Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming

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Abstract

The responses of CD8+ T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8+ T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8+ T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.

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Fig. 1: Spatiotemporal dynamics of naive CD8+ T cells undergoing intrahepatic priming.
Fig. 2: Transcriptomic and chromatin accessibility analyses of CD8+ T cells undergoing intrahepatic priming.
Fig. 3: Intrahepatically primed, dysfunctional CD8+ T cells can be rescued by IL-2, but not by anti-PD-L1 antibodies.
Fig. 4: Therapeutic potential of IL-2 treatment for restoration of T cells during chronic HBV infection.

Data and availability

The RNA-seq and ATAC-seq data on sorted hepatic CD8+ T cells have been deposited in the ArrayExpress database under the accession codes E-MTAB-7462 and E-MTAB-7461, respectively. All other data are available in the main text or the supplementary materials.

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Acknowledgements

We thank F. V. Chisari for critical comments and suggestions, and for providing transgenic mouse lineages 1.3.32, MUP-core 50, Cor93 and Env28 TCR that were produced in his laboratory at The Scripps Research Institute in La Jolla; A. Fiocchi, M. Freschi, M. Mainetti, M. Raso and G. Sitia for technical support; M. Silva for secretarial assistance; E. Lugli, A. Mondino, L. Pace, R. Pardi and S. Trifari for critical reading of the manuscript and the members of the Iannacone laboratory for discussions. Confocal immunofluorescence histology was carried out at Alembic, an advanced microscopy laboratory established by the San Raffaele Scientific Institute and the Vita-Salute San Raffaele University. Flow cytometry was carried out at FRACTAL, a flow cytometry resource and advanced cytometry technical applications laboratory established by the San Raffaele Scientific Institute. We would like to acknowledge the PhD program in Basic and Applied Immunology and Oncology at Vita-Salute San Raffaele University, as G.D.S., F.C., V.F. and V.B. conducted this study as partial fulfilment of their PhD in Molecular Medicine within that program. M.I. is supported by European Research Council (ERC) Consolidator Grant 725038, Italian Association for Cancer Research (AIRC) grant 19891 and 22737, Italian Ministry of Health (MoH) grant RF-2018-12365801, Lombardy Foundation for Biomedical Research (FRRB) grant 2015-0010, the European Molecular Biology Organization Young Investigator Program, and a Career Development from the Giovanni Armenise-Harvard Foundation; A.P.B. is the recipient of EMBO Long-Term Fellowship ALTF 694-2016; F.M. is the recipient of Marie Curie Intra-European Fellowship (IEF) for Career Development SEP-210371319; F.A. is the recipient of a Fondazione Umberto Veronesi postdoctoral fellowship; M.K. is supported by the Italian Ministry of Education grant SIR-RBSI14BAO5; L.G.G. is supported by the Italian MoH grant RF-2013-02355209 and the Lombardy Open Innovation grant 229452; R.O. is supported by ERC Starting Grant 759532, Italian Telethon Foundation SR-Tiget Grant Award F04, Italian MoH grant GR-2016-02362156, AIRC MFAG 20247, Cariplo Foundation Grant 2015-0990 and the EU Infect-ERA 126.

Author information

A.P.B. and G.D.S. designed and performed experiments, analysed data, performed the statistical analyses, prepared the figures and edited the paper; P.D.L., F.M., P.Z., V.F., E.B., L.G., M.K., F.A. performed experiments and analysed data; F.C. generated RNA-seq and ATAC-seq data with help from V.B.; G.B. and E.L. analysed RNA-seq and ATAC-seq data; R.O. supervised F.C., G.B., E.L. and V.B. and prepared figures; N.L.B., K.K., P.T.F.K. and A.B. performed experiments on HBV-infected patients, analysed data, prepared the figures and edited the manuscript; C.B. and F.G. helped with experiments involving Kupffer cells; A.C. and L.N. generated the lentiviral vectors encoding IL-2; G.G.-A. generated recombinant adeno-associated viruses; W.V.B. and D.D.P. generated rLCMV vectors; M.K., R.O. and L.G.G. provided funding, conceptual advice and edited the manuscript; M.I. designed and coordinated the study, provided funding, analysed the data, and wrote the paper.

Correspondence to Matteo Iannacone.

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Competing interests

M.I., L.G.G., R.O., A.C. and L.N. are inventors on patents filed, owned and managed by Telethon Foundation and San Raffaele Scientific Institute on LV technology related to the work presented in this manuscript (UK patent application 1907493.9).

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Peer review information Nature thanks Barbara Rehermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Naive CD8+ T cells that recognize hepatocellular antigen are activated locally and expand, but do not develop effector function.

a, Schematic of the experimental setup. Five million Env28 TN cells were transferred into C57BL/6 × BALB/c F1 (WT) or HBV replication-competent transgenic (HBV Tg, C57BL/6 × BALB/c F1) recipients. Livers were collected and analysed 5 days after Env28 TN cell transfer and sera from the same mice were collected daily from day 0 to 5 after transfer. b, c, Absolute numbers of total (b) and IFNγ-producing (c) Env28 T cells in the livers of the indicated mice. d, ALT levels detected in the sera of the indicated mice at the indicated time points. n = 4. e, Schematic of the experimental setup. Five million Cor93 TN cells were transferred into C57BL/6 (WT) or MUP-core recipients. Mice were splenectomized and treated with anti-CD62L antibody 48 h or 4 h before cell transfer, respectively. Untreated wild-type mice that received 5 × 106 Cor93 TN cells were used as controls. Where indicated, mice were injected with 2.5 × 105 infectious units of non-replicating rLCMV-core 4 h before Cor93 TN cell transfer. Liver-draining lymph nodes68 (dLN) and non-draining inguinal lymph nodes (ndLN) were collected at 4 h and 1 day after transfer. f, Representative flow cytometry plots 4 h after Cor93 TN cells transfer. Numbers indicate the percentage of cells within the indicated gate. g, h, Quantification of the absolute numbers of cells recovered from the ndLN (g) and dLN (h) of the indicated mice 4 h and 1 day (d1) after Cor93 TN cell transfer. n = 3. i, Confocal immunofluorescence micrographs of liver sections from wild-type mice, wild-type mice transduced with rLCMV-core, MUP-core mice, and R26-ZsGreen mice injected with 2.5 × 105 infectious units of non-replicating rLCMV-cre. Scale bars, 100 μm. Note that, because HBV core protein did not accumulate at detectable levels in Kupffer cells and hepatic dendritic cells after rLCMV-core injection, we confirmed the tropism of this vector by injecting rLCMV-cre into R26-ZsGreen mice, which express the fluorescent protein ZsGreen after Cre-mediated recombination. j, MFI of CD69 expression on Cor93 T cells in the liver, blood, lung and bone marrow of the indicated mice 4 h after Cor93 TN cell transfer. n = 4. Data are mean ± s.e.m. and representative of at least three independent experiments. ***P < 0.001, two-tailed t-test (b, c) or one-way ANOVA with Bonferroni post-test (g, h, j). Mouse drawings were adapted from ref. 69. Source data

Extended Data Fig. 2 Spatiotemporal dynamics of naive CD8+ T cells after intrahepatic priming.

Five million fluorescent Cor93 TN cells were transferred into MUP-core mice or wild-type mice transduced with rLCMV-core. Mice were splenectomized and treated with anti-CD62L antibody 48 h or 4 h before Cor93 TN transfer cell, respectively. a, Left, confocal immunofluorescence micrographs of liver sections from the indicated mice at the indicated time points after Cor93 TN cell transfer, showing the distribution of Cor93 T cells (green) relative to portal tracts (highlighted by anti-cytokeratin 7 (CK-7)-antibody-mediated staining of bile ducts in red). Sinusoids are highlighted by anti-LYVE-1+ antibodies (white). Scale bars, 100 μm. Right, immunohistochemical micrographs of liver sections from the indicated mice at the indicated time points after Cor93 TN cell transfer, showing the distribution of leukocyte infiltrates relative to portal tracts (highlighted by anti-CK-7-antibody-mediated staining of bile ducts in brown). Scale bars, 100 μm. b, Distribution of the distances of each Cor93 T cell from the centre of the closest portal triad at the indicated time points. n = 3 mice. Data are representative of at least three independent experiments. Source data

Extended Data Fig. 3 Kupffer cells, but not dendritic cells, promote CD8+ T cell effector differentiation after rLCMV injection.

a, Schematic of the experimental setup. Five million Cor93 TN cells were transferred into C57BL/6 (WT) recipients. Mice were splenectomized and treated with anti-CD62L antibodies 48 h or 4 h before cell transfer, and injected with 2.5 × 105 infectious units of non-replicating rLCMV-core 4 h before Cor93 TN cell transfer. Where indicated, mice were treated with clodronate liposomes (CLL) 48 h before Cor93 TN cell transfer. b, Confocal microscopy of liver sections from control mice (top) and clodronate liposome-treated mice (bottom) Kupffer cells are depicted in red in all panels, and sinusoids are depicted in grey only in the left panels. Scale bars, 100 μm. c, Absolute numbers of CD11c+MHC-IIhigh dendritic cells (DCs) in the livers of the indicated mice. d, e, Absolute numbers of total (d) and of IFNγ-producing (e) Cor93 T cells in the livers of the indicated mice 5 days after Cor93 TN cell transfer. n = 4 mice (control) and 3 mice (CLL). f, Confocal immunofluorescence micrographs of liver sections from the indicated mice 5 days after Cor93 TN cell transfer. Scale bars, 100 μm. g, Schematic of the experimental setup. Wild-type mice were lethally irradiated and reconstituted with CD11c-DTR bone marrow (BM). One million Cor93 TN cells were transferred into recipients. Mice were injected with 2.5 × 105 infectious units of non-replicating rLCMV-core 4 h before Cor93 TN cell transfer. Indicated mice were treated with 400 ng of diphtheria toxin (DT) 3 days before, 1 day before and 1 day after T cell transfer. Livers were collected and analysed 5 days after Cor93 TN cell transfer. h, Representative flow cytometry plots in the liver of control (left) or diphtheria-toxin-treated (right) mice. i, CD11c+MHC-II+ dendritic cells (expressed as percentage of the total intrahepatic leukocyte population, IHL) in the livers of the indicated mice. n = 3. j, k, Absolute numbers of total (j) and IFNγ-producing (k) Cor93 T cells in the livers of the indicated mice 5 days after Cor93 TN cell transfer. n = 3 (control and WT + rLCMV-core), 4 (control + DT) and 5 (WT + rLCMV-core + DT). Data are mean ± s.e.m. and representative of three independent experiments. **P < 0.01, ***P < 0.001, two-tailed t-test (d, e, i) or one-way ANOVA with Bonferroni post-test (ik). Mouse drawings were adapted from ref. 69. Source data

Extended Data Fig. 4 A strong reduction in the levels of hepatocellular core antigen expression is per se not sufficient to induce effector differentiation.

a, Schematic of the experimental setup. One million Cor93 TN cells were transferred into C57BL/6 (WT) or MUP-core recipients. Indicated wild-type mice were injected with 3 × 1010 viral genomes of AAV-core 15 days before Cor93 TN cell transfer. Livers were collected and analysed 5 days after Cor93 TN cell transfer. b, Representative confocal immunofluorescence micrographs of a liver section from an AAV-core-injected mouse 15 days after virus injection. Transduced hepatocytes are depicted in green and nuclei in grey. Scale bar, 50 μm. n = 3 mice. ce, Absolute numbers of total (c) and IFNγ-producing (d) Cor93 T cells in the livers of the indicated mice 5 days after Cor93 TN cell transfer. e, ALT levels detected in the sera of the indicated mice. n = 3 (WT and MUP-core) and 5 (AAV-core). f, Schematic of the experimental setup. One million Cor93 TN cells were transferred into 8- or 4-week-old (wo) MUP-core mice. Livers were collected and analysed 5 days after Cor93 TN cell transfer. g, Expression of HBV core antigen (HBcAg) in the livers of the indicated mice was analysed by western blotting. h, Quantification of the western blot shown in g. Core expression, normalized to the housekeeping nuclear protein H3, is expressed as arbitrary units (A.U.). n = 1 (WT) and 3 (MUP-core 8wo and MUP-core 4wo). i, Immunohistochemical micrographs of liver sections from the indicated mice, showing core antigen expression (brown). Scale bars, 50 μm. CV, central vein; PV, portal vein. n = 3 mice. j, k, Absolute numbers of total (j) and of IFNγ-producing (k) Cor93 T cells in the livers of the indicated mice 5 days after Cor93 TN cell transfer. n = 4 mice. l, ALT levels detected in the sera of the indicated mice. n = 4. Data are mean ± s.e.m. and representative of two independent experiments. *P < 0.05, **P < 0.01, one-way ANOVA with Bonferroni post-test (ce) or two-tailed t-test (h, jl). Mouse drawings were adapted from ref. 69. Source data

Extended Data Fig. 5 Genomic landscape of naive CD8+ T cells undergoing intrahepatic priming.

a, Box plots showing expression levels (log2(RPKM)) in the indicated experimental condition of genes belonging to the categories described in Fig. 2a. Box plots are as in Fig. 3f. Naive (n = 2), WT + rLCMV-core (n = 3), MUP-core (day1 and 3, n = 2; day 7, n = 3). b, Box plots showing ATAC-seq signal intensity (log2(CPM)) in the indicated experimental condition of peaks belonging to the categories described in Fig. 2c. Box plots are as in Fig. 3f. Naive (n = 2), WT + rLCMV-core (day 1 and 7, n = 2; day 3, n = 3), MUP-core (day 1 and 3, n = 2; day 7, n = 3). P values in a and b were determined by two-sided Mann–Whitney U-test. c, Bar plot showing the number of inducible ATAC-seq peaks (logFCCPM > 2.5, FDR < 0.001 versus Cor93 TN) in the indicated conditions. ATAC-seq peaks with higher intensity signal in Cor93 T cells from WT + rLCMV-core (logFCCPM > 1.5, FDR < 0.1) or from MUP-core mice (logFCCPM < −1.5, FDR < 0.1) are shown in blue and red, respectively. Differences in peak signal intensities were evaluated fitting a negative binomial generalized linear model on the dataset and then performing a quasi-likelihood F-test. The Benjamini–Hochberg procedure was applied to correct for multiple tests. Naive (n = 2), WT + rLCMV-core (day 1 and 7, n = 2; day 3, n = 3), MUP-core (day 1 and 3, n = 2; day 7, n = 3).

Extended Data Fig. 6 GO analysis of intrahepatically primed CD8+ T cells.

Heat map showing the NES value associated to the seed GO categories (identified by REVIGO) found enriched in the indicated time points by GSEA. Colour legends indicate NES, with positive values (in blue) reflecting enrichment of GO categories in hepatic CD8+ T cells isolated from wild-type mice injected with rLCMV-core, and negative values (in red) reflecting enrichment of GO categories in hepatic CD8+ T cells isolated from MUP-core mice.

Extended Data Fig. 7 Although priming by hepatocytes initiates a unique dysfunctional program, hepatocellular antigen persistence may gradually trigger an additional exhaustion signature.

ad, Left, number of top 100 genes from Cor93 T cells recovered from the livers of wild-type mice transduced with rLCMV-core (a, c) or of MUP-core (b, d) mice reaching log2(RPKM) >1 in the indicated conditions in RNA-seq data from splenic LCMV-specific effector or exhausted CD8+ T cells30 (a, b) or splenic LCMV-specific exhausted CD8+ T cells31 (c, d). Right, box plots showing the expression levels of top 100 genes from Cor93 T cells recovered from livers of WT + rLCMV-core mice (a, c) or MUP-core mice (b, d) in the indicated conditions in RNA-seq data from splenic LCMV-specific effector or exhausted CD8+ T cells30 (a, b) or splenic LCMV-specific exhausted CD8+ T cells31 (c, d). Naive (n = 2), effector (n = 2), exhausted (n = 2). e, f, Left, number of top 100 genes in Cor93 T cells isolated from livers of wild-type + rLCMV-core mice (e) or from MUP-core mice (f) expressed (log2(normalized data) > 65th percentile of the full distribution) in the indicated conditions in microarray data from tolerant self-antigen-specific CD8+ T cells32. Right, box plots showing the expression levels of genes retrieved in the dataset among the top 100 genes in Cor93 T cells isolated from the livers of wild-type + rLCMV-core (e) or MUP-core (f) mice in the indicated conditions in microarray data from tolerant self-antigen-specific CD8+ T cells32. Only genes for which microarray probes were retrieved were kept for these analyses. Naive (n = 3), tolerant (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Wilcoxon rank-sum test. All box plots are represented as in Fig. 3f, and dots represent the expression distribution of the set of 100 genes. gi, Enrichment plot showing the results of a GSEAPreanked analysis (Kolmogorow–Smirnov statistics) performed on genes expressed in CD8+ T cells from wild-type + rLCMV-core or MUP-core mice (gene lists ranked by log(FCRPKM)) and using as gene set a curated list of genes induced in exhausted CD8+ T cells (n = 2) but not in effector CD8+ T cells (n = 2) as compared to naive cells (n = 2)30. NES and P values are reported for each time point.

Extended Data Fig. 8 IL-2c substantially rescues the transcriptional program of dysfunctional CD8+ T cells.

Heat map showing expression values (log2(RPKM)) of genes hypo-expressed (top) or hyper-expressed (bottom) in Cor93 CD8+ T cells from livers of MUP-core mice at day 5, which are rescued by treatment with IL-2c.

Extended Data Fig. 9 Therapeutic restoration of intrahepatically primed, dysfunctional CD8+ T cells by IL-2.

a, Schematic of the experimental setup. One million Cor93 TN cells were transferred into HBV Tg mice. Indicated HBV Tg mice received IL-2c treatment 1 day after CD8+ T cell transfer. Livers were collected and analysed 5 days after Cor93 TN cell transfer. Sera were collected before and 5 days after Cor93 TN cell transfer. b, Absolute numbers of IFNγ-producing Cor93 T cells in the livers of the indicated mice. n = 3 (control), 4 (IL-2c). c, ALT levels detected in the sera of the indicated mice. n = 3 (control), 4 (IL-2c). d, HBV DNA quantification (expressed as fold reduction over pre-treatment levels) in sera of the indicated mice before and 5 days after Cor93 TN cell transfer. n = 5. e, HBV DNA quantification by Southern blot analysis in the liver of the indicated mice. Bands corresponding to the expected size of the integrated transgene, relaxed circular (RC), double-stranded linear (DS), and single-stranded (SS) HBV DNAs are indicated. n = 5 mice. f, Representative immunohistochemical micrographs of liver sections from the indicated mice showing HBV core antigen expression (brown). Scale bars, 100 μm. n = 5 mice. Data are mean ± s.e.m. and representative of at least two independent experiments. *P < 0.05, ***P < 0.001, two-tailed t-test. Mouse drawings were adapted from ref. 69. Source data

Extended Data Fig. 10 Summary of the main findings.

Top, priming by Kupffer cells—which are not natural targets of HBV—leads to differentiation into bona fide effector cells that form dense, extravascular clusters of rather immotile cells scattered throughout the liver. Middle, priming by hepatocytes—which are the natural targets of HBV—leads to local activation and proliferation but lack of differentiation into effector cells; these dysfunctional cells express a unique set of genes including some belonging to GO categories linked to tissue remodelling and they form loose, intravascular clusters of motile cells that coalesce around portal tracts. Bottom, CD8+ T cells primed by hepatocytes can be rescued by IL-2 treatment.

Supplementary information

Supplementary Figure

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Reporting Summary

Supplementary Table 1 A list of datasets generated in this study and sequencing information

Supplementary Table 2 Differentially expressed genes from RNA-seq analyses on hepatic Cor93 CD8+ T cells before and after (1, 3 and 7 days) adoptive transfer into rLCMV-core-transduced WT mice or MUP-core mice. Differential gene expression was evaluated fitting a negative binomial generalized linear model on the dataset and then performing a quasi-likelihood (QL) F-test. The Benjamini-Hochberg procedure was applied in order to correct for multiple tests. Sample size: naïve (n = 2), WT+rLCMV-core (n = 3), MUP-core (day1 and day3, n = 2; day7, n = 3).

Supplementary Table 3 Differential ATAC-seq peaks of hepatic Cor93 CD8+ T cells before and after (1, 3 and 7 days) adoptive transfer into rLCMV-core-transduced WT mice or MUP-core mice. Differences in peak signal intensities were evaluated fitting a negative binomial generalized linear model on the dataset and then performing a quasi-likelihood (QL) F-test. The Benjamini-Hochberg procedure was applied in order to correct for multiple tests. Sample size: naïve (n = 2), WT+rLCMV-core (day1 and day7, n = 2; day3, n = 3), MUP-core (day1 and day3, n = 2; day7, n = 3).

Supplementary Table 4 Motif enrichment analyses on differential ATAC-seq peaks in hepatic Cor93 CD8+ T cells before and after (1, 3 and 7 days) adoptive transfer into rLCMV-core-transduced WT mice or MUP-core mice. Motif enrichment was calculated using cumulative binomial distributions. The Benjamini-Hochberg procedure was applied in order to correct for multiple tests. n = 2 (Naïve) or 3 (WT+rLCMV-core and MUP-core) biologically independent samples.

Supplementary Table 5 Differentially expressed genes from RNA-seq analyses on hepatic or splenic Cor93 CD8+ T cells before and after (1, 3 and 7 days) adoptive transfer into rLCMV-core-transduced WT mice. Cor93 CD8+ T cells from the liver of rLCMV-core-injected mice that were not splenectomized nor treated with anti-CD62L upregulated up to 82.8% of the genes induced in Cor93 CD8+ T cells isolated from the spleen of the same mice, including critical effector-related molecules such as Gzma, Gzmb, Gzmc, Gzmk, Il2, Ifng and many others. A negative binomial generalized linear model followed by quasi-likelihood (QL) F-test and Benjamini-Hochberg procedure for multiple tests correction was used for differential gene expression analysis. n = 3 biologically independent samples.

Supplementary Table 6 Enriched Gene Ontologies in RNA-seq data of hepatic Cor93 CD8+ T cells before and after (1, 3 and 7 days) adoptive transfer into rLCMV-core-transduced WT mice or MUP-core mice. n = 2 (Naïve) or 3 (WT+rLCMV-core and MUP-core) biologically independent samples.

Supplementary Table 7 Lists of 100 inducible genes (versus Cor93TN) with highest differential expression in hepatic Cor93 CD8+ T cells 7 days after adoptive transfer into rLCMV-core-transduced WT mice or MUP-core mice.

Supplementary Table 8 Expression levels of genes from Table S7 in datasets from LCMV-specific exhausted CD8+ T cells (refs.30,31) and from tolerant self-Ag-specific CD8+ T cells (ref.32). Numbers of expressed genes from Table S7 in datasets from LCMV-specific exhausted CD8+ T cells (refs.30,31) and from tolerant self-Ag-specific CD8+ T cells (ref.32).

Supplementary Table 9 Differentially expressed genes from RNA-seq analyses on Cor93 CD8+ T cells before and after (5 days) adoptive transfer into WT + rLCMV-core or MUP-core mice upon treatment with vehicle or IL-2c. A negative binomial generalized linear model followed by quasi-likelihood (QL) F-test and Benjamini-Hochberg procedure for multiple tests correction was used for differential gene expression analysis. Sample size: WT + rLCMV-core (n = 3); MUP-core (n = 3); MUP-core+IL-2c (n = 2).

Supplementary Table 10 Clinical and virological details of chronic HBV patients. Patients were categorized into EASL 2017 standard phases using the clinical and virological criteria outlined in the EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection9: i) HBeAg+ chronic infection (eAg+CInf): normal ALT (< 40 IU/L), HBeAg positive and high HBV DNA; ii) HBeAg+ chronic hepatitis (eAg+CHep): elevated ALT, HBeAg positive; iii) HBeAg- chronic hepatitis (eAg-CHep): elevated ALT, anti-HBe positive; and iv) HBeAg- chronic infection (eAg-CInf): normal ALT, anti-HBe positive, low HBV DNA. Patients were followed for at least 1 year with virological and clinical parameters collected every 6 months. The ALT and virological parameters shown in Supplementary Table 10 are the ones present at the time of PBMC isolation. Patients were classified as immunotolerant (IT) if they had HBeAg+ chronic infection (eAg+CInf); alternatively, they were classified as immune active (IA) if they showed signs of immunological activity, that is eAg+CHep or eAg-CHep.

Video 1 Multiphoton intravital microscopy in the livers of rLCMV-core-transduced WT or MUP-core mice 3 days after Cor93 TN transfer. Cor93 T cells (green) form dense, poorly perfusable clusters that are scattered throughout the liver and are composed of largely immotile cells in WT + rLCMV-core livers (left movies); by contrast Cor93 T cells (green) form looser, intravascular clusters that coalesce around portal tracts and are composed of more motile cells in MUP-core livers (right movies). Blood vasculature is visualized by the intravenous injection of nontargeted Quantum Dots 655 (white). Scale 50 μm (first movie set) or 20µm (second movie set); time in min:s. For four-dimensional analysis of cell migration, 14-15 z-stacks (spacing 4 µm) of 520 µm x 240 µm xy sections were acquired every 27 s for 40 min. Data are representative of at least 5 independent experiments.

Video 2 Multiphoton intravital microscopy in the livers of rLCMV-core-transduced WT or MUP-core mice 3 days after Cor93 TN transfer. Videos show tracks (yellow) of Cor93 T cells in the livers of rLCMV-core-transduced WT (left movies) or MUP-core mice (right movies). Blood vasculature is visualized by the intravenous injection of nontargeted Quantum Dots 655 (white). Scale 10 μm (first movie set) or 20 µm (second movie set); time in min:s. For four-dimensional analysis of cell migration, 12-17 z-stacks (spacing 4 µm) of 260 µm x 140 µm xy sections were acquired every 23 to 30 s for 38 min to 1 h.

Video 3 Multiphoton intravital microscopy in the livers of rLCMV-core-transduced WT or MUP-core mice 7 days after Cor93 TN transfer. Cor 93 T cell clusters (green) in WT mice transduced with rLCMV-core (left movies) start to disaggregate as cells move out from the liver into the peripheral blood; by contrast Cor 93 T cell clusters (green) in MUP-core mice (right movies) remain in place, possibly reflecting Ag persistence. Scale 40 μm (first movie set) or 20 µm (second movie set); time in min:s. For four-dimensional analysis of cell migration, 10-18 z-stacks (spacing 4 µm) of 520 µm x 200 µm xy sections were acquired every 18 to 32 s for 25 min.

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