Galectin-9 bridges human B cells to vascular endothelium while programming regulatory pathways.

Humoral immunity is reliant on efficient recruitment of circulating naïve B cells from blood into peripheral lymph nodes (LN) and timely transition of naive B cells to high affinity antibody (Ab)-producing cells. Current understanding of factor(s) coordinating B cell adhesion, activation and differentiation within LN, however, is incomplete. Prior studies on naïve B cells reveal remarkably strong binding to putative immunoregulator, galectin (Gal)-9, that attenuates BCR activation and signaling, implicating Gal-9 as a negative regulator in B cell biology. Here, we investigated Gal-9 localization in human tonsils and LNs and unearthed conspicuously high expression of Gal-9 on high endothelial and post-capillary venules. Adhesion analyses showed that Gal-9 can bridge human circulating and naïve B cells to vascular endothelial cells (EC), while decelerating transendothelial migration. Moreover, Gal-9 interactions with naïve B cells induced global transcription of gene families related to regulation of cell signaling and membrane/cytoskeletal dynamics. Signaling lymphocytic activation molecule F7 (SLAMF7) was among key immunoregulators elevated by Gal-9-binding, while SLAMF7's cytosolic adapter EAT-2, which is required for cell activation, was eliminated. Gal-9 also activated phosphorylation of pro-survival factor, ERK. Together, these data suggest that Gal-9 promotes B cell - EC interactions while delivering anergic signals to control B cell reactivity.

Analysis of Galectin-Binding Receptors on B Cells.

The reported roles of the ß-galactoside-binding lectin family, known as galectins, in disease development have been advancing at a remarkable pace. Galectins and their glycan counter-receptor ligands are now considered key functional determinants in malignant and metastatic progression, tumor immune evasion, autoimmunity, and immune homeostasis. Their influence in these processes is elicited through coordinated expression in tumor, immune and stromal cellular compartments. While analysis of galectin levels in related research efforts is routinely performed through immunoassays and RT-qPCR, detection, and identification of glycan counter-receptor ligands in their native form on the cell surface has lagged. In this report, we present methods to detect and identify glycan counter-receptor ligands to galectin (Gal)-3 and Gal-9-two galectins at the crosshairs of cancer and immunology research. As a model, we will describe (1) isolation of human B-cell subsets from fresh tonsil tissue, (2) assaying of Gal-3/-9-binding activities on human B cells, and (3) identifying Gal-3/-9 ligands on human B-cell surfaces. These methods, of course, can be implemented on any cell type to provide a cellular and molecular context capable of transmitting a galectin-mediated phenotype. Establishing a galectin-binding activity on specific counter-receptor ligand(s) can help unearth potential critical determinants capable of delivering cellular signals required for disease progression. These advances open new avenues of research investigation that result in novel therapeutic targets and approaches.