baCD3/CD28-TK⁺ human lymphocytes induce severe GvHD in NOD/scid mice. NOD/scid mice were conditioned, infused intraperitoneally with 20 × 10⁶ PBLs or aCD3- or baCD3/CD28-TK⁺ lymphocytes and followed for human chimerism and incidence of severe GvHD. (A) Human chimerism was assessed weekly by flow cytometry after staining for human CD3 and mouse CD45. Quadrants and percentages were set according to isotype control staining. The inserts report the percentages of cells for each quadrant. Results from a single representative mouse per condition are shown over weeks 1, 2, and 4. (B) At week 1, engrafting cells were analyzed by flow cytometry for CD8 and CD4 or LNGFR expression (top dot plots). After gating for CD3, cells were also analyzed for CD45RA and CCR7 expression (middle dot plots) or for CD27 and CD28 expression (bottom dot plots). Quadrants and percentages were set according to isotype-control staining. The inserts report the percentages of cells for each quadrant. Results from a single representative mouse per condition are shown. (C) Mice were followed for severe GvHD-free survival over time. Severe GvHD was defined as weight loss greater than 5% concomitant to human chimerism greater than 10%. In the study 25 mice were infused with PBLs, 27 with aCD3/CD28-TK⁺, and 27 with baCD3/CD28-TK⁺ lymphocytes. Cells were derived from 7 human donors. For overall results on engraftment and incidence of severe GvHD, see Table 1.