ROLE OF BLYS IN PERIPHERAL B CELL SELECTION AND SURVIVAL

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DESCRIPTION (provided by applicant): The tumor necrosis factor family member, BLyS, plays a key role in the homeostasis and selection of peripheral B cells. The relative roles played by expansion, survival, and differentiation rates in these effects, as well as how BLyS influences cells within each peripheral maturation subset, remains unknown. Through studies of normal and mutant mice, we have shown that BLyS controls peripheral B cell numbers in at least two ways: by varying the proportion of cells that successfully complete transitional development, and by serving as the primary determinant of longevity among mature B cells. The proposed studies will further examine the mechanisms through which BLyS influences peripheral B cell differentiation, selection, and lifespan. In the first aim, we will assess whether BLyS mediated effects solely reflect enhanced survival, or if they also involve direct influences on differentiation. We will address this question through cytofluorimetric and in vivo labeling studies of Bcl-xl transgenic mice homozygous for the A/WySnJ defect, as well a analysis of hosts reconstituted with either A/WySnJ or normal stem cells transfected with viral constructs containing Bcl-xl. We have recently discovered that BLyS receptor expression shifts with maturation and can be regulated by BcR signaling, thus linking BLyS-mediated survival with BcR-driven selection. In the second aim, we will examine BLyS receptor expression and BLyS-responsiveness in all immature and mature B cell subsets following treatment in vitro with anti-Ig or antigen. In addition, we will further characterize BcR-mediated BLyS receptor expression with kinetic and dose response studies, as well as studies where the potential coupling of other Ig isotypes or costimulatory molecules with Bcmd/BR3 expression is assessed. In the third aim, We will examine how the coupling of BcR and Bcmd/BR3 is related to the requisite of BcR expression for mature B cell viability, and for successful transitional cell maturation. We will determine whether ectopically expressed Bcmd/BR3 affords B cell survival in mice where BcR expression is conditionally ablated, and whether BcR ablation yields down-regulation of Bcmd/Br3. We will also determine whether constitutive Bcmd/BR3 expression thwarts transitional selection; whether the degree of BcR stimulation required for B cell maturation can be altered by varying available BLyS levels. In the fourth aim, we will examine how Bcmd/BR3 influences clonotype diversity and composition in the emerging repertoire responsive to influenza hemagglutinin (HA), through limiting dilution and fine specificity analyses of HA specific responses in A/J vs. A/WySnJ mice.