Suboptimal stimulation by weak agonist epitope variants does not drive dysfunction of HIV-1-specific cytotoxic T lymphocyte clones

Abstract

Objective: To assess whether weakly recognized epitope variants induce anergy in HIV-1-specific CD8 T lymphocyte (CTL) clones as a mechanism of dysfunction.

Design: HIV-1-specific CTL clones were exposed to suboptimally recognized epitope variants, and screened for anergy and other T-cell dysfunction markers, and subsequent capability to kill target cells bearing index epitope.

Methods: In addition to the optimally recognized index epitope, two suboptimally recognized epitope variants were selected based on titration curves for killing of peptide-labeled target cells by three HIV-1-specific CTL clones targeting the epitopes SLYNTVATL (Gag 77-85, A02-restricted), RPAEPVPLQL (Rev 66-75, B07-restricted), and KRWIIMGLNK (Gag 263-272, B27-restricted). Consequences of suboptimal stimulation were assessed by cytokine secretion, gene expression, and capacity to kill index epitope-labeled target cells upon rechallenge.

Results: Suboptimal recognition of epitope variants reduced cytokine production by CTL similarly to reduction in killing of target cells. Gene expression profiles after suboptimal stimulation demonstrated no patterns consistent with T-cell dysfunction due to anergy, exhaustion, or apoptosis. Preexposure of CTL to epitope variants had no discernable impact on their subsequent capacity to kill index epitope-bearing target cells.

Conclusion: Our data explore the hypothesis that poorly recognized epitope variants not only facilitate HIV-1 evasion of CTL recognition, but also induce CTL dysfunction through suboptimal signaling causing anergy. However, the results do not suggest that suboptimal signaling induces anergy (or exhaustion or apoptosis), indicating that the major role of CTL epitope variation is likely viral escape.

Figure 1.. Identification of altered peptide ligand epitopes spanning a range of reduced agonism for triggering cytolysis by CTL clones.

For each CTL clone, two epitope variants were selected to span a broad range of reduced recognition as defined by triggering of target cell killing across varying peptide concentrations. For the remainder of this study these were termed index (), variant 1 (), or variant 2 (), where the index was the best recognized and variant 2 was the least recognized epitope sequence. A. For clone S00001-SL9-3.23T, these were SLYNTVATL (index), SLFNTVAVL (variant 1), and SLFNTIATL (variant 2); results are representative of four independent experiments. B. For clone S42758-RL10-3.22, these were RPAEPVPLQL (index), RPVEPVPLQL (variant 1), and RPTEPVPFHL (variant 2); results are representative of three independent experiments. C. For clone S00076-KK10-10.37, these were KRWIIMGLNK (index), KRWIILGLNK (variant 1), and KKWIILGLNK (variant 2); results are representative of three independent experiments.

Figure 2.. CTL epitope variants that are weak agonists for triggering killing are similarly weak agonists for triggering cytokine release functions.

The concentrations of secreted IFN-γ, TNF-α, IL-2, MIP-1α and MIP-1β after six hours of stimulation with varying concentrations of index (), variant 1 (), or variant 2 () peptide are shown for CTL clones S00001-SL9-3.23T (A), S42758-RL10-3.22 (B), and S00076-KK10-10.37 (C). The plots indicate average values with error bars representing one standard deviation for three independent experiments.

Figure 3.. Gene expression changes induced by suboptimal stimulation.

Stimulation-induced differential transcription is expressed as fold-Δ (when baseline expression was readily detectable) or ΔMFI (when baseline expression was very low or undetectable) relative to unstimulated controls after 6 hours of stimulation with varying concentrations of index (), variant 1 (), or variant 2 () peptide are shown for CTL clones S00001-SL9-3.23T (A), S42758-RL10-3.22 (B), and S00076-KK10-10.37 (C). The indicated relative levels of transcripts for proteins associated with activation/metabolism (black labels), exhaustion (red labels), apoptosis (green labels), and anergy (blue labels) reflect average values from three independent experiments. * indicates undetectable or minimally detectable expression.

Figure 4.. Suboptimal stimulation of CTL does not cause aberrant transcription of markers of T cell dysfunction.

Selected data from Figure 3 are plotted in detail for changes in mRNA levels (relative to unstimulated controls) for CD25, Tim-3, Bim, DGK-α, and Egr2 for CTL clones S00001-SL9-3.23T (A), S42758-RL10-3.22 (B), and S00076-KK10-10.37 (C) for index (), variant 1 (), or variant 2 () peptides. Plots represent average values with error bars representing one standard deviation for three independent experiments.

Figure 5.. Suboptimal pre-stimulation of HIV-1-specific CTL does not reduce subsequent cytolytic function.

CTL clones S00001-SL9-3.23T (A), S42758-RL10-3.22 (B), and S00076-KK10-10.37 (C) were pre-exposed to the indicated concentrations of index (), variant 1 (), or variant 2 () peptide presented by irradiated APC matched by the restricting HLA type. After 48 hours, killing of ⁵¹Cr-labeled target cells pulsed with the index epitope was assayed. Target cells for clones S00001-SL9-3.23T, S42758-RL10-3.22, and S00076-KK10-10.37 were T1, Jurkat, and autologous EBV-transformed B cells respectively. Dotted horizontal lines represent the levels of killing by control CTL pre-exposed to APC without peptide. Graphs are representative examples from three independent experiments for each CTL clone.