Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection

Abstract

During some persistent viral infections, virus-specific T-cell responses wane due to the antigen-specific deletion or functional inactivation (i.e., exhaustion) of responding CD8 T cells. T-cell exhaustion often correlates with high viral load and is associated with the expression of the inhibitory receptor PD-1. In other infections, functional T cells are observed despite high levels of pathogen persistence. The reasons for these different T-cell fates during chronic viral infections are not clear. Here, we tracked the fate of virus-specific CD8 T cells in lymphocytic choriomeningitis virus (LCMV)-infected mice during viral clearance, the persistence of wild-type virus, or the selection and persistence of a viral variant that abrogates the presentation of a single epitope. Viral clearance results in PD-1(lo) functional virus-specific CD8 T cells, while the persistence of wild-type LCMV results in high PD-1 levels and T-cell exhaustion. However, following the emergence of a GP35V-->A variant virus that abrogates the presentation of the GP33 epitope, GP33-specific CD8 T cells remained functional, continued to show low levels of PD-1, and reexpressed CD127, a marker of memory T-cell differentiation. In the same animals and under identical environmental conditions, CD8 T cells recognizing nonmutated viral epitopes became physically deleted or were PD-1(hi) and nonfunctional. Thus, the upregulation of PD-1 and the functional inactivation of virus-specific T cells during chronic viral infection is dependent upon continued epitope recognition. These data suggest that optimal strategies for vaccination should induce high-magnitude broadly specific T-cell responses that prevent cytotoxic T-lymphocyte escape and highlight the need to evaluate the function of vaccine-induced T cells in the context of antigens presented during virus persistence.

FIG. 1.

Transfer of different numbers of virus-specific TCR-Tg cells, followed by LCMV infection, results in different patterns of viral infection. (A) Mice received the indicated number of CD8 TCR-Tg cells and were depleted of CD4 T cells 2 days prior to infection with LCMV clone 13. Serum virus titers were determined 8 days p.i. by plaque assay. A dagger indicates that all of the mice receiving this dose of cells died at 9 to 10 days p.i. in five separate experiments. (B) LCMV kinetics in individual mice that received <20,000 donor cells and had high initial viremia followed by persistence (group I), mice that received >20,000 donor cells and had low initial viremia and control (group II), or mice that had low initial viremia but the establishment of a persistent infection with high virus levels (group III). Virus levels in serum were determined by plaque assay at the indicated times p.i. Five representative mice from each group are shown.

FIG. 2.

Fate and function of virus-specific CD8 T cells in LCMV-infected mice. Groups are the same as those described in the legend to Fig. 1B. Splenocytes from LCMV-infected mice were isolated at >30 days p.i. and analyzed by flow cytometry following staining with the indicated tetramers ex vivo or after 5 h of restimulation in vitro with the indicated peptide, followed by intracellular staining for IFN-γ. The physical presence (tetramer) and function (IFN-γ) of donor TCR-Tg CD8 T cells (A) and endogenous polyclonal virus-specific CD8 T cells (B) are shown. Percentages indicate the fraction of CD8 T cells. Data are representative of five separate experiments (group I, n = 48; group II, n = 11; group III, n = 37).

FIG. 3.

PD-1 and CD127 expression on virus-specific CD8 T cells is dependent on antigen persistence. Groups are the same as those described in the legend to Fig. 1B. Lymphocytes of spleen and bone marrow were isolated from mice at day 55 p.i. and stained with the indicated reagents. PD-1 (A) and CD127 (B) expression on the indicated tetramer (D^bGP33 or D^bGP276) were shown by representative plots gated on CD8 T cells and by percent expression (average ± standard deviation). Data are representative of mice from each group (n = 5).

FIG. 4.

Altered function of CD8 T cells during different patterns of viral infection. Lymphocytes isolated from spleen and bone marrow (BM) of mice generated as described in the legend to Fig. 3 were stimulated with GP33 or GP276 peptide for 5 h in vitro, followed by intracellular staining for IFN-γ. The MFI of IFN-γ expression in cells capable of making IFN-γ responsive to corresponding peptide was shown as the average ± standard deviation (n = 5).

FIG. 5.

Selection of GP35V→A CTL escape variant virus explains differences in GP33-specific CD8 T cells in persistently infected mice. Groups are the same as those described in the legend to Fig. 1B. (A) Serum was obtained from six mice each from group I and group III, and virus levels were determined by plaque assay. Six plaques were selected and grown to high titer in BHK cells and then were subjected to isolation and sequencing. Shown are the nucleotide sequences obtained as well as the resulting changes in amino acid coding sequences. Fifty percent inhibitory concentrations are derived from Puglielli et al. (36). (B) GP35V→A variant LCMV does not elicit a D^bGP33 CD8 T-cell response. Mice were infected 8 days prior with the wild-type sequence clone 13 or the GP35V→A variant LCMV. Virus-specific CD8 T cells were quantitated by staining splenocytes with MHC-I tetramers as indicated. Percentages indicate the fraction of CD8 T cells. Data are from representative mice (n = 6 for each group).