Modulation of autoimmune pathogenesis by T cell-triggered inflammatory cell death

T cell-mediated autoimmunity encompasses diverse immunopathological outcomes; however, the mechanisms underlying this diversity are largely unknown. Dysfunction of the tripartite linear ubiquitin chain assembly complex (LUBAC) is associated with distinct autonomous immune-related diseases. Cpdm mice lacking Sharpin, an accessory subunit of LUBAC, have innate immune cell-predominant dermatitis triggered by death of LUBAC-compromised keratinocytes. Here we show that specific gene ablation of Sharpin in mouse Treg causes phenotypes mimicking cpdm-like inflammation. Mechanistic analyses find that multiple types of programmed cell death triggered by TNF from tissue-oriented T cells initiate proinflammatory responses to implicate innate immune-mediated pathogenesis in this T cell-mediated inflammation. Moreover, additional disruption of the Hoip locus encoding the catalytic subunit of LUBAC converts cpdm-like dermatitis to T cell-predominant autoimmune lesions; however, innate immune-mediated pathogenesis still remains. These findings show that T cell-mediated killing and sequential autoinflammation are common and crucial for pathogenic diversity during T cell-mediated autoimmune responses.

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Flow cytometry data was collected by using FACS Diva software ver6.0 (Becton Dickinson). On western blot analyses, chemiluminescent images were acquired using Image reader LAS-4000mini ver2.1 (FUJIFILM). Signal intensity on MTT analyses was measured by SoftMax Pro software ver5.4 (Molecular Devices). Cell survival data on iCelligence system were acquired by using RTCA iCelligence Software (ACEA Biosciences). H/E and MT images were collected using cellSens standard Software (Olympus), and Immunofluorescence images were collected using BZ-II viewer application (Keyence) or FV10-ASW ver1.6 (Olympus). Raw microarray data from GEO database (Accession number GSE108793) was collected using Affymetrix GeneChip Command Console Software 4.0 (Affymetrix), and the data was background corrected and normalized using Microarray Suite ver5.0 (MAS5) algorithm in Affymetrix Expression Console Software 1.4 (Affymetrix).

Data analysis
Graphpad Prism software 5.0c was used for statistical analyses of cell number, a ratio of two types of cells, percentage or MFI of cells analyzed by FACS, survival data, disease severity data, gene expresssion data, and MTT assay data. FlowJo software (Tomy Digital Biology, version 9.9.6) was used for all analyses of flow cytometry data. Image Gauge ver4.22 (FUJIFILM) was used for chemiluminescent images from LAS-4000 Image reader. ViiA7 RUO Software ver1.2.3 (Thermo Fisher Scientific) was used for RNA expression analysis. Recording data of cell survival using iCelligence system was analyzed by RTCA Data Analysis Software 1.0 (ACEA Biosciences). Immunofluorescence images were processed using the BZ-II image analysis application (Keyence). Microarray data was analyzed by using GeneSpring GX (Agilent Technologies). a heatmap for each transcript value was plotted using Excel (Microsoft). Figures in the manuscript were arranged and converted into PDF files by using PowerPoint software (Microsoft).
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/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

October 2018
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 list of figures that have associated raw data -A description of any restrictions on data availability Microarray data that support the findings of this study have been deposited in GEO with the accession codes GSE108793, which are accessible using token "gzatqeosbdibpqn" while they remain in private status. The authors declare that the other data supporting the findings of this study are available within the paper and its supplementary information files.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
At least 3 animals of each genotype were analyzed.
Data exclusions A few animals were excluded from survival curves due to fight wounds, malocclusion, dystocia, or genotype-independent hypotrophy.

Replication
All findings were confirmed by at least two independent experiments including at least 3 animals of each genotypes or at least three independent in vitro assays.
Randomization For in vivo experiments, animals were allocated to different experimental groups based on gender and age to equal distribution.

Blinding
In general, all animal experiments were under a blinded condition because mice were identified only by mice ID but not genotype during data collection.
Reporting for specific materials, systems and methods 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.

Wild animals
This study did not involve the wild animals.

Field-collected samples
This study did not involve samples collected from the field.

Ethics oversight
All animal studies were approved by Animal Research Committee, Graduate School of Medicine, Kyoto University.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

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.

Methodology Sample preparation
Single cell suspenstions of thymocytes, splenocytes, and lymph node cells were prepared from dissected tissues by grinding with 5ml syringe piston (Terumo) on a 70 μm cell strainer (Falcon). Cells infiltrating the liver were isolated by mechanical dissociation using a 5ml syringe piston (Terumo) on a 100 μm cell strainer (Falcon), followed by purification on a 33% Percoll solution (Sigma) (800g, 30 min at room temp). Skin-resident cells were isolated separately from the dermis or epidermis. After shaving, the skin was immersed in 0.5 g/ml Dispase II (Wako) diluted in PBS and left at 4°C overnight. The epidermal layer was then peeled from the dermis. Epidermal tissues were treated at 37°C for 10 min with 0.05% DNase I (Roche) containing trypsin solution. Dermal tissues were incubated with 0.13 units/ml Liberase TM (Roche) and 100 μg/ml DNase I (Roche) diluted in plain RPMI and then rotated at 37°C for 40 min. Before staining for the purifications of naive T cells or Tregs by FACSAria, isolated single cell suspensions were mixed with antibody-conjugated MACS beads to remove CD8+ and CD19+ cells. Red blood cell lysis was