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Purified anti-mouse CD20 antibodies (SA271G2, Ultra-LEAF™, BioLegend, Heidelberg, Germany) were injected intravenously, 0.25 mg/mouse at week 16 (point 0, p0) and then twice with 10 d intervals.

Low passage T2 cells (source DSMZ) were electroporated with crRNA-tracrRNA-Cas9 complexes targeting human CD80 and CD86 (see sequences above) using the CA148 program in SE buffer at 0.4 Mio cells / 20 μl in a 16-well strip cuvette. Alt-R crRNA was obtained from IDT, Cas9-NLS protein from Q3 Macrolab (Berkeley). Cells were then expanded for 7 days before sorting for CD80- CD86- live cells using an AriaIII sorter (Beckmann). Cells were then expanded for 7 days before resorting for highly pure CD80- CD86- cells. For the co-culture assays with T2 wt and T2 CD80 CD86 KO cells, T_eff cells (day 3 post first stimulation with T2 wt + NY-ESO-1 9 V peptide) with or without SNX9 were washed and reseeded at 1mio cells per ml in 10 U/ml IL2 in human serum medium as above. After 24 h of resting at 37 °C, the cells were co-incubated with T2 wt or T2 KO cells at an E:T ratio of 1:2 in presence of 100 nM NY-ESO-1 9 V peptide. Additionally, either 10 μg/ml human IgG1 (Ultra-LEAF, Biolegend 403502) or Ipilimumab (clinical grade, Yervoy, BMS) was added to the culture. After 14 h of incubation at 37 °C, the co-culture was stained for CD25 in addition to CD8 and Zombie NIR. Fixed stainings (IC fix, eBiosciences) were acquired on a Cytoflex (Beckmann Coulter).

FLS were seeded into 24-well plates (2x10⁴ cells/well) and stimulated with pSpA, RA or OA SF pool at dilution 1:10 in DMEM medium without fetal bovine serum for 72 h. In some experiments, the FLS were stimulated for 48h with pSpA pool in the absence or the presence of anti-IL-6 receptor (tocilizumab, 200 μg/ml), anti-TNF (infliximab, 200 μg/ml), TNF inhibitor (etanercept, 100 μg/ml), anti-IL-17 (sekukinumab, 100 μg/ml) or anti-human S100A8/S100A9 mouse IgG (Ultra-LEAF, Biolegend, San Diego, CA, USA, 5 μg/ml). To study the signaling pathways, FLS were pretreated with JNK (SP600125), ERK (PD98059), or p38 MAPK (SB203580) inhibitors (all from Calbiochem, San Diego, CA, USA,10 μM), or JAK1/3 inhibitor (tofacitinib, 1000 nM), or 0.1% DMSO (diluent control) for 1 h and additionally stimulated with pSpA pool, as previously described. Different concentrations of IL-6 (5-500 pg/ml, BD Biosciences, San Diego, CA, USA) or S100A8 (1-10 ng/ml, Biolegend, San Diego, CA, USA) were also assayed. In addition, experiments using FLS stimulated with SF treated with hyaluronidase (50 μg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) (SpA-H), or proteinase K (500 μg/ml, Promega, Madison, WI, USA) (SpA-P) were performed.