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Research Article
Immunobiology of naïve and genetically modified HLA-class-I-knockdown human embryonic stem cells
Tobias Deuse, Martina Seifert, Neil Phillips, Andrew Fire, Dolly Tyan, Mark Kay, Philip S. Tsao, Xiaoqin Hua, Joachim Velden, Thomas Eiermann, Hans-Dieter Volk, Hermann Reichenspurner, Robert C. Robbins, Sonja Schrepfer
Journal of Cell Science 2011 124: 3029-3037; doi: 10.1242/jcs.087718
Tobias Deuse
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Martina Seifert
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Neil Phillips
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Andrew Fire
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Dolly Tyan
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Mark Kay
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Philip S. Tsao
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Xiaoqin Hua
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Joachim Velden
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Thomas Eiermann
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Hans-Dieter Volk
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Hermann Reichenspurner
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Robert C. Robbins
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Sonja Schrepfer
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  • For correspondence: schrepfer@stanford.edu
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    Fig. 1.

    Characterization of hESC and hESCKD. Normalized surface molecule expression was assessed by flow cytometry. HLA I and β2-microglobulin were modestly expressed on hESCs and were successfully knocked down after 7 days in hESCKD. Both hESC and hESCKD were negative for HLA II and co-stimulatory molecules. They were also negative for SSEA-1 but positive for SSEA-4. Prominent expression of Oct-4, Sox-2, SSEA-3 and Tra-1-60 was confirmed by immunofluorescence (right-hand panel).

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    Fig. 2.

    Transplantation of hypoantigeneic hESC. (a) HLA I knockdown in hESCKD was followed by flow cytometry and showed levels between 1% and 12% of those of naïve hESC between days 7 and 42 (means ± s.e.m.). (b) A total of 106 hESCs or hESCKD were transplanted into the gastrocnemius muscle of either immunocompetent Balb/c or severely immunocompromised SCID-beige mice. All Balb/c mice rapidly rejected the hESC transplants, whereas the cells survived in SCID-beige recipients. Rejection of hESCKD was markedly attenuated and four out of ten hESCKD grafts achieved long-term survival. (c) On day 5, BLI signals from hESCs in SCID-beige and hESCKD in Balb/c were similarly strong, whereas signals from hESCs in Balb/c were negligible. (d) Balb/c cellular immune activation on the same day was significantly stronger after hESC rather than hESCKD transplantation. *P<0.05 compared with hESCs; †P<0.001 compared with hESCs. (e) The percentage of Treg cells (CD25+ Foxp3+ cells) among the CD4+ population in inguinal lymph nodes was monitored over time. The Treg fraction increased after both hESC and hESCKD transplantation (*P<0.05 compared with native Balb/c) but remained elevated only in the hESCKD group (§P<0.01 compared with hESC after 14 days).

  • Fig. 3.
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    Fig. 3.

    Histology of hESC transplants. Intramuscularly-injected hESCs were identified according to their morphology in hematoxylin and eosin (a,b) and their Fluc-positivity (c). At 5 days after transplantation, hESC grafts showed markedly more dense infiltrates of MAC2+ macrophages and CD3+ lymphocytes than hESCKD grafts (d). Only a few KLRA1+ NK cells were found after hESC or hESCKD transplantation. (e) Cell numbers of all three cell populations were significantly lower within hESCKD rather than hESC grafts. †P<0.001 compared with hESCs.

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    Fig. 4.

    Antibody response after hESC transplantation. (a) At 5 days after transplantation into Balb/mice, flow cytometry showed a significantly stronger hESC-specific antibody production for hESCs compared with hESCKD. *P<0.05 compared with both before transplantation and with hESCKD transplantation. (b) Luminex single-antigen assays revealed HLA class I-specific antibody induction by hESCs but not hESCKD, which was massively boosted by hESC re-injection. *P<0.05 compared with native Balb/c; §P<0.05 compared with hESCs. HLA class II antibody induction was not observed. (c,d) In recipients of hESC grafts, the mean Z-score of hESC-specific class I antibodies was significantly higher than that of non-hESC-specific antibodies and all of the six hESC-specific HLA-A, -B and -C antibody Z-scores were above a threshold of 3 s.d. #P<0.05. The recipients of hESCKD did not significantly induce hESC-specific class I antibody production, and recipients of both groups did not develop relevant hESC-specific class II antibodies.

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    Fig. 5.

    Allogeneic cytotoxic killing of hESC. Human PBMCs were separated into lymphocytes (CD3+ CD56−) and NK cells (CD3− CD56+). IFN-γ (a) and IL-4 (b) Elispot assays revealed that only hESCs, and not hESCKD, significantly induced allogeneic lymphocyte activation in vitro. *P<0.05 compared with responder lymphocytes. NK cell activation (c) and CD107a surface expression (d) was provoked by either PMA plus ionomycin stimulation or K562 incubation. *P<0.05 and †P<0.001 compared with responder NK cells. Both hESCs and hESCKD did not induce significant NK cell activation.

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Research Article
Immunobiology of naïve and genetically modified HLA-class-I-knockdown human embryonic stem cells
Tobias Deuse, Martina Seifert, Neil Phillips, Andrew Fire, Dolly Tyan, Mark Kay, Philip S. Tsao, Xiaoqin Hua, Joachim Velden, Thomas Eiermann, Hans-Dieter Volk, Hermann Reichenspurner, Robert C. Robbins, Sonja Schrepfer
Journal of Cell Science 2011 124: 3029-3037; doi: 10.1242/jcs.087718
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Research Article
Immunobiology of naïve and genetically modified HLA-class-I-knockdown human embryonic stem cells
Tobias Deuse, Martina Seifert, Neil Phillips, Andrew Fire, Dolly Tyan, Mark Kay, Philip S. Tsao, Xiaoqin Hua, Joachim Velden, Thomas Eiermann, Hans-Dieter Volk, Hermann Reichenspurner, Robert C. Robbins, Sonja Schrepfer
Journal of Cell Science 2011 124: 3029-3037; doi: 10.1242/jcs.087718

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