Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA-E/human CD46 double-transgenic pig limbs with human blood

Autor: Eckhard Wolf, Anjan K. Bongoni, Robert Rieben, Gisella Puga Yung, David Ayares, Mihai A. Constantinescu, Amandine Pradier, Riccardo Sfriso, Natacha Madelon, Jorg Dieter Seebach, Maria Papaserafeim, Nikolai Klymiuk, David Kiermeir, Andrea Bähr, Esther Voegelin, Hansjörg Jenni
Jazyk: angličtina
Rok vydání: 2018
Předmět:
Cytotoxicity
Immunologic
0301 basic medicine
Histocompatibility Antigens Class I/genetics
Membrane Cofactor Protein/genetics/immunology
medicine.medical_treatment
Cell
030230 surgery
Animals
Genetically Modified
Heterografts/immunology
0302 clinical medicine
HLA Antigens
Leukocytes
Cytotoxicity
610 Medicine & health
ddc:616
education.field_of_study
medicine.diagnostic_test
Chemistry
HLA Antigens/genetics
Killer Cells
Natural
medicine.anatomical_structure
Extremities/blood supply
Killer Cells
Natural/immunology
Leukocytes/metabolism
Heterografts
Xenotransplantation
Transplantation
Heterologous
Immunology
Population
Peripheral blood mononuclear cell
Cytotoxicity
Immunologic/immunology
Flow cytometry
Membrane Cofactor Protein
03 medical and health sciences
medicine
Animals
Humans
Leukocytes
Mononuclear/immunology
education
Transplantation
Heterologous/methods
Transplantation
Histocompatibility Antigens Class I
Endothelial Cells/immunology
Endothelial Cells
Extremities
Molecular biology
In vitro
030104 developmental biology
Animals
Genetically Modified/immunology
Leukocytes
Mononuclear
Ex vivo
Zdroj: Xenotransplantation, Vol. 25, No 1 (2018)
ISSN: 0908-665X
DOI: 10.7892/boris.107320
Popis: Background: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. Methods: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. Results: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. Conclusion: sEx vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.
Databáze: OpenAIRE
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