Lck-interacting membrane protein (LIME), one of TRAPs, was previously identified as a binding partner of Lck. LIME expression is mostly confined to hematopoietic cells such as T, B and mast cells. In T cells, in response to TCR stimulation, LIME is ty ...
Lck-interacting membrane protein (LIME), one of TRAPs, was previously identified as a binding partner of Lck. LIME expression is mostly confined to hematopoietic cells such as T, B and mast cells. In T cells, in response to TCR stimulation, LIME is tyrosine phosphorylated by Lck and associates with signaling proteins such as Lck, PI3K, Grb2, Gads, and SHP-2. As LIME overexpression led to the MAPK and IL-2 promoter activation, LIME was suggested as the positive regulator of TCR signaling. However, the functions of LIME in other hematopoietic cells than T cells have not been clarified yet.In the first part of this thesis, I describe characterization of the functions of LIME using B cell line and show that LIME is a transmembrane adaptor required for BCR-mediated B-cell activation. LIME was found to be expressed in mouse splenic B cells. Upon BCR-cross-linking, LIME was tyrosine-phosphorylated by Lyn and associated with Lyn, Grb2, PLC-γ2, and PI3K. Reduction of LIME expression by the introduction of siRNA resulted in the disruption of BCR-mediated activation of MAPK, calcium flux, NF-AT, PI3K, and NF-kB. Taken together, these results establish that LIME is an essential transmembrane adaptor linking BCR ligation to the downstream signaling events that lead to B-cell activation.
Having established the biochemical functions of LIME in B cell line, in the second part, I was interested in investigating the immunological functions of LIME at the animal level. For this purpose, I characterized the functions of B cells from LIME-/- mice. In LIME-/- mice, B cell development was found as normal from bone marrow to periphery. BCR-mediated early activation signaling was only marginally affected and B cell activation by several mitogens including BCR stimulation was not affected in LIME-/- B cells suggesting that LIME is not involved in the activation of naïve B cells. Subsequently, LIME expression was found to increase markedly in GC B cell compared to naïve B cells by T cell-dependent signals, CD40+ IL-4. Moreover, immunoglobulin levels of LIME-/- mice immunized with T-dependent (TD) antigen but not T-independent (TI) antigen were significantly reduced compared to wild type mice. These results suggest that LIME functions in the germinal center after help from T cells.
To clearly show that the observed defect in TD-antigen induced B cell response is due to intrinsic defect of LIME-/- B cells but not defect in the help from T cells, RAG2-/- mice was used as the host of wild type or LIME-/- B cells. In RAG2-/- mice transferred with LIME-/- B cells, antigen-specific immunoglobulin production was significantly diminished compared to those transferred with wild type B cells. Consistently, TD antigen-specific plasma cell number was dramatically reduced in RAG2-/- mice transferred with LIME-/- B cells. Moreover, the number and percentage of antigen-specific plasma cells were dramatically reduced in RAG2-/- mice transferred with LIME-/- B cells. These findings show that the absence of LIME led to the defect either in the differentiation to or the function of antigen-specific plasma cells and this defect is intrinsic to B cells. However, TD-antigen-dependent germinal center formation and germinal center reaction including affinity maturation and class switching occurred normally in LIME-/- mice. In addition, the number and percentage of Ag-specific GC B cells and memory B cells were normal in LIME-/- mice suggesting that LIME function is confined specifically in the plasma cells.
In summary, I defined LIME as a transmembrane adapter mediating BCR-induced activation signaling and a key regulator of TD antigen-derived differentiation to and function of plasma cells in germinal center.