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

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

San Luis

Congreso; LXXI Reunión de la Sociedad Argentina de Inmunología; 2023

Sociedad Argentina de Inmunología

Leukocyte-specificprotein 1 (LSP1) is a 52kDa cytoplasmic F-actin binding phosphoproteinexpressed within human, murine leukocytes, and endothelial cells. It serves asa crucial regulator of actin cytoskeleton remodeling and potentially plays arole in antigen processing within endomembrane compartments of dendritic cells(DCs). Previous findings from our research highlighted that Lsp1-/- DCsexhibit impaired antigen presentation to CD4+ T cells in comparison to DCs fromwild 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 totheir WT counterparts. As a result, we investigated whether this impairedantigen presentation in Lsp1-/-DCs stems from alterations in antigen uptake or processing. To address this, wederived DCs in vitro from bone marrow precursors with Flt3-L. For assessinguptake of particulate antigens, DCs were co-cultured for 1 hour withYellow-Green fluorescent microspheres. Subsequently, cells underwent two PBSwashes, followed by staining with distinct antibodies and subsequent flowcytometry 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 wereanalyzed by flow cytometry at 1, 2, 3, and 4-hour intervals. Despite thepreviously noted reduced antigen uptake in Lsp1-/-DCs compared to their Lsp1+/+counterparts, these cells surprisingly exhibited consistent degradationkinetics.Contrastingly,in exploring degradation of particulate antigens, DCs were co-cultured withOVA-bound microspheres for an hour. Following two PBS washes, cells werecultured for 1, 2, 3, and 4-hour periods. After these intervals, DCs werefixed, permeabilized, and subjected to anti-OVA staining to gauge intactprotein or incomplete proteolysis. Strikingly, at the 1-hour mark, Lsp1-/- DCs demonstrateddiminished OVA degradation (p<0.05) compared to their Lsp1+/+ counterparts, though both groups reached similarvalues at the 2-hour mark. These trends persisted at 3 and 4-hour intervals.Thesefindings suggest that the impaired antigen presentation in Lsp1-/- DCs may, in part, result from impaired uptake ofsoluble 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 intracellularcompartments participating in the uptake and degradation of soluble versusparticulate antigens.@font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-469750017 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin-top:0cm;margin-right:0cm;margin-bottom:8.0pt;margin-left:0cm;line-height:107%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-fareast-language:EN-US;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:11.0pt;mso-ansi-font-size:11.0pt;mso-bidi-font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-font-kerning:0pt;mso-ligatures:none;mso-fareast-language:EN-US;}.MsoPapDefault{mso-style-type:export-only;margin-bottom:8.0pt;line-height:107%;}div.WordSection1{page:WordSection1;}