Mouse strains and treatments
Female mice from autoimmune (NZB/W F1 and BcN/LmoJ) and non-autoimmune (C57BL/6) strains were bred at the animal facility of the Deutsches Rheuma-Forschungszentrum (DRFZ) and C57BL/6 mice with experimental autoimmune myasthenia gravis (EAMG) were kept at the animal facility of the Charité - Universitätsmedizin Berlin under defined pathogenic-free conditions and treatments were approved (approval number G130/08, G113/10 and G0172/13) by the Landesamt für Gesundheit und Soziales (LaGeSo) Berlin, Germany. All animal experiments were performed by certified personnel and all efforts were made to minimize suffering. For dsDNA analysis, mice with positive dsDNA antibody titer (between 5 and 7 months old) and age-matched controls were chosen. Experimental autoimmune myasthenia gravis was induced by three intraperitoneal injections of torpedo acetylcholine receptor (AChR) in Freund’s complete adjuvant into 3-month-old C57BL/6 mice at 4-week intervals. Mice were sacrificed for analysis 17 days after the last injection at an age of 6 months and after conformation of active EAMG by positive anti-AChR antibody titer and disease score . For distinction of autoreactive LLPCs and SLPCs, 6-month-old BcN/LmoJ mice (another mouse model with genetic predisposition for SLE, which was backcrossed to the C57BL/6 strain and thus is also known as the B6.Sle123 strain ) with positive dsDNA antibody titer were fed 5-ethynyl-2′-deoxyuridine (EdU) (0.3 mg/ml) for 2 weeks.
Labeling of dsDNA
Five hundred micrograms of dsDNA (activated dsDNA from calf thymus (Sigma-Aldrich Chemie GmbH, Munich, Germany)) in 500 μl phosphate-buffered saline (PBS) was reduced with 10 μl 0.5 mol/l dithiothreitol for 30 min at room temperature. Subsequently, the labeling solution was rebuffered with Amicon Ultra (10 kDa) in borate buffer (pH9.5) with 10 mg/ml DMSO containing 20 μl digoxigenine (dig-NHS, Sigma-Aldrich) and incubated for 1 h at room temperature in the dark. After labeling, reaction-free dig was removed in a PD10 desalting column equilibrated with PBS/0.05 % azide and fractions containing high concentrations of labeled dsDNA were collected and pooled.
Sample preparation and immunofluorescence staining for histology
Spleen and bone marrow samples were embedded in O.C.T. compound medium (Sakura Alphen aan den Rijn, The Netherlands) in cryomold vessels, snap frozen in liquid nitrogen for 10 min and stored at −80 °C. Kryosections of 8 μm thickness were prepared and subsequently fixated with acetone at −20 °C for 10 min, air dried and stored at −80 °C.
For blocking, sections were rehydrated with RPMI 1640 medium (Gibco, Waltham, MA, USA) containing 10 % fetal calf serum (FCS) and anti-FcyRII/III antibody (clone 2.4G2, DRFZ). Samples for antigen-blocking control additionally contained unlabeled dsDNA (7 μl of 1 mg/ml to 70 μl). After 1 h, dsDNA-dig was centrifuged to remove DNA aggregates and immediately added to the sample. Sections were incubated overnight at 4 °C. The following day, samples were washed and incubated with anti-Ig light chain kappa (IgL)-rPE (clone 187.1, DRFZ) for plasma cell identification . The secondary staining solution containing anti-dig-Cy5 (prepared and labeled at DRFZ) was applied for 30 min. Stained samples were mounted with fluoromount (Dako, Glostrup, Denmark) and analyzed by confocal laser scanning microscopy (Zeiss LSM 710, Zeiss, Oberkochen, Germany).
For parallel staining of pathogenic and protective PCs, samples were incubated with Ova-FITC (Sigma-Aldrich, labeled at DRFZ) together with secondary staining solution.
For identification of Ig-class of dsDNA PCs, sections were incubated with anti-IgA-bio (goat, SouthernBiotech, Birmingham, AL, USA), anti-IgG-FITC (rabbit, SouthernBiotech) and anti-IgM-Pacific blue (clone M41, DRFZ) instead of anti-IgL and subsequently incubated with streptavidin-rPE (BD Biosciences, Franklin Lakes, NJ, USA).
For distinction of proliferating SLPCs and non-proliferating LLPCs, incorporated EdU was detected with the click-it® EdU kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction.
Ninety-six-well microtiter plates (Merck Millipore, Billerica, MA, USA) were precoated with methyl-bovine serum albumin (BSA) (Sigma-Aldrich) and subsequently coated with calf thymus DNA (Sigma-Aldrich) as previously described . Single-cell suspensions were prepared in RPMI 1640 medium supplemented with 10 % FCS (Invitrogen, Waltham, MA, USA), penicillin, streptomycin, and glutamine (complete medium). The cells were then pipetted onto the plates and incubated at 37 °C for 3 h in a 5 % CO2-containing incubator. Afterward, cells were washed away and plates were incubated with biotin-labeled goat anti-mouse IgM or IgG (SouthernBiotech) followed by ExtrAvidin-Alkaline Phosphatase (Sigma-Aldrich). The spots were developed with NBT/BCIP (Thermo Fisher Scientific, Waltham, MA, USA) and enumerated by an automatic ELISpot Reader (AID Autoimmun Diagnostika, Strassberg, Germany) using the ELISpot Reader software (AID Autoimmun Diagnostika).
Ninety-six-well microtiter plates (Merck Millipore) were coated with goat anti-mouse IgG and IgM (SouthernBiotech) respectively or together. For fluorescence acquisition of dsDNA-specific PCs the probes were incubated with dsDNA-dig followed by anti-dig-A594 and for total PCs with anti-IgG and IgM-bio (SouthernBiotech) followed by streptavidin-FITC (BD Biosciences). ELISpot analysis were performed blinded and independently to histologic analysis.
Blood was drawn from the left heart ventricle directly after cervical dislocation of the mouse. Serum was prepared and analyzed as previously described . Briefly, 96-well microtiter plates were precoated with methyl-BSA (Sigma-Aldrich; 1 mg/mL) at 37 °C for 3 h and subsequently coated with calf thymus DNA (Sigma-Aldrich; 1 mg/mL) at 4 °C overnight. After blocking with PBS/3 % BSA, serum was added and incubated overnight at 4 °C. The next day, the ELISA was developed by addition of biotin-labeled goat anti-mouse IgM or IgG (SouthernBiotech), ExtrAvidin-peroxidase (Sigma-Aldrich) and TMB substrate (Thermo Fisher Scientific).
Data analysis and statics
Histology data were analyzed with Zen2012 (Zeiss), ELISpot data with ELISpot6 (AID Autoimmun Diagnostika), and graphs were prepared with Prism 5 (Graphpad Software Inc., San Diego, CA, USA). Results are expressed as means ± standard deviation (SD) and groups were compared by unpaired Student’s t test. Correlation between matched data points from histologic and ELISpot analysis were calculated via Pearson test. P values <0.05 were considered significant differences or correlation respectively and are indicated by one star (*), values <0.01 with two stars (**) and values <0.001 with three stars (***).