Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy

Lee J, Sheen JH, Lim O, Lee Y, Ryu J, Shin D, Kim YY, Kim M.
Source: Scientific Reports
Publication Date: (2020)
Issue: 20;10(1): :17753
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit

Raji cell culture and transfection

 Raji cells were maintained in RPMI 1640 (Gibco, A10491-01) supplemented with 10% heat-inactivated FBS (Gibco, 16000–044), Antibiotic–Antimycotic (Gibco, 15240) and ß-mercaptoethanol (Gibco, 21985–023). A total of 7.5 µg of Cas9 protein (Toolgen, TGEN_CP1; gRNA screening experiment in Supplementary fgure S1, Clontech, 632640; all other experiments) and 7.5 µg of gRNA per reaction were mixed and incubated at room temperature for 10  min to form the Cas9/gRNA complex. For multiplex genome editing, 7.5 µg of gRNA in total were used. 4×10^5 Raji cells were transfected with the Cas9/ gRNA complex by 4D-Nucleofector X Unit (Lonza, AAF-1002X) using the SG Cell Line 4D-Nucleofector X Kit S (Lonza, V4XC-3032) with the program DS-104.

Human T cell transfection and expansion

 Cryopreserved human PBMCs from healthy donors were  thawed, and CD3+ cells were isolated using human CD3 MicroBeads (Miltenyi Biotec, 130–050-101). Upon T  cell isolation, the CD3+ T cells were initially cultured in X-VIVO15 (Lonza, BE02-060Q) supplemented with  Dynabeads Human T-Activator CD3/CD28 (Gibco, 111.31D), 200 IU/mL IL-2 (Novartis, 502519AF) and 5% human plasma (Valley Biomedical, HP1050) in a culture bag (NIPRO, 87–352). On day 1, 37.5  µg of Cas9 protein and 37.5 µg of gRNA per reaction were mixed and incubated at room temperature for 10 min to form  Cas9/gRNA complex. For multiplex genome editing, 37.5 µg of gRNA in total were used. Te CD3+ T cells were taken out from the culture bag and 4×10^6 cells were transfected with Cas9/gRNA complex by 4D-Nucleofector  X Unit (Lonza, AAF-1002X) using P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, V4XP-3024) with the program DN-100. Following transfection, the cells were cultured at 37 °C in 5% CO2, and fresh culture medium,  X-VIVO15 (Lonza, BE02-060Q) supplemented with 200  IU/mL IL-2 (Novartis, 502519AF) and 5% human  plasma (Valley Biomedical, HP1050), was added every 2 to 3 days to reach a density of 1×106 cells/mL. Te cell number and viability were documented using the automated fuorescence cell counter (NanoEntek, ADAMMC). The Dynabeads Human T-Activator CD3/CD28, which was provided in the culture media at the beginning  of the ex vivo expansion was not replenished further.


As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting a chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-? and TNF-a in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.