Mouse model of RBC alloimmunization. (A) Mice (n = 38, 6 separate experiments) were given intravenous transfusion of buffy-coated/granulocyte-depleted huGPA RBCs (equivalent to 1-2 packed units) with CpG-ODN adjuvant, followed by weekly transfusions of red cells alone for another 3 weeks. The presence of IgG-specific alloanti-huGPA in plasma from mice was then measured using diluted plasma (1 in 4) and huGPA transgenic RBCs followed by analysis using flow cytometry and is expressed in fluorescent units on the y-axis. Some mice (n = 17) did not receive any transfusions (“control”). The square box represents the cohort with the highest levels of alloantibodies, whereas the oval box includes the group with background control levels of alloanti-huGPA. (B) Red cell survival studies were performed using approximately 50 μL of PKH-26-labeled huGPA red cells transfused into mice with the highest levels of alloanti-huGPA (square box in panel A and line with ● in panel B, n = 5) and into mice with background levels of alloanti-huGPA (oval in panel A and line with ○ in panel B, n = 5). Control B6 mice were transfused with labeled wild-type red cells to show normal red cell survival (…, n = 3). Percentage survival of transfused cells is shown on the y-axis, and time in days is shown on the x-axis. Error bars represent SEM. Recipients with highest levels of alloanti-huGPA in addition to shortened transfused allogeneic RBC survival were designated “responders,” and those with background alloantibody levels plus normal RBC survival were called “nonresponders.”