Analysis of the immunoglobulin heavy chain gene variable region of CD5-positive diffuse large B-cell lymphoma.

To clarify the cell origin of CD5+ diffuse large B-cell lymphoma (DLBCL), we analyzed and compared the variable region of the immunoglobulin heavy chain gene (VH gene) in eight cases of CD5+ DLBCL and 23 cases of other CD5+ B-cell neoplasms; 10 cases of chronic lymphocytic leukemia (CLL), one case of small lymphocytic lymphoma, one case of hairy cell leukemia, and 11 cases of mantle cell lymphoma. CD5+ DLBCL were comprised of two cases of de novo lymphoma of nodal origin, five cases of de novo lymphoma of extranodal origin, and one case of Richter transformation. Whereas all cases of mantle cell lymphoma except one showed a germ line or low mutation frequency of the rearranged VH gene, the rearranged VH genes in both CD5+ CLL and CD5+ DLBCL were heterogeneous. The degree of somatic mutation of CD5+ CLL and CD5+ DLBCL ranged between approximately 0 to 15.0% and 0.7 to 12.9%, respectively. High frequency of expression of the VH4 family in both CD5+ CLL and CD5+ DLBCL was found. Moreover, none of the three cases of CD5+ DLBCL examined exhibited intraclonal diversity. These findings may be common characteristics of the rearranged VH gene of CD5+ CLL and CD5+ DLBCL and suggested that the cell origin of CD5+ DLBCL was the same as that of CD5+ CLL.     

中国人群慢性淋巴细胞白血病免疫球蛋白重链可变区基因组成及突变分析

目的 研究中国慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者免疫球蛋白重链可变区(immunoglobulin variable heavy chain region,IGVH)基因各个片段的组成以及突变情况.方法 应用多重PCR技术扩增64例CLL患者的IGVH基因片段,纯化PCR扩增产物后直接测序,应用IMGT/V-QUEST及IGBlast数据库分析,明确VH基因有无突变和突变位置,以及VH、DH,JH家族各个成员组成情况.结果 64例CLL患者中,VH3家族31例(48%)、VH4家族26例(41%)、VH1家族4例(6%)、VH2家族2例(3%)、VH7家族1例(2%).44例患者发生体细胞突变,占CLL患者的69%;20例无突变,占CLL患者的31%,其中6例(9%)VH基因的同源性为100%.VH4家族中有9例无突变(9/26,35%);VH3家族中8例无突变(8/31,26%);VH1家族中1例无突变(1/4,25%).在所检测的CLL标本中,VH4-34是最常见的VH基因片段,检测出8例(13%),其中无突变3例;其次为VH4-59,检测出7例(11%),无突变者3例.DH基因中DH3家族最常见,有25例(39%);其中11例(11/20,55%)出现在无突变组中.无突变组中最常见的DH基因片段为DH3-10和DH3-22,各为4例(4/20,20%).JH基因中JH6家族最常见,检测出23例(36%),其中9例出现在无突变组中(9/20,45%).结论 中国CLL患者IGVH基因家族表达比例和突变情况与西方国家存在显著差异,推测可能与不同的种族和环境中的抗原选择有关,其预后意义有待进一步探讨.     

Histological and immunoglobulin VH gene analysis of interfollicular small lymphocytic lymphoma provides evidence for two types

Interfollicular small lymphocytic lymphoma (I-SLL) has not been well characterized and its relationship to small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL) is uncertain. Moreover, two different proliferation center growth patterns have been described with respect to reactive germinal centers. In this study, we evaluate the histological and immunophenotypic features of 13 cases of I-SLL and immunoglobulin heavy chain variable (VH) gene sequences from 10 cases. Immunophenotypic analyses indicate that cases showing either growth pattern have the same CD5-positive B cell phenotype typical of SLL or CLL. Sequence analysis revealed the use of VH, D, and J gene segments often found in CLL, although there may be more frequent use of J6. Similar to recent studies of CLL, there were approximately equal numbers of cases with either mutated or unmutated VH genes without evidence of ongoing mutation, consistent with I-SLL having either a na?ve or memory B cell origin. Interestingly, the mutational status of the I-SLL VH genes seemed to correlate with the two different histological growth patterns. These studies support the proposal that I-SLL represents SLL/CLL and suggest the recently proposed two types of CLL originating from either memory or na?ve B cells may have different histological patterns of growth in lymph nodes that show architectural preservation.     

Characterization of a human monoclonal autoantibody directed to cardiolipin/beta 2 glycoprotein I produced by chronic lymphocytic leukaemia B cells.

We determined the specificity and sequence of immunoglobulin molecules synthesized by monoclonal B cells from a patient with chronic lymphocytic leukaemia (CLL) who presented with a number of clinical and biological autoimmune symptoms. Heterohybrids obtained by fusion of CLL cells with the mouse X63-Ag 8.653 myeloma produced IgM lambda MoAbs directed to the cardiolipin/beta 2 glycoprotein I (beta 2GPI) complex and ssDNA. They were devoid of polyreactivity. Nucleotide sequence analysis of the variable domain of the mu chain indicated the utilization of the VH4 71.2 gene or one allotypic variant, DXP4 and JH3 segments. The lambda light chain used the single gene from the V lambda 8 subfamily, J lambda 3 and C lambda 3 genes. The VH gene displayed 11 nucleotide changes in comparison with its putative germline counterpart. However, these nucleotide changes correspond to variations observed in other published VH4 sequences, suggesting gene polymorphism rather than somatic mutation. DXP4 and JH3 were also in germline configuration. The VL gene exhibited a single replacement mutation in CDR1. These data suggest that the monoclonal CLL B cells in this patient retained VH and VL genes in germline configuration although they secreted a pathogenic anti-cardiolipin antibody associated with clinical symptoms, vasculitis and thrombosis, which may be provoked by antibodies to the phospholipid/beta 2GPI complex.     

Over‐expression of BACH2 is related to ongoing somatic hypermutation of the immunoglobulin heavy chain gene variable region of de novo diffuse large B‐cell lymphoma

The basic region–leucine zipper (bZip) factor BTB, CNC homology 2 (BACH2) is known to have important roles in class switch recombination and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. In this study, we investigated the relationship between the expression of BACH2 and the status of SHM of the Ig heavy chain gene variable region (IgHV) for SHM in diffuse large B‐cell lymphoma (DLBCL). We examined 20 cases of DLBCL, 13 of which were germinal center B‐cell (GCB) DLBCL and 7 were non‐GCB DLBCL. Seven cases were negative, 6 were positive (cytoplasmic expression) and 7 were strongly positive (both nuclear and cytoplasmic expression) for BACH2. Confirmed mutation (CM) was identified in 8 cases and the CM index (number of confirmed mutations per 10 subclones) was distributed from 0 to 5. A CM index of 7 strongly positive (over‐expression) cases with BACH2 were distributed from 0 to 5, and that of 7 negative and 6 positive cases were distributed from 0 to 1. Over‐expression of BACH2 was statistically related to CM index (P = 0.008). In conclusion, over‐expression of BACH2 is critical for ongoing SHM of IgHV in DLBCL, and our data suggest that BACH2 may play an essential role for SHM of the Ig gene in B‐cell lymphoma.     

Epstein-Barr virus-positive Hodgkin/Reed-Sternberg-like B cell in non-hodgkin lymphoma: nucleotide sequence of the amplified immunoglobulin heavy-chain variable region gene by the single-cell polymerase chain reaction technique.

We examined nucleotide sequences of Epstein-Barr virus (EBV)-positive Hodgkin/Reed-Sternberg (HRS)-like B cells in a case of diffuse large B-cell lymphoma (DLBCL) and a case of adult T-cell lymphoma (ATL) for single-cell polymerase chain reaction of the immunoglobulin heavy-chain gene variable region (VH gene). HRS-like B cells were scattered in the area irrelevant to the lymphoma infiltrates of DLBCL and in the lymphoma area of ATL. HRS-like B cells were positive for CD20 and CD30 but negative for CD15. EBV presented in HRS-like B cells in both cases but not in any lymphoma cells. VH genes of five HRS-like B cells analyzed in DLBCL were polyclonal and showed in-frame sequences with 0% to 2.8% somatic mutation frequency. In an ATL, VH genes of five HRS-like B cells analyzed were polyclonal and somatically mutated. Four cells carried in-frame rearrangements with 3.5% to 17.7% mutation frequency. One of the VH genes has a one-codon deletion. From the fifth cell, an out-of-frame rearrangement with an insertion and a deletion was obtained. Thus, we showed polyclonal EBV-positive HRS-like B cells in both DLBCL and ATL and that whereas EBV-positive, HRS-like B cells in DLBCL exhibited unmutated and mutated VH gene, those in ATL were found to have a somatically mutated VH gene with/without deletions and/or insertions. The HRS-like B cells may appear because of active EBV infection in a patient who is immunosuppressed from the primary lymphoma.     

Analysis of the immunoglobulin heavy chain gene of secondary diffuse large B-cell lymphoma that subsequently developed in four cases with B-cell chronic lymphocytic leukemia or lymphoplasmacytoid lymphoma (Richter syndrome)

The immunoglobulin heavy chain gene (IgH gene) was analysed in four cases of B-cell Richter syndrome, in order to determine whether a secondary diffuse large B-cell lymphoma (DLBCL) could arise from the same clone as the initial B-cell chronic lymphocytic leukemia (B-CLL) and lymphoplasmacytoid lymphoma (LPL) or be a de novo event, and whether secondary DLBCL shows an intraclonal microheterogeneity. Both the initial B-CLL and secondary DLBCL in two cases expressed CD5 antigen. Both samples of the initial B-CLL or LPL and the secondary DLBCL in three cases were examined for comparison. The polymerase chain reaction-amplified IgH gene of secondary DLBCL in two cases (CD5+ case and CD5- case) were different from those of the initial B-CLL, revealing a new malignant clone. The other case (CD5-) showed that secondary DLBCL had a sequence identical to the initial LPL, indicating the same clonal origin. The variable region of the IgH gene of secondary DLBCL (CD5+ two cases and CD5- two cases) exhibited a 0.5-9.0% somatic mutation range and no intraclonal microheterogeneity.     

Richter syndrome in B-cell chronic lymphocytic leukemia

Richter syndrome (RS) is well known as a secondary high‐grade lymphoma, mostly diffuse large B‐cell lymphoma (DLBCL) developed in patients with B‐cell chronic lymphocytic leukemia (B‐CLL). In this review, we describe clinicopathological, histological, immunophenotypical and genetic findings of RS. The patients with RS, regardless of transformation of pre‐existing clone or de novo malignant clone, were resistant to conventional combined chemotherapy and died within months of diagnosis. Molecular techniques can provide convincing results for the clonal relationship of RS to pre‐existing B‐CLL. When RS carries a same rearrangement band or a same sequence as B‐CLL by Southern blotting or nucleotide sequence analyses of immunoglobulin heavy and/or light chain genes, it is suggested to that RS transforms from original B‐CLL. These analyses have showed that approximately two‐thirds of RS cases evolved from a B‐CLL clone. How and where does the B‐CLL clone evolve to RS? The genetic alteration of transforming B‐CLL clone into RS has been addressed. Abnormalities of chromosomes 11 and 14 were most frequently involved in RS, but non‐specific. In addition, RS does not include chromosomal translocation between Ig locus and oncogenes or rearrangements of bcl‐6 gene, both of which were found in some de novo DLBCL. Several candidates, such as mutation of p53 gene and abnormalities of cyclin dependent kinase inhibitor, have been proposed to play an important role in the transformation of a part of B‐CLL. However, there is still uncertainly as to how B‐CLL progresses or develops into RS.     

经典型Richter综合征转化前后IgVH基因克隆重排及突变分析

目的 检测经典型Richter综合征的IgVH基因克隆重排及突变状态,分析转化前后IgVH基因的分子特征及其与预后的关系,并对可能涉及的分子机制进行初步探讨.方法 用基因扫描和测序分析IgVH基因.另外用免疫组织化学LAB-SA法检测两种肿瘤中zeta链结合蛋白激酶70(ZAP70)、p53、干扰紊调节因子(IRF)-4等可能潜在危险因子的表达,并结合随访资料进行生存率分析,筛选危险因子.结果 (1)B-CLL/DLBCL克隆相关(18/23,78.3%),克隆不相关(5/23,21.7%);(2)在16例克隆相关中,12例转化前B-CLL及转化后DLBCL携带未突变IgVH基因;(3)转化前后IgVH基因使用是非随机的,在克隆相关的病例中,B-CLL/DLBCL最常使用VH3-23、VH3-74、VH1-2,各占11.1%;(4)转化后DLBCL仅部分表达CD5(32.1%)和CD23(14.3%)及ZAP70(23.8%),绝大多数表达p53(80.6%)和IRF-4(82.6%);(5)17例转化后DLBCL患者中位生存时间为7个月;(6)统计学分析生存时间与B-CLl/DLBCL转化前后克隆相关与否、IgVH基因的突变状态、ZAP70、p53、IRF-4的表达不相关.结论 (1)转化后DLBCL中克隆相关与克隆不相关的比例为2:1;(2)克隆相关的DLBCL多由携带未突变型IgVH基因的B-CLL患者转化而来;(3)发生转化的B-CLL中IgVH基因使用的偏向性提示了抗原在转化中的可能作用;(4)转化后DLBCL与普通DLBCL在IgVH基因的使用、突变状态,免疫表型及预后的不同,提示其可能是一种新的DLBCL亚类.     

Diffuse large B cell lymphoma expressing the natural killer cell marker CD56

The expression of the natural killer (NK) cell antigen, CD56, in hematological malignancies is rare. However, there are several reports that some hematological malignancies, such as T/NK cell lymphoma, multiple myeloma (MM) and acute myeloid leukemia (AML), express this molecule. In B cell non-Hodgkin's lymphomas (NHL), however, very limited number of cases have been reported to express CD56 molecule. Although one study has recently described that half of microvillous B cell lymphoma (MVL), an uncommon subset of large cell lymphoma, expressed CD56, there have been no reports about most common type of B-NHL, diffuse large B cell lymphoma (DLBL) other than a mention of weak CD56 expression in one of 83 DLBL. We herein presented the first case of diffuse large B cell lymphoma expressing CD56 clearly. The immunophenotype determined by immunostaining and flow cytometric analysis was CD10+, CD19+, CD20+, CD45RO-, CD3- and CD56+. On immunohistochemical study, neither bcl-2 nor TIA-1 was positive for tumor cell. Monoclonal immunoglobulin heavy chain (IgH) gene rearrangement was detected, and the sequence analysis of the variable region of IgH (VH) suggested that this tumor was derived from antigen selected post germinal center B cell. Conventional combination chemotherapy (CHOP) was administered, and the patient has still been in complete remission for 10 months.     

Large cell lymphoma transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma. A flow cytometric analysis of seven cases

We studied 7 cases of large cell transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) immunophenotyped by multiparameter flow cytometry. The 6 women and 1 man ranged in age from 45 to 91 years. All had previous or concurrent evidence of CLL/SLL. Morphologic features and sites of involvement of the diffuse large B-cell lymphoma (DLBCL) were heterogeneous; 2 cases had paraimmunoblastic morphologic features. Six DLBCLs had an immunophenotype consistent with CLL: CD19+, CD5+, CD23+, and FMC7 negative (3 cases) or very dim (2 cases); 1 case was not studied for FMC7. CD20 was dim in 3 of these, moderate to bright in 2, and variable in 1. Surface immunoglobulin was dim in 2 cases and moderate or bright in 4. Five of 6 expressed CD38. Comparison with the immunophenotypes of the previous or coexistent CLL/SLL (4 of 6 cases) revealed minor modulations in antigen expression but no major alterations. The seventh DLBCL lacked CD5 expression, but otherwise had immunophenotypic features similar to CLL. These findings indicate that DLBCL arising in CLL/SLL tends to retain a CLL immunophenotype, in contrast with de novo CD5+ large cell lymphomas that uncommonly express such a phenotype.     

Immunoglobulin VH genes in thymic MALT lymphoma are biased toward a restricted repertoire and are frequently unmutated

Thymic MALT lymphoma shows certain distinctive features among MALT lymphomas, such as expression of IgA isotype, consistent lack of API2-MALT1 gene fusion, and very strong association with autoimmune disease, especially Sjogren's syndrome. To help clarify the nature of the clonal lymphoid infiltrates, we analysed the usage and somatic hypermutation of the Ig heavy chain variable region (V(H)) genes in nine different cases. The V(H) rearrangement was potentially functional in all cases and was restricted to the V(H)3 family. V(H) usage was biased toward V(H)3-30 (five cases) and V(H)3-23 (three cases) segments, which have both been frequently expressed by autoimmune B cells. Somatic hypermutation was absent in five cases. Fewer than the expected replacement mutations were found in the framework regions in two cases, indicating a negative antigen selection pressure. Ongoing mutation was absent in all cases. D segment usage was varied, whereas J(H) segment usage was restricted to J(H)4. The observed patterns of V(H) usage and mutations suggested that specific antigens may play a pathologically relevant role in the genesis or progression of thymic MALT lymphoma.     

The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma

Masir N, Jones M, Lee A M, Goff L K, Clear A J, Lister A, Marafioti T & Mason D Y
(2010) Histopathology 56 , 617–626 The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma Aims: To investigate the relationship between Bcl‐2 protein expression and cell proliferation at single‐cell level in B‐cell lymphomas using double‐labelling techniques. Methods and results: The relationship between Bcl‐2 protein expression and cell proliferation was explored in 124 cases of B‐cell lymphoma using double immunofluorescence labelling for Bcl‐2 and Ki67. In follicular lymphoma, marginal zone lymphoma and a subset of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), neoplastic cells tended to lose Bcl‐2 when they are in cell cycle. This pattern is usually maintained in both follicular lymphoma and CLL/SLL when they undergo high‐grade transformation. In mantle cell lymphoma, diffuse large B‐cell lymphoma and a subset of CLL/SLL, the inverse relationship (between Bcl‐2 and Ki67) was not observed, i.e. the proliferating cells tended to show co‐expression of Bcl‐2. Conclusions: In low‐grade lymphomas, including those that are transformed, Bcl‐2 expression is lost when cell proliferate. However, in more aggressive tumours (i.e. mantle cell and de novo diffuse large B‐cell lymphomas) the inverse Bcl‐2/Ki67 relationship was not observed. It would be of interest to explore the clinical implications in lymphoma of the presence and absence of the inverse Bcl‐2/Ki67 pattern.     

Flow cytometric analysis of surface light chain expression patterns in B-cell lymphomas using monoclonal and polyclonal antibodies

Light chain (LC) expression by flow cytometry (FC) in B cell non-Hodgkin lymphomas (B-NHLs) can occasionally be detected with one anti-LC antibody but not with another. We retrospectively analyzed 564 four-color FC files from B-NHLs, assessing LC staining with monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs). Discrepancies in LC expression between mAbs and pAbs were present in 9.2% of cases, mainly in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; 11.1%), diffuse large B-cell lymphoma (DLBCL; 10.2%), follicular lymphoma (9.5%), and mantle cell lymphoma (11.1%) and most frequently in body fluids. Equal proportions of cases were LC+ only with pAbs (4.8%) or mAbs (4.4%). Negative LC expression with both antibodies was present in 7.5% of cases, most frequently in DLBCL (21.6%) and body fluids (27.6%). Evaluation with both mAbs and pAbs increases the sensitivity for LC detection, with no single reagent outperforming the other, although CLL/SLL preferentially showed LC expression with pAbs.     

Detection of Merkel cell polyomavirus in chronic lymphocytic leukemia T-cells

Chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL) is the most common B-cell leukemia/lymphoma, effecting > 15,000 patients/year. There has been a proposed limited antigenic etiology, at least in some cases, of CLL/SLL based upon immunoglobulin heavy chain stereotypy found across unrelated cases, suggesting viral source may provide such antigenic stimulation. With an established epidemiological link between CLL/SLL and Merkel cell carcinoma (MCC), there has been some interest in investigating a possible leukemogenic role of Merkel cell polyomavirus (MCPyV), which is found in 80% of MCC cases. Recent studies have shown that MCPyV is present in lymphocytes in a small percentage of CLL/SLL cases, but the specific tropism for lymphocytes has not been well-established. In this study, we used quantitative PCR to investigate the presence of MCPyV in fluorescence activated cell sorted purified B- and T-cells from 23 CLL/SLL cases. Three of 23 cases (13%) had detectable MCPyV in T-cells, and none of the cases had detectable MCPyV in B-cells. These findings suggest that MCPyV may have tropism for T-cells in addition to previously reported neoplastic B-cells.     

胃肠道黏膜相关边缘区B细胞淋巴瘤和弥漫性大B细胞淋巴瘤中API2-MALT1融合基因表达的差异

目的 比较t(11;18)(q21;q21)/API2-MALX1融合基因在胃肠道黏膜相关边缘区B细胞淋巴瘤(MALN淋巴瘤)和弥漫性大B细胞淋巴瘤(DLBCL)中的发生情况,探讨t(11;18)(q21;q21)与胃肠道MALT淋巴瘤和DLBCL间演进的关系.方法 收集57例胃肠道MALT淋巴瘤(包括38例胃和19例肠),32例胃肠道DLBCL(包括28例胃和4例肠)和7例胃DLBCL同时合并MALT淋巴瘤成分,用荧光原位杂交(FISH)检测API2-MALT1融合基因.使用的探针包括API2-MALT1双色融合易位探针和MALT1双色分离重排探针.结果 在MALT淋巴瘤中有21.1%(12/57,包括10例胃和2例肠)发现API2-MALT1融合基因,而在32例DLBCL和7例DLBCL与MALT淋巴瘤混合的病例中均未检测出API2-MALT1融合基因.两组经统计学比较差异有统计学意义(X~2=9.383,P=0.001).结论 API2-MALT1融合基因是MALT淋巴瘤中特异的遗传学异常,而不见于DLBCL或DLBCL与MAIX淋巴瘤共存的病例中,提示至少在胃肠道API2-MALT1阳性的MALT淋巴瘤一般不会发生大细胞转化,而胃肠道DLBCL可能为原发或由t(11;18)阴性的部分MALT淋巴瘤转化而来.     

Multiple lymphomatous polyposis of the gastrointestinal tract is a heterogenous group that includes mantle cell lymphoma and follicular lymphoma: analysis of somatic mutation of immunoglobulin heavy chain gene variable region.

Multiple lymphomatous polyposis (MLP) is characterized by multiple polyps involving long segments of the gastrointestinal (GI) tract. MLP is thought to represent mantle cell lymphoma (MCL) of the GI tract; however, some cases of follicular lymphoma (FL) of the GI tract are found with a multiple polypoid appearance. In the present study, to clarify the cellular origin of MLP, clonal immunoglobulin heavy chain (IgH) gene rearrangement of four cases with MLP was amplified by polymerase chain reaction (PCR) and analyzed for the presence of somatic mutation. The IgH variable (VH) region sequences of three cases (CD5+ CD10- cyclin D1+) showed a little somatic mutation compared with the closest published germline. The other case (CD10+ CD5- cyclin D1-) was highly mutated and showed intraclonal heterogeneity (ongoing somatic hypermutation). These data indicate that three of the cases with MLP are derived from pregerminal center B cells (mantle zone B cells) and one case with MLP from germinal center B cells. Our study suggests that MLP is a heterogenous group that includes MCL and FL.     

Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.