Supplementary Figure 1 Recombinant LIGHT-VTP induces pericyte contractility and endothelial cell activation. (a) Western blot showing purification steps for full length murine LIGHT-VTP (CGKRK) protein: (1) protein marker; (2) fusion protein expression in bacterial lysate; (3) column flow through; (4) column washing; (5) eluate from Ni-NTA column; (6) protein after dialysis; (7) released protein after tobacco edge virus (TEV) protease cleavage; (8) purified LIGHT-CGKRK at approximately 20 kda (boxed); arrow points at bovine serum albumin (BSA) which was added for stability. (b) Histological quantification of CD31 + vessel diameters in untreated mice (n=4), or mice treated bi-weekly for 2 weeks with VTP (n=3) or 20 ng LIGHT-VTP (LV, n=3). **P=0.009, *P=0.003 (Student s t-test). Scale bar, 50 µm. (c) Histological analysis of ICAM expression in untreated (n=4) and LV treatment groups (n=6). *P=0.003 (Student s t-test). Scale bar, 50 µm. (d) Immunohistochemical quantification of pericyte (αsma + ) expression of contractile markers (calp, calponin; cald, caldesmon, arrows) and tumor-associated collagen I (col I) in treatment groups. Caldesmon, n=7 untr mice, n=5 LV treated mice; calponin, n=5 untr mice, n=7 LV treated mice; collagen I, n=9 untr mice, n=6 LV treated mice. ***P=0.0001, **P=0.008, *P=0.04, Student s t-test. Scale bars, 50 µm. Data are presented as means ± s.d. and representative of three independent experiments.
Supplementary Figure 2 CCL21 + TLSs are induced by LIGHT-VTP or transfer of LIGHT-stimulated macrophages. (a) Histological analysis of LV-induced TLS structures in RIP1-Tag5 tumors before and after laser microdissection and pressure catapulting (LMPC). Scale bar, 100 µm (upper), 150 µm (lower). (b) Immunohistochemical analysis of CCL21 expression in untreated or LV treated tumors in conjunction with MECA79 + HEV (middle) or CD68 + macrophage (right) stainings. Dashed lines show TLS. Depicted are CCL21-positive endothelial cells (arrows), HEV structure (filled arrow head) and macrophages associated with a blood vessel (open arrow head). Scale bar, 50 µm. (c) Relative mrna in macrophage-depleted, naïve (left) or ConA-activated (right) splenocytes, n=3 C3H donor mice. (d) Schematic presentation of adoptive macrophage transfers (AdT) into tumor-bearing RIP1-Tag5 mice. (e) Quantification of B cells within 100 µm of MECA79 + vessels in tumors after macrophage transfer, *P=0.02, n=3 (Student s t-test). (f) Histological analysis of CCL21 expression in tumors after transfer of PBS- or LV-stimulated macrophages (PBS MФ, LV MФ), or LV MФ transfer into T cell-depleted mice (αcd4/8). Dashed lines show TLS. Depicted are CCL21-positive endothelial cells (arrows) and macrophages (open arrow heads). Scale bar, 50 µm. Data are presented as mean ± s.d. and are representative of one experiment (c, e, f), two independent (b) and three independent experiments (a).
Supplementary Figure 3 LIGHT-VTP treatment induces infiltration of immune cells into RIP1-Tag5 tumors. (a) Representative FACS plots showing gating strategy for Fig. 4a including Fluorescence Minus One (FMO) controls. (b) Macroscopic tumor appearance shown for individual RIP1-Tag5 mice at 30 weeks of age, representative images for untreated and LV treatment groups are shown.
Supplementary Figure 4 PDL-1 expression in RIP1-Tag5 tumors and therapeutic effects of checkpoint-blockade treatments. (a) Histological analysis of PD-L1 expression in untreated RIP1-Tag5 tumors (n=4) and after a 2 week treatment with bi-weekly injections of 20 ng LIGHT-VTP (LV, n=3). Scale bar, 50 µm. Data are presented as mean ± s.d. (b) Survival analysis of RIP1-Tag5 mice treated with LV and control rat IgG2a antibody (n=8), αctla-4 (n=6) or αpd-1 (n=5) as single treatment modalities. (c) Macroscopic RIP1-Tag5 tumor appearance after 7 weeks of treatment with LV and control antibodies (LV+control abs) or LV combined with checkpoint inhibitors (LV+αCTLA-4/αPD-1). Representative images from individual mice are shown. Data represent two independent experiments.
Supplementary Figure 5 LIGHT-VTP and checkpoint blockade induce effector T cells in tumors and draining lymph nodes. (a) Schematic outline of long term treatment of RIP1-Tag5 mice from 23 to 30 weeks with combination therapies. (b) FMO controls for the markers Ki67 and GrzB, representative contour plots. Corresponding data are shown in Fig. 6b. (c) FMO controls for the markers FoxP3 and CD25. Corresponding data are shown in Fig. 6c. (d) FACS quantification of % CD25 + FoxP3 + T reg cells of CD3 + CD4 + T cells in pancreatic tumor-draining lymph nodes of RIP1-Tag5 mice which were left untreated, or treated with LIGHT-VTP and control antibodies (LV+ctrl) or LV+αCTLA-4/αPD-1 antibodies (LV+αCTLA-4/αPD-1), n=3-6 mice (pooled into one group because of limited size of pancreas draining lymph nodes). (e) Ratio of intratumoral Ki67 + GrzB + CD4 + or CD8 + effector T cells to CD25 + FoxP3 + T reg cells of CD3 + CD4 + T cells, n=4 for all CD4 + T cell analyses, n=5 for CD8 + T cells in LV+ctrl tumors, n=4 for CD8 + T cells in LV αctla-4/αpd-1 tumors, *P=0.021, **P=0.001 (Student s t-test). (f) Quantification of Ki67 + GrzB + effector CD4 + and CD8 + T cells of CD3 + T cells in tumor-draining lymph nodes from various treatment groups, and representative plots, n=3-6 mice (pooled into one group). Data represent two independent experiments.
Supplementary Figure 6 LIGHT-VTP and checkpoint blockade induce a switch from naive T cells to memory T cells. (a) FACS quantification of % CD4 + or CD8 + naïve (CD44 low CCR7 +, upper graph) or memory (CD44 + CCR7 -, lower graph) T cells of CD3 + T cells from untreated tumors (n=7), or tumors of LIGHT-VTP+control antibodies (LV+ctrl, n=5) or LV+αCTLA-4/αPD-1 treated mice (n=4 mice), and representative plots. *P=0.05, **P=0.01, Student s t-test. (b) Quantification of % intratumoral CD4 + or CD8 + proliferating (Ki67 + ) T cells of memory (CD44 + CCR7 - ) CD3 + T cells from different treatment groups and representative plots. *P=0.02 (Student s t- test). (c) FMO controls for CD44 and CCR7, representative FACS plots. (d) FMO controls for CD44, CCR7 and Ki67 stainings, representative FACS plots. Data are presented as mean ± s.d. and represent two independent experiments.
Supplementary Figure 7 LIGHT-VTP combination immunotherapies enhance survival and reduce tumor angiogenesis. (a) Treatment scheme for bi-weekly LIGHT-VTP (LV) injections combined with adoptive T cell transfers (AdT) or anti-tag/cpg-odn vaccine with or without checkpoint blockade. (b) Survival analyses comparing LV+Tag-activated TCR T cell transfer (Tag T) (n=6, mean survival 34±2 weeks) with LV+ConA-activated C3H T cell transfer (ConA T) (n=5, 30±2 weeks). The LV group is shown as reference (original data in Fig. 4d). **P=0.008, *P=0.05 (log-rank test). Data are representative for one experiment. (c) Macroscopic tumor appearance at 29 weeks of age in RIP1-Tag5 groups treated with LV+vaccine or LV triple therapy + vaccine. Representative images from individual mice are shown.
Supplementary Table 1: Data presented in Fig. 4A in tabular form including P-values Marker TLS- TLS+ P-value % Total cells CD45 2.43±1.08 8.12±1.49 0.0007 CD3 0.72±0.49 3.24±1.56 0.0128 CD4 0.36±0.26 1.44±0.64 0.0136 CD8 0.15±0.11 1.04±0.75 0.0325 B220 0.21±0.29 0.24±0.09 0.8543 CD11b 0.08±0.04 0.33±0.08 0.0008 DX5 0.12±0.09 0.39±0.07 0.0051 CD11c 0.01±0.01 0.03±0.01 0.0382
Supplementary Table 2: Total intratumoral T cells [%] Figure Marker Treatment % total ± SD P-value 6b CD8 LV + Ctrl 3.7 ± 1.4 0.00016 LV + αctla-4/αpd-1 17.8 ± 4.6 CD4 LV + Ctrl 2.6 ± 0.3 0.005 LV + αctla-4/αpd-1 7.0 ± 4.2 6c CD8 Ki67 GrzB Untr 0.115 ± 0.075 0.494 a ; 0.004 b LV + Ctrl 0.148 ± 0.060 0.022 LV + αctla-4/αpd-1 1.009 ± 0.540 CD4 Ki67 GrzB Untr 0.015 ± 0.007 0.855 a ; 0.001 b LV + Ctrl 0.014 ± 0.007 0.001 LV + αctla-4/αpd-1 0.137 ± 0.049 6d CD4 CD25 FoxP3 6e CD8 CD44 + CCR7 - (memory) CD4 CD44 + CCR7 - (memory) CD8 CD44 low CCR7 + (naïve) CD4 CD44 low CCR7 + (naïve) Untr 0.088 ± 0.064 0.065 a ; 0.004 b LV + Ctrl 0.206 ± 0.135 0.206 LV + αctla-4/αpd-1 0.332 ± 0.155 Untr 2.6 ± 1.5 0.754 a ; 0.014 b LV + Ctrl 2.3 ± 1.5 0.020 LV + αctla-4/αpd-1 11.4 ± 7.7 Untr 2.4 ± 1.2 0.438 a ; 0.014 b LV + Ctrl 3.3 ± 2.5 0.065 LV + αctla-4/αpd-1 8.5 ± 5.3 Untr 0.357 ± 0.151 0.081 a ; 0.585 b LV + Ctrl 1.330 ± 1.341 0.172 LV + αctla-4/αpd-1 0.298 ± 0.172 Untr 0.075 ± 0.113 0.181 a ; 0.540 b LV + Ctrl 0.362 ± 0.517 0.251 LV + αctla-4/αpd-1 0.035 ± 0.024 6f CD8 CD44 + Untr 0.973 ± 0.499 0.587 a ; 0.011 b CCR7 - Ki67 LV + Ctrl 0.821± 0.420 0.015 LV + αctla-4/αpd-1 4.980 ± 3.359 CD4 CD44 + Untr 0.503 ± 0.113 0.086 a ; 0.002 b CCR7 - Ki67 LV + Ctrl 0.860 ± 0.487 0.011 LV + αctla-4/αpd-1 3.835 ± 2.179 a compared to (LIGHT-VTP +control antibodies, LV+ctrl); b compared to LIGHT-VTP+αCTLA-4/αPD-1 checkpoint inhibitors (LV+αCTLA-4/αPD-1).
Supplementary Table 3: Antibodies used for FACS analyses Antibody Clone Color Source CD3 ebio500a2 PerCp-eFlour 710 ebiosciences CD3 145-2C11 BB515 BD Biosciences B220 RAC3-6B2 PECy7 BD Biosciences CD4 Gk1.5 BUV737, APC BD Biosciences/BioLegend CD4 L3T4 PE BD Biosciences CD8 53-6-7 APC-Cy7 BD Biosciences CD11c N418 PerCp-Cy5.5 BioLegend CD11b M1/70 APC-Cy7 BioLegend CD45 30F-11 PECF594 BD Biosciences CD44 IM7 PECy7 BioLegend CCR7 4B12 PE BioLegend CTLA4 UC10-4F10-11 PE BD Biosciences PD1 RMPI-30 APC BioLegend GrzB GB11 AF647 BD Biosciences Ki67 B56 FITC BD Biosciences FoxP3 150D APC BioLegend DX5 CD49b PE BD Biosciences CD25 PC61 PECy7 BD Biosciences
Supplementary Table 4: Primer sequences used for transcript detection by quantitative PCR Gene Forward primer (5 3 ) Reverse primer (5 3 ) CCL-2 TTA AAA ACC TGG ATC GGA ACC AA GCA TTA GCT TCA GAT TTA CGG GT CCL-3 CAA GCT TTC TCA GCG CCA TAT G ACG ATG AAT TGG CGT GGA AT CCL-5 GCA AGT GCT CCA ATC TTG CA AGT TGA TGT ATT CTT GAA CCC ACT TCT CCL-19 CCT GGG AAC ATC GTG AAA GC TAG TGT GGT GAA CAC AAC AGC CCL-21 GTG ATG GAG GGG GTC AGG A GGG ATG GGA CAG CCT AAA CT CXCL-1 GGA TTC ACC TCA AGA ACA TCC AG GTG TGG CTA TGA CTT CGG TTT G CXCL-9 GAG GAA CCC TAG TGA TAA GGA ATG C TCT TCA GTG TAG CAA TGA TTT CAG TTT CXCL-10 CCT TCA CCA TGT GCC ATG C TCT TAC ATC TGA AAT AAA AGA GCT CAG GT CXCL13 GGC CAC GGT ATT CTG GAA GC GGG CGT AAC TTG AAT CCG ATC TA HPRT ACA CCT GCT AAT TTT ACT GGC TGG AAA AGC CAA ATA CAA AGC CTA AAC A IFNγ ATC TGG CTC TGC AGG ATT TTC A TCA AGT GGC ATA GAT GTG GAA GAA IL1β AAC CTG CTG GTG TGT GAC GTT GAG GTG GAG AGC TTT CAG CTC A IL6 AAG TAG GGA AGG CCG TGG TT TCC TCT CTG CAA GAG ACT TCC AT IL7 GGA ATT CCT CCA CTG ATC CTT GT CTT TGT CTT TAA TGT GGC ACT CAG L19 ATT GGG ATC CAA CCA GAC CTT AGG CTA CAG AAG AGG CTT GCC LTα GACTCTCTGGTGTCCGCTTCT GGTACCCAACAAGGTGAGCAG LTβ GGA GCA CAG GCT CAG AAA AGA GGG TGA GGG CAA GAT GCA TNFα ACA AGG CTG CCC CGA CTA CTT GAC GGC AGA AGG AGG TT VE- Cadherin CAG AAT TAA GCA CTG ACA CAT CAT AGC GCG CAT GCT CAT AGC AAG AGT