In vivo distribution of HA-1 CTLs and ex vivo analysis of subcutaneous macrotumors. (A) NOD/scid mice with subcutaneous MDA-MB 231 macrotumors were treated with a single dose of 30 × 10⁶ HA-1 CTLs (clone 1.7) intravenously. HA-1 CTLs were quantified by flow cytometry in lung, liver, spleen, bone marrow, peripheral blood, and the solid tumor at 1, 3, 7, 14, or 21 days after CTL transfer. Depicted is the percentage of viable human CTLs in relation to all measured viable cells in the individual tissues. Bars correspond to means plus or minus SEM (4 mice per measurement time point). (B) HA-1 mRNA expression in single-cell suspensions of MDA-MB 231 macrotumors freshly explanted from 2 individual untreated (ie, without HA-1 CTL infusion) NOD/scid mice, mouse bone marrow, and cultured MDA-MB 231 and 518A2 cells determined with human (top row) and mouse (bottom row) HA-1–specific primers. Weak mouse HA-1 mRNA signals in the explanted tumor samples indicate contamination with mouse hematopoietic cells. (C) Single-cell suspensions of MDA-MB 231 macrotumors freshly explanted from 3 individual untreated NOD/scid mice were lysed by allo–HLA-A2 and HA-1 CTLs. X-axis represents percentage of specific lysis (means plus or minus SEM); y-axis, CTL clones in 4 different effector-target ratios. (D-F) Biopsies of MDA-MB 231 subcutaneous macrotumors explanted from untreated NOD/scid mice were embedded in collagen type I matrix and coincubated with the HA-1 CTL clones 1.7, 2.12, and 3HA15 for 3 days. Shown is a representative example of a human CD8 staining. HA-1 CTLs are present only in the surrounding collagen type I matrix and the tumor border. Arrowheads indicate CD8⁺ CTLs; bar represents 100 μm.