The Efficient Engraftment of Human Hematopoiesis in the Balb/c Strain Is Mounted by Balb/c-Specific SIRPA Polymorphism That Enhances Binding Affinity to Human CD47,

Abstract 4008

Various immunodeficient mouse lines for human hematopoietic assay have been developed. It is well recognized that in addition to disruption of the lymphoid system, the strain background such as NOD and Balb/c is critical to achieve efficient xenotransplantation. We have reported that in the NOD strain, NOD-specific polymorphism of SIRPA allows the mouse SIRPA to bind human CD47, preventing activation of host macrophages to engulf human hematopoietic cells and/or to cytokine production, and therefore can inhibit graft rejection of human cells (Takenaka et al., Nature Immunology, 2007). Here we tested whether the efficient xenotransplantation capability in the Balb/c strain is decided also by the SIRPA-CD47 self-recognition system. We have also reported that the efficiency of human to mouse xenotransplantation in hematopoiesis can be measured by maintenance of human hematopoiesis in vitro in chimeric long-term culture (LTC) assay, where human hematopoietic cells are cultured in the presence of murine macrophages. Different doses of macrophages were seeded on MS-5 stromal cells with lineage negative cord blood cells, and 4–5 weeks after the initiation of culture, the number of colony-forming cells (CFCs) was counted. In this system the addition of B6-derived murine macrophages severely suppressed human LTC-initiating cells (LTC-IC), whereas replacement of B6-derived macrophages with NOD-derived macrophages significantly enhanced the maintenance of human LTC-IC in vitro. Interestingly, Balb/c-derived macrophages had an intermediate effect between those of B6 and NOD-derived macrophages. These results are reasonably corresponding to the efficiency of human hematopoietic cell engraftment in immunodeficient mice of each background strain. To test if this effect is derived from binding of murine SIRPA and human CD47, we performed protein-binding assays of these molecules. B6 SIRPA never binds to human CD47. NOD SIRPA showed a very high binding affinity, while Balb/c SIRPA showed an intermediate affinity (Kd=2.501 for NOD, 3.076E+2 for Balb/c, and 4.974E+10 for B6). We then cloned the SIRPA cDNA from Balb/c and NOD strains, and enforced to express these molecules in HeLa cells by using lentivirus vectors. HeLa cells with enforced NOD and Balb/c SIRPA also showed the similar strong and intermediate affinity to human CD47, respectively, indicating that strain-specific SIRPA decides its binding capability with human CD47. We then enforced to express SIRPA derived from B6, NOD, or Balb/c strains into B6 macrophages, and tested their inhibitory effect on the human LTC-IC assay. Again, the addition of macrophages with enforced B6 SIRPA into cultures inhibited human LTC-IC, whereas those with enforced NOD SIRPA promoted maintenance of human LTC-IC. The addition of macrophages with Balb/c SIRPA again showed an intermediate effect. Because we have also reported the high binding affinity to human CD47 in the NOD strain is caused by alteration of amino-acid sequences of the IgV domain of SIRPA caused by the NOD-specific single nucleotide polymorphisms (SNPs), DNA sequencing of the SIRPA domain in each strain was performed. We found 2 independent Balb/c-specific SNPs. One of the SNPs causes the exchange of the 29^th amino acid leucine with valine (L29V). We then transduced the SIRPA with L29V mutation into HeLa cells, performed the binding assay for human CD47, and found that cells expressing L29V SIRPA had the affinity equivalent to HeLa cells transduced with Balb/c SIRPA. Thus, the Balb/c-specific L29V polymorphism of SIRPA allows moderate binding to human CD47, and SIRPA signaling evoked by this binding maintains human LTC-IC activity presumably by inhibiting Balb/c macrophages to engulf cultured human cells. These data collectively suggest that the efficiency of mouse to human xenotransplantation is decided by “macrophage tolerance” based on the binding affinity of human CD47 to strain-specific murine SIRPA. Our findings should be useful to establish a novel immunodeficient strain with more efficient xenotransplantation capability for human cells.