Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (p < 0.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56brightCD16−CD57−), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6− fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bethiEomeslo(CXCR6−) and T-betloEomeshi(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bethiEomeslo, suggesting its lineage was closer to CXCR6− peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-betloEomeshi NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity.

A regulatory subset of B cells has been found to modulate immune responses in autoimmunity, infection and cancer but has not been investigated in the setting of human persistent viral infection. IL-10 is elevated in patients with chronic hepatitis B virus infection (CHB)3 but its cellular sources and impact on antiviral T cells have not been addressed. We investigated the role of IL-10 and regulatory B cells in the pathogenesis of CHB. Serum IL-10 levels were studied longitudinally in patients with CHB undergoing spontaneous disease flares. There was a close temporal correlation between IL-10 levels and fluctuations in viral load or liver inflammation. Blockade of IL-10 in vitro rescued polyfunctional virus-specific CD8 T cell responses. To investigate the potential contribution of regulatory B cells, their frequency was measured directly ex vivo and after exposure to stimuli relevant to HBV (CpG or HBV antigens). IL-10-producing B cells were enriched in patients, and their frequency correlated temporally with hepatic flares, both after stimulation and directly ex vivo. Phenotypically, these cells were predominantly immature (CD19+CD24hiCD38hi) ex vivo; sorted CD19+CD24hiCD38hi cells suppressed HBV-specific CD8 T cell responses in an IL-10-dependent manner. In summary, these data reveal a novel IL-10-producing subset of B cells able to regulate T cell immunity in CHB.

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56bright NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/− TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/− TGF-β blockade.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

The generation of T cells from pluripotent stem cells (PSCs) is attractive for investigating T cell development and validating genome editing strategies in vitro. X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the IL2RG gene and characterised by the absence of T and NK cells in patients. IL2RG encodes the common gamma chain, which is part of several interleukin receptors, including IL-2 and IL-7 receptors. To model X-SCID in vitro, we generated a mouse embryonic stem cell (ESC) line in which a disease-causing human IL2RG gene variant replaces the endogenous Il2rg locus. We developed a stage-specific T cell differentiation protocol to validate genetic correction of the common G691A mutation with transcription activator-like effector nucleases. While all ESC clones could be differentiated to hematopoietic precursor cells, stage-specific analysis of T cell maturation confirmed early arrest of T cell differentiation at the T cell progenitor stage in X-SCID cells. In contrast, genetically corrected ESCs differentiated to CD4 + or CD8 + single-positive T cells, confirming correction of the cellular X-SCID phenotype. This study emphasises the value of PSCs for disease modelling and underlines the significance of in vitro models as tools to validate genome editing strategies before clinical application.

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