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Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout)

Yamamoto, Takayuki, Iwase, Hayato, Patel, Diyan, Jagdale, Abhijit, Ayares, David, Anderson, Douglas, Eckhoff, Devin E., Cooper, David K. C., Hara, Hidetaka·2020·Scientific Reports
DOI 10.1038/s41598-020-66311-3PMC PMC7300119
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Abstract

Triple-knockout (TKO) pigs (with added protective human transgenes) are likely to be optimal sources of organs for clinical organ xenotransplantation because many humans have minimal or no natural antibody to TKO pig cells. However, Old World monkeys (OWMs) have naturally-existing antibodies directed to TKO cells. We measured anti-pig IgM/IgG binding, and complement-dependent cytotoxicity to wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pig peripheral blood mononuclear cells (PBMCs) using sera from humans, several OWMs, and two New World monkeys (NWMs). Furthermore, we compared survival of GTKO (n = 5) and TKO (n = 3) pig kidneys in baboons. OWMs had significantly greater IgM binding and cytotoxicity to TKO PBMCs than humans or NWMs. Mean anti-TKO IgM was significantly higher in OWMs and significantly lower in NWMs than in humans. Cytotoxicity of OWM sera to TKO PBMCs was significantly greater than of human serum, but there was no significant difference between human and NWM sera. The median survival of TKO pig kidneys (4 days) in baboons was significantly shorter than that of GTKO kidneys (136 days) (p < 0.05). Even though considered ideal for clinical xenotransplantation, the presence of naturally-existing antibodies to TKO pig cells in OWMs complicates the transplantation of TKO pig kidneys in OWMs.

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Figure 1 Mean levels (±SEM) of anti-WT, GTKO, and TKO pig IgM and IgG in various primate species (A), and summarized as human, OWM, and NWM (B). (A) Comparison of binding of preformed xenoreactive antibodies in various primate species (human [n = 9], baboon [n = 6], rhesus [n = 6], cynomolgus [n = 6], African green [n = 6], capuchin [n = 9] and squirrel monkey [n = 6]) to WT (left), GTKO (middle), and TKO (right) pig PBMCs. IgM (top) and IgG (bottom). Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01). (B) Comparison of binding of preformed xenoreactive antibodies in humans [n = 9], OWMs [n = 24], and NWMs [n = 15] to WT (left), GTKO (middle), and TKO (right) pig PBMCs. IgM (top) and IgG (bottom). Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01).
Figure 1 Mean levels (±SEM) of anti-WT, GTKO, and TKO pig IgM and IgG in various primate species (A), and summarized as human, OWM, and NWM (B). (A) Comparison of binding of preformed xenoreactive antibodies in various primate species (human [n = 9], baboon [n = 6], rhesus [n = 6], cynomolgus [n = 6], African green [n = 6], capuchin [n = 9] and squirrel monkey [n = 6]) to WT (left), GTKO (middle), and TKO (right) pig PBMCs. IgM (top) and IgG (bottom). Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01). (B) Comparison of binding of preformed xenoreactive antibodies in humans [n = 9], OWMs [n = 24], and NWMs [n = 15] to WT (left), GTKO (middle), and TKO (right) pig PBMCs. IgM (top) and IgG (bottom). Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01).CC BY · PubMed Central
Figure 2 Comparison of mean serum CDC (at serum concentrations of 50% to 3.125%) of human (n = 9), baboon (n = 6), rhesus (n = 6), cynomolgus (n = 6), African green (n = 6), capuchin (n = 9), and squirrel (n = 6) monkeys against WT, GTKO, and TKO pig PBMCs. Cytotoxicity is expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%).
Figure 2 Comparison of mean serum CDC (at serum concentrations of 50% to 3.125%) of human (n = 9), baboon (n = 6), rhesus (n = 6), cynomolgus (n = 6), African green (n = 6), capuchin (n = 9), and squirrel (n = 6) monkeys against WT, GTKO, and TKO pig PBMCs. Cytotoxicity is expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%).CC BY · PubMed Central
Figure 3 Mean (±SEM) complement-dependent cytotoxicity (CDC, at 25% serum concentration) of sera from various primate species (A), summarized as humans, OWMs, and NWMs (B) to WT, GTKO, and TKO pig PBMCs. (A) Comparison of mean CDC of human [n = 9], baboon [n = 6], rhesus [n = 6], cynomolgus [n = 6], African green [n = 6], capuchin [n = 9], and squirrel [n = 6]) against WT (left), GTKO (middle), and TKO (right) pig PBMCs. Results are expressed as mean ± SEM. The dotted line represents cut-off value (6.4%), below which there is no killing. (**p < 0.01). (B) Comparison of mean CDC of human [n = 9], OWM [n = 24], and NWM [n = 15] sera against WT (left), GTKO (middle), and TKO (right) pig PBMCs. Results are expressed as mean ± SEM. The dotted line represents cut-off value (6.4%). (*p < 0.05, **p < 0.01).
Figure 3 Mean (±SEM) complement-dependent cytotoxicity (CDC, at 25% serum concentration) of sera from various primate species (A), summarized as humans, OWMs, and NWMs (B) to WT, GTKO, and TKO pig PBMCs. (A) Comparison of mean CDC of human [n = 9], baboon [n = 6], rhesus [n = 6], cynomolgus [n = 6], African green [n = 6], capuchin [n = 9], and squirrel [n = 6]) against WT (left), GTKO (middle), and TKO (right) pig PBMCs. Results are expressed as mean ± SEM. The dotted line represents cut-off value (6.4%), below which there is no killing. (**p < 0.01). (B) Comparison of mean CDC of human [n = 9], OWM [n = 24], and NWM [n = 15] sera against WT (left), GTKO (middle), and TKO (right) pig PBMCs. Results are expressed as mean ± SEM. The dotted line represents cut-off value (6.4%). (*p < 0.05, **p < 0.01).CC BY · PubMed Central
Figure 4 Correlation of human (n = 9, black), OWM (n = 24, blue), and NWM (n = 15, red) serum IgM (upper) and IgG (lower) antibody binding with serum complement-dependent cytotoxicity (CDC, at 25% serum concentration) to WT (left), GTKO (middle), and TKO (right) pig PBMCs. On the y axis, the dotted line represents cut-off value (6.4%) below which there is no cytotoxicity. On the x axis, the dotted line represents cut-off value (IgM:1.2, IgG:1.1) below which there is no binding. (*p < 0.05, **p < 0.01). (Note the difference in scale on the x axis between IgM and IgG. Note the difference in scale on the x axis between TKO and others).
Figure 4 Correlation of human (n = 9, black), OWM (n = 24, blue), and NWM (n = 15, red) serum IgM (upper) and IgG (lower) antibody binding with serum complement-dependent cytotoxicity (CDC, at 25% serum concentration) to WT (left), GTKO (middle), and TKO (right) pig PBMCs. On the y axis, the dotted line represents cut-off value (6.4%) below which there is no cytotoxicity. On the x axis, the dotted line represents cut-off value (IgM:1.2, IgG:1.1) below which there is no binding. (*p < 0.05, **p < 0.01). (Note the difference in scale on the x axis between IgM and IgG. Note the difference in scale on the x axis between TKO and others).CC BY · PubMed Central
Figure 5 Comparison of the ratio of complement-dependent cytotoxicity (CDC, at 25% serum concentration) of TKO pig PBMCs to IgM binding in human (n = 9), OWM (n = 24), and NWM (n = 15) sera. Results are expressed as mean ± SEM. (**p < 0.01; N.S. = not significant).
Figure 5 Comparison of the ratio of complement-dependent cytotoxicity (CDC, at 25% serum concentration) of TKO pig PBMCs to IgM binding in human (n = 9), OWM (n = 24), and NWM (n = 15) sera. Results are expressed as mean ± SEM. (**p < 0.01; N.S. = not significant).CC BY · PubMed Central
Figure 6 Human, OWM, and NWM IgM and IgG binding and complement-dependent cytotoxicity (CDC, at 25% serum concentration) to WT, GTKO, and TKO pig PBMCs. Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01; N.S. = not significant). On the y axis, the dotted line represents cut-off value of binding (relative GM: IgM 1.2, IgG 1.1). For CDC on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (Note the difference in scale on the y axis between IgM and IgG).
Figure 6 Human, OWM, and NWM IgM and IgG binding and complement-dependent cytotoxicity (CDC, at 25% serum concentration) to WT, GTKO, and TKO pig PBMCs. Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01; N.S. = not significant). On the y axis, the dotted line represents cut-off value of binding (relative GM: IgM 1.2, IgG 1.1). For CDC on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (Note the difference in scale on the y axis between IgM and IgG).CC BY · PubMed Central
Figure 7 Pre-transplant IgM (upper) and IgG (middle) binding and CDC (at 25% serum concentration) (lower) of recipient baboons receiving pig kidney grafts to GTKO (left) and TKO (right) pig PBMCs. Results are expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value of binding (relative GM) (IgM:1.2, IgG:1.1). For CDC, on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (*p < 0.05, **p < 0.01; ns = not significant).
Figure 7 Pre-transplant IgM (upper) and IgG (middle) binding and CDC (at 25% serum concentration) (lower) of recipient baboons receiving pig kidney grafts to GTKO (left) and TKO (right) pig PBMCs. Results are expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value of binding (relative GM) (IgM:1.2, IgG:1.1). For CDC, on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (*p < 0.05, **p < 0.01; ns = not significant).CC BY · PubMed Central
Figure 8 Baboon survival (A) and rejection-free survival (B) between the TKO and GTKO groups. (A) Baboon survival in the TKO group (red line) (N = 3) was significantly shorter than in the GTKO group (black line) (N = 5) (p < 0.05). (B) Rejection-free pig kidney graft survival in the TKO group was also significantly shorter than in the GTKO group (p < 0.05).
Figure 8 Baboon survival (A) and rejection-free survival (B) between the TKO and GTKO groups. (A) Baboon survival in the TKO group (red line) (N = 3) was significantly shorter than in the GTKO group (black line) (N = 5) (p < 0.05). (B) Rejection-free pig kidney graft survival in the TKO group was also significantly shorter than in the GTKO group (p < 0.05).CC BY · PubMed Central
Figure 9 Pre-transplant IgM (left), IgG (middle) binding and CDC (at 25% serum concentration) (right) of recipient baboons receiving pig kidney grafts to either TKO pig PBMCs (TKO group) or GTKO pig PBMCs (GTKO group). Results are expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value (relative GM) (IgM:1.2, IgG:1.1). For CDC, on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (*p < 0.05, **p < 0.01; ns = not significant).
Figure 9 Pre-transplant IgM (left), IgG (middle) binding and CDC (at 25% serum concentration) (right) of recipient baboons receiving pig kidney grafts to either TKO pig PBMCs (TKO group) or GTKO pig PBMCs (GTKO group). Results are expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value (relative GM) (IgM:1.2, IgG:1.1). For CDC, on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (*p < 0.05, **p < 0.01; ns = not significant).CC BY · PubMed Central
Figure 10 Mean levels (±SEM) of IgM/IgG binding and CDC against GTKO, GTKO/β4 NT2KO, and TKO pig PBMCs in baboon sera (n = 6). Results are expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value of binding (relative GM) (IgM:1.2, IgG:1.1). For CDC, on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (**p < 0.01; ns = not significant).
Figure 10 Mean levels (±SEM) of IgM/IgG binding and CDC against GTKO, GTKO/β4 NT2KO, and TKO pig PBMCs in baboon sera (n = 6). Results are expressed as mean ± SEM. On the y axis, the dotted line represents cut-off value of binding (relative GM) (IgM:1.2, IgG:1.1). For CDC, on the y axis, the dotted line represents cut-off value of cytotoxicity (6.4%). (**p < 0.01; ns = not significant).CC BY · PubMed Central

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Research works printed but not linked: 20

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Authors
Yamamoto, T., Iwase, H., Patel, D., Jagdale, A., Ayares, D., Anderson, D., Eckhoff, D. E., Cooper, D. K. C., & Hara, H.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-06-17
Volume
10
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-020-66311-3
PMC
PMC7300119
Cited by
110
References
0
Language
en
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