Description): The goal of the investigators is to elucidate the mechanisms that restrict the diversity of the perinatal antibody repertoire and to gain insight into the role these mechanisms play in limiting the response of the infant to infection and to vaccination. In both human and mouse, the ability to respond to antigen develops in a stepwise, programmed fashion which is mirrored by a parallel restriction in antibody diversity. One focus of regulation is a limitation in the structure (length) of the third H chain complementarity determining region (HCDR3). This region forms the center of the antigen binding site and plays a critical role in antigen specificity. Human fetal B cell progenitors restrict the length of HCDR 3 by preferential use of short DH and JH elements and by control of N region addition. Although by the time of birth these restrictions may no longer be apparent in VDJC-mu transcripts, gamma H chain transcripts may continue to show a fetal HCDR3 pattern. The investigators propose to evaluate the development of HCDR3 length distributions in first and second trimester fetal tissues, the cord blood of preterm infants, and the peripheral blood of normal and septic pre-term infants, while taking into account serum IgG concentrations and immunization schedules. In order to elucidate mechanisms that underlie control of HCDR3 length, they will create gene targeted mice wherein the most JH and DH gene segment (DFL16.1) has been lengthened by 6 codons, prejudicing the repertoire towards structures seen in human, but not mice. These alterations should prejudice initial VD rearrangement towards generations of long HCDR3 intervals while retaining physiologic hydropathicity profiles. This mutated gene will also be inserted into the DH locus of mice wherein the most JH proximal DH gene segment (DQ52) has been replaced by a loxP site. In these mice, cre-mediated recombination will remove the intervening DH gene segments, forcing sole use of mutated DFL16.1, while leaving the rest of the H chain locus intact. HCDR3 length distribution will be monitored during B cell development in neonates (which lack N nucleotides) and 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 lambda5 knock-out mice. Finally, the ability of the mutant mice to respond to model T-independent (PC and DEX) and T-dependent (NP) antigens, and to challenge with Streptococcus pneumoniae will be assessed. This comprehensive examination of the normal development of systemic immunity might yield significant fundamental knowledge for the study of susceptibility to infectious diseases and of responsiveness to vaccines.
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