CD4+ T-cell Proliferation Assay

We assessed CD4⁺ T-cell proliferation, as previously described (13 (link), 15 (link)). CFSE-labeled PBMCs were treated in culture medium for six days with sorted CD27⁺ MPs at ratios of 2000:1, 200:1, 20:1, 1:1 and 1:10 (PBMC: sorted CD27⁺ MPs). Lymphoproliferation was measured by flow cytometry analyses of CD4⁺ TLs with anti-CD4-APC-H7 and anti-CD3-PE (BD Biosciences) antibodies. Aqua Live/Dead viability dye (Thermo Fisher Scientific, MA, Waltham) was added to exclude dead cells. Lymphoproliferation was normalized between donors. For each HD tested, a proliferation index of 1 was assigned to the lymphoproliferation observed in the absence of MPs. Lymphoproliferation is expressed proportionally, as the fold-induction relative to lymphoproliferation in the absence of MPs.