Texmacs serum free medium

Bead-isolated CD4⁺HLA-G⁺ T cells from patients with AD (AD-ACLF, n=3) were tested for their suppressive capacities in co-cultures with allogeneic PBMCs isolated from HCs. Prior to co-culture, allogeneic PBMCs were stained with 10 μM cell proliferation dye (CPD) eFluor 670 (eBioscience, Hatfield, UK) as per manufacturer’s protocol. Cells were cultured at different responder:HLA-G⁺ suppressor ratios (16:1, 8:1, 4:1 and 2:1) in TexMACS serum-free medium (Miltenyi Biotec) in the presence of anti-CD3 monoclonal antibody stimulation (α-CD3, 0.5 μg/mL) (eBioscience) for 5 days at 37°C in 5% CO₂. Proliferation was then measured on gated CD3⁺ T cells by dilution of the CPD-eFluor 670 dye using flow cytometry. Suppressive capacity was measured as percentage of suppression calculated as: [100−(% proliferation of responders:suppressors/% proliferation of responders only)×100].30 (link)

Data from a set of 32 patients were validated in parallel by independent handling, processing, staining, flow cytometry data collection, and analysis. The validation dataset was generated by a technician working in unrelated research themes (A.B.). A very different protocol was used to quantify CD4 T_HD cells in the validation set compared to the discovery cohort. For the validation dataset, isolated PBMCs were resuspended in TeXmacs serum‐free medium (Miltenyi) and plated on 6‐well cell culture plates. Myeloid cells were allowed to adhere overnight, and non‐adherent cells were collected, centrifuged, and resuspended and T cells stained with the appropriate antibodies for flow cytometry analyses. ROC analysis was used to establish the cut‐off value for the relative percentage of CD4 T_HD cells to discriminate G1 versus G2 patients in the validation cohort. Post hoc Cohen's kappa coefficient test was used to test the agreement between the discovery cohort versus the validation cohort on classification of G1/G2 patients.

In order to increase the frequency of PAP-specific T cells, cryopreserved PBMCs were thawed in CTL thaw solution (RPMI 1640 medium containing 10% v/v CTL thaw solution (SLS/Lonza, UK) and 0.02% v/v Benzonase™ (Millipore, Merck Life Science UK Limited, Gillingham, UK). PBMCs (2 × 10⁶ cells/mL in a 24-well plate) were cultured in TexMACS™ serum-free medium (Miltenyi Biotec, UK) supplemented with 5% v/v human pooled serum (Sigma-Aldrich, Millipore, Merck Life Science UK Limited, Gillingham, UK) and stimulated with 1 µg/mL of ILL 9 mer peptide or 10 µg/mL of MutPAP42mer peptide for 4 days at 37 °C, after which recombinant human IL-2 (rhIL-2, 10 IU/mL) and rhIL-15 (10 ng/mL, R&D Systems, Biotechne, Abingdon, UK) were added and PBMCs incubated for a further 6 days at 37 °C. PBMCs were washed and resuspended in TexMACS™ medium containing 10 IU /mL rhIL-2 and incubated for a further 2 days at 37 °C. PBMCs were washed in TexMACS™ medium prior to Dextramer™ staining and cytotoxicity assays.