The goal of this application is to elucidate one of the key mechanisms for antibody repertoire diversification, namely, regulation of the composition of the third complementarity determining region of the H chain (HCDR3). HCDR3 forms the center of the antigen binding site and controls antigen specificity. In the human fetus, HCDR3 is limited by preferential use of DHQ52 and control of N region addition. Through evaluation of DJ transcripts and rearrangements, the hypothesis that these differences are the product of two distinct progenitor populations, on common in the fetus and the other in the adult, will be tested. CD34+CD19- cells from fetal liver, and from the cord blood of pre-term and term infants will be examined and the stage at which the pattern changes to the one found in the adult will be determined. The second hypothesis holds that charged or hydrophobic HCDR3s are inherently deleterious and subject to negative selection. In human and mouse each of the six DH reading frames (rfs) has a characteristic hydropathicity signature - charged, hydrophobic, or neutral. Although the mechanisms may differ between species, the final product is almost always neutral. The functional significance of this preference will be tested by mutating the JH distal DH gene segment, DFL16/1. First, an inverted DSP2.2 gene segment will be embedded into DFL16.1 rf 1 in charged orientation. Second, single nucleotide insertions at the ends of DFL16.1 will force use of hydrophobic rf2. These mutants will targeted into BALB/c ES cells that contain a loxP site next to JH. Through cre-mediated recombination, the intervening DH gene segments will be deleted, forcing sole use of the mutated DFL16.1 element. These alterations should prejudice initial VDJ rearrangement towards generation of DCR 3 intervals with charged or hydrophobic amino acids, respectively, while maintaining normal patterns of VH rearrangement. If use of hydrophobic or charged HCDR3 is deleterious, then during B-cell development there should be diminution of VDJ rearrangements with the altered HCDR3s. B-cell development will be monitored in neonates (which lack N nucleotides) and in adult mice. In order to further elucidate the role of: a) N region addition; and b) the pre-B-cell receptor complex in regulating the diversity of HCDR3, the mutant mice will be crossed with TdT -/- and l5 -/- mice. Finally, the ability of the mutant mice to respond to T-independent (PC and DEX) and T-dependent (NP) antigens and to challenge with Streptococcus pneumoniae will be assessed. This analysis should yield insight into the mechanisms that regulate antigen receptor repertoire diversification, a process that influences susceptibility to infectious and autoimmune diseases, responsiveness to vaccines, and recovery after bone marrow transplant.
