Volume 1, Issue 3, Pages 191-199 (March 2012) VEGF-C Promotes Immune Tolerance in B16 Melanomas and Cross-Presentation of Tumor Antigen by Lymph Node Lymphatics  Amanda W. Lund, Fernanda V. Duraes, Sachiko Hirosue, Vidya R. Raghavan, Chiara Nembrini, Susan N. Thomas, Amine Issa, Stéphanie Hugues, Melody A. Swartz  Cell Reports  Volume 1, Issue 3, Pages 191-199 (March 2012) DOI: 10.1016/j.celrep.2012.01.005 Copyright © 2012 The Authors Terms and Conditions

Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure 1 VEGF-C Protect against Preexisting Antitumor Immunity in B16-F10 Melanomas (A) Location of tumor inoculation with associated draining lymph node (dLN, axillary, and brachial) and contralateral non-dLN. (B) Lymphatic vessels in the tumor (left) and dLN (right) at day 12. Bar, 50 μm. (C) OVA vaccination scheme with lipopolysaccharide (LPS) adjuvant and 500 μg anti-VEGFR3 (mF4-31C1 #). (D) Growth profiles for control OVA and OVA/VEGF-C+ B16 tumors after vaccination and anti-VEGFR3 treatment. (E–G) Representative plots (E) and quantification (F) of OVA-specific CD8α+ T cells infiltrating the tumor (left) or draining lymph node (right) and in the blood (G). Shown are medians and whiskers show range. ∗p ≤ 0.05, ∗∗p ≤ 0.01, n ≥ 8. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure 2 CD8+ T Cells Activated in Lymph Nodes Draining VEGF-C+ Tumors Are Dysfunctional and Apoptotic (A) Scheme for OT-I adoptive transfer. After 9 days tumor growth, 106 naive CD8+ OT-I cells were injected i.v. with 500 μg mF4-31C1 (#). (B–E) Representative plots (B and D) and quantification (C and E) of CFSE-labeled OT-I cells (CD45.2+CD8α+) 3 days after transfer in the tumor (B and C) or draining LN (D and E). (F) IFN-γ production (left) and annexin V (AnV, right) binding by transferred OT-I cells with proliferation. (G) IFN-γ (left) and AnV (right) as % OT-I cells present within the draining LN. Shown are medians and whiskers show range. ∗p ≤ 0.05, ∗∗p ≤ 0.01, n ≥ 8. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure 3 Lymphatic Endothelial Cells Interact with CD8+ T Cells In Vivo and Cross-Present Tumor Antigen (A) Immunofluorescence showing OVA-specific T cells interacting with LECs (arrows) in the stroma of OVA and OVA/VEGF-C+ tumors. Bar, 100 μm. (B and C) Representative plots (B) and quantification (C) of OT-I CD8+ T cell proliferation in draining lymph nodes (dLN) of tumor-bearing mice after adoptive transfer. β2m−/−:wt (β2m:wt) chimeric mice lack MHC I expression in hematopoietic but not stromal cells. (D and E) Representative plots (D) and quantification (E) of ex vivo OT-I cell proliferation after coculture with cells isolated dLN of VEGF-C+ tumors. Splenocytes from naive mice with SIINFEKL were used as a positive control. (F) MHC I-SIINFEKL complex (25d1.16) on LECs (gp38+CD31+) in the tumor and dLN. Shown are means ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, n ≥ 8. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure 4 Lymphatic Endothelial Cells Cross-Present Antigen to Induce Dysfunctional T Cell Activation (A and B) Representative plots (A) and geometric means (B) of MHCI/SIINFEKL complex (25d1.16) on cultured lymph node (LN) LECs, dendritic cells (BMDCs), and B16-F10 tumor cells after 24 hr pulse with NPssCOVA250-264 or SIINFEKL. (C and D) Representative plots (C) and quantification (D) of OT-I CD8+ T cell proliferation after coculture with antigen-pulsed LECs. (E) IFN-γ production (by intracellular cytokine staining) in OT-I cells stimulated by antigen-presenting BMDCs or LECs. (F and G) Representative plots (F) and quantification (G) of Annexin V (AnV) binding on OT-I cells after coculture with freshly sorted tumor-draining LN LECs or splenocytes reexposed to SIINFEKL antigen ex vivo. Shown are means ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, n ≥ 3. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure S1 VEGF-C Expression Enhances Metastasis, Lymphangiogenesis, Draininage, and DC Trafficking, Related to Figure 1 (A) Cell proliferation reported as a fold change in cell number over 24 hr. (B) Cell migration over 20 hr in 3D insert cultures. Data represent mean ± SEM. (C) VEGF-C protein levels in vivo after 12 days of tumor growth measured by ELISA of whole tumor lysate and normalized to control. (D and E) (D) B16-F10 tumor growth profiles and (E) OVA tumor growth profiles. (F) Tumor metastases in the dLN (melanin+). Scale bar, 5mm. (G) Analysis of peri-tumoral lymphangiogenesis. lymphatic vessel density analyzed as % LYVE1+ pixels. Analyses made using Image J software. (H) Lymphatic function as assessed by FITC-dextran 70kD measured in dLNs 30 min after intratumoral injection. (I) Dendritic cell (CD45+CD11c+ bead+) trafficking from the tumor to the dLN 24 hr after intratumoral injection of 500nm fluorescent beads. Data represent mean ± SEM. Box plots, whiskers max to min. ∗p ≤ 0.05, n ≥ 6. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure S2 VEGF-C Recruits a Regulatory Immune Cell Infiltrate, Related to Figure 1 (A) CD45+ immune cell infiltrates at the tumor periphery by day 12 (CD45, green) and lymphatics (LYVE-1, red). Scale bar, 50 μm. (B) Tumor infiltrating lymphocytes (TILs) (CD3ε+). (C and D) (C) Tumor and LN infiltrating regulatory T cells (CD4+CD25+FoxP3+) and (D) percentage of CD8α+ TILs specific for the melanoma antigen Trp2 (H2Kb-Trp2 Pentamer). Data represent mean ± SEM. Box plots, whiskers max to min. ∗p ≤ 0.05, n ≥ 6. (E) Antigen-presenting cell subsets within the tumor were analyzed by flow cytometry and reported as a percentage of all CD45+ tumor-infiltrating cells. (F) CD11b+VEGF-C+ immune infiltrates in the peritumoral space by day 12. CD11b+ myeloid cells (red) and intracellular VEGF-C staining (green). Scale bar, 100 μm. Data represent mean ± SEM. ∗P ≤ 0.05 as determined by Mann Whitney non-parametric t test; n ≥ 5. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure S3 VEGF-C Prevents CD8α Antigen Specific T Cell Activation In Vivo, Related to Figure 2 (A and B) (A) IFN-γ production and (B) activation (CD25 and CD69) of tumor infiltrating CD45.2+CD8α+ OT-I five days following transfer into tumor-bearing hosts. Unactivated T cells used as negative control for CD25 and CD69 expression. (C and D) (C) CD25 and (D) CD69 activation marker expression on donor CD45.2+antigen-specific OT-I day three and five following adoptive transfer into tumor-bearing hosts. Data represent mean ± SEM. ∗P ≤ 0.05 as determined by one-way ANOVA; n ≥ 4. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions

Figure S4 Sorting Scheme for LN Stromal Cells and MHCI Expression in Chimeras, Related to Figures 3 and 4 (A) Sorting scheme for LN stromal cells gated first on the CD45− population after magnetic CD45 depletion and then separated into 4 subpopulations: fibroblastic reticular cells (FRC; gp38+CD31−), lymphatic endothelial cells (LEC; gp38+CD31+), blood endothelial cells (BEC; gp38−CD31+) and a double negative population. (B) MHC class I expression by indicated cell subsets in β2m:wt chimeric mice, which were designed to express MHC I molecules only by non-hematopoietic cells. (C) Purity of sorted cell populations. (D and E) Confirmation of (D) LYVE-1 and (E) VEGFR3 expression on LECs in freshly isolated LN stromal cells and day 7 LN cultures by flow cytometry. LYVE-1 staining used the same antibody as in immunofluorescence, with donkey anti-rabbit, VEGFR-3 (R&D Systems), with chicken anti-goat secondary antibodies in AF647. Cell Reports 2012 1, 191-199DOI: (10.1016/j.celrep.2012.01.005) Copyright © 2012 The Authors Terms and Conditions