Initial In Vitro Investigation of the Human Immune Response to Corneal Cells from Genetically Engineered Pigs

Hara, Hidetaka; Koike, Naoko; Long, Cassandra; Piluek, Jordan; Roh, Danny S.; SundarRaj, Nirmala; Funderburgh, James L.; Mizuguchi, Yoshiaki; Isse, Kumiko; Phelps, Carol J.; Ball, Suyapa; Ayares, David; Cooper, David K.C.

doi:10.1167/iovs.10-6947

Cited by 46 publications

(55 citation statements)

References 59 publications

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“…Blood was also drawn from wild-type (outbred Large White Landrace, n=3) pigs and from α1,3-galactosyltransferase gene-knockout pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pigs, n=4), both from Revivicor, Blacksburg, VA [36]. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described [37]. …”

Section: Methodsmentioning

confidence: 99%

“…The response of primate cells to the activated pAECs was determined as previously described [37, 38]. In our preliminary study, since it was difficult to define the suppressive capacity of 2C10R4 among species when GTKO/CD46 pAECs were used as stimulators (because the MLR response to GTKO/CD46 pAECs was significantly lower than to wild-type pAECs) (data not shown), wild-type pAECs were used as stimulators.…”

Section: Methodsmentioning

confidence: 99%

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In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

Lee

Satyananda

Iwase

et al. 2015

Transplant Immunology

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Background The CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs. Methods The binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators. Results There was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs. Conclusions Since both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

Lee

Satyananda

Iwase

et al. 2015

Transplant Immunology

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“…Corneas from deceased humans (blood type O) that were not suitable for clinical transplantation were provided by the Pittsburgh Center for Organ Recovery and Education (CORE) with the approval of the University of Pittsburgh Committee for Oversight of Research Involving the Dead (CORID No.231) , and in accordance with the guidelines of the Declaration of Helsinki for research involving the use of human tissues 16 .…”

Section: Methodsmentioning

confidence: 99%

“…Surface expression of Gal and NeuGc, and human IgM/IgG binding to CECs and AECs were detected by flow cytometry (BD LSR II; BD Biosciences), as previously described 16 . Serum samples from healthy human volunteers (n=7, including all ABO blood types) were pooled.…”

Section: Methodsmentioning

confidence: 99%

“…Gal is mainly expressed on the stromal keratocytes (and weakly on the stroma), with no expression on the corneal epithelium or endothelium in naïve status (i.e., when the cornea is not activated). However, WT pig corneal endothelial cells (CECs) develop Gal epitopes in certain situations (e.g., during in vitro culture 16 , or when exposed to inflammatory cytokines after xenotransplantation) 17 . In contrast to Gal, NeuGc is expressed on corneal epithelium and endothelium in addition to stromal keratocytes 18 .…”

Section: Introductionmentioning

confidence: 99%

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Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation

Lee

Miyagawa

Long

et al. 2016

Cornea

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Purpose Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (i) to document the lack of NeuGc expression on corneas and aortas, and cultured endothelial cells (aortic [AECs]; corneal [CECs]) of GTKO/NeuGcKO pigs, and (ii) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. Methods Wild-type (WT), GTKO, and GTKO/NeuGcKO pig were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and to determine human IgM and IgG binding to tissues. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. Results Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither human nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared to binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. Conclusions The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.

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The first comprehensive description of the expression profile of genes involved in differential body growth and the immune system of the Jeju Native Pig and miniature pig

et al. 2018

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Initial In Vitro Investigation of the Human Immune Response to Corneal Cells from Genetically Engineered Pigs

Abstract: These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.

Cited by 46 publications

References 59 publications

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation

The first comprehensive description of the expression profile of genes involved in differential body growth and the immune system of the Jeju Native Pig and miniature pig

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