BALB/c- Specific L29V Polymorphism In SIRPA Determines The Efficient Engraftment Of Human Hematopoiesis In Xenogeneic Model

It has been shown that in xenotransplantation of human cells into immunodeficient mice, the mouse strain background is critical. For example, the non-obese diabetic (NOD) strain is most efficient, the BALB/c is moderate, and the C57BL/6 is inefficient for human cell engraftment. We have shown that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows NOD SIRPA to bind human CD47, and the resultant “don’t eat me” signaling by this binding prevents host macrophages to engulf human grafts, thereby inhibiting rejection. We then developed the C57BL/6.Rag2^nullIl2rg^null strain harboring NOD-specific Sirpa, named the BRGS mouse. The efficiency of human cell engraftment in BRGS mice was equal to that of NOD.Rag2^nullIl2rg^null mice. These findings clearly show that in addition to depletion of lymphocytes, inactivation of phagocytosis via the CD47-SIRPA interaction is one of the critical determinants to establish an efficient xenogeneic transplantation system. Here we tested whether the efficient xenotransplantation capability of the BALB/c strain is also mediated by the SIRPA-CD47 self-recognition system. BALB/c SIRPA was capable of binding to human CD47 at an intermediate level between those of C57BL/6 SIRPA and NOD SIRPA. Consistent with its binding activity, BALB/c-derived macrophages exhibited a moderate inhibitory effect on human long-term culture-initiating cells in in vitro cultures, and showed moderate phagocytic activity against human hematopoietic stem cells. DNA sequencing of the SIRPA IgV domain located in the CD47 binding site in each strain was performed. We identified 2 SNPs unique to BALB/c mice, and 3 SNPs unique to both BALB/c and NOD mice. Among these 5 SNPs, BALB/c strain has a Val residue at the 29^th amino acid, whereas all other mouse strains have a Leu residue. Introduction of C57BL/6 SIRPA carrying the L29V mutation into C57BL/6 conferred the binding affinity to human CD47 equivalent to that of BALB/c SIRPA, indicating that L29V mutation at least contributes to the enhanced binding of BALB/c SIRPA to human CD47. Thus, BALB/c-specific L29V polymorphism in SIRPA renders to be able to recognize human CD47. Our data strongly suggest that the mouse strain effect on xenogeneic engraftment might be ascribed mainly to “macrophage tolerance” based on the binding affinity of strain-specific polymorphic murine SIRPA with human CD47. This information should be useful to develop a novel immunodeficient strain with superior efficiency for xenogeneic transplantation of human cells.