Generation of Human Regulatory T Cells Using NOD/SCID/γ _C^NULL Mice Model.

We have reported a NOD/SCID/γ_c^null (NOG) mice model, which enable efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells. Using this model, we investigated whether various subpopulations of T cells were generated in this unique murine microenvironment.

Freshly collected cord blood was depleted of phagocytes with Silica ® followed by CD34 positive selection using auto MACS ®. The purity of CD34 positive cells always exceeded 98%. These cells were transplanted into irradiated NOG mice intravenously. About 3 months after the transplantation, human T cells in peripheral blood, bone marrow and spleen were analyzed by flow cytometry. As we have reported previously, more than half of the human cells seen in the spleen were human CD3⁺ T cells and as many as 30% of them expressed CD4 and CD25 without activation markers such as CD69. To examine if these CD4⁺ CD25⁺ cells have regulatory activity, CD4⁺ CD25⁻ cells were stimulated with anti-human CD3 antibody along with irradiated autologous antigen presenting cells in the presence of limiting dose of CD4⁺ CD25⁺ cells. The inhibition of proliferation by CD4⁺ CD25⁺ cells was analyzed by ³H-thymidine uptake. CD4⁺ CD25⁺ cells successfully suppressed the CD4⁺ CD25⁻ T cell proliferation and RT-PCR analysis revealed the expression of Foxp3, a marker for regulatory T cells, specifically in the CD4⁺ CD25⁺ cell population. These results suggest that regulatory T cells can develop from hematopoietic stem cells in our NOG mice model. As human T cells appear first in the thymus of NOG mice, these regulatory T cells are considered to arise in the murine thymus. Our model provides a new and versatile tool to investigate development and function of human regulatory T cells, which are often difficult to study because of complicated history of infection or genetic differences among individuals.