T-cell migration across the blood-brain barrier is a crucial step in the pathogenesis of EAE, an animal model for MS. Live cell imaging studies demonstrated that P-selectin glycoprotein ligand-1 (PSGL-1) and its endothelial ligands E-and P-selectin mediate the initial rolling of T cells in brain vessels during EAE. As functional absence of PSGL-1 or E/P-selectins does not result in ameliorated EAE, we speculated that T-cell entry into the spinal cord is independent of PSGL-1 and E/P-selectin. Performing intravital microscopy, we observed the interaction of WT or PSGL-1 −/− proteolipid protein-specific T cells in inflamed spinal cord microvessels of WT or E/P-selectin −/− SJL/J mice during EAE. T-cell rolling but not T-cell capture was completely abrogated in the absence of either PSGL-1 or E-and P-selectin, resulting in a significantly reduced number of T cells able to firmly adhere in the inflamed spinal cord microvessels, but did not lead to reduced T-cell invasion into the CNS parenchyma. Thus, PSGL-1 interaction with E/P-selectin is essential for T-cell rolling in inflamed spinal cord microvessels during EAE. Taken together with previous observations, our findings show that T-cell rolling is not required for successful T-cell entry into the CNS and initiation of EAE.Keywords: Blood-brain barrier r Experimental autoimmune encephalomyelitis r P-selectin r P-selectin glycoprotein ligand-1 r T-cell rolling Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionIn MS, and in its animal model, EAE, neuro-Ag-specific CD4 + (where CD is cluster of differentiation) T cells breach the bloodbrain barrier (BBB) and gain access to the CNS, where they cause inflammation, demyelination, and BBB breakdown leading to the clinical manifestation of these diseases. T-cell migration across the BBB is a multi-step process mediated by the sequential interaction of different adhesion and/or signaling molecules on the T cells and Correspondence: Prof. Britta Engelhardt e-mail: bengel@tki.unibe.ch the highly specialized endothelium of the BBB [1]. The discovery that α4β1-integrin plays a critical role in T-cell trafficking across the BBB during EAE has led to the development of a humanized monoclonal anti-α4-integrin Ab (natalizumab) for the treatment of MS [2].In vivo live cell imaging studies have provided direct evidence that the multistep extravasation of encephalitogenic T cells across the spinal cord microvasculature during initiation of EAE is unique [3]. Initiation of T-cell interaction with the spinal cord microvascular endothelium takes place in the absence of rolling and is rather mediated by α4-integrin-mediated G-protein-independent capture and subsequent G-protein-dependent arrest of the T cells in spinal cord microvessels [3]. Once neuroinflammation iswww.eji-journal.eu 2288 Karthik Sathiyanadan et al. Eur. J. Immunol. 2014. 44: 2287-2294 ongoing, it is mostly T-cell rolling with a few alternative capture events also detected, which characterize th...

1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of l-alanine over its canonical substrate l-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from l-serine toward l-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2–S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position. Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-014-8339-1) contains supplementary material, which is available to authorized users.

Preclinical data shows that intravesical instillation of Ty21a/Vivotif®, a commercial vaccine against typhoid fever, is an effective alternative option to standard Bacillus-Calmette-Guérin (BCG) immunotherapy for nonmuscle-invasive bladder cancer (NMIBC). Here we characterized the inflammatory effects of Ty21a on the bladder and investigated the immune mechanisms underlying tumor-regression towards the use of this bacterial vaccine in NMIBC patients. MB49 bladder tumor-bearing mice had significantly improved survival after intravesical instillations of Ty21a doses of 10 6 to 10 8 colony-forming units. By immunohistochemistry and morphology, both BCG and Ty21a instillations were associated with bladder inflammation, which was decreased with the use of low, but ,effective doses of Ty21a. Flow cytometry analysis showed a significant infiltration of T cells, natural killer (NK) cells, and myeloid cells, compared with controls, after a single dose of Ty21a, whereas this was only observed after multiple doses of BCG. The induced myeloid cells were predominantly neutrophils and Ly6C + CD103 + dendritic cells (DC), the latter being significantly more numerous after instillation of Ty21a than BCG. Ex vivo infection of human leukocytes with Ty21a, but not BCG, similarly significantly increased DC frequency. CD4 + and CD8 + T cells, but not NK cells nor neutrophils, were required for effective bladder tumor regression upon Ty21a treatment. Thus, the generation of antitumor adaptive immunity was identified as a key process underlying Ty21a-mediated treatment efficacy. Altogether, these results demonstrate mechanisms behind intravesical Ty21a therapy and suggest its potential as a safe and effective treatment for NMIBC patients.

Connexin37 (Cx37) and Cx40 form intercellular channels between endothelial cells (EC), which contribute to the regulation of the functions of vessels. We previously documented the participation of both Cx in developmental angiogenesis and have further shown that loss of Cx40 decreases the growth of different tumors. Here, we report that loss of Cx37 reduces (1) the in vitro proliferation of primary human EC; (2) the vascularization of subcutaneously implanted matrigel plugs in Cx37−/− mice or in WT using matrigel plugs supplemented with a peptide targeting Cx37 channels; (3) tumor angiogenesis; and (4) the growth of TC-1 and B16 tumors, resulting in a longer mice survival. We further document that Cx37 and Cx40 function in a collaborative manner to promote tumor growth, inasmuch as the injection of a peptide targeting Cx40 into Cx37−/− mice decreased the growth of TC-1 tumors to a larger extent than after loss of Cx37. This loss did not alter vessel perfusion, mural cells coverage and tumor hypoxia compared to tumors grown in WT mice. The data show that Cx37 is relevant for the control of EC proliferation and growth in different tumor models, suggesting that it may be a target, alone or in combination with Cx40, in the development of anti-tumoral treatments.

Bacillus Calmette-Guérin (BCG) instillations for the treatment of non-muscle-invasive bladder cancer patients can result in significant side effects and treatment failure. Immune checkpoint blockade and/or decreasing tumor-infiltrating myeloid suppressor cells may be alternative or complementary treatments. Here, we have characterized immune cell infiltration and chemoattractant molecules in mouse orthotopic MB49 bladder tumors. Our data show a 100-fold increase in CD45+ immune cells from day 5 to day 9 tumors including T cells and mainly myeloid cells. Both monocytic myeloid-derived suppressor-cells (M-MDSC) and polymorphonuclear (PMN)-MDSC were strongly increased in day 9 tumors, with PMN-MDSC representing ca. 70% of the myeloid cells in day 12 tumors, while tumor associated macrophages (TAM) were only modestly increased. The kinetic of PD-L1 tumor expression correlated with published data from patients with PD-L1 expressing bladder tumors and with efficacy of anti-PD-1 treatment, further validating the orthotopic MB49 bladder-tumor model as suitable for designing novel therapeutic strategies. Comparison of chemoattractants expression during MB49 bladder tumors grow highlighted CCL8 and CCL12 (CCR2-ligands), CCL9 and CCL6 (CCR-1-ligands), CXCL2 and CXCL5 (CXCR2-ligands), CXCL12 (CXCR4-ligand) and antagonist of C5/C5a as potential targets to decrease myeloid suppressive cells. Data obtained with a single CCR2 inhibitor however showed that the complex chemokine crosstalk would require targeting multiple chemokines for anti-tumor efficacy.