SUPPLEMENTARY FIG. S2. Expression of single HLA loci in shns- and shb 2 m-transduced MKs. Expression of HLA class I antigens (HLA-ABC) as well as

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1 Supplementary Data Supplementary Methods Flow cytometric analysis of HLA class I single locus expression Expression of single HLA loci (HLA-A and HLA-B) by shns- and shb 2 m-transduced megakaryocytes (MKs) was assessed with PE-conjugated anti-hla-a2 antibody (BioLegend, London, UK) or purified anti-hla-a1,36 and anti- B7,27,42 antibodies (One Lambda, Canoga Park, CA) in combination with PerCP/Cy5.5-labeled anti-igm (BioLegend). Supplementary Results Expression of single HLA loci in shns- and shb 2 m- transduced MKs To assess whether antigens of different HLA loci are equally influenced by b 2 m silencing, expression of HLA-A01, -A02, and -B07 loci was analyzed with specific antibodies and flow cytometric analysis. As seen for general HLA class I expression detected by anti-hla-abc (w6/32), single locus expression of HLA-A01, -A02, and -B07 was also significantly downregulated (Supplementary Fig. S2). These data indicate that antigens of different HLA loci are equally affected by b 2 m silencing. Furthermore, these results coincide with the results of our mouse model for platelet (PLT) refractoriness, which showed that human HLA-universal PLTs survive the presence of both anti-hla-a and anti-hla-b antibodies (see also Supplementary Table S2). Biodistribution analysis of genetically engineered human MKs or PLTs in mouse tissues To evaluate the safety of the infusion of HLA-universal MKs, we analyzed the biodistribution of human cells in tissues of mice on day 13 postinfusion. Antibodies detecting human (h)cd41 and hcd42a or hcd42a and hcd61 were used to detect human MKs and PLTs, respectively. To exclude false positive events due to autofluorescence or antibody background staining of mouse cells, gates were set on the basis of fluorescence characteristics of cells from negative control mice (Supplementary Figs. S3 and S4). The data show that, based on significantly higher mean fluorescence intensity (MFI) values resulting from specific hcd41/cd42a/cd61 staining, human MKs and PLTs could be clearly distinguished from mouse cells. On day 13 after MK infusion, no human cell population could be detected in peripheral blood (PB), lung, spleen, or BM of treated mice compared with mice without MK infusion (Supplementary Figs. S3 and S4). These data show that after 13 days there were no residual human cells present in the tissues of mice that received human MK infusions. SUPPLEMENTARY FIG. S1. Schematic setup of the in vivo experiments using a mouse model for platelet (PLT) refractoriness. NOD/SCID/IL-2Rcc / (NSG) mice were injected intravenously with specific anti-hla antibodies (ab) (to make them refractory to human PLTs) followed by infusion of shns- or shb 2 m-expressing megakaryocytes (MKs). Mice that received antibody only were used as negative control (NC) and mice that received shns- or shb 2 m-expressing MKs only were used as positive control. As additional control, shns- or shb 2 m-expressing MKs were infused together with nonspecific anti- HLA antibodies; these conditions are not included in the scheme.

2 SUPPLEMENTARY FIG. S2. Expression of single HLA loci in shns- and shb 2 m-transduced MKs. Expression of HLA class I antigens (HLA-ABC) as well as expression of HLA-A01, -A02, and -B07 loci was analyzed with specific antibodies and by flow cytometric analysis. Means and SD from three independent experiments are shown. Levels of significance are expressed as p values (*p < 0.05, **p < 0.01, ***p < 0.001).

3 SUPPLEMENTARY FIG. S3. Biodistribution analysis of human MKs in mouse PB, lung, spleen, and bone marrow (BM) on day 13 postinfusion. NOD/SCID/IL-2Rcc / (NSG) mice that received genetically engineered human MKs (expressing shns or shb 2 m, n = 8) were sacrificed on day 13 postinfusion. PB, lung, spleen, and BM were harvested and analyzed for the presence of human cells compared with PB harvested on day 1 postinfusion. Tissues from mice without human cell infusion were used as negative control (NC; n = 2). Human PLTs were identified on the basis of the expression of human (h)cd41 and hcd42a, using flow cytometry. Gates were set on the basis of the flow cytometric characteristics of cells from NC mice. Graphs show representative results from all experiments.

4 SUPPLEMENTARY FIG. S4. Biodistribution analysis of human PLTs in mouse PB, lung, spleen, and BM on day 13 postinfusion. NOD/SCID/IL-2Rcc / (NSG) mice that received genetically engineered human MKs (expressing shns or shb 2 m, n = 8) were sacrificed on day 13 postinfusion. PB, lung, spleen, and BM were harvested and analyzed for the presence of human cells compared with PB harvested on day 1 postinfusion. Tissues from mice without human cell infusion were used as negative control (NC; n = 2). Human PLTs were identified on the basis of the expression of human (h)cd42a and hcd61, using flow cytometry. Gates were set on the basis of the flow cytometric characteristics of cells from NC mice. Graphs show representative results from all experiments.

5 Supplementary Table S1. Schematic Presentation of the shrna Sequence Targeting b2m Transcripts (shb2m)

6 Supplementary Table S2. HLA Typing of Megakaryocytes and Platelets, and Anti-HLA Antibodies Used in In Vitro and In Vivo Experiments HLA type of MKs and PLTs Donor-specific anti-hla antibodies Nonspecific antibody control Experiments 1 HLA-A *03 *32 Anti-HLA-A3 Anti-HLA-A25,26,34 CDC assay HLA-B *15 *44 HLA-C *03 *05 2 HLA-A *25 *31 Anti-HLA-A25,26,34 Anti-HLA-A3 CDC assay HLA-B *18 *27 HLA-C *02 *12 3 HLA-A *02 *03 Anti-HLA-A2 Anti-HLA-A1,36 CDC assay and animal model for PLT refract. HLA-B *07 *44 Anti-HLA-B7,27,42 HLA-C *05 *07 4 HLA-A *02 *03 Anti-HLA-A2 n.d. Animal model for PLT refract. HLA-B *07 *18 HLA-C *07 *07 5 HLA-A *01 *03 Anti-HLA-A1,36 Anti-HLA-A2 Animal model for PLT refract. HLA-B *08 *40 Anti-HLA-A3 HLA-C *03 *07 6 HLA-A *01 *26 Anti-HLA-A1,36 Anti-HLA-A3 Animal model for PLT refract. HLA-B *08 *37 Anti-HLA-A25,26,34 HLA-C *06 *07 7 HLA-A *01 *03 Anti-HLA-ABC (w6/32) Anti-HLA-A2 ADCC assay HLA-B *08 *40 HLA-C *03 *07 8 HLA-A *02 *03 Anti-HLA-ABC (w6/32) Anti-HLA-A23,24 ADCC assay HLA-B *07 *44 Anti-HLA-A2 HLA-C *05 *07 9 HLA-A *02 *03 Anti-HLA-ABC (w6/32) Anti-HLA-A23,24 ADCC assay HLA-B *07 *18 Anti-HLA-A2 HLA-C *07 *07 ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; MKs, megakaryocytes; n.d., not done; PLTs, platelets; PLT refract., platelet refractoriness.