Early induction and maintenance of Env-specific T-helper cells following human immunodeficiency virus type 1 infection

Abstract

Mounting evidence points to a role for CD4+ T-helper (Th) cell activities in controlling human immunodeficiency virus type 1 (HIV-1) infection. To determine the induction and evolution of Th responses following acute infection, we prospectively analyzed Env- and Gag-specific Th responses longitudinally for 92 patients with acute (n = 28) or early (n = 64) HIV-1 infection (median, 55 days postinfection [DPI]). The probability of detecting HIV-1-specific lymphoproliferative responses was remarkably low, and when present, the responses were more likely to be Gag specific than Env specific (16 versus 5%). Env-specific responses were significantly more common in patients presenting at <30 DPI than in those presenting at 30 to 365 DPI (21 versus 0.5%, P = 0.001). By contrast, Gag-specific responses occurred with similar frequencies among subjects presenting at <30 DPI and 30 to 365 DPI (13 versus 17%, P = 0.6). After treatment, and regardless of the duration of infection before therapy, Gag-specific Th responses predominated. Furthermore, some acutely infected subjects lost detectable Env-specific Th proliferative responses, which failed to reemerge upon treatment. Detailed analysis for one such subject revealed Env-specific lymphoproliferation at 11 DPI but no detectable Env-specific lymphoproliferation or ex vivo gamma interferon (IFN-gamma) secretion at multiple subsequent time points. Env-specific CD4+ T-cell clones from 11 DPI recognized six epitopes in both conserved and variable regions within gp120 and gp41, exhibited major histocompatibility complex-restricted cytotoxicity, and secreted high levels of antiviral cytokines. T-cell receptor clonal transcript analyses and autologous virus sequencing revealed that Th cells induced during acute infection were maintained and there were no Th escape mutations. Subsequent analysis for this subject and six of seven others revealed detectable IFN-gamma-secreting cells, but only following in vitro gp160 stimulation. In summary, we conclude that Env-specific Th responses are elicited very early in acute infection and may precede Gag-specific responses. The inability to detect Env-specific Th responses over time and despite antiretroviral therapy may reflect low frequencies and impaired proliferative capacity, and viral escape is not necessary for this to occur.

FIG. 1.

HIV-1 Gag and Env-specific Th responses of 62 untreated subjects with acute or early HIV-1 infection. All assays were performed with fresh PBMC. Each symbol represents the measurement for an individual subject at enrollment. The net counts per minute for the positive Env-specific responses ranged from 1,111 to 7,696 (median, 3,479), and for the positive Gag-specific responses, they ranged from 1,128 to 26,956 (median, 3,917).

FIG. 2.

Characterization of Env-specific Th responses of subject 1212. (A) Effect of combination ART on plasma viremia (RNA copies/milliliter) and CD4⁺ T-cell count (no. of cells/cubic millimeter) for subject 1212. The subject presented on day 11, and treatment was initiated on day 16 of infection. (B) Effects of ART on HIV-1-specific Th responses measured by lymphoproliferation and indicated by S.I. All assays were performed with fresh PBMC. (C) Env- specific CD4⁺ T-cell clones established from peripheral blood taken on day 11 of infection. Clones map to epitopes in gp120 and gp41 (clones 1 to 7, PI20; clone 8, DP20; and clone 9, VD20). (D) Mapping of immunodominant epitope region PI20 by ELISPOT assay. (E) Env- specific clones exhibit cytotoxic activity when incubated with autologous EBV-transformed lymphoblastoid cell lines pulsed with a specific peptide at an E:T ratio of 20 to 1. (F) Secretion of IFN-γ, IL-4, and IL-10 by Env-specific CD4⁺ T-cell clones after stimulation with the envelope or control peptide (clone 1, PI20; clone 9, VD20; clone 10, RD20; clone 11, NN20; and clone 12, TF20). A level of cytokine secretion in antigen-stimulated cultures exceeding two times that of the unstimulated cultures was considered a positive response.

FIG. 3.

Genetic variability in regions encoding Th epitopes and the evolutionary changes in autologous viral sequences. (A) Amino acid variability in Th epitopes. Entropy measurements of amino acid variability mapped across the gp160 coding sequence by using 244 full-length subtype B sequences obtained from the Los Alamos HIV sequence database are shown. The thick blue line indicates the V1-to-V5 variable region, and the thick red line denotes the six Th epitopes recognized by patient 1212. The full gene plot illustrates a moving average of variability at adjoining three-amino-acid windows. Two of the epitopes (RD20 and NN20) mapped to regions of high variability, while the other epitopes mapped to more conserved regions in the protein. (B) Site-by- site analysis of variability. The variability at each amino acid position within the six Th epitopes was examined by plotting the site-by-site variability. (C) Evolutionary changes at the Th gp120 epitopes in autologous viral sequences sampled at 16 and 683 DPI from patient 1212. Identity to amino acid in the consensus sequence is indicated by a dot. Numbers at the ends of the sequences indicate the proportion of clones encoding the given variant. Compartments sampled included monocytes, resting CD4⁺ T cells, activated CD4⁺ T cells, and plasma.

FIG. 4.

Longitudinal tracking of Env-specific Th cells from subject 1212. PBMC from six time points between 16 DPI and 27 months posttreatment (weeks 0, 24, 40, 78, 104, and 120 posttreatment) were tested in a batch analysis for lymphoproliferation (S.I. are shown) and IFN-γ secretion by ELISA in response to stimulation with peptide PI20 for 120 h. PBMC from these time points were also stimulated with the peptide for 10 days, and IFN-γ ELISPOT assays were performed with CD8-depleted PBMC poststimulation. Env-specific IFN-γ SFC were detected by stimulated ELISPOT only.

FIG. 5.

Longitudinal tracking of Env-specific clonal transcripts from subject 1212. (A) Analysis of TCRBV expression in clone 1212 no. 1 by multiplex RT-PCR and agarose gel electrophoresis. The outside lane (M) contains the molecular weight markers (100-bp ladder). The arrowhead indicates the single distinct band visualized in this gel. The size and location of the band indicate that the clone expressed TCRBV13. Clones 2, 3, 4, 5, and 6 also expressed TCRBV13 (data not shown). (B) Direct DNA sequencing was performed with the amplified products from clones 1, 2, 3, 4, and 5 by using the TCRBC primer. Four unique TCRBV CDR sequences were detected, with clones 1 and 2 sharing identical sequences. Nucleotide mismatches between the CDR sequences for clones 1 and 2 versus clones 3, 4, and 5 are indicated in bold. (C) Detection of clone 1212 no. 1 transcripts before and after treatment. RT-PCR was performed with CD4⁺-enriched PBMC from weeks 0 and 24 by using primers specific for the Vβ in the clone and Cβ. On each of the amplified DNA samples, two elongations of 10 cycles were performed, initiated by either the fluorescent dye-labeled Jβ or the clonotype primer. The two reaction products were electrophoresed, and the fluorescent profiles were analyzed by GeneScan software. Shown are histograms for spectratyping analysis of CDR3 lengths with the fragment length depicted on the x axis and fluorescent intensity on the y axis. Priming with the Jβ primer depicts a single clonal expansion in clone 1, polyclonal profile in CD4⁺-enriched PBMC from subject 1212 at weeks 0 (16 DPI) and 24 posttreatment and from subject 1243. Priming with the clonotypic primer depicts the presence of the clone transcripts in clone 1, CD4⁺-enriched PBMC from subject 1212 at time points week 0 (16 DPI) and week 24 posttreatment, but not in the CD4⁺-enriched PBMC from subject 1243. The negative control (no cDNA) sample showed no amplification with either Jβ or clonotypic primers.