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

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Abstract

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. Rearranged heavy and light chain genes 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). Rabbits were immunized to make classical anti-DNP 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 determined DNA sequences of rearranged VH and VL in single cells from developing antigen-specific GC at several time points after immunization. The changes at the DNA level that may lead to affinity maturation occur by both gene conversion and somatic hypermutation (D. Sehgal et al., 2000). In the course of the 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. We have shown the development of different potential heavy and light chain pairs through gene conversion that affects amino acids in complementarity determining regions (CDRs). Our observations help to account for the rabbit's known ability to produce heterogeneous high affinity anti-DNP antibodies. We speculate that if cells with sequences altered by gene conversion or receptor revision that no longer react with the immunizing Ag are a by-product of the GC reaction, the rabbit splenic or human tonsillar GC could produce new members of the B-cell repertoire and thus play a role in adults similar to that of the gut associated lymphoid tissues of young rabbits (R. Mage et al, 1999; D. Seghal et al., 2000).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 (R. Pospisil et al, 2000; R. Mage et al., 2000).