Lymphocyte activation gene-3 : the expression and function in the immune system

Lymphocyte activation gene-3 (LAG-3) is the structural homologue of a well-known TCR co-receptor CD4. Recently, it has been suggested that LAG-3 might play a role in modulation of immune response, by being a negative regulator of T cell activation. To further characterize its expression and function in the immune system, monoclonal Abs were generated against mouse LAG-3. The availability of different mAbs allowed the identification of LAG-3 on various immune subpopulations. The main finding of this work is that, unlike previously thought, LAG-3 expression is not limited to activated T and NK cells, but can also be induced on B cells and DCs. B cells were found to express LAG-3 in a T cell dependent manner; LAG-3 was expressed on B cells in the presence of activated T cells, but not upon stimulation with thymus independent stimuli like CpG or LPS. Furthermore, requirements for LAG-3 induction on B cells were defined; this event requires B cells proliferation and is mediated by a soluble factor released by activated T cells. Among DCs, two populations, CD8- (‘myeloid’) and plasmacytoid DCs, expressed high levels of surface LAG-3 upon stimulation with LPS and CpGs, respectively. To evaluate the potential role of LAG-3 in APC function, LAG-3 deficient and OT-II transgenic mice were used. Antigen presentation assay showed, that LAG-3 presence on the surface of APC can affect T cell responses. T cells stimulated with OVA peptide presented by LAG-3 deficient B cells proliferated poorly compared to T cell stimulated with OVA-pulsed LAG-3 positive B cells. This finding suggests a novel co-stimulatory function of LAG-3 on APCs. In addition, experiments re-evaluating published data concerning the role of LAG-3 on T cells were performed. In agreement with previous reports, the ectopic expression of LAG-3 on the surface of T cell line exerted an inhibitory effect on T cell activation induced by relevant antigen. However, in disagreement with recent report (by Huang et al. 2004), the standard regulatory cell assays performed with normal as well as LAG-3 deficient CD4+CD25+ cells, suggested no involvement of LAG-3 in regulatory T cell function. Furthermore, the experiments demonstrating the surface expression on murine CD4 and CD8 T cells in vivo are presented. LAG-3 was identified on homeostatically expanding T cells after transfer into lymphopenic mice. To investigate structural aspects of the LAG-3 molecule, soluble LAG-3 (sLAG-3), composed of four extracellular domains was produced. For this purpose Drosophila expression system was employed. The conditions were developed for production of two forms of sLAG-3, the original glycosylated form and its non-glycosylated version. Biochemical analysis of soluble protein showed that in solution LAG-3 forms stable dimers, which suggests the presence of such dimers on cell surface. Because heterogenous glycosylation of WT form likely prevents protein crystallization, attempts are now made to crystallize non-glycosylated form of sLAG-3 alone or in complex with its ligand, MHC class II. The detailed structural information would significantly enhance our knowledge about LAG-3 and allow for rational drug design, if desired.