Subjects, Materials, and Methods
Donors Frozen peripheral blood mononuclear cells (PBMC) from healthy donors were screened for tetramer-positive responses to HLA*A0201 complexed with an Epstein-Barr virus (EBV) epitope, a cytomegalovirus (CMV) epitope, or an influenza A (FLU) epitope. HIV-1–infected subjects in this study were recruited from the AIDS clinic at the University of Alabama at Birmingham, as well as from the State University of New York at Stony Brook
Isolation of PBMC PBMC were isolated from fresh heparinized blood by use of ficoll-hypaque density gradient centrifugation or by use of Accuspin tubes (Sigma) and usually were frozen in 90% fetal calf serum (FCS) and 10% dimethyl sulfoxide for later analysis
TetramersHLA-A2 tetramers were synthesized as described elsewhere, using the following epitopes: from the EBV lytic cycle protein, BMLF1, GLCTLVAML; CMV matrix phosphoprotein, pp65, NLVPMVATV; FLU matrix, M1, GILGFVFTL; HIV gag SLYNTVATL; and HIV pol ILKEPVHGV. HLA-A3 tetramers containing the HIV gag epitope RLRPGGKKKHIV and HLA-B8 tetramers containing the HIV nef epitope FLKEKGGL were sythesized as described elsewhere.
Tetramer staining and phenotypingTetramer (0.5–1 μg) was added to 1–2×106 PBMC in PBS containing 1% FCS and 0.1% sodium azide (fluorescence-activated cell sorter [FACS] buffer). Cells were incubated in the dark for 20 min at room temperature. Antibodies were added to a final concentration, as suggested by the manufacturer (see below), and cells were incubated in the dark at 4°C for 20 min. Antibody clones were as follows: CD45RA-FITC clone HI100, CD28-APC clone CD28.2, CD27-APC Clone MT21 (Pharmingen), and CD8-PerCP Clone SK1 (Becton Dickinson). Cells were washed with cold FACS buffer and were resuspended in 400–500 μL of FACS buffer containing 1% formaldehyde. Cells were acquired on a FACS Calibur (Becton Dickinson) instrument within 24 h of staining and were analyzed by use of Cellquest software (Becton Dickinson). For analysis of tetramer-positive cells, the lymphocyte gate, R1, was taken from forward and side scatter plots. R1-gated cells were plotted for CD8 expression versus side scatter, and the R2 gate was drawn around the CD8+ cells. R1- and R2-gated cells were plotted for tetramer staining versus side scatter, and the R3 gate was drawn around the tetramer-positive cells. Tetramer-positive cells were plotted for CD27 versus CD45RA expression or for CD28 versus CD45RA expression. Quadrants for determining negative and positive populations were taken from CD8-gated cells plotted for expression of the relevant markers. Reproducibility of the 2 stains was determined from multiple independent stainings of cells from a healthy donor with a CMV-specific tetramer and the cocktails used in this study. Assays performed on repeated identical samples demonstrated that the range in staining for any combination of 2 antibodies, whether it represented 80% or 10% of the tetramer-positive cells, was about ±3%. Full phenotyping when cells are mixed for CD27 expression uses the fact that, for CD8+ cells, the CD27− cells are all contained within the CD28− cells (data not shown), the full subset distribution is determined by subtracting the CD45RA+CD27− cells from the CD45RA+CD28− cells and the CD45RA−CD27− cells from the CD45RA−CD28− cells.
Measurement of PVL and absolute CD4+ T cell counts T lymphocyte subgroups were quantified by means of flow cytometry. Plasma was processed, stored at −70°C, and subsequently assayed for HIV RNA (Amplicor and UltraSensitive assays; Roche)