Supplementary Figure 1: Specificity of Anti-TIGIT Antibody 1G9. TIGIT-Specific Antibodies Were Generated in TIGIT-/- Mice (Clone 1G9)

Supplementary Figure 1: Specificity of anti-TIGIT antibody 1G9. TIGIT-specific antibodies were generated in TIGIT-/- mice (clone 1G9). (A) 1G9 was titrated in an

ELISA against recombinant mouse TIGIT or a control protein. (B) Anti-TIGIT antibody

1G9 was used to stain P815 cells transfected with mouse TIGIT (solid line) or the

parental untransfected cell line as control (shaded histogram) and samples were analyzed

by flow cytometry. (C) Primary T cells from B6 or TIGIT-/- mice were activated for 48h

and stained using anti-TIGIT antibody 1G9 (solid line) or an isotype control (shaded

histogram) and analyzed by flow cytometry. Samples are gated on the CD4+ population.

Representative examples of 5 (A) or over 10 (B and C) independent experiments are

shown.

Supplementary Figure 2: TIGIT acts on APCs and on T cells. (A) CD4+ T cells were

isolated and labeled with CFSE. Wild type B6 or TIGIT-/- CD4+ T cells were stimulated with anti-CD3 in the presence of irradiated APCs from wild type B6 or TIGIT-/- mice.

Proliferation was analyzed after 60h using flow cytometry. (B) CD45.1 B6 recipients

received 105 CD62L+CD4+ 2D2 or 2D2 x TIGIT-/- cells i.v. one day before s.c.

immunization with 100μg MOG35-55 peptide in CFA. On day 7 spleens and lymph nodes

(LN) were harvested and frequencies and absolute numbers of transferred CD45.2+ T

cells were determined by flow cytometry (B; n=4). (C-E) TIGIT-specific antibodies were

generated in Armenian hamsters (clone 4D4). (C) 4D4 was titrated in an ELISA against

recombinant mouse TIGIT or a control protein. (D) P815 cells transfected with mouse

TIGIT (solid line) or the parental cell line (shaded histogram) were stained with anti-

TIGIT antibodies and analyzed by flow cytometry. (E) Primary T cells from B6 or TIGIT-/- mice were activated for 48h and stained with anti-TIGIT antibody 4D4 (solid

line) or isotype control (shaded histogram) and analyzed by flow cytometry (gated on

CD4+ cells).

Supplementary Figure 3: TCR activation and survival pathways are differentially

activated upon TIGIT engagement. Expression values of all analyzed genes (A) or genes contained in the TCR activation (B) and T cell survival (C) gene sets are displayed.

Normalized log2 expression values in wild type CD4+ T cells treated with agonistic anti-

TIGIT (B6 4D4) are plotted against the mean of the isotype and TIGIT-/- controls

(controls). Plots were produced in the statistical environment R (http://www.r- project.org/). Molecules driving the association are marked in red and their names are given. Overall, the T cell survival gene set shows higher expression in the B6 4D4

sample (most genes are on the right side of the diagonal), while the T cell activation

molecules are expressed to a higher extent in the control samples (more genes on the left

side of the diagonal line).

Supplementary Figure 4: Signaling pathways downstream of TIGIT. (A and B)

Expression of genes involved in the TCR activation (A) and T cell survival (B) were

differentially expressed in wild type CD4+ T cells stimulated with agonistic anti-TIGIT

antibody compared to the controls (isotype and TIGIT-/- controls). Molecules are

represented as nodes (see legend for different shapes), and the biological relationships

between different nodes are indicated. The node color indicates if a gene is up- (green) or

down- (red) regulated (p-value smaller or equal to 0.1, and fold change greater or equal to

2 1.2, or smaller or equal to 0.8 was regarded as significant). Complexes formed by molecules that were both up- and down-regulated are shaded in red and green. White

nodes represent molecules that are not part of the data set. Figures generated within

Ingenuity. (C and D) CD4+ T cells were sorted from B6 and TIGIT-/- mice and stimulated

with plate-bound anti-CD3 and anti-CD28 plus 4D4 or isotype control Ab. After 24h

RNA was isolated and expression of genes involved in TCR activation (C) or T cell

survival (D) was assessed by quantitative RT-PCR (triplicate wells, one of three

experiments).

3 100 100 100

80 80 80

60 60 60 % of Max % of Max % of Max 40 40 40 20 20 20

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

! 100 40! 80! 30! 60!

20! 40!

10! 20!

0! 0! ! 3! 4! 5! ! 3! 4! 5! 2 0!102! 10! 10! 10! 0!10! 10! 10! 10!

800 600 600 400 400 # Cells

200 200

0 0 100 101 102 103 104 100 101 102 103 104 FL1-H: CFSE 1000 800

800 600 600 # Cells # Cells 400 400

200 200

0 0 100 101 102 103 104 100 101 102 103 104

100 100 100 100 100 " 80 80 80 80 80 60 60

60 60 60 % of Max % of Max 40 40 % of Max % of Max % of Max 40 40 40 20 20 20 20 20 0 0 0102 103 104 105 0102 103 104 105 0 0 0 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 100 101 102 103 104 FL2-H FL2-H FL2-H ! A 14

8 controls 6

2 2 4 6 8 10 12 14 B6 4D4 stimulated B log(Expression) of T-cell activation genes in B6 4D4 vs. controls

Ptprc 12 Actr3 Cd3d Actr3 Cd3g Lat Cd28 10 Cd247 Cdc42 Lcp2 Rasgrp1 Zap70 Pdk1Cd3e Nfatc3

8 Fyn Grb2Ctla4 Cdk2 Mapk1 Ccna2 NrasNfatc1 Sos2Ralbp1

controls Pic3ca Malt1Cd28 Nfatc1 Relb Grb2 Vav1 6 Pic3ca Map3k1 Lcp2 Vav2 Vav3 Nfat5 Mapk8 Fadd Nfatc1Pik3r1 Card11

4 Rasgrp1 Rras Ppp3ca Mapk8 Pik3cb Mapk1 Cdk6 Pik3cg 4 6 8 10 12 B6 4D4

C log(Expression) of T-cell survival genes in B6 4D4 vs. controls

10 Myc Nfkb1

Rac2 Bax Rac1 8 Mcl1 Rela Jun Il2ra

controls Rel Bcl-XL

6 Il7r Relb Casp7 Fos Bad Nfkb2 Stat5a Jak1

4 Il2rb Akt3 Il15ra Jak3 Il7r Il15ra Bcl-XL Map3k8 Mdk Casp9 4 6 8 10 B6 4D4

! ! ! ! ! ! Supplementary Table I

fold change fold change (B6 4D4 vs. (B6 4D4 vs. Name Affimetrix ID controls) p-value Name Affimetrix ID controls) p-value

Luc7l2 1436767_at -2.548 0.023 Amigo2 1434601_at 1.793 0.023 Nfil3 1418932_at -2.081 0.042 Elf2 1428045_a_at 1.786 0.007 Ttc39b 1452009_at -1.996 0.047 RIKEN cDNA 4930513F16 1429874_at 1.730 0.014 Pura 1436844_at -1.858 0.030 Il7r 1448576_at 1.730 0.002 Slc25a24 1452717_at -1.856 0.049 Ccdc21 1432391_at 1.723 0.007 Araf 1440764_at -1.828 0.013 Zfp653 1435481_at 1.706 0.001 1441233_at -1.803 0.040 Pik3ip1 1428332_at 1.647 0.040 Arl6 1417331_a_at -1.738 0.028 Eif3s6i 1455811_at 1.640 0.003 Nktr 1437660_at -1.727 0.001 Glt25d1 1433496_at 1.634 0.001 Rbpj 1418114_at -1.671 0.050 Trmt1 1437314_a_at 1.603 0.040 Zfp826 1428748_at -1.657 0.035 LOC433762 1431213_a_at 1.596 0.030 Wdr41 1433620_at -1.640 0.002 Il7r 1448575_at 1.586 0.024 Cetn4 1436617_at -1.638 0.028 RIKEN cDNA 1810044D09 1441871_at 1.577 0.025 Hibch 1451511_at -1.632 0.034 Gclm 1439050_at 1.555 0.045 Hells 1453361_at -1.623 0.032 Acy1 1419173_at 1.536 0.031 Ar 1455647_at -1.621 0.017 Gramd3 1428737_s_at 1.515 0.019 1456864_at -1.609 0.017 Eif2ak2 1422005_at 1.486 0.002 Lyst 1434674_at -1.608 0.023 Sec22a 1416320_at 1.485 0.002 Zfp799 1437873_at -1.582 0.007 Immt 1429533_at 1.477 0.001 Ddx6 1447789_x_at -1.547 0.014 RIKEN cDNA 5830468F06 1432288_at 1.476 0.009 Mnt 1449661_at -1.547 0.016 Naa35 1453263_at 1.473 0.028 Ptk2 1423059_at -1.546 0.020 RIKEN cDNA 2810408M09 1452931_at 1.470 0.044 Idh3a 1422500_at -1.540 0.029 Ifit3 1449025_at 1.470 0.036 Samd8 1434402_at -1.539 0.027 Polrmt 1452835_a_at 1.466 0.036 Fbxo45 1428742_at -1.531 0.013 Ly6e 1439773_at 1.451 0.012 Rpl27a 1437729_at -1.529 0.056 RIKEN cDNA 2900018N21 1432824_at 1.450 0.000 Tpp2 1421893_a_at -1.528 0.009 Nfkbiz 1457404_at 1.442 0.050 Cul4a 1426060_at -1.522 0.174 H2-Ob 1422201_at 1.440 0.000 RIKEN cDNA E330009D23 1443779_s_at -1.520 0.008 Abcg1 1423570_at 1.438 0.034 Kctd20 1416323_at -1.517 0.010 Epm2aip1 1434106_at 1.432 0.199 Scai 1456357_at -1.516 0.010 Magt1 1419459_a_at 1.431 0.029 Rc3h2 1438268_at -1.516 0.028 Irf8 1448452_at 1.429 0.025 B3gnt2 1420852_a_at -1.507 0.023 Fabp1 1448764_a_at 1.409 0.001 Apc 1435543_at -1.506 0.006 Dpm1 1419353_at 1.404 0.000 RIKEN cDNA 4921513D23 1445862_at -1.500 0.000 Zfp398 1439979_at -1.490 0.018 Tbl1xr1 1450739_at -1.486 0.007 Fam33a 1437480_at -1.482 0.001 Strn3 1441266_at -1.481 0.005 Hipk2 1424863_a_at -1.475 0.035 RICKEN cDNA E130012A19 1426980_s_at -1.474 0.004 Nfat5 1439805_at -1.473 0.024 Zmym3 1455017_a_at -1.472 0.043 Hipk2 1428433_at -1.471 0.003 NA 1457053_at -1.466 0.066 NA 1458718_at -1.465 0.037 Arl13b 1437021_at -1.460 0.002 Zfp191 1430651_s_at -1.457 0.001 Vps41 1437901_a_at -1.457 0.033 RIKEN cDNA 2210018M11 1430028_at -1.455 0.011 Sacs 1434958_at -1.442 0.046 Ralgapa1 1428594_at -1.441 0.016 RIKEN cDNA E230008J23 1439571_at -1.435 0.002 Fam149b 1435686_at -1.434 0.008 Phf3 1428999_at -1.433 0.047 Strada 1428188_at -1.433 0.031 Nudt6 1456945_at -1.422 0.007 1439169_at -1.420 0.000 RIKEN cDNA C230085N15 1457656_s_at -1.420 0.036 Ms4a4d 1418990_at -1.420 0.006 Vps37a 1419177_at -1.406 0.005 C1d 1416886_at -1.404 0.007 Nnt 1456573_x_at -1.404 0.002