T-cell receptors (TCRs) upon binding to peptide-MHC ligands transduce signals in T lymphocytes.Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (γδε) and ζ subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/ CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 ζ chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.

Macrophage subpopulations in the mouse express a lectin‐like receptor, sialoadhesin (originally named sheep erythrocyte receptor, SER), which selectively recognizes sialoglycoconjugates and is likely to be involved in cellular interactions of stromal macrophages in haematopoietic and lymphoid tissues. In this report we describe the purification and ligand specificity of sialoadhesin isolated from mouse spleen. Purified sialoadhesin, a glycoprotein of 185 kd apparent Mr, agglutinated sheep or human erythrocytes at nanomolar concentrations in a sialic acid‐dependent manner. Low angle shadowing and electron microscopy showed that sialoadhesin consisted of a globular head region of approximately 9 nm and an extended tail of approximately 35 nm. To investigate the specificity for sialic acid, we studied the interaction of sialoadhesin with derivatized human erythrocytes, glycoproteins, and glycolipids. In conclusion, sialoadhesin specifically recognizes the oligosaccharide sequence Neu5Ac alpha 2‐‐‐‐3Gal beta 1‐‐‐‐3GalNAc in either sialoglycoproteins or gangliosides. These findings imply that specific sialoglycoconjugates carrying this structure may be involved in cellular interactions between stromal macrophages and subpopulations of haematopoietic cells and lymphocytes.

The discovery of a targeted therapeutic compound along with its companion predictive biomarker is a major goal of clinical development for a personalized anticancer therapy to date. Here we present evidence of the predictive value of TLR3 expression by tumor cells for the efficacy of Poly (A:U) dsRNA in 194 breast cancer patients enrolled in a randomized clinical trial. Adjuvant treatment with double-stranded RNA (dsRNA) was associated with a significant decrease in the risk of metastatic relapse in TLR3 positive but not in TLR3-negative breast cancers. Moreover, we show the functional relevance of TLR3 expression by human tumor cells for the antitumor effects mediated by dsRNA in several preclinical mouse models carried out in immunocompromised animals. These 2 independent lines of evidence relied upon the generation of a novel tool, an anti-TLR3 antibody (40F9.6) validated for routine detection of TLR3 expression on paraffin-embedded tissues. Altogether, these data suggest that dsRNA mediates its therapeutic effect through TLR3 expressed on tumor cells, and could therefore represent an effective targeted treatment in patients with TLR3-positive cancers. Cancer Res; 71(5);