Frequencies of ex vivo-activated human immunodeficiency virus type 1-specific gamma-interferon-producing CD8+ T cells in infected children correlate positively with plasma viral load

Abstract

HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-gamma)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A*0201-positive HIV-infected children. The IFN-gamma production in response to stimulation with two HLA-A*0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/10(6) peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-gamma. Most importantly, we found that the frequencies of IFN-gamma-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-gamma-producing HIV-specific CD8+ T-cell subset is dependent upon continuous antigenic stimulation.

FIG. 1.

(A and B) IFN-γ production by HIV-1-specific CD8⁺ T cells of HLA-A∗0201-negative and HLA-A∗0201-positive children stimulated with peptides SL9 and IV9. Fresh PBMC were stimulated overnight with either peptide SL9 (A) or peptide IV9 (B) at 1 μg/ml. SFC were enumerated in a 16-h ELISPOT assay. Mean values from triplicate wells are shown. (C) Peptide SL9 induces IFN-γ production by CD8⁺ T lymphocytes. Total PBMC and CD8-depleted PBMC from the HIV-infected patient EM78 were stimulated overnight with peptide SL9 at 1 μg/ml. SFC were enumerated in a 16-h ELISPOT assay. Mean values (and standard deviations) from triplicate wells are shown. CD8⁺ CD3⁺ T cells were 54 and 0.8% in total and CD8-depleted PBMC, respectively.

FIG. 2.

IFN-γ production by HIV-1-specific CD8⁺ T cells of HLA-A∗0201-positive patients following stimulation with free peptide and peptide bound to HLA-tetramers. (A) Fresh PBMC were stimulated overnight with either peptide SL9 at 1 μg/ml or HLA-A∗0201-SL9 tetramer at 1 μg/ml. (B) Fresh PBMC were stimulated overnight with peptide IV9 at 1 μg/ml or HLA-A∗0201-IV9 tetramer at 1 μg/ml. SFC were enumerated in a 16-h ELISPOT assay, and results are expressed as SFC per 10⁶ PBMC. Mean values from triplicate wells and the linear regression curves are shown.

FIG. 3.

Frequency of HIV-1-specific CD8⁺ T cells determined by ELISPOT and tetramer assays. Results from 17 HLA-A∗0201-positive patients are shown for the SL9 (A) and the IV9 (B) epitopes. SFC numbers and tetramer binding cells are expressed as percentages of CD8⁺ T cells. The linear regression curves are shown.

FIG. 4.

Correlations between frequencies of HIV-1-specific CD8⁺ T cells and plasma viral load. The frequencies of SL9-specific CD8⁺ T cells (A and C) and IV9-specific CD8⁺ T cells (B and D) are represented as function of the plasma viral load. (A and B) ELISPOT assay; (C and D) tetramer assay. The linear regression curves are shown.