Perforin-independent rejection of transplanted human stem cells

Abstract

The NOD/SCID mouse model is one of the most established model systems for the analysis of human stem cells in vivo. The lack of mature B and T cells renders NOD/SCID mice susceptible to transplantable human stem and progenitor cells. One remaining functional component of the immune system in NOD/SCID mice is natural killer (NK) cells. We rationalized that by eliminating NK cell-mediated cytotoxicity in this model system engraftment of human haematopoietic stem cells could be improved. Thus perforin-deficient NOD/SCID mice (PNOD/SCID) were generated, which display a complete lack of NK cell-mediated cytotoxicity. To test the engraftment potential of human stem cells in PNOD/SCID mice, we compared the repopulating potential of human haematopoietic stem cells in these mice with the repopulating potential in NOD/SCID mice. Upon injection with varying numbers of mononuclear cells from human cord blood, the number of engrafted PNOD/SCID mice was lower (34.8%) than the number of engrafted NOD/SCID mice (64.7%). Similarly, injection of purified CD34(+) human cord blood cells led to engraftment in 32.3% PNOD/SCID versus 60% in NOD/SCID mice. Surprisingly, these results show that the inactivation of cytotoxic activity of NK cells in PNOD/SCID mice did not result in better engraftment with human haematopoietic stem cells. A potential reason for this observation could be that compensatory activation of NK cells in PNOD/SCID mice induces high levels of soluble factors resulting in an environment unfavourable for human stem cell engraftment.

Fig. 1

Lack of natural killer cell-mediated cytotoxicity in PNOD/SCID mice. Two animals of each of the three mouse strains C57/BL6 (B6), NOD/SCID (NOD) and PNOD/SCID (PNOD) were injected with 0·1 mg/0·1 ml poly-IC. Splenocytes were isolated 24 h later and used as effector cells in a cytotoxicity assay using ⁵¹Cr-labelled YAC-1 and RMA-S cells as targets.

Fig. 2

Engraftment of human mononuclear cord blood cells in PNOD/SCID and NOD/SCID mice. PNOD/SCID and NOD/SCID mice were injected with 2·5 × 10⁶ or 3·0 × 10⁶ mononuclear cells from human cord blood. Mice were killed 3–6 weeks after transplantation. Frequencies of human CD45⁺ haemopoietic cells in the murine bone marrow were measured by flow cytometry.

Fig. 3

Engraftment of human haemopoietic stem cells in PNOD/SCID and NOD/SCID mice. PNOD/SCID and NOD/SCID mice were injected with varying numbers of CD34⁺ cells from human cord blood. Mice were killed 6–13 weeks after transplantation. Frequencies of human haemopoietic cells in the murine bone marrow were measured by flow cytometry. The dotted line separates engrafted from not-engrafted mice.