MyD88^–/– mice have impaired adaptive immune responses to HY antigens. HY-specific tetramers were used to quantify the number of anti-HY CD8⁺ T cells in response to full-thickness male allografts. Fourteen days after transplantation, spleen cells were harvested from MyD88^+/+ and MyD88^–/– females, and T cells were purified with magnetic microbeads, stained with KCSRNRQYL HY tetramer (HD^b) conjugated to phycoerythrin and FITC-conjugated anti-CD8, and then analyzed by flow cytometry. (a) Representative flow cytometric plots of HY tetramer staining, showing base-line purified naive T cells in MyD88^+/+ and MyD88^–/– mice; and plots of spleen cells from MyD88^+/+ and MyD88^–/– recipients of MyD88^+/+ and MyD88^–/– skin grafts, respectively, 14 days after transplantation, demonstrating a reduced number of CD8⁺ tetramer-positive cells in MyD88^–/– recipients. Positive control from an HY transgenic animal and specificity control using KAVYNFATC HD^b tetramer are shown. (b) Histogram plot of pooled data from three experiments demonstrating that MyD88^–/– mice manifested an impaired generation of anti-HY CD8⁺ T cells in response to transplantation, compared with WT mice (1.52% vs. 15.79%, P = 0.01).