In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naïve T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naïve T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.

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There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8+ T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products.

Lymphangiogenesis occurs in inflammation and wound healing, yet its functional roles in these processes are not fully understood. Consequently, clinically relevant strategies for therapeutic lymphangiogenesis remain underdeveloped, particularly using growth factors. To achieve controlled, local capillary lymphangiogenesis with protein engineering and determine its effects on fluid clearance, leukocyte trafficking, and wound healing, we developed a fibrin-binding variant of vascular endothelial growth factor C (FB-VEGF-C) that is slowly released upon demand from infiltrating cells. Using a novel wound healing model, we show that implanted fibrin containing FB-VEGF-C, but not free VEGF-C, could stimulate local lymphangiogenesis in a dose-dependent manner. Importantly, the effects of FB-VEGF-C were restricted to lymphatic capillaries, with no apparent changes to blood vessels and downstream collecting vessels. Leukocyte intravasation and trafficking to lymph nodes were increased in hyperplastic lymphatics, while fluid clearance was maintained at physiological levels. In diabetic wounds, FB-VEGF-C-induced lymphangiogenesis increased extracellular matrix deposition and granulation tissue thickening, indicators of improved wound healing. Together, these results indicate that FB-VEGF-C is a promising strategy for inducing lymphangiogenesis locally, and that such lymphangiogenesis can promote wound healing by enhancing leukocyte trafficking without affecting downstream lymphatic collecting vessels.

Increased density of tumor-associated lymphatic vessels correlates with poor patient survival in melanoma and other cancers, yet lymphatic drainage is essential for initiating an immune response. Here we asked whether and how lymphatic vessel density (LVD) correlates with immune cell infiltration in primary tumors and lymph nodes (LNs) from patients with cutaneous melanoma. Using immunohistochemistry and quantitative image analysis, we found significant positive correlations between LVD and CD8+ T cell infiltration as well as expression of the immunosuppressive molecules inducible nitric oxide synthase (iNOS) and 2,3-dioxygénase (IDO). Interestingly, similar associations were seen in tumor-free LNs adjacent to metastatic ones, indicating loco-regional effects of tumors. Our data suggest that lymphatic vessels play multiple roles at tumor sites and LNs, promoting both T cell infiltration and adaptive immunosuppressive mechanisms. Lymph vessel associated T cell infiltration may increase immunotherapy success rates provided that the treatment overcomes adaptive immune resistance.

• Antigen cross-presentation is regulated by the activity of deubiquitylase YOD1 that influences the control of viral infections.• The enhanced crosspresentation depends on the proteasomal activity and the acidification status of APCs but is independent of functional TAP1. IntroductionCD8 ϩ T-cell responses constitute a critical component of host defense against intracellular pathogens. Antigen-specific CD8 ϩ T cells are primed by antigen presenting cells (APCs), such as dendritic cells (DCs), which process and present antigens derived from intracellular pathogens either by becoming infected themselves (direct presentation) or by phagocytosis of infected cells (cross-priming). 1,2 However, infected DCs may be compromised in their function and be less efficient at inducing an immune response, 3-5 making the cross-presentation pathway all the more important. 6 Pathogen-derived antigens are then presented to naive CD8 ϩ T cells by DCs via class I MHC molecules. The ensuing activation and expansion of CD8 ϩ T cells of the appropriate specificity allows them to kill the infected target cells in the acute phase of response. After the pathogen is cleared, the pool of effector cells contracts. A small pool of self-renewing memory cells survives to respond to an eventual infection with the same pathogen. 7 Although the intricacies of direct presentation are fairly well understood, the mechanisms involved in cross-presentation are not fully resolved and are probably diverse. 1,2,8 DCs are particularly good at presenting exogenous antigens to CD8 ϩ T cells, attributes that derive from their morphology, strategic location in vivo, efficient antigen uptake, low lysosomal proteolysis, and from the presence of specific protease inhibitors. 9 These traits allow them to retain antigen and carry it to the draining lymph node (LN) where immune responses are induced. 10,11 DCs take up not only soluble, but also particulate antigens released by apoptotic or pathogen-damaged cells and process them to cross-prime CD8 ϩ T cells. 12,13 The latter event is particularly relevant for the generation of immune responses against pathogens that do not directly infect APCs. 8 The endoplasmic reticulum-associated degradation machinery is responsible for degradation and disposal of misfolded proteins from the ER, and its possible relevance for the processing of exogenous antigens is now apparent. 14,15 Thus, p97, a crucial player in this pathway, has been implicated in cross-presentation. 14 A dominant-negative form of p97 impairs processing of exogenously provided chicken ovalbumin (Ova) and its subsequent presentation to Ova-specific CD8 ϩ T cells. The deubiquitylating enzyme YOD1 interacts with p97 and is involved in the dislocation reaction. 16 Cells that express a mutant form of YOD1 devoid of deubiquitylating activity (YOD1-C160S) accumulate various polyubiquitylated dislocation intermediates that would otherwise have been cleared from the ER. If the ER-to-cytosol pathway were indeed an important component of cross-presentation, then expre...

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The human microbiome is the collective of microbes living in symbiosis on and within humans. Modulating its composition and function has become an attractive means for the prevention and treatment of a variety of diseases including cancer. Since the initiation of the human microbiome project in 2007, the number of academic publications and active patent families around the microbiome has grown exponentially. Screening patent databases can be useful for the early detection and the tracking of new technology trends. However, it is not sufficient to assess portfolio sizes because emerging players with small but high-quality patent portfolios will be missed within the noise of large but low-quality portfolio owners. Here we used the consolidated database and software tool PatentSight to benchmark patent portfolios, and to analyze key patent owners and innovators in the microbiome space. Our study shows how in-depth patent analyses combining qualitative and quantitative parameters can identify actionable early indicators of technology and investment trends from large patent datasets.