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

Congreso; LXXI REUNIÓN SAI.; 2023

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<0.05) compared to their Lsp1+/+ counterparts, though both groups reached similar values at the 2-hour mark. These trends persisted at 3 and 4-hour intervals.These findings suggest that the impaired antigen presentation in Lsp1-/- DCs may, in part, result from impaired uptake of soluble antigens rather than alterations in degradation kinetics. Conversely, the capture of particulate antigens by Lsp1-/- DCs appears unaffected, with differences instead noted in degradation kinetics. This discrepancy could arise from distinctions in the intracellular compartments participating in the uptake and degradation of soluble versus particulate antigens.