J. B. Natvig

Bm1-Bm5 classification of peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and disturbance in the B cell subpopulations in patients with primary Sjogren's syndrome

Analyses of B cells in the bone marrow and secondary lymphoid tissues have revealed a broad range of cell surface markers defining B cell subpopulations, but only a few of these have been used to analyze B cell subpopulations in peripheral blood (PB). We report here the delineation of circulating PB B cell subpopulations by staining for CD19, CD38, and IgD in combination with CD10, CD44, CD77, CD95, CD23, IgM, and the B cell memory marker CD27. The utility of this approach is shown by the demonstration of disturbances of circulating B cell subpopulations in patients with autoimmune disease. Five mature B cell (Bm) subpopulations were identified in normal PB that were comparable with the tonsillar Bm1, Bm2, early Bm5, Bm5 subpopulations and, surprisingly, to the germinal center (GC) founder cell subpopulation (Bm2′ and Bm3δ–4δ), suggesting that some GC founder cells are circulating. No PB B cells resembled the Bm3 and Bm4 GC cells. Remarkably, some cells with the CD38−IgD+ phenotype, previously known as naive Bm1 cells, expressed CD27. The CD38−IgD+ subpopulation therefore includes both naive Bm1 cells and IgD+ memory B cells. This new classification of B cell developmental stages reveals disturbances in the proportions of B cell subpopulations in primary Sjögren’s syndrome (pSS) patients compared with healthy donors and rheumatoid arthritis patients. Patients with pSS contained a significantly higher percentage of B cells in two activated stages, which might reflect a disturbance in B cell trafficking and/or alteration in B cell differentiation. These findings could be of diagnostic significance for pSS.

Rheumatoid Inflammatory T‐Cell Clones express mostly Th1 but also Th2 and Mixed (Th0‐Like) Cytokine Patterns

This study was performed in order to characterize whether T cells from rheumatoid synovial inflammation belong to the Th1‐ or Th2‐like functional subsets. Cytokine production was studied in 26 CD4+αβ+ and 2 CD8+αβ T‐cell clones from the synovial fluid, the synovial membrane and peripheral blood of 5 patients. Fifteen of the CD4+ clones were raised against various mycobacterial antigens and 11 CD4+ clones and 2 CD8+ clones were raised unspecifically using PHA and/or IL‐2. The specificities of these clones are not known. In the mycobacterial antigen‐specific group, all CD4+’αβ T‐cell clones produced IFN‐γ at high levels, while the production of IL‐4 was generally absent or low (< 1 ng/ml), consistent with a Thl‐like profile. Some of these clones, however, also produced various amounts of IL‐10 which has been regarded as a Th2 product but can be produced also in lower amounts by Thi cells. One HSP‐65‐specific clone produced levels of IL‐4 and IL‐10 in the same order as that of IFN‐γ, thus appearing to be Th0‐like. Among the 11 unspecific CD4+ clones, 7 showed a Thl‐like pattern but with lower levels of IFN‐γ than the antigen‐specific clones. However, three clones did not produce any IFN‐γ activity but produced IL‐4 and one of them also produced distinct amounts of IL‐10, compatible with a Th2‐like pattern. In addition, one of the clones also showed an almost equally strong IFN‐γ and IL‐4 production, thus most likely representing a Th0‐like clone.

Elution and Characterization of Lymphocytes from Rheumatoid Inflammatory Tissue

Lymphocytes were eluted from the synovial tissue of 19 patients with classical rheumatoid arthritis. The tissue was minced and dissociated by treatment with crude collagenase and DNase, The cell suspension obtained was filtered and incubated in plastic culture flasks overnight at 37°C, The cells that did not adhere to the plastic surface were harvested and the lymphocytes further purified by the Ficoll‐Isopaque gradient centrifugation technique. The lymphocyte yield varied from 0.64 to 32 × 106 cells. Differential counts showed on the average 85% lymphocytes, 12% macrophage‐like cells, and variable proportions of polymorphonuclear granulocytes, unclassified cells, and dead cells. An average of 77% of the cells were viable as assessed by the trypan blue exclusion test. This cell suspension was investigated for lymphocyte populations, T lymphocytes were predominant in all experiments (mean, 73.6%), The mean percentage of B lymphocytes was 9.7%, whereas the proportion of Fc‐receptor‐bearing lymphocytes was on the average 6.0%,

Same Idiotypc of B‐Lymphocyte Membrane IgD and IgM. Formal Evidence for Monoclonality of Chronic Lymphocytic Leukemia Cells

Idiotypic antisera were raised in rabbits against serum M‐cnmponents in two patients with chronic lymphocytic leukemia (CLL) In one of the patients the leukemic cells had membrane‐bound IgG. in the other IgM and IgD. The M‐components were IgG and IgM. respectively. By immunofluorescence technique the CLL cells were shown to be positive when stained with idiotypic antiserum prepared against the corresponding M component, whereas normal lymphocytes, as well as cells from other CLL patients, were negative. These findings represent strong evidence for the monoclonality of the malignant B cells in CLL The fact that the CLL cells had membrane‐bound Ig with the same idiotypic determinants as the corresponding serum M‐component indicated that the latter had been synthesized and secreted by the malignant clone of B cells. In further experiments redistribution of membrane‐bound Ig on CLL cells bearing both IgM and IgD was induced with idiotypic antiserum This indicated that IgM and IgD on the same cells share idiotypic specificity and therefore have the same variable region and. presumably, the same antibody specificity

Augmented numbers of HLA-DR-positive T lymphocytes in the synovial fluid and synovial tissue of patients with rheumatoid arthritis and juvenile rheumatoid arthritis: in vivo-activated T lymphocytes are potent stimulators in the mixed lymphocyte reaction.

Twenty to 40 % of T cells from synovial fluid and synovial tissue and 3–11 % of the peripheral blood T lymphocytes from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) expressed HLA‐DR antigens as detected by monoclonal anti‐HLA‐DR antibodies in direct immunofluorescence. Synovial fluid and synovial tissue T lymphocytes had a stimulating capacity comparable to that of non‐T cells in the allogeneic primary mixed lymphocyte reaction (MLR). The MLR was inhibited by monoclonal anti‐HLA‐DR antibodies. This is, to our knowledge, the first report on in‐vivo‐activated T lymphocytes as stimulator cells in MLR. The results suggest that T cells from synovial fluid and synovial tissue are locally activated by stimuli so far unidentified.

Isolation and Characterization of Amyloid‐Related Serum Protein SAA as a Low Molecular Weight Protein

With direct immunoprecipitation or gel filtration under dissociating conditions, amyloid‐related serum protein SAA has been isolated as a low molecular weight protein from the serum of two patients with rheumatoid arthritis but without known amyloidosis. The isolated protein SAA showed antigenic identity and an amino acid composition that was similar, but not identical, with isolated fibril protein AA., Molecular weight estimations suggest that protein SAA is approximately 50%, larger than protein AA and has a molecular weight of 14,000–15,000 daltons Preliminary results indicate that protein SAA from a patient with amyloidosis has a similar small molecular weight subunit.

In situ characterization of mononuclear cells in rheumatoid tissues, using monoclonal antibodies. No reduction of T8-positive cells or augmentation in T4-positive cells.

The reactivity of monoclonal mouse anti-human antibodies specific for mononuclear cell surface antigens were studied by the indirect immunofluorescence technique in frozen synovial tissue sections from patients with rheumatoid arthritis (RA). Most of the proliferating synovial lining cells were positive for HLA-DR antigens and monocyte-specific antigens, since they reacted with the OKIa1 and OKM1. Cells positive for HLA-DR and monocyte antigens were also seen scattered or in small nests in the synovial stroma, probably representing synovial cells or monocytes/macrophages. Some of the HLA-DR-positive cells may also be B lymphocytes or activated T cells. Endothelial cells were also HLA-DR antigen-positive. Monoclonal antibodies with specificity for all T cells (OKT3), for helper/inducer cells (OKT4), and for suppressor/cytotoxic cells (OKT8) reacted with cells often located in follicle-like structures around vessels. Cells with the T4 phenotype tended to be located in the centre of the follicles, whereas the T8 positive cells were more peripherally situated. In most instances fewer cells were positive for the T8 than for the T4 marker. In some instances there was as many T8-positive cells as T4-positive cells. Complete lack of T-lymphocyte subpopulations was not seen.

Human Monoclonal Antibodies against Blood Group Antigens Preferentially Express a VH4‐21 Variable Region Gene‐Associated Epitope

An anti‐idiotypic antibody has been raised which recognizes human immunoglobulins with cold agglutinin activity of anti‐I/i specificity. The pattern of reactivity of the antibody indicates that the structural basis for the epitope is located in the VH4‐21 gene segment of the VHIV family, which is preferentially utilized by these cold reactive antibodies.

Evidence of Similar Idiotypic Determinants on Different Rheumatoid Factor Populations

Strong idiotypic antigens were detected both in monoclonal and polyclonal rheumatoid factors (RF) by applying idiotype‐specific antisera. Shared idiotypic antigens were found between some polyclonal and monoclonal RF as well as between certain IgG and IgM RF.

Nucleotide Sequence Analysis of Rheumatoid Factors and Polyreactive Antibodies derived from Patients with Rheumatoid Arthritis Reveals Diverse Use of VH and VL Gene Segments and Extensive Variability in CDR‐3

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogrens syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain ‘restrictions’ do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.