Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.

BACKGROUND: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning. RESULTS: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes). CONCLUSIONS: Therefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.

Tmem176B and Tmem176A are associated with the immature state of dendritic cells.

DCs play a central role in the development of innate and adaptive immunity but also in the induction and maintenance of immune tolerance. Identification of factors that govern DC activation, their maturation state, and their capacity to induce proinflammatory or tolerogeneic responses therefore represents a crucial aim of research. We previously identified a new molecule, Tmem176B (which we named TORID initially), as highly expressed in a model of allograft tolerance in the rat. We showed that its overexpression in rat DCs blocked their maturation, suggesting a role for this molecule in the maturation process. To characterize the function of Tmem176B further, we used a split-ubiquitin yeast, two-hybrid system to identify interacting partners and found that Tmem176B associated with itself but also with Tmem176A, a membrane protein similar to Tmem176B. Interestingly, these two molecules showed similar mRNA expression patterns among various murine tissues and immune cells and were both down-regulated following DC maturation. In addition, we showed that in using RNAi, these molecules are both involved in the maintenance of the immature state of the DCs. Taken together, these data suggest that Tmem176B and Tmem176A associate to form multimers and restrain DC maturation. Therefore, these two molecules may represent valid targets to regulate DC function.