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 tag 1 . Plasmids were transfected into CHO cells and protein surface expression verified using an anti--gD mAb 1 (5B6). Primary human cell assays PBMC from healthy volunteers was obtained after informed consent. Immature monocyte-derived DCs (iMDDCs) were generated as described 2 . 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 × 10 5 ) were cultured in the absence (medium alone) or presence of allogenic iMDDCs and MDDCs or autologous purified CD11c + cells. Proliferation was determined by [ 3 H]-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
Transcript
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
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’
