A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon

Abstract

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.

FIG. 1

CTL lytic activity for HIV-infected patients (P1 through P5 and P7) and a representative control (C4). Fresh PBMCs were stimulated for 2 weeks with vP1291 (encoding HIV Gag, Pro, and Env) and mixed with autologous B-lymphocyte cell lines (BLCL) infected with vSC8 (control for Gag), vDK1 (Gag), vP1170 (control for Env), or vP1174 (Env) at the indicated E:T ratios (x axis). The percent release of ⁵¹Cr-labeled targets relative to spontaneous release in a 5-h assay is depicted (y axis). The spontaneous release of ⁵¹Cr was <20% for all experiments.

FIG. 2

ELISPOT analysis for HIV-positive patients (P1 through P7) and controls (C1 through C7). Fresh or thawed PBMCs were stimulated with the indicated antigen and incubated for 18 h. After being developed, the SFC were counted under a stereomicroscope.

FIG. 3

SL9 HLA A*0201-restricted tetramer staining of thawed PBMCs from HLA-A2-positive patients. Cells were gated by using three parameters (forward and side light scatter and CD3⁺ T cells) in order to focus on the CD3⁺ T lymphocyte population. In addition to the HIV-infected cohort (P1 through P7), two types of control individuals were included (control A was HIV negative and HLA-A2 positive; control B was HIV positive and HLA-A2 negative). The percentage of CD8⁺ T cells that stained with the SL9 tetramer is given in the upper right quadrant of each dot plot.

FIG. 4

Direct comparison of the ELISPOT and tetramer assays. (A) Both the percentage of CD8⁺ T cells and the number of positive cells per million PBMCs are shown. (B) The correlation between the numbers of PBMCs as detected by the SL9 tetramer assay (x axis) or the SL9 ELISPOT assay (y axis) is graphically represented. For the tetramer assay, the forward and side light scatter gates were enlarged to include PBMCs as counted in the hemocytometer, thereby allowing more direct comparison with the “nongated” ELISPOT assay.

FIG. 5

Direct comparison of the tetramer and intracellular cytokine assays. PBMCs derived from individual P8 were either stained with the SL9 tetramer (A) or stimulated with the SL9 peptide for 6 h and stained for the presence of IFN-γ (B). Results are given in terms of the percentage of CD8⁺ T cells that stained with either the tetramer or IFN-γ.