Phenotype and fate of liver-resident CD8 T cells during acute and chronic hepacivirus infection

Abstract

Immune correlates of hepatitis C virus (HCV) clearance and control remain poorly defined due to the lack of an informative animal model. We recently described acute and chronic rodent HCV-like virus (RHV) infections in lab mice. Here, we developed MHC class I and class II tetramers to characterize the serial changes in RHV-specific CD8 and CD4 T cells during acute and chronic infection in C57BL/6J mice. RHV infection induced rapid expansion of T cells targeting viral structural and nonstructural proteins. After virus clearance, the virus-specific T cells transitioned from effectors to long-lived liver-resident memory T cells (TRM). The effector and memory CD8 and CD4 T cells primarily produced Th1 cytokines, IFN-γ, TNF-α, and IL-2, upon ex vivo antigen stimulation, and their phenotype and transcriptome differed significantly between the liver and spleen. Rapid clearance of RHV reinfection coincided with the proliferation of virus-specific CD8 TRM cells in the liver. Chronic RHV infection was associated with the exhaustion of CD8 T cells (Tex) and the development of severe liver diseases. Interestingly, the virus-specific CD8 Tex cells continued proliferation in the liver despite the persistent high-titer viremia and retained partial antiviral functions, as evident from their ability to degranulate and produce IFN-γ upon ex vivo antigen stimulation. Thus, RHV infection in mice provides a unique model to study the function and fate of liver-resident T cells during acute and chronic hepatotropic infection.

Fig 1. RHV clearance is associated with the expansion of T cells and the formation of CD8 T_RM cells in the liver.

(A) The course of RHV viremia in 6–8 weeks old C57BL/6J mice. A few mice cleared the viremia earlier than most others, between days 10 to 17 p.i. The dotted line indicates the limit of quantification (LOQ) of RT-PCR assay. VGE is virus genomes equivalent per ml of serum. (B) Titers of anti-NS3 IgG antibodies were measured using LIPS assay in the serum samples of infected mice. (C) Serial analysis of total liver-infiltrating leukocytes (LIL), NK (NK1.1⁺), and T (CD3⁺) cells in the liver of infected mice. (D) Absolute numbers of CD4 and CD8 T cells per liver in the RHV infected mice. The numbers on the graph indicate the fold increase relative to the naive mice. Black asterisks indicate significant differences between CD4 and CD8 T cells at the same time point, and colored asterisks indicate significant differences between CD8 or CD4 T cells between different time points. (E) Serial analysis of CD4 T cell differentiation to compare the proportion of FoxP3⁺ Tregs and T-bet⁺ CD4 T cells in the liver of infected mice. (F) The expansion of CD8 T cells coexpressing tissue-retention markers CD69 and LFA-1 in the liver after RHV infection. (G) Representative flow plots of liver-infiltrating CD8 T cells expressing CD62L (blue) overlayed on total CD8 T cells (red) gated for the expression of CD69 and LFA-1 in infected mice on different days after RHV infection. The data are shown for individual mice or as mean ± SEM of 4–11 mice done in >2 independent experiments. All statistical analysis was done in GraphPad PRISM 9.0.0. using a two-tailed unpaired t-test, and only significant p-values are shown, where p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

Fig 2. Specificities of RHV-specific T cells and construction of MHC class I and II tetramers.

(A) RHV infected mice were euthanized on day 17 p.i. to analyze virus-specific T cell responses targeting different RHV proteins in livers (red bars) and spleens (green bars) using IFN-γ ELISPOT assays. Mononuclear cells were stimulated with viral peptides representing different structural and nonstructural proteins (2 μg/mL) for 40–48 h. Each data represents cells pooled from the livers or spleens of 2–3 mice. SFU is spot-forming units. (B) Comparison of total RHV-specific T cells in the livers and spleens of infected mice. (C) ICS analysis to confirm the specificities of selected immunodominant class I and class II epitopes identified in the NS3 protein of RHV. Flow plots show the percentage of CD8 and CD4 T cells producing IFN-γ following 5-hrs ex vivo stimulation with the selected peptides (5 μg/ml). (D) Representative flow plots show the frequencies of intrahepatic CD8 and CD4 T cells that specifically bind the NS3₉₆₈ MHC class I (H-2D^b) and NS3₁₂₆₅ MHC class II (H-2A^b or I-A^b) tetramers. (E) Intracellular cytokine [IFN-γ, TNF-α, and IL-2 (as blue color)] and expression of LFA-1, and transcription factor (T-bet) staining of intrahepatic T cells following 5-h stimulation with the NS3₉₆₈ or NS3₁₂₆₅ peptides. (F) Comparison of NS3₉₆₈ or NS3₁₂₆₅ tetramer labeled T cells and proportion of total T cells producing IFN-γ after ex vivo stimulation with NS3₉₆₈ or NS3₁₂₆₅ peptides of individual mice (n = 8). (G). Relative frequencies of NS3₉₆₈ tetramer-specific CD8 T cells in the liver and PBMC of nine individual RHV infected mice. All statistical analyses used a two-tailed unpaired t-test (F-G, paired t-test), and only significant p-values are shown, where p-values <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****). Fold changes were calculated using means ± standard errors of the means [SEM].

Fig 3. Serial analysis of RHV-specific CD8 T cells in the liver.

(A) The frequencies of NS3₉₆₈-specific CD8 T cells in the liver (red) and spleen (blue) of RHV infected mice. Asterisks show statistically significant differences. Blue asterisks depict the differences between frequencies of Tet+ cells in the liver and spleen. (B) The total number of NS3₉₆₈-specific CD8 T cells in the liver after RHV infection. (C) Expression of the surface markers of T cell activation, tissue residency, and memory on NS3₉₆₈-specific CD8 T cells and NS3₉₆₈-negative CD8 T cells in the liver of RHV infected mice. The baseline expression values of these markers in naïve mice are shown as black circles for comparison. The x-axis displays the % of cells expressing the T cell markers on the NS3₉₆₈- specific (filled red circle) or NS3₉₆₈-negative CD8 T cells (empty circle). Each data represents 1–2 mice, and >6 mice were analyzed in >2 independent experiments for each time point. Statistical analyses to compare the expression of T cell marker on tetramer+ and tetramer- CD8 T cells were done using a two-tailed paired t-test, and only significant p-values are shown, where p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****). (D) Simple linear regression analysis between the titers of RHV viremia on days 10 and 14 p.i. and NS3₉₆₈-specific CD8 T cells (left panel) and the CX3CR1⁺ NS3₉₆₈-specific CD8 T cells (right panel) in corresponding mice (n = 14). (E). Coexpression of T cell markers of tissue-residency, CD69 and LFA-1, and CD62L and TOX, on NS3₉₆₈-specific CD8 T cells from livers of 4 mice (displayed as different colors), and from spleens of the same mice (displayed as black) on day 17 p.i. (F and G). tSNE analysis shows the diversity and changes in the phenotype of NS3₉₆₈-specific CD8 T cells during RHV infection and clearance. FlowSOM was used to cluster NS3₉₆₈-specific CD8 T cells into 15 subpopulations based on the relative expression of different T cell markers. tSNE plots show the expression of T cell markers on different subpopulations (F) and the dynamics of these markers in these populations over time (G). The tSNE analysis was done by pooling the data of liver-infiltrating NS3₉₆₈-specific CD8 T cells of 4–6 mice for each time point.

Fig 4. Phenotype and function of RHV-specific T cells and analysis of memory T cell response during RHV reinfection.

(A) The frequencies and total numbers of NS3₁₂₆₅ specific CD4 T cells in the livers of RHV infected mice. (B) Kinetics of T-bet, CXCR3, and PD-1 expression on NS3₁₂₆₅-specific and non-specific CD4 T cells in the liver of mice. The baseline expression values of these markers in naïve mice are shown as black-filled circles. Each data represents 1–2 mice, and >6 mice were analyzed in >2 independent experiments for each time point. Statistical analyses to compare the expression of T cell marker on Tet⁺ and Tet^- CD4 T cells were done using a two-tailed paired t-test, and only significant p-values are shown, where p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****). (C) The % of CD8 T cells and (D) CD4 T cells producing IFN-γ, TNF-α, IL-2, and IL17 after 5-hrs ex vivo stimulation with NS3₉₆₈ and NS3₁₂₆₅ peptides at 5μg/ml concentration and analyzed by intracellular cytokine staining. (E) The proportions of NS3₉₆₈-specific CD8 T cells producing IFN-γ versus TNF-α during effector (days 10, 14, and 17 p.i.) and memory (days 29 and 53 p.i.) phases. (F). RHV-cleared mice were reinfected >80 days after the first infection with a 10-times higher dose (10⁵ VGE/mice) of mouse-adapted RHV variant to analyze the nature of viremia and recall T cell response on days 3, 5, and 10 p.i. (G) RHV reinfection was associated with a significant increase in the total number of NS3₉₆₈-specific CD8 T cells and NS3₁₂₆₅-specific CD4 T cells in the liver but not in the spleen. (H) Changes in the NS3₉₆₈-specific CD8 T cells in the liver and spleen of mice after RHV reinfection. Black, light red, red, and intense-red circles depict the NS3₉₆₈-specific CD8 T cells for uninfected, day 3, day 5, and day 10 time points, respectively. Asterisks show statistically significant differences: black for uninfected vs. day 3 p.i., light red for day 3 vs. day 5 p.i., and red for day 5 vs. day 10 p.i. Statistical analyses were done using a two-tailed unpaired t-test, and only significant p-values are shown, where p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

Fig 5. Chronic RHV infection leads to T cell exhaustion and the development of severe liver diseases.

(A) RHV viremia in undepleted (acute) and CD4-depleted (chronic) mice. (B) The frequencies of NS3₉₆₈-specific CD8 T cells in mice with acute and chronic infection. (C) ICS analysis of liver-infiltrating CD8 T cells stimulated ex vivo using NS3₉₆₈ peptide indicates that the NS3₉₆₈-specific CD8 T cells in chronically infected mice are exhausted since they fail to produce IFN-γ or express degranulation marker CD107A during the early and late phases of infection. (D) Representative flow plots showing the exhausted nature of CD8 T cells in acute and two chronic mice infected for 80 days p.i. (E). Comparison of cell surface markers and transcription factors on NS3₉₆₈-specific CD8 T cells on day 14, 80 and 300 p.i. in mice with acute and chronic infection. A more extensive flow cytometry panel compared the NS3₉₆₈-specific CD8 T cells in cleared and chronic mice on day 300 p.i. (lower panels). Statistical analyses were done using a two-tailed unpaired t-test, NS means nonsignificant and significant p-values are shown, where p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****). (F). Comparative liver histology of mice with acute and chronic RHV infection (day 300 p.i.). Cleared mouse (a, b, c). (a) Normal portal tracts outlined by collagen (red) and are regularly distributed (Picrosirius Red). (b) Low magnification view of normal-appearing parenchyma (H&E). (c) Higher magnification of hepatocytes with normal age-related hepatocyte variation and clear cytoplasmic spaces due to glycogen accumulation. Chronically infected mouse: (d, g) Increased fibrosis emanating from portal tracts with evidence of bridging and sinusoidal fibrosis. The tumor-bearing liver at the top left corner lacks portal tracks and has lipid vacuoles. (e, h) A hepatocellular carcinoma at the top of the image has a irregular border (line) with the non-neoplastic but inflamed liver with regularly spaced portal tracts below. (f) Higher magnification of lymphocytic infiltrate in the portal region. (i) Higher magnification of the hepatocellular carcinoma with loss of sinusoidal architecture, prominent pleomorphism of hepatocytes, and lipid vacuolation.

Fig 6. The phenotype and transcriptome of RHV-specific liver CD8 T_RM cells.

(A). Comparative expression of cell surface markers on the NS3₉₆₈-specific CD8 T cells from the liver (T⁺_RM) and spleen (T⁺_S) of RHV cleared mice. Asterisks show statistically significant differences between the expression of T cell markers on NS3₉₆₈-specific CD8 T cells in the liver and spleen, where blue asterisks depict the differences between day 14 p.i. and red asterisks depict the differences between day 80 p.i. (B) NS3₉₆₈-specific CD8 T cells from the liver (T⁺_RM) and spleen (T⁺_S) of RHV cleared mice, and CD62L⁺ CD8 T cells (P^NM) from the spleen of naïve mice were labeled and sorted using BD Influx Cell Sorter to prepare RNA-seq libraries. (C). PCA analysis of the normalized read counts for all samples. (D). Pooled data of all T⁺_RM and T⁺_S samples were compared to identify the 100 most over- and under-expressed DEGs. Thirty-six DEGs that showed consistent log2 fold change gene expression patterns between all samples of the groups are shown together with P^NM samples. (E). The heatmap shows log2 fold change expression pattern of DEGs between RHV-specific T⁺_RM and T⁺_S analyzed in this study compared to the pattern of 33 core-signature DEGs reported for CD8 T⁺_RM cells of skin, lung, gut, and liver. (F). The heat map shows log2 fold change expression pattern of 63 DEGs that were the most up- or down-regulated between T⁺_RM and T⁺_S of RHV-cleared mice. (G). The volcano plot shows selected significantly differentially expressed genes including T cell transcription factors between T⁺_RM and T⁺_S cells.