Activation of Antigen-Specific Cytotoxic T Lymphocytes by β2-Microglobulin or TAP1 Gene Disruption and the Introduction of Recipient-Matched MHC Class I Gene in This information is current as Allogeneic Embryonic Stem Cell-Derived of October 2, 2021. Dendritic Cells Yusuke Matsunaga, Daiki Fukuma, Shinya Hirata, Satoshi Fukushima, Miwa Haruta, Tokunori Ikeda, Izumi Negishi, Yasuharu Nishimura and Satoru Senju Downloaded from J Immunol 2008; 181:6635-6643; ; doi: 10.4049/jimmunol.181.9.6635 http://www.jimmunol.org/content/181/9/6635 http://www.jimmunol.org/ References This article cites 41 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/181/9/6635.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Activation of Antigen-Specific Cytotoxic T Lymphocytes by ␤ 2-Microglobulin or TAP1 Gene Disruption and the Introduction of Recipient-Matched MHC Class I Gene in Allogeneic Embryonic Stem Cell-Derived Dendritic Cells1 Yusuke Matsunaga,*§ Daiki Fukuma,† Shinya Hirata,* Satoshi Fukushima,*§ Miwa Haruta,*§ Tokunori Ikeda,*§ Izumi Negishi,‡ Yasuharu Nishimura,* and Satoru Senju2*§ A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. ␤ ␤ b d Downloaded from The TAP1 or 2-microglobulin ( 2m) gene was disrupted in 129 (H-2 )-derived ES cells and then expression vectors for the H-2K d ␤ d ؊/؊ d ␤ or 2m-linked form of K ( 2m-K ) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1 /K ؊/؊ ␤ d d b ␤ ES-DC and 2m / 2m-K ES-DC. As intended, both of the transfectant ES-DC expressed K but not the intrinsic H-2 ؊/؊ ␤ d ؊/؊ d b ␤ haplotype-derived MHC class I. 2m / 2m-K and TAP1 /K ES-DC were not recognized by pre-activated H-2 -reactive b ␤ ؊/؊ ␤ d ؊/؊ d CTL and did not prime H-2 reactive CTL in vitro or in vivo. 2m / 2m-K ES-DC and TAP1 /K ES-DC had a survival ؉/؊ ␤ d ؉/؉ d d ␤ advantage in comparison to 2m / 2m-K ES-DC and TAP1 /K ES-DC, when transferred into BALB/c mice. K -restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective http://www.jimmunol.org/ b ␤ ؊/؊ ␤ d of the cell surface expression of intrinsic H-2 haplotype-encoded MHC class I. 2m / 2m-K ES-DC were significantly more ؉/؊ ␤ d ␤ efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than 2m / 2m-K ES-DC. The modification of ␤ the 2m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated. The Journal of Immunology, 2008, 181: 6635–6643. n efficient means for the activation of the CTL reactive alloreactive T cells and to prime Ag-specific CTL is comparable to to tumor Ags is crucial for T cell-mediated antitumor that of conventional bone marrow-derived DC (BM-DC). Genet- 3 A immunotherapy (1). Dendritic cells (DC) are potent T ically modified ES-DC can be readily generated by introducing by guest on October 2, 2021 cell stimulators and cellular vaccination using Ag-loaded DC has expression vectors into ES cells and the subsequent induction of proven to be an efficient means for priming CTL specific to Ags (2, their differentiation into ES-DC (9). The transfection of ES cells 3). This laboratory and others have established methods to gener- can be done by electroporation with plasmid vectors and the use of ate DC from mouse and human embryonic stem (ES) cells (4–8). virus-based vectors is not necessary. Once an ES cell clone with The capacity of ES cell-derived DC (ES-DC or esDC) to simulate proper genetic modification is established, it then serves as an in- finite source for genetically modified DC. Mouse models have demonstrated that vaccination with genetically engineered ES-DC *Department of Immunogenetics and †Department of Oral and Maxillofacial Surgery, Kumamoto University, Graduate School of Medical Sciences, Kumamoto, Japan; expressing tumor Ags (10) and T cell-attracting chemokines (11) is ‡Department of Dermatology, Gunma University Graduate School of Medicine, very effective for the induction of antitumor immunity. § Gunma, Japan; and Japan Science and Technology Agency, CREST, Tokyo, Japan In the future, the clinical application of ES-DC technology will Received for publication July 10, 2008. Accepted for publication August 20, 2008. require a solution to the problem of histoincompatibility between The costs of publication of this article were defrayed in part by the payment of page patients to be treated and the ES-DC. Specifically, the HLA allele charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mismatch may cause a rapid immune response and rejection of the 1 This work was supported in part by Grants-in-Aid 16590988, 17390292, 17015035, inoculated cells (12), although a discrepancy in the minor histo- 18014023, 19591172, and 19059012 from the Ministry of Education, Culture, Sports, compatibility Ag can also be a cause of alloreaction (13). The Science and Technology (MEXT), Japan; the Program of Founding Research Centers present study addressed this problem by using the strategy of mod- for Emerging and Reemerging Infectious Diseases launched as a project commis- sioned by MEXT, Japan; Research Grant for Intractable Diseases from Ministry of ification of the genes that control the cell surface expression of Health and Welfare, Japan; and grants from Japan Science and Technology Agency MHC class I, i.e., ␤ -microglobulin (␤ m) and TAP. TAP1Ϫ/Ϫ and (JST), the Uehara Memorial Foundation, and the Takeda Science Foundation. 2 2 ␤ mϪ/Ϫ ES cell clones were generated from the ES cell lines de- 2 2 Address correspondence and reprint requests to Dr. Satoru Senju, Department of b Immunogenetics, Graduate School o f Medical Sciences, Kumamoto University, rived from the 129 mouse (H-2 ) embryo. The expression vectors d ␤ d ␤ d Honjo 1–1-1, Kumamoto, Japan. E-mail address: [email protected] for H-2K and 2m-linked form of H-2K ( 2m-K ) were then Ϫ Ϫ Ϫ Ϫ 3 / ␤ / Abbreviations used in this paper: DC, dendritic cell; ES cell, embryonic stem cell; introduced into TAP1 and 2m ES cell clones, respec- ␤ ES-DC, embryonic stem cell-derived DC; BM-DC, bone marrow-derived DC; 2- ␤ tively. Subsequently, these genetically modified ES cells were sub- microglobulin, 2m; HA, hemagglutinin; RSV, respiratory syncytical virus; PEF, pri- mary mouse embryonic fibroblast; CMTR, chloromethyl-benzoyl-amino-tetramethyl- jected to a differentiation culture to generate ES-DC. The MHC rhodamine; CMFDA, chlorometylfluorescein diacetate; iPS cell, induced pluripotent class I molecules encoded by the genes in the H-2b haplotype were stem cell; Luc, luciferase; IRES, Internal ribosomal entry site. either absent or at very low levels on the cell surface of these Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 genetically modified ES-DC. The effect of the alteration of cell www.jimmunol.org 6636 GENETIC MODIFICATION OF ES-DC TO AVOID ALLOREACTION β +/- Hi gh dose G418 S S 2m ES cells α α selection 2 S S 1 N-terminal S S α3 S S β2m β2m+/- ES cells β2m-/- ES cells CAG promoter Linker Transfection of β2m-linked Kd IRES β2mβ H-H-2K2K dd PuPu ro roRpApA β2m+/-/β2m-Kd β2m-/-/β2m-Kd 2m ES cells ES cells C-terminal Differentiation D H-2Db H-2Kb H-2Kd β2m+/-/β2m-Kd ES-DC β2m-/-/β2m-Kd ES-DC β2m+/-/β2m-Kd ES-DC B ES cells β2m+/- β2m-/- Mutant allele 1.6 kb β2m-/-/β2m-Kd Wild type allele 290 bp ES-DC Downloaded from ␤ ␤ FIGURE 1. Generation of ES-DC deficient in the intrinsic 2m gene and expressing recipient-matched MHC class I linked to 2m. A, Overview of the ␤ d ␤ ϩ/Ϫ ␤ d ␤ Ϫ/Ϫ ␤ d ␤ Ϫ/Ϫ method for generation of ES-DC-expressing 2m-linked K , 2m / 2m-K ES-DC and 2m / 2m-K ES-DC. 2m ES cells were generated from ␤ ϩ/Ϫ ␤ ϩ/Ϫ ␤ Ϫ/Ϫ ␤ d 2m ES cells by high dose G418 selection. 2m and 2m ES cells were introduced with cDNA for the 2m-linked form of K and subsequently ␤ ϩ/Ϫ ␤ d ␤ Ϫ/Ϫ ␤ d ␤ ␤ Ϫ/Ϫ differentiated to generate 2m / 2m-K ES-DC and 2m / 2m-K ES-DC. B, The absence of wild-type 2m gene in the 2m ES cell clone was ␤ d confirmed by genomic PCR.