Establishment of a New Immunodeficient Mouse Line Harboring NOD-Type Sirpa with Highly-Efficient Human Hematopoietic Cell Engraftment.

Abstract 2301

To evaluate stem cell potential of human cells in xenotransplant models, a variety of immunodeficient mouse lines have been developed. Depletion of lymphoid cells including T, B and NK cells by introducing with Il2rg^null, and SCID or RAG^null mutations is necessary to avoid rejection of human cells in these models. Interestingly, in mice having these immunodeficiencies, the NOD strain shows even better engraftment of human cells as compared to C57BL/6 or Balb/c strains. Recently, we found that in xenograft rejection, the innate phagocytic reaction of mouse macrophages could occur because murine signal regulatory protein alpha (mSIRPA) on macrophages cannot bind to human CD47 (hCD47). However, NOD-specific polymorphism of mSIRPA allows NOD-type SIRPA to bind hCD47, resulting in inhibition of phagocytic reaction against human cells in this strain at least in vitro. Here, we have established a new immunodeficient BRGS mouse line, C57BL/6.Rag2^nullIl2rg^null mice with NOD-type SIRPA. To test the reconstitution activity of human hematopoiesis in vivo, we transplanted 5 × 10³ CD34⁺CD38⁻ human cord blood cells intrafemorally into C57BL/6.Rag2^nullIl2rg^null (C57BL/6-RG), BRGS or NOD.Rag1^nullIl2rg^null (NOD-RG) mice. At 8 weeks after transplantation, human CD45⁺ cells were not detectable in C57BL/6-RG mice in the bone marrow. Both BRGS and NOD-RG showed successful reconstitution, and their frequency of human CD45⁺ cells in the bone marrow was 59.9 % and 55.8% in average, respectively. The frequency of human CD45⁺ cells was maintained at least until 24 weeks after transplantation. Percentages of CD19⁺ B cells, CD33⁺ myeloid cells and CD34⁺CD38⁻ cells that contain the majority of human hematopoietic stem cells (HSCs) were almost equal between the BRGS and the NOD-RG strains. In the spleen, the majority of human cells were CD19⁺ B cells expressed surface immunoglobulin light chain λ/κ, reflecting their normal maturation. In the thymus, CD4⁺CD8⁺ thymic precursors, and CD4⁺ and CD8⁺ single positive T cells were present, and they expressed surface T cell receptor (TCR)-ab or TCR-gd. These data show that replacement of the C57BL/6-Sirpa with the NOD-Sirpa is sufficient for the C57BL/6-RG strain to gain the human cell engraftment ability equal to the NOD-RG strain. In addition, the BRGS strain has normal complement activity, in contrast to the NOD strain that does not have C5, a component necessary for complement-dependent cytotoxic (CDC) activity. We injected 8 × 10⁵ cells of Raji cells into BRGS or NOD-RG mice, and administered rituximab, an anti-CD20 antibody that has both CDC and ADCC activities, to test the in vivo efficacy of rituximab on transplanted Raji cells. After injection of rituximab, percentages of human CD45⁺ Raji cells were significantly decreased in BRGS mice (15.1 %), whereas percentages of Raji cells in NOD-RG mice were only slightly reduced by rituximab treatment (79.2 %). These data clearly show that the CDC activity of antibodies can operate in the BRGS strain.

Collectively, this study formally proves that the excellent transplantability of human grafts in the NOD strain is explained simply by a single gene mutation, NOD-specific polymorphism of SIRPA, and that the BRGS strain should be very useful in future xenotransplant experiments of human stem cells.