*Purpose: Triple-knockout (TKO) pig RBCs (pRBCs) may be an alternative source for clinical blood transfusion because almost humans have no preformed antibody to TKO pRBCs. However, we are still unclear how important the species incompatibility of CD47/SIRP-α affects the result. Old World NHPs (e.g., baboons, rhesus monkeys) have antibodies to TKO pig cells, but capuchin (New World NHPs) monkeys immunologically mimic humans in respect to their response to TKO pig cells. The aim of this study was to determine survival of (i) TKO pRBCs (xenotransfusion) and (ii) of allogeneic monkey RBCs in capuchin monkeys (allotransfusion) after xenotransfusion in capuchin monkeys.

*Methods: After 25% of the blood volume was removed from capuchin monkey (n=2), the same volume of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled TKO pRBCs was transfused into these monkeys (xenotransfusion). Seven weeks after xenotransfusion, CFSE-labeled allogeneic monkey RBCs were transfused. No immunosuppressive therapy was administered. The percentage of donor blood, complete blood count, IgM/IgG binding, and complement-dependent cytotoxicity (CDC) to TKO pRBCs or allogeneic monkey RBCs were measured after xenotransfusion and allotransfusion. Spleen mononuclear cells were isolated from recipient capuchin monkeys at euthanasia to evaluate direct and indirect phagocytosis of TKO pRBCs or allogeneic monkey RBCs by flow cytometry.

*Results: After xenotransfusion, survival of TKO pRBCs was for 5 or 7 days (Fig. A red) even though direct phagocytosis of TKO pRBCs (5.5%) was significantly greater than of allogeneic monkey RBCs (1.6%) (p<0.01). After allotransfusion, survival of RBCs was significantly longer at >28 days (Fig. A black). After xenotransfusion, IgM/IgG binding (Figs. B and C) and CDC (Fig. D) to TKO pRBCs increased after days 5 and 7, but there was no further increase after allotransfusion. After xenotransfusion Indirect phagocytosis of TKO pRBCs (10.5%) was significantly greater than of allogeneic monkey RBCs (2.7%) (p<0.05).

*Conclusions: (1) TKO pRBCs survived in capuchin monkeys for 5 or 7 days without any immunosuppressive therapy. (2) Loss of TKO pRBCs by direct phagocytosis (due to the species incompatibility of CD47/SIRP-α) is less than by indirect phagocytosis and CDC. (3) TKO pRBCs xenotransfusion in primates does not sensitized to subsequent allotransfusion. (4) TKO pRBCs may be life-saving if no human RBCs are immediately available.

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