LSP1-/- DENDRITIC CELLS EXHIBIT IMPAIRED SOLUBLE ANTIGEN UPTAKE AND DELAYED PARTICULATE ANTIGEN DEGRADATION KINETICS

NICOLÁS DANIEL DHO; LUZ MARÍA PALACIOS; BELKYS MALETTO; MARÍA INÉS CRESPO; GABRIEL MORÓN

San Luis

Congreso; LXXI Reunión Científica Anual de la Sociedad Argentina de Inmunología SAI; 2023

Sociedad Argentina de Inmunología SAI

Leukocyte-specific protein 1 (LSP1) is a 52kDa cytoplasmic F-actin bindingphosphoprotein expressed within human, murine leukocytes, and endothelialcells. It serves as a crucial regulator of actin cytoskeleton remodeling andpotentially plays a role in antigen processing within endomembranecompartments of dendritic cells (DCs). Previous findings from our researchhighlighted that Lsp1-/- DCs exhibit impaired antigen presentation to CD4+ T cellsin comparison to DCs from wild type (WT) mice, for both soluble and particulateantigens. Specifically, in the case of soluble antigens, we observed diminisheduptake in Lsp1-/- DCs as opposed to their WT counterparts. As a result, weinvestigated whether this impaired antigen presentation in Lsp1-/- DCs stems fromalterations in antigen uptake or processing.To address this, we derived DCs in vitro from bone marrow precursors with Flt3-L. For assessing uptake of particulate antigens, DCs were co-cultured for 1 hourwith Yellow-Green fluorescent microspheres. Subsequently, cells underwent twoPBS washes, followed by staining with distinct antibodies and subsequent flowcytometry analysis. Intriguingly, Lsp1-/- DCs displayed statistically non-significantdifferences compared to Lsp1+/+ DCs in this regard. To evaluate degradation ofsoluble antigens, DCs were incubated with OVA-AF 647 (a fluorochromeunaffected by pH changes) and OVA-FITC for 30 minutes. Afterward, cells werewashed with PBS and were placed in RPMI media and were analyzed by flowcytometry at 1, 2, 3, and 4-hour intervals. Despite the previously noted reducedantigen uptake in Lsp1-/- DCs compared to their Lsp1+/+ counterparts, these cellssurprisingly exhibited consistent degradation kinetics. Contrastingly, in exploringdegradation of particulate antigens, DCs were co-cultured with OVA-boundmicrospheres for an hour. Following two PBS washes, cells were cultured for 1,2, 3, and 4-hour periods. After these intervals, DCs were fixed, permeabilized,and subjected to anti-OVA staining to gauge intact protein or incompleteproteolysis. Strikingly, at the 1-hour mark, Lsp1-/- DCs demonstrated diminishedOVA degradation (p