Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies, II

We have previously described complement-independent killing of human B lymphocytes by two IgM MoAbs derived from the VH4-34 (VH4.21) gene. Analysis of 17 independently derived VH4-34-encoded MoAbs shows that B cell toxicity is not limited to the two described MoAbs, but is a general property shared by a subset of MoAbs derived from the VH4-34 gene. As observed by two independent microscopy techniques, giant membrane pores were formed on target B cells within 10–15 min of exposure to cytotoxic VH4-34-derived MoAbs. Toxicity by individual MoAb correlated directly to its B cell binding intensity measured by FACS, i.e. stronger the binding greater the killing. Sequence analysis showed that V_H region in germ-line or in near germ-line configuration was necessary but not sufficient for B cell binding. In addition, a particular sequence motif enriched in basic amino acids in the CDR3 may be required to supplement the reactivity mediated by the V_H region of the MoAb molecule. VH4-34-encoded antibodies that fulfil the above sequence requirements have cold agglutinin activity towards the i antigen of cord erythrocytes. In vivo, such anti-i/anti-B cell antibodies are rarely detected in healthy adults, but serum levels are dramatically elevated in selective pathological conditions, such as systemic lupus erythematosus and infectious mononucleosis. This strict regulation may be related to the novel and rapid mechanism of human B cell toxicity demonstrated by antibodies encoded by a single human V_H gene.     

VH gene usage in humans: biased usage of the VH6 gene in immature B lymphoid cells

Preferential usage of JH-proximal VH genes has been demonstrated in immature murine B cell repertoires. To determine whether this phenomenon is also evident in human repertoires, we studied utilization of VH6, the most JH-proximal human VH gene. Examination of VH gene usage in a panel of precursor B cell acute lymphoblastic leukemia samples indicated that 15% of the IgH rearrangements utilized VH6. VH6 is a single-member family in a total repertoire of 100-200 VH genes; thus, if usage were purely random, one would expect VH6 rearrangement frequency to be less than 1%. Analysis of VH gene usage in normal lymphoid tissues also revealed biased usage of VH6. VH6 was preferentially utilized in 16- to 24-week-old fetal liver as compared to adult peripheral blood mononuclear cells or spleen. Possible implications of the conservation of preferential usage of JH-proximal genes in both immature murine and human repertoires are discussed.     

Endogenous VH gene family expression in immunoglobulin-transgenic mice: evidence for selection of antibody repertoires

VH gene family expression in single cells of the emergent, available and actual B cell repertoires of C57BL/6 mice was compared to that of two immunoglobulin (Ig)-transgenic B6 lines (B6-Sp6 and M54). We found that less than 5% of bone marrow cells of transgenic mice express endogenous VH genes and that the vast majority (95%) of the peripheral, mature B cell repertoire in these animals is composed of cells expressing the VHJ558 transgenic family. Unimmunized transgenic mice, however, diversify VH gene family usage by 'background' Ig-secreting cells in the spleen, greater than 50% of which express endogenous VH genes. The pattern of endogenous VH gene family expression in the actual repertoire of B6-Sp6 mice is indistinguishable from that of normal B6 mice. In contrast, actual repertoires of M54 mice differ by a 4- to 5-fold higher representation of the VHQ52 family. These results demonstrate a powerful positive selection of B cells into the secretory compartments of unimmunized animals, show that actual and available repertoires differ very markedly, and suggest that V region interactions participate in the selection of 'natural antibody' repertoires.     

Immunoglobulin VH gene expression in human B cell lines and tumors: biased VH gene expression in chronic lymphocytic leukemia

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.     

Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires

The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1–6 and for the constant region genes Cμ and Cγ. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.     

B cell lymphoproliferative disorders and VH4-34 gene encoded antibodies

The VH4-34 represents an unusual Ig heavy chain variable region gene given that it is conserved and overexpressed despite its autoreactivity. Besides RBC 'I/i' recognition, a subset of VH4-34 encoded Igs bind and kill human B-lymphocytes via interaction with a cytoskeletally-associated ligand similar in structure to the cord RBC 'i' antigen. In vivo, secretion of VH4-34 gene encoded antibodies is minimal in healthy individuals. The turn on signal occurs in few clinical conditions such as, systemic lupus erythematosus, AIDS and infectious mononucleosis. Here we show that secretion of VH4-34 Abs is also switched on in hepatitis C and nasopharyngeal carcinoma; but not in diseases such as HPV-associated cervical carcinoma, multiple sclerosis and sarcoidosis. All syndromes with increased VH4-34 Igs appear to be associated with B cell hyperproliferation and B cell lymphotropic viruses, particularly EBV. The significance of the tightly controlled secretion of an autoreactive, conserved Ig gene is discussed.     

Characterization of murine monoclonal antibodies against the Ro52 autoantigen

Immunization of BALB/c mice with purified recombinant human Ro52 protein resulted in three anti-Ro52 MoAbs termed 2E7, 4C6 and 4F11. All anti-Ro52 MoAbs specifically reacted with recombinant human Ro52 protein, and also with Ro52 protein in total extracts of all human cell lines analysed, including the epithelial cell line HeLa, the B cell line Raji, the bladder carcinoma cell line RT112, and a fibroblast cell line derived from patients with xeroderma pigmentosum. The anti-Ro52 MoAbs were able to immunoprecipitate the recombinant human Ro52 protein expressed in wheat germ extract, but failed to precipitate hY RNAs from cell extracts. The staining pattern of the MoAbs strongly differed between the RT112 cells and the fibroblast cell line. RT112 cells displayed an intense cytoplasmic staining and in addition distinct fine nuclear speckles. In contrast, in the fibroblast cell line no cytoplasmic staining but only staining of distinct nuclear speckles was observed. Using deletion mutants of Ro52 the epitopes recognized by the anti-Ro52 MoAbs 2E7, 4C6 and 4F11 were partially mapped. All three MoAbs appeared to recognize distinct epitopes, that are located in the regions of Ro52 bordered by amino acids 136–164, 208–363 and 136–190, respectively. These MoAbs can be of great use in studying the cellular processes in which the Ro52 protein is involved.     

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.     

All VH11 genes expressed in peritoneal lymphocytes encode anti-bromelain-treated mouse red blood cell autoantibodies but other VH gene families contribute to this specificity

We have studied the relationship between B cell reactivity to bromelain-treated autologous mouse erythrocytes (BrMRBC) and expression of the VH11 gene family in splenic, peritoneal and pleuropericardial cell populations from normal C57BL/6 mice. B lymphocytes producing antibodies to BrMRBC were selectively enriched or depleted from normal populations by rosette formation with BrMRBC, followed by centrifugation over density gradients. This selection method, based on the presence of functional receptors (membrane IgM), is harmless for the cells and allowed subsequent cloning in agar (colony-forming unit-B). The utilization of the 10 VH gene families was then scored in mRNA colony blot assays. The analysis of greater than 650 anti-BrMRBC clones and greater than 350 VH11-expressing colonies indicates that about half of those antibody reactivities are encoded by VH11 genes. Furthermore, it appears that all VH11-expressing B cells in the peritoneal cavity produce anti-BrMRBC antibodies.     

Skewed B cell VH family repertoire in Bcl-2-Ig transgenic mice.

The proto-oncogene Bcl-2 is normally expressed in B lineage cells in a stage specific manner and extends cell survival. Deregulated Bcl-2 expression has been shown to cause a major expansion in surface IgM and IgD positive B cells. In this report, the influence of deregulated expression of Bcl-2 on the VH repertoire of B cells was studied. This was accomplished by stimulating B cells from both adult and fetal Bcl-2-Ig transgenic mice and their normal littermates using the polyclonal activator lipopolysaccharide. Activated cells were then analyzed by in situ hybridization using radiolabeled C mu and VH gene probes. The D-proximal VH families 7183 and Q52 were preferentially expressed in the adult transgenic mice compared to their normal littermates. VH 7183 and Q52 were also over-represented in fetal transgenic mice but not to a greater extent than that observed with normal fetuses. These results demonstrate that the overproduction of Bcl-2, which prolongs cell survival independent of affecting proliferation, substantially alters the VH gene repertoire.     

Serological biochemical and functional characterisation of three different HLA-DR monoclonal antibodies derived from C57BL6 mice

C57BL6 mice which do not express I-E gene products were immunised with EBV transformed human B cell lines to generate MoAbs. Three hybridoma supernatants which initially reacted with the immunising donor cell but not a T cell line lacking Class II antigens were further investigated. I-D SDS-PAGE patterns of molecules precipitated by the three supernatants from a cell membrane lysate were characteristic of HLA-Class II alpha and beta chains. Two-dimensional analysis established the specificity of the supernatants as HLA-DR specific. This was confirmed by the reaction patterns with Class II mutant deletant cell lines. In both ELISA and cytotoxicity one reacted with all lymphoblastoid cell lines tested, one reacted with all except two that were DR7 homozygous and the third reacted strongly only with cells that were DR3. All three antibodies were cytotoxic to both peripheral blood lymphocytes and EBV transformed B cell lines. The DR3 specific MoAb (IgG2a) was suitable as a typing reagent. The DR3 reactive MoAb specifically inhibited stimulation by a Dw3 HTC and the other two MoAbs inhibited all HTCs tested. These findings are consistent with the view that certain determinants responsible for the Dw specificities are carried on the DR molecules.     

Antibodies Directed Against Monomorphic and Evolutionary Conserved Self Epitopes may be Generated in 'Knock-Out' Mice. Development of Monoclonal Antibodies Directed Against Monomorphic MHC Class I Determinants

Beta-2 microglobulin (β₂m) gene ‘knock-out’ mice (C1D) were primed wilh purified H-2K^b and H-2D^b molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2^b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2^b, H-2^d, as well as H-2^k cells by FACS analysis. The MoAbs stained to two β₂m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous β₂m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β₂m suggesting that xenogeneic β₂m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26°C, however, staining was reduced five-fold when RMA-S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β₂m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epilopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.     

In vitro augmentation of human natural cytotoxic activity.

Stimulation of human blood lymphocyte preparations with mitomycin C-treated lymphoid cell lines produced increased levels of cytotoxicity against both NK-susceptible and NK-resistant target cell lines. The greatest effect was seen following stimulation by the B lymphocyte-derived lines, Bri8 and raji. K562 also stimulated high levels of activity while the T lymphocyte-derived lines, CCRF/CEM and MOLT 4, produced smaller increases activity was also found in PHA- and MLC-stimulated populations. Stimulation by lymphoid cell lines gave increased cytotoxic activity against all five cell lines when used as target cells and the pattern of target cell susceptibility was maintained, with K562, CCRF/CEM and MOLT 4 being more susceptible than Bri8 and Raji. No direct correlation was found between the level of cytotoxic activity and the level of 3H-thymidine uptake in stimulated effector cell populations. The B cell lines stimulated high levels of isotopic uptake, while the T cell lines gave no significant stimulation. Similarly, the level of 3H-thymidine incorporation following PHA and MLC stimulation showed no direct correlation with the level of cytotoxic activity. Stimulation of lymphocyte transformation did not appear to be necessary for the induction of cytotoxic activity, although the largest increases in cytotoxicity occurred in populations showing high isotope incorporation. No correlation was found between the target cell susceptibility of the different cell lines and their ability to stimulate cytotoxicity.     

T cells recognize the VH complementarity-determining region 3 of the idiotypic protein of B cell non-Hodgkin's lymphoma

The idiotypic protein expressed by B lymphoma cells is a clone-specific tumor antigen which may be suitable for immune targeting by T cells. In this study, we cloned the immunoglobulin heavy chain variable gene (VH) of the idiotypic protein from a patient with B cell lymphoma and used a synthetic peptide of 22 amino acids corresponding to the VH complementarity-determining region (CDR)-3 of the idiotypic protein to investigate whether autologous T cells could recognize this unique peptide. We demonstrated that autologous T cells possessing both CD4⁺ and CD8⁺ phenotypes could be propagated. The T cells were able to proliferate, secrete cytokines, and lyse autologous cells presensitized with the specific peptide in a major histocompatibility complex-dependent manner. Moreover, these CDR3 peptide-primed T cells were also able to kill autologous lymphoma cells. Our results therefore offer new perspectives for specific therapeutic vaccination for the treatment of B cell lymphoma.     

Transfer of the human X chromosome to human-Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack HPRT activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in HAT medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of HAT-resistant clones was 32 × 10^–6 when 10⁷ cells were incubated with 10⁸ HeLa chromosomes. Potential reversion of the hybrid cells in HAT medium was less than 5 × 10^–7 The 16 isolated cell lines all contained activity of the human X-linked marker enzymes HPRT, PGK, -Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Generation of a novel CD8+ cytotoxic T lymphocyte that requires soluble factor to lyse autologous antigen-presenting cells

We reported the existence of high and low responders to the streptococcal cell wall antigen (SCW) in the human population. To analyze the mechanism of the low responsiveness to SCW at the cellular level, we established SCW-specific CD4⁺ T cell lines. During the course of generation of a SCW-specific CD4⁺ T cell line restricted by HLA-DQ from a low responder, we obtained autoreactive CD8⁺ cytotoxic T lymphocytes as a cell line (HYCD8). They proliferated in the presence of autologous monocytes and IL-2, without SCW. HYCD8 lysed autologous monocytes and Epstein-Barr virus-transformed B lymphoblastoid cell line (BLCL). This cytotoxic activity was specifically inhibited by an anti-HLA class I framework monoclonal antibody and restricted by HLA-B52 or B54 specificity, as judged by killing activity against panel cells and HLA class I-transfected BLCL. It was unique to HYCD8 that the HLA class I-restricted cytotoxicity was observed only in the presence of soluble factor with low molecular mass (< 10⁴ Da) produced mainly by B cells, which could not be replaced by known cytokines and their mixtures. We thus describe novel HLA class I-restricted cytotoxic CD8⁺ T cells that kill antigen-presenting cells in a soluble factor-dependent manner.     

Altered VH6-D-JH repertoire in human insulin-dependent diabetes mellitus and autoimmune idiopathic thrombocytopenic purpura.

We have characterized the peripheral B cell repertoire in T cell-mediated insulin-dependent diabetes mellitus (IDMM) and in B cell-mediated autoimmune idiopathic thrombocytopenic purpura (AITP). The VH6-containing repertoire in adult patients with IDDM or AITP and healthy control subjects was investigated by PCR amplification using VH6- and JH-specific primers. Nucleotide sequence analysis of VH6-D-JH rearrangements showed an abnormally high frequency of somatic mutations in non-functional rearrangements from diabetic (3. 58 %) as well as AITP patients (3.18 %), compared to controls (0.4 % and 1.43 %, respectively; p < 0.05). In contrast, the mutation frequency among functional rearrangements was 2.4 - 3 times lower in patients compared to controls ( p < 0.05). Detailed analysis of the VH6 genes carrying mutations showed that the underlying mechanism for this observation is probably different for the two diseases. Analysis of D- and JH gene usage revealed additional deviations from the normal pattern. Taken together, these results suggest defects in the mechanisms controlling selection of the B cell repertoire in patients with IDDM or AITP.     

IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies

VH4-34 gene encoded autoantibodies are elevated in systemic lupus erythematosus (SLE) and in other diseases associated with B-cell hyperproliferation/dysfunction. One of the autoantigens recognized by VH4-34-encoded antibodies are branched/linear poly N-acetyl lactosamine chains. Since the anti-carbohydrate response in humans is dominated by the IgG2 subclass, here we tested whether VH4-34 encoded IgG showed similar subclass segregation. Serum samples from SLE, infectious mononucleosis, nasopharyngeal carcinoma and hepatitis-C were analyzed. Levels of VH4-34-encoded IgM and IgA isotypes were also tested. VH4-34-IgM and IgA were elevated in all four clinical conditions. VH4-34-IgG was detected in the IgG1 and IgG3 subclass but not in the IgG2 and IgG4 subclass. Interestingly, VH4-34-IgG3 was also detected in serum samples of normal healthy adults. These observations are discussed in context of the VH4-34 gene regulation. VH4-34 repertoire development is of interest since it is the only human VH gene profoundly overrepresented in the naïve repertoire but counter-selected for antibody secretion. VH4-34 B-cell could thus become a unique tool to inspect germinal center independent/dependent pathways of subclass and isotype-specific antibody secretion.     

Three New Cross-Reacting Idiotopes as Markers for the Products of Two Distinct Human VH3 Genes Expressed in the Early Repertoire

This article describes characterization of three new cross-reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing V_H3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) V_H3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti-idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fcα, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti-idiotypic MoAbs were shown to be V_H-specific reagents by the independence of their reactivity upon the L-chain type, or the untigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for V_H3 gene-products was demonstrated by its sole cross-reactivity with V_H3 proteins. Each anti-Id had a different reactivity pattern with a panel of MoAbs utilizing different V_H3 genes. By relating the V_H sequences of the tested V_H3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47-encoded proteins, although 3H1 had weak cross-reactivities with a few other V_H3 gene-products. 7B4 appeared to be specific for antibodies utilizing DP46-related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize V_H3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46-related gene products, B6 and D12 additionally recognized non DP46-related proteins and were thus different to 7B4.     

Acute exposure to morphine suppresses cytotoxic T-lymphocyte activity

Chronic exposure (11 days) to morphine has previously been shown to suppress splenic and peritoneal cytotoxic T-lymphocyte activity through μ-opioid receptors. The present study was undertaken to assess the effects of varying the frequency of exposure to morphine on cytotoxic T-lymphocyte activity in C3H/HeN mice immunized with C57BL/6 splenocytes. Mice subchronically treated with morphine (50.0 mg/kg) showed no measurable suppression of splenic natural killer activity or splenic or peritoneal cytotoxic T-lymphocyte activity. However, mice treated acutely with 50.0 mg/kg of morphine exhibited a significant suppression of peritoneal but not splenic cytotoxic T-lymphocyte activity. Naltrexone pretreatment of mice receiving an acute dose of morphine blocked the suppression, implicating the involvement of opioid receptors. Using column depletion chromatography, peritoneal exudate cells mediating cytotoxic T-lymphocyte activity were both CD4⁺CD8⁻ and CD4 CD8⁺. Collectively, the results suggest that the duration of opioid (morphine) exposure differentially affects peritoneal cytotoxic T-lymphocyte activity. These results may have important implications regarding immunity to viral infections in individuals who abuse drugs such as heroin.