2 β mercaptoethanol

10 mL of blood was collected into heparin tubes, red blood cells were lysed utilizing hypotonic salt solution, washed with endotoxin-free PBS (Thermo Fisher Scientific), filtered through a 40 μm cell strainer, counted for viable cells utilizing trypan-blue staining, resuspended in complete RPMI supplemented with 2 mM L-glutamine, 25 mM HEPES buffer, 1% penicillin-streptomycin solution, 50 mg/mL gentamicin sulfate, 1% nonessential amino acids, 2% essential amino acids, 1% sodium pyruvate (all reagents Thermo Fisher Scientific), 50 μM 2-beta-mercaptoethanol (Sigma Aldrich, Saint Louis, MO), and 10% (v/v) Bovine Viral Diarrhea Virus-negative-Fetal Bovine Serum (PAA Laboratories, Etobicoke, ON, Canada) (cRPMI). Peripheral blood mononuclear cells (PBMC) were plated at 10⁶ cells/well into 96-well, tissue culture-treated, round bottom plates.

In order to assess peripheral immune responses, blood samples were collected at zero, eight, twelve- and eighteen-weeks post-vaccination. Thirty mL of whole blood were collected via venipuncture of the jugular vein and placed into a conical tube containing 3 mL of 2x acid citrate dextrose (ACD) to prevent coagulation. PBMCs were then isolated as described previously (7 (link)). PBMC were counted utilizing the Muse^® Count and Viability Kit (Luminex) on the MUSE^® detection system (Luminex). Live cell numbers were used to adjust cell suspensions to a concentration of 1 × 10⁷ cells per mL of complete RPMI 1640 (cRPMI) (Gibco Life Tech, Thermo Fisher Scientific) media consisting of 20% heat-inactivated fetal bovine serum (FBS) (HyClone™ Cytiva, Marlborough, MA), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 nM glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 1% essential amino acids (Sigma Life Science, St. Louis, MO), 50 μM 2-beta mercaptoethanol (Sigma Aldrich), and 1% HEPES buffer (Gibco Life Tech, Thermo Fisher Scientific).

Arthritic hind paws were removed at the hairline and homogenised in Qiagen RLT buffer (Qiagen, Germantown, MD) with 1% 2-betamercaptoethanol (Sigma-Aldrich, St Louis, MO) and stored at −80°C. The Ambion Magmax prep protocol was used to extract total RNA. RNA quantity was assessed using the Nanodrop and RNA quality was assessed using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNAseq libraries were prepared using the Illumina TruSeq Sample Prep Kit (Illumina, Inc., San Diego, CA). 120 ng of total RNA was used for input. Barcode adapters were added to samples in such a way as to allow pooling of samples in flow cell lanes in a randomized pattern relative to sample annotation and source. Samples were sequenced using an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA) at a multiplexing level sufficient to generation 10-million to 25-million reads per sample. Following generation of sequence, reads were aligned to the mouse genome version NCBI m37 using TopHat (http://tophat.cbcb.umd.edu/). Quality control of sequencing data was accomplished by using the ShortRead package in R (http://www.r-project.org/). Aligned reads were mapped to Ensembl transcript models and converted to reads per kilobase of transcript per million mapped reads (RPKM) values using Cufflinks (http://cufflinks.cbcb.umd.edu/).