Abstract NG02: Lymphatic vessels: Balancing immune priming and immune evasion in melanoma
Authors: Lane Ryan S. , Femel Julia , Booth Jamie , Loo Christopher , Nelson Nicholas , Tsujikawa Takahiro , Thibault Guillaume , Lund Amanda W. 
Published: July 2017

Abstract
Lymphatic vessel remodeling and lymphangiogenesis is correlated with melanoma progression and lymph node metastasis. While lymphatic vessels provide an important route for disseminating tumor cells, they are also a crucial interface between a developing malignancy and the host immune response. Rather than acting as passive conduits, lymphatic vessels directly regulate their transport function and facilitate leukocyte trafficking for efficient induction of adaptive immunity in downstream draining lymph nodes. We recently published that in the absence of dermal lymphatic vessels, the tumor microenvironment of murine melanoma remains completely uninflamed and fails to induce a robust T-cell response (1). Consistently, TCGA analysis of human cutaneous metastatic melanoma identified positive correlations between LEC gene expression and immune genes, including a T cell-inflamed signature, indicating a relationship between lymphatic vessel remodeling and local immunity. Furthermore, others have recently demonstrated that lymphatic vessel density (LVD) in combination with intratumoral T cell function stratified best overall survival in nonmetastatic and metastatic colorectal cancer (2). In contrast to this, however, many reports independently correlate peritumoral LVD with lymph node metastasis and some poor prognosis (3). Furthermore, our previous work demonstrated that vascular endothelial growth factor C (VEGFC)-driven lymphangiogenesis in the context of murine melanoma drove increased leukocyte infiltration but associated with poor CD8+ T cell priming in draining lymph nodes (4). We therefore hypothesize that lymphatic vessels are (A) required for induction of adaptive immunity but (B) acquire immunosuppressive activity as a function of the accumulation of local cytotoxic immunity. Furthermore, we predict that LVD may be a relevant biomarker of in situ immune responsiveness and response to therapy (5).
To test the first part of this hypothesis (A), we have continued our published work and used a cutaneous model of viral infection to demonstrate the requirement for lymphatic vessel drainage in cutaneous immunity. Following cutaneous vaccinia infection we demonstrate that in the absence of lymphatic vessel transport, both cellular and humoral adaptive immune responses fail to initiate, leading to enhanced cutaneous immunopathology and persistent viral load. The complete absence of primed CD8+ and CD4+ T cells in cutaneous tissue following challenge mirrors our observations in melanoma and is consistent with correlations of intratumoral lymphocytes and LVD both by our group in cutaneous metastatic melanoma as well as by others. This unequivocal requirement for a functional lymphatic vasculature in the priming of cutaneous immunity further supports the prediction that LVD may be a novel biomarker of immune reactivity within tumor parenchyma. To test this, we simultaneously evaluated immune and vascular components in human primary melanoma samples using a multiplex-immunohistochemistry-based approach. Tissue regions that include tumor/stroma borders and show high CD8+ T-cell infiltrates are selected for analysis, followed by tissue segmentation, and automated detection of cell populations within intratumoral regions and bordering stroma. Interestingly, those tumors with enhanced hematopoietic infiltrate (CD68 and CD8) also appear to demonstrate increased vasculature, both blood (CD31 and CD34) and lymphatic (D2-40 and Prox1). Preliminary data demonstrate that lymphatic vessels, blood vessels, and CD8+ T cells are significantly enriched at the tumor-stroma border in primary melanoma and positively correlate with one another, indicating that lymphatic vessels may be a dynamic component of the “T cell-inflamed” microenvironment.
While we demonstrate that lymphatic vessels are necessary for immune induction, we further hypothesized (B) that in the context of an ongoing immune response lymphatic vessels adapt their function to promote immune resolution. The adaptive resistance hypothesis proposes that upon accumulation of local cytotoxic immunity, both tumors as well as stromal components adapt and acquire therapeutically relevant immunosuppressive function. We demonstrate that peripheral, tumor-associated lymphatic endothelial cells (LEC; CD45-CD31+gp38+) acquire expression of immunoregulatory proteins, most notably programmed death receptor ligand-1 (PD-L1) and major histocompatibility complex II (MHCII), coincident with CD8+ T-cell infiltration in an interferon gamma (IFNg)-dependent manner. Adoptive transfer of activated CD8+ T cells induced higher expression of PD-L1 by LECs in B16 F10 tumors, while neutralization of IFNg reduced levels to that of naïve skin. Furthermore, conditional knockout of the IFNgR (Lyve1-Cre) prevented upregulation of PD-L1 on tumor-associated LECs. Notably, lymph node LECs constitutively express PD-L1 and this expression contributes to the attenuation of self-reactive CD8+ T-cell responses (6). Importantly, in the absence of IFNgR on peripheral LECs we observed significantly enhanced accumulation of antigen-specific CD8+ T cells in cutaneous tissue. Thus, cutaneous lymphatic vessels, while necessary for immune induction, acquire immunodulatory properties in a context-dependent manner and may participate in immune evasion within tumor microenvironments.
In conclusion, our work across multiple model systems provides strong experimental evidence to indicate that the lymphatic vasculature is an important, active component of the antitumor immune response and may represent a biomarker to stratify patient response and survival for effective clinical immunotherapy. These data indicate a need to revisit the passive paradigm of lymphatic vessel involvement in tumor progression and metastasis to a more active model whereby lymphatic vessels are both required for antitumor immunity but functionally evolve in response to accumulating cytotoxicity to drive immune evasion.
References:
1. Lund AW et al. Lymphatic vessels regulate immune microenvironments in human and murine melanoma. J Clin Invest 2016;126:3389-402. doi: 10.1172/JCI79434.
2. Mlecnik B et al. The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis. Sci Transl Med 2016;8:327ra26.
3. Pasquali S et al. Lymphatic biomarkers in primary melanomas as predictors of regional lymph node metastasis and patient outcomes. Pigment Cell Melanoma Res 2013;26:326-37.
4. Lund AW et al. VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics. Cell Rep 2012;1:191-9.
5. Lund AW. Rethinking lymphatic vessels and antitumor immunity. Trends Cancer 2016;2:548-51. doi: 10.1016/j.trecan.2016.09.0056. Tewalt EF et al. Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells. Blood 2012;120:4772-82.
Citation Format: Ryan S. Lane, Julia Femel, Jamie Booth, Christopher Loo, Nicholas Nelson, Takahiro Tsujikawa, Guillaume Thibault, Amanda W. Lund. Lymphatic vessels: Balancing immune priming and immune evasion in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG02. doi:10.1158/1538-7445.AM2017-NG02