C. I. Mockridge

Mechanisms and clinical significance of BIM phosphorylation in chronic lymphocytic leukemia

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIM(EL) and BIM(L), in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIM(EL) only. In CLL, anti-IgM-induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIM(EL) serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIM(EL)/BIM(L) phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIM(EL) phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.

Surface IgM of CLL cells displays unusual glycans indicative of engagement of antigen in vivo

Surface IgM (sIgM) has a key influence on the clinical behavior of chronic lymphocytic leukemia (CLL). We now report that it exists in 2 forms with different N-glycosylation patterns in the mu-constant region. One glycoform is similar to normal B cells in bearing mature complex glycans common to most cell-surface glycoproteins. The other is an immature mannosylated form more characteristic of mu chains in the endoplasmic reticulum. Unmutated CLL (U-CLL) expresses a higher proportion of mannosylated surface mu chains than mutated CLL. Normal B cells express only the mature glycoform but can express the immature form after persistent engagement of sIgM, suggesting that glycan modification is a consequence of antigen exposure. CLL cells express variable proportions of the mannosylated form and can revert to the mature form after incubation in vitro. Both glycoforms are able to signal after sIgM engagement in vitro, leading to enhanced tyrosine phosphorylation. These findings support the concept that CLL cells are continuously exposed to antigen in vivo, driving the N-glycosylation pattern of expressed sIgM toward a mannosylated form, especially in U-CLL. Strikingly, this is reminiscent of follicular lymphoma, where mannosylated Ig is expressed constitutively via N-glycosylation sites in the variable region, suggesting a functional asset for this glycoform.

Disturbances in peripheral blood B cell subpopulations in autoimmune patients.

A variety of cell surface markers are being used to identify B cell subpopulations in peripheral blood. Currently at least eight subpopulationshave been identified. Analyses of healthy individuals indicate that in general the various B cell subpopulations exist in relatively similar ratios in unrelated individuals. It has been demonstrated that B lymphocyte homeostasis is disturbed during infection and autoimmune disease. In this review we compare the distribution of B cell subpopulations in the peripheral blood of patients with systemic lupus erythematosus, rheumatoid arthritis and primary Sjogrens syndrome with each other, and with healthy individuals. The different autoimmune diseases have distinct changes in the B cell subpopulations. Understanding the nature of these B subpopulation signatures will potentially impact understanding the mechanisms of disease, diagnosis and therapy.

Tracking of the V4–34 (VH4–21) gene in human tonsil reveals clonal isotype switch events and a highly variable degree of somatic hypermutation

The V4–34 (VH4–21) gene has been found to encode certain IgM autoantibodies, and is mandatory for pathological IgM anti‐erythrocyte antibodies of I/i specificity. The gene is also commonly used by normal IgM‐positive B lymphocytes, but its involvement in B cells which have undergone class switching to IgG or IgA is less clear. In order to track V4–34 gene usage and class switching events during a normal immune response, we have probed RNA in a limited area of human tonsil. Results indicate that the V4–34 gene undergoes class switching to IgG or IgA, with the progeny either remaining unmutated or containing large numbers of somatic mutations. Mutational patterns indicate possible ‘hot spots’, and some mutations appear deleterious. At the level of individual B cells, we have tracked a clonal isotype switch event from IgM to IgA, with each retaining close to germ‐line configuration. In addition, we have followed a clonal switch from a mutated IgM to IgG, with no further accumulation of somatic mutations. These data indicate that the V4–34 gene is involved in a maturing immune response, and that the routes to production of IgG or IgA antibodies are various.

Upregulation of FcγRIIb on monocytes is necessary to promote the superagonist activity of TGN1412

The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (PBMCs) do not respond to soluble TGN1412 but do respond following high-density (HD) preculture. We show for the first time that this response is dependent on crystallizable fragment gamma receptor IIb (FcγRIIb) expression on monocytes. This was unexpected because, unlike B cells, circulating monocytes express little or no FcγRIIb. However, FcγRIIb expression is logarithmically increased on monocytes during HD preculture, and this upregulation is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell FcγRIIb expression is unchanged by HD preculture, but B cells can support TGN1412-mediated T-cell proliferation when added at a frequency higher than that in PBMCs. Although low-density (LD) precultured PBMCs do not respond to TGN1412, T cells from LD preculture are fully responsive when cocultured with FcγRIIb-expressing monocytes from HD preculture, which shows that they are fully able to respond to TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by FcγRIIb is critical for the superagonist activity of TGN1412 after HD preculture, and this may contribute to CRS in humans because of the close association of FcγRIIb-bearing cells with T cells in lymphoid tissues.

Sequence analysis of V4–34‐encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE)

SLE is an autoimmune disease characterized by the presence of autoantibodies against double‐stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V4–34 heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti‐dsDNA antibodies. In order to probe the nature of the V4–34‐encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single‐cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both VH and VL genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For VH, all were derived from V4–34 as expected, and the isotype‐switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, VL (12 κ and 3 λ) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated VH sequences were associated with unmutated VL sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity‐determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of VH ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the VL–JL junctions in 4/15. These findings provide insight into the V4–34–VL gene combinations used by B cells in patients with SLE which might have clinical relevance.

Anti-mouse FcγRIV antibody 9E9 also blocks FcγRIII in vivo.

To the editor: Monoclonal antibodies (mAbs), such as the anti-CD20 mAb rituximab, have transformed the treatment of malignant disease and are now a first-line treatment of many hematologic conditions. Although in many cases their precise mechanism of action is not fully elucidated, where target