Supplementary Tables
Gene
Rat HPRT
Rat IL-22BP
Rat IL-22R1
Rat ALDH1A2
Human ALDH1A2
Mouse IL-22BP
Mouse GAPDH
Gene
Rat IL-22BP
Human IL-22BP
Forward
Reverse
CCTTGGTCAAGCAGTACAGCC
GGCAAAGGGATCTCTCTGTTCT
GTCCAGCAACTTTGAAAACATCTT
GGGGGTTCAAGATGTCTGGA
GAGGAGTTTGTGAGAAGAAGCGT
TCA GCAGCAAAGACAGAAGAAAC
CTACAGCAACAGGGTGGTGG
TTCGCTGATGACACAAACATGA
TAGGAGAGGCAGTGTAGTTCGC
CGGGTCTCCACAGTCAGGTT
GGAGAGTCAGGCTTGCTTCAC
CTGTGGGCTCAATGAAAAACC
GTGTCTCCAGCCCAACTCTCA
TATGGGGGTCTGGGATGG
Forward
Reverse
ATGCCTAAGCACTGCTTTC
GGCTTCCTCATCAGTTTCTTCC
CACACATCTCTCCTTGCTTC
TTCCACACATCTCTCTTCACTTCTC
Supplementary Results
rat IL-22BP (A.U)
Supplementary Figure S1
20
15
10
5
0
MLN
CLN
ALN
Supplementary Figure S1A: IL-22BP expression in rat lymph nodes. IL-22BP gene
expression was analyzed by RT-qPCR. Bars represent mean ± SEM ratio of IL-22BP gene to
HPRT expression as determined by the 2-ΔΔCt method of relative quantification (n=3). MLN:
Mesenteric Lymph Nodes; CLN: Cervical Lymph Nodes; ALN: Axillary Lymph Nodes.
mouse IL-22BP (A.U)
8
6
4
2
0
MLN CLN ALN Spleen
Supplementary Figure S1B: IL-22BP expression in mouse SLOs. IL-22BP gene expression
was analyzed by RT-qPCR. Bars represent mean ± SEM ratio of IL-22BP gene to HPRT
expression as determined by the 2-ΔΔCt method of relative quantification (n=2). MLN: Mesenteric
Lymph Nodes; CLN: Cervical Lymph Nodes; ALN: Axillary Lymph Nodes.
Supplementary Figure S2
Supplementary Figure S2: Cell sorting of rat MLN cDCs. MLN were harvested from adult
SPD rats, dilacerated using 26G needles and digested for 25 min with Collagenase D, in the
presence of DNAse I. Cells were collected and low density cells were prepared using a 14,5%
Nycodenz gradient. Cells were then stained with anti-TCRαβ, anti-CD45R, anti-CD103 and antiCD4 and sorted on a FACS Aria with the gating strategies illustrated above. Purity was routinely
>95%. The figure is representative of 3 independent experiments.
Supplementary Figure S3
A
B
+
Supplementary Figure S3: Spleen CD4 cDC were isolated. IL-22BP expression was analyzed
by RT-PCR. (A) PCR products were sequenced. Alignment of the rat short isoform with its
human counterpart revealed 80% identity (B) Alignment of the putative protein encoded by the
short isoform with the human IL-22BP short isoform shows 73% identities.
human IL-22BP (A.U)
Supplementary Figure S4
500
GM-CSF+IL-4
GM-CSF
GM-CSF+TGF-
GM-CSF+TGF-+IL-13
400
300
200
100
0
d0
d2
d4
d6
d8
LPS
Supplementary Figure S4: Human monocytes from peripheral blood of healthy donors were
differentiated into DCs in complete medium with the indicated ligands for 6 days. LPS was added
on day 6. Cells were collected at the indicated times during differentiation and IL-22BP gene
expression analyzed by RT-qPCR. Each point represents the ratio of IL-22BP gene to HPRT
expression as determined by the 2-ΔΔCt method of relative quantification. Data are representative
of two independent experiments.
Supplementary Figure S5
1.0
0.0
MDDC
0.5
Monocytes
human ALDH1A2 (A.U)
1.5
Supplementary Figure S5A: Human MDDC express RALDH2. Human monocytes from
peripheral blood of healthy donors were differentiated into DCs in complete medium with GMCSF and IL-4 for 6 days. RALDH2 gene expression was analyzed by RT-qPCR and normalized
relative to HPRT expression. Bars represent mean ± SEM ratio of RALDH2 gene to HPRT
expression as determined by the 2-ΔΔCt method of relative quantification (n=3).
human ALDH1A2 (A.U)
30
20
10
0
0h 2h 4h 8h 20h 48h 6d
Supplementary Figure S5B: Kinetic of RALDH2 expression during human MDDC
differentiation. Human monocytes from peripheral blood of healthy donors were differentiated
into DCs in complete medium with GM-CSF and IL-4 for 6. At indicated time points cells were
harvested and RALDH2 gene expression was analyzed by RT-qPCR. Each point represents the
ratio of RALDH2 gene to HPRT expression as determined by the 2-ΔΔCt method of relative
quantification. Data are representative of 2 independent experiments.
human IL-22BP (A.U)
1.5
1.0
0.5
+ RAR inh
Medium
0.0
MDDC
Supplementary Figure S5C: RA signalling is not required for constitutive expression of IL22BP by MDDC. Human monocytes from peripheral blood of healthy donors were differentiated
into DCs in complete medium with GM-CSF and IL-4 for 6 days, in the presence or not of RARα
inhibitor BMS 195314. IL-22BP gene expression was analyzed by RT-qPCR and normalized
relative to HPRT expression. Bars represent mean ± SEM ratio of IL-22BP gene to HPRT
expression as determined by the 2-ΔΔCt method of relative quantification (n=4).
RA (d0)
+ RARα inhibitor
medium
RA
d0
d1
d2
d3
d4
d5
CD1d
CD103
Supplementary Figure S5D: Effects of RARα inhibitor on MDDC differentiation in the
presence of RA. Human monocytes from peripheral blood of healthy donors were differentiated
into DCs in complete medium with GM-CSF and IL-4 for 6 days. The RARα inhibitor BMS
195314 was added in the culture well at the indicated day during differentiation. Cell surface
markers were analyzed by flow cytometry. Grey histograms, isotype control staining; empty
histrograms, antibody staining. Data are representative of 2 independent experiments.
10
RAR inh d5
RAR inh d3
RAR inh d0
RAR inh d1
0
RA
5
medium
human IL-22BP (A.U.)
15
RA d0
Supplementary Figure S5E: Effects of RARα inhibitor on IL-22BP expression by MDDC.
Human monocytes from peripheral blood of healthy donors were differentiated into DCs in
complete medium with GM-CSF and IL-4 for 6 days. When indicated, the RARα inhibitor BMS
195314 was added in the culture well. IL-22BP gene expression was analyzed by RT-qPCR . Bars
represent mean ± SEM ratio of IL-22BP gene to HPRT expression as determined by the 2-ΔΔCt
method of relative quantification (n=2).
Supplementary Figure S6
35
10
ON cultured
0
Fresh
5
MLN cells
rat IL-22BP (A.U)
15
MLN DCs
Supplementary Figure S6: IL-22BP expression is lost when rat MLN-DCs undergo
spontaneous maturation. Total MLN-DCs were isolated and analyzed for IL-22BP gene
expression by RT-qPCR, before and after ON culture in complete culture medium. Bars represent
mean ± SEM ratio of IL-22BP gene to HPRT expression as determined by the 2-ΔΔCt method of
relative quantification (n=2).
0.012
0.010
0.008
0.006
0.004
+ RA d3
Medium
0.000
+ RA d3
0.002
Medium
mouse IL-22BP (A.U)
Supplementary Figure S7
Unstimulated LPS 24h
Supplementary Figure S7: IL-22BP expression is induced by RA and down-regulated by
LPS in mouse BMDC. BMDC were differentiated by GM-CSF from total bone marrow cells
cultured in complete medium for 8 days, in the presence or not of RA added at day 3. When
indicated, cells were harvested and cultured 24h in complete medium, with or without LPS.
BMDC were analyzed for IL-22BP gene expression by RT-qPCR. Bars represent mean ± SEM
ratio of IL-22BP gene to HPRT expression as determined by the 2-ΔΔCt method of relative
quantification (n=2).