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Supplementary Materials and Methods
Cell lines
TIGIT, PVR, PVRL2, PVRL3, DNAM, and CD96 full length cDNAs were cloned into
the pRK5 vector containing an N-terminal gD tag1. Plasmids were transfected into CHO
cells and protein surface expression verified using an anti--gD mAb1 (5B6).
Primary human cell assays
PBMC from healthy volunteers was obtained after informed consent. Immature
monocyte-derived DCs (iMDDCs) were generated as described2. Primary human NK
cells, naïve CD4+CD45RA+ or memory CD4+CD45RO+ cells were sorted by flow
cytometry to a purity > 98%. T cells were purified by negative selection (CD4 T cell
isolation kit, Miltenyi Biotech) to a purity of >95%. Cells were resuspended in complete
RPMI 1640 medium with 10% FBS, penicillin and streptomycin, and 2 mM glutamine. T
cells (2 × 105) were cultured in the absence (medium alone) or presence of allogenic
iMDDCs and MDDCs or autologous purified CD11c+ cells. Proliferation was determined
by [3H]-thymidine incorporation, carried out in triplicate. Cytokine concentrations from
cell culture supernatants were measured by LUMINEX for human IL-6, IL-18 and with
specific ELISA for IL-23 (R&D Systems). FACS antibodies are listed in Supplementary
Table 2.
RNA isolation and RT-PCR.
Sorted or transfected cell RNA was isolated with RNeasy mini kit (Qiagen). Real-time
RT-PCR was conducted on an ABI 7500 Real-Time PCR system (Applied Biosystems)
with primers and probes as descried in Supplementary Table 3. All assays were done in
duplicates and data was normalized to RPL19 and expressed as fold induction of over
unstimulated cells.
Nature Immunology: doi:10.1038/ni.1674
Supplementary Figure Legends
Supplementary Figure 1 (a) The previously published expression profile of TIGIT
mRNA in non-immune tissues and immune cell subsets is shown for the reader’s
convenience3. TIGIT is referred to as FLJ39873 in this reference and the expression was
determined based on the Affymetrix GeneCHIP™ HGU133P 240070_at probe. The
expression in non-immune tissues is represented as the highest mean expression in any
non-immune tissue. The isolation of immune cell subsets and the methods for selecting
immune-specific genes is described in the previous publication3. (b) Alignment of
human, rhesus, dog and mouse TIGIT protein sequences. Shading indicates amino acid
identity in two or more species. The signal sequence,, two N-glycosylation sites, and
putative ITIM are boxed. The Ig V-set domain is underlined with a solid line and the
transmembrane domain is underlined with a dashed line. Genbank accession numbers are
XP_001107698 (rhesus), XP_545108 (dog), EU675310 (human) and EU675311 (mouse).
Identities with the human sequence are 88% (rhesus), 67% (dog) and 58% (mouse). No
orthologues have been identified in non-mammalian species.
Supplementary Figure 2 Co-expression of TIGIT with FoxP3 and GITR. (a) 293 cells
(gray fill) or 293 cells stably transfected with TIGIT were incubated with anti-TIGIT
(10A7, solid line) or isotype control (dashed line). Data shown is one representative of
five independent experiments. (b) 293 cells stably transfected with TIGIT were
incubated with anti-TIGIT (10A7) with (dashed line) or without (solid line) PVR-Fc.
Isotype control, gray fill. Data shown is one representative of five independent
experiments. (c) Total human PBMC were stained with anti-CD4 and anti-GITR (left).
GITR+CD4+ T cells were gated and analyzed for the expression of TIGIT and FoxP3 by
flow cytometry (right). Data shown is one representative of two independent
experiments.
Supplementary Figure 3 Binding of biotinylated Fc-fusion proteins (indicated below
histograms) to CHO-K1 cells stably transfected with indicated receptors (indicated at left
of histograms). Histograms are gated on receptor-expressing cells. Binding of ligand (25
Nature Immunology: doi:10.1038/ni.1674
µg/ml) was detected with PE-streptavidin. Open histogram: TIGIT-expressing CHO-K1
cells. Shaded histogram: parental CHO-K1 cells. Data shown is representative of greater
than three experiments.
Supplementary Figure 4 (a) Binding affinity of the TIGIT-PVR interaction was
determined in a radioligand cell-binding assay using 125I-TIGIT-Fc and PVR-expressing
CHO-K1 cells. 125I-TIGIT-Fc (0.2 nM) was competed with unlabeled TIGIT-Fc (500 -
0.122 nM). Kd was determined by analysis with NewLigand 1.05 software and averaged
over 4 assays. The results of one representative assay are shown with Kd and standard
error indicated. (b) CHO transfectants expressing gD-tagged CD226 or TIGIT were
stained with 10 µg/ml biotin-PVR-Fc alone (solid line) or with a 10-fold molar excess of
anti-PVR (D171; dotted line). Isotype control, shaded histogram. Histograms are gated
on the gD-positive population, and PVR-biotin binding was detected with PE-
streptavidin. (c) Binding of 100 nM PVR-Fc to Octet biosensors loaded with CD226-Fc
or TIGIT-Fc without (red) or with (green) pre-incubation with equimolar D171. Yellow
trace indicates buffer treatment.
Supplementary Figure 5 Expression of CD226. (a) Surface expression of CD226 on
resting and anti-CD3 plus anti-CD28 activated (day 1 and 2) sorted naïve CD4+CD45RA+
(top) or memory CD4+CD45RO+ (bottom) T cells. Data shown is one representative of
four donors from two independent experiments. (b) Fold induction in CD226 mRNA in
sorted T cell populations activated with anti-CD3 plus anti-CD28 for 1 or 2 days, and
sorted CD56+ NK cells activated with IL-2 plus IL-15 for one day, compared to
unstimulated cells. Data shown is one representative of four independent experiments. (c)
CD226 mRNA was measured by qRT-PCR in total CD4+, total CD8+, CD4+CD45RO+,
CD4+CD25hi (Treg), NK and CD11c+ (DC) cells sorted from PBMCs. Expression is
shown relative to naïve CD4+CD45RA+ cells. Data shown is one representative of three
donors from two independent experiments. (d) Total human PBMCs were stained with
anti-CD4, anti-CD25, and anti-CD226. Plot is gated on CD4+ cells. FACS plot shown is
one representative of 2 donors from two independent experiments, and RT-PCR is an
average of 3 donors from three independent experiments. (e) CD4+CD25– (Tnaive) and
Nature Immunology: doi:10.1038/ni.1674
CD4+CD25hi (Treg) cells were isolated from PBMC and left unstimulated (Resting) or
stimulated with anti-CD3 plus anti-CD28 for 2 days (Activated). TIGIT and CD226
mRNA was measured by qRT-PCR and are represented as fold change over expression in
resting CD4+CD25– cells. Data are an average of two donors representative from two
independent experiments.
Supplementary Figure 6 (a) PVR expression on monocytes (CD14+), iMDDC and
MDDC, as determined by anti-PVR (solid line) and isotype control antibody (grey
shaded). Data shown is one representative of three independent experiments. (b) PVR
expression on resting CD11c+ human DC and activated CD11c+ cells (LPS) determined
by anti-PVR (solid line) and isotype control antibody (grey shaded). Data shown is one
representative of two independent experiments. (c) PVRL2 expression on MDDC
determined by anti- PVRL2 (solid line) and isotype control antibody (grey shaded). Data
shown is one representative of two independent experiments. (d) Human CD4+ T cells
were co-cultured with allogeneic iMDDC or TNF-matured MDDC in the presence of 100
µg/ml TIGIT-Fc or isotype control where indicated. When indicated, 10 µg/ml 10A7 or
anti-PVR (TX21) were also included in the culture. T cell proliferation was determined
by [3H]-thymidine incorporation on day 4. * P < 0.05. Data shown is one of two
independent experiments.
Supplementary Figure 7 TIGIT affects DC production of other proinflammatory
cytokines. MDDC were matured with TNF or LPS for 24 h, in the presence of TIGIT-Fc
or human IgG1 isotype control as indicated (n=3), and IL-6, IL-18 and IL-23 amounts
determined. Data shown is the average of three donors from two independent
experiments.
Supplementary References
1. Osheroff, P.L., et al. Receptor binding and biological activity of mammalian expressed sensory and motor neuron-derived factor (SMDF). Growth factors (Chur, Switzerland) 16, 241-253 (1999).
2. Caparros, E., et al. DC-SIGN ligation on dendritic cells results in ERK and PI3K activation and modulates cytokine production. Blood 107, 3950-3958 (2006).
Nature Immunology: doi:10.1038/ni.1674
3. Abbas, A.R., et al. Immune response in silico (IRIS): immune-specific genes identified from a compendium of microarray expression data. Genes Immun 6, 319-331 (2005).
Supplementary Tables
Antibodies Item # Supplier
phosphotyrosin 4G10 Upstate
p-p38 #P1491 Sigma
p-ERK #MAB1018 R&D
ERK #AF1576 R&D
PVR #AF2530 R&D
β-actin #RB-9421-P0 NeoMarkers
Supplementary Table 1 Antbodies for immunoblot
Nature Immunology: doi:10.1038/ni.1674
Gene Primer/probe sequences
Human TIGIT Forward Primer: 5’-TGC CAG GTT CCA GAT TCC A-3’
Reverse primer: 5’-ACG ATG ACT GCT GTG CAG ATG-3’
Probe: 5’-AGC CAT GGC CGC GAC GCT-3’ (FAM, TRAMA)
Murine IL-10 Forward primer: 5’-TGA GTT CAG AGC TCC TAA GAG AGT-3’ Reverse primer:, 5’-AAA GGA TCT CCC TGG TTT CTC-3’ Probe: 5’-TCC CAA GAC CCA TGA GTT TCT TCA CA-3’ (FAM, TRAMA)
Murine IL-12p35 Forward primer: 5’-TCT GAA TCA TAA TGG CGA GAC T-3’
Reverse primer: 5’-TCA CTC TGT AAG GGT CTG CTT CT-3’
Probe: 5’-TGC GCC AGA AAC CTC CTG TGG-3’ (FAM, TRAMA)
Murine IL-
12/23p40
Forward primer: 5’-ACA TCT ACC GAA GTC CAA TGC A-3’
Reverse primer: 5’-GGA ATT GTA ATA GCG ATC CTG AGC-3’
Probe: 5’-TGC ACG CAG ACA TTC CCG CCT-3’ (FAM, TAMRA)
Human RPL19 Forward primer: 5’-GCG GAT TCT CAT GGA ACA-3’
Reverse primer: 5’-GGT CAG CCA GGA GCT TCT TG-3’
Probe: 5’-CAC AAG CTG AAG GCA GAC AAG GCC C-3’ (FAM, TAMRA)
Murine RPL19 Forward primer: 5’-GTA CCT GAA GGT CAA AGG GAA T-3’
Reverse primer: 5’-CTG CCT TCA GCT TGT GGA T-3’
Probe: 5’-AAA ACA AGC GCA TCC TCA TGG AGC-3’ (FAM, TAMRA)
Supplementary Table 2 Sequences of primers and probes for qRT-PCR Human CTLA4
(Assay ID#: Hs03044418_m1) and CD226 (Assay ID#: Hs00170832_m1) primers and
probes were from ABI (applied Biosystems).
Nature Immunology: doi:10.1038/ni.1674
Supplementary Table 3 Antibody clones for flow cytometry
Antibodies Clone name Supplier
CD4 SK3 BD Biosciences
CD8 SK1 BD Biosciences
CD11c B-ly6 BD Biosciences
CD25 M-A251 BD Biosciences
CD45RA L48 BD Biosciences
CD45RO UCHL1 BD Biosciences
CD56 B159 BD Biosciences
CD80 L307.4 BD Biosciences
CD83 HB15e BD Biosciences
CD86 IT2.2 BD Biosciences
CD96 NK92.39 Cell Science
HLA-DR TU36 BD Biosciences
CD112 TX31 MBL International
CD155 TX21 MBL International
CD155 D171 Lab Vision Corp.
CD226 TX25 MBL International
CD226 DX11 AbD Sterotec
GITR DT5D3 Miltenyi Biotec
Anti-Human
FOXP3 hFoxy eBiosciences
Anti-Murine CD11c HL3 BD Biosciences
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Figure 1
b
a
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Figure 2
CD4
GIT
R
Isot
ype
FoxP3
TIG
IT
a
c
Cel
l cou
nt
44
12
68
0.2
0.11
0
20
40
60
80
100 b
Cel
l cou
nt
TIGIT TIGIT
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Figure 3
CHO-TIGIT
CHO-PVRL2
CHO-PVR
CHO-CD96
CHO-CD226
CHO-PVRL3
hIgG1-Fc TIGIT-Fc PVR-Fc PVRL2-Fc PVRL3-Fc CD96-Fc CD226-Fc
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Fig 4a
010-1 100 101 102 103
Total (nM)
Bou
nd/T
otal
Bound (pM)
Bou
nd/F
ree
0 80 160 240 320 400
0.12
0.08
0.04
0
0.12
0.08
0.04
Kd = 3.05 nM (5.7%)
CHO-CD226 CHO-TIGIT
PVR-Fc
TIGIT-FcCD226-Fc0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Bin
ding
(nm
)
0 200 400 600 800
Time (seconds)
0 100 200 300 400 500 600
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
Cel
l cou
nt
b
c
Time (seconds)
Bin
ding
(nm
)
PVR-Fc
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Fig 5
CD8+CD4+CD45RA+ CD4+CD45RO+ NK
CD
226
mR
NA
(fold
indu
ctio
n)
bm
RN
A (f
old
indu
ctio
n)
e
CD45RA
CD45RO
CD
226
CD
226
Day 0 Day 1 Day 2
1.3
87.4
37.1
30.4 77.9
62.8
a
CD
226
mR
NA
(fold
indu
ctio
n)
CD
226
mR
NA
(fold
indu
ctio
n)
CD
226
mR
NA
(fold
indu
ctio
n)
0 1 2 0 1 2 0 1 2 0 1Time (day) Time (day) Time (day) Time (day)
CD226
CD
25
CD4+dc
CD
226
mR
NA
(fold
indu
ctio
n)
CD4+CD8+ NK DC
CD45RA
+
CD45RO
+T reg T naiv
e
T reg
TIGIT CD226T naiv
e
T reg
RestingActivated
0
2
4
6
0 0 0
2
4
6
1
2
3
4
5
10
15
0
2
4
6
0
4
8
12
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Figure 6
a
PVR
CD14+ iMDDC MDDCC
ell c
ount
iMDDC
Cel
l cou
nt
b
PVR
CD11c+ +LPS
Cel
l cou
nt
PVRL2
c
d*
Prol
ifera
tion
(c.p
.m.) Isotype + Isotype
TIGIT-Fc + IsotypeTIGIT-Fc + ∝-TIGITTIGIT-Fc + ∝-PVR
Nature Immunology: doi:10.1038/ni.1674
Yu - Supplementary Fig 7
TNF LPSIso
type
TIGIT-F
c
IL-6
(ng/
ml)
IL-1
8 (p
g/m
l)
IL-6
(ng/
ml)
IL-1
8 (p
g/m
l)IL
-23
(pg/
ml)
Isotyp
eTIG
IT-Fc
Isotyp
eTIG
IT-Fc
Isotyp
eTIG
IT-Fc
Isotyp
eTIG
IT-Fc
0
100
200
300
0
5
10
0
5
10
0
1
2
0
1
2
Nature Immunology: doi:10.1038/ni.1674