Journal: Cell reports
Article Title: Essential role of TOSO/FAIM3 in intestinal IgM reverse transcytosis.
doi: 10.1016/j.celrep.2021.110006
Figure Lengend Snippet: Figure 2. TOSO receptor is involved in IgM reverse transcytosis (A) Mono- (Caco-2 cells alone) and co-cultures (Caco-2 and Raji cells) were pre-incubated apically with 10 mg blocking Abs directed against potential receptors prior to addition of various IgM for 90 min at 37C. The concentration of transported IgM was evaluated by ELISA (n = 4). Vertical bars show the mean value ± SEM. (B and C) One-way ANOVA followed by Bonferroni post hoc test was used. Detection of TOSO on M-like cells (GP2+ cells) in vitro assessed by immunofluo- rescence (top view) (B) and flow cytometry (C). Scale bars represent 100 mm, except for (B), top and bottom left, where they represent 10 mm. (D) TOSO (Alexa Fluor 488) expression increases under co-culture conditions and is bound by IgM-APC(allophycocyanin). Here, two commercial IgMs with different paratopes (Invitrogen) were used. Data are representative of three distinct experiments. (E and F) Tissue section showing a PP obtained from an intestine in mice (E) and patient biopsy samples taken from terminal ileum (F). Tissues were im- munolabeled with PE-UEA-1 and FITC-TOSO (E) and human PE-GP2 and FITC-TOSO (F) at room temperature (RT) for 2 h. Colocalization between GP2 or UEA-1 with TOSO was confirmed (n = 2). In all images, dotted lines delineate the FAE separating the intestinal lumen and the lymphoid tissue (side view). Scale bars, 100 mm. (G) After siRNA knockdown, IgM transport was quantified in the inverted in vitro model of FAE, and transported IgM was evaluated by ELISA. Vertical bars show the mean value ± SEM; n = 3 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used (***p < 0.005). (H) Immunofluorescence staining of murine PPs after intestinal loop experiments with and without anti-TOSO antibody injection into the loops. The white line marks the edges of the PP. Images are representative of observations made from three distinct experiments. Scale bar, 100 mm. (I) Quantification of Cy3 signal in PPs using ImageJ (Fiji). Four fields were acquired per section, and three to four sections per loop were imaged for each mouse. Data are shown as means ± SEM. *p < 0.05 (two-way ANOVA with Tukey correction). (J) Quantification of double-positive Cy3-AF488 signal in PPs. Four fields were acquired per section, and three to four sections per loop were imaged for each mouse. Data are shown as means ± SEM. *p < 0.05 (two-way ANOVA with Tukey correction).
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human Dectin-1/CLEC 7A polyclonal Ab (pAb) (goat IgG) R&D Systems Cat# MAB1859; RRID:AB_2081791 anti-human pIgR Ab (goat IgG) R&D Systems Cat# AF2717; RRID:AB_2164821 anti-human CD170 mAb (SIGLEC-5) (mouse IgG1) R&D Systems Cat# MAB1072; RRID:AB_2239301 Anti-human CD71 mAb (mouse IgG1) Clinisciences Cat# 11-235-C100; RRID:AB_10755321 Anti-FAIM3 Ab (rabbit IgG) LifeSpan BioSciences Cat# LS-C375684-50 anti-human FAIM3 (TOSO) Biolegend Cat# 398102; RRID:AB_2814380 Anti-CD351 antibody Abcam Cat# ab170491 anti-human GP2 mAb MBLbio Cat# D277-3; RRID:AB_10598500 anti-human GP-2-PE MBLbio Cat# D277-5; RRID:AB_11160953 mouse anti-human CA19.9 Thermo Scientific Cat# MA5-12421; RRID:AB_10980397 anti-DC-SIGN BV421 BD Biosciences Cat# 564127; RRID:AB_2738610 anti-CD1a APC-Vio770 Miltenyi Biotec Cat# 130-100-224; RRID:AB_2656026 anti-CD14 PE Miltenyi Biotec Cat# 130-113-149; RRID:AB_2725977 anti-CD11b PE-Vio770 Miltenyi Biotec Cat# 130-113-803; RRID:AB_2819369 anti-HLA-DR BV510 BD Biosciences Cat# 740173; RRID:AB_2739926 anti-CD80-PE-Vio770 Miltenyi Biotec Cat# 130-102-885; RRID:AB_2659270 anti-CD86-FITC Miltenyi Biotec Cat# 130-113-571; RRID:AB_2751132 anti-CD40- PE Miltenyi Biotec Cat# 130-105-376; RRID:AB_2660764 anti-IgM-APC Southern Biotech Cat# 9020-11; RRID:AB_2687522 Experimental models: Mouse 8-week-old female BALB/c mice Charles River Strain Code: 028 Experimental models: Cell lines Caco-2 cells obtained from Dr. Maria Rescigno Rescigno et al., 2001 human Burkitt’s lymphoma cell line Raji B American Type Culture Collection Raji.
Techniques: Incubation, Blocking Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Cytometry, Expressing, Co-Culture Assay, Knockdown, Staining, Injection
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