The human T‐cell proliferative response to triple‐knockout pig cells in mixed lymphocyte reaction

Wang, Zheng‐Yu; Morsi, Mahmoud; Nguyen, Huy Quoc; Bikhet, Mohamed H; Burnette, KaLia S.; Ayares, David; Cooper, D. K. C.; Hara, Hidetaka

doi:10.1111/xen.12619

Cited by 7 publications

(10 citation statements)

References 9 publications

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“…The T‐cell proliferative response to cells from an α1,3‐galactosyltransferase gene‐knockout (GTKO) pig (that does not express the important Gal xenoantigen) is weaker than that to cells from a wild‐type (genetically‐ un modified) pig. However, perhaps surprisingly, the response to TKO pig cells is not significantly different from that to GTKO pig cells 27 …”

Section: In Vitro Studies On Cells From Triple‐knockout Pigsmentioning

confidence: 76%

“…However, perhaps surprisingly, the response to TKO pig cells is not significantly different from that to GTKO pig cells. 27 The observation that many humans have no or minimal antibody binding or cytotoxicity to TKO pig cells clearly suggests that this genetic manipulation will be beneficial in clinical xenotransplantation. However, a complicating factor for studies in NHPs is that all Old World NHPs, including baboons, do have antibodies against TKO pig cells ( Figure 4).…”

Section: In Vitro S Tud Ie S On Cell S From Triple-k No Ckout Pi G Smentioning

confidence: 99%

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Pig kidney xenotransplantation: Progress toward clinical trials

Cooper

Hara

Iwase

et al. 2020

Clinical Transplantation

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Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ‐source pigs has largely been directed to two major aims—(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement‐ and coagulation‐regulatory proteins. Conventional (FDA‐approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non‐immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait‐list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.

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Section: In Vitro Studies On Cells From Triple‐knockout Pigsmentioning

confidence: 76%

Section: In Vitro S Tud Ie S On Cell S From Triple-k No Ckout Pi G Smentioning

confidence: 99%

Pig kidney xenotransplantation: Progress toward clinical trials

Cooper

Hara

Iwase

et al. 2020

Clinical Transplantation

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“…As an indicator of the adaptive cellular immune response, the reactivity of recipient T cells to pig antigens must also be assessed to avoid potential deleterious adaptive cellular immunity. This is accomplished by measuring the proliferative response of bulk PBMCs (including CD4 + and CD8 + T‐cell populations) to pig PBMCs by the mixed lymphocyte reaction 14‐17 …”

Section: Assessing the Immunologic Suitability Of Potential Recipientsmentioning

confidence: 99%

“…The post‐transplant immunologic responsiveness of the recipient to the xenograft will be monitored in part by using the same methods as those employed during pre‐transplant screening, including assays for elicited anti‐pig antibody and recipient PBMC proliferative reactivity to pig xenoantigens 5,7,14,16,17,23 . Assays involving both pig RBCs and PBMCs would need to be performed.…”

Section: Monitoring Of the Recipient Post‐transplantationmentioning

confidence: 99%

Immunological selection and monitoring of patients undergoing pig kidney transplantation

Lucander

Nguyen

Foote

et al. 2021

Xenotransplantation

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Pig kidney xenotransplantation has the potential to alleviate the current shortage of deceased and living human organs and provide patients with end‐stage renal disease with a greater opportunity for long‐term survival and a better quality of life. In recent decades, advances in the genetic engineering of pigs and in immunosuppressive therapy have permitted the resolution of many historical obstacles to the success of pig kidney transplantation in nonhuman primates. Pig kidney xenotransplantation may soon be translated to the clinic. Given the potential risks of kidney xenotransplantation, particularly of immunologic rejection of the graft, potential patients must be carefully screened for inclusion in the initial clinical trials and immunologically monitored diligently post‐transplantation. We provide an overview of the immunological methods we believe should be used to (i) screen potential patients for the first clinical trials to exclude those with a higher risk of rejection, and (ii) monitor patients with a pig kidney graft to determine their immunological response to the graft.

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“…The prevention of antibody-mediated hyperacute rejection of organ xenografts in nonhuman primates (NHPs) through the disruption of the GGTA1 gene and/or the expression of human complement regulatory proteins in genetically engineered donor pigs has marked a major milestone in the development of xenotransplantation (1,2). The cellular immune response to cell and organ xenografts is also vigorous (3)(4)(5)(6) and genetic engineering of donor pigs to mitigate anti-pig cellular immunity has emerged as an important goal in the field (7)(8)(9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

et al. 2021

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The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger’s sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

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The human T‐cell proliferative response to triple‐knockout pig cells in mixed lymphocyte reaction

Cited by 7 publications

References 9 publications

Pig kidney xenotransplantation: Progress toward clinical trials

Pig kidney xenotransplantation: Progress toward clinical trials

Immunological selection and monitoring of patients undergoing pig kidney transplantation

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

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