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. 2006 Dec;38(10):3184-8.
doi: 10.1016/j.transproceed.2006.10.158.

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes

K Haga  1 , N A Lemp, C R Logg, J Nagashima, E Faure-Kumar, G G Gomez, C A Kruse, R Mendez, R Stripecke, N Kasahara, J C Cicciarelli
Affiliations

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes

K Haga et al. Transplant Proc. 2006 Dec.

Erratum in

  • Transplant Proc. 2007 Jan-Feb;39(1):314. Kasahara, N A [corrected to Kasahara, N]

Abstract

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

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Figures

Fig 1
Fig 1
HLA knockdown by lentiviral shRNA vectors. The control lentiviral vector expresses the DsRed marker gene only (DsRed only lentivirus), while the A0201 shRNA-DsRed lentiviral vector expresses an HLA-A0201 allele-specific shRNA in addition to DsRed, and the ABC shRNA-DsRed lentiviral vector expresses a pan-Class I HLA-ABC-specific shRNA in addition to DsRed. Cell surface HLA expression was detected by FACS analysis using a fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A antibody (FITC-anti-A2 Ab) or anti-HLA-ABC antibody (FITC-anti-ABC Ab). Note that with no lentivirus infection and isotype control antibody (Ab control), the events are all in the lower left quadrant. DsRed-only lentivirus infection with control antibody causes the entire population (highlighted in red) to shift only along the DsRed axis (Y-axis) to the upper left quadrant with no shift along the FITC axis (X-axis). DsRed-only lentivirus-infected cells stained with FITC-conjugated HLA antibodies show a shift in both DsRed fluorescence (Y axis) and FITC fluorescence (X-axis) to the upper right quadrant (also highlighted in red). In contrast, with HLA-A0201- or HLA-ABC-specific shRNA-DsRed infection in the presence of FITC antibodies, there is only a shift up the DsRed axis and very little shift along the FITC axis, indicating that there is nothing for the anti-HLA antibodies to bind to even when present and demonstrating that successful knockdown has been achieved.
Fig 2
Fig 2
Alloreactive CTL-mediated cytotoxicity after lentivirus-mediated shRNA knockdown of HLA expression. 293T target cells transduced with control lentivirus vectors expressing DsRed only (Lenti-DsRed only) continue to express HLA-A2 and B7,Cw7 and show <40% viability 48 hours after incubation with alloreactive CTL that had been activated against stimulator cells expressing HLA-A2/B44/C5. In contrast, significantly increased viability is observed in target cells transduced with pan-specific (Lenti-ABC-shRNA-DsRed) or allele-specific (Lenti-A0201-shRNA-DsRed) vectors (P < .05 for both compared to control, as indicated). Results are expressed as mean values ± SEM in percent viability.
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