Home » C3 » Then, cells were incubated with mouse anti-Armenian hamster IgG2/3 FITC, followed by incubation with Dynabeads coupled with anti-rat IgG and anti-mouse IgG (Invitrogen)

Then, cells were incubated with mouse anti-Armenian hamster IgG2/3 FITC, followed by incubation with Dynabeads coupled with anti-rat IgG and anti-mouse IgG (Invitrogen)

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Then, cells were incubated with mouse anti-Armenian hamster IgG2/3 FITC, followed by incubation with Dynabeads coupled with anti-rat IgG and anti-mouse IgG (Invitrogen). not induce anti-dsDNA autoantibodies. GKA50 a Splenic DCs from aged [NZWxBALB/c]F1 control (control DCs) or aged BWF1 mice (autoimmune DCs) were sorted and injected i.v. (4??106/mouse) into groups of small control [NZWxBALB/c]F1 mice at days 0 and 20 (black arrows). Serum was obtained every eight or ten days after the first injection over the course of 75?days and tested for anti-dsDNA auto-antibodies by standard ELISA. White circles: sera from young [NZWxBALB/c]F1 mice treated with control DCs (mice decreases the growth and differentiation of T cells as well as plasmablast generation [11]. DC functions, distribution, phagocytosis, cytokine secretion, and migration have been found altered in lupus and other autoimmune diseases [12, 13], indicating that these cells participate in the maintenance of health. Several studies have underlined significant DC abnormalities both in humans [14] and in lupus-prone mice [15]. Jin et al. exhibited that plasmacytoid DCs (pDCs) from SLE patients lacked TLR9 expression, failed in the induction of PGC1A regulatory T cell differentiation, and produced high levels of IL-10 [14]. The same phenomenon was reported in [NZBNZW]F1 (BWF1) mice, where DCs present an altered phenotype and migratory behavior [15]. We sought to determine the nonredundant functions of pathogenic autoimmune DCs in BWF1 mice, a polygenic and spontaneous autoimmune disease setting. BWF1 mice develop lupus starting at the age of 6?months, characterized by high levels of proteinuria and elevated serum autoantibody GKA50 titers [16]. By adoptively transferring autoimmune DCs obtained from the spleens of aged autoimmune BWF1 mice into young healthy BWF1 mice, we exhibited that purified DCs from an autoimmune context were able to trigger humoral autoimmune responses. Moreover, autoimmune DCs from aged BWF1 mice induced the growth and differentiation of plasmablasts and CD5+ B cells in the peripheral blood of pre-autoimmune mice and participated in the induction of Th1 responses. These results reveal that autoimmune DCs from aged BWF1 mice exhibit functional characteristics that allow them to trigger B cell hyperactivation and promote an exacerbated humoral response in SLE. Materials and methods Mice and disease evaluation Female lupus-prone [NZBNZW]F1 (BWF1) mice were purchased from your Jackson Laboratory (Bar Harbor, ME, USA). All mice used in this study were housed in the animal facility of Fundacin Ciencia & Vida. Animal work was carried out under the institutional regulations of the Fundacin Ciencia & Vida and was approved locally by the ethical review committee of the Facultad GKA50 de Ciencias, Universidad de Chile. BWF1 female mice aged 2?months old represented small mice, while 8?-month-old mice with severe proteinuria (i.e., 500?mg/dl protein) and high antibody titers against GKA50 double-stranded DNA (dsDNA) represented aged autoimmune mice. Age-matched [NZWBALB/c]F1 female mice were used as controls. Proteinuria was measured on a monthly basis during the first 6?months of age by a standard semi-quantitative test using a Combur GKA50 Test N (Roche Diagnostics, Germany). After 6?months of age, proteinuria was measured every week to detect premature lupus. Autoantibodies against dsDNA were measured in serum samples by a standard ELISA using calf thymus DNA. Briefly, 650?ng/ml dsDNA was used to coat ELISA plates (Nalge Nunc International, USA) in an overnight incubation. Antigen-coated plates were subsequently blocked for 1?h with phosphate-buffered saline (PBS) containing 1.5% bovine serum albumin (BSA) and then incubated for 1?h at room temperature with sample sera (1:250 dilution). The plates were then washed with PBS-0.05% Tween 20 and incubated for 1?h with a peroxidase-labeled goat anti-mouse IgG antibody (Dako, USA). The color was developed by adding the TMB substrate kit (BD Bioscience, USA), and the absorbance at 450?nm (OD 450?nm) was measured using a plate reader (Jenway, UK). Antibodies Monoclonal antibodies (mAbs) against mouse CD86 FITC (GL1), CD138 PE (281-2), CD45R/B220 PE-Cy7 (RA3-6B2), CD4 PE (RM4-5), CD19 FITC or eFluor 780 (6D5), IL-10 PE (JES5-16E3), CD1d APC (1B1), CD69 (H1.2F3), IgM PE-Cy7 (RMM-1), purified CD16/32 (93), NK1.1 Alexa Fluor 488 (PK136), CD49b PE (DX5), CD11b APC (N1/70), and PDCA-1 APC (927) were purchased from BioLegend (San Diego, CA, USA). mAbs against mouse CD5 PE-Cy7 (53-7.3), CD11c PE (N418), IFN- FITC (XMG1.2), CD62L PE (MEL-14), CD25 APC (PC61.5), CD273 PE (PD-L2) (TY25), CD3 FITC (17A2), purified CD3 (145-2C11), and CD279 FITC (PD-1) (J43) were purchased from eBioscience (San Diego, CA, USA). mAbs against mouse IgD FITC (11-26c.2a), I-Ad FITC or.