Fig 2: In vivo allogeneic transplantation of B2M−/− cyiPSC-derived cartilage. (A) Allogeneic B2M+/+ cyiPSC-derived cartilage (top row) and B2M−/− cyiPSC-derived cartilage (bottom row) were transplanted into osteochondral defects created in the joint surface of the distal femur in the knee joints of monkeys. The transplanted sites were analyzed histologically 4 weeks after the transplantation. Left, Safranin O-fast green-iron hematoxylin staining. Middle, HE staining. Right, magnification of the boxed regions in the middle panels. (B) Immunohistochemical analysis of the semiserial sections in (A) using anti-CD3 antibody (top two rows) and anti NKG2A antibody (bottom two rows). Magnification of the boxed regions in the third column is shown in the fourth column. Scale bars, 100 μm. HE, hematoxylin and eosin.

Fig 3: A correlation between LC3B mRNA level and sensitivity of Sal in melanoma. (a) The mRNA levels of LC3B were analyzed using real-time PCR assay, and normalized with the amount of B2M mRNA. (b) Linear regression analysis was performed to identify the potential correlation between LC3B expression and inhibition rate of Sal treatment (1 μM, 7 days) on clinical melanoma samples. (c) Model of the action of Sal in SK-Mel-19 cells. Sal which disrupted the balance of Na+ and K+ triggered ER stress and a decrease of mitochondrial ΔΨ, thus leading to UPR and damaged mitochondria. To keep its stability balance and normal function, cells produced autophagosomes to degrade those damaged organelles. However, Sal not only induced autophagy, but also inhibited the fusion between autophagosomes and lysosomes, causing an aberrant autophagic flux. Finally, accumulations of autophagosomes lead to autophagic cell death.

Fig 4: Transcriptional targets of IFN response, including MHC-I expression, are upregulated in CLAD basal cells compared with donor controls.(A) Volcano plot of gene expression changes in CLAD (n = 4) compared with control (n = 3) basal cells showing upregulation of MHC-I–expressing genes. (B) Hallmark pathway analysis of 855 preranked genes (Supplemental Data File 2) showing pathways significantly different (Padj < 0.05) in CLAD compared with control basal cells. (C) GSEA plot showing enrichment of IFN-α and IFN-γ response genes in CLAD basal cells. (D) Violin plots demonstrating upregulation of IFN response genes in CLAD compared with control basal cells. (E) Immunofluorescence staining of control (top) and CLAD (bottom) airways showing increased expression of MHC-I (red), as measured by antibody staining of B2M, in KRT5+ basal cells (green) from CLAD airway tissue compared with control. Yellow in merged panel shows colocalization of MHC-I expression present in CLAD but not donor controls. Scale bar: 30 μm. (F) Quantification of MHC-I expression in basal cells in every airway across 3 CLAD and 3 control samples. (G) Immunoblotting showing increased protein expression of B2M in epithelial-enriched protein lysates from CLAD compared with control airway tissue. Differential gene expression (A–D) was performed using the Wilcoxon rank sum test with Bonferroni correction to generate adjusted P values. Statistical analysis for protein quantification (F) was performed using the unpaired parametric t test. Data are shown as mean ± SEM. **P < 0.01.

Fig 5: Inactivation of B2M gene in cyiPSCs using the CRISPR/Cas9 system. (A) Schematic representation of the target sequence recognized by the guide RNA. The target sequence for the guide RNA is underlined. The arrow indicates the site of the double strand break. (B) Locations of the sequences corresponding to the primers used for the PCR amplification. (C) PCR amplification with primers 1 and 2 using genomic DNA extracted from B2M+/+ or B2M−/− cyiPSCs. The PCR products were separated on an agarose gel. The expected size of the PCR product from B2M+/+ genomic DNA is 1205 bp. (D) PCR amplification with primers 3 and 4 using genomic DNA extracted from B2M+/+ or B2M-/− iPSCs. The PCR products were digested with restriction enzymes. Top, Schematic representation of the expected PCR products with primers 3 and 4 from the wild-type allele and locations of the sites recognized by the restriction enzymes BstXI, MfeI, and HincII. Bottom, PCR products digested with the restriction enzymes were separated on an agarose gel during electrophoresis. RE (−), without digestion. Asterisks indicate fragments of different size between B2M+/+ and B2M−/− cyiPSCs. (E) The PCR product from B2M−/− genomic DNA was subjected to direct sequencing. B2M, β2 microglobulin; iPSCs, induced pluripotent stem cells; polymerase chain reaction (PCR).