Interleukin-2 production by polyfunctional HIV-1-specific CD8 T cells is associated with enhanced viral suppression

Abstract

Background: Assays to measure the induction of HIV-1-specific CD8 T-cell responses often rely on measurements of indirect effector function such as chemokine and cytokine production, which may not reflect direct elimination of an invading pathogen. Assessment of the functional ability of CD8 T cells to suppress HIV-1 replication has been viewed as a surrogate marker of an effectual immune response. To further investigate this, we measured the capacity of virus-specific CD8 T cells to inhibit HIV-1 replication in an in vitro suppression assay.

Methods: We expanded 15 epitope-specific CD8 T-cell lines from peripheral blood mononuclear cells of chronically HIV--infected progressors (n = 5) and controllers (n = 4) who were not on antiretroviral therapy. Cell lines were tested for their ability to produce effector molecules (CD107a, IL-2, IFN-γ, TNF-α, perforin) and suppress virus replication in autologous CD4 T cells.

Results: CD8 T-cell lines from both progressors and controllers had largely similar effector function profiles as determined by intracellular cytokine staining. In contrast, we observed that CD8 T-cell lines derived from controllers show enhanced virus suppression when compared with progressors. Virus suppression was mediated in an major histocompatibility complex-dependent manner and found to correlate with a polyfunctional IL-2 CD8 T-cell response.

Conclusions: Using a sensitive in vitro suppression assay, we demonstrate that CD8 T-cell-mediated suppression of HIV-1 replication is a marker of HIV-1 control. Suppressive capacity was found to correlate with polyfunctional IL-2 production. Assessment of CD8 T-cell-mediated suppression may be an important tool to evaluate vaccine-induced responses.

Figure 1. ICS analysis of multiple effector functions elicited by ex vivo and in vitro expanded CD8 T-cells

PBMCs from controllers and progressors were stimulated with HIV-1 peptides and analyzed by ICS for production of CD107a, IFN-γ, IL-2, TNF-α, and perforin. Representative flow cytometry plots depict (A) ex vivo and (B) in vitro expanded CD8 T-cell responses in subject C5168. Top and bottom panels represent the unstimulated (negative control) and B27-KK10 stimulated response respectively. Lymphocytes were gated by forward and side scatter, viable cells, CD3⁺ cells, and then CD8⁺ cells. Numbers indicate the percent of CD8 T-cells positive for the indicated function. (C) Total ex vivo and in vitro CD8 T-cell responses detected in HIV-1 infected progressors (ex vivo, n=6; in vitro, n=9; open symbols) and controllers (ex vivo, n=6; in vitro, n=6; closed symbols); analysis of ex vivo immune response was not completed for subject P5766 due to limited sample availability. Each dot represents a single epitope-specific response. Horizontal bars indicate median response. Significant differences (p≤0.05) between ex vivo and in vitro response are indicated by asterisks.

Figure 2. Polyfunctionality of ex vivo and in vitro expanded HIV-1 specific CD8 T-cell responses

Data generated by ICS was analyzed using SPICE software for coincident production of IL-2, perforin, IFN-γ, CD107a, and TNF-α by epitope-specific CD8 T-cells. Pie charts denote the proportion of CD8 T cells producing 1 (gray), 2 (purple), 3 (yellow), 4 (blue), or 5 (red) functions. (A) Ex vivo and (B) in vitro expanded CD8 T-cell responses were averaged within each group. Arcs identify cell populations that are positive for IL-2 (black arc), perforin (orange), and IFN-γ (green), and CD107a (maroon). Bar graphs depict the relative frequency of responses measured (C) ex vivo and (D) after in vitro expansion. Responses for all 31 possible subsets are shown for both groups—progressors (pink bars) and controllers (dark blue bars). Plus signs denote a positive response for the effector function listed to the left—2 indicates IL-2, P indicates perforin, G indicates IFN-γ, C indicates CD107a, and T indicates TNF-α.

Figure 3. Enhanced HIV-1 suppression mediated by epitope-specific CD8 T-cells derived from controllers

CD4 T-cells (targets) were infected with HIV-1_NL4-3 and co-cultured with expanded CD8 T-cell lines (effectors) at multiple E:T ratios for seven days. Supernatant from days 0, 1, 3, 5, and 7 were analyzed for production of luciferase using the TZM-bl reporter cell line. Background luciferase production in uninfected primary cells was approximately 1000 relative luciferase units (RLU). All experiments were run in duplicate. (A–D) Representative iVSA data is shown for a CD8 T-cell line derived from a progressor (cell line P7 in A, B) and a controller (cell line C6 in C, D). Targets cells used were autologous (left panels) or non-autologous (right panels). (E) Antiviral suppression by CD8 T-cell lines derived from progressors (P, n=9) compared to lines derived from controllers (C, n=6) at indicated E:T ratios. Each cell line is denoted by a unique symbol. Black bars indicate median percent suppression. Mann-Whitney test was used to compare the median percent suppression between groups at each E:T ratio. NS, not significant.