Igg from rat serum

Neutrophils were depleted by intraperitoneal injection of 100 μg of anti-mouse Ly6G (clone 1A8, BioXcell, West Lebanon, NH, USA) 24 hours before infection i.p. with 1 × 10⁶ CFU B. abortus virulent strain S2308. The neutrophils depletion was maintained with applications of anti-mouse Ly6G antibodies at intervals of 2 days each dose for 7 days. In these experiments, 100 μg of an isotype control antibody (IgG from rat serum, Sigma-Aldrich, St. Louis, MO, USA) was administered as control. After 1 week of infection, mice were sacrificed, spleens were harvested and CFU counting was performed as described above. Neutrophil depletion was confirmed by flow cytometry analysis of spleen cells from depleted and control mice. The neutrophil population was analyzed by staining 1x10⁶ cells for 30 min on 4°C with fluorescent antibodies against Ly6G (PE, clone 1A8, BD Biosciences). Stained cells were acquired in Attune Flow Cytometer (Applied Biosystems, Waltham, MA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

RNEU_420-429 (PDSLRDLSVF), the immunodominant peptide of HER2/neu for MHC class I, and NP_118-126 (RPQASGVYM), a peptide derived from a nuclear protein as a control, were obtained from Hokkaido System Science (Sapporo, Japan). An APC-anti-mouse CD8a monoclonal antibody (mAb), PE-anti-mouse IFN-γ mAb, and Fixable Viability Stain 520 were obtained from BD Biosciences (Franklin Lakes, NJ, USA). A therapeutic anti-PD-1 mAb (clone: RMP1-14) and anti-CTLA-4 mAb (clone: 9H10) were obtained from BioXcell (Lebanon, NH, USA). IgG from rat serum, administered as a therapeutic control, was obtained from Sigma-Aldrich (St. Louis, MO, USA). The antibodies (Abs) used for immunostaining were as follows: anti-CD8 mAb (ab209775, 1:2000, Abcam, Cambridge, United Kingdom), anti-CD4 mAb (ab183685, 1:1000, Abcam), anti-FOXP3 Ab (polyclonal, NB100-39002, 1:800, Novus Biologicals, Centennial, CO, USA), and Histofine Simple Stain Mouse MAX-PO (Nichirei Biosciences, Tokyo, Japan).

Five-week-old female C57BL/6 mice were purchased from Charles River Japan, Inc. (Yokohama, Japan). They were maintained in the animal facility of Tokushima University under specific pathogen-free conditions according to the guidelines of the ethics committee of our university [6 (link)]. The present study was approved by Institutional Animal Care and Use Committee of the Tokushima University (Permission Number: 08128). The anti-mouse PDGFR blocking antibodies APA5 and APB5 were kindly provided by Dr. Nishikawa (Kyoto University). MLE12 cells were purchased from the American Type Culture Collection (Manassas, VA). Bovine serum albumin, IgG from rat serum, and PDGF-AA and PDGF-BB were purchased from Sigma-Aldrich (St. Louis, MO). BLM was purchased from Nippon Kayaku Co. (Tokyo, Japan).