IG GENETICS--ONTOGENY AND DIFFERENTIATION OF CELLS OF THE RABBIT IMMUNE SYSTEM

为了产生针对a的保护性抗体

In order to develop protective antibodies against a wide range of potentially infectious pathogens, the young rabbit must diversify a limited initial repertoire by somatic mechanisms (the high copy number primary repertoire). The majority of rabbit B cells produce heavy-chain variable regions by rearranging the VHa allotype-encoding VH1 gene. Thus in normal rabbits the majority of serum immunoglobulins bear VHa allotype (due to VH1 FR1 and FR3 sequences). The young rabbit appendix is a site of diversification of rearranged VH genes by gene-conversion-like and somatic hypermutation mechanisms. The newly generated B cells probably undergo selection processes that involve self and foreign antigens and superantigens. We find preferential expansion and survival of B cells in normal and VH-mutant ali / ali rabbits based on their heavy chain FR1 and FR3 sequences (VHa allotype). This selection may involve "superantigen"-like interactions with endogenous as well as exogenous ligands (Pospisil and Mage 1998 a,b,c). 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. We extended our investigations of CD5-Ig interaction to human B cells using B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from B-CLL patients. CD5+ B cells develop early in ontogeny and are maintained throughout life by self-renewal. By flow cytometry, human IgG binds CD5+CD19+ B cells and this interaction can be inhibited by anti-CD5 antibodies. Immobilized immunoglobulin isolates CD5 molecules from lysates of CD5 expressing cell lines. Human immunoglobulin binds to purified recombinant CD5. The binding maps to the CD5-D2 domain whereas CD5 epitopes recognized by monoclonal antibodies are localized in the D1 domain. Immunoglobulins of different VH families demonstrated different effectiveness as inhibitors of anti-CD5 staining of CLL cells and appendix and tonsil tissue sections. We propose that interactions of VH framework regions with CD5 may maintain, select or expand autoimmune or transformed B cells and also contribute to skewing of the normal human VH repertoire [ (Pospisil and Mage 1998 a,b,c) and R. Pospisil, G. J. Silverman, G. E. Marti and R.G. Mage, "CD5 on Normal and Malignant Human B Lymphocytes is a Potential Selecting Ligand for B-Cell Surface Immunoglobulin" (manuscript in preparation)]. 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. Rabbits were immunized to make classical anti-DNP hapten antibody responses in order to study clonal VH and VL region diversification in splenic germinal centers during antibody responses known to exhibit affinity maturation. Individual cells from germinal centers were collected by micromanipulation. The rearranged genes for antibody heavy and light chains in single cells were PCR-amplified and sequenced. We asked whether gene conversion, somatic hypermutation or both occur in rabbit splenic germinal centers (GC) during responses to a protein antigen (F1 capsular antigen of Yersinia pestis) or to the hapten dinitrophenol (DNP). We determined DNA sequences of rearranged VH and VL in single cells from developing DNP-specific GC at several time points after immunization with DNP-BGG. The changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation (Mage, 1998 and Schiaffella E, Sehgal D, Anderson AO and Mage RG "Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits", ms in preparation). In the course of the above study of anti-DNP responses, we found adult splenic B cells with rearranged VDJ that were germline or close to germline in sequence. This challenged the attractive idea that the rabbit, like the chicken develops its B cell repertoire early in life and depends upon self renewing cells in the periphery to maintain its B lymphocyte pool throughout life. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. Although a major population of B lymphocytes may be generated early in life, diversified extensively and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although in rabbits considerable repertoire diversity is generated in the neonatal period, mechanisms for continued generation of B cell receptor diversity are retained in adult life where they may confer survival advantage (Mage, 1998 and Sehgal D, Schiaffella E, Anderson AO, and Mage, RG "Analyses of Single B Cells by PCR Reveals Rearranged VH with Germline Sequences in Spleens of Immunized Adult Rabbits: Implications for B Cell Repertoire Maintenance and Renewal" ms submitted for publication).