A novel subset of NK cells expressing high levels of inhibitory FcgammaRIIB modulating antibody-dependent function

NK cells can kill antibody-coated target cells following engagement of FcgammaRIIIA, the major activating FcgammaR expressed by these cells. The presence of FcgammaRIIC (CD32C) has also been reported, but its contribution to the FcgammaR-dependent effector functions of NK cells remains debated. We demonstrate here that inhibitory FcgammaRIIB is also expressed by a small subset of CD56+/NKp46+ NK cells and can efficiently down-modulate their FcgammaR-dependent effector function. Immunofluorescence analyses of NK cells from 52 healthy donors showed the presence of CD56bright/FcgammaRII(-) (5.2%+/-3.4), CD56dim/FcgammaRII(lo/-) (94.1%+/-3.4), and CD56dim/FcgammaRIIbright (0.64%+/-0.72) cells. QRT-PCR and protein analyses performed on isolated FcgammaRIIbright NK cells indicated that FcgammaRIIB is strongly expressed by these cells but not by FcgammaRII(lo/-) cells. In addition, FcgammaRIIbright cells showed a weaker antibody-dependent degranulation when incubated with IgG-coated target cells compared with FcgammaRII(lo/-) NK cells, although a strong FcgammaRIIIA expression was detected in both cells. Furthermore, the addition of anti-FcgammaRII Fab paralleled a higher degranulation of FcgammaRIIbright NK cells, indicating a direct role for FcgammaRIIB in this down-modulating effect. Thus, it is proposed that FcgammaRIIBbright NK cells represent a new NK cell compartment able to down-modulate NK cell functions triggered by the engagement of activating FcgammaR.     

Allelic polymorphisms in the FcγRIIC gene can influence its function on normal human natural killer cells

Natural killer (NK) cells are important in host defense against viruses and tumors and can induce death of virally infected cells following engagement of cell surface receptors. Human NK cells express receptors for the Fc portion of IgG which stimulate antibody-dependent cell-mediated cytotoxicity and induce cytokine production. We have shown that NK cells from certain individuals can express, in addition to CD16 (FcgammaRIIIa), isoforms of CD32 (FcgammaRIIc1-4). Expression of CD32 on NK cells is dependent on an allelic polymorphism of the FcgammaRIIC gene. We analyzed the expression and function of CD32 on NK cells from 31 normal donors. Fourteen of the 31 (45%) donors expressed CD32 on their NK cells. Molecular characterization of FcgammaRIIc isoforms expressed by the CD32+ donors revealed that the majority of donors expressed the FcgammaRIIc1 isoform. Interestingly, 3 of the 14 positive donors did not express FcgammaRIIc1, and we identified a novel isoform, FcgammaRIIc5, expressed by these individuals. The expression of this isoform was correlated to a second allelic polymorphism that controls exon splicing. One of the three was found to express FcgammaRIIb on the NK cells. Biochemical analysis revealed that CD32+ donors of both types expressed a 40-kDa protein, specifically immunoprecipitated by anti-CD32 monoclonal antibodies. Functionally, only individuals expressing the FcgammaRIIc1 isoform were able to trigger reverse antibody-dependent cell-mediated cytotoxicity via CD32 whereas a CD32+ individual expressing the FcgammaRIIb isoform was unable to trigger this function. These results demonstrate that the presence of multiple allelic polymorphisms in the FcgammaRIIC gene determine the expression and function of CD32 on NK cells.     

A novel mutation in the FcgammaRIIIA gene (CD16) results in active natural killer cells lacking CD16

We report a novel mutation in FcgammaRIIIA (the transmembrane-form CD16) on natural killer (NK) cells in a patient with polyneuropathy. She had no history of recurrent infections. Her NK cells expressed no detectable CD16; however, her NK cytotoxic activity was normal, suggesting that CD16 expression and cytotoxic activity are independent of one another. Molecular analysis revealed a deletion of a single adenine base in exon 4 of CD16 at nucleotide 550. This deletion generates a STOP codon in an extra-cellular domain of the FcgammaRIIIA gene, thereby truncating the CD16 molecule. The patient's NK cells were not recognized by the anti-CD16 monoclonal antibodies 3G8 and Leu11c. Whether the development of her polyneuropathy is associated with this novel mutation is unclear.     

Expression of Fc receptors for IgG in peripheral blood leucocytes from hepatitis C virus infected individuals

The immune response represents a fundamental element in the control of Hepatitis C virus (HCV) infection. Viral and cellular host factors may modulate this response. In the present study, we characterized immune complexes (cryoprecipitates) isolated form HCV-infected patients and evaluated the expression of Fc receptors for IgG (FcgammaR) in peripheral blood leucocytes of these patients. Twelve HCV (+) patients and 12 healthy control individuals were selected for this study. For each group, sera samples were collected for cryoglobulins isolation and characterization and EDTA-anticoagulated venous blood samples were collected for flow cytometry analysis of FcgammaR, CD64 (FcgammaRI), CD32 (FcgammaRII) and CD16 (FcgammaRIII) expression. Presence of HCV RNA in serum and cryoprecipitates was analysed by RT-PCR. Results show that 50% of HCV-infected patients present high levels of cryoglobulins mainly constituted by IgG. Three out of 5 cryoglobulins analyzed by RT-PCR were positive for HCV-RNA. Expression of CD64 was observed mainly in monocytes (80%), CD32 in monocytes, B lymphocytes and neutrophils (> 90%) and CD16 in NK cells and neutrophils (85% and 95% respectively). No differences were observed in the percentage of FcgammaR expression when comparing HCV-infected patients with healthy controls. On the contrary, density of expression of CD32 in monocytes and neutrophils cell populations of HCV patients was significantly lower than that observed in healthy controls (p < 0.05). We concluded that low density expression of FcgammaRII in HCV-infected patients may have implications in the physiopatholgy of HCV infection.     

Abnormalities in the T and NK lymphocyte phenotype in patients with Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by spontaneous chromosomal instability with predisposition to immunodeficiency and cancer. In order to assess the cellular basis of the compromised immune response of NBS patients, the distribution of functionally distinct lymphocyte subsets in peripheral blood was evaluated by means of double-colour flow cytometry. The study involved the 36 lymphopenic patients with a total lymphocyte count < or =1500 microl (group A) and seven patients (group B) having the absolute lymphocyte count comparable with the age-matched controls (> or =3000 microl). Regardless of the total lymphocyte count the NBS patients showed: (1) profound deficiency of CD4+ and CD3/CD8+ T cell subsets and up to fourfold increase in natural killer (NK) cells, almost lack of naive CD4+ T cells expressing CD45RA isoform, unchanged percentage of naive CD8+ cell subset (CD8/CD45RA+) but bearing the CD8 receptor of low density (CD8low); (2) normal expression of CD45RA isoform in the CD56+ lymphocyte subset, profound decrease in alpha beta but up to threefold increase in gamma delta-T cell-receptor (TCR)-positive T cells; (3) shift towards the memory phenotype in both CD4+ and CD8+ lymphocyte subpopulations expressing CD45RO isoform (over-expression of CD45RO in terms of both the fluorescence intensity for CD45RO isoform and the number of positive cells); and (4) an increase in fluorescence intensity for the CD45RA isoform in NK cells population. These results indicate either a failure in T cell regeneration in the thymic pathway (deficiency of naive CD4+ cells) and/or more dominant contribution of non-thymic pathways in lymphocyte renewal reflected by an increase in the population of CD4+ and CD8+ memory cells, gamma delta-TCR positive T as well as NK cell subsets.     

Surface and cytoplasmic expression of CD45 antigen isoforms in normal and malignant myeloid cell differentiation.

The CD45 family contains protein tyrosine phosphatase (PTPase) activity and is expressed in one or more of its isoforms on all lymphohematopoietic cells. Considerable work has focused on CD45 expression by lymphoid cells, but minimal work has involved granulocytes. Granulocytic, or myeloid, cell differentiation is accompanied by a number of morphologic and immunophenotypic changes. This study used flow cytometric and immunocytochemical methods in conjunction with morphologic assessment to investigate the expression of CD45 isoforms during differentiation of normal and malignant granulocytic cells. On normal bone marrow cells, the quantity of surface CD45 did not change during earlier stages but did increase significantly at the terminal stages (bands and polymorphonuclear leukocytes [PMNs]). CD45RO (the low relative molecular mass [Mr] isoform) was very dimly expressed on immature cells but became increasingly brighter beginning at approximately the myelocyte stage. The high Mr isoform (CD45RA) was virtually absent from the cell surface at all stages. Only a small percentage (3-15%) of PMNs expressed surface CD45RA. However, there was a cytoplasmic pool of each isoform associated with membrane-bound granules found throughout differentiation, with remarkable increases in expression at the terminal stages. In the case of acute myeloid leukemias (AMLs), most cases expressed surface CD45RA with, or without, CD45RO, regardless of their French-American-British (FAB) classification. This appeared to be a stable process at diagnosis and relapse in individual patients and may therefore serve as a diagnostic aid. The biologic significance of this aberrant expression of CD45RA by malignant cells is unknown but raises important questions regarding the cellular processes of phosphorylation/dephosphorylation in normal and malignant cells.     

FcγRIII (CD16)‐mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32)

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK‐cell‐associated antibody‐dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)‐dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV‐1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16‐mediated NK‐cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV‐1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV‐1 infection. As a result, the regulation of NK‐cell‐associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV‐1‐associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII‐mediated NK‐cell functions maintaining a well‐balanced immune response.     

Increased miR‐223 expression in T cells from patients with rheumatoid arthritis leads to decreased insulin‐like growth factor‐1‐mediated interleukin‐10 production

We hypothesized that the aberrant expression of microRNAs (miRNAs) in rheumatoid arthritis (RA) T cells was involved in the pathogenesis of RA. The expression profile of 270 human miRNAs in T cells from the first five RA patients and five controls were analysed by real‐time polymerase chain reaction. Twelve miRNAs exhibited potentially aberrant expression in RA T cells compared to normal T cells. After validation with another 22 RA patients and 19 controls, miR‐223 and miR‐34b were over‐expressed in RA T cells. The expression levels of miR‐223 were correlated positively with the titre of rheumatoid factor (RF) in RA patients. Transfection of Jurkat cells with miR‐223 mimic suppressed insulin‐like growth factor‐1 receptor (IGF‐1R) and transfection with miR‐34b mimic suppressed cAMP response element binding protein (CREB) protein expression by Western blotting. The protein expression of IGF‐1R but not CREB was decreased in RA T cells. The addition of recombinant IGF‐1‐stimulated interleukin (IL)‐10 production by activated normal T cells, but not RA T cells. The transfection of miR‐223 mimic impaired IGF‐1‐mediated IL‐10 production in activated normal T cells. The expression levels of SCD5, targeted by miR‐34b, were decreased in RA T cells after microarray analysis. In conclusion, both miR‐223 and miR‐34b were over‐expressed in RA T cells, but only the miR‐223 expression levels were correlated positively with RF titre in RA patients. Functionally, the increased miR‐223 expression could impair the IGF‐1‐mediated IL‐10 production in activated RA T cells in vivo, which might contribute to the imbalance between proinflammatory and anti‐inflammatory cytokines.     

Differential effects of infliximab on absolute circulating blood leucocyte counts of innate immune cells in early and late rheumatoid arthritis patients

Anti-tumour necrosis factor (TNF) biologics have revolutionized therapy of rheumatoid arthritis (RA). We compared the effects of infliximab on numbers of circulating leucocyte subsets in early RA (disease/symptom duration of ≤1 year) and late RA patients (>1 year). A control group consisted of early RA patients treated with a combination of methotrexate (MTX) and methylprednisolone. Blood samples were obtained at baseline (pre-therapy) from all RA patients, divided into three groups: (i) late RA receiving infliximab/MTX, (ii) early RA-infliximab/MTX, (iii) early RA-steroid/MTX, and also from follow-up patients at 2 and 14 weeks. Significant differences in absolute counts of monocytes and granulocytes were observed between healthy controls and RA patients. At baseline CD14(bright) monocytes and CD16(+) granulocytes were increased in both early RA and late RA patients. CD4(+) T cells, CD8(+) T cells and B cells were all increased at baseline in early RA, but not in late RA. At 2 weeks following infliximab treatment decreased granulocytes were observed in both early and late RA and decreased natural killer (NK) cells in late RA. CD16(+) granulocytes and NK cells were also decreased at 14 weeks post-infliximab in early RA. Biotinylated infliximab was used to detect membrane-associated TNF (mTNF)-expressing leucocytes in RA patients. CD16(+) granulocytes, NK cells and CD14(dim) monocytes all expressed higher levels of mTNF in RA patients. In summary infliximab is associated with decreased CD16(+) granulocyte and NK cell counts, possibly through binding of mTNF. Differential effects of infliximab between early and late RA suggest that pathogenic mechanisms change as disease progresses.     

Monoreactive and Polyreactive Rheumatoid Factors Produced by in Vitro Epstein-Barr Virus-Transformed Peripheral Blood and Synovial B Lymphocytes from Rheumatoid Arthritis Patients

The CD5 membrane molecule, initially identified as an exclusive T-cell marker, also defines a phenotypically and functionally distinct B-lymphocyte population. In normal individuals, CD5+ B cells are committed to secrete 'natural' polyreactive (auto)antibodies, that is antibodies, mainly IgM, endowed with multiple antigen-binding capabilities, including rheumatoid factor (RF) activity. At variance with this, in rheumatoid arthritis (RA) as well as in other autoimmune conditions, monoreactive autoantibodies binding with high affinity and specificity to a given self antigen have been isolated and the cells from which they originate differently related to the CD5+ B-lymphocyte subset. Here, we studied the proportions of CD5+ B cells and the characteristics, in terms of polyreactivity and monoreactivity, of RF produced by B lymphocytes in RA patients with classified disease activity. Our results suggest that patients with a more severe disease activity have higher proportions of CD5+ B cells and higher frequencies of B lymphocytes committed to secrete RF, with the characteristics of polyreactive antibodies. On the other hand, we did not find a significant difference between the proportions of peripheral B cells producing monoreactive RF in patients with high- versus patients with low-activity RA. However, in two highly active RA patients, we found that synovial fluid, compared with peripheral blood, was significantly enriched for (IgG and IgA) monoreactive RF-producing B cells.     

类风湿关节炎患者外周血和滑液中CD4~+CD25~+调节性T细胞的水平变化

了解具有抑制功能的CD4+CD25+调节性T细胞(Treg)在类风湿关节炎(RA)中的水平变化。分离32例RA患者及35例正常对照者外周血和15例RA关节滑液中的单个核细胞,用荧光抗体标记细胞膜表面CD4、CD25分子和细胞内Foxp3转录因子,进行流式细胞分析,同时用RT-PCR方法测定单个核细胞中Foxp3 mRNA水平。实验发现RA外周血中CD4+CD25hT细胞比例(1.90±1.68)与健康人(1.81±1.79)无明显差异,而RA关节滑液中CD4+CD25+和CD4+CD25hT细胞含量却明显增高(14.98±12.52,8.94±9.67,P<0.01)。RA患者外周血单个核细胞中Foxp3+/CD4+T细胞比值(2.35±2.06)较正常人(7.25±3.98)明显降低(P<0.01),RA外周血中Foxp3 mRNA含量较正常人Treg减少,而RA关节液中Foxp3 mRNA含量较RA外周血更为低下(P<0.01)。RA患者存在CD4+CD25+Treg的异常改变,其外周血和关节液中具有抑制作用的Treg含量明显降低提示RA患者Treg数量减少及抑制功能下降可能是RA自身免疫反应亢强不能控制的原因之一。RA关节液中CD4+CD25hT细胞增高考虑与RA炎症反应造成T细胞过度活化有关。     

CD161 receptor participates in both impairing NK cell cytotoxicity and the response to glycans and vimentin in patients with rheumatoid arthritis

We investigated the role of natural killer (NK) cells and CD161, their primary C-type-lectin-like receptor in rheumatoid arthritis (RA). Samples were compared with healthy donors (HD), dermatomyositic (DM), polymyositic (PM), and osteoarthritic (OA) patients. RA, PM, and DM NK cell cytotoxicities significantly decreased relative to the HD and OA NK cells (p < 0.0001). These results correlated with an increased expression of NK cell inhibitory receptor CD161, in active disease RA patients. We demonstrated that NK cells are able to respond to mutated citrullinated vimentin (MCV), an RA-specific autoantigen, leading to increases in both PAD4 enzyme and CD161 mRNA expression. MGAT5 glycosidase involvement was detected in GlcNAc metabolism within the synoviocytes of RA patients. Our findings reveal a functional relationship between CD161 expression and NK cell cytotoxicity as well as reactivity to glycans and MCV, thus providing new insight into the pathogenesis of RA and confirming the involvement of surface glycosylation.     

Phenotypical and biochemical characterization of a variant CD45R expression pattern in human leukocytes.

As a result of a regulatory polymorphism a variant pattern of CD45R expression can be found in 8% of healthy individuals. We have previously reported that all resting T cells of these individuals react with CD45RA monoclonal antibodies (mAb) that are directed to the high-molecular mass isoforms (220 and 205 kDa) of the leukocyte common antigen (CD45). In addition, their T cells remain CD45RA+ after in vitro activation. In this report we studied the regulatory CD45R polymorphism in more detail. Flow cytometry studies revealed that CD45RA antigens are weakly expressed on normal monocytes but are absent from granulocytes. In individuals displaying the variant CD45R pattern both monocytes and granulocytes were CD45RA+. In controls and variant T cells CD45RA mAb precipitated two chains with molecular masses of about 220 and 205 kDa. In activated T cells from controls (CD45RA-) neither p220 nor the p205 chain of CD45RA could be detected. However, activated T cells from variant individuals (CD45RA+) did not express p220 but expressed p205 of CD45RA. These data indicate that there is a failure to down-regulate p205 CD45RA in individuals displaying the variant CD45R expression pattern. Since their T cells lost p220 CD45RA during activation the data also suggest that the two isoforms detected by CD45RA mAb can be regulated independently.     

Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.     

Loss of activation-induced CD45RO with maintenance of CD45RA expression during prolonged culture of T cells and NK cells.

The results of the present study show that activation-induced changes in CD45RA and CD45RO expression on T cells and natural killer (NK) cells are not unidirectional for all cells during a 5-week culture period. T cells and NK cells were generated from a resting subpopulation of peripheral blood mononuclear cells (PBMC) defined by sedimentation at Percoll high buoyant densities (p greater than 1.0640 g/ml) and unresponsiveness to IL-2. T cells were activated by a combination of PHA, sheep erythrocytes and IL-2-conditioned medium (IL-2-CM), and NK cells were activated by co-culture with gamma-irradiated malignant melanoma (MM-170) cells and IL-2-CM. Both T-cell and NK-cell cultures were maintained by subculture in IL-2-CM. NK cells and the CD45R(Abright)RO(dim/neg) subpopulation of T cells gained CD45RO following activation and this was accompanied by a two-fold decrease in CD45RA expression. In different cultures, CD45RO expression was not stable on 28-80% of T cells and 10-55% of NK cells. Cells with decreased CD45RO expression showed increased expression of CD45RA. Instability of CD45RO expression on cultured T cells and NK cells occurred at a time following the period of rapid cell growth when the cells were entering a quiescent phase. Both the CD4+ and CD8+ T-cell subpopulation showed similar changes in CD45 isoform expression. In contrast to the results obtained with the CD45R(Abright)RO(dim/neg) resting T cells, the CD45RO(bright)RA(dim/neg) subpopulation of resting T cells when activated and cultured under identical conditions retained CD45RO expression and remained CD45RAdim/neg. Thus a significant proportion of resting CD45R(Abright)RO(dim/neg) T cells is not related in a differentiation sequence to resting CD45RObrightRAdim/neg T cells, and therefore resting CD45RAbrightROdim/neg T cells and resting NK cells may be heterogeneous with respect to their activation history.     

Expression of Activation Markers on CD4+ and CD8+ Cells from Synovial Fluid, Synovial Tissue, and Peripheral Blood of Patients with Inflammatory Arthritides

We studied the expression of the Tac antigen, the transferrin receptor (Tfr-R), HLA class II antigens (DR, DQ, DP), CD30, and Act 1 on purified CD4+ and CD8+ cells isolated from synovial fluid (SF), synovial tissue (ST), and peripheral blood (PB) of patients with rheumatoid arthritis (RA) and with non-RA inflammatory arthritides (not ST). Subfractionated T cells of PB from healthy individuals served as controls. SF CD4+ cells from RA and non-RA arthritides expressed the Tac antigen much more frequently than corresponding CD8+ cells (54 and 58% versus 16 and 17%). In contrast, SF CD8+ cells of both patient groups expressed the HLA class II antigens rather more frequently than the corresponding CD4+ cells (88 and 68% versus 72 and 40%). Tfr-R expression was low on CD4+ and CD8+ SF T cells from both patient groups. SF T cells did not express CD30, and their expression of Act 1 did not differ from that of normal PB T cells. The RA ST findings were similar to those of RA SF. The overall expression of activation markers on PB T cells of patients was slightly higher than on those of normal controls, and the RA group was slightly higher than the non-RA group. The results show that intra-articular T cells in arthritis are activated and that CD4+ and CD8+ subsets differ in their expression of Tac antigen and HLA class II antigens. There were also similar patterns of activation markers on both CD4+ and CD8+ SF cells from RA and non-RA arthritis patients, suggesting that several types of arthritis display a similar immunopathogenesis in the joints.     

FcγRⅡB1介导的信号传导异常与SLE患者B细胞的过度活化

目的:观察系统性红斑狼疮(SLE)患者B细胞表面功能分子表达的特征及其功能状态,评价以FcγRⅡB1(CD32)为代表的B细胞自身抑制调节机制在SLE发病中的作用。方法:采用Ficoll密度梯度离心法分离出人外周血单个核细胞(PBMC),并以免疫磁珠法(MACS)分离纯化B细胞。采用荧光分光光度法检测B细胞受不同激活物刺激后细胞内钙([Ca2 ]i)的反应。用ELISA法检测B细胞与刺激物共同培养后所分泌IgG的量。采用流式细胞术及间接免疫荧光染色法,检测B细胞膜表面CD32、CD19及IgM的表达水平。结果:(1)以羊抗人μ链的F(ab′)2片段及完整IgG分别刺激SLE患者B细胞时,其[Ca2 ]i反应的比值显著低于类风湿性关节炎(RA)患者(P<0.05)及正常人对照(P<0.01)。(2)分别用葡萄球菌A蛋白(SPA)单独刺激与SPA和羊抗人μ链的完整IgG抗体共同刺激SLE患者的B细胞所分泌的IgG的比值,明显低于RA患者及正常人对照组(P<0.05)。(3)SLE患者与RA患者及正常对照组B细胞上CD19、CD32及IgM的表达无统计学意义(P>0.05)。结论:SLE患者B细胞上CD32抑制性信号传导的异常,可能是导致B细胞过度活化的重要机制。     

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.