Anti pe beads

Splenocytes from anemic mice were processed into single cell suspensions by grinding between sterile frosted glass slides in RBC lysis buffer and filtering through nylon mesh as we have reported elsewhere (19 (link)). As described above, CECs were purified by negative selection using biotin-conjugated antibodies and streptavidin linked magnetic beads (Miltenyi Biotec) according to our previous reports (19 (link)). Isolated CECs from anemic mice (1 × 10⁷) first stained with the CFSE-dye then injected intravenously into the tail vein of recipient mice 24 h prior infection. Control mice were administered through the tail vein with 1 × 10⁷ mature red blood cells. The single cell suspension of splenocytes was processed in the absence of RBC lysis buffer then mature RBCs and CECs were isolated by positive selection using biotin-conjugated anti-TER119 antibody (Thermo Fisher Scientific). In the next step, we used the anti-CD71 antibody on PE followed by anti-PE beads (Miltenyi Biotec) to exclude CECs.

BM cells were harvested from the femurs and tibias of LMC and M-Traf3^-/- mice (8-12-wk-old). B cells, T cells, and RBCs were depleted from BM cells using anti-B220, anti-CD19, anti-CD90.2, and anti-Ter119 magnetic beads (Miltenyi Biotec), and then Gr1+ BM cells were purified from the B220-CD19-CD90.2-Ter119- fraction using anti-Gr1-PE followed by anti-PE beads (Miltenyi Biotec). The purity of the resultant Gr1+ populations was verified to be greater than 92% of B220-CD19-CD3-Ter119-CD11b+Gr1+ as determined by FACS. Purified CD11b+Gr1+ cells were stimulated with 20 ng/ml GM-CSF at 37°C and total cellular protein lysates were prepared at different time points (0, 6 and 12 h) as previously described (29 (link)) for measurements of TRAF3 degradation by Western blot analysis.

Memory B cells were isolated from cryopreserved lymphocytes isolated from tonsils using phycoerythrin (PE)-Cy7-labeled CD19 microbeads (BD Biosciences), followed by staining with anti-PE beads (Miltenyi Biotec) and depletion of cells carrying IgM, IgD, and IgA by cell sorting on a FACSAria (BD Biosciences). Cells were immortalized under clonal conditions with Epstein-Barr virus as described previously (55 (link)). After 2 weeks, the culture supernatants were screened for the presence of ClfA001-specific MAbs using a 384-well-based ELISA. Positive cultures were expanded in complete RPMI medium and selected for their ability to bind to ClfA genotypes 001, 002, and 004 with high affinity. The VH and VL sequences were retrieved by reverse transcription-PCR (RT-PCR).

Splenocytes were collected from the C57BL/6J mice and red blood cells were lysed with ACK lysing buffer (Gibco, now Thermo Fisher Scientific, Waltham, MA, USA), followed by isolation using the naïve CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with some modifications. In brief, the splenocytes were stained with a biotinylated non-CD4⁺ T cell antibody cocktail provided in the naïve CD4⁺ T cell isolation kit together with phycoerythrin (PE)-conjugated anti-CD8 (clone 53-6.7), anti-CD19 (clone 6D5), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD44 (clone IM7), and anti-CD73 (clone TY/11.8) antibodies for 20 min at 4 °C. The stained splenocytes were washed with MACS buffer (0.5% bovine serum albumin (BSA), 2 mM ethylenediaminetetraacetic acid (EDTA) in Dulbecco’s phosphate-buffered saline (DPBS)) and incubated with anti-biotin beads (Miltenyi Biotec) and anti-PE beads (Miltenyi Biotec) for 20 min at 4 °C. Naïve CD73⁻CD4⁺ T cells were negatively isolated using an LS column (Miltenyi Biotec), and the isolated CD4⁺CD62L^hi cells (>95%) were used for in vitro T cell culture. The monoclonal antibodies were purchased from BD Biosciences (San Jose, CA, USA), eBioscience (now Thermo Fisher Scientific, Waltham, MA, USA) or Biolegend (San Diego, CA, USA).

HLA‐DRB1*01:01 restricted B. Anthracis Protective Antigen peptide 713–732 (KLPLYISNPNYKVNVYAVT) and HLA‐DRB1*04:01 restricted HIV‐1 Gag p24 peptide 164–183 (AFSPEVIPMFSALSEGATPQ) were custom synthesized to greater than 95% purity (GenScript). Peptide loaded biotinylated monomers were obtained from the National Institutes of Health Tetramer Core Facility. The peptide‐loaded monomers were subsequently cross‐linked with PE‐labeled streptavidin (Invitrogen) or BV421‐labeled streptavidin (BioLegend) for at least 4 h at 4°C to produce peptide tetramers. PBMCs (150‐250 × 10⁶) were thawed and rested at 37°C for 2 h before staining with Live/Dead Zombie viability dye. Tetramer staining was carried out for 1 h (HIV‐1) or 2 h (B. anthracis) at room temperature using 10 μg/mL of tetramer. Cell surface antibodies were added in the last 20 min of the tetramer stain. Tetramer tagged cells were magnetically labelled by adding anti‐PE beads (Miltenyi Biotec) and enriched by passing through a magnetic column (Miltenyi Biotec). Cells were acquired on a Fortessa X‐20 flow cytometer (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).