Single-cell analysis of human MAIT cell transcriptional, functional and clonal diversity

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize microbial metabolites through a semi-invariant T cell receptor (TCR). Major questions remain regarding the extent of human MAIT cell functional and clonal diversity. To address these, we analyzed the single-cell transcriptome and TCR repertoire of blood and liver MAIT cells and developed functional RNA-sequencing, a method to integrate function and TCR clonotype at single-cell resolution. MAIT cell clonal diversity was comparable to conventional memory T cells, with private TCR repertoires shared across matched tissues. Baseline functional diversity was low and largely related to tissue site. MAIT cells showed stimulus-specific transcriptional responses in vitro, with cells positioned along gradients of activation. Clonal identity influenced resting and activated transcriptional profiles but intriguingly was not associated with the capacity to produce IL-17. Overall, MAIT cells show phenotypic and functional diversity according to tissue localization, stimulation environment and clonotype.

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Software and code
Policy information about availability of computer code Data collection Flow cytometry data were collected using BD FACSDiva Software (v8.0.1). Sequencing data were collected using HiSeq Control Software  For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

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Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Sequencing data generated in this study have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO SuperSeries accession number GSE194189. The Bioconductor org.Hs.eg.db annotation package and ENSEMBL_MART_ENSEMBL BioMart database are publicly available.
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Sample size
Statistical methods were not used to predetermine sample size. scRNA-seq and flow cytometry experiments included ≥ 3 donors per group (tissue or stimulation condition). Initial blood-liver (Exp 1) and stimulation (Exp 3) scRNA-seq experiments were followed by validation experiments (Exp 2 and 4) -the findings of these were highly concordant.
Data exclusions No samples were excluded from scRNA-seq, ATAC-seq or flow cytometry datasets.
scRNA-seq cell exclusions: cells with low unique molecular identifier counts, low gene counts and/or a high percentage of mitochondrial reads were assumed to be dead/dying cells and were removed. For Exp 1 and 2, cells labelled as empty droplets or damaged cells by DropletQC were removed (damaged cells in Exp 4 also removed). For Exp 2 and 4, only cells called as consensus singlets by hashtag demultiplexing (cellhashR) were retained. Cells with two TCRα and two TCRβ chains, or more than two TCRα and/or TCRβ chains, were assumed to be doublets and discarded.

Replication
Initial blood-liver (Exp 1) and stimulation (Exp 3) scRNA-seq experiments were followed by validation experiments (Exp 2 and 4) -the findings of these were highly concordant. Genes of interest from Exp 1 and 3 were validated at the protein level by CITE-seq (Exp 2 and 4) and flow cytometry. As described in the methods, pySCENIC transcription factor regulon analysis was performed 100 times and results aggregated to define high-confidence regulons.

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ATAC-seq was performed once with three donors.
Activation markers and cytokines identified by scRNA-seq were validated by flow cytometry in 14 donors (three independent experiments) and 11 donors (two independent experiments), respectively. Stimulation of sorted CD56-and CD56+ MAIT cells was performed once with three donors. All attempts at replication were successful.
Randomization For blood-liver scRNA-seq experiments, randomization to groups was not possible since liver patients were compared with healthy donors.

For stimulation experiments, cells from each donor were present in all conditions.
Blood-liver scRNA-seq experiments (Exp 1 and 2) were each performed in two batches -donors were randomly distributed across batches. Stimulation scRNA-seq experiments (Exp 3 and 4) were each performed as a single batch. Donor was included as a covariate in differential expression analyses.

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Experiments and analyses were not performed blinded as the same investigator performed sample collection, sample processing, data generation and data analysis.
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Validation
All antibodies are commercially available and validation statements can be found on the manufacturers' websites using the catalogue number or in the Antibody Registry database (https://antibodyregistry.org) using the RRID. Antibodies were titrated to achieve optimal separation between negative and positive populations.

Flow Cytometry
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Sample preparation
Liver tissue collection and processing (Exp 1 and 2): Liver tissue (n = 7) and matched blood (n = 6) were obtained from patients undergoing liver resection at the Churchill Hospital, Oxford, UK and the University Hospital Basel, Basel, Switzerland (Supplementary Table 1). Patients had no chronic liver disease, active excess alcohol consumption (> 14 g/day), infection, immunosuppression or family history of liver disease.
Disease-free liver tissue was collected from the resection margin, cut into small pieces with a scalpel, and ground through a 70 μm cell strainer. Surface staining and cell sorting for scRNA-seq and scTCR-seq (Exp 1-4): TotalSeq-C hashtag antibodies (BioLegend) were used in Exp 2 and 4. Hashtag antibody dilutions were prepared according to the manufacturer's instructions. Namely, antibody vials were centrifuged at 10,000g, 30 s, 4 °C, before antibody dilution in