Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. In man and mouse, V-gene diversification by somatic hypermutation in germinal centers of secondary lymphoid organs leads to affinity maturation. In previous studies we showed that gene conversion and somatic hypermutation both occur in rabbit splenic germinal centers (GC) during specific immune responses. Rabbits were immunized to make classical anti-DNP antibody responses in order to study clonal VH and VL region diversification during antibody responses known to exhibit affinity maturation. The development of different potential heavy and light chain pairs through gene conversion that affects amino acids in complementarity determining regions (CDRs) may account for the rabbit's known ability to produce heterogeneous high affinity anti-DNP antibodies. In order to determine whether antibody affinity increases in clonally related sequences that undergo gene conversion, we are employing phage display to analyze Fab from individual germinal centers. Antigen-specific splenic germinal centers were identified by immunohistochemistry and Leica LMD microdissection was used to recover individual germinal centers from serial tissue sections of spleens of immunized rabbits (2). Generation and analyses of phage display libraries from individual germinal centers are in progress. The young rabbit appendix is a dynamic primary lymphoid organ where a broad B cell preimmune repertoire is produced mainly by a gene conversion-like process. The variety of combining sites generated within individual clones from 3 to 5 weeks of age (3) suggest that some clonal expansion and selection, known to require gut flora, may be driven through indirect effects of microbial components (superantigens) rather that solely by their recognition as specific foreign antigens. This may also include interactions between B cell receptor framework regions and endogenous superantigens such as CD5. Although the function of CD5 on B cells is unknown, our studies in the rabbit, suggested that CD5 interaction with VH framework regions of surface immunoglobulins may contribute to survival and expansion of B cells. A detailed analysis of the development and selection of rabbit appendix B lymphocytes is in progress. In normal rabbits 3 days after birth, B cells start to form small follicles. As detected by flow cytometry and immunohistochemistry they are CD79a positive and express IgM but lack the CD5 antigen. By day 8, appendix B cells start to express CD5 and by 2 weeks, the majority of appendix B cells are CD5 positive. These findings contrast to the spleen and PBL where most B cells (IgM+ CD79a+) already express CD5 3 days after birth. There are differences between the kinetics of B cell development in normal compared to mutant ali/ali rabbits. Since mutant ali/ali rabbits lack normal VH framework regions associated with the deleted VH1a2 gene, their B cell development is delayed. B-cell expansion seems to correlate with development of B cells that have undergone gene conversion and consequent expression of VH framework regions more similar to those encoded by the missing VH1a2 gene. B cell development is not uniform in all appendix follicles of ali/ali rabbits. VHa2+ B cells start to develop in some follicles but not in the others. Once VHa2 positive follicles develop, their B cells also start to up-regulate CD5. B cell growth and expansion in such follicles is also accompanied by apoptotic death suggesting that both positive and negative selection processes are affecting B cell repertoire formation. Developmental switches in glycosylation distinguish three populations of B cells in the chicken bursa of Fabricius (new immigrants, gene converted, and post bursal exiting). We are investigating whether comparable defined stages of development can be identified by studies of cell-surface glycosylation. In particular the appearance of CD15 (Lewis-x), CD15s (Sialyl Lewis-x ), CD15u (Sulfated Lewis-x) and of CD5 on surfaces of developing B cells is being followed during development in appendix and spleen. In normal 1 week-old rabbits CD15s was found on 10% of B cells. The percentage declined gradually to 2% by 4 weeks of age. These may represent newly immigrating B cells in the early phase of appendix development. Comparisons of normal with ali/ali rabbits, in which B cell development is delayed, showed low levels of CD15s+ B cells compared to age matched normal a2/a2 rabbits and a delay in appearance of CD15+ B cells (2.6% and 43% at 1 and 4 week of age in ali/ali compared to 36.3% and 67.7% in a2/a2 rabbits). We have been further studying whether the membrane microdomains, termed lipid rafts, might play a role in BCR signalling during B cell development in appendix follicles. The earliest event of a signal transduction cascade involves the phosphorylation of the immunoreceptor tyrosine based activation motifs of Ig alpha and Ig beta by the src family kinase Lyn in lipid rafts. It has been proposed that the BCR in resting cells is excluded from rafts and that after antigen binding and oligomerization, the BCR translocates into rafts, where it is phosphorylated by Lyn, initiating a signaling cascade. We have detected CD79a, VHa2 immunoglobulin molecules and Lyn in rafts isolated from splenocytes as early as 3 days after birth. BCR translocation into rafts is delayed in the appendix and starts around 1 week of age, at about the same time as B cells start to up regulate the expression of CD5.
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