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Cord blood and fetal liver B cells were immortalized using Epstein-Barr virus, and IgM antibodies from the resulting lines and clones were examined for their binding to a variety of auto-antigens and micro-organisms by ELISA and fluorescence assays. Auto-antigens tested included Fc of IgG, ssDNA and dsDNA, cardiolipin, histones 1-4, collagens type I and II, thyroglobulin, cytoskeletal components, and a tissue section screen. Of 71 cell lines tested, all but 19 showed some autoreactivity. All 32 fetal liver lines reacted to some self-antigens. In cord blood clones, 16 out of 26 bound to auto-antigens. Many of the clones reacted with more than one auto-antigen and were 'polyreactive'. Some of the cord blood clones bound to extracts of micro-organisms, showing specificity for both endogenous and exogenous antigens. The high frequency of CD5+ B cells in the cord blood (greater than 50%) and fetal liver (greater than 70%) argues for many of these clones being derived from this subset. Therefore, our data support the concept that many 'early' B cells produce polyreactive IgM which can bind to a variety of different auto-antigens and micro-organisms. These IgM antibodies are similar to those described by others as 'natural antibodies'.

We have examined the frequencies of T gamma delta cells in blood, synovial fluids, and synovial membranes of patients with rheumatoid arthritis (RA) and in blood from age-matched controls. Immunocytochemical and immunohistochemical techniques were used with monoclonal antibodies BB3 and A13 to define a major and minor blood subset of T gamma delta cells respectively. Together, these antibodies identify the majority (if not all) of the peripheral blood T gamma delta cells. Significantly lower levels of T gamma delta cells were found in the blood of RA patients compared with controls, whilst higher but not significant numbers were found in the synovial fluids of paired samples. Scattered T gamma delta cells were found only in some synovial membranes with a distribution similar to the T alpha beta cells. Analysis of the two different T gamma delta-cell subsets indicated a ratio of BB3 to A13 of about 5:1 in control and RA blood. However, this ratio was less than 1:1 in the RA synovial fluids and membranes. The migratory nature of the A13+ cells could account for their predominance in these sites. The possible pathological significance of these cells in the rheumatoid synovial fluid and synovial membranes is discussed.

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The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein-Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population.

The influence of genetic factors on the expression of CD5+ B lymphocytes and their relationship to a broad spectrum of autoantibodies was investigated in a study of 12 patients with rheumatoid arthritis (RA) and 52 of their healthy first-degree relatives.

During the past few years, heterogeneity within B lymphocyte populations has become increasingly apparent. Several studies have demonstrated functional, physiological, and developmental heterogeneity. Studies in the mouse suggested that two separate lineages of B cells may exist (1) . One lineage, is defined by cell surface expression of the Ly-1 antigen, characteristic of T lymphocytes, and the secretion of polyspecific antibodies reactive with a variety of self antigens. The second lineage lacks the Ly-1 antigen and produces antibodies, mainly, to exogenous antigens (1). It is not clear, however, if the distinction is also reflected in antibody variable region gene use by the Ly-1 + and Ly-1 -B cell subsets. This question can be approached by analysis ofvariable region gene segment rearrangements, at the DNA level or through serological markers expressed on the protein products.In humans, the majority of B lymphocytes in fetal liver, cord blood, and a minority in adult peripheral blood and lymph nodes express surface CD5 molecule, the human equivalent of mouse Ly-1 (2) . 109In the present study, we have used a number ofmAbs that recognize cross-reactive idiotypes (CRI)t associated with individual V or V, gene family products to examine IgV gene use in CD5+ and CD5 -B lymphocytes . Materials and MethodsPreparation and Sorting of B Lymphocytes. B Lymphocytes were enriched from heparinized blood of three full-term donors by centrifugation over Ficoll-Hypaque and rosetting using SRBCs (3). The enriched B cell populations were stained simultaneously with fluorescein-conjugated anti-CD20 (clone Leu-16) and PE-labeled anti-CD5 (clone Leu-1) and sorted using a FACStar® (Becton Dickinson Immunocytometric Systems, Mountain View, CA). Lymphocytes displaying green fluorescence only (CD20', CD5 -cells) and those displaying green and red fluorescence (CD20', CD5' cells) were gated and sorted as CD5 -and CD5+ B cells, respectively.'Abbreviation used in this paper: CRI, cross-reactive idiotypes.J . Exp. Med .