LV, Lymphatic vessel; BV, Blood vessel, E, epithelium. the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection. transmigration of activated T cells (6) and monocytes (7, 8) across EC monolayers. Moreover, ALCAM on DCs interacting with T cell-expressed CD6 was shown to provide T cell co-stimulation (9). In line with the latter findings, two recent studies reported that ALCAM-deficient (ALCAM?/?) mice are partially protected from T cell-mediated inflammation in murine models of asthma (10) and food allergy (11). In ECs ALCAM was shown to mediate migration, tube formation and barrier function of blood vascular and lymphatic ECs (LECs) (2, 12, T863 13). Moreover, our group recently demonstrated a role for ALCAM in the formation of both vascular networks (12, 14) and in tumor angiogenesis (14), whilst another study reported that ALCAM regulates the integrity of the blood brain barrier (13). Given the involvement of ALCAM in leukocyte trafficking, (lymph)angiogenesis, and the induction of T cell-mediated immune responses, therapeutic blockade of ALCAM with monoclonal antibodies could represent a promising approach for treating immune-mediated inflammatory disorders. A pathologic condition that involves all of the above-mentioned processes is allograft rejection. Corneal allografts are among the most commonly transplanted tissues and are typically well tolerated (15, 16). Under normal conditions the cornea is avascular due to the expression of potent anti-(lymph)angiogenic factors (15, 16). However, the presence of inflammation-induced neovascularization in the recipient’s cornea prior to transplantation is nowadays well recognized to significantly increase the risk of allograft rejection (17C19). Under such pre-vascularized conditions, blood vessels mediate leukocyte recruitment, and lymphatic vessels provide the exit routes for alloantigen-presenting dendritic cells (DCs), which migrate to draining lymph nodes to induce T-cell mediated allograft rejection (15, 16). Particularly the presence of inflammation-induced lymphatic vessels in the recipient cornea was shown to significantly increase the risk of corneal allograft rejection (17C19). In this study we reformatted a previously described single-chain variable fragment (scFv) antibody with blocking activity toward human ALCAM (20) into a bivalent Fc fusion protein (I/F8-Fc) and validated its ability to bind and block murine T863 ALCAM and (lymph)angiogenis. (A,B) A cell-free scratch was introduced into T863 confluent monolayers of (A) human LECs or (B) HUVECs and the impact of I/F8-Fc or KSF-Fc control antibody on VEGF-A-induced scratch closure was analyzed after 24 and 12 h, respectively (C) Blocking ALCAM with I/F8-Fc reduced tube formation of human LECs. (D,E) A cell-free scratch was introduced into confluent monolayers of (D) murine MS-1 cell or (E) murine primary dermal LECs and the impact of I/F8-Fc on scratch closure was analyzed after 24 and 27 h, respectively. Data from 1 out of 3 to Rabbit polyclonal to IkB-alpha.NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA (MIM 164014), or RELB (MIM 604758) to form the NFKB complex.The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA or NFKBIB, MIM 604495), which inactivate NF-kappa-B by trapping it in the cytoplasm. 4 4 similar experiments are shown in (ACE). (FCI) Effects on T cell activation. WT or ALCAM?/? BM-DCs were pulsed with OVA peptide in presence of LPS and co-incubated with CD4+ OTII cells in presence of I/F8-Fc or KSF-Fc control antibody. (F) FACS analysis demonstrating ALCAM and CD6 expression in BM-DCs and OTII cells, respectively. (G,H) Impact of I/F8-Fc treatment on T cell proliferation. (G) Representative FACS plots showing CFSE-dilution, as a readout of T cell proliferation. (H) Quantitation of proliferating cells. (I) T cell-mediated IFN- production was quantified in the cell culture supernatants. Data from 1 out of 4 similar experiments (= 6 replicates) are shown in F-I. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM. ALCAM Blockade Reduces T Cell Activation studies revealed that ALCAM supports T cell activation by binding to the costimulatory molecule CD6 (9). In a competition ELISA T863 I/F8-Fc T863 significantly and dose-dependently reduced murine CD6-Fc binding to plate-bound murine ALCAM (Figures S3A,B). We next performed DC-T cell co-culture assays involving CD4+ T cells isolated from TCR-transgenic OTII mice (22) and WT or ALCAM?/? bone marrow-derived DCs (BM-DCs) pulsed with peptide derived from ovalbumin (OVA) (Figures 2FCI). FACS analysis confirmed constitutive expression of CD6 in CD4+ OTII T cells and of ALCAM in BM-DCs (Figure 2F). Similarly to two recent reports (10, 11), we found that proliferation (Figures 2G,H) and IFN- production (Figure 2I) of OTII T cells were significantly reduced in co-cultures involving ALCAM?/? DCs. Moreover, treatment of co-cultures involving WT DCs with I/F8-Fc reduced T cell proliferation and IFN- production to similar levels as observed in co-cultures.