Lsp1-/- DENDRITIC CELLS EXHIBIT DELAYED ANTIGEN DEGRADATION DUE TO AN IMPAIRED UPTAKE

DHO, NICOLÁS DANIEL; PASCUAL, MARÍA MERCEDES; PISTORESI, MARÍA CRISTINA; MALETTO, BELKYS A; CRESPO, MARÍA INÉS; MORÓN, GABRIEL

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias SAIC-SAI&FAIC-SAFIS; 2022

Sociedad Argentina de Inmunología

Leukocyte-specific protein 1 (LSP1) is a 52kDa cytoplasmic F-actin binding phosphoprotein expressed in all human and murine leukocytes and endothelial cells. LSP1 is an important regulator of actin cytoskeleton remodelling. We have previously shown that Lsp1-/- dendritic cells (DCs) have a defective antigen presentation to CD4+ T cells compared to DCs from wild type (WT) mice and that MHC class II dynamics is similar between Lsp1+/+ and Lsp1-/- DCs. We thus studied whether this defective antigen presentation in Lsp1-/- DCs is due to either an alteration in the uptake or in the processing of Ag, using DCs in vitro derived from bone marrow precursors with Flt3-L. For antigen degradation, DCs were co-cultured with OVA-DQ (which fluoresces after being degraded by proteases) for 15min, 1, 2, 3 and 4h. After these times DCs were fixed, permeabilizated and stained with anti-CD107a, a marker of lysosomes, and analyzed by in cell imaging system (INCell Analyzer 2500HS). We found that Lsp1-/- DCs showed significant lower levels of OVA-DQ degradation (p<0.01) at 1h and higher levels at 2h (p<0.0001) to then been similar at 3h and 4h than Lsp1+/+ DCs. Regarding to CD107a, we observed Lsp1-/- DCs a significant lower amount at 15 min (p<0.0001) than WT DCs, which then equals at 1h. To assess antigen uptake, DCs were co-cultured with OVA-AF647 during 30, 60 or 90 minutes and then analyzed by flow cytometry. We observed that at every time point, Lsp1-/- DC population contained a lower proportion of cells with OVA-AF647 (p<0.001, p<0.0001 and p<0.0001 respectively) than Lsp1+/+ ones. After 30 minutes, Lsp1-/- DCs showed a lower mean fluorescence intensity (p<0.001) to then reach similar values than Lsp1+/+ DCs.These results suggest that altered antigen presentation in Lsp1-/- DCs could be, at least in part, a consequence of an impaired antigen capture responsible of a delayed degradation.