

Combined in vitro differentiation and cell sorting-based isolation of highly pure mouse bone marrow-derived basophils.

Basophils constitute a rare population of granulocytes with key functions in allergies, immunodeficiencies and cancer. The scarcity of basophils in the human blood and tissues constitutes a considerable limit for the study of these cells. Interleukin-3 (IL-3) is a cytokine that stimulates both the differentiation and the expansion of basophils from bone marrow (BM) precursors by positively regulating the expression of the IL3Ra receptor. We have standardized an in vitro differentiation protocol of mouse basophils (mBaso) from BM precursors through culture in presence of IL-3 for 10 days followed by cell sorting. At the end of the 10-day differentiation, a considerable number of mBaso can be obtained and cell sorting procedures further improved the isolation of an extraordinarily pure (>98%) and vital Fc{varepsilon}R1+ CD11c- c-kit- mBaso population. Phenotypic analysis revealed that terminally differentiated (day 10) unsorted mBaso cultured for 24 hours showed a decrease in basophilic lineage (c-kit-) and an increase of mastocytic lineage (c-kit+) and reduced the expression of basophil markers Fc{varepsilon}RI, CD49b and CD200R either in absence of stimuli or following activation with the alarmin IL-33, indicating cell dedifferentiation. In contrast, terminally differentiated and Fc{varepsilon}R1+ CD11c- c-kit- sorted mBaso do not dedifferentiate in mast cells when placed in culture, and responded to IL-33 stimulation by up-regulating the activation marker CD63 without down-modulation of Fc{varepsilon}RI and CD200R3. These evidences highlight that in vitro differentiation followed by cell sorting is a useful method to obtain elevated numbers of highly pure mBaso that preserve their lineage markers and thus are suitable for conducting the desired functional studies.

cell biology↗

bioRxiv2019

Activation of the liver X receptors alters CD4+ T cell membrane lipids and signalling through direct regulation of glycosphingolipid synthesis

The liver X receptor (LXR) is a key transcriptional regulator of cholesterol, fatty acid, and phospholipid metabolism. Dynamic remodeling of immunometabolic pathways, including lipid metabolism, is a crucial step in T cell activation. Here we explored the role of LXR-regulated metabolic processes in primary human CD4+ T cells, and their role in controlling plasma membrane lipids (glycosphingolipids and cholesterol) which strongly influence T cell immune signaling and function. Crucially, we identified the glycosphingolipid biosynthesis enzyme glucosylceramide synthase (UGCG) as a direct transcriptional LXR target. LXR activation by agonist GW3965 or endogenous oxysterol ligands significantly altered the glycosphingolipid:cholesterol balance in the plasma membrane by increasing glycosphingolipid levels and reducing cholesterol. Consequently, LXR activation lowered plasma membrane lipid order (stability), and an LXR antagonist could block this effect. LXR stimulation also reduced lipid order at the immune synapse and accelerated activation of proximal T cell signaling molecules. Ultimately, LXR activation dampened pro-inflammatory T cell function. Finally, compared to responder T cells, regulatory T cells had a distinct pattern of LXR-target gene expression corresponding to reduced lipid order. This suggests LXR-driven lipid metabolism could contribute to functional specialization of these T cell subsets. Overall, we report a novel mode of action for LXR in T cells involving the regulation of glycosphingolipid and cholesterol metabolism, and demonstrate its relevance in modulating T cell function.

immunology↗