Lsp1-/- DENDRITIC CELLS EXHIBIT IMPARIED SOLUBLE ANTIGEN UPTAKE AND DELAYED PARTICULATE ANTIGEN DEGRADATION KINETICS.

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

San Luis

Congreso; LXXI REUNION ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA; 2023

Sociedad Argentina de Inmunología

Leukocyte-specific protein 1 (LSP1) is a 52kDa cytoplasmic F-actin binding phosphoprotein expressed within human, murine leukocytes, and endothelial cells. It serves as a crucial regulator of actin cytoskeleton remodeling and potentially plays a role in antigen processing within endomembrane compartments of dendritic cells (DCs). Previous findings from our research highlighted that Lsp1-/- DCs exhibit impaired antigen presentation to CD4+ T cells in comparison to DCs from wild type (WT) mice, for both soluble and particulate antigens. Specifically, in the case of soluble antigens, we observed diminished uptake in Lsp1-/- DCs as opposed to their WT counterparts. As a result, we investigated whether this impaired antigen presentation in Lsp1-/- DCs stems from alterations 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 hour with Yellow-Green fluorescent microspheres. Subsequently, cells underwent two PBS washes, followed by staining with distinct antibodies and subsequent flow cytometry analysis. Intriguingly, Lsp1-/- DCs displayed statistically non-significant differences compared to Lsp1+/+ DCs in this regard. To evaluate degradation of soluble antigens, DCs were incubated with OVA-AF 647 (a fluorochrome unaffected by pH changes) and OVA-FITC for 30 minutes. Afterward, cells were washed with PBS and were placed in RPMI media and were analyzed by flow cytometry at 1, 2, 3, and 4-hour intervals. Despite the previously noted reduced antigen uptake in Lsp1-/- DCs compared to their Lsp1+/+ counterparts, these cells surprisingly exhibited consistent degradation kinetics.Contrastingly, in exploring degradation of particulate antigens, DCs were co-cultured with OVA-bound microspheres 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 incomplete proteolysis. Strikingly, at the 1-hour mark, Lsp1-/- DCs demonstrated diminished OVA degradation (p