Anti human cd4 microbeads

Peripheral blood mononuclear cells (PBMC) were isolated from healthy controls and HLA-B27⁺ SpA patients (all fulfilling Assessment of SpondyloArthritis international Society classification criteria for axial SpA (27 (link))) by Ficoll (GE Healthcare) density gradient centrifugation. The characteristics of patients and controls (unmatched for age) are shown in Supplementary Table SII). Patients were considered as resistant to conventional treatment if they had a Bath Ankylosing Spondylitis disease activity score > 3/10 despite non-steroidal anti-inflammatory drug and biotherapy usage. CD14⁺ cells were first removed from PBMC by positive magnetic selection, using anti-human CD14 microbeads and AutoMacs Pro Separator (Miltenyi). Then, CD4⁺ T cells were magnetically sorted from the CD14^- fraction using anti-human CD4 microbeads and AutoMacs Pro Separator (Miltenyi) and cultured during 6 days with rhIL-2 (10 ng/mL), soluble anti-CD28 (2 µg/mL) and coated-anti-CD3 (10 µg/mL) in the presence or absence of rhIL-27 (40 ng/mL) in complete medium. At day 6, supernatants were harvested for measure of cytokine levels by ELISA and cells were stimulated with PMA (10ng/mL), ionomycin (1µg/mL) and BFA (2µg/mL) for 6 hours for intracellular cytokine staining.

CIK cells were obtained from ex vivo expansion of PBMC for 14 days. CD4^-CIK and CD4⁺ T cells were isolated using anti-human CD4 microbeads (Miltenyi Biotec, Germany) and co-cultured with A549, H520, or K562 cells for 4 h. Cytotoxicity assays were performed by measuring the optical density (OD) value from the release of lactate dehydrogenase (LDH) in the culture supernatant through the specific lysis of target cells.

Human PBMCs were obtained as above, and CD4⁺ T cells enriched using anti-human-CD4 microbeads (Miltenyi Biotec) before sorting naive CD4⁺ CD45RA⁺ CD25⁻T cells by flow cytometry using an Influx II cell sorter (BD Bioscience, San Diego, CA, USA). Naive T cells were co-cultured with allogenic moDC in StemXvivo (R&D Systems) serum-free medium in roundbottom plates containing interleukin-2 (10 ng ml⁻¹, Biolegend) and anti-CD3 antibody (clone OKT3, 0.2 µg ml⁻¹, Biolegend). In all, 5 × 10³ DCs were plated per 1 × 10⁵ T cells in the presence of either 100 µg ml⁻¹ anti-TGFβ (1D11), 20 µg ml⁻¹ anti-integrin β8 (clone 37e1B5,38 100 µg ml⁻¹ isotype control IgG (MOPC-21), or 5 ng ml⁻¹ active TGFβ (Peprotech).

A 2‐h adhesion step was used to separate monocytes from peripheral blood mononuclear cells (PBMCs) at 37°C. Adherent monocytes were treated with a combination of IL‐4 (PeproTech, AF‐200‐04‐20, 20 ng/mL) and granulocyte‐macrophage colony stimulating factor (GM‐CSF) (PeproTech, AF‐300‐03‐20, 20 ng/mL) after nonadherent cells were removed. The cultures were incubated in 1640 medium (HyClone) supplemented with 10% fetal bovine serum. Every 48 h, half‐volume adjustments were made, and fresh 20 ng/mL IL‐4 and 20 ng/mL GM‐CSF supplements were added; the cells were cultured for 7 days to obtain DCs. Afterward, the cell purity was determined by flow cytometry; additionally, the isotype control served as the adverse control.
PBMCs were collected using density gradient centrifugation, as previously described, and CD4⁺ T‐cells were subsequently purified using antihuman CD4 MicroBeads on a magnetic activated cell sorting (MACS) magnetic rack (Miltenyi Biotec). Purified CD4⁺ T‐cells were resuspended in 1640 medium (HyClone) containing 10% fetal bovine serum (Gibco) at a concentration of 1 × 10⁶/mL. Pure mouse antihuman CD3 antibody (BD Pharmingen, 553057, 10 μg/mL) and purified mouse antihuman CD28 antibody (BD Pharmingen, 553294, 5 μg/mL) were added to the mixture to activate CD4⁺ T‐cells.