Conventional understanding of CD4 T cell development is that the MHC class II molecules on cortical thymic epithelial cell are necessary for positive selection, as demonstrated in mouse models. Clinical data, however, show that hematopoietic stem cells reconstitute CD4 T cells in patients devoid of MHC class II. Additionally, CD4 T cells generated from human stem cells in immunocompromised mice were restricted to human, but not mouse, MHC class II. These studies suggest an alternative pathway for CD4 T cell development that does not normally exist in mice. MHC class II is expressed on developing human thymocytes, indicating a possible role of MHC II on thymocytes for CD4 T cell generation. Therefore, we created mice in which MHC class II is expressed only on T lineage cells. Remarkably, the CD4 compartment in such mice is efficiently reconstituted with unique specificity, demonstrating a novel thymocyte-driven pathway of CD4 T cell selection.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly 149 of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly 149 is a necessary prerequisite step for release of the protein.

Vimentin, an intermediate filament protein induced during epithelialto-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin β3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin β3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin β3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.

The gene LamC2 encoding the γ2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the α3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75–90%) was seen in the CD25+CD44+ and CD25+CD44−subsets of the CD4−CD8− double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin α3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.