CD1a promotes systemic manifestations of skin inflammation

Inflammatory skin conditions are increasingly recognised as being associated with systemic inflammation. The mechanisms connecting the cutaneous and systemic disease are not well understood. CD1a is a virtually monomorphic major histocompatibility complex (MHC) class I-like molecule, highly expressed by skin and mucosal Langerhans cells, and presents lipid antigens to T-cells. Here we show an important role for CD1a in linking cutaneous and systemic inflammation in two experimental disease models. In human CD1a transgenic mice, the toll-like receptor (TLR)7 agonist imiquimod induces more pronounced splenomegaly, expansion of the peripheral blood and spleen T cell compartments, and enhanced neutrophil and eosinophil responses compared to the wild-type, accompanied by elevated skin and plasma cytokine levels, including IL-23, IL-1α, IL-1β, MCP-1 and IL-17A. Similar systemic escalation is shown in MC903-induced skin inflammation. The exacerbated inflammation could be counter-acted by CD1a-blocking antibodies, developed and screened in our laboratories. The beneficial effect is epitope dependent, and we further characterise the five best-performing antibodies for their capacity to modulate CD1a-expressing cells and ameliorate CD1a-dependent systemic inflammatory responses. In summary, we show that a therapeutically targetable CD1a-dependent pathway may play a role in the systemic spread of cutaneous inflammation.


Statistics
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Software and code
Policy information about availability of computer code Data collection BD Zeiss Zen 2011 SP7 FP3 (black), FACS Diva v8.0 Data analysis FlowJo v10.4.2, GraphPad Prism version 6.00, (FIJI is just) Image J 1.52p, LEGENDplex™ Data Analysis Software Cloud Suite, UCSF Chimera 1.15 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.

Data
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Life sciences study design
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Sample size
Sample size was determined based on our previous studies and those in the literature (Kim et al Nat Immunol. 2016 Oct;17(10): 1159-1166 and is indicated in the legend. Where appropriate following previous experience, in experiments designed to test whether a particular manipulation leads to a reduction in the severity of disease compared to the control group, the number of animals in each group has been calculated to ensure a 90% chance of detecting a change that is statistically significant at the 5% level. Power analysis was performed for research output including ear thickness induced by imiquimod application where for example ear thickness increase reached 0.2mm in the CD1a transgenic group and where we would like to be able to reliably detect a reduction in the wildtype group to 0.09mm, power analysis indicates a minimum group size of n=4. However other read outs including cytokine production required larger sample sizes to capture variation and expression changes for the measurement tested and the sample sizes are chosen because they provide sufficient confidence to assess the experimental results.

Replication
The majority of experiments were repeated at least 3 times with independent donors/mice, as noted in the figure legends, with few being performed twice. All attempts at replication were successful, individual data points shown.
Randomization Comparison of wild-type and CD1a-transgenic mice could not be fully randomized. Additionally due to cage grooming effects, it is not possible to randomise topical treatments within cages. Allocation of samples to the different experimental groups was based on mouse genotyping results. As part of the experimental design, biological samples from different genotypes were tested on any experimental day (where feasible) in order to avoid any technical confounders. In addition, all biological samples from different experimental groups were subject to the same protocols and treatment.

Blinding
Flow cytometry data were collected objectively using automated plate readers to avoid bias. Human participants were recruited sequentially; blinding was not required as there was no intervention.
No blinding was possible in the mouse studies, because experiments required repeated injections of different antibodies. A micrometer was used to empirically measure/quantify inflammation (ear thickness) rather than relying on a scoring system. As allocation of samples to the different experimental groups required assessment of mouse genotyping results -blinding was not possible. However, samples from different experimental groups were processed on each experimental day. All samples were subject to same treatment and protocol.
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.  Newly generated OX16, OX77a, OX110, OX111, OX116 and OKT6 and commercially available SK9, HI149, NA1/34 antibodies were conjugated to Alexa-674 using AF-647 Antibody labelling kit (Life Technologies A20186).

Validation
Antibodies that are commercially available from Biolegend, Bio-Techne, eBioscience (Thermo Fisher Scientific), Abcam and Miltenyi Biotec, were validated for specificity to human or mouse antigens and for the application of flow cytometry. Validation statements are found on the manufacturer's websites: CD3 (741064 BD Pharmingen), Application: Flow cytometry (Qualified). This antibody was developed for use in flow cytometry. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity.

Authentication
The cell lines were not authenticated, other than for expression of the transfected protein (CD1a).

Mycoplasma contamination
Cell lines were routinely tested for mycoplasma and were negative Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
CD1a transgenic mice were generated by the Wellcome Trust Centre for Human Genetics, Oxford on a C57BL/6J background. Transgenic offspring were identified by PCR using transgene specific primers and bred as individual lines with wild-type C57BL/6J mice. All mice were bred in a specific pathogen-free facility. In individual experiments, hemizygous transgenic mice were matched for age (experiments performed at 6-10 weeks old), sex and background strain with wild-type litter mates/counterparts used as matched controls. Mice were maintained in a pathogen-free facility in individually ventilated cages in an ambient temperature-and humiditycontrolled room with a 12 h light/12 h dark cycle under standard housing conditions with continuous access to food and water.
In the generation of anti-CD1a antibodies, female Balb/C Mice 11-13 weeks old were immunized with syngeneic cells transfected with human CD1a and mouse β2M and Female New Zealand White rabbits 12-16 weeks old were immunized with syngeneic cells transfected with human CD1a and rabbit β2M. Mice were housed in conventional cages with temperatures maintained between 19-230C and a humidity of around 55% +/-10%. Rabbits were housed in floor pens with temperatures in the room maintained

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between 16-200C with the same humidity. For both mice and rabbits. 12 hours light 12 hours dark are cycled on the system from 7 to 7.

Wild animals
The study did not involve wild animals Field-collected samples The study did not involve samples collected from the field

Ethics oversight
All experiments undertaken in this study were done so with the approval of the UK Home Office.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Population characteristics
Human cells were isolated from individuals of different, age (25-50), balanced gender distribution of healthy controls.

Recruitment
Blood samples were provided by healthy volunteers, from The Weatherall Institute of Molecular Medicine, or who visited the Churchill Hospital Department of Dermatology, Oxford. Samples were de-identified to limit preselection bias. Participants were recruited sequentially; blinding and randomization were not required as there was no intervention.

Ethics oversight
PBMCs were isolated from healthy adult donors under local ethics approval (National Research Ethics Service Committee South Central, Oxford C, 14/SC/0106).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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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.

Sample preparation
Human blood samples: PBMCs were isolated with Lymphoprep density gradient from healthy adult donors and T cells purified using anti-CD3 magnetic bead sorting following the manufacturer's protocol (MACS, Miltenyi). Mouse Tissue processing: Mice were sacrificed and tissues taken 24 h after final imiquimod challenge. Ears, cervical lymph nodes (cLN) and spleen were collected for immunophenotyping or imaging. Cell suspensions of spleen and cLN, were obtained by passing the tissues through a 70 μm strainer and washed with RPMI containing 10% FCS. Spleen cell suspension and whole blood red blood cells were removed by incubation with RBC lysis solution (eBioscience). Ear skin tissue was washed in HBSS to remove excess imiquimod, split ventrally, diced into <0.5mm pieces and digested with 1 mg/mL collagenase P (Roche) and 0.1 mg/mL DNaseI (Sigma-Aldrich) DMEM for 3-5x30mins with agitation, dispase 5mg/mL was added to the final 30min digest step. A single cell suspension was obtained upon washing with DMEM containing 10% FCS through a 30 μm strainer prior to analysis by flow cytometry.

Instrument
For flow cytometric analysis BD Fortessa was used.

Software
Flow Jo V10 was used for all flow cytometry analysis Cell population abundance Sorted CD1a restricted T cell lines constituted more than 80% CD1a restricted cells. Purity was assessed based on subsequent functional analysis in response to CD1a coated beads or CD1a expressing cells and on TCR sequencing results of paired TCR. CD1a restricted lines were selected for analysis.

Gating strategy
CD1a gating performed on CD1a transgenic mice using wildtype litter mates as a negative control for anti-CD1a antibody staining as used in figure 1A.
Gating strategy for CD1a+ thymocytes provided in figure S1B.