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

Nature Biotechnology volume 35, pages 765772 (2017) | Download Citation


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.

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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


  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. Center for Stem Cell Biology and Engineering, Department of Molecular, 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


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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.

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.

Corresponding author

Correspondence to David W Russell.

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