Human placental trophoblast cells contribute to maternal–fetal tolerance through expressing IL-35 and mediating iTR35 conversion

During pregnancy, trophoblast cells sustain the maternal–fetal tolerance via expressing and secreting various chemokines and cytokines. Our previous study revealed the expression of interleukin-35 (IL-35) in human first-trimester trophoblasts. Here we show that IL-35 is expressed in both human first-trimester primary trophoblast cells and a trophoblast cell line. Trophoblast cells inhibit the proliferation of human naive conventional T cells (Tconv cells) and convert suppressed Tconv cells into iTR35 in an IL-35-dependent manner. Mechanistically, trophoblast cell derived IL-35 mediates its function through phosphorylation of STAT1 and STAT3. In vivo studies confirm that mice with immunologically spontaneous abortion have lower levels of IL-35 and iTR35 cells at the maternal–fetal interface, and neutralizing anti-IL-35 mAb enhances abortion rates. Meanwhile, exogenous IL-35 induces iTR35 and prevents immunological abortion. Our findings thus suggest that trophoblast cells have a critical function in preserving maternal–fetal tolerance via secreting IL-35 during pregnancy.

Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
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
The softwares to collect the data:Bio-rad CFX Manager; Bio-Rad Microplate Reader; Olympus fluorescence microscope eBioscience Human High Sensitivity Panel BD Biosciences FACS Calibur flow cytometer.

Data analysis
The softwares to analyse the data: Graphpad Prism 5; Image J; FCS express V3; Flow Jo V10.
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.

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 The data that support the fndings of this study are available from the corresponding author upon reasonable request 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. 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
We used standard sample sizes reported in the literature previously in mouse studies. The sample size of animal experiments according to similar studies in the field was chose and at least 3 mice per group was used.
Data exclusions No data were excluded.

Replication
All key experiments and analysis were performed for at least three times. All necessary required controls were included in our experiments.
Randomization Mice were randomly assigned to groups

Blinding
The investigators were blinded to group allocation during data collection and analysis.

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

Study description
Briefly describe the study type including whether data are quantitative, qualitative, or mixed-methods (e.g. qualitative cross-sectional, quantitative experimental, mixed-methods case study).

Data collection
Provide details about the data collection procedure, including the instruments or devices used to record the data (e.g. pen and paper, computer, eye tracker, video or audio equipment) whether anyone was present besides the participant(s) and the researcher, and whether the researcher was blind to experimental condition and/or the study hypothesis during data collection.

Timing
Indicate the start and stop dates of data collection. If there is a gap between collection periods, state the dates for each sample cohort.

Data exclusions
If no data were excluded from the analyses, state so OR if data were excluded, provide the exact number of exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.

Non-participation
State how many participants dropped out/declined participation and the reason(s) given OR provide response rate OR state that no participants dropped out/declined participation.

Randomization
If participants were not allocated into experimental groups, state so OR describe how participants were allocated to groups, and if allocation was not random, describe how covariates were controlled.

Ecological, evolutionary & environmental sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sampling strategy
Note the sampling procedure. Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient.

Data collection
Describe the data collection procedure, including who recorded the data and how.
Timing and spatial scale Indicate the start and stop dates of data collection, noting the frequency and periodicity of sampling and providing a rationale for

Blinding
Describe the extent of blinding used during data acquisition and analysis. If blinding was not possible, describe why OR explain why blinding was not relevant to your study.
Did the study involve field work?

Yes No
Field work, collection and transport

Field conditions
Describe the study conditions for field work, providing relevant parameters (e.g. temperature, rainfall).

Location
State the location of the sampling or experiment, providing relevant parameters (e.g. latitude and longitude, elevation, water depth).

Access and import/export
Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and in compliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority, the date of issue, and any identifying information).

Disturbance
Describe any disturbance caused by the study and how it was minimized.

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.

Authentication
All of the cell lines were authenticated by short tandem repeat (STR)-profiling.

Mycoplasma contamination
All cell lines tested negative for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines are used in the study.

Palaeontology Specimen provenance
Provide provenance information for specimens and describe permits that were obtained for the work (including the name of the issuing authority, the date of issue, and any identifying information).

Specimen deposition
Indicate where the specimens have been deposited to permit free access by other researchers.

Dating methods
If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.
Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

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

Laboratory animals
Eight-week-old CBA/J females as well as BALB/c and DBA/2J males were purchased from Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS). Mice were housed on a 12-hour light/dark cycle and given ad libitum access to food and water. All animal protocols were approved by the Animal Investigation Committee of The Second Hospital of Shandong University.

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve field-collected samples.

Ethics oversight Outcomes
Describe how you pre-defined primary and secondary outcome measures and how you assessed these measures.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.
For "Initial submission" or "Revised version" documents, provide reviewer access links. For your "Final submission" document, provide a link to the deposited data.

Files in database submission
Provide a list of all files available in the database submission.
Genome browser session (e.g. UCSC) Provide a link to an anonymized genome browser session for "Initial submission" and "Revised version" documents only, to enable peer review. Write "no longer applicable" for "Final submission" documents.

Methodology Replicates
Describe the experimental replicates, specifying number, type and replicate agreement.

Sequencing depth
Describe the sequencing depth for each experiment, providing the total number of reads, uniquely mapped reads, length of reads and whether they were paired-or single-end.

Antibodies
Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot number.

Peak calling parameters
Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files used.

Data quality
Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.

Software
Describe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community repository, provide accession details.

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 Cells were harvested and stained for cell-surface markers with monoclonal antibodies against LAG-3, CD73 and CTLA-4 or their specific isotype controls. For the intracellular staining of IL-35, cells were first pre-incubated with Cell Activation Cocktail (R&D systems), and then fixed and permeabilized with intracellular fixation & permeabilization buffer set (eBioscience) according to the manufacturer's instructions. Then the cells were incubated with mouse or human EBI3 mAb and analyzed using a BD Biosciences FACS Calibur flow cytometer. Data analysis was performed using FCS express V3 or Flow Jo V10. Cell population abundance Cell sorting was not used in our studies.

Gating strategy
The gating strategy is shown in Supplementary material, and the boundaries for "positive" and "negative" cells are indicated based on control antibodies.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

Magnetic resonance imaging
Experimental design