CD81 interacts with the T cell receptor to suppress signaling

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling

The role of CD3 delta interacting proteins in T-Cell receptor assembly and signaling

The signals initiated from the T cell receptor (TCR)-CD3 complex receptors are not only important for the correct functioning but also for the differentiation and survival of T cells. While CD3 chains play an essential role in the assembly of and signaling by the TCR and preTCR complexes, the logic behind the redundancy of these signaling subunits has not been addressed. In this study, we aimed to identify novel proteins that interact with the CD3delta subunit of the TCR complex. We performed a membrane based split ubiquitin yeast two hybrid screen. This novel genetic screening system allows the identification of interaction partners of membrane-associated proteins. We used the CD3delta chain as bait and screened a Jurkat cell cDNA library to identify CD3 interacting proteins. We confirmed the in-vivo relevance of these interactions by co-immunoprecipitation in human tissue culture cell lines. We further examined the functional significance of two CD3delta interacting proteins: BCAP31 and CD81.We demonstrate that BCAP31, previously shown to associate with B cell receptor, interacts with CD3delta in the endoplasmic reticulum (ER) and plays a role in TCR assembly. We also identified the tetraspanin superfamily member CD81, previously shown to have a role in early T cell development, as a CD3delta interaction partner. In order to investigate the functional significance of this interaction, we silenced CD81 expression in T lymphocyte cell lines by shRNA mediated expression arrest and performed functional assays. We demonstrate that CD81 is important for TCR/CD4 co-receptor mediated signaling of T lymphocytes, possibly inhibiting the initiation of TCR signals

Cryopreservation of spleen and lymph nodes as a source of mononuclear cells to be used for the development of monoclonal antibody producing hybridoma cells

In the present study, spleen and lymph nodes of mice were cryopreserved as a whole tissue and after thawing, membrane integrity of mononuclear cells was determined by trypan blue exclusion and PI staining. T and B lymphocytes, macrophages and dendritic cells have been isolated from both cryopreserved tissue and analyzed by Flow cytometry. BALB/c mice were immunized with Hepatitis e antigen (HBeAg) and spleen and lymph nodes of mice were cryopreserved for 3 to 10 months. The cells obtained from both tissue were applied to hybridoma technology to understand if the cells keep their viability and functionality. The cells were isolated and fused with F0 mouse myeloma cells and several antibody producing hybrid cells were developed. Results have shown that cryopreserved spleen and lymph nodes of mice can be efficiently used in hybridoma technology for the successful generation of monoclonal antibody producing hybrid cells