Early interferon therapy for hepatitis C virus infection rescues polyfunctional, long-lived CD8+ memory T cells

Abstract

The majority of acute hepatitis C virus (HCV) infections progress to chronicity and progressive liver damage. Alpha interferon (IFN-alpha) antiviral therapy achieves the highest rate of success when IFN-alpha is administered early during the acute phase, but the underlying mechanisms are unknown. We used a panel of major histocompatibility complex class I tetramers to monitor the phenotypic and functional signatures of HCV-specific T cells during acute HCV infection with different infection outcomes and during early IFN therapy. We demonstrate that spontaneous resolution correlates with the early development of polyfunctional (IFN-gamma- and IL-2-producing and CD107a(+)) virus-specific CD8(+) T cells. These polyfunctional T cells are distinguished by the expression of CD127 and Bcl-2 and represent a transitional memory T-cell subset that exhibits the phenotypic and functional signatures of both central and effector memory T cells. In contrast, HCV-specific CD8(+) T cells in acute infections evolving to chronicity expressed low levels of CD127 and Bcl-2, exhibited diminished proliferation and cytokine production, and eventually disappeared from the periphery. Early therapeutic intervention with pegylated IFN-alpha rescued polyfunctional memory T cells expressing high levels of CD127 and Bcl-2. These cells were detectable for up to 1 year following discontinuation of therapy. Our results suggest that the polyfunctionality of HCV-specific T cells can be predictive of the outcome of acute HCV infection and that early therapeutic intervention can reconstitute the pool of long-lived polyfunctional memory T cells.

FIG. 1.

Longitudinal phenotyping of virus-specific T cells during acute HCV infection. Representative longitudinal tetramer staining and phenotyping data from patient R1 with acute resolving HCV infection prior to and after detection of viremia (A) and patient T4 with chronic evolution (B). Tetramer frequency is represented as percentage of CD3⁺ CD8⁺ T cells. Black represents HLA-A2/HCV-1073 tetramer-positive cells, and red represents total CD3⁺ CD8⁺ T cells in the same donor. Percentages of tetramer-positive cells in each quadrant are shown. (C) Summary of longitudinal phenotypic changes in CD127 and PD-1 expression on HCV-tetramer-positive cells during acute HCV at the earliest and latest follow-up time points prior to commencement of therapy. Each symbol represents one HCV tetramer as indicated in the legend according to the following key: A2-1073, HLA-A2/NS3-1073; B8-1395, HLA-B8/NS3-1395; A1-1436, HLA-A1/NS3-1436; B7-41, HLA-B7/core-41; A2-1406, HLA-A2/NS3-1406. Patient numbers are included. Wks, weeks.

FIG. 2.

Spontaneous resolution of acute HCV infection is associated with upregulation of CD127 and Bcl-2 and early development of polyfunctional HCV-specific T cells. Representative data from patients R1 and R2 using HLA-A2/NS3-1073 (A2-1073) and HLA-B8/NS3-1395 (B8-1395) tetramers, respectively, are given. (A) Longitudinal expression of CD127 and intracellular Bcl-2 in tetramer-positive cells in patients R1 and R2; tetramer frequency is represented as the percentage of CD3⁺ CD8⁺ T cells in the upper right quadrant. Black represents tetramer-positive cells, and red represents total CD3⁺ CD8⁺ T cells in the same donor; percentages of tetramer-positive cells in each quadrant are shown. (B) CFSE proliferation of HCV-specific T cells in the absence or presence of the specific peptide; plots are gated on CD3⁺ CD8⁺ T cells. Numbers in the upper-left quadrant represent the percentage of proliferating (CFSE^lo) tetramer-positive cells. (C) Longitudinal analysis of cytokine production and CD107a degranulation following specific peptide stimulation represented as percent specific expression of each individual cytokine/marker by viable, CD3⁺ CD8⁺ T cells. IFN-γ MFI is represented as a line graph. The asterisk indicates long-term follow-up experiments performed on a different date. (D) Polyfunctionality of HCV-specific T cells, gated on viable CD3⁺ CD8⁺ IFN-γ⁺ cells. Red represents three functions (IFN-γ⁺, IL-2⁺, and CD107a⁺), blue represents two functions (IFN-γ⁺ and CD107a⁺), green represents two functions (IFN-γ⁺ and IL-2⁺), and yellow represents one function (IFN-γ⁺); numbers on the pie chart represent the percentage of each population. Tet, tetramer; Wks, weeks.

FIG. 3.

Chronically evolving acute HCV infection is associated with low-level expression of CD127 and Bcl-2 and effector functions. Representative data from patient C1 using HLA-A2/NS3-1073 (A2-1073) and HLA-A2/NS3-1406 (A2-1406) tetramers (A) and patient C2 using HLA-B7/core-41 (B7/core-41) tetramer (B) are shown. Top panel depicts tetramer frequency relative to HCV RNA viral load. Middle panel represents longitudinal expression of CD127 and intracellular Bcl-2 in HLA-A2/NS3-1073 tetramer positive (Tet+) cells in patient C1 (A) and HLA-B7/core-41 tetramer-positive cells in patient C2 (B); proliferation of HCV-specific T cells in response to NS3-1073 peptide in patient C1 or core-41 peptide in patient C2 is depicted as a bar graph representing the percentage of proliferating (CFSE^lo) tetramer-positive (Tet+) cells. The bottom panel represents longitudinal cytokine production and CD107a degranulation following stimulation with a mixture of NS3-1073 and NS3-1406 peptides for patient C1 (A) or core-41 peptide for patient C2 (B), represented as percent specific expression in viable CD3⁺ CD8⁺ T cells.

FIG. 4.

CD127 expression delineates a unique polyfunctional T-cell subset. CD3⁺ T cells from PBMCs of patient R2 at week 17 PDV were sorted into CD127^neg and CD127^hi cells and tested for their proliferative capacity and cytokine production. (A) Pre- and postsorting purity of cells stained with HLA-B8/NS3-1395 tetramer, CD127, and Bcl-2. Black represents HLA-B8/NS3-1395 tetramer-positive cells, and red represents total CD3⁺ CD8⁺ T cells; overall tetramer frequency is represented in the upper right quadrant, and percentages of tetramer-positive cells in each quadrant are shown. (B) CFSE proliferation of CD127^hi cells gated on CD3⁺ CD8⁺ T cells. The percentage of proliferating (CFSE^lo) tetramer-positive cells is represented in the upper left quadrant; the percentage of total cells in each quadrant is represented in the bottom left quadrant. Postsorting, CD127^neg cells were too few to perform this assay. ND, not detected. (C) Cytokine production and CD107a degranulation of CD127^neg and CD127^hi cells stimulated with NS3-1395 peptide. Pie chart represents polyfunctional analysis for CD127^hi cells, gated on viable CD3⁺ CD8⁺ IFN-γ⁺ cells. Red represents three functions (IFN-γ⁺, IL-2⁺, and CD107a⁺), blue represents two functions (IFN-γ⁺ and CD107a⁺), green represents two functions (IFN-γ⁺ and IL-2⁺), and yellow represents one function (IFN-γ⁺); numbers on the pie chart represent the percentage of each population. The frequency of IFN-γ-producing cells in the CD127^neg population was too low to perform the polyfunctional analysis.

FIG. 5.

Early treatment with PEG-IFN-α rescues CD127⁺ Bcl-2⁺ HCV-specific T cells. Longitudinal phenotypic characterization and proliferative response of HCV tetramer-positive T cells in patient T1 using HLA-A1/NS3-1436 tetramer (A1-1436 Tet) and treated between weeks 15 to 27 (A), patient T2 using HLA-B7/core-41 tetramer (B7-Core 41 Tet) and treated between weeks 32 and 54 (B), and patient T3 using HLA-A2/NS3-1073 tetramer (A2-1073 Tet) and treated between weeks 32 and 48 (C). The shaded area represents the period of PEG-IFN-α therapy. The top panel depicts the respective tetramer(s) frequency relative to HCV RNA viral load. The bottom panel represents the expression of CD127 and Bcl-2 on tetramer-positive cells and proliferation of HCV-specific T cells in the presence of their specific peptides depicted as bar graphs representing the percentage of proliferating (CFSE^lo) tetramer-positive cells. Tet+, tetramer positive.

FIG. 6.

Early treatment with PEG-IFN-α rescues polyfunctional HCV-specific T cells. Longitudinal analysis of cytokine production, IFN-γ MFI, and CD107a degranulation following specific peptide stimulation and polyfunctionality in patient T1 (A and B), patient T2 (C and D), and patient T3 (E and F). The shaded areas represent the PEG-IFN-α administration period between weeks 15 to 27 in patient T1, weeks 32 to 54 in patient T2, and weeks 32 to 48 in patient T3. For polyfunctionality, red represents three functions (IFN-γ⁺, IL-2⁺, and CD107a⁺), blue represents two functions (IFN-γ⁺ and CD107a⁺), green represents two functions (IFN-γ⁺ and IL-2⁺), and yellow represents one function (IFN-γ⁺); numbers on the pie chart represent the percentage of each population. Polyfunctional analysis was not performed before therapy when the frequency of IFN-γ-producing cells was mostly lower than the cutoff threshold (0.02%) for polyfunctionality analysis. A1-1436, HLA-A1/NS3-1436; B7-core 41, HLA-B7/core-41; A2-1073, HLA-A2/NS3-1073; Tet, tetramer.

FIG. 7.

Transient restoration of CD127 and Bcl-2 expression and limited polyfunctionality in HCV-specific T cells during therapy, followed by loss upon viral recurrence in patient T4. Longitudinal phenotypic characterization and proliferative response of HLA-A2/NS3-1073 (A2-1073) tetramer-positive T cells in patient T4 are shown. (A) The top panel represents the A2/1073 tetramer frequency relative to HCV RNA viral load. The bottom panel represents the expression of CD127 and Bcl-2 on tetramer-positive cells; CFSE proliferation of HCV-specific T cells in the presence of NS3-1073 peptide is depicted as bar graphs representing the percentage of proliferating (CFSE^lo) tetramer-positive cells. (B) Longitudinal analysis of cytokine production, IFN-γ MFI, and CD107a degranulation following NS3-1073 peptide stimulation. The shaded areas represent the period of administration of PEG-IFN-α therapy between weeks 21 to 36. (C) Polyfunctionality of HCV peptide-specific T cells in each patient. Red represents three functions (IFN-γ⁺, IL-2⁺, and CD107a⁺), blue represents two functions (IFN-γ⁺and CD107a⁺), green represents two functions (IFN-γ⁺ and IL-2⁺), and yellow represents one function (IFN-γ⁺); numbers on the pie chart represent the percentage of each population. The frequency of IFN-γ-producing cells at some points prior to therapy was lower than the cutoff threshold (0.02%) for the polyfunctionality analysis. Tet, tetramer.