Purpose: The sclera is mainly made of collagen and fibroblasts. The aim of this study was to analyze whether immune cells are present in the healthy human sclera. Methods: Ten human anterior episcleral or stromal tissue samples from globe donors were immunohistochemically examined using confocal microscopy. The expression of the macrophage markers CD68, CD163 and CD11b, CD45 (a general leukocyte marker), MHCII (expressed by antigen-presenting cells [APCs]), CD11c (dendritic cell marker), lymphatic endothelium hyaluronan receptor-1 (LYVE1; expressed on lymphatic endothelium and macrophage subsets), chemokine receptor 7 (CCR7, a homing receptor for leukocytes), CXCL12 (expressed by activated leukocytes), CCR2 (a marker for inflammatory monocytes), and glial fibrillary acidic protein (GFAP; expressed by astrocytes) was analyzed and quantified. Results: In the episclera, a high number of cells (≥40 cells/mm²) were immunoreactive for CD68, CD45, MHCII, CCR7, LYVE1, and CD11b. Lower numbers (<20 cells/mm²) were positive for CXCL12, CCR2, and GFAP. The episclera showed a significantly higher number of cells compared to the stroma (P = 0.008). MHCII+ cells could be double positive for CCR7, CD45, CD11c, or CD11b and seldom CXCL12. Macrophages were most likely from the M1 type (CD68+, CD163−). Conclusions: The healthy human sclera contains several macrophage populations, which can function as APCs, with the highest density being present in the episclera. Most cells express macrophage markers and may function as APCs. The presence of these cells might indicate that scleral immune cells are important for maintaining physiological functions in the eye and may potentially contribute to blood vessel homeostasis.

Lymphangiogenesis is a very early step in lymphatic metastasis. It is regulated and promoted not only by the tumor cells themselves, but also by cells of the tumor microenvironment, including cancer associated fibroblasts, mesenchymal stem cells, dendritic cells, or macrophages. Even the extracellular matrix as well as cytokines and growth factors are involved in the process of lymphangiogenesis and metastasis. The cellular and noncellular components influence each other and can be influenced by the tumor cells. The knowledge about mechanisms behind lymphangiogenesis in the tumor microenvironmental crosstalk is growing and offers starting points for new therapeutic approaches.

Purpose: Avascular tissues can be used to identify antilymph- or antihemangiogenic factors. The human sclera—the outer covering layer of the eye, lacks lymphatic vessels and contains only a superficial network of blood vessels and was used here to identify endogenous antiangiogenic factors. Methods: Expression levels of a panel of 96 known pro- and antiangiogenic factors were analyzed in 12 scleral or conjunctival control samples from normal human donors using real-time PCR. In vitro, scleral homogenate was cocultured with blood- and lymphatic endothelial cells (BECs and LECs) and immunohistochemistry was performed of scleral fibroblasts and BECs. Results: Three antiangiogenic factors were significantly upregulated in the human sclera compared to the conjunctiva, including FBLN5 (fibulin 5), SERPINF1 (serpin peptidase inhibitor, clade F, member 1 = pigment epithelium derived factor) and TIMP2 (Tissue inhibitor of metalloproteinases 2). Six proangiogenic factors were significantly downregulated in the sclera, including FLT4 (Fms-related tyrosine kinase 4=VEGF-R3), HGF (hepatocyte growth factor), KIT (CD117 / c-kit), PROX1 (prospero homeobox 1), SEMA3F (semaphorin-3F) and TGFA (transforming growth factor alpha). In vitro, scleral homogenate inhibited the growth of both BECs and LECs. Immunohistochemistry labeling of three major antiangiogenic factors from scleral tissue confirmed TIMP3 and PEDF expression both in scleral fibroblasts and in blood endothelial cells, whereas TIMP2 was not detectable. Conclusion: Balancing anti- and proangiogenic factors actively regulates human scleral avascularity, inhibits endothelial cell growth in vitro, and thus may help maintaining the vascular privilege of the inner eye.

Conjunctival melanoma (CM) accounts for 5% of all ocular melanomas and arises from malignantly transformed melanocytes in the conjunctival epithelium. Current therapies using surgical excision in combination with chemo- or cryotherapy still have high rates for recurrences and metastatic disease. Lately, novel signal transduction-targeted and immune checkpoint inhibitors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein-1 (PD-1) receptor inhibitors, BRAF- or MEK-inhibitors for systemic treatment of melanoma have improved the outcome even for unresectable cutaneous melanoma, improving patient survival dramatically. The use of these therapies is now also recommended for CM; however, the immunological background of CM is barely known, underlining the need for research to better understand the immunological basics when treating CM patients with immunomodulatory therapies. Immune checkpoint inhibitors activate tumor defense by interrupting inhibitory interactions between tumor cells and T lymphocytes at the so-called checkpoints. The tumor cells exploit these inhibitory targets on T-cells that are usually used by dendritic cells (DCs). DCs are antigen-presenting cells at the forefront of immune response induction. They contribute to immune tolerance and immune defense but in the case of tumor development, immune tolerance is often prevalent. Enhancing the immune response via DCs, interfering with the lymphatic pathways during immune cell migration and tumor development and specifically targeting tumor cells is a major therapeutic opportunity for many tumor entities including CM. This review summarizes the current knowledge on the function of lymphatic vessels in tumor growth and immune cell transport and continues to compare DC subsets in CM with related melanomas, such as cutaneous melanoma and mucosal melanoma.