Availability of a diversely avid CD8+ T cell repertoire specific for the subdominant HLA-A2-restricted HIV-1 Gag p2419-27 epitope

Abstract

HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p24(19-27), TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer and correspondingly distinct functional dependencies on the CD8 coreceptor. The lack of dominant clonotypes was not affected by the stage of maturation of the priming dendritic cells. Cultures primed by dendritic cells transduced to present endogenous TV9 were also incapable of clonal maturation. Thus, a diffuse TCR repertoire appeared to be an intrinsic characteristic of TV9-specific responses. These data indicate that subdominance is not a function of poor immunogenicity, cognate TCR repertoire availability, or the potential avidity properties thereof, but rather suggest that useful responses to this epitope are suppressed by competing CD8+ T cell populations during HIV-1 infection.

FIGURE 1

A, CD8⁺ TV9-specific responses in four selected patients by IFN-γ ELISPOT assay. One hundred thousand live PBMCs were incubated overnight with the TV9 peptide at concentrations ranging from 0.01 to 1000 ng/ml. B, Relative binding of TV9, the TV9 variant (9I), the HIV-2 TV9 homolog (8L), SL9, and the flu MP (GL9) peptides to HLA-A*0201 determined by the T2 stabilization assay. GL9 served as the positive control. The fluorescence ratio was calculated as described in the Materials and Methods. Assays were performed in triplicate, and the values represent the mean ± SD. This experiment was repeated twice with essentially identical results.

FIGURE 2

Characteristics of ex vivo-primed TV9-specific CD8⁺ T cell cultures. A, Tetramer-binding cells in TV9−2, TV9−4.1, and TV9−5 over time. T cells were stained 6 days after restimulation. Numbers in the upper right quadrants are the percentages of CD8⁺tetramer⁺ T cells. B, Tetramer-binding cells in SL9-, p41-, 3F-, and YV9-specific CD8⁺ T cell cultures. Cells were stained with cognate tetramers and anti-CD8 mAb. C, Parallel cultures primed with iDCs or mDCs from donor 1 (TV9−1i and TV9−1m, top two rows) and donor 3 (TV9−3i and TV9−3m, bottom two rows). Cultures were monitored for >50 days. D, Cytotoxicity against T2 cells in the presence of 1 μg/ml cognate peptide by TV9−1i and TV9−1m. This experiment was repeated four times with identical results. E, Functional avidity of TV9−1i and TV9−1m determined by IFN-γ secretion at the E:T ratio of 1:10. EC₅₀s were 6.6 × 10⁻⁹ and 7.3 × 10⁻⁹ M, respectively. F, Scatter plots showing induction of CD107a/b expression and IFN-γ production by TV9−1i and TV9−1m after stimulation by C1R-A2^wt cells pulsed with 10 μg/ml cognate peptide at the E:T ratio of 1:1. Numbers in the upper right quadrants represent the percentages of CD107a/b⁺ IFN-γ-secreting cells. Nonspecific activation with an irrelevant peptide (YV9) resulted in <1% CD107a/b⁺ IFN-γ-secreting T cells in both cultures (data not shown).

FIGURE 3

Priming of TV9-specific CTLs by DCs transduced with the pNL43 E⁻ vector. A, CD86 and CD80 expression by iDCs from donor 7 4 days after transduction (top panels). Transduction was visualized by intracellular p24 expression. A parallel culture of nontransduced iDCs is shown in the bottom panels. B, Reactivity of TV9−7t CD8⁺ T cells to SL9, TV9, and IV9 as determined by IFN-γ ELISPOT assay after one (d8) and two (d16) stimulations with transduced DCs. C, Transduced iDCs from donor 2; details as for A above. D, Tetramer-binding TV9−2t CD8⁺ T cells on days 26 and 54. E, Cytotoxicity of TV9−2t against T2 cells in the presence of cognate peptide. T2 cells without peptide were included as a negative control.

FIGURE 4

Participation of the CD8 coreceptor in tetramer binding and functional activation of TV9-specific T cells. A, TV9-specific cultures stained with wild-type or CD8^null tetramers and anti-CD8 mAb. B, Lysis of C1R-A2^wt or C1R-A2^CD8null cells pulsed with a range of TV9 concentrations by TV9−1, TV9−2, TV9−3, and TV9−5 at the E:T ratio of 2.5:1. Experiments were repeated at least twice for TV9−1, TV9−2, and TV9−5 with similar results. C, Flow cytometric analysis of IL-2, TNF-α, and IFN-γ production and degranulation (CD107a/b) by TV9−3 after a 4-h stimulation with C1R-A2^wt or C1R-A2^CD8null cells loaded with a saturating concentration of TV9 (10 μg/ml). The number in each quadrant represents the percentage of cells within that quadrant. Plots are gated on live CD8⁺ T cells.

FIGURE 5

TCR Vβ composition of tetramer-binding cells in four representative TV9-specific cultures. Each culture was stained with tetramer, anti-CD8 mAb, and a TCR Vβ-specific mAb at a concentration shown a priori not to interfere with tetramer binding. Panels on the left show CD8⁺ tetramer⁺ cells in TV9−2, TV9−4, TV9−1, and TV9−2t cultures. Panels on the right are gated on CD8⁺ cells, and the inset numbers represent the percentages of tetramer⁺ cells that stain with each depicted TCR Vβ-specific mAb.

FIGURE 6

Characterization of TV9-specific CD8⁺ T cells sorted according to different TCR Vβ usage from the TV9−2 culture. A, TV9−2(Vβ3⁺) and TV9−2(Vβ8⁺) T cells stained with the wild-type (top panels)orCD8^null (bottom panels) tetramers and anti-CD8 mAbs. B, Lysis of C1R-A2^wt (top panel) and C1R-A2^CD8null cells (bottom panel) over a range of TV9 peptide concentrations by the parental TV9−2 culture and sorted TV9−2(Vβ3⁺) or TV9−2(Vβ8⁺) cells. The E:T ratio was 2.5:1. Lysis of C1R cells alone or pulsed with the irrelevant peptide YV9 (negative controls) was <7%. Specific lysis was calculated by subtracting lysis in the negative control from the overall lysis for each E:T ratio. Data are representative of three independent experiments and presented as mean ± SEM of triplicate assays. C, Secretion of IFN-γ by TV9-specific CTLs after stimulating for 48 h with C1R-A2^wt (top panel) and C1R-A2^CD8null (bottom panel) cells over a range of TV9 peptide concentrations. The E:T ratio used was 1:10. D, Suppression of HIV-1 JR-CSF replication in three acutely infected, allogeneic, HLA-A2-matched, CD8-depleted PBMC cultures by TV9−2, TV9−2(Vβ3⁺), and TV9−2(Vβ8⁺) CTLs measured 9 days postinfection. The concentrations of p24 in infected PBMC-1, PBMC-2, and PBMC-3 in the absence of CTLs were 311, 210, and 184 ng/ml, respectively. The difference in the median inhibition against the three cultures between the parental TV9−2 cells and the low-avidity Vβ3⁺ T cells was determined to be statistically significant by the Student t test with Holm's adjustment for pairwise comparison (p = 0.006). Similarly, the difference between the high-and low-avidity T cell subclones was also significant (p = 0.01).

FIGURE 7

A, Cross-recognition of variants of TV9 by a representative TV9-specific culture (TV9−1) determined by cytotoxicity at various E:T ratios. T2 cells pulsed with 1 μg/ml TV9, 9I, and 8L peptides were used as targets. B, Lysis of JA2/R7/Hyg cells by three TV9-specific cultures (TV9−1, TV9−2, and TV9−5) and a clone (clone 3.2) isolated from TV9−2.