Expansion of OT-I T cells in response to transfusion of OVA-RBCs. OT-I splenocytes (20 × 10⁶) were adoptively transferred into B6.Thy1.1 mice. One day later, mice underwent transfusion with 100 μL OVA-RBCs, 100 μL HEL-RBCs, or soluble OVA. One microgram soluble OVA was used because this was the amount of OVA contained on 100 μL OVA-RBCs (see Figure 1). To control for any changes in OVA immunoreactivity as a result of the crosslinker, the soluble OVA used was LC-SPDP-modified OVA. There were 5 animals in each experimental group; 2 animals in the control group received no transfusions. Three days after transfusion, splenocytes were isolated from recipient animals, and OT-I T cells were visualized by staining with anti-CD8 and tetramer (A-E) or anti-CD8 and anti-Thy 1.2 (F-J). The legitimacy of the gates used to identify OT-I cells was confirmed by staining splenocytes from mice that had received no OT-I T cells (E,J). Total numbers of OT-I T cells were calculated by counting the number of splenocytes recovered from each animal and multiplying by the resulting percentages of CD8⁺ tetramer⁺ cells. Representative flow cytometry plots from individual animals are presented (A-J). Five animals were included in each group that underwent transfusion, and the numerical averages for each group are presented in Table 1. Standard deviations were calculated by combining the results of all animals in a given group using total numbers of cells. Probabilities were determined as 2-tailed values generated using an unpaired t test. This experiment was performed 4 times with similar results.