Phenotypic and functional profile of HIV-inhibitory CD8 T cells elicited by natural infection and heterologous prime/boost vaccination

Abstract

Control of HIV-1 replication following nonsterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8(+) T-cell-mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8(+) T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically defined CD8(+) T-cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8(+) T-cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by recombinant adenovirus type 5 (rAd5) boosting elicited CD8(+) T-cell-mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically infected virus controllers, this activity correlated with HIV-1-specific CD107a or macrophage inflammatory protein 1beta (MIP-1beta) expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8(+) T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8(+) T-cell response that may be optimized in the search for an efficacious HIV-1 vaccine.

FIG. 1.

DNA prime/rAd5 boost elicits CD8⁺ T cells capable of mediating HIV-1 inhibition in vitro. CD8⁺ T-cell antiviral activity against five Env reporter viruses expressing transmitted/founder envelopes or NL4-3 is shown for 23 seronegative donors, 40 DNA/rAd5 vaccinees, 21 HIV-1-positive chronically infected donors, and 16 HIV-1-positive virus controllers. The inhibition is calculated as the log₁₀ reduction in virus infection, measured as viral luciferase-produced relative light units (RLU) compared to autologous infectivity controls. A threshold for positive antiviral activity was established as 3 standard deviations above the mean of the seronegative inhibition values. (The dotted line shows a 0.8-log reduction.)

FIG. 2.

Impact of HLA alleles on CD8⁺ T-cell-mediated virus inhibition. Cohorts were divided based on the expression of HLA-B alleles associated with virus control in vivo (HLA B*57 and B*27). Increased antiviral activity associated with the presence of controlling alleles for two viruses (CXCR4-tropic NL4.3-LucR.T2A and CCR5⁺ CXCR4-dualtropic NL-LucR.T2A-WEAU.ecto) are indicated by asterisks (P < 0.05).

FIG. 3.

Env- and Gag-specific CD8⁺ T cells in vaccinees correlate with HIV-1 inhibition in vitro. Expression of cytokine and degranulation markers was measured in PBMCs from 40 vaccinees by polychromatic flow cytometry. Total CD8⁺ T-cell responses for each function are shown following Env (A) and Gag (B) peptide stimulation. Asterisks denote functions for which statistically significant differences between vaccinees with antiviral activity (red symbols) and vaccinees without antiviral activity (blue) (P < 0.05, Wilcoxon's rank sum test) were found.

FIG. 4.

Effector function in HIV-1-infected subjects with antiviral activity. Expression of cytokine and degranulation markers was measured in 15 virus controllers (A) and 15 chronically infected subjects (B) by flow cytometric analysis. Total CD8⁺ T-cell responses for each function are shown following Env (blue symbols), Gag (red), or Nef (green) peptide stimulation. Shaded bars indicate interquartile range.

FIG. 5.

CD107a and MIP-1β expression independently correlate with CD8⁺ T-cell-mediated virus inhibition. CD8⁺ cells from 40 vaccinees were examined for coexpression of three different effector functions by flow cytometric analysis. CD8⁺ T-cell responses are shown following Env (A) and Gag (B) peptide stimulation. Asterisks denote effector functions for which there are statistically significant differences between vaccinees with (red symbols) or without (blue) antiviral activity (*, P < 0.05; **, P < 0.01, by Wilcoxon's rank sum test). Shaded bars indicate interquartile range.

FIG. 6.

Differentiation state of HIV-specific CD8⁺ T cells from subjects with antiviral activity. CD8⁺ T cells from 15 virus controllers, 15 HIV-1⁺ chronic donors, and 20 vaccinees with positive CD8⁺ T-cell-mediated antiviral activity were analyzed for expression of the differentiation markers CD45RO, CCR7, and CD28. Cells that produced at least one function in response to HIV-1 peptide stimulation were categorized as early memory (CD45RO⁻ CCR7⁺ CD28⁺), central memory (CM; CD45RO⁺ CCR7⁺ CD28⁺), transitional memory (TM; CD45RO⁺ CCR7⁻ CD28⁺), effector memory (EM; CD45RO⁺ CCR7⁻ CD28⁻), or terminal effector (TE; CD45RO⁻ CCR7⁻ CD28⁻). Pie charts show the fraction of HIV-1-specific cells expressing the indicated phenotype.

FIG. 7.

Virus inhibition mediated by subsets of CD8⁺ T cells. CD8⁺ T cells from 10 donors positive for antiviral activity (4 vaccinees and 6 viral controllers) were sorted by flow cytometry into four of five possible subsets for each donor (sorts performed in each of three different experiments focused on different subsets). Bars represent the mean ± standard error of the mean for those sorts where sufficient cells were isolated for the assay: naïve, n = 6; early memory (CD45RO⁻ CCR7⁻ CD27⁺), n = 4; central memory (CD45RO⁺ CD27⁺), n = 9; effector memory (CD45RO⁺ CD27⁻), n = 8; and terminal effector (CD57⁺), n = 10. (A) Total inhibitory activity per culture of 45,000 cells; (B) Inhibitory activity normalized by the fraction of HIV-1-specific T cells (determined by intracellular staining) contained within each subset.