Impaired NFAT nuclear translocation results in split exhaustion of virus-specific CD8+ T cell functions during chronic viral infection

Abstract

In persistent viral infections, the host's immune system is challenged by the constant exposure to antigen, potentially causing continuous activation of CD8(+) T cells with subsequent immunopathology. Here we demonstrate, for experimental chronic lymphocytic choriomeningitis virus and human HIV infection, that upon prolonged in vivo exposure to antigen, TCR-triggered Ca(2+) flux, degranulation, and cytotoxicity are maintained on a cellular level, whereas cytokine production is severely impaired because of a selective defect in activation-induced NFAT nuclear translocation. During chronic infection, this differential regulation of pathways leading to diverse effector functions may allow CD8(+) T cells to sustain some degree of local viral control by direct cytotoxicity while limiting systemic immune pathology by silencing cytokine production.

Fig. 1.

Functional assessment of CD8⁺ T cells. (A) C57BL/6 mice were infected i.v. with 200 pfu (l.d.) or 10⁶ pfu (h.d.) LCMV Docile, and 20 days later, gp33- or np396-induced degranulation (CD107) or IFN-γ, TNF-α, and IL-2 production were analyzed for splenocytes. (B) Monoclonal anti-PD-L1 or -CTLA-4 antibodies were added during stimulation. Plots are gated on CD8⁺ T cells. One of three similar experiments is shown.

Fig. 2.

Phenotypical and functional assessment of TCR tg CD8⁺ T cells. (A) Spleen cells were stained for Ly5.1 and CD43 (gated on CD8⁺ T cells) or for CD62L and CD127 (gated on CD8⁺Ly5.1⁺ T cells). Spleen cells were stimulated with gp33 peptide and degranulation, and IFN-γ production (B) or TNF-α production (C) was assessed. Plots are gated on TCR tg CD8⁺ T cells (B) or CD8⁺ T cells (C). (D) Phorbol 12-myristate 13-acetate/ionomycin or gp33 stimulation 16 days after infection. Plots are gated on TCR tg CD8⁺ T cells.

Fig. 3.

Cytotoxic function of LCMV-specific CD8+ T cells. (A) In vitro cytotoxicity. At day 8, 16, 50, or 90 after infection, CD8⁺ T cells were purified from the spleens of adoptively transfused l.d.- or h.d.-infected mice and adjusted for equal numbers of TCR tg CD8⁺ T cells for ⁵¹Cr release assays (gp33-pulsed: closed circles, mock-pulsed: open circles). Specific lysis was determined after 16 h. Effector-to-target cell ratios indicate the ratio between Ly5.1⁺ TCR tg CD8⁺ T cells and target cells. (B) In vivo cytotoxicity. Gp33 peptide-loaded (CFSE^lo) and unloaded (CFSE^hi) target cells were adoptively transferred into infected (day 16) or naïve recipient mice. Target cells were quantified in blood 15 min, 4 h, or 16 h after transfer. Representative plots of three mice per group and of three experiments are shown.

Fig. 4.

Chronic LCMV infection in C57BL/6 and PKOB mice. (A) CD8⁺ T cell function in C57BL/6 and PKOB mice. C57BL/6 and PKOB mice were infected with 10⁶ pfu LCMV Docile, and gp33- and np396-specific CD8⁺ T cells were analyzed at day 19 in spleen for degranulation and IFN-γ production (gated on CD8⁺ T cells). (B) Ex vivo quantification of gp33⁺APCs. CD11c⁺ DCs were purified to high purity from spleens, were either mock treated (Left) or pulsed with gp33 peptide (Right), serially diluted, and incubated with 2 × 10⁵ purified naïve CFSE-labeled Ly5.1⁺ TCR tg CD8⁺ T cells. CFSE dilution profiles were analyzed after 3 days (plots are gated on CD8⁺ Ly5.1⁺ cells). One representative plot from three mice and three independent experiments is shown.

Fig. 5.

TCR signaling in LCMV-specific CD8+ T cells from h.d. or l.d. infected mice. (A) Signaling requirements for degranulation and IFN-γ production. Spleen cells from adoptively transfused and infected mice (day 20) were stimulated with gp33 peptide in the presence of EGTA, cyclosporine A, calphostin C, or PD98059 and degranulation and IFN-γ production were analyzed. Plots are gated on Ly5.1⁺ TCR tg CD8⁺ T cells. One representative staining of two mice and three independent experiments is shown. (B) Ca²⁺ flux analysis. Spleen cells were isolated, labeled with Fura-red, and stained for Ly5.1 followed by incubation with mock- or gp33-pulsed C57BL/6 peritoneal macrophages; arrows indicate the start of analysis of Ca²⁺ flux in the presence of gp33-pulsed macrophages. Plots are gated on Ly5.1⁺ T cells. Representative plots of two mice and three independent experiments are shown. (C–E) Ly5.1 TCR tg CD8⁺ T cells were sorted from adoptively transfused and h.d.- or l.d.-infected mice or from naïve TCR tg mice. Sorted cells were incubated in presence (+) or absence (−) of gp33 peptide for 16 h. Nuclear and cytoplasmic extracts were prepared and assayed in Western blots for presence of NFAT2 (C; molecular mass, 90, 110, or 140 kDa), p-NFAT2 (D), or NF-κB (E). One of two or three similar experiments is shown.