*4.7. Flow Cytometry Analysis*

Cells from blood, spleen, and lymph nodes were incubated with 1 μL per sample of mouse anti-Rat CD32 (clone D34-485, 20 μg/mL, BD biosciences, San Jose, CA, USA) diluted in staining buffer (0.5% bovine serum albumin (BSA) and 0.01% sodium azide in PBS), to prevent unspecific bindings. Then, cells were immunostained for 30 min on ice for the presence of T cells, B cells, NK cells, and myeloid cells by the following surface antibodies: anti-TCR-PerCP (clone R73, 4 μg/mL), anti-CD4-APC (clone OX-35, 4 μg/mL), anti-CD8a-V450 (clone OX-8, 8 μg/mL), anti-CD45R-PE (clone His24, 8 μg/mL), anti-CD161a-FITC (clone10/78, 2 μg/mL), anti-CD11b/c-PE-Cy7 (clone OX-42, 1.6 μg/mL), anti-CD40-FITC (clone HM40-3, 40 μg/mL), anti-CD86-PE (clone 24F, 16 μg/mL) all from BD biosciences. Cells stained with the corresponding isotype antibodies were used as control. Cells were washed 4 times in PBS and fixed with paraformaldehyde 1%. Samples were analyzed on a FACSCanto flow cytometer (Becton Dickinson), with acquisition of 10.000 events in gate for each sample, and data analyzed with FlowJo software.

### *4.8. Plasma Cytokine Quantification*

Plasma cytokines were quantified at Eve Technologies Corp. (Calgary, AB, Canada) using the Rat Cytokine Array/Chemokine Array 27-Plex Discovery Assay. Plasma samples, 30 μL, were diluted twice in PBS prior analysis, according to the manufacturer's indications. The sensitivity of the assay for the markers analyzed ranged from 0.1 to 15.7 pg/mL.

#### *4.9. Isolation of MSC, Primary Culture and Phenotypic Characterization*

Rat MSC were isolated form CIA and control femurs, as previously described [41]. Briefly, bone marrow was flushed with PBS and bone debris were removed by filtering the cell suspension through a 100 μm cell strainer. After red blood cells lysis, cells were counted and seeded in minimum

essential medium alpha modification (α-MEM) supplemented with 10% MSC-qualified fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA MA, USA) and 1% penicillin G-streptomycin (P/S; Invitrogen, Carlsbad, CA, USA), at 106/100 mm plate. MSC were selected by adherence to plastic surface, expanded and cryopreserved for forward use. Isolated and cultured MSC identity was confirmed by multicolor flow cytometry based on classical cell surface markers CD29<sup>+</sup> (anti-CD29-APC, HMβ1-1, BioLegend, San Diego, CA, USA), CD90<sup>+</sup> (anti-CD90-PE, MRC OX-7, Immunotools, Friesoythe, Germany) and CD45− (anti-CD45-FITC, MRC OX-30, Immunotools, Friesoythe, Germany) staining, and by ability of cells to differentiate into the osteogenic, chondrogenic and adipogenic lineages. Cells from CIA and control animals were maintained in a humidified incubator, at 37 ◦C and 5% CO2, and used for experiments between passages 4 and 8.
