Fig. 4: Multiplex CRISPR editing generates Tregs that retain canonical phenotype and function.
A Gene editing strategy using CRISPR/Cas9 to disrupt HLA class I and class II by targeting B2M and/or CIITA, respectively. HLA-E was inserted with a short linker sequence into B2M exon 2, generating an HLA-E-B2M fusion protein. For H/C Tregs the additional CIITA KO was performed by splice site disruption using the adenine base editor ABE-8.20-m. B Timeline of cell isolation, gene-editing, and surface protein expression measurement. C Representative contour plots of editing outcome by HLA-A,B,C (PE-Cy7), HLA-DR,DP,DQ (FITC), and HLA-E (APC) staining. D Summary of editing outcome for two biological replicates showing frequency of HLA class-I and class-II negative cells and E of HLA-E positive and HLA-A,B,C negative knock-in cells among live lymphocytes. F Treg identity was confirmed by staining of CD25 and intracellular staining of FoxP3, as well as G intracellular cytokine (TNFα, IFNγ, IL-2) expression after PMA/Ionomycin stimulation with conventional T cells as controls. H Timeline of suppression assay: CFSE-stained human CD3+ cells (2.5 × 104) with and without either autologous or the different allogeneic Treg conditions (at 1:1, 1:4, and 1:8 Treg:Tresp ratios) were stimulated with αCD3αCD28-coated beads at a 1:1 ratio of beads to total cells (Treg+Tresp). I CFSE dilution after 5 days incubation was measured by flow cytometry, and a division index was calculated. Data are presented as mean values ± SD. J Targeted DNA-methylation analysis of the 15 CpGs within the FOXP3-TSDR using bisulfite amplicon sequencing. Data were normalized by subtracting the methylation frequency of the WT Treg sample. Mean of two biological replicates is shown. Panel A Created in BioRender. Wagner, D. (2025) https://BioRender.com/ydoiabq. Source data are provided as a Source data file.
