Identification of the CTL-1B9 minimal minor H epitope. (A) Interferon-γ production from CTL-1B9 against HLA-A*2402–transduced 293T cells transfected with plasmid encoding full-length BCL2A1 cDNA cloned from either the recipient (Rt) from whom CTL-1B9 was isolated or his donor (Do). Rt B-LCL and Do B-LCL were used as positive and negative controls, respectively. Secreted interferon-γ was measured by ELISA and is expressed in arbitrary units (AUs) corresponding to optical density at 630 nm. Results are typical of 2 experiments and data are the mean plus or minus SD of triplicates. (B) A peptide reconstitution assay was conducted to determine the minimal epitope for CTL-1B9. Nonameric peptide (DYLQCVLQI), 2 nonameric peptides shifted by one amino acid to N- or C-terminus, N- and C-terminal extended decameric peptides, and its allelic counterpart (DYLQYVLQI) were synthesized and tested by adding to antigen-negative donor B-LCL at 10 nM in a standard ⁵¹Cr release assay. Results are typical of 2 experiments and data are the mean plus or minus SD of triplicates. (C) Titration of the candidate minor H peptide by epitope reconstitution assay. Chromium-labeled donor B-LCLs were distributed to wells of 96-well round-bottomed plates, pulsed with serial dilutions of the indicated peptides for 30 minutes at room temperature, and then used as targets for CTL-1B9 in a standard ⁵¹Cr release assay. A cysteinylated peptide (indicated by an asterisk) was included as an alternative form of the potential epitope. Results are typical of 2 experiments. (D) Tracking of ACC-1^C–specific T cells in the recipient's peripheral blood. In order to longitudinally analyze the kinetics of the ACC-1^C–specific CTLs in peripheral blood from the patient from whom CTL-1B9 was established, a real-time quantitative PCR was conducted. Complementary DNAs of peripheral blood mononuclear cells from the donor and patient before and after HSCT were prepared from the patient. Real-time PCR analysis was performed using a TaqMan assay as described previously.⁹  The primers and fluorogenic probe sequences spanning the CTL-1B9 complementarity-determining region 3 (CDR3) were used to detect T cells carrying the CDR3 sequences identical to that of CTL-1B9. The primers and fluorogenic probe sequences spanning constant region of TCR beta chain (TCRBC) mRNA were used as internal control. Samples were quantified with the comparative CT method. The delta CT value was determined by subtracting the average CT value for TCRBC from the average CTL-1B9 CDR3 CT value. The standard curve for the proportion of CTL-1B9 among TCRαβ⁺ T cells was composed by plotting mean delta CT values for each ratio, and the percentages of T cells carrying the CDR3 sequence identical to CTL-1B9 were calculated by using this standard curve. During this period, quiescent chronic GVHD, which required steroid treatment, developed; however, involvement of immune reaction to ACC-1^C minor H antigen was unlikely since its frequency increased even after resolution of most chronic GVHD symptoms. c-GVHD, chronic GVHD; CSA, cyclosporine A; PSL, prednisolone; WBC, white blood cell count.