Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover

Abstract

The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)-B27 patients. To understand further the nature of CD8+ T cell-mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27-restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10-specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines.

Figure 1.

Association between Gag-specific CD8⁺ T cell responses and viral load in HLA-B27 individuals. (A) Total CD8⁺ T cells responses specific for the Gag protein were assessed in IFN-γ ELISPOT assays by stimulation with overlapping peptides of PBMCs from 47 antiretroviral therapy–naive HIV-infected donors, including 11 HLA-B27 donors. Magnitude (in SFU per 10⁶ PBMCs) of responding cells specific for Gag was plotted as a function of cVL for all 47 donors, the 11 HLA-B27 donors, and the 36 non–HLA-B27 donors. Correlations were determined using the Spearman's rank test. (B) Subdivision of Gag-specific responses from HLA-B27 donors according to peptide pools. Median responses for the 11 HLA-B27 donors are shown for each pool, and correlations between T cell frequencies and cVL were calculated. (C) Frequency of T cells responding to p24 pool 3 in HLA-27 and non–HLA-B27 donors. Horizontal bars represent means. The p-value was calculated by the Mann-Whitney U test. (D) Representative examples of HLA-B27 and HLA-B57 donor responses to individual peptides (n = 11) included in p24 pool 3. Peptide 11 covers Gag residues 261–275. Gag-specific responses were assessed in duplicate, and CD8⁺ T cell–depleted PBMCs were used to verify that observed responses were CD8⁺ T cell mediated.

Figure 2.

Phenotypic and functional assessment of B27-KK10–specific CD8⁺ T cells. (A) Representative staining for the expression of cell surface markers on B27-KK10–specific CD8⁺ T cells. HIV-specific cells identified using MHC class I–peptide complexes (black) are superimposed on the whole CD8⁺ T cell population in the same donor (red). Plots are gated on CD3⁺CD8⁺ cells. (B) Comparative marker expression between B27-KK10–specific CD8⁺ T cells (open diamonds) and other HIV-specific CD8⁺ T cells (closed circles). Horizontal bars show medians. (C) Representative example of simultaneous multifunctional assessment of B27-KK10–specific CD8⁺ T cells using nine-color flow cytometry. Cells were stimulated for 6 h in the presence of cognate peptide before intracellular staining. Percentages of cells in the different quadrants are shown. Plots are gated on CD3⁺CD8⁺ cells. (D) The pie charts depict the background-adjusted multifunctional behavior (one to five functions: CD107a, IFN-γ, TNF-α, IL-2, and MIP-1β) of B27-KK10–specific CD8⁺ T cell populations (n = 8) versus other HIV-specific CD8⁺ T cell populations (n = 7, including 2xA3-RY10, 2xB7-GL9, and 3xA2-SL9). For simplicity, responses are grouped by the number of functions (indicated by the numbers inside the pie charts and matched to the colored bars in E). The p-value was calculated by the permutation test. (E) Detailed functional responsiveness was analyzed for the B27-KK10–specific CD8⁺ T cell populations compared with the other HIV-specific CD8⁺ T cell populations. Each bar shows the mean percentages of cells displaying a particular combination of functions within the total of functional cells. p-values were calculated by the Mann-Whitney U test. (F) IFN-γ fluorescence of B27-KK10–specific CD8⁺ T cells and other HIV-specific CD8⁺ T cells upon stimulation with cognate peptides. Representative profiles are shown (left), and the IFN-γ MFI ratios (for antigen-specific CD8⁺ T cells upon stimulation/no activation) are presented for all populations studied (right). Horizontal bars represent means.

Figure 3.

High turnover of B27-KK10–specific CD8⁺ T cell clonotypes. (A) Clonal composition of several CD8⁺ T cell populations specific for B27-KK10 or other epitopes. Clonotypic analysis was performed on FACS-sorted viable HIV-specific CD8⁺ T cells. The percent frequencies of single clonotypes within B27-KK10 or other HIV epitope–specific CD8⁺ T cell populations are shown; each bar represents one population from one donor and is subdivided into distinct clonotypes represented according to frequency. 6 B27-KK10 and 9 out of 13 other HIV epitope–specific CD8⁺ T cell populations are shown. Donor identification is provided under the bars, and a detailed clonotypic analysis (with sequences) is shown in Table I and Table S1. (B and C) Analysis of clonal dominance over two time points. The percent frequencies of HIV-specific CD8⁺ T cells, identified by cognate MHC class I–peptide multimeric complexes, within the total CD8⁺ population and clonal composition, represented as the frequency of individual clonotypes within sorted HIV-specific CD8⁺ T cell populations, are shown for four B27-KK10 (C) and four other HIV peptide–specific CD8⁺ T cell populations (B) separated by the indicated intervals. Donor identification and specific responses are indicated; a detailed clonotypic analysis with sequences is shown in Table S1.

Figure 4.

Changes in CD57 expression on B27-KK10–specific CD8⁺ T cell clonotypes. (A) Clonal analysis (including TCRBV usage, CDR3 aa sequence, TCRBJ usage, and percent frequency) of the B27-KK10– and A2-SL9–specific CD8⁺ T cell populations present in patient 04.064 at two time points (representing a total of 292 TCRB sequences). Representative examples of B27-KK10 pentamer and anti-TCRBV costaining on CD8⁺ T cells (B), and of anti-TCRBV and CD57 costaining on B27-KK10–gated CD8⁺ T cells (patient 04.064, year 5; C) are shown. Numbers represent percentages of CD57⁺ cells within the BV⁺/pentamer⁺ populations. (D) CD57 expression on TCRBV⁺ B27-KK10– or A2-SL9–specific CD8⁺ T cell populations for patient 04.064. (E) CD57 expression on TCRBV⁺ B27-KK10– or other HIV-specific CD8⁺ T cell populations for the seven patients studied. Clonotypes that are dominant at one of the two time points (for B27-KK10) or the two time points (for the other) are presented; the arrows indicate the time of dominance. The p-value was calculated by the paired t test. (F) The percent frequency of B27-KK10–specific CD8⁺ T cells and the expression of TCRBV-characterizing dominant clonotypes before (Pre) or after (Post) 9 d of in vitro expansion in the presence of KK10 peptide and IL-2.

Figure 5.

Disappearance of one B27-KK10–specific CD8⁺ T cell clonotype and loss of control of HIV replication. (A) 5-yr follow-up (pVL, CD4 counts, and percent frequency of B27-KK10–specific CD8⁺ T cells) in one patient (02.011) with sudden disease progression at year 2. (B) Clonal analysis (TCRBV usage, CDR3 aa sequence, TCRBJ usage, and percent frequency) of B27-KK10–specific CD8⁺ T cell populations is shown for years 1 and 2 (representing a total of 150 TCRB sequences). (C) CD57 expression on B27-KK10–specific CD8⁺ T cells. Percentages of pentamer⁺ cells that express CD57 are indicated. (D) KK10 viral sequence of the dominant (i.e., >80%) HIV-1 strain over time. (E) CD38 expression on B27-KK10–specific CD8⁺ T cells at years 1 and 2.

Figure 6.

CD8⁺ T cell functional avidity and control of viral replication. (A) Representative example of peptide titrations in IFN-γ ELISPOT assays (normalized SFC per 10⁶ PBMCs) to assess functional avidity of KK10 or other HIV epitope–specific CD8⁺ T cells. Functional avidity is defined as the concentration required to achieve half-maximal recognition of the wild-type peptide (EC₅₀). Error bar values are 95% confidence intervals. (B) Functional avidity of CD8⁺ T cell populations specific for HLA-A–restricted (including 2, 3, 11, 26, and 31) or HLA-B–restricted (including 7, 8, 27, 35, and 51) HIV epitopes (n = 20), as well as for B27-KK10. Horizontal bars represent means. p-values were calculated by the Mann-Whitney U test. (C) Correlation between functional avidity of immunodominant HIV-specific CD8⁺ T cell responses and HIV cVL. Immunodominant (n = 16, closed symbols) and the highest subdominant (n = 12, open symbols) responses are shown, as is a linear regression trend line. p-values and r values between cVL, pVL, CD4 count, and functional avidity are shown for the immunodominant responses. Spearman's rank test was used to determine correlations.