Richard Pospisil

CD5 Is A Potential Selecting Ligand for B-Cell Surface Immunoglobulin: A Possible Role in Maintenance and Selective Expansion of Normal and Malignant B Cells

Although the function of CD5 on B cells is unknown, previous studies suggested that CD5 interaction with VH framework regions of surface immunoglobulins (Igs) may contribute to survival and expansion of B cells. Here we used B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from normal and B-CLL patients to study CD5-Ig interactions. Immobilized Ig binds and permits isolation of CD5 from lysates of CD5-expressing cell lines. Immunoglobulins or Fab fragments of different VH families varied in their effectiveness as inhibitors of anti-CD5 staining of CLL cells, appendix and tonsil tissue sections. Human Ig also binds to purified recombinant CD5. We show here for the first time that the unconventional Ig-CD5 interaction maps to the extracellular CD5-D2 domain whereas conventional epitopes recognized by anti-CD5 antibodies are localized in the Dl domain of CD5. We propose that interactions of VH framework regions with CD5 as a ligand may maintain, select or expand normal, autoimmune or transformed B cells and also contribute to skewing of the normal VH repertoire.

Characterization of antigen-binding protein in earthworms Lumbricus terrestris and Eisenia foetida.

Injection of antigen into the annelid worms Lumbricus terrestris (LT) and Eisenia foetida (EF) results in a marked increase of coelomic fluid protein concentration and the formation of a protein which binds the stimulating antigen (3). In this report we show that the increases in total protein concentration after first and second doses of antigen were higher and were achieved earlier in LT than in EF, while the accumulation of antigen-binding protein in coelomic fluid was similar in both species. Antigen-binding protein isolated by affinity chromatography retained its original binding activity. Its molecular weight in coelomic fluid as well as after isolation was 56 kD when analyzed by SDS-PAGE and immunoblotting. Under reducing conditions, two bands with mol./wt. 31 and 33 kD appeared which did not reveal detectable binding activity. This suggests that the 56 kD binding protein of annelids is composed of two disulphide-linked polypeptide chains both of which participate in antigen-binding site formation.

Characterization of rabbit CD5 isoforms.

Previously described polyclonal or monoclonal antibodies (mAb) to rabbit CD5, raised against expressed recombinant protein or peptides, recognize CD5 on most rabbit B cells. The mAb KEN-5 was originally reported to recognize rabbit CD5. However, KEN-5 binds almost exclusively to T cells and only to a minor population of B cells. We show here that by Enzyme-linked Immunosorbent Assay (ELISA), KEN-5 binds to recombinant rabbit CD5. This interaction is partially inhibited by polyclonal goat anti-CD5 antibody. In addition, immunoprecipitations from lysates of surface biotinylated rabbit lymphocytes with KEN-5 or our anti-CD5 mAb isolate molecules that migrate identically on gels with the same approximate relative molecular mass of 67,000 M(r). By flow cytometric analyses of individual cells from spleen, thymus and appendix, KEN-5 recognizes CD5-like molecules mainly on T cells and on 3-6% of IgM(+) B cells. Immunohistochemical staining of splenic and appendix tissues and confocal immunofluorescent imaging confirm and extend results from flow cytometric analyses. Quantitation of fluorescent colocalization indicates that staining by KEN-5 colocalizes with staining by anti-CD5 on small percentage lymphocytes in splenic tissue sections. As CD5 has both N- and O-linked glycosylation, we hypothesised that differential binding of KEN-5 to T cells and B-cells may be explained by different glycan structures on the CD5 present on T compared to B cells. This hypothesis is supported by ELISA data that show that deglycosylation diminishes the binding of KEN-5 to recombinant rabbit CD5. Screening KEN-5 on an array with 406 glycans was inconclusive. Although we did not identify a strongly binding glycan structure, the data are suggestive that the epitope recognized by KEN-5 may be influenced by glycan structures. The epitope this mAb recognizes may either be the glycan itself, or more likely, is influenced by neighboring glycan structure. Our findings suggest that development, selection and function of different B- and T-cell subsets or their preferential survival may be directly or indirectly dependent on different glycan structures associated with CD5 or CD5-like molecules expressed on T cells compared to B cells.

Investigations of a Rabbit (Oryctolagus cuniculus) Model of Systemic Lupus Erythematosus (SLE), BAFF and Its Receptors

B-cell activation factor belonging to the tumor necrosis factor family (BAFF) is a major contributor to survival of B lymphocytes during development and maturation. A relationship between circulating BAFF levels and disease activity has been reported in patients with the autoimmune disease Systemic Lupus Erythematosus (SLE). Clinical trials targeting BAFF or its receptors are currently in progress. In order to further characterize a rabbit (Oryctolagus cuniculus) model of SLE, we investigated the expression of BAFF and its receptors in non-inbred, pedigreed rabbits derived from breeding and selection based on autoantibody responses. We immunized rabbits related to previous groups that developed autoantibodies and inflammatory responses after immunizations with peptides synthesized on multiple antigen-branched polylysine backbones. Blood and sera collected before immunization and after boosts were used for health monitoring, analyses of serum autoantibody responses by ELISA and immunofluorescence. Peripheral blood mononuclear cells (PBMC) were studied by flow cytometry and were the source of mRNA for quantitative PCR analyses. We hypothesized that BAFF mRNA expression and serum BAFF levels measured indirectly through BAFF receptor binding might increase in autoantibody-producing rabbits. Immunized rabbits developed elevated levels of leucocyte populations, anti-nuclear, anti-dsDNA and other autoantibodies. BR3 mRNA levels in total PBMC decreased and BAFF levels remained low and unchanged in most immunized rabbits. By flow cytometry, percentages of BAFF positive cells decreased. Percentages of transmembrane activator and CAML interactor (TACI) decreased in most rabbits from all the immunized groups. The rabbit is an important model for human autoimmune and infectious diseases, and a high quality draft rabbit genome assembly was recently completed. Human disease models developed in non-inbred pedigreed animals are better able to reflect the complexities of diseases such as SLE with familial patterns of inheritance. Although no consistent pattern of elevated expression of BAFF mRNA or protein was found in the rabbits studied, the data collected and reported here build upon previous data to refine understanding of a rabbit model of SLE.

Expression and localization of rabbit B-cell activating factor (BAFF) and its specific receptor BR3 in cells and tissues of the rabbit immune system

Rabbits are widely used for vaccine development, and investigations of human infectious and autoimmune diseases such as Systemic Lupus Erythematosus (SLE). For these applications, we cloned, sequenced and expressed rabbit B-cell Activating Factor (BAFF), and localized BAFF in cells and tissues of the rabbit immune system. The rabbit homolog of the human BAFF binding site (miniBR3 peptide) within the BAFF-specific receptor BR3 was synthesized. This 26-residue core domain binds to recombinant rabbit BAFF protein. Flow cytometric analyses using purified recombinant rabbit BAFF combined with real-time PCR findings revealed that BAFF detected on peripheral blood B-cells from normal rabbits is probably complexed to BAFF receptors rather than produced by the B-cells. BAFF was detected in developing appendix of young rabbits by immunohistochemical staining suggesting that BAFF plays a role during the period following birth when rabbit B-cell development and pre-immune antibody repertoire diversification and selection is occurring.