Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells

Nature Biotechnology volume 35, pages 765–772 (2017)Cite this article

Subjects

Abstract

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: AAV-mediated knock in of HLA-E.
Figure 2: Inducible HLA-E expression without other class I molecules.
Figure 3: Protection of HLA-E-expressing cells from NK cell-mediated lysis.
Figure 4: Allogeneic CD8+ T cells do not recognize HLA-engineered cells.

Similar content being viewed by others

References

  1. Taylor, C.J., Peacock, S., Chaudhry, A.N., Bradley, J.A. & Bolton, E.M. Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types. Cell Stem Cell 11, 147–152 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Nishikawa, S., Goldstein, R.A. & Nierras, C.R. The promise of human induced pluripotent stem cells for research and therapy. Nat. Rev. Mol. Cell Biol. 9, 725–729 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Taylor, C.J. et al. Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 366, 2019–2025 (2005).

    Article  PubMed  Google Scholar 

  4. Braciale, T.J. Antigen processing for presentation by MHC class I molecules. Curr. Opin. Immunol. 4, 59–62 (1992).

    Article  CAS  PubMed  Google Scholar 

  5. Arce-Gomez, B., Jones, E.A., Barnstable, C.J., Solomon, E. & Bodmer, W.F. The genetic control of HLA-A and B antigens in somatic cell hybrids: requirement for beta2 microglobulin. Tissue Antigens 11, 96–112 (1978).

    Article  CAS  PubMed  Google Scholar 

  6. Riolobos, L. et al. HLA engineering of human pluripotent stem cells. Mol. Ther. 21, 1232–1241 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lu, P. et al. Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin. Stem Cell Rev. 9, 806–813 (2013).

    Article  CAS  Google Scholar 

  8. Wang, D., Quan, Y., Yan, Q., Morales, J.E. & Wetsel, R.A. Targeted disruption of the β2-microglobulin gene minimizes the immunogenicity of human embryonic stem cells. Stem Cells Transl. Med. 4, 1234–1245 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Feng, Q. et al. Scalable generation of universal platelets from human induced pluripotent stem cells. Stem Cell Reports 3, 817–831 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bix, M. et al. Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice. Nature 349, 329–331 (1991).

    Article  CAS  PubMed  Google Scholar 

  11. Liao, N.S., Bix, M., Zijlstra, M., Jaenisch, R. & Raulet, D. MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity. Science 253, 199–202 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. Zarcone, D., Tilden, A.B., Friedman, H.M. & Grossi, C.E. Human leukemia-derived cell lines and clones as models for mechanistic analysis of natural killer cell-mediated cytotoxicity. Cancer Res. 47, 2674–2682 (1987).

    CAS  PubMed  Google Scholar 

  13. Pazmany, L. et al. Protection from natural killer cell-mediated lysis by HLA-G expression on target cells. Science 274, 792–795 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Braud, V.M. et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391, 795–799 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Lee, N. et al. HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc. Natl. Acad. Sci. USA 95, 5199–5204 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lilienfeld, B.G., Crew, M.D., Forte, P., Baumann, B.C. & Seebach, J.D. Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation 14, 126–134 (2007).

    Article  PubMed  Google Scholar 

  17. Miller, J.D. et al. Analysis of HLA-E peptide-binding specificity and contact residues in bound peptide required for recognition by CD94/NKG2. J. Immunol. 171, 1369–1375 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Horowitz, A. et al. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry. Sci. Transl. Med. 5, 208ra145 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Russell, D.W. & Hirata, R.K. Human gene targeting by viral vectors. Nat. Genet. 18, 325–330 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Khan, I.F., Hirata, R.K. & Russell, D.W. AAV-mediated gene targeting methods for human cells. Nat. Protoc. 6, 482–501 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chamberlain, J.R. et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303, 1198–1201 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Li, L.B. et al. Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell 11, 615–619 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Khan, I.F. et al. Engineering of human pluripotent stem cells by AAV-mediated gene targeting. Mol. Ther. 18, 1192–1199 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ichiryu, N. & Fairchild, P.J. Immune privilege of stem cells. Methods Mol. Biol. 1029, 1–16 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Pegram, H.J., Andrews, D.M., Smyth, M.J., Darcy, P.K. & Kershaw, M.H. Activating and inhibitory receptors of natural killer cells. Immunol. Cell Biol. 89, 216–224 (2011).

    Article  PubMed  Google Scholar 

  26. Gong, J.H., Maki, G. & Klingemann, H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8, 652–658 (1994).

    CAS  PubMed  Google Scholar 

  27. Brooks, A.G., Posch, P.E., Scorzelli, C.J., Borrego, F. & Coligan, J.E. NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor. J. Exp. Med. 185, 795–800 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Brodin, P., Kärre, K. & Höglund, P. NK cell education: not an on-off switch but a tunable rheostat. Trends Immunol. 30, 143–149 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Huang, Y. et al. NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing. J. Immunol. 175, 3753–3761 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Rouas-Freiss, N., Gonçalves, R.M., Menier, C., Dausset, J. & Carosella, E.D. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc. Natl. Acad. Sci. USA 94, 11520–11525 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zappacosta, F., Borrego, F., Brooks, A.G., Parker, K.C. & Coligan, J.E. Peptides isolated from HLA-Cw*0304 confer different degrees of protection from natural killer cell-mediated lysis. Proc. Natl. Acad. Sci. USA 94, 6313–6318 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Koller, B.H., Marrack, P., Kappler, J.W. & Smithies, O. Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science 248, 1227–1230 (1990).

    Article  CAS  PubMed  Google Scholar 

  33. Zijlstra, M. et al. Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature 344, 742–746 (1990).

    Article  CAS  PubMed  Google Scholar 

  34. Mandal, P.K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. DeSandro, A., Nagarajan, U.M. & Boss, J.M. The bare lymphocyte syndrome: molecular clues to the transcriptional regulation of major histocompatibility complex class II genes. Am. J. Hum. Genet. 65, 279–286 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Street, S.E.A. et al. Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gammadelta T cells. J. Exp. Med. 199, 879–884 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gadola, S.D., Moins-Teisserenc, H.T., Trowsdale, J., Gross, W.L. & Cerundolo, V. TAP deficiency syndrome. Clin. Exp. Immunol. 121, 173–178 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pietra, G. et al. HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes. Proc. Natl. Acad. Sci. USA 100, 10896–10901 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hansen, S.G. et al. Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E. Science 351, 714–720 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Pietra, G., Romagnani, C., Manzini, C., Moretta, L. & Mingari, M.C. The emerging role of HLA-E-restricted CD8+ T lymphocytes in the adaptive immune response to pathogens and tumors. J. Biomed. Biotechnol. 2010, 907092 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Eichelberger, M., Allan, W., Zijlstra, M., Jaenisch, R. & Doherty, P.C. Clearance of influenza virus respiratory infection in mice lacking class I major histocompatibility complex-restricted CD8+ T cells. J. Exp. Med. 174, 875–880 (1991).

    Article  CAS  PubMed  Google Scholar 

  42. Hou, S., Doherty, P.C., Zijlstra, M., Jaenisch, R. & Katz, J.M. Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells. J. Immunol. 149, 1319–1325 (1992).

    CAS  PubMed  Google Scholar 

  43. Spriggs, M.K. et al. Beta 2-microglobulin-, CD8+ T-cell-deficient mice survive inoculation with high doses of vaccinia virus and exhibit altered IgG responses. Proc. Natl. Acad. Sci. USA 89, 6070–6074 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ware, C.B. et al. Derivation of naive human embryonic stem cells. Proc. Natl. Acad. Sci. USA 111, 4484–4489 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  PubMed  Google Scholar 

  46. Kotini, A.G. et al. Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells. Nat. Biotechnol. 33, 646–655 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Leach, L.L., Buchholz, D.E., Nadar, V.P., Lowenstein, S.E. & Clegg, D.O. Canonical/β-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 56, 1002–1013 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Sambrook, J. & Russell, D.W. Molecular Cloning: a Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, 2001).

  49. Papapetrou, E.P. & Sadelain, M. Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector. Nat. Protoc. 6, 1251–1273 (2011).

    Article  CAS  PubMed  Google Scholar 

  50. Lauterbach, N., Voorter, C.E.M. & Tilanus, M.G.J. Molecular typing of HLA-E. Methods Mol. Biol. 882, 143–158 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Aicheler, R.J. & Stanton, R.J. Functional NK cell cytotoxicity assays against virus infected cells. Methods Mol. Biol. 1064, 275–287 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Terasaki, P.I. & McClelland, J.D. Mirodroplet assay of human serum cytotoxins. Nature 204, 998–1000 (1964).

    Article  CAS  PubMed  Google Scholar 

  53. Vernon, R.B. et al. Reversal of diabetes in mice with a bioengineered islet implant incorporating a type I collagen hydrogel and sustained release of vascular endothelial growth factor. Cell Transplant. 21, 2099–2110 (2012).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants DK55759 and HL007093 to D.W.R. 1S10OD010652-01 to the UW Shared IVIS Core Facility, and HL007093 to A.G.C. as well as California Institute for Regenerative Medicine grants DR3-07438, TG2-01161, TG2-01151, CL1-00521 and FA1-00616 to D.O.C. We also acknowledge support from the Garland Initiative for Vision, The Foundation Fighting Blindness Wynn-Gund Translational Research Acceleration Program and the UCSB Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the US Army Research Office. The authors thank R. Stolitenko for technical assistance, D. Geraghty (FHCRC) for providing HLA-E plasmids, D. McDonald (FHCRC) for cytogenetic analyses, D. Youngs (Bloodworks Northwest) for help in performing CDC assays, P. Treuting, B. Johnson and Y.-T. Tien (University of Washington) for histology and immunohistochemistry analysis, W. Loomis for assistance in IVIS image analysis, S. Quaratella (Medtronic Inc.) for providing PVA sponges, M. Gerace and D. Yadock (FHCRC) for providing CD8+ T and CD56+ NK cells (funding provided by the Cooperative Centers of Excellence in Hematology: NIH Grant DK106829)) and C. Levy (FHCRC) for help with graphing programs.

Author information

Authors and Affiliations

  1. Department of Medicine, University of Washington, Seattle, Washington, USA

    Germán G Gornalusse, Roli K Hirata, Sarah E Funk, Laura Riolobos, Gabriel Manske, Donna Prunkard, Aric G Colunga & David W Russell

  2. Department of Molecular, Center for Stem Cell Biology and Engineering, Cellular, and Developmental Biology, University of California, Santa Barbara, USA

    Vanda S Lopes & Dennis O Clegg

  3. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Laïla-Aïcha Hanafi & Cameron Turtle

  4. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    David W Russell

Authors
  1. Germán G Gornalusse
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roli K Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarah E Funk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laura Riolobos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vanda S Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gabriel Manske
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Donna Prunkard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Aric G Colunga
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Laïla-Aïcha Hanafi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dennis O Clegg
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Cameron Turtle
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. David W Russell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.G.G. and D.W.R. wrote the manuscript. G.G.G. and D.W.R. designed the experiments. G.G.G. and R.K.H. prepared AAV vectors stocks and performed gene editing. G.G.G. performed flow cytometry, hematopoietic differentiation, chromium release assays, CDC assays and data analysis. S.E.F. conducted in vivo experiments, PCR analysis and Southern blots. D.P. and R.K.H. performed flow sorting experiments. G.G.G., S.E.F. and G.M. did molecular cloning. G.M. performed RNA analysis. L.R. designed both the HLA-E dimer and trimer open reading frames, conducted preliminary studies with HLA-E vectors and optimized chromium release assay protocols. V.S.L., D.O.C. and G.G.G. performed RPE differentiation experiments and immunocytochemistry. L.-A.H. and C.T. did flow cytometry to detect costimulatory receptors. A.G.C. and L.R. each designed and built one of the AAV vectors. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to David W Russell.

Ethics declarations

Competing interests

D.W.R. is on the Advisory Board of Horizon Discovery and co-founder of Universal Cells, Inc. D.O.C. is co-founder of Regenerative Patch Technologies, LLC.

Integrated supplementary information

Supplementary Figure 1 Generation of B2M-/- ESCs.

(a) Maps of the B2M alleles in cells with the indicated B2M genotypes. Probes and restriction enzymes used in Southern blots are indicated (H, Hind III; B, Bgl II; G, BsrG I). Dark blue regions show overlap with the gene editing vectors used to produce each allele. (b) B2M flow cytometry of different mixed colonies of ESCs and their subcloning results showing the bias against obtaining pure populations of normal B2M-/- cells. (c) Flow cytometry showing a lack of B2M expression in two B2M-/- ESC clones after IFN-γ treatment. (d) Karyotypic analysis of these same two B2M-/- clones.

Supplementary Figure 2 Trace B2M expression in B2M-edited clones.

(a) Pan-HLA- ABC flow cytometry of IFN-γ-treated B2M+/+ and B2M-/- H1 ESCs created with vectors AAV-B2M-EHyTKpA and AAV-B2M-ETKNpA6 (blue tracings) and isotype controls (red tracings) showing low level expression. (b) The sequence of an mRNA isolated from B2M-/- H1 ESCs showing transcription of the edited allele through the pA site, the location of an initiation codon (underlined) in the loxP site, and in-frame continuation of the reading frame into downstream B2M exon 1 sequences. (c) Modifications made in vectors AAV- B2M-HyTK and AAV-B2M-TKN that eliminate trace B2M expression. The B2M gene is shown after Cre-mediated excision of the HyTK or TKN genes present in either vector, with the loxP-encoded ATG start codon shown above, and the downstream stop codons that prevent translation (asterisks) in all three reading frames shown below.

Supplementary Figure 3 Retinal pigmented epithelium (RPE) cell differentiation.

RPE cells derived from B2M+/+ H9 ESCs (panels a-c) and B2M-/Edimer ESCs (panels d-f) were visualized by bright field microscopy (a, d) and immunofluorescence microscopy for retinal markers PMEL (b, e) and MITF (c, f) after 42 days of differentiation. Bright field images demonstrate the level of pigmentation. PMEL+ and MITF+ cells are shown in green, with DAPI stained nuclei in blue. Scale bar = 50 μm.

Supplementary Figure 4 Hematopoietic potential and NK cell-mediated lysis of ESC- derived CD45+ cells.

(a) Flow cytometry analysis of CD45 expression after hematopoietic differentiation of Elf-1 ESCs with the indicated B2M genotypes. Data were acquired from suspension cells on day 38 of differentiation. Results for B2M-/Etrimer c5 shown in Fig. 3B. (b) B2M-/- ESCs produce fewer hematopoietic cells. Kinetics of suspension cell production during hematopoietic differentiation of ESCs with the indicated B2M genotypes. Y-axis denotes number of live suspension cells generated per 5x106 undifferentiated ESCs. The results from two independent differentiation experiments are shown with numbers between parentheses. (c) Flow cytometry analysis of NKG2A and NKG2C receptors on NK cells derived from donor 2. Percents were calculated by subtracting the isotype control frequencies. (d) Chromium release assay with NK cells from donor 2 and ESC-derived CD45+ cells with the indicated B2M genotypes showing B2M-/Edimer expression partially prevents lysis by NK cells with low NKG2A expression levels. Data are represented as mean + SD (n=3). (e) Chromium release assay with NK cells from donor 1 and ESC- derived CD45+ cells showing that B2M-/- and B2M-/Edimer(pre-Cre) cells had similar susceptibility to NK-mediated lysis. Data are represented as mean + SD (n=3). (f) Chromium release assay as in (d) but with NK cells from donor 3 cultured at a low IL-2 dose (100 U/ml). Asterisks indicate p<0.05 for pair-wise comparison between the indicated cells (ANOVA followed by the post hoc Tukey HSD test). (g) Change in luciferase expression in B2M-/Edimer(pre-Cre) (HLA class I-negative control) and B2M-/Etrimer teratomas measured from day 13 to day 19 after implantation, with NK-92 cells administered to half the animals on days 13, 15 and 17. P-values were determined in each group (with or without NK-92 cells) by paired Student’s t-test (n=3 mice for both groups that received NK-92 cells and n=4 for both groups that did not). An unpaired Student’s t-test (p=0.096) was also applied to the same data to compare the relative growth of the B2M-/Etrimer and B2M-/Edimer(pre-Cre) teratomas in mice that received NK-92 cells to their relative growth in mice that did not receive NK-92 cells. (h) Examples of luciferase imaging in mice from (g), half of which received NK-92 cells as noted. “Pre” indicates B2M-/Edimer(pre-Cre) genotype, “-/E” indicates B2M-/Etrimer genotype. Red circles indicate measured areas.

Supplementary Figure 5 HLA molecule and costimulatory receptor expression.

(a) Flow cytometry analysis of HLA-ABC and HLA-DR expression in IFN-γ-stimulated B2M+/+ Elf-1 EBs used for priming CD8+ T cells as shown in Figure 4A. (b) Costimulatory receptor profile of B2M+/+ Elf-1 EBs. Isotype controls in red and specific antibodies in blue. (c) Costimulatory receptor profile for ESC-derived CD45+ cells with the indicated B2M genotypes.

Supplementary Figure 6 Differential growth of B2M+/+ and B2M-/Etrimer ESC-derived teratomas when challenged with allogeneic CD8+ T cells in vivo.

(a) Luciferase signal measured on day 1 was used to normalize the data. Each graph shows the results from an individual mouse. In all panels, blue and red lines show the growth of B2M-/Etrimer and B2M +/+ teratomas respectively. The three bottom panels show teratoma growth in mice that did not receive CD8+ cells. (b) B2M-/Etrimer and B2M+/+ teratoma growth rates (luciferase measurement increase per day) were measured before and after allogeneic primed CD8+ T cell infusions from three different donors (n=9 teratomas per genotype). Horizontal black bars indicate the means. P values were calculated by paired Student’s t-tests. (c) Quantitative measurement of CD8+ T cells in B2M-/Etrimer and B2M+/+ teratomas (n=5 mice, each with both types of teratoma; each line represents one mouse). Results were calculated as % of CD8 signal in the total area examined. The p value was calculated by paired Student’s t-test.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 2. (PDF 1663 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gornalusse, G., Hirata, R., Funk, S. et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol 35, 765–772 (2017). https://doi.org/10.1038/nbt.3860

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3860

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing