De novo gene mutations in normal human memory B cells.

In the past years, the genomes of thousands of tumors have been elucidated. To date however, our knowledge on somatic gene alterations in normal cells is very limited. In this study, we demonstrate that tetanus-specific human memory B lymphocytes carry a substantial number of somatic mutations in the coding regions of the genome. Interestingly, we observed a statistically significant correlation between the number of exome mutations and those present in the immunoglobulin heavy variable regions. Our findings indicate that the majority of these genomic mutations arise in an antigen-dependent fashion, most likely during clonal expansion in germinal centers. The knowledge that normal B cells accumulate genomic alterations outside the immunoglobulin loci during development is relevant for our understanding of the process of lymphomagenesis.

Chlamydia psittaci-negative ocular adnexal marginal zone B-cell lymphomas have biased VH4-34 immunoglobulin gene expression and proliferate in a distinct inflammatory environment.

Ocular adnexal marginal zone B-cell lymphomas (OAMZLs) arise in the connective tissues of the orbit or in the mucosa-associated lymphoid tissue of the conjunctiva. Here, we present the immunological and genetic analyses of 20 primary Chlamydia psittaci (Cp)-negative OAMZLs. Analysis of the immunoglobulin variable heavy chain (IgV(H)) gene usage demonstrated a significant preference for V(H)4-34. A combined analysis across all previously published OAMZLs confirmed that this is a general feature of OAMZL, in particular of the Cp-negative group. Our series of OAMZLs did not express the characteristic rheumatoid factor V(H)DJ(H) rearrangements that were previously found in salivary gland- and gastric-marginal zone B-cell lymphomas (MZBCLs). We did not detect the MZBCL-specific chromosomal translocations, t(11;18) API2-MALT1 (mucosa-associated lymphoid tissue1) and t(14;18) IgH/MALT1. Two cases contained a premature stop codon in the A20 gene (TNFAIP3) and one case harbored the activating MYD88 hotspot mutation L265P. Variable nuclear expression of BCL10, NFκB1 (p50) and NFκB2 (p52) suggests that other additional genetic abnormalities affecting the NFκB pathway exist within this group of lymphomas. OAMZL showed variable expression of the chemokine receptor CXCR3 and integrin α4ß7 by the tumor B cells, and low interferon-γ and interlukin-4 mRNA levels in the tissue, indicative of an inflammatory environment with features in between those previously found in cutaneous and other extranodal MZBCL. The strongly biased usage of V(H)4-34 in Cp-negative OAMZLs suggests involvement of a particular stimulatory (auto-) antigen in their development.

Molecular pathways in follicular lymphoma.

Follicular lymphoma (FL) is one of the most common B-cell non-Hodgkin's lymphomas. The initiating genetic event found in approximately 90% of FL is the t(14;18), causing constitutive expression of the antiapoptotic BCL-2 protein. The exact secondary alterations leading to full FL development are still poorly defined. In this review, we address (i) the genetic pathways associated with tumorigenesis and progression of FL, (ii) the role of micro-environmental factors with emphasis on B-cell receptor ligands and (iii) lymphoma models in mice and what they teach us about lymphomagenesis in man.

The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma.

The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow (BM) microenvironment, supporting the proliferation and survival of malignant plasma cells. An interesting candidate signal is hepatocyte growth factor/scatter factor (HGF), since its receptor Met is expressed on MM cells, while HGF is produced by BM stromal cells and by some MM cell lines, enabling para- or autocrine interaction. To explore this hypothesis, we studied the biological effects of HGF stimulation on MM cell lines and on primary MMs. We observed that Met is expressed by the majority of MM cell lines and by approximately half of the primary plasma cell neoplasms tested. Stimulation of MM cells with HGF led to the activation of the RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathways, signaling routes that have been implicated in the regulation of cell proliferation and survival. Indeed, functional studies demonstrated that HGF has strong proliferative and anti-apoptotic effects on both MM cell lines and primary MM cells. Furthermore, by applying specific signal-transduction inhibitors, we demonstrated that MEK is required for HGF-induced proliferation, whereas activation of PI3K is required for both HGF-induced proliferation and for rescue of MM cells from apoptosis. Taken together, our data indicate that HGF is a potent myeloma growth and survival factor and suggest that the HGF/Met pathway is a potential therapeutic target in MM.

Biased Iglambda expression in hypermutated IgD multiple myelomas does not result from receptor revision.

Normal IgM(-)IgD(+) CD38(+) B cells and IgM(-)IgD(+) multiple myelomas (MM) are characterized by Cmu deletion, biased Iglambda expression and hypermutated IgV regions. The predominant Iglambda usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal expansion in the germinal center environment. Here, four cases of IgDlambda MM were studied to address the question of light chain receptor revision in a 'single cell' model. Detailed analyses of both IGK and IGL alleles of each case were performed by Southern blotting, (RT-) PCR, and sequencing. The expressed IgV genes were extensively mutated and Cmu deletion was confirmed in two cases. In addition, in the four MM a total of six non-functional deletional IGK rearrangements were identified, which proved to be unmutated. We conclude that IgD myelomas indeed originate from (post) germinal center B cells in which, in spite of the fact that they are hypermutated, there is no evidence of receptor revision.

Immunoglobulin diversification in B cell malignancies: internal splicing of heavy chain variable region as a by-product of somatic hypermutation.

In this study we describe alternative splicing of somatically mutated immunoglobulin (Ig) variable heavy chain (V(H)) genes in three distinct primary B cell non-Hodgkin's lymphomas (B-NHL). In two V4-34 expressing lymphomas, ie a post-germinal center type B cell chronic lymphocytic leukemia (B-CLL) and a follicular lymphoma (FL), internally spliced V(H) gene transcripts were found in which a sequence stretch of 116 bp between the framework region 1 (FR1) and complementarity determining region 2 (CDR2) had been deleted. We provide evidence that for this alternative IgV(H) mRNA processing a known cryptic 5' splice donor site and a previously unidentified cryptic 3' splice acceptor site were used. Site-directed mutagenesis showed that the cryptic 3' splice acceptor site had been activated by specific somatic point mutations. The B-CLL further harbored a triplication of the rearranged JH3 gene segment including the putative N region and part of the JH3-JH4 intron sequence. This triplication probably took place via a repeated mechanism of DNA double strand break followed by homologous recombination, a mechanism which was recently proposed also involved in the somatic hypermutation process and is compatible with the post-germinal center derivation of this B-CLL. Finally, in a V4-34 expressing diffuse large B cell lymphoma, we observed alternative IgV(H) mRNA processing using the same cryptic 5' splice donor site and the normal splice acceptor site of the CH1-C(mu) exon. The significance of alternative IgV(H) processing in B cell malignancies and as a potential mechanism of somatic Ig diversification is discussed.

Variable heavy-chain gene analysis of follicular lymphomas: subclone selection rather than clonal evolution over time.

To investigate B-cell receptor evolution in follicular lymphomas (FLs), immunoglobulin variable heavy chain (V(H)) gene regions of 3 FLs were analyzed at different time points. One FL with a high somatic mutation load and intraclonal V(H) gene diversity was investigated in situ. V(H) gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells isolated from frozen tissue sections by laser microdissection. Interestingly, the mutation pattern of the prevalent subclone in the relapse biopsy was virtually identical to that of a subclone isolated by microdissection from the presentation biopsy 9 years earlier. In a second FL, proof was obtained that the subclone that dominated the relapse sample had already been present in the initial biopsy. The finding that subclones found in the relapses of these FLs had not evolved over time but were preexistent, challenges the concept of antigen-driven B-cell receptor evolution during disease course.

Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.

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)

VH gene analysis of primary cutaneous B-cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.

Primary cutaneous B-cell lymphomas are B-cell non-Hodgkin's lymphomas that arise in the skin. The major subtypes discerned are follicle center cell lymphomas, immunocytomas (marginal zone B-cell lymphomas), and large B-cell lymphomas of the leg. In this study, we analyzed the variable heavy chain (VH) genes of 7 of these lymphomas, ie, 4 follicle center cell lymphomas (diffuse large-cell lymphomas) and 3 immunocytomas. We show that all these lymphomas carry heavily mutated VH genes, with no obvious bias in VH gene usage. The low ratios of replacement versus silent mutations observed in the framework regions of 5 of the 7 lymphomas suggest that the structure of the B-cell antigen receptor was preserved, as in normal B cells that are selected for antibody expression. Moreover, evidence for ongoing mutation was obtained in 3 immunocytomas and in one lymphoma of large-cell type. In addition, in 1 immunocytoma, both IgG- and IgA-expressing clones were found, indicative of isotype switching. Our data provide insight into the biology of primary cutaneous B-cell lymphomas and may be of significance for their classification.

Expression of connective tissue growth factor in human renal fibrosis.

Chronic renal failure may occur in etiologically diverse renal diseases and can be caused by hemodynamic, immunologic and metabolic factors. Initial damage may evoke irreversible scarring, which involves production of a number of proinflammatory and fibrogenic cytokines, including platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta). Connective tissue growth factor (CTGF), a cytokine of the family of growth regulators comprising sef10, cyr61, CTGF and nov, has recently been described in association with scleroderma and other scarring conditions. We investigated CTGF mRNA expression in 65 human renal biopsy specimens of various renal diseases by in situ hybridization. In control human kidney CTFG mRNA was mainly expressed in visceral epithelial cells, parietal epithelial cells, and some interstitial cells. Connective tissue growth factor was strongly up-regulated in the extracapillary and severe mesangial proliferative lesions of crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis and diabetic nephropathy. An increase in the number of cells expressing CTGF mRNA was observed at sites of chronic tubulointerstitial damage, which correlated with the degree of damage. in the tubulointerstitial area the majority of the CTGF mRNA positive cells coexpressed alpha-smooth muscle actin, and were negative for macrophage markers. Our results indicate that CTGF may be a common growth factor involved in renal fibrosis.

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.

Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.

Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics.

To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.

Associations between HLA frequencies and pathogenic features of human immunodeficiency virus type 1 infection in seroconverters from the Amsterdam cohort of homosexual men.

HLA-disease associations may be important for understanding the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. Therefore, 106 homosexual men from the Amsterdam Cohort Study on AIDS with a known date of HIV-1 seroconversion were serologically typed for HLA. Several significant associations between HLA type and pathogenic features of HIV-1 infection were observed: Subjects with fever and skin rash during primary HIV-1 infection showed an increased frequency of HLA-B62 (relative risk [RR], 5.8; P = .005). The frequency of HLA-B35 was increased in subjects with a rapid decline in CD4+ T lymphocytes (RR, 3.2; P = .021). Kaplan-Meier survival analysis revealed a significant association between HLA-B35 and a decrease in CD4+ cells to < 200/microL (P = .01). The strongest association was found between HLA-DR1 and AIDS-related Kaposi's sarcoma (RR, 22.5; P < .001), also confirmed in survival analysis (P = .001). In AIDS patients with only opportunistic infections, increased frequencies of HLA-DR3 (P = .011) and -DQ2 (P = .007) were observed. Finally, the occurrence of syncytium-inducing HIV-1 variants was significantly associated with HLA-DQ2 (P = .01).

Expression of the IL-6 gene induces differentiation of a human monoclonal EBV-transformed B cell line.

To study the role of interleukin (IL)-6 as a growth and differentiation factor for Epstein-Barr virus (EBV)-transformed B lymphocytes, we transfected the cDNA coding for human IL-6 in a monoclonal IgG1-secreting EBV B cell line. Two independent clones were selected that constitutively secreted high amounts of IL-6. These clones showed enhanced levels of IL-6 and tumor necrosis factor alpha secretion when compared to non-IL-6 transfected controls. Moreover, they could efficiently be recovered from low cell density cultures in limiting dilutions when plated on a feeder layer of heterologous EBV B cells. IL-6-induced phenotypical changes comprised a significant rise in immunoglobulin secretion levels and enhanced membrane expression of CD25 (the beta chain of the IL-2 receptor) and of the B cell differentiation antigen CD40. IL-6-dependent down modulation of CD38 and of the adhesion structure VLA4 were also observed. Our data support the notion that IL-6 can serve as an growth and differentiation factor for EBV B cells.

Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells.

In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.

Comparison of human B cell antigen receptor complexes: membrane-expressed forms of immunoglobulin (Ig)M, IgD, and IgG are associated with structurally related heterodimers.

We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.