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. 2009 Apr;83(7):3138-49.
doi: 10.1128/JVI.02073-08. Epub 2009 Jan 21.

Differential neutralization of human immunodeficiency virus (HIV) replication in autologous CD4 T cells by HIV-specific cytotoxic T lymphocytes

Huabiao Chen  1 , Alicja Piechocka-Trocha, Toshiyuki Miura, Mark A Brockman, Boris D Julg, Brett M Baker, Alissa C Rothchild, Brian L Block, Arne Schneidewind, Tomohiko Koibuchi, Florencia Pereyra, Todd M Allen, Bruce D Walker
Affiliations

Differential neutralization of human immunodeficiency virus (HIV) replication in autologous CD4 T cells by HIV-specific cytotoxic T lymphocytes

Huabiao Chen et al. J Virol. 2009 Apr.

Abstract

Defining the antiviral efficacy of CD8 T cells is important for immunogen design, and yet most current assays do not measure the ability of responses to neutralize infectious virus. Here we show that human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) clones and cell lines derived from infected persons and targeting diverse epitopes differ by over 1,000-fold in their ability to retard infectious virus replication in autologous CD4 T cells during a 7-day period in vitro, despite comparable activity as assessed by gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. Cell lines derived from peripheral blood mononuclear cells stimulated in vitro with peptides representing targeted Gag epitopes consistently neutralized HIV better than Env-specific lines from the same person, although ineffective inhibition of virus replication is not a universal characteristic of Env-specific responses at the clonal level. Gag-specific cell lines were of higher avidity than Env-specific lines, although avidity did not correlate with the ability of Gag- or Env-specific lines to contain HIV replication. The greatest inhibition was observed with cell lines restricted by the protective HLA alleles B*27 and B*57, but stimulation with targeted Gag epitopes resulted in greater inhibition than did stimulation with targeted Env epitopes even in non-B*27/B*57 subjects. These results assessing functional virus neutralization by HIV-specific CD8 T cells indicate that there are marked epitope- and allele-specific differences in virus neutralization by in vitro-expanded CD8 T cells, a finding not revealed by standard IFN-gamma ELISPOT assay currently in use in vaccine trials, which may be of critical importance in immunogen design and testing of candidate AIDS vaccines.

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Figures

FIG. 1.
FIG. 1.
HIV-specific CD8 T cells can potently suppress replication of primary HIV isolates in autologous CD4 cells. (A) Bulk CD8 T cells expanded in vitro from an elite controller (subject 013196g) by stimulation of PBMCs with CD3:CD4-bispecific monoclonal antibody inhibit HIV R5 replication in autologous CD4 T cells at the indicated effector/target cell ratios. The control of uninfected CD4 T cells showed that there were no autologous viruses grown out from the tested subject during the period of the assay. (B) Bulk CD8 T cells directly isolated from peripheral blood of the same subject by positive selection with anti-CD8 antibody-coated magnetic beads suppressed HIV X4 replication in autologous CD4 T cells at a 1:1 ratio of CD8 to CD4 T cells. (C) The Gag epitope KK10-specific, HLA-B*27-restricted CD8 T-cell clone recognized pNL4-3 wild-type virus but did not recognize an engineered escape variant which contains R-to-K and L-to-M mutations within the KK10 epitope and thus abrogates HLA class I binding with the peptide. (D) The B*57-restricted cell line specific for the epitope IW9 in Gag inhibited the R5 and CRF_01 viruses over time, whereas it had no demonstrable effect on HIV SE virus, which contains a single-amino-acid substitution, A-to-P, known to alter the peptide processing.
FIG. 2.
FIG. 2.
CD8 T-cell clones differ in antiviral efficacy depending on antigen specificity. (A and B) HIV-specific CD8 T-cell clones were isolated from HIV-infected individuals' peripheral blood by limiting dilution. The B27-KK10-specific clone was isolated from an elite controller with 1,060 SFC per million PBMCs assessed directly ex vivo in an IFN-γ ELISPOT assay, a B57-TW10-specific clone from a chronic progressor who did not recognize TW10 peptide in the IFN-γ ELISPOT assay at that time, a Cw3-RL9-specific clone from an elite controller with 1,360 SFC per million PBMCs, an A3-ATK-specific clone from an HIV-infected individual during a time the IFN-γ ELISPOT assay was technically not available, and a B57-HW9-specific clone from an elite controller with 1,640 SFC per million PBMCs. These clones were tested in a standard IFN-γ ELISPOT assay (A) and a 4-h chromium release assay with peptide-pulsed autologous or HLA-matched B-LCL targets (B). (C) The same clones were tested for antiviral function using autologous CD4 T cells infected with different primary HIV isolates. Peptide-specific CD8 T-cell clones differed in their antiviral efficacy with similar potency against the R5, SE, and JGC HIV isolates. Data are expressed as log inhibition units, calculated as −log10 (p24 with CTL/p24 without CTL) at day 7 in culture.
FIG. 3.
FIG. 3.
Different antiviral efficacies of epitope-specific CD8 T-cell lines enriched in vitro from PBMCs. (A) The generated cell lines were stimulated in vitro in the presence of peptides representing epitopes in an IFN-γ ELISPOT assay. There was no difference among all lines in terms of the magnitude (SFC per million cells) or specificity (% response) of response specific for Gag (n = 14) versus Env epitopes (n = 12) and calculated as the percentage of the epitope-specific response of the overall HIV-specific CD8 T-cell response. Statistical comparisons were made using the Mann-Whitney test. (B) Example of differences in the antiviral efficacy of epitope-specific CD8 T-cell lines generated from a single subject (013646a) against X4 and R5 viruses during a 7-day period. The control uninfected CD4 T cells showed that there were no autologous viruses grown out from the tested subjects during the period of the assay. (C) Summary of data demonstrating different antiviral efficacies for Gag- and Env-specific cell lines against X4 and R5 in log units of inhibition after 7 days of culture. Inhibition was evaluated in multiple assays for each cell line at least twice (mean ± standard deviation).
FIG. 4.
FIG. 4.
Enrichment for Gag-specific responses enhances neutralization, whereas enrichment for Env-specific responses diminishes neutralization. PBMCs were stimulated in vitro with the cognate HIV Env or Gag epitope (Table 1). After a single round of stimulation starting with bulk PBMCs to establish epitope-specific cell lines (setup), the resultant cell lines were assessed for HIV-specific activity by IFN-γ ELISPOT and in the viral inhibition assay. After progressive enrichment of the setup lines for Gag or Env specificities using targeted epitopes as a stimulus (enriched), assays were repeated. (A) The progressive increase in magnitude (SFC per million cells) and specificity (% responses) for Gag-specific responses enhanced their ability to inhibit viral replication, as measured by log inhibition units (n = 11). (B) The progressive increase in magnitude and specificity for Env-specific responses diminished their ability to inhibit viral replication (n = 12). Statistical comparisons were made using the Wilcoxon matched-pairs test.
FIG. 5.
FIG. 5.
Gag-specific CD8 T-cell lines are more effective than Env-specific lines in control of HIV replication. (A) Significant differences in antiviral efficacy against both R5 and X4 virus were observed between Gag-specific (n = 14) and Env-specific (n = 12) cell lines, using either an R5 virus (left panel) or X4 virus (right panel). (B) Similar significant differences in the ability to inhibit virus replication were detected when we compared Gag-specific (n = 13 for R5; n = 12 for X4) and Env-specific (n = 7 for either R5 or X4) cell lines for which the autologous viral sequences were homologous to the experimental R5 or X4 virus. (C) Antiviral efficacy against both R5 and X4 virus appeared greater for HLA B*27/B*57-restricted lines (n = 5) compared to non-HLA B*27/B*57-restricted lines (n = 21). (D) Among non-HLA B*27/B*57-restricted cell lines, we observed that Gag-specific (n = 10) responses inhibited R5 or X4 virus replication significantly better than Env-specific (n = 11) responses. Statistical comparisons were made using the Mann-Whitney test.
FIG. 6.
FIG. 6.
Gag-specific cell lines are of higher avidity than Env-specific cell lines. (A) An example demonstrates that the indicated cell lines differed markedly in functional avidity by peptide titration in the IFN-γ ELISPOT assay. (B) Summary data demonstrate that Gag-specific responses (n = 10) were of higher avidity than Env-specific responses (n = 8). Statistical comparisons were made using the Mann-Whitney test. (C) Avidity did not correlate with the ability to control viral replication for either Gag-specific responses (left panel; n = 10) or Env-specific responses (right panel; n = 8). Correlation statistics were analyzed using the Spearman correlation.
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