List of Abbreviations

B7-H3 B7 superfamily member-H3, a potent inhibitor of T cell activation (also known as CD276) BMP4 Bone Morphogenetic Factor-4 CD45 Leukocyte common antigen CD45RA RA-isoform of the leucocyte common antigen expressed mainly on Naïve T cells CFSE Carboxyfluorescein succinimidyl ester, a fluorescent dye to track cell proliferation CRTAM Cytotoxic and Regulatory T cell molecule EBI3 Epstein-Barr virus induced gene 3 EGF Epidermal Growth Factor EGFR1 Epidermal Growth Factor Receptor-1 (also ERBB1) EGFR2 Epidermal Growth Factor Receptor-2 (also ERBB2) FDR False Discovery Rate FGF4 Fibroblast Growth Factor-4 FOXP3 Forkhead box P3, a transcription factor expressed by regulatory T cells GM-CSF Granulocyte monocyte-colony stimulating factor GSEA Gene set enrichment analysis HLA Human Leukocyte Antigen IL Interleukin KIR2DS1 Killer Immunoglobulin-like Receptor 2DS1; activating NK receptor ligand HLA-C2 molecules LIF Leukemia Inhibitory Factor LILBR1 Leukocyte immunoglobulin-like receptor subfamily B member 1 (Also ILT-2) HLA-G receptor LPS Lipopolysaccharide MFI Mean Fluorescence Intensity MHC Major Histocompatibility Complex PMA Phorbol Myristate Acetate, a mitogen used to activate NK cells TACSTD2 Tumor-associated calcium signal transducer-2, used as a marker for villous trophoblasts TGF-β Tumor Growth Facor-beta TNFα Tumor Necrosis Factor-alpha VEGF Vascular Endothelial Growth Factor Cells and cell lines used dNK Decidual Natural Killer cells dT Decidual T cells dMϕ Decidual macrophages DSC Decidual Stromal Cells EVT Extravillous Trophoblasts, HLA-G+ JEG3 HLA-G expressing choriocarcinoma cell line, a model for extravillous trophoblast pNK Peripheral blood Natural Killer cells pT Peripheral blood T cells Treg Regulatory T cells VT Villous Trophoblasts 221 721.221 MHC class I negative EBV transfected B cell line, classical targets for NK cells

Supplemental Methods

Co-cultures

To establish untouched (free of antibody staining) EVT and VT cultures for functional assays, for each

sample the percentage CD45-HLA-G+ EVT was determined. Samples with >9% CD45-HLA-G+ cells were

stained for CD45 and sorted for viable CD45- large trophoblast cells (Fig. S8D). CD45-HLA-G+ EVT 50.000

cells (calculated based on percentage and total cell number) were plated in 48 well cell culture plates

(Costar) pre-coated with fibronectin (100μl 20ng/ml 45min, BD), in DMEM/F12 (Gibco) supplemented

with 10% NCS, pen/strep and glutamine, insulin, transferrin, selenium (Gibco), 5ng/ml EGF (Peprotech)

and 400 units human gonadotropic hormone (Sigma) (James et al., Hum. Reprod., 2005). Trophoblasts

were incubated 2h/37oC. Non-adherent VT were harvested, counted and plated (50.000 cells), while

adherent cells were washed 2 times to remove non-adherent VT. Cultures resulted in 50-80% HLA-G+

EVT and >80% HLA-G- VT. EVT or VT were incubated with 200.000 dNK cells from the same pregnancy

sample or 200.000 pNK cells from unrelated blood donors in X-Vivo10 medium (Lonza), supplemented

with 5% human AB serum and 40ng/ml IL-15 (Peprotech). Similar co-cultures were setup with 100.000

CD4+ and CD8+ dT or pT supplemented with 100 units IL-2 (eBioscience) or CD14+ dMϕ or monocytes.

All co-cultures consisted of 200µl DMEM/F12 plus supplements and 200µl x-Vivo plus supplements in a

48 well plate.

Statistics

All data was analyzed using GraphPad prism software. To determine differences between two unpaired

groups a non-parametric Mann Whitney test was performed and for two paired groups a non-

parametric Wilcoxon signed rank test. All data is depicted as median with interquartile range, *<0.05;

**<0.01.

Flow Cytometry

The following conjugated mouse anti-human antibodies were used for FACS analysis: CD3-Percp, CD4-

Pacific Blue, CD14-PE-Cy7, CD45-APC, CD45-Alexa700, CD45RA-Alexa700, CD56-Alexa700, CD56-

Alexa488 (Biolegend), CD25-PE (BD), CD8-Pacific Orange (life technologies), FOXP3-Alexa488

(eBioscience) EGFR1-FITC (Serotec) and HLA-G-PE (MEM-G9, Abcam). Cells were washed and stained for

30 minutes at 4oC and analyzed on a FACS CaliburTM (Immunocytometry Systems; Becton Dickinson) or

LSR-II flow cytometer (BD). Data was analyzed using FACS Diva software. Cell sorting was performed on

a MoFlo Astrios high performance cell sorter (DAKO-Cytomation) or a FACS-ARIA III cell sorter (BD).

Cytokine analysis

Cell culture supernatants were snap frozen at -80 °C and all cytokines were analyzed using a multiplex

cytokine assay (Magpix, Bio-Rad). Galectin-1 was measured using a sandwich ELISA (Ouyang et al.,

Blood, 2013). Reagents for the Galectin-1 ELISA were kindly provided by Jing Quyang Department of

Medical Oncology, Dana-Farber Cancer Boston, MA, USA.

Supplemental Methods (Continued)

Imaging

For confocal imaging, EVT were cultured for 3 days on fibronectin coated cover slips. EVT were stained

with anti-HLA-G (MEM-G9, Abcam) for 30 min, and a goat-anti-mouse-Alexa488 (Life Technologies) for

30 min. Cells were fixed with 1% paraformaldehyde for 10 min and permeabilized using PBS/0.01%

Triton X and further stained with Phalloidin-Alexa647 (Molecular Probes) and Hoechst

(Immunochemistry Technologies). Acquisition was performed on the Zeiss LSM510 microscope at 40x

magnification. Zen software was used for analysis and image preparations. Light microscopy was

performed on unstained cells on a Nikon Eclipse Ti microscope at 60x magnification.

Trophoblast RNA preparation and microarray hybridization

Trophoblast preparations were stained for EGFR1-FITC (Serotec), HLA-G-PE (MEM-G9, Abcam) and

CD45-APC (BD). CD45-EGFR1+HLA-G- VT and CD45-HLA-G+ EVT were sorted on a FACS ARIA (BD). For

both cell types >95% purity was obtained (FACS sort gating strategy supplemental Figure 7C). Total RNA

was directly isolated with a Stratagene® Absolutely RNATM Microprep Kit according to manufactures

instructions. RNA was also made from JEG-3 (2 technical replicates) and decidual stromal cell lines at

passage 2 obtained from 4 distinct samples (Blanco et al., Hum. Reprod., 2008). RNA was analysed on

nanodrop and Bioanalyzer to determine RNA yield and RNA integrity. RNA was subjected to one round

of amplification and biotinylation using Ambion’s® MessageAmpTM III RNA Amplification Kit. Biotinylated

aRNA was hybridized to human genome U133 Plus 2.0 chips. All was processed at Harvard University’s

Microarray core facility. Microarray data are available in the ArrayExpress database

(www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-3217.

Microarray data analysis

Raw microarray expression data were normalized with the expression file creator module from the

GenePattern software package http://genepattern.broadinstitute.org (Reich et al., Nat.Genet., 2006).

Unsupervised hierarchical clustering analysis was performed with GenePattern using a Pearson

correlation and all data were visualized with the multiplot module from GenePattern. Gene signatures

of differentially expressed genes in VT and EVT were generated based on a five-fold difference gene

expression difference across all patient-paired samples. Gene Set Enrichment Analysis (GSEA) was

performed within GenePattern software (Subramanian et al., PNAS., 2005). Signal2Noise was used for

ranking genes. Gene sets derived from the Biological Process Ontology (BP:GO) in the Molecular

Signatures Database (mSigDB) v4.0, that includes 825 gene sets, were used to identify differential

regulated functional gene sets between VT and EVT.

Fraction Median Range

HLA-G+ EVT

Purity (before sort)

Cell yield (after sort)

RNA

8.8 %

1.5 x105

1.7 μg

0.5 – 29 %

0.3 – 7.4 x105

1.2 - 5.2 μg

HLA-G- VT

Purity (before sort)

Cell yield (after sort)

RNA

77%

1.1 x 106

5.1 μg

30 - 91%

0.4 - 3.3 x106

2.1 - 8.7 μg

Supplemental Table S1. EVT and VT purity and yield

Supplemental Table 2. Functional gene sets up-regulated in EVT

Rank Gene Set Name FDR q-val

1 Positive regulation of lymphocyte activation 0.001

2 Positive regulation of immune system process 0.001

3 Positive regulation of multicellular organismal process 0.001

4 Positive regulation of T cell activation 0.001

5 Lymphocyte activation 0.002

6 Regulation of immune system process 0.002

7 Adaptive immune response 0.002

8 Response to virus 0.003

9 Regulation of lymphocyte activation 0.003

10 B cell activation 0.008

11 Cell activation 0.007

12 Positive regulation of protein metabolic process 0.007

13 Leukocyte activation 0.007

14 JAK-STAT cascade 0.007

15 T cell activation 0.007

16 Response to other organism 0.007

17 Positive regulation of cellular protein metabolic process 0.007

18 Immune effector process 0.007

19 Adaptive immune response 0.007

20 Regulation of RAS protein signal transduction 0.009

Gene sets highlighted in grey depict gene sets associated with immune activation and regulation

Rank Gene set name FDR q-val

1 INORGANIC_ANION_TRANSPORT 0.004

2 AXONOGENESIS 0.004

3 GENERATION_OF_NEURONS 0.012

4 CELLULAR_MORPHOGENESIS_DURING_DIFFERENTIATION 0.013

5 ANION_TRANSPORT 0.011

6 NEUROGENESIS 0.009

7 NEURITE_DEVELOPMENT 0.008

8 NEGATIVE_REGULATION_OF_MAP_KINASE_ACTIVITY 0.008

9 DETECTION_OF_CHEMICAL_STIMULUS 0.011

10 G_PROTEIN_SIGNALING__ADENYLATE_CYCLASE_ACTIVATING_PATHWAY 0.010

11 NEURON_DIFFERENTIATION 0.011

12 ADENYLATE_CYCLASE_ACTIVATION 0.020

13 TRANSMEMBRANE_RECEPTOR_PROTEIN_TYROSINE_KINASE_SIGNALING_PATHWAY 0.022

14 NEGATIVE_REGULATION_OF_TRANSFERASE_ACTIVITY 0.024

15 EPIDERMAL_GROWTH_FACTOR_RECEPTOR_SIGNALING_PATHWAY 0.026

16 INNATE_IMMUNE_RESPONSE 0.025

17 NEURON_DEVELOPMENT 0.024

18 EXCRETION 0.024

19 INSULIN_RECEPTOR_SIGNALING_PATHWAY 0.023

20 AXON_GUIDANCE 0.022

Supplemental Table 3. Functional gene sets up-regulated in VT

Supplemental Figure S1.

A

B

C

%EV

T %

VT

%C

D4

5+

Supplemental Figure S1. The fraction of EVT does not vary with gestational age A) Percentage of CD45-EGFR1dimHLA-G+ EVT, B) CD45-HLA-G-EGFR1+ VT (gated within live gate and CD45- fraction) and C) Percentage of CD45+ cells (gated with the live cell gate) with each sample plotted against gestational age. The fraction of EVT did not appear to vary with gestational age. However the fraction of VT decreased and the fraction of CD45+ cells increased with gestational age.

Supplemental Figure S2.

Cytokeratin-7 Vimentin

EV

T

VT

C

D4

5+

Ig

G

A E

VT

V

T

HLA-C (DT9) HLA-E (3D12) MHC Class I (W6/32)

B

Supplemental Figure S2. Phenotype of EVT and VT A) Representative FACS plots of intracellular Cytokeratin-7 (left panels) and Vimentin (right panels) expression within EVT, VT and CD45+ cells compared to IgG controls. EVT and VT express Cytokeratin-7 but not Vimentin, whereas CD45+ cells express Vimentin but not Cytokeratin-7. B) Representative FACS plots of HLA-C (mAb DT9), HLA-E (mAb 3D12) and total MHC class I (mAb W6/32) expression on EVT (top panels) and VT (bottom panels). EVT express HLA-C, HLA-E and MHC class I whereas VT are MHC negative.

Supplemental Figure S3.

A

B

Supplemental Figure S3. No proliferation of HLA-G+ EVT or EGFR1+ VT A) Representative FACS plots of EGFR1, EGFR2 and LIF expression on fresh VT, VT cultured for 2 days

and th ematching isotype controls. B) Representative FACS plots of Ki67 (top panels) and IgG controls (bottom panels) staining in

trophoblast preparations at day 1 and day2. C) Representative FACS plots of HLA-G expression and CFSE intensity in trophoblast preparations

cultured on fibronectin for 3 days. No proliferation is observed in the adherent and non-adherent fractions.

EGFR1 LIFR EGFR2

− IgG − fresh − day 2

Ki67

HLA

-G

IgG

Day1 Day2

0% 0%

0%

0%

0.9%

0%

0%

0.6%

M1

HLA

-G

CFSE

Non-adherent Adherent

CFSE

0% 0.3%

2.9% 1.0%

counts

− non-adherent − adherent HLA-G- − adherent HLA-G+

C

counts

Supplemental Figure S4.

B

A

t-te

st p-v

alu

e

EVT

256

DSC

72

t-te

st p-v

alu

e

EVT

90

816

VT

807

44

All data (54676)

EVT signature (328)

VT signature Apps et al. (897)

EVT signature Apps et al. (860)

VT signature Telugu et al. (1520)

EVT signature Telugu et al. (1096)

Supplemental Figure S4. Expression profiling of VT and EVT A) Volcano plots were generated based on the mean expression value of an individual probe’s fold change and the p-value associated with reproducibility of these changes between the DSC and EVT samples. The unique gene signature based on a >4-fold difference for EVT (328 probes, red dots) were highlighted in the volcano plots. B) Volcano plots between VT and EVT. The unique gene signatures for VT (897 green dots) and EVT (840, pink dots) generated by Apps et al., 2011b, are depicted on the volcano plot. C) The unique gene signatures generated by Telugu et al., 2013 were selected for genes with a 4-fold difference and are depicted on the volcano plot for VT (1520 blue dots) and EVT (1096, orange dots).

t-te

st p

-valu

e

Fold Change

C EVT

637

878

VT

883

218

VT EVT DSC JEG3 VT < Fold change > EVT

A) Growth factors and cytokines

TNFSF10

TGFβ1

EBI3

TGFB2

IGFB3

FST

GAL

TYMP

CGA

JAG1

BMP7

F5

FAM3B

-40 -20 0 20 40

FGFR3

CCR1

IL10RA

IL2RB

LAIR2

PTH1R

FLT4

CSF1R

IL1RAP

IL1R2

FGFRL1

ERBB2

IL1R1

NRP2

CCR7

PRLR

BMPR1A

LGR5

-80 -20 0 20 40 -40 -60

B) Growth factors and cytokine receptors

VT EVT DSC JEG3 VT < Fold change > EVT

Supplemental Figure S5.

Relative expression values

-3 +3

Supplemental Figure S5. Expression profiling of VT and EVT A) Heat maps depict the expression of A) growth factors and cytokines, B) receptors for growth factors and cytokines, C) cell adhesion molecules, D) and extracellular matrix components in VT, EVT, JEG3 and DSC. The fold change of each probe between VT and EVT is depicted in a graph. Data show many differences between VT and EVT as well as the absence of many EVT specific probes in JEG3.

CD9

ITGA5

GPC4

MCAM

AMIGO2

FBLIM1

FAT2

CDH3

CADM1

SSPN

EPCAM

SELL

ITGB8

VT EVT DSC JEG3

-30 -20 -10 0 10 20 30

VT < Fold change > EVT

C) Cell Adhesion Molecules

LAMA4

MUC4

COL27A1

MMP12

IMPG1

ACAN

MMP2

PRG2

MFAP2

ADAM19

MMP3

FN1

SPON2

ADAM12

HAPLN3

ADAM8

ECM1

COL4A1

ACPP

FREM2

PRSS12

MMP1

ADAMTS19

MMP7

COL14A1

FRAS1

FBN2

-40 -20 0 20 40

D) Extracellular matrix

60

Supplemental Figure S5 (continued).

VT EVT DSC JEG3 VT < Fold change > EVT

Relative expression values

-3 +3

Supplemental Figure S5. Expression profiling of VT and EVT A) Heat maps depict the expression of A) growth factors and cytokines, B) receptors for growth factors and cytokines, C) cell adhesion molecules, D) and extracellular matrix components in VT, EVT, JEG3 and DSC. The fold change of each probe between VT and EVT is depicted in a graph. Data show many differences between VT and EVT as well as the absence of many EVT specific probes in JEG3.

ITGA5

ITGA6

ITGA6

ITGAE

ITGAV

ITGB1

ITGB1

ITGB1

ITGB4

ITGB4

ITGB5

ITGB5

ITGB5

ITGB5

ITGB8

Supplemental Figure S6.

αL

αM

αX

αD

α1

α2

α3

α4

α5

α6

α7

α8

α9

α10

α11

β3

β5

β6

β8

αV

αIIb

β7 αE

β2

− EVT − VT − EVT and VT − Not expressed

β4

Adapted from Luo et al. Ann. Rev. Immunol. 2007

β1

B A VT EVT DSC JEG3

C

VT EVT

M1

ITGA5 ITGA5

ITGA6 ITGA6

ITGB1 ITGB1

ITGB4 ITGB4

ITGB5 ITGB5

Relative expression values

-3 +3

Supplemental Figure S6. Integrin expression in VT and EVT A) Heat maps depict the relative expression values of integrin expression by VT, EVT, JEG3 and DSC. EVT uniquely expresses ITGA5 whereas VT uniquely expresses ITGB5 and ITGB8. Both VT and EVT express ITGA6, ITGAE, ITGAV, ITGB1, ITGB4. Furthermore, ITGA2, ITGA3, ITGA4, ITGA11, ITGB3, were expressed in DSC but not in VT, EVT and JEG3, whereas ITGA7, ITGA8, ITGA9, ITGA10 ITGAD, ITGAL, ITGAM, ITGAX, ITGB2, ITGB6, ITGB7 were not expressed by any of the cells (data not shown). B) Representative FACS plots of ITGA5, ITGA6, ITGB1, ITGB4 and ITGB6 (purple histograms) compared to isotype controls (green lines) demonstrate an increase in ITGA5 and ITGB1 expression on EVT whereas VT express higher levels of ITGB5. C) Schematic representation of integrin alpha and beta pairs. Specific integrin (pairs) present in EVT are depicted in red, for VT in blue, and integrin (pairs) present in both VT and EVT are green.

TGFB1 Transforming Growth Factor Beta 1

EBi3 Epstein Barr virus induced gene 3

TGFB2 Transforming Growth Factor Beta 2

FST Follistatin

INHBA Inhibin, beta A

CRTAM Cytotoxic and Regulatory T cell Molecule

IL-8 Interleukin-8

CD276 B7-H3

ELF4 E74-like factor 4

PREX1 Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac Exchange factor 1

SPHK1 Sphingosine Kinase-1

Supplemental Figure S7.

TGFB1

EBi3

TGFB2

TGFB2

TGFB2

FST

FST

FST

INHBA

CRTAM

IL8

IL8

CD276

CD276

CD276

ELF4

ELF4

PREX1

SPHK1

VT EVT DSC JEG3

0 10 20 30 Fold change EVT/VT

B

A

Relative expression values

-3 +3

Supplemental Figure S7. Expression of core genes associated with the immune activating gene sets A) Heat maps depict expression of the core genes associated with the immune activating gene sets that were upregulated in EVT. Heat maps depict expression of the core genes in VT, EVT, JEG3 and DSC. The fold change of each probe between VT and EVT is depicted in a graph. Data demonstrate the absence of many EVT specific probes in JEG3. B) Description of the gene symbols from A.

B) Macrophages

A) Lymphocytes

D) Trophoblasts I

HLA

-G

FSC

SSC

CD45 EGFR1

CD

8

FSC

SSC

CD4 CD45

CD

14

CD45

NK

CD45-

CD45+CD14- CD45+CD14-

NK

EVT

18%

VT

78%

0.5% 78%

VT (3 days on FN) EVT (3 days on FN)

85%

SSC

FSC CD45

CD

14

CD45

HLA

-DR

C

D5

6

CD45+CD14+

E) Trophoblasts II

CD45+ CD45-

Live gate

Live gate

Live gate

Supplemental Figure S8.

Supplemental Figure S8. FACS sort strategy for all major leucocyte subsets and VT and EVT

A) Decidual lymphocytes were stained for CD45-Alexa700, CD14-PE-Cy7, CD56-Alexa488, CD4-Pacific Blue and CD8-

PE. A live gate was set based on forward and side scatter (FSC and SSC) profile and duplicates were avoided. dNK

were sorted based on CD45+CD14-CD56+ staining; CD4+ and CD8+ dT were sorted based on CD45+CD14-CD56-

CD4+CD8- and CD45+CD14-CD56-CD4-CD8+ staining respectively. CD3 was not included to avoid non-specific

activation of T cells. For all leucocyte types >95% purity was obtained. B) Macrophages were stained for CD45-

Alexa700, CD14-PE-Cy7 and CD56-Alexa488. A live gate was set based on FSC and SSC profile and duplicates were

avoided. Macrophages were sorted based on CD45+CD14+ staining. All macrophages are positive for HLA-DR. A >95%

purity was obtained. C) Viable macrophages can be obtained after 3 day of cell culture. For VT and EVT purification,

the following two FACS sort strategies were employed. D) For RNA isolation and microarray analysis, trophoblasts

were stained for EGFR1-FITC, HLA-G-PE and CD45-APC. CD45-EGFR1dimHLA-G+ EVT and CD45-EGFR1+HLA-G- VT were

directly sorted and for both cell types >95% purity was obtained. E) For trophoblast-leucocyte co-culture experiments

VT and EVT that were untouched by antibody staining were obtained in the following way: A small fraction of the

trophoblast preparation was stained for EGFR1-FITC, HLA-G-PE and CD45-APC to determine the percentage CD45-

EGFR1dimHLA-G+ EVT. The remaining trophoblasts were stained for CD45-APC. A live gate was set based on FSC and

SSC profile and duplicates were avoided. Total trophoblasts were sorted based on CD45- staining. 50.000 EVT

(number based on sorted cell number and %CD45-EGFR1dimHLA-G+ EVT) were plated for 2 hours on fibronectin. Non-

adherent VT were removed, counted and 50.000 VT were plated. Adherent EVT were washed thoroughly and

cultures resulted in 50-80% HLA-G+ EVT and >80% HLA-G- VT after 3 days of culture on FN.

SSC

FSC CD45

CD

14

Live gate C) Macrophages day 3

Hoechst HLA-G Phalloidin Composite

VT

EVT1

EV

T2

EVT + CD4+ dT EVT + dNK EVT + CD8+ dT

EVT + Monocytes

EVT

VT EVT + CD4+ pT EVT + pNK EVT + CD8+ pT

A

B EVT + dMϕ

Supplemental Figure S9.

Supplemental Figure S9. Images of EVT and of EVT - leucocyte co-cultures A) Confocal images of VT and 2 different EVT. Cells are stained with the nuclear stain Hoechst (blue), HLA-G Alexa-488 (green) and phalloidin Alexa-647 (red). Single colors as well as a composite image are shown. All scale bars indicate 10 μM. B) Representative light microscopic images of VT and EVT and co-cultures of EVT with NK, CD4 and CD8+ T cells and CD14+ macrophages from peripheral blood (left) and decidua (right). Photos were taken at 60x magnification. Cell types and cell culture conditions are indicated on the image.

6.4% 6.3%

1.3% 2.6%

0.5% 1.3%

0.2% 0.3%

96%

22% 16%

97%

CD

25

CD4

FO

XP

3

CD

25

C

A B

Supplemental Figure S10.

CD4

CD25

CD4+ pT CD4+ pT +EVT CD4+ (-CD25HI) pT

CD25 F

OX

P3

CD

25

CD4

FO

XP

3

CD25

CD4+CD25HI pT CD4+CD25HI pT +EVT

CD4+ (-CD25HI) pT +EVT

Supplemental Figure S10. EVT increase the proportion of CD4+CD25HI FOXP3+ Treg A) Total CD4+ pT, B) CD4+ pT depleted of CD25HI cells and C) purified CD4+CD25HI pT were cultured alone or in the presence of EVT. FACS analysis depicts CD4 and CD25 expression (Top panels) and CD25 and intracellular FOXP3 expression (bottom panels). Both CD4+ pT and purified CD4+CD25HI pT co-cultured with EVT have a marked increase in CD4+CD25HI FOXP3+ cells compared to the cells cultured alone. In contrast CD4+ pT depleted of CD25HI cells that were co-cultured with EVT do not increase the population of CD4+CD25HI FOXP3+ cells, demonstrating that EVT increase specifically increase FOXP3 expression in CD4+CD25HI cells or promote expansion of CD4+CD25HI FOXP3+ cells. Numbers indicate the percentage of gated cells with total cells depicted. D) CD4+ pT (left) or CD4+CD25hi pT (right) were labelled with CFSE and incubated with or without EVT, VT or anti-CD3/CD28 beads. Only the pT cells incubated with anti-CD3/CD28 beads show proliferation at day 3 (indicated by the loss of CFSE). Depicted data is one representative experiment of three independent replicates.

CD4+CD25hi pT

CD4+CD25hi pT

+EVT

CD4+CD25hi pT

+VT

CD4+ pT

CD4+ pT

+EVT

CD4+ pT

+VT

CD4+CD25hi pT

+aCD3/28

CD4+ pT

+aCD3/28

CFSE CFSE

D

Supplemental Figure 11.

C D

A B -

Monocytes

dMϕ dMϕ (allo)

Supplemental Figure 11. Co-culture with VT or EVT does not change cytokine secretion by dMϕ Monocytes (blue) and dMϕ (red) were incubated in the absence of stimulation or with VT, EVT or for 3 days as described in Methods. dMϕ were added to VT and EVT cultures in a sample matched setting (macrophages, VT and EVT were obtained from the same pregnancy) and un-matched allogeneic setting (macrophages and trophoblast were obtained from different pregnancies). Cell culture supernatants were analyzed for A) IL-10, B) IL-1β, C) TNFα and D) IL-6.

CD8+ pT

CD8+ dT

CD4+ pT

CD4+ dT

A B

Supplemental Figure S12.

Supplemental Figure S12. CD4+ and CD8+ pT and dT do not secrete IFNγ during co-culture with VT or EVT A) CD8+ and B) CD4+ T cells from peripheral blood (blue) and decidua (red) were incubated in the absence of stimulation or with anti-CD3/anti-CD28 beads, VT or EVT for 3 days as described in Methods. Cell culture supernatants were analyzed for IFNγ.