The lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II ligand evolutionarily related to CD4, is expressed exclusively in activated T and NK lymphocytes and seems to play a role in regulating the evolving immune response. We first determined that surface LAG-3 expression on activated human T cells is upregulated by certain cytokines (IL-2, IL-7, IL-12) and not by others (IL-4, IL-6, IL-10, TNF-alpha, TNF-beta, IFN-gamma). Surface LAG-3 expression correlated with intracellular IFN-gamma production in both CD4+ and CD8+ T-cell subsets. We then analyzed the 5' transcription control sequences of LAG-3. A DNase I hypersensitive site induced in T cells following cellular activation was found in the region including the transcriptional start site, showing that DNA accessibility is a mechanism which restricts LAG-3 expression to activated T cells. Transcription is initiated at three sites. A GC box, 80 base pairs (bp) upstream of the major transcription start site, forms a minimal promoter which is regulated by two upstream regions containing positive and negative regulatory elements with multiple protein binding sites as shown by footprinting analysis. In particular, a GATA/c-Ets motive was identified in a short segment homologous to the mouse CD4 distal enhancer, suggesting that LAG-3, which is embedded in the CD4 locus, may be controlled by some CD4 regulatory elements. Finally, a 100 bp region downstream of the transcription start site was shown to be involved in the cell-specific control of LAG-3 expression. Understanding this highly regulated expression may help to determine the intriguing role of this activation-induced MHC class II ligand.

The threshold, extent and termination of TCR activation is controlled in part by inhibitory co‐receptors expressed on activated T cells. The lymphocyte activation gene product (LAG‐3), a ligand for MHC class II molecules co‐caps with the CD3/TCR complex and inhibits cell proliferation and cytokine secretion in response to CD3 signaling. We first investigated whether LAG‐3 is localized in activated T cells in detergent‐resistant membrane rafts enriched in glycosphingolipids and cholesterol. We showed that both LAG‐3 and MHC class II are present in the cell fraction of glycosphingolipid‐rich complexes (GSL complexes) before the assembly of the immunological synapse by CD3/TCR complex cross‐linking. Using the LAG‐3 intracytoplasmic region as bait in the yeast two‐hybrid cloning system, we next identified a novel protein termed LAP for LAG‐3‐associated protein. LAP is encoded by a 1.8‐kb RNA message in lymphocytes and encodes a 45‐kDa protein that is expressed in most tissues. We showed that LAP binds specifically in vitro and in vivo to the Glu‐Pro (EP) repeated motif present in the LAG‐3 intracytoplasmic region. LAP also binds to the EP motif of another functionally important receptor, the PDGFR. Thus, LAP is a candidate molecule for a new type of signal transduction and/or coupling of clustered rafts to the microtubule networks that could explain how negative signaling of co‐receptors may occur through molecules devoid of any immunoreceptor tyrosine‐based inhibitory motif consensus sequence.

Previous studies indicated that signaling through lymphocyte activation gene-3 (LAG-3), a MHC class II ligand, induced by multivalent anti-receptor antibodies led to unresponsiveness to TCR stimulation. Here, lateral distribution of the LAG-3 molecules and its topological relationship (mutual proximity) to the TCR, CD8, CD4, and MHC class I and II molecules were studied in the plasma membrane of activated human T cells in co-capping experiments and conventional fluorescence microscopy. Following TCR engagement by either TCR-specific mAb or MHC-peptide complex recognition in T-B cell conjugates, LAG-3 was found to be specifically associated with the CD3-TCR complex. Similarly, following CD8 engagement LAG-3 and CD8 were co-distributed on the cell surface while only a low percentage of CD4-capped cells displayed LAG-3 co-caps. In addition, LAG-3 was found to be associated with MHC class II (i.e. DR, DP and DQ) and partially with MHC class I molecules. The supramolecular assemblies described here between LAG-3, CD3, CD8 and MHC class II molecules may result from an organization in raft microdomains, a phenomenon known to regulate early events of T cell activation.

SUMMARYWe recently described the inhibition of host B lymphocytes by Ixodes ricinus tick saliva. In this study, we characterized the factor responsible for this activity and examined the modulation of lipopolysaccharide (LPS)-and Borrelia burgdorferi outer surface protein (Osp)-induced proliferation of naive murine B lymphocytes by an enriched fraction of this factor. The B-lymphocyte inhibitory activity was destroyed by trypsin treatment, indicating that a proteinaceous factor was responsible for this activity. The removal of glutathione-S-transferase (GST) from tick salivary glands extracts (SGE) showed that this B-cell inhibitory protein (BIP) was not a GST. Gel filtration liquid chromatography indicated that BIP has a native molecular weight of » 18 000. An enrichment protocol, using a combination of anion-exchange and reverse-phase liquid chromatography, was established. BIP-enriched fractions did not suppress T-cell proliferation. Delayed addition of BIP-enriched fractions, up to 7 hr after LPS addition, inhibited the proliferation of isolated B cells. BIP-enriched fractions dramatically inhibited both OspA-and OspC-induced proliferation of isolated B cells. These results strongly suggest that BIP may facilitate B. burgdorferi transmission by preventing B-cell activation, and also highlights the potential of BIP as a therapeutic agent in B-cell maladies.