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The B cell Ag-receptor complex is composed of membrane immunoglobulin (mIg) and the mb-1/B29 heterodimer. In order to obtain insight into the architecture of the B cell receptor complex, we have looked for conditions that disrupt all disulfide bridges in the complex without affecting the noncovalent interaction between the mIg heavy chain and one or both members of the associated heterodimer. We show that in the presence of the reducing agent beta-mercaptoethanol the m mu, m delta, and m gamma heavy chains remain selectively associated with the B29 members. Our findings implied that if isotype-related differences exist between the mIg-associated dimers, they may reside in B29 and not, as initially suggested, in mb-1. However, sequence analyses of B29 gene transcripts from B cells expressing mIgM, mIgD, or mIgG only revealed no differences in their nucleotide composition. Thus, in spite of their close physical interaction with mIg heavy chain classes, which are significantly distinct in the C-terminal regions, no isotype-specific forms of B29 seem to exist.

The diagnosis severe combined immunodeficiency was made in a male infant at the age of 18 wk. Known causes of severe combined immunodeficiency were excluded. The activity of total 5'-nucleotidase (E.C. 3.1.3.5) in the PBMC was found to be strongly decreased. Analysis of the peripheral blood revealed a lymphocytosis, mainly of CD8+ T cells. These lymphocytes expressed high levels of CD29, CD38, CD45RA, and MHC class II molecules but no CD25, CD26, CD27, or CD28 Ag. The cells proliferated poorly to all T cell stimulants tested and no helper activity for IgM secretion could be induced. In contrast to the poor proliferative responses, high levels of TCR-induced cytolytic activity, without lymphokine-activated killer-cell outgrowth, were induced by CD3 mAb. Analysis of TCR-beta gene rearrangements indicated that two clonal populations constituted the majority of the E-rosette+ peripheral blood fraction. Moreover, the vast majority of the CD8+ cells were found to react with a mAb to V beta 3. Polymerase chain reaction on cDNA from peripheral blood cells with primers that amplify TCR V beta elements showed, in agreement with the fluorescence data, an overrepresentation of V beta 3 but absence of usage of approximately 50% of the other V beta elements. Thus, in a severe combined immunodeficiency patient, CD8+ T cells with limited T cell receptor usage and restricted effector functions were found. The observed alterations in the 5'-nucleotidase levels may be secondary to the outgrowth of this population.

In a man aged 56 years with dysphagia, an oesophageal biopsy was found to contain an adenocarcinoma. In view of the discrepancy between the clinical and the histological pictures, the biopsy was repeated; in a second biopsy no carcinoma was demonstrable. DNA microsatellite analysis proved that the first biopsy originated from another person. The scheduled oesophageal resection was cancelled and the patient was reassured. A woman aged 77 years underwent gastrectomy because of biopsy samples with adenocarcinoma. However, no tumour was found in the resected stomach. DNA microsatellite analysis showed that the biopsy samples indeed originated from the patient. Unfortunately, mix-up of patient specimens occasionally occurs. Especially a discrepancy between the clinical picture and the histological diagnosis must raise the suspicion of a specimen mix-up. In such cases, DNA microsatellite analysis can give a rapid and reliable answer whether a mix-up has indeed taken place

The expansion of follicular lymphomas (FLs) resembles, both morphologically and functionally, normal germinal center B-cell growth. The tumor cells proliferate in networks of follicular dendritic cells and are believed to be capable of somatic hypermutation and isotype switching. To investigate the relation between somatic mutation and heavy chain isotype expression, we analyzed the variable heavy (V(H)) chain genes of 30 FL samples of different isotypes. The V(H) genes of the FLs were heavily mutated (29.3 mutations on average). In addition, isotype-switched lymphomas contained more somatic mutations than immunoglobulin M-positive lymphomas (33.8 mutations per V(H) gene versus 23.0, respectively). In all but one of the FLs, the ratios of replacement versus silent mutations in the framework regions were low, independent of the absolute number of somatic mutations and the level of intraclonal variation. Analysis of relapse samples of 4 FLs showed no obvious increase in somatic mutation load in most FLs and a decrease in intraclonal variation in time. In 3 of 4 cases, we obtained evidence for selection of certain subclones, rather than clonal evolution. Our findings question if intraclonal variation is always a reflection of ongoing somatic hypermutation. This may have implications for the concept of antigen-driven lymphomagenesis. (Blood. 2000;95:2922-2929)

B chronic lymphocytic leukemia (B-CLL) are clonal populations of mIgM+ or mIgM+/mIgD+ CD5+ B cells that appear to be arrested in the follicular mantle-zone B-cell stage. Functional analyses have shown two groups of B-CLL that can be distinguished based on their capacity to proliferate in response to B-cell antigen receptor complex (BCR) cross-linking. To investigate the molecular basis for this phenomenon, we have analyzed both architecture and functional properties of BCR complexes on these two groups of B-CLL. Both groups were found to express structurally similar BCR. However, protein tyrosine kinase (PTK) activity associated with and specific for BCR constituents was strongly diminished in nonresponsive B-CLL. Moreover, the PTK-dependent assembly of Shc/Grb2 complexes, which may couple the BCR to p21ras, was absent in these B-CLL. Finally, of all PTKs tested, the expression of PTK syk was found to be considerably lower in nonresponsive B-CLL. Thus, absence of mitogenic responses upon BCR cross-linking in particular B-CLL was found to be strictly correlated with diminished induction of BCR-associated PTK activity and lower levels of PTK syk. Because nonresponsive B-CLL closely resembles tolerant autoreactive B cells both functionally and biochemically, distinction between B-CLL with respect to functional properties in vitro may be determined by differences in antigen encounter in vivo

Surface expression of membrane(m) IgM molecules requires the association of two disulphide-linked transmembrane (TM) glycoproteins, encoded by the B-cell-specific genes mb-1 and B29. We have shown that mIgM, mIgD and mIgG are associated with structurally related heterodimers on primary human B cells. Transfection studies in murine plasmacytoma cells, however, have demonstrated mb-1-independent expression of both mIgD and mIgG. The recent finding that mIgD is expressed on these cells through glycosyl-phosphatidylinositol (GPI) linkage may be interesting in view of the function of mIgD on primary B cells. We therefore investigated whether GPI linkage serves as an additional mechanism for expression of mIgD and the other mIg isotypes on primary human B cells. However, we were unable to demonstrate the release of mIg molecules upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) in either immunofluorescence analysis or Ig heavy (H) chain-specific enzyme-linked immunosorbent assay (ELISA). We conclude that primary human B cells, which constitutively express the mb-1 and B29 genes, do not express the mIg isotypes in a GPI-linked manner.

The complement receptor 2 (CR2 or CD21) can be found in non-covalent association with the B lymphocyte specific CD19 complex at the surface of mature human B cells. Upon ligation of the B cell antigen receptor complex (BCR), members of the CR2-CD19 complex may associate with membrane immunoglobulin (mlg). Moreover, CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein-Barr virus, are known to have profound effects on B cell activation. We here show that CD19 is tightly linked to the non-receptor src kinase Lyn and that the CD19 glycoprotein itself serves as a substrate for a yet undefined serine/threonine kinase present within the complex. In the process of antigen recognition, mlg and the CR2-CD19 complex may bind different sites of a complement-opsonized antigenic particle. We hypothesize that in this process, approximation to the BCR allows CD19-associated Lyn kinase to phosphorylate potential substrates within the antigen-receptor complex, thereby effecting its coupling to the intracellular compartment

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s)