T-cell receptor-mediated anergy of a human immunodeficiency virus (HIV) gp120-specific CD4(+) cytotoxic T-cell clone, induced by a natural HIV type 1 variant peptide

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection triggers a cytotoxic T-lymphocyte (CTL) response mediated by CD8(+) and perhaps CD4(+) CTLs. The mechanisms by which HIV-1 escapes from this CTL response are only beginning to be understood. However, it is already clear that the extreme genetic variability of the virus is a major contributing factor. Because of the well-known ability of altered peptide ligands (APL) to induce a T-cell receptor (TCR)-mediated anergic state in CD4(+) helper T cells, we investigated the effects of HIV-1 sequence variations on the proliferation and cytotoxic activation of a human CD4(+) CTL clone (Een217) specific for an epitope composed of amino acids 410 to 429 of HIV-1 gp120. We report that a natural variant of this epitope induced a functional anergic state rendering the T cells unable to respond to their antigenic ligand and preventing the proliferation and cytotoxic activation normally induced by the original antigenic peptide. Furthermore, the stimulation of Een217 cells with this APL generated altered TCR-proximal signaling events that have been associated with the induction of T-cell anergy in CD4(+) T cells. Importantly, the APL-induced anergic state of the Een217 T cells could be prevented by the addition of interleukin 2, which restored their ability to respond to their nominal antigen. Our data therefore suggest that HIV-1 variants can induce a state of anergy in HIV-specific CD4(+) CTLs. Such a mechanism may allow a viral variant to not only escape the CTL response but also facilitate the persistence of other viral strains that may otherwise be recognized and eliminated by HIV-specific CTLs.

FIG. 1

Functional response of Een217 cells to HIV-1 gp120-derived peptides. (A) Proliferation of Een217 cells in response to DR4-expressing L cells pulsed with the indicated concentrations of the agonist antigenic ligand (PV22) or natural peptide variants from the CDC42, EL1, Z3, and HXB2 strains of HIV-1. Note that the HXB2-derived sequence can induce the proliferation of Een217 cells but is less potent than the PV22 peptide. (B) Cytotoxic activity of Een217 cells toward ⁵¹Cr-loaded DR4⁺ L cells pulsed with the indicated concentrations of peptides. In this experiment, the cells were cocultured for 6.5 h with an effector-to-target ratio of 20:1 as described elsewhere (57). Note that the HXB2 peptide is at least 100 times less efficient than the PV22 peptide in this assay. Each determination was made in triplicate. The results shown are representative of three independent experiments.

FIG. 2

Induction of Een217 T-cell anergy by pretreatment with the HXB2 natural variant peptide. Een217 cells were cocultured for 48 h with APCs alone or APCs pulsed with 10 nM peptide, rested for 48 h, and restimulated with 10 nM PV22 in a standard proliferation assay. Each determination was made in triplicate. The percentage of inhibition obtained with each peptide is indicated. The data presented are representative of three independent experiments.

FIG. 3

Inhibition of PV22-induced Een217 T-cell cytotoxicity by pretreatment of the cells with the HXB2 peptide. (A) Cytotoxic activity of Een217 cells, pretreated with culture medium alone for 48 h, toward ⁵¹Cr-loaded DR4⁺ L cells pulsed with the indicated concentrations of the PV22 peptide. (B) Cytotoxicity of Een217 cells, pretreated with the indicated concentrations of the HXB2 peptide for 48 h, and then stimulated with ⁵¹Cr-loaded DR4⁺ L cells pulsed with 10 nM PV22. The specific lysis measurement obtained with 10 nM PV22 (A) is represented by the asterisk in panel B. The data presented in panels A and B were derived from the same experiment.

FIG. 4

Modulation of the TCR-CD3 complex (A) and CD4 (B) surface expression levels induced by stimulation of Een217 T cells for 48 h with the indicated concentrations of the PV22-, HXB2-, and CDC42-derived peptides. The data are presented as relative expression index, representing the ratio of the mean fluorescence intensity of each surface antigen in peptide-stimulated cells to that measured in nonstimulated Een217 cells (mean fluorescence intensities for CD3 and CD4 surface expression in nontreated cells were 8.97 and 4.54, respectively). In all cases, the proportion of TCR-CD3- and CD4-positive cells remained unchanged, indicating that the surface density of these molecules decreased following peptide treatment.

FIG. 5

Induction of Zap-70 tyrosine phosphorylation and association with the TCRζ chain in response to PV22 and HXB2 stimulation of Een217 cells. (A) Immunoprecipitation of Zap-70 from APCs alone or Een217 cells stimulated for 5 min with DR4-expressing L cells pulsed overnight with the indicated concentrations of PV22 and HXB2. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with antiphosphotyrosine antibodies (αPTyr; upper panel). Note that PV22 induced the phosphorylation of Zap-70 and its association with the fully phosphorylated form of TCR ζ, which appears as a p21/p23 doublet. The membrane was stripped and reblotted with a Zap-70-specific MAb (αZap-70) to ensure that equal amounts of Zap-70 were present in all immunoprecipitates (lower panel). As expected, Zap-70 was absent from anti-Zap-70 immunoprecipitates in assays performed on APCs alone (first lane of each panel). (B) Cell lysates of APCs or Een217 cells stimulated as described above were incubated in presence of ∼5 μg of the purified Zap(SH2)₂-GST fusion protein. The Zap(SH2)₂-GST-bound material was precipitated with glutathione-Sepharose beads and analyzed by immunoblotting with antiphosphotyrosine antibodies. The results presented were reproduced in four independent experiments. IgH, immunoglobulin heavy chain.

FIG. 6

Inhibition of PV22-induced phosphorylation of TCRζ by pretreatment of Een217 cells with the HXB2 peptide. Een217 cells were preincubated for 48 h with APCs alone or APCs pulsed with 1, 10, or 100 nM HXB2 or CDC42 peptide. The Een217 cells were recovered, rested for 48 h, and restimulated for 5 min with APCs alone (lane 2) or APCs pulsed with 10 μM of PV22 (lanes 3 to 9). The TCRζ chain was then isolated from cellular lysates by using the Zap(SH2)₂-GST fusion protein and analyzed by immunoblotting with antiphosphotyrosine antibodies. The tyrosine-phosphorylated p21/23 protein originated from the Een217 T cells, as T-cell-free APCs, used as a negative control (lane 1), did not contain this phosphopeptide. Note that HXB2 attenuated the PV22-induced ζ phosphorylation, whereas the CDC42 peptide, which does not induce Een217 T-cell anergy, did not. Identical results were obtained in two independent experiments.

FIG. 7

Prevention and reversion of HXB2-induced Een217 T-cell anergy by exogenous IL-2. Een217 T cells were pretreated with either medium alone or 10 nM peptide (in the presence of DR4-transfected L cells) for 48 h, separated from the APCs, rested for 48 h, and finally stimulated with 10 nM PV22 peptide as before. The anergy assay (A) was performed in the absence of IL-2. The addition of IL-2 (50 U/ml) during the first peptide stimulation (B) or during the 48-h rest period (C) prevented or reverted the induction of anergy by the HXB2 peptide. All determinations were made in triplicate.