Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection

Abstract

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.

FIG. 1.

Multispecific CD8⁺ T-cell responses during acute resolving HBV infection. (A and B) PBMC of six patients with acute HBV infection were analyzed for CD8⁺ T-cell responses to four immunodominant HBV epitopes in the early phase (A) and after resolved infection (B) with HLA-A2 multimers. Multimer data are shown as the percentages of multimer-positive cells among all CD8⁺ T cells. Early and late time points for each patient are defined in Table 1. (C) Representative density plots of each patient showing multimer-positive CD8⁺ T-cell responses.

FIG. 2.

Phenotypic characterization of HBV-specific CD8⁺ T cells. For each patient, surface staining of HBV-specific CD8⁺ T cells was performed using an HBV multimer with a strong ex vivo response (Table 1) and antibodies to CD127 (A), CD38 (B), CCR7 (C), or CD27 (D). The phenotype of HBV-specific CD8⁺ T cells was analyzed in the early phase (gray bars) and after resolved infection (black bars). Data are shown as the percentages of all multimer-positive CD8⁺ T cells. Representative density plots of each surface staining from patients 1 and 3 during acute (early) and after resolved infection (late) gated on CD8⁺multimer-positive cells are shown on the right.

FIG. 3.

Courses of acute resolving HBV infection. Clinical and virological data are shown for three patients at several time points during and after acute HBV infection. (A, B, and C) Viral load (VL), ALT levels, and dominant multimer response. GE, genome equivalents; U/l, units per liter. (D, E, and F) Kinetics of CD38 expression of HBV-multimer-positive CD8⁺ T cells. (G, H, and I) Kinetics of CCR7 expression of HBV-multimer-positive CD8⁺ T cells. (J, K, and L) Kinetics of CD127 expression of HBV-multimer-positive CD8⁺ T cells. Data are shown as the percentages of multimer-positive CD8⁺ T cells specific for the respective surface marker. (M, N, and O) Expansion in the proliferative capacity of multimer-specific CD8⁺ T cells. Calculation of the expansion was performed as described in Materials and Methods. “n.d.,” not done.

FIG. 4.

Functional capacities of HBV-specific CD8⁺ T cells. (A and B) PBMC of five patients with acute HBV infection were analyzed for HBV-specific CD8⁺ T-cell IFN-γ production after stimulation with four immunodominant HBV epitopes in the early phase (A) and after resolved infection (B). HBV-specific IFN-γ production of CD8⁺ T cells was examined by intracellular cytokine staining. Data are shown as the percentages of peptide-specific IFN-γ-producing CD8⁺ T cells in relation to the frequency of the respective multimer-positive CD8⁺ T cells. (C) Representative plots of multimer and IFN-γ staining for patient 2 performed at the clinical onset of infection (early) and after resolution (late). The displayed numbers indicate the percentages of multimer-positive or IFN-γ-producing CD8⁺ T cells. The negative control results were <0.03% in all cases and are not shown. (D) The proliferative capacity of HBV-specific CD8⁺ T cells was studied in the early phase of infection (gray bars) and after resolved infection (black bars). PBMC were stimulated using the dominant epitope from each patient as shown in Table 1 and were cultured for 14 days prior to staining with the corresponding multimer. Cells were gated on multimer-positive CD8⁺ cells. Calculation of the expansion was performed as described in Materials and Methods.

FIG. 5.

PD-1 expression on HBV- and Flu-specific CD8⁺ T cells. Virus-specific CD8⁺ T cells were detected via multimer staining and analyzed for PD-1 expression. (A) PD-1 expression on HBV-specific CD8⁺ T cells was analyzed at clinical presentation of acute hepatitis B (early) and after resolved infection (late). Data are shown as the mean PD-1 fluorescence results (⋄, patient 1; ▪, patient 2; ▴, patient 3; ×, patient 4; ○, patient 6). Expression of PD-1 on HBV-specific CD8⁺ T cells (B), whole CD8⁺ T cells (C), and influenza virus-specific CD8⁺ T cells (D) of patient 2 is demonstrated by overlay histograms of both time points. The expression of PD-1 on unstimulated (E) and stimulated (F) PBMC from one young, healthy donor served as a positive control.