Re-programming mouse liver-resident invariant natural killer T cells for suppressing hepatic and diabetogenic autoimmunity

Invariant NKT (iNKT) cells comprise a heterogeneous group of non-circulating, tissue-resident T lymphocytes that recognize glycolipids, including alpha-galactosylceramide (αGalCer), in the context of CD1d, but whether peripheral iNKT cell subsets are terminally differentiated remains unclear. Here we show that mouse and human liver-resident αGalCer/CD1d-binding iNKTs largely correspond to a novel Zbtb16+Tbx21+Gata3+MaflowRorc– subset that exhibits profound transcriptional, phenotypic and functional plasticity. Repetitive in vivo encounters of these liver iNKT (LiNKT) cells with intravenously delivered αGalCer/CD1d-coated nanoparticles (NP) trigger their differentiation into immunoregulatory, IL-10+IL-21-producing Zbtb16highMafhighTbx21+Gata3+Rorc– cells, termed LiNKTR1, expressing a T regulatory type 1 (TR1)-like transcriptional signature. This response is LiNKT-specific, since neither lung nor splenic tissue-resident iNKT cells from αGalCer/CD1d-NP-treated mice produce IL-10 or IL-21. Additionally, these LiNKTR1 cells suppress autoantigen presentation, and recognize CD1d expressed on conventional B cells to induce IL-10+IL-35-producing regulatory B (Breg) cells, leading to the suppression of liver and pancreas autoimmunity. Our results thus suggest that LiNKT cells are plastic for further functional diversification, with such plasticity potentially targetable for suppressing tissue-specific inflammatory phenomena.


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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. induced LiNKTR1 cells and pMHCII-NP-induced TR1 CD4+ T-cells. The raw RNAseq and scRNAseq data files have been uploaded to the GEO database (accession number: GSE168488). Bulk RNA reads were aligned to the GENCODE release 16 of the Mus musculus genome (mm10 assembly). scRNAseq fastq files were processed using either the mouse mm10 or the human GRCh38 human reference transcriptomes.
Based on previous studies in similar biological systems. Sample sizes were as large as possible with tight controls of gender, age, dosing regiments, and experimental conditions within and between experiments, to limit experimental variability. In vitro experiments typically involved smaller sample sizes than in vivo experiments and all statistically significant differences are reported with the corresponding P values.
No data/mice were excluded from analyses. All analyzed samples are reported.
Most data sets were replicated in independent experiments and the number of experiments that were pursued are specifically stated in the Figure legends. In addition, the manuscript used several redundant disease model systems as well as read-outs (e.g. similar disease but in a different genetic background) that further substantiate the robustness of the conclusions.
Randomization into individual treatment groups was pre-determined. Mice were entered into the study when they reached a pre-determined disease score that ensured sustained disease progression and disease chronicity in all the mice. This information is provided in Methods and Figure legends.
Investigators were not blinded to therapeutic outcome as the investigators responsible for treating the mice where generally also those responsible for scoring the outcome of the corresponding experiments. However, they were replicated by different investigators using different read-outs. All cytokine/chemokine determinations were blinded. To minimize potential bias in the scoring of therapeutic effects, pathological scores were evaluated independently by two different investigators (one of whom was not involved in treating the mice or group allocation) and we report the average values, as noted in the manuscript. RNAseq data were generated and allocated to the corresponding groups blindly. CHO and Ad-293 T cells were commercially available and were not authenticated in our laboratories upon receipt. They were used for pMHC production or adenovirus titration. However, we regularly sequence transduced CHO cell lines (the transduced genes), to confirm identity in terms of transgene specificity.
Cell lines used for pMHC production were free of mycoplasma contamination.
No commonly misidentified cell lines were used in the study. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Recruitment
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Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
No wild animals were used in this study.
No field collected samples were used in this study.
The reported studies were approved by the institutional animal care committee of the Cumming School of Medicine at the University of Calgary.
The liver isolates studied herein were obtained from 4 patient samples: IHL-453 (61 year-old female with alcoholic liver disease and cirrhosis with past medical history of obesity and type 1 diabetes); IHL-465 (58 year-old female with nonalcoholic steatohepatitis, cirrhosis and an hepatocellular carcinoma, past medical history of hypertension and type 2 diabetes); IHL-484 (38 year-old male with Wilson's disease); and IHL-486 (63 year-old male with resolved hepatitis C virus infection and hepatocellular carcinoma, with past medical history of dyslipidemia).
Patients were recruited at the University of Alberta under informed consent. There was no specific selection for samples of any sex or disease type. Liver explants from which enough T-cell frozen material was available were selected, regardless of other variables.
The studies described herein were approved by the institutional ethic boards of both the University of Alberta and the University of Calgary Murine iNKT cells (TCR"int+/aGalCer/CD1d tetramer+) were sorted by flow cytometry from the livers, PCLNs and spleens of aGalCer/CD1d-NP-or Cys-NP-treated NOD.c3c4 mice. B-cells were isolated from PCLN, MLN, spleen, liver and lung mononuclear cell suspensions from aGalCer/CD1d-NP-or Cys-NP-treated mice by cell sorting upon staining with PEconjugated anti-CD19 mAb or using an Easysep CD19 Positive Selection kit II (Stem Cell Technologies). Briefly, mice were bled to completion by severing the heart and abdominal aortas. Liver cell suspensions were subjected to 37.5% isotonic Percoll gradient (Percoll, Sigma-Aldrich) centrifugation in the presence of heparin (10 U/ml) and mononuclear cells prepared as described above. Lungs were cut into small pieces and digested in RPMI-1640 medium containing 10% FCS, DNAseI (200 U/ ml) and Collagenase IV (100 $g/ml) at 37oC for 90 mins. All the pieces were homogenized into a single cell suspension, washed and hemolyzed. Single cell suspensions from liver, lungs and spleen were stained with aGalCer/CD1d tetramer (3 to 5 $g/ml, at room temperature for 1h) and anti-murine TCR" and B220 mAbs. For iNKTs, TCR" int+/B220-/tetramer+ cells and for B cells, TCR"-/B220+ cells were sorted using a FACSAria III instrument (BD Biosciences). Dead cells were excluded from analysis by staining with 7ADD Viability dye from BD biosciences. The percent purity of the iNKT and B-cell preparations were: 88.3+1.9 and 89.9+0.76 for liver iNKTs of control vs. treated mice for bulk RNAseq; 94.6+2.2 and 91.7+0.96 for liver iNKTs of