α il 35

CD4⁺ T cells were seeded at 2.5×10⁵ cells/well on 24-well plates (Starlab, Milton Keynes, UK) and cultured for 48 hours in the presence of 25% sera derived from patients or HCs (n=15 per group). The effect of IL-35 present in the sera (n=12) on driving an HLA-G-positive phenotype was tested through sera pretreatment with 0.5 µg/mL anti-IL-35 neutralising antibody (α-IL-35) (Bio-Techne, Abingdon, UK) prior to culture isolated CD4⁺ T cells for 45 min at room temperature. Similarly, controls were carried out in the presence or absence of anti-IL-10 neutralising antibody (α-IL-10, at 1 µg/mL) (Bio-Techne). The phenotype of the cells following sera conditioning was screened using flow cytometry.

Following 48-hour sera treatment, CD4⁺HLA-G⁺ generated in response to AD sera were collected and incubation with carboxyfluorescein succinimidyl ester-labelled PBMCs and α-CD3 stimulation (0.5 µg/mL) (eBioscience) in the presence or absence of either α-CTLA-4 (10 µg/mL) (eBioscience), α-HLA-G (10 µg/mL) (Miltenyi Biotec) or α-IL-35 (0.5 µg/mL) (Bio-Techne, Abingdon, UK) neutralising antibodies. Cells were co-cultured for 5 days to allow measurement of proliferation in CD3⁺ responder T cells. Supernatants were collected to assess cytokine secretion in the T helper 1 (Th1)/T helper 2 and Th17 pathways using multiplex cytokine detection system (Meso Scale Discovery System, Rockville, USA) (see online supplemental methods).