Rat igg2a

Pancreas, Peyer’s patches, large intestine, gastrointestinal ducts (stomach, small intestine, and large intestine) including luminal contents, submandibular glands and sublingual glands were fixed in 4% paraformaldehyde (Wako) and embedded in paraffin or followed by 15% sucrose cryoprotection at 4 °C overnight and embedded in OCT compound (Sakura Finetek Japan). Large intestine without luminal content was fixed in 4% paraformaldehyde and embedded in paraffin. After embedding, 5-μm sections were cut and stained with the following antibodies: for GP2 detection, anti-mGP2 (MBL, 2F11-C3, #D278-3, 1:200) and Alexa 555-conjugated anti-rat IgG (BioLegend, Poly4054, #405420, 1:200). To confirm that GP2 signal was correct, serial sections were stained with isotype control (rat IgG2a, BioLegend, RTK2758, #400501, 1:100) and Alexa 555-conjugated anti-rat IgG in all samples; for Peyer’s patch FAE, fluorescein isothiocyanate (FITC)-conjugated UEA-1 (Vector Laboratories, # FL-1061, 1:100). DAPI (4′,6-diamidino-2-phenylindole; Dojindo, #D523, 1:1000) was used for counter staining. Observations were performed under a fluorescence microscope (BZ-9000; Keyence). Hematoxylin (Wako) and eosin (Wako) staining was performed by using standard procedures.

Cell migration assays were performed using Transwell chambers (pore size 5 μm, Costar, Corning 3421, Corning, NY, USA). THP-1 cells (2 × 10⁶ in 200 μl) were re-suspended in serum-free RPMI 1640 medium and added to the upper chamber. Supernatant from infected 786-O cells was mixed with 1 μg/ml neutralizing antibody against human GM-CSF (502203, BVD2-23B6, Biolegend) or control IgG2a (400515, Rat IgG2a, Biolegend) and incubated for 30 min. Then, 600 μl of medium containing the supernatant was added to the lower chamber as the chemoattractant. After 3 h or 6 h of incubation at 37 °C in a 5% CO2 humidified atmosphere, the migrated cells in the lower chamber were counted.

The mice were injected i.p. with 4 µg per mouse a monoclonal anti-mouse IL-1α antibody (Ab) (BioLegend, Germany) or isotype control Ab (rat IgG2a, BioLegend, Germany) in 100 µl of NS 24 h before TBI (total body irradiation) and 24 h after BMT.

Cells were stained for PE-conjugated anti-human PD-L2 (Cat#329606, Biolegend) or APC-conjugated anti-human PD-L1 antibody (Cat#329708, Biolegend) at 1:100 dilution on ice for 30 mins and analyzed by BD FACSCalibur. Isotype controls are mouse IgG2a, κ (Cat#400213, Biolegend) and mouse IgG2b, κ (Cat#400322, Biolegend), respectively. GL261 cells overexpressing PD-L1 and/or PD-L2 were stained for PE-conjugated anti-mouse PD-L2 (Cat#107205, Biolegend) and/or APC-conjugated anti-mouse PD-L1 antibody (Cat#124312, Biolegend) at 1:100 dilution on ice for 30 minutes and sorted using a BD FACSAria III cell sorter. Isotype controls were Rat IgG2a, κ (Cat#400508, Biolegend) and Rat IgG2b, κ (Cat#400612, Biolegend), respectively.

Each cell was collected and washed with flow cytometry buffer (PBS with 2% FBS and 0.1% NaN3). 2 × 10⁵ cells were stained with the primary antibody: an anti-canine CD20 antibody (4E1-7, 4E1-7-B, 4E1-7-C, or 1E-4-B), a PE-labeled anti-CD21 antibody (Bio-Rad Laboratories, Inc., Berkeley, CA), or an isotype control (rat IgG_2a, BioLegend, San Diego, CA, USA), followed by a secondary antibody: PE-labeled anti-rat IgG (SouthernBiotech, Birmingham, AL), Dylight 649-labeled anti-rat IgG (BioLegend), or Alexa 647-labeled anti-dog IgG (Jackson ImmunoResearch, West Grove, PA, USA).
To determine the subclass of 4E1-7, NRK/cCD20 cells were first stained with the anti-canine CD20 antibody (4E1-7), and then stained with either biotin-labeled anti-rat IgG-κ, anti-rat IgG-λ, anti-rat IgG1, anti-rat IgG_2a, or anti-rat IgG_2b antibody, followed by incubation with streptavidin-PE (Thermo Fisher Scientific Inc.). Results obtained using a BD Accuri C5 flow cytometer (BD Biosciences) were analyzed using FlowJo v.10 software (Tree Star Inc., Ashland, OR, USA).
Binding affinity of each antibody was determined by flow cytometry. Each cell was stained by the serially diluted antibody, as described above. Kd value was determined using the Nonlinear Regression Michaelis–Menten curve fit by JMP14.0 software (JMP Japan, Tokyo, Japan).