Figure 4

Human c-mpl⁺ HSC/PC have increased T-lineage repopulation capability compared with the corresponding c-mpl^-- HSC/PC. Small 1 mm³ fragments of human HLA-B8^-- fetal thymus and fetal liver were co-implanted, adjacent to one another, under the renal capsule of scid/scid (SCID) mice, generating one SCID-hu Thy/Liv graft per mouse. Viable, robust 8-week old Thy/Liv grafts (n = 21) were exposed to sublethal irradiation (400 Rads) in vivo, then injected with either 60,000 or 30,000 sorted human HLA-B8⁺ CD34⁺CD38^--/dimc-mpl⁺ or CD34⁺CD38^--/dimc-mpl^-- ABM cells and allowed to engraft for 8 weeks. Three grafts were reserved as non-injected controls. After 8 weeks, the Thy/Liv grafts were harvested, and single cell suspensions prepared from these grafts were stained with PI and a panel of fluorescent-conjugated antibodies to T-lineage markers and HLA-B8, with all appropriate isotype controls. 100,000 events from each graft were acquired on a FACSCalibur™ flow cytometer. Representative examples of CD34⁺CD38^--/dimc-mpl⁺-injected grafts (n = 4), CD34⁺CD38^--/dimc-mpl^---injected grafts (n = 7), and non-injected grafts (n = 3) are shown. A. Using FlowJo^® software, a pseudocolor dot plot of Forward Scatter vs. Side Scatter was generated and a Human Lymphoid Cell Gate was applied to isolate engrafted human mononuclear cell (MNC) events. B. Viable (PI^--) human MNC events were then plotted on CD2-PE vs. HLA-B8-APC dot plot, and a CD2-PE⁺ gate was applied to isolate all viable CD2-PE⁺ MNC events. C. The total CD2-PE⁺ MNC events were next plotted on an HLA-B8-APC histogram plot to apply HLA-B8^-- and HLA-B8⁺ gates. The HLA-B8^-- and HLA-B8⁺ gates were generated from the three non-injected Thy/Liv grafts combined. The HLA-B8^-- gate was defined to include 99.0% of the left-most viable MNC events of the non-injected grafts on an HLA-B8-APC log histogram plot. The HLA-B8⁺ gate was drawn from the end of the HLA-B8^-- gate to the far right of the histogram plot. The percentage of viable (PI^--) CD2-PE⁺ HLA-B8⁺ events, out of the total viable CD2-PE⁺ MNC population, is shown for the representative examples of the three treatment arms. D. CD4-FITC vs. CD8-PE pseudocolor dot plot of viable (PI^--) Human Lymphoid Cell gated events. Gates were applied to depict the thymocyte subsets (CD4⁺CD8⁺, CD4⁺CD8^--, CD4^--CD8⁺). E. Total viable (PI^--) CD4⁺CD8^-- MNC events were plotted on an HLA-B8-APC log histogram plot and the HLA-B8⁺ gate was applied. The percentage of viable donor-derived CD4⁺CD8^--HLA-B8⁺ events out of the total viable CD4⁺CD8^-- MNC events is shown for the representative examples of the three treatment arms. F. Total viable (PI^--) CD4^--CD8⁺ MNC events were plotted on an HLA-B8-APC log histogram plot, the HLA-B8⁺ was applied, and the percentage of viable, donor-derived CD4^--CD8⁺HLA-B8⁺ MNC events out of the total viable CD4^--CD8⁺ MNC population in the Thy/Liv graft was calculated.