Introduction: Alternatives to human red blood cells (RBCs) might be lifesaving if a blood transfusion is urgently required (e.g., acute blood loss trauma) or in chronic hematologic diseases requiring frequent transfusions (e.g., sickle cell disease). Triple-knockout (TKO) pigs could be a source of RBCs for clinical transfusion because many humans have no preformed antibodies to TKO pRBCs. However, we are still unclear how species incompatibility of CD47/SIRP-α affects the outcome. Old World nonhuman primates (NHPs, e.g., baboons) have antibodies to TKO pig cells, but New World NHPs (e.g., capuchin or squirrel monkeys) more closely mimic humans in their response to TKO pig cells. We report the first successful experimental model using capuchin monkeys for TKO pRBC xenotransfusion.

Methods: In vitro: Sera from (i) healthy human volunteers (n=12), (ii) kidney transplant waitlist patients (n=19), (iii) capuchin monkeys (n=12), and (iv) squirrel monkeys (n=25) were tested by flow cytometry for binding of IgM and IgG to RBCs from TKO pigs (presented as relative geometric mean [rGM]).
In vivo: After 25% of the blood volume was removed from the capuchin monkey (n=2), the same volume of CFSE-labeled TKO pRBCs was transfused. Seven weeks after xenotransfusion, using the same protocol, CFSE-labeled allogeneic capuchin monkey RBCs were transfused. No immunosuppressive therapy was administered. Throughout the experiments, the following parameters were measured - complete blood count, the percentage of RBCs that were of donor (pig or monkey) origin, and IgM/IgG binding and complement-dependent cytotoxicity (CDC) to TKO pRBCs and allogeneic monkey RBCs. At euthanasia, spleen mononuclear cells were isolated from the recipient monkeys to evaluate direct and indirect phagocytosis of TKO pRBCs or allogeneic monkey RBCs by flow cytometry.

Results: In vitro: Binding of serum IgM/IgG to TKO pRBCs was demonstrated in (i) only one of 12 (IgM 1/12 [8.3%], IgG 1/12 [8.3%]) healthy human serum samples, (ii) very few kidney transplant waitlist patients (IgM 2/19 [10.5%], IgG [0%]), (iii) 6 of 12 (50%) capuchin monkeys, and (iv) very few squirrel monkeys (IgM 1/25 [4%], IgG 2/25 [8%]).

In vivo (xenotransfusion followed by allotransfusion in capuchin monkeys [n=2]): After xenotransfusion, (i) survival of TKO pRBCs was for 5 or 7 days whereas allogeneic monkey RBCs survived for >28 days (p<0.01); (ii) direct phagocytosis of TKO pRBCs (5.5%) was significantly greater than of allogeneic monkey RBCs (1.6%) (p<0.05); (iii) indirect phagocytosis of TKO pRBCs (10.5%) was significantly greater than of allogeneic monkey RBCs (2.7%) (p<0.05); (iv) IgM/IgG binding and CDC to TKO pRBCs increased after days 5 and 7, but there was no further increase after allotransfusion,

Conclusions: Although deletion of expression of xenoantigens extended pRBC survival compared to historic controls, novel genetic methods are needed to introduce human ‘protective’ transgenes into pRBCs to enable longer survival after xenotransfusion. (2) Because of their low incidence of preformed anti-TKO antibodies, squirrel monkeys may be preferred to capuchin monkeys as potential recipients of TKO RBC transfusion.