Immunoglobulin Genetics-Ontogeny Cell Differentiation

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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. We asked whether gene conversion, somatic hypermutation or 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. Individual cells from splenic germinal centers were collected by micromanipulation. The rearranged genes for antibody heavy and light chains in single cells were PCR-amplified and sequenced. The changes at the DNA level that may lead to affinity maturation occur by both gene conversion and somatic hypermutation (2). 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. We speculate that if cells with sequences altered by gene conversion or receptor revision that no longer react with the immunizing antigen 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 (1). 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 (1)