Lymphatic vessel density is associated with CD8⁺ T cell infiltration and immunosuppressive factors in human melanoma.

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 <sup>+</sup> 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

Human melanoma-specific CD8(+) T-cells from metastases are capable of antigen-specific degranulation and cytolysis directly ex vivo.

The relatively low frequencies of tumor Ag-specific T-cells in PBMC and metastases from cancer patients have long precluded the analysis of their direct ex vivo cytolytic capacity. Using a new composite technique that works well with low cell numbers, we aimed at determining the functional competence of melanoma-specific CD8(+) T-cells. A multiparameter flow cytometry based technique was applied to assess the cytolytic function, degranulation and IFNγ production by tumor Ag-specific CD8(+) T-cells from PBMC and tumor-infiltrated lymph nodes (TILN) of melanoma patients. We found strong cytotoxicity by T-cells not only when they were isolated from PBMC but also from TILN. Cytotoxicity was observed against peptide-pulsed target cells and melanoma cells presenting the naturally processed endogenous antigen. However, unlike their PBMC-derived counterparts, T-cells from TILN produced only minimal amounts of IFNγ, while exhibiting similar levels of degranulation, revealing a critical functional dichotomy in metastatic lesions. Our finding of partial functional impairment fits well with the current knowledge that T-cells from cancer metastases are so-called exhausted, a state of T-cell hyporesponsiveness also found in chronic viral infections. The identification of responsible mechanisms in the tumor microenvironment is important for improving cancer therapies

Tumor-associated factors are enriched in lymphatic exudate compared to plasma in metastatic melanoma patients.

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137 <sup>+</sup> and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response

The neuronal alpha(6) subunit forms functional heteromeric acetylcholine receptors in human transfected cells

We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells

Lymphatic vessel density is associated with CD8+ T cell infiltration and immunosuppressive factors in human melanoma

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. © 2018, © 2018 The Author(s). Published with Taylor & Francis Group