Anti cd45ro fitc

CD45-RO + frequencies were assessed directly ex vivo by surface staining at 4 °C in PBS with antiCD45RO-FITC, CD3-PE and CD4-APC (all from BD Biosciences). For post-stimulation cultures, dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of regulatory markers: CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience/Thermofisher Foxp3 staining buffers according to the manufacturer's instructions. For analysis of cytokine expression, PMA/ionomycin-restimulated cells were fixed with 3% paraformaldehyde in PBS for 12 min followed by a 5-minute wash with PBS under centrifugation. Fixed cells were permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with IL-17-PE, IFNγ-e450, IL-21-APC, CD3-PERCP, CD4-FITC. For all studies cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star version 8.8.6). All antibodies were purchased from ebioscience/Thermofisher or BD Biosciences and expression quantified relative to the appropriate isotype control.

Peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). Isolated PBMCs were stained with anti-CD8-AF700 (Affymetrix, San Diego, CA, USA); anti-CD4-eF450 (Thermo Fisher Scientific, Breda, The Netherlands); and anti-CD45RO-FITC, anti-CD127-AF647, anti-CD25-PE, and anti-CCR7-PE-CY7 (BD Biosciences, Breda, The Netherlands) and sorted using FACS (MoFlo Astrios, Beckman Coulter, Woerden, The Netherlands). Memory Tregs (_MTregs) (CD4⁺CD8⁻CD45RO⁺CD127⁻CD25^high), naïve T (T_NAÏVE) cells (CD4⁺CD8⁻CD45RO⁻CCR7⁺), and effector memory T (T_EM) cells (CD4⁺CD8⁻CD45RO⁺CCR7⁻) were sorted (Figure 1A).
The purity of the sorted populations, was >95% for all samples. Samples were subsequently lysed using QIAzol lysis reagent (Qiagen, Venlo, The Netherlands) and stored at −80°C.

Cryopreserved PBMNCs were thawed, incubated with an Fc receptor blocking reagent (ChromPure Mouse IgG, Jackson ImmunoResearch Suffolk, UK) to inhibit non-specific binding before incubated with the following, pre-titrated antibodies (all from BD Biosciences, San Jose, CA): anti-CD3 V450, anti-CD45RO FITC, anti-PD-1 (CD279) PE, and anti-CD8 AF700. Flow cytometry was acquired by a LSRFortessa (BD Biosciences, San Diego, CA) and data analyzed using FlowJo version 10.2 (Tree Star Inc., Ashland, OR). Compensation was performed using BD CompBeads (BD Biosciences). Applying gating strategies based on unstained controls and for PD-1 the fluorescence minus one (FMO), the expression of PD-1 was measured on CD3⁺/CD45RO⁺/CD8⁺ singlets and given by the median fluorescense intensity (MFI) (Supplementary Figure 4).

Specific cell lines against T1BT* and T1BT*-Y peptide constructs were generated from naïve CD4 T cells from a HLA-DRβ1*04:01:01 donor using as APCs autologous monocyte derived dendritic cells (DCs) as described by Moser and colleagues [47] (link). CD4 T cells were purified using a naïve CD4 T cell isolation kit as manufacturer instructions (Miltenyi Biotec, Germany). DCs were pulsed with 3.75 mM of peptides T1BT* or T1BT*-Y. DCs were matured with TNF-α (25 ng/mL). Cells were co-cultured at 37°C, 5% CO₂ for 14 days and re-stimulated at 1∶10 ratio with mature dendritic cells pulsed with 3.75 mM of the same peptide used to prime. Seven days after the cells were stained with fluorescent DR4/T*-1; DR4/QNT-5 and DR4/QNT-Y tetramers assembled by stepwise addition of streptavidin-PE (Caltag, Life technologies – California, USA) to biotinylated HLA-DR4–peptide complexes at a final molar ratio of 1∶4 as described [41] (link). T cells were incubated with tetramers for 4 hours at 37°C followed by 20 min incubation with cell surface markers: anti-CD3 PE-Cy7, anti-CD4 PE-Texas red (Beckman Coulter, Palo Alto CA. USA), anti-CD45RO FITC, anti-CD62-L PECy5 (all from BD Biosciencies. San Diego CA. USA) on ice before washing with ice-cold buffer. Tetramer and Ab binding were determined using an FACS Aria II flow cytometer (BD Biosciences).

PBMC were washed with PBS and stained with anti-CD3-PE-Cy7 (Biolegend), anti-CD4-PerCP (BD Pharmingen), anti-CD8-PE (BD Pharmingen), anti CD28-APC (Biolegend), anti CD45RO-FITC (BD Pharmingen), anti-CD20-PerCP (Biolegend), anti-CD27-APC-Cy7(Biolegend) and anti-IgD-FITC (BD Pharmingen) antibodies for 20 min, 4 °C in the dark. After washing with PBS, cells were analyzed using a FACS Canto II cytometer and FACSDiva software (BD).