Human T-cell development in NSγ mice. (A-E) The development of human T cells was characterized in NSγ thymuses. In some thymuses, the human CD45⁺ CD3⁺ T cells included CD4⁺ CD8⁺ “double-positive” T-cell progenitors (A), whereas other thymuses contained phenotypically mature “single-positive” T cells (B). High percentages of CD4⁺ CD8⁺ T-cell progenitors were observed in thymuses across a range of transplant cell doses (C). Similarly, high copy numbers of human TRECs could be detected in thymus cell preparations across a range of transplant cell doses (D). In 8 of 9 mice that expressed TRECs, at least 25% of the CD3⁺ cells were CD4⁺ CD8⁺ T-cell progenitors (E). (F-M) Human CD3⁺ T cells isolated from NSγ spleens were tested for their responsiveness to mitogens. In spleen cell preparations, the human CD3⁺ cells had matured into CD4⁺ or CD8⁺ “single-positive” T cells, which expressed CD28 (F-G). The T cells were costimulated using immobilized antibodies directed against CD3 and CD28 and cultured with (n = 6) or without irradiated HUVEC cells (20 Gy; n = 4). After 7 days, human CD45⁺ CD3⁺ T cells were present in the cultures, and expressed the CD25 (IL-2Rα) activation antigen (H-I). Both CD4⁺ and CD8⁺ were detected; however, most frequently, there was a net loss of T cells in the cultures (J,L). Cultures established CD3⁺ CB T cells contained similar CD4⁺ and CD8⁺ cells, but the numbers of cells increased dramatically in 7 days (K,M).