Ziva Misulovin

Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V (D)J -recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2 (ref. 4). We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.

A monoclonal antibody to the DEC-205 endocytosis receptor on human dendritic cells ☆

DEC-205 is a multilectin receptor for adsorptive endocytosis, expressed in mouse dendritic cells (DC) and some epithelia. DEC-205 is homologous to the macrophage mannose receptor (MMR). A cDNA for murine DEC-205 was used to identify 3 overlapping human DEC-205 clones from a lymphocyte library. The human homologue is a transmembrane protein of 1722 aminoacids with 10 externally disposed C-type lectin domains having 77% identity to the mouse counterpart. The NH(2) terminal cysteine-rich and fibronectin type II domains were expressed and used to immunize mice. A hybridoma, MG38, which specifically recognized the immunogen was obtained from a DEC-205 knockout mouse. The antibody precipitated a 205 kD protein from metabolically labeled, monocyte-derived DCs. MG38 labeled mature monocyte-derived DCs but showed weak or no labeling of other peripheral blood mononuclear cells. In tissue sections, MG38 identified DEC-205 on thymic cortical epithelium and DCs in the thymic medulla and tonsillar T cell areas. In contrast, an anti-MMR antibody stained DEC-205 negative, macrophages in the thymus cortex, the trabeculae of the thymus and tonsil, as well as efferent lymphatics in the tonsil. Therefore, the MG38 anti-DEC-205 antibody is useful for identifying DCs and reveals clear differences in sites where MMR and DEC-205 are expressed in lymphoid tissues.

AID from bony fish catalyzes class switch recombination

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

A rapid method for targeted modification and screening of recombinant bacterial artificial chromosome

Modification of bacterial artificial chromosomes (BACs) has been a useful method to produce genomic DNA fragments for studying gene expression and function in vitro and in vivo. The original technique involved restrictions for BAC modification and required multiple cloning steps to target sequences into the shuttle vector. Selection and screening of BAC recombinants was accomplished by drug resistance and Southern blotting. We have developed a PCR-based method for producing the modified shuttle vectors and for screening for BACs carrying homologous integrants. The combination of these techniques allows for rapid and easy targeted BAC sequence deletion or insertion.