Journal: Stem Cell Research & Therapy

Article Title: Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity

doi: 10.1186/s13287-024-03810-4

Figure Lengend Snippet: Generation of HyPSCs. A No HLA-E expression was observed in HLA class Ia/II DKO cells. The expression of HLA-E in HLA class Ia/II DKO cells was analyzed by flow cytometry. B Overexpression of B2M increased HLA-G expression in HLA class Ia/II DKO cells. Flow cytometry was used to analyze the expression of B2M and HLA-G in HLA class Ia/II DKO cells transfected with HLA-G gene alone (left) or with HLA-G gene and B2M gene (right). C HyPSCs expressed HLA-G, B2M, PD-L1, and PD-L2 on their cell surface. The expression of transgene was analyzed by flow cytometry in WT hiPSCs and HyPSCs. Light gray histograms represent isotype IgG control and dark gray histograms indicate specific antibody staining. D HyPSCs expressed RapaCasp9. The expression of RapaCasp9 was analyzed by a Jess western blot system (ProteinSimple) using anti-FKBP12 antibody in WT hiPSCs (lane 1) and HyPSCs (lane 2). β-Actin was an internal loading control (lane 3 for WT hiPSCs and lane 4 for HyPSCs). The asterisk indicates a nonspecific band resulting from anti-FKBP12 antibody. MW molecular weight markers

Article Snippet: For the detection of the expression of B2M, HLA-G, PD-L1, and PD-L2 molecules, the cells were stained with APC-labeled anti-PD-L1 antibody (1:20; clone MIH1; Invitrogen, 17–5983-42), FITC-labeled anti-PD-L2 antibody (1:10; clone MIH18; Miltenyi Biotec, 130–098-528), PECy7-labeled anti-B2M antibody (1:20; clone 2M2; BioLegend, 316,318), and PE-labeled anti-HLAG antibody (1:100; clone MEM-G/9; Abcam, Cambridge, England, ab24384).

Techniques: Expressing, Flow Cytometry, Over Expression, Transfection, Control, Staining, Western Blot, Molecular Weight