Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes

Abstract

Liver infections with hepatotropic viruses, such as hepatitis B virus and hepatitis C virus are accompanied by viral persistence and immune failure. CD8+ T cells are crucial mediators of the intrahepatic antiviral immune response. Chronic infections of the liver and other organs correlate with T-cell exhaustion. It was previously suggested that high antigen load could result in T-cell exhaustion. We aimed at elucidating the impact of different intrahepatic antigen loads on the quality of CD8+ T-cell-mediated immunity by employing an infection-free transgenic mouse model expressing ovalbumin (Ova) as the target antigen. Adoptive transfer of OT-I cells induced a transient intrahepatic immune response toward both high and low Ova levels. However, antigen clearance was achieved only in mice expressing low antigen levels. In contrast, T cells exposed to high antigen levels underwent exhaustion and became depleted, causing antigen persistence. Moreover, when functional T cells were exposed to high intrahepatic antigen levels, a complete transition toward exhaustion was observed. Thus, this study shows that the antigen expression level in the liver correlates inversely with T-cell immunity in vivo and governs the efficiency of immune responses upon antigen presentation.

Figure 1

Adoptive transfer of antigen-specific T cells induces acute hepatitis. (a) Analysis of ALT activity in Ova × CreER^T2 mouse plasma on the indicated days upon adoptive transfer of 5 × 10⁶ naïve OT-I cells. ALT activity below 40 U L⁻¹ was considered to be physiological. (b) qRT-PCR-based analysis of Ova expression within the liver of Ova × CreER^T2 mice on day 13 upon adoptive transfer of OT-I cells. The means of n ≥ 4 mice/group ± SD are shown. The kinetic analysis was performed twice, analysis of day 3 was done more than 6 times.

Figure 2

The differentiation of antigen-specific T cells depends on the amount of antigen-presenting cells in the liver. 5 × 10⁶ OT-I cells were adoptively transferred into Ova × CreER^T2 mice that were Tam-induced or untreated. On days 3 and 13, the total numbers of non-parenchymal liver cells (a) and antigen-specific Thy1.1+ OT-I cells (b) were calculated. (c–e) Phenotypic characterization of Thy1.1+ OT-I cells isolated from recipient mouse livers. The expression of the activation marker CD69, as reflected by the geometric mean fluorescence intensity (MFI), was evaluated by flow cytometry on days 3 and 13, after adoptive transfer (c). Analysis of PD-1 (d) and Lag-3 (e) exhaustion marker expression on days 3 and 13 after adoptive transfer of liver localized Thy1.1+ OT-I cells. The PD-1 and Lag-3 expression levels are depicted as the percentage of positive cells, as determined by flow cytometry. The expression levels were compared with those of non-specific CD8+ T cells. The means of n = 3–8 mice ± SD (bar diagrams) and the representative histograms are depicted from one of the four performed experiments.

Figure 3

T-cell effector function upon adoptive transfer. (a) On day 3 after adoptive transfer, the OT-I cells were isolated from the liver and stimulated in vitro with Ova peptide to induce effector cytokine expression. Seven hours after stimulation, the cells were stained for the effector cytokines IFNγ (upper panel) and TNFα (lower panel). The expression levels were compared with those of Ova single transgenic control mice. (b) Quantification of IFNγ/TNFα double positive OT-I cells isolated from the livers and spleens of Ova × CreER^T2 mice on day 3. (c) Investigation of cytokine expression in OT-I cells on day 13 following adoptive transfer. Cells from the liver were treated and analyzed as described in (a). IFNγ (upper panel) and TNFα (lower panel) release was investigated. (d) Quantification of the IFNγ/TNFα double positive OT-I cells in the livers and spleens of the respective recipient groups is shown. (e) In vivo cytotoxic activity of OT-I cells in the periphery of adoptively transferred recipient mice. OT-I recipients were challenged with Ova-pulsed (CFSE^hi) and control (CFSE^lo) splenocytes on days 13 (left panel) and 27 (right panel). Eighteen hours after transfer, the CFSE-labeled cell populations in the spleen were analyzed by flow cytometry to investigate the specific lysis percentage. Results from n ≥ 5 mice/group ± SD from two independent experiments are shown.

Figure 4

The capacity of OT-I cells primed inflow antigen conditions and subsequently exposed to high antigen loads. (a) The experimental scheme. (b) Serum ALT levels upon adoptive transfer of 5 × 10⁶ naïve OT-I cells into low antigen-expressing mice on days -10 and 3 upon induction of a high antigen load. The intrahepatic Ova expression levels were assessed with qRT-PCR 14 days following Tam application (c). At the same time, the numbers of Thy1.1+ OT-I cells in the liver (left panel) and spleen (right panel) were calculated (d). Mean of n ≥ 3 mice/group ± SD is shown for one of two independent experiments.