Alexafluor700 anti hla dr

Peripheral CD4⁺ T cells were enriched with the EasySep Human CD4⁺T cells kit (STEMCELL Technologies) from the blood of patients, heterozygous relatives, and control donors. Enriched CD4⁺ T cells were stained with the following antibodies: APC-Cy7-anti-CD4, PE-anti-CD25, PerCP-Cy5.5-anti-CD127, AlexaFluor700-anti-HLA-DR (all from BioLegend) and with AlexaFluor647-anti-Helios (BioLegend), Alexa Fluor 488-anti-FOXP3, and eV605-anti-CD3 (both from eBioscience) after fixation and permeabilization of T cells (gating strategy is shown in figs. S23 and S24). After staining enriched CD4⁺ T cells with APC-Cy7-anti-CD4, PE-anti-CD25, and Alexa Fluor 647-anti-CD127 (BioLegend), CD4⁺CD127⁺T_conv cells and CD4⁺CD25^hiCD127^−/lo T_reg cells from patients, heterozygous relatives, and HDs were sorted using a FACSAria flow cytometer (Becton Dickinson, Mountain View, CA). CD4⁺CD25⁺CD127⁺ Tconv cells were labeled with CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) (InVivogen). Cocultures of T_reg and T_conv at 1:1, 1:4, 1:8, and 1:16 ratios were stimulated with the T_reg Suppression Inspector Human kit (Miltenyi Biotec) at a 1 bead/1 cell ratio. Proliferation of the viable T_conv was analyzed by CFSE dilution at day 3.5.