Early Recovery of CD4 T Cell Receptor Diversity after “Lymphoablative” Conditioning and Autologous CD34 Cell Transplantation

Example of sequencing clones in batches of 20 until it became obvious that no new distinct sequences would be found.

Persistence of CMV- and EBV-specific CD8 T cell clones, using allophycocyanin (APC)-labeled HLA-viral peptide tetramer analysis. Both patients were positive for HLA-A∗0201; thus, we used NLVPMVATV (CMV peptide) conjugated to HLA-A∗0201-APC tetramer and GLCTLVAML (EBV peptide) conjugated to HLA∗0201-APC tetramer (both purchased from Beckman Coulter) to stain CMV- and EBV-specific clones in blood mononuclear cell specimens by flow cytometry. The cells also were stained by CD3-phycoerythrin antibody and CD8-fluorescein isothiocyanate (FITC) antibody. The density dot plots, showing tetramer-APC fluorescence on the x-axis and CD8-FITC fluorescence on the y-axis, were gated on CD3⁺CD8⁺ lymphocytes. The CMV tetramer staining of the cells from the MS patient (who was CMV-seronegative) served as a negative control for setting the border between tetramer-positive and tetramer-negative cells. The percentages of the tetramer-positive cells (of total CD8 T cells) are shown, indicating that even though the percentages varied, the same EBV-specific CD8 clones in both patients and the same CMV-specific CD8 clone in the CMV-seropositive patient were present both pretransplantation and posttransplantation.