MOLECULAR REQUIREMENTS FOR ANTIBODY CLASS SWITCHING

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Our long-term objectives are to elucidate the mechanistic requirements, B cell stage specificity and functional properties of the DNA recombinase system responsible for antibody (Ab) class switching. Our novel strategy is largely based on the use of immunoglobulin (Ig) heavy chain constant (CH) region gene switch (S) regions as recombination substrates engineered in retroviruses. Amphotropic retroviruses harboring a variety of S region substrates will be introduced into a variety of switch- competent B cells: 300-18 pre-B and 1.29 mature B cell lines, EBV- immortalized germinal center B cells, long-term cultures of differentiated Ab secreting B cells, stimulated splenic and Peyer's patch B cells. The recombinagenic effects of mitogenic (LPS) and differentiation (LPS and II-4) stimuli on the B cell specific recombination of S substrate retroviruses will be determined. Genetic techniques with counter-selectable retroviruses harboring a chimeric hygromycin-thymidine kinase (Hytk) gene will be employed in attempts to isolate B cells which have up and down-modulated their constitutive switch-recombinase activity. Such switch-variant lines would be invaluable not only for deciphering recombinase regulation but also as potentially critical reagents for cloning the recombinase activity. In a second aim, the role of BSAP/NFSmu-B1 (a B cell specific transcription/switch region binding factor) in switch-recombination will be investigated. The recombinagenic activity of high affinity BSAP binding sites inserted in S substrates and the affects of establishing and inhibiting BSAP activity on recombinase function will be determined in various B cell backgrounds. In a third aim, BSAP accessory or partner proteins which could modulate its DNA binding site specificity, affect S region accessibility and/or represent components of the switch- recombinase will be cloned by screening lambda-gt11 cDNA expression libraries of recombinase positive B cells with a glutathione S- transferase-BSAP fusion protein. Finally, a gene rescue-selection strategy will be developed to clone genes encoding B cell specific components of the switch-recombinase. A mammalian expression vector cDNA library prepared from recombinase positive B cell mRNA will be introduced by liposome-mediated transfection into a recombinase negative, antibody secreting hybridoma line, A39Rgamma1.1tk-, harboring a recombinationally insert switch-retrovector. Bromodeoxyuridine selection will permit the isolation of rare transfectants which have lost S-retrovector thymidine kinase gene function via switch-deletion mediated by the fusion of flanking Smu and Sgamma2b sequences. These BudR resistant cells would be candidate recipients of switch-recombinase activating genes, which would greatly facilitate their cloning and functional characterization. These objectives will enable us to decipher the molecular basis and developmental regulation of a key process in the maturation of antibody mediated immune responses yielding a variety of essential effector cell specificities.