Hepatocytes undergo punctuated expansion dynamics from a periportal stem cell niche in normal human liver

Background & Aims: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. Methods: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver. Results: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations. Conclusions: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver. Impact and implications: The liver is mainly composed of hepatocytes, but we know little regarding the source of these cells or how they multiply over time within the disease-free human liver. In this study, we determine a source of new hepatocytes by combining many different lab-based methods and computational predictions to show that hepatocytes share a common cell of origin with bile ducts. Both our experimental and computational data also demonstrate hepatocyte clones are likely to expand in slow waves across the liver in a specific trajectory, but often lie dormant for many years. These data show for the first time the expansion dynamics of hepatocytes in normal liver and their cell of origin enabling the accurate measurment of changes to their dynamics that may lead to liver disease. These findings are important for researchers determining cancer risk in human liver

Clonal transitions and phenotypic evolution in Barrett esophagus

BACKGROUND & AIMS: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma but our understanding of how it evolves is poorly understood. We investigated BE gland phenotype distribution, the clonal nature of phenotypic change, and how phenotypic diversity plays a role in progression. METHODS: Using immunohistochemistry and histology, we analyzed the distribution and the diversity of gland phenotype between and within biopsy specimens from patients with nondysplastic BE and those who had progressed to dysplasia or had developed postesophagectomy BE. Clonal relationships were determined by the presence of shared mutations between distinct gland types using laser capture microdissection sequencing of the mitochondrial genome. RESULTS: We identified 5 different gland phenotypes in a cohort of 51 nondysplastic patients where biopsy specimens were taken at the same anatomic site (1.0-2.0 cm superior to the gastroesophageal junction. Here, we observed the same number of glands with 1 and 2 phenotypes, but 3 phenotypes were rare. We showed a common ancestor between parietal cell-containing, mature gastric (oxyntocardiac) and goblet cell-containing, intestinal (specialized) gland phenotypes. Similarly, we have shown a clonal relationship between cardiac-type glands and specialized and mature intestinal glands. Using the Shannon diversity index as a marker of gland diversity, we observed significantly increased phenotypic diversity in patients with BE adjacent to dysplasia and predysplasia compared to nondysplastic BE and postesophagectomy BE, suggesting that diversity develops over time. CONCLUSIONS: We showed that the range of BE phenotypes represents an evolutionary process and that changes in gland diversity may play a role in progression. Furthermore, we showed a common ancestry between gastric and intestinal-type glands in BE

Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver

Blocking the Tim-3 pathway can increase the frequency of IFNγ and TNFα-producing HBV-specific CD8 T cells.

<p>(a) Representative dot plot showing recovery of HBV-specific CD8 T cells responses (IFN-γ, TNF-α) in a patient with CHB. PBMC were stimulated with peptides and cultured for 10 days in the presence of soluble Tim-3 FC chimera, PDL1/L2 blocking antibody or both. Summary data of the effect of blocking Tim-3 on IFN-γ (b) and TNF-α (c) production by CD8 T cells in response to HBV peptides.</p

Effect of antiviral treatment on Tim-3 expression and response to Tim-3 blockade.

<p>(a) Tim-3 expression on global CD3 CD4+ T cells and CD3 CD8+ T cells. Each dot represents an individual data point (12 healthy, 26 CHB, 6 CHB on antivirals with undetectable viral load); horizontal lines represent the mean. (b) Percent of CD4 (square), CD8 (triangle) T cells expressing Tim-3 (left y axis) and HBV load circle, right y axis) plotted longitudinally for 3 patients starting antiviral therapy. (c) PBMC sampled at the indicated time points from these three individuals were stimulated for ten days with HBV OLP with control IgG, Tim-3 Fc chimera, anti-PDL1/PDL2 antibodies or both. Bars represent the % of HBV-specific CD8 T cells producing IFN-γ or TNF-α following blockade after subtracting the frequency detectable without any blockade.</p

Patient clinical characteristics.

*<p>Median and range shown.</p>**<p>NA not applicable.</p

Clinical characteristics of patients followed up longitudinally.

<p>Clinical characteristics of patients followed up longitudinally.</p

Tim-3 expressing HBV-specific T cells are dysfunctional.

<p>PBMC derived from HLA A2+ patients with CHB were stained with a panel of HLA-A2/HBV multimers and then were stimulated overnight with a pool of HBV peptides of matched specificity to the multimers, followed by intracellular staining for IFN-γor TNF-α. (a) Representative histograms showing levels of Tim-3 (black line) or isotype binding (grey shading) on CD8 T cells binding HLA-A2/HBV peptide multimers or producing IFN-γ upon encounter with HBV peptides. (b) Compiled data from 10 patients with CHB. (c) Tim-3 expression on CD8 T cells binding HLA-A2/HBV peptide multimers or producing TNFa upon stimulation with HBV peptides. (d) FACS plots and (e) summary data showing the induction of caspases (FLICA) and 7AAD in CD8 and CD4 T cells with or without the addition of galectin-9. Active caspases, indicating apoptosis, were determined using a fluorescent-labelled inhibitor of polycaspases (FAM-VAD-FMK, FLICA), and death was identified by 7AAD stain. Early apoptotic events are indicated in the lower right quadrant (FLICA+7AAD−), late apoptotic events in the right upper quadrant (FLICA+7AAD+) and necrotic cells in the left upper quadrant (7AAD+FLICA-). ‘Total death’ was estimated by summing events in these 3 quadrants.</p

Tim-3 expression is increased on HBV-specific T cells.

<p>HLA-A2+ healthy controls and CHB patients were stained <i>ex vivo</i> with HLA-A2 multimers presenting the CMV-pp65 epitope NLVPMVATV (HLA-A2/NLVP) or a combined panel of 6 HBV-multimers (HLA-A2/core 18–27, envelope 183–191, envelope 348-, envelope 335-,polymerase 455-, polymerase 502-) and with anti-Tim-3 mAb or its isotype. Representative FACS plots from two patients with CHB showing staining for HBV (a) and CMV (b) multimers and Tim-3 expression on gated multimer–specific CD8 T cells compared to an isotype control mAb. (c) Compiled data showing the frequency of virus-specific (multimer+) and global CD8 T cells expressing Tim-3 directly <i>ex vivo</i> in 24 CHB patients. (d) Compiled data showing the frequency of HBV and CMV-specific CD8 T cells expressing Tim-3 directly <i>ex vivo</i> in healthy controls (n = 6), patients with CHB (n = 24) and patients who had resolved HBV (n = 6). (e) <i>Ex-vivo</i> staining in 17 individuals with CHB in whom paired responses could be analysed with both HBV and CMV multimers.</p

Clinical details of patient who underwent liver biopsy and galectin-9 quantification from serum samples.

*<p>Median and range shown.</p>**<p>NA not applicable.</p