Susceptibility of B-cell lymphoma to human antibodies encoded by the V4-34 gene.

Our previous studies have shown that mAbs derived from the human V4-34 gene bind and kill human B-lymphocytes via membrane disruption. This study demonstrates the cytotoxicity of two V4-34 encoded mAbs, 216 and Z2D2, towards human B-cell lymphoma. In vitro, 216 and Z2D2 are cytotoxic to a variety of B-cell lymphomas obtained from patient biopsies. In vivo, increased survival was observed with both mAbs in a lymphoma model developed in scid mice with human B-cell line Nalm-6. Studies in mice show that these mAbs are well tolerated with minimum side effects. Since 216 and Z2D2 show increased toxicity towards cycling cells, V4-34 mAb-based therapy can be additive with drugs that block cell-cycle progression. Stem cells that are V4-34 mAb ligand negative would not be depleted. Together, these studies recommend an evaluation of the two completely human mAbs in a phase I trial for B-cell lymphoma.

Recognition of auto- and exoantigens by V4-34 gene encoded antibodies.

The antigenic specificities of 24 V4-34-encoded monoclonal antibodies were compared with the amino acid sequence. The specificities were divided into three categories, red blood cells, B lymphocytes and auto/exoantigens. Six anti-I monoclonal antibodies, with multiple substitutions in their VH region, did not bind B lymphocytes or auto/exoantigens. Reactivity to these two antigens segregated with the 16 anti-i monoclonal antibodies, which were derived from the near germline V4-34 gene. All anti-i monoclonal antibodies bound B lymphocytes, albeit with varying intensities. B-cell binding correlated with basic amino acids in the VH-CDR3. Reactivity to auto/exoantigens was demonstrated only by a subset anti-i monoclonal antibodies and did not correlate with B-lymphocyte or i-antigen binding. These anti-ssDNA reactive monoclonal antibodies had basic amino acids in the VH-CDR3, strongly supporting the suggested role of arginine in DNA binding. However, an arginine-rich CDR3 was not enough to ensure DNA reactivity, since six other anti-i monoclonal antibodies that fulfilled this criteria did not bind ssDNA. Thus it is possible that the anti-DNA reactivity of V4-34-encoded monoclonal antibodies is mediated by the classic antigen-binding groove generated by the CDRs of the heavy/light chains. In contrast, anti-B-cell/i-antigen reactivity is mediated, unconventionally, by the V4-34 protein with a dominant influence of the VH-CDR3.

VH4-34 encoded antibodies in systemic lupus erythematosus: a specific diagnostic marker that correlates with clinical disease characteristics.

OBJECTIVE: To determine the clinical significance of elevated serum levels of VH4-34 encoded antibodies (VH4-34 Ab) with respect to the diagnosis and clinical characteristics of systemic lupus erythematosus (SLE). METHODS: Ninety-five patients with SLE and 344 controls were studied. The controls included 34 healthy individuals, 282 patients with nonautoimmune diseases, and 28 patients with autoimmune diseases other than SLE. VH4-34 Ab levels were measured by inhibition ELISA using anti-idiotope monoclonal antibody (9G4). SLE disease activity, severity, and damage were assessed by visual analog scales, Systemic Lupus Activity Measure, Lupus Severity of Disease Index, and Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. RESULTS: Fifty-two of 95 patients with SLE had elevated levels of VH4-34 Ab compared to 18 of 344 controls (5%), giving a sensitivity of 55% and a specificity of 95% for elevated VH4-34 Ab as a serologic test for SLE. The positive predictive value of elevated VH4-34 under these conditions was 74-85%. In this study, anti-dsDNA was not VH4-34 encoded. Significant correlations between VH4-34 and disease activity and severity indices were observed (r = 0.29-0.50). The relative risk for severe disease in SLE patients with VH4-34 antibody level in the highest tertile compared to the lowest tertile was 5.25. Twenty-five of 29 patients with lupus nephritis and 6 of 6 patients with central nervous system (CNS) lupus had elevated VH4-34 Ab. CONCLUSION: With a specificity of 94-95%, the VH4-34 antibody assay may prove valuable as a confirmatory diagnostic test for SLE. In patients with known SLE, serum VH4-34 Ab levels correlate with overall disease severity and activity, but not damage, and with nephritis and CNS lupus.

Cytotoxicity of murine B lymphocytes induced by human VH4-34 (VH4.21) gene-encoded monoclonal antibodies.

We have previously described specific binding and cytotoxicity of human B lymphocytes by VH4-34 gene-derived anti-i cold agglutinin (CA) mAbs. Here we demonstrate that the carbohydrate ligand recognized by human VH4-34 anti-i CA mAbs is also expressed on murine B lymphocytes. Similar to human B cells, binding of murine B lymphocytes by VH4-34-derived anti-i CA mAbs leads to rapid cytotoxicity of target cells as tested both in vitro and in vivo. Moreover, the mechanism leading to murine B cell death is also similar to human B cells, since morphologically identical membrane pores were detected within 15 min of mAb exposure by scanning electron microscopy. The conservation of the carbohydrate ligand across species provides an ideal system to study the function of human VH4-34 gene derived Abs in immune regulation.

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.

Heavy chain variable gene usage by human B-1 lymphocytes and polyreactive autoantibodies.

To evaluate the role of B-1 cells and polyreactive autoantibodies in the development of adult immune repertoire, it is necessary to assess their immunoglobulin heavy chain variable-gene usage. We thus screened 28 independently derived human polyreactive MAbs from fetal and adult splenic B lymphocytes for their VH-region usage. We demonstrate that the polyreactivity of the IgM antibodies secreted by B-1 cells is not the result of the expression of particular variable region gene families. All six VH families are represented roughly in proportion to their estimated family size. Furthermore, the representation of the six families appears similar in polyreactive MAbs derived from fetal or adult lymphocytes.

VH4-34 (VH4.21) gene expression in the chronic arthritides of childhood: studies of associations with anti-lipid A antibodies, HLA antigens, and clinical features.

OBJECTIVE: To determine if the germ line gene VH4-34 (VH4.21) encodes the antimonophosphoryl lipid A (MPL) polyspecific antibodies found in oligoarticular arthritis of childhood. METHODS: Sera from a range of rheumatic diseases of childhood were assayed for VH4-34 derived antibodies by ELISA using the antiidiotype monoclonal antibody 9G4. Results were compared to assays for anti-MPL antibodies, C4d, and Bb, and for HLA type, joint count, and sedimentation rate. RESULTS: VH4-34 derived antibodies were elevated in all diseases studied except rheumatoid factor positive polyarticular disease. In oligoarticular arthritis, VH4-34 gene expression correlated with C4d concentration, and VH4-34 encoded globulins were more concentrated in synovial fluid than in blood. No association was found with HLA type. An association between VH4-34 expression and IgG anti-MPL was found in sera from patients from Cincinnati but not from Stanford. No other evidence supported a direct association between VH4-34 derived and anti-MPL antibodies in these children. CONCLUSION: The expression of VH4-34 is increased in several rheumatic diseases of childhood, but, as in adults, not in rheumatoid arthritis. VH4-34 expression is not associated with HLA type. The polyspecific autoantibody nature of some VH4-34 derived antibodies may explain the wide range of the unusual antibodies found in oligoarticular arthritis.

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

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, 3H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20+ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing with greater at 4 degrees C than 37 degrees C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular process and independent of complement, suggests a novel mechanism of all death via membrane perturbations.

Anti-endotoxin human monoclonal antibody A6H4C5 (HA-1A) utilizes the VH4.21 gene.

Human IgM monoclonal antibody A6H4C5 was manufactured by Centocor (Malvern, PA) and used in clinical trials as HA-1A (Centoxin). In vitro, A6H4C6 binds to lipid A and rough-strain, gram-negative bacteria endotoxin. Further analysis of A6H4C5 has shown that it is a polyreactive, cold agglutinin that utilizes the VH4.21 gene segment in germline configuration. It is also a human antibody that binds to human B cells. We have characterized several other independently derived VH4.21 human monoclonal antibodies with the same characteristics as A6H4C5. This group of antibodies may represent a conserved host immune response.

Human CD5+ B lymphocytes (B-1 cells) decrease in peripheral blood during pregnancy.

Pregnancy is a unique immunologic state where a natural homeostasis exists between antigenically different tissues. Several earlier studies have addressed the fluctuations in the number and/or function of lymphocytes, including B cells during pregnancy, but changes within the subsets of B lymphocytes, conventional (CD5-) and B-1 (CD5+), have not been addressed. Here we demonstrate that the frequency of B-1 cells decreases dramatically during pregnancy, whereas the frequency of conventional B cells remains relatively constant. The missing B-1 cells return to pre-pregnancy levels 8-10 weeks after parturition. The polyreactive autoantibodies secreted by B-1 cells have been implicated in autoimmunity and immune regulation. The possible role of B-1 cells during pregnancy will be discussed in that context.

FACS analysis of peritoneal lymphocytes in ovarian cancer and control patients.

In this study different lymphocyte populations in the malignant ascites of 10 patients with ovarian carcinoma and in the peritoneal fluid of 8 control patients (tubal ligation and benign conditions) were analyzed. A panel of monoclonal antibodies against the CD markers of lymphocytes was used to stain different populations and the cells were analyzed on a FACS II (fluorescence-activated cell sorter). The mean percentage of B lymphocytes in the peritoneal cavity of the OVCA patients was 0.18 +/- 0.5% and in the control patients 0.05 +/- 0.07%. There was no significant difference between the two groups. In the OVCA group and in the controls the percentage of T lymphocytes (CD5+) was 23.5% and 17.1% respectively with no significant difference between the groups. These results indicate that B lymphocytes are not present in the human peritoneal cavity. The small numbers of B cells found in this study could be due to contamination with peripheral blood. The human peritoneal cavity contains a cell population which differs from that present in peripheral blood. Significant numbers of B lymphocytes have been reported in the peritoneal cavity of mice. The difference between the lymphocyte population of the human peritoneal cavity and that of rodents implies that data on characterization and function of B lymphocytes in the mouse peritoneal cavity would not be applicable to humans.

Human antilipid A monoclonal antibodies bind to human B cells and the i antigen on cord red blood cells.

We describe two independently derived human mAb, A6(H4C5) and 216, initially selected for their reactivity to the lipid A domain of bacterial LPS, which also react with the following Ag: the i Ag present on cord RBC, a ligand on human B lymphocytes, and to certain autoantigens, defining these mAb as polyreactive. Both mAb have specific affinity for a carbohydrate epitope consisting minimally of a disaccharide with an acyl substitution at the 2-carbon position. Structural examination of the diverse Ag recognized by the two antibodies reveals the presence of this carbohydrate structure required for antibody binding. A6(H4C5) and 216 are IgM in isotype, but differ in their L chain expression. Molecular analysis shows that both the mAb are encoded by a highly conserved VH4 gene, designated VH4-21. This gene encodes a number of autoantibodies, particularly cold agglutinins. Specific recognition of lipid A and of a carbohydrate epitope on B lymphocytes by the two human mAb suggests a dual function for the highly conserved VH4-21 gene in antibacterial response and in B cell development and regulation.

Lipopolysaccharide and peptidoglycan share binding sites on human peripheral blood monocytes.

p73, a binding site for lipopolysaccharide (LPS) on human peripheral blood monocytes was identified using the radiolabeled photoaffinity cross-linker sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). The 125I-labeled conjugate of SASD and LPS (125I-labeled ASD-LPS) was bound to monocytes and UV cross-linked, and the cellular extracts were analyzed with two-dimensional SDS-PAGE and autoradiography. In addition to the major binding site on human monocytes at 73 kDa, isoelectric point 5.95, there were multiple minor binding sites that recognized both smooth and rough LPS. Binding of 125I-labeled ASD-LPS to monocytes is concentration dependent, decreased in the absence of calcium and magnesium, and inhibited by either excess LPS or the low-molecular-weight soluble isolate of bacterial cell wall peptidoglycan (sPGN). However, sPGN only minimally stimulates tumor necrosis factor (TNF) secretion by human peripheral blood mononuclear cells. In contrast, the relatively insoluble high-molecular-weight peptidoglycan significantly stimulates TNF secretion.

One-step enrichment of nucleated red blood cells. A potential application in perinatal diagnosis.

We describe a discontinuous triple density gradient to obtain a 25-fold enrichment of nucleated red blood cells from mononuclear cells, granulocytes, and mature red blood cells in a single centrifugation step.

The ontogeny and functional characteristics of human B-1 (CD5+ B) cells.

We demonstrate that, on average, greater than 90% of B lymphocytes in fetal spleen express CD5 at gestational ages of 17-23 weeks. Similarly, CD5+ B cells (B-1 cells) are the major B cell subset in umbilical cord blood. These findings depend on the optimization of fluorochrome conjugated anti-CD5 reagents for multiparameter fluorescent-activated cell sorter (FACS) analysis. From infancy through childhood the percentage of B-1 cells gradually diminishes in both spleen and peripheral blood. Stable adult levels, 25-35% of the total B cell population, are reached in late adolescence. The decrease in the percentage of B-1 cells in spleen is accompanied by an increase in conventional (CD5-) B cells, keeping the percentage of total B cells per mononuclear cells relatively constant. In contrast, in peripheral blood, the concentration of both B-1 cells and total B cells decreases, while T cells increase. At the functional level, we show that polyreactive IgM autoantibodies are produced by FACS-sorted CD5high B cells, but not by CD5- B cells from adolescent spleen. In contrast, fetal splenic CD5high and CD5- B cells appear functionally uniform, both producing IgM autoantibodies that are typical of B-1 cells. The apparent level of CD5- B cells in fetal spleen, on average 10% of total B cells, may still result from limitations of our reagent. The prominence of B-1 cells in fetal spleen and cord blood, the gradual reduction of B-1 cells with increasing age, and its characteristic repertoire, all suggest a role for this cell type in immunologically immature hosts.

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies.

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.

Effects of anti-lipid A human monoclonal antibody on lipopolysaccharide-induced toxicity to the kidney.

Studies were done to evaluate the effects of the human monoclonal anti-lipid A IgM antibody A6(H4C5) on several components of the hemodynamic and renal toxicity of the cell wall lipopolysaccharide of E. coli 0111:B4. Antibody (0.25 to four mg./kg. BW) was administered 0.5 hour before, or premixed for one hour with, lipopolysaccharide (0.05 mg./kg., a 14 to 18% lethal dose), and the following measurements made over 0.5 to 3.5 hours of study: systemic arterial blood pressure, renal plasma flow, and glomerular filtration. The proximal tubular cell cytotoxicity of 90 mg./kg. of the cephalosporin cephaloridine was also quantified in similarly treated animals sacrificed 48 hours later. While one mg./kg. of antibody prevented the reduction by the lipopolysaccharide of renal plasma flow, it did not prevent the nephrotoxic synergy with cephaloridine, and four times the antibody dose did not prevent lipopolysaccharide-induced hypotension or reduced glomerular filtration. These amounts of this antibody protect leukopenic rabbits against the lethality of the slow onset bacteremic model of Pseudomonas conjunctivitis. It is suggested that the incompleteness of protection in this study may be the result of the sensitivity of the assay methods used and/or the acute endotoxemia produced in these animals.

Biology and virology of the human malignant lymphomas: 1st Milford D. Schulz Lecture.

Permanent cell lines have been established from twelve diffuse histiocytic lymphomas (SU-DHL-1 to -12), three American Burkitt's lymphomas (SU-AmB-1 to -3), two acute lymphoblastic leukemias (SU-ALL-1 and -2), and three diffuse undifferentiated lymphomas (SU-DUL-1, -2, and -3). The cultured cells displayed neoplastic characteristics, as manifested by heterotransplantability in congenitally athymic nude mice and by the presence of cytogenetic abnormalities in early passage generations. Functional and marker studies revealed that the three American Burkitt's lymphomas, as well as several of the diffuse histiocytic and undifferentiated lymphomas, were of B-lymphocytic origin, whereas the two acute lymphoblastic leukemias were both of T-lymphocytic origin. Two of the cell lines, SU-DHL-1 and -2, appeared to be of true histiocytic origin; two others exhibited no markers and were designated as "null" cells. All ten of the DHL cell lines studied to date, as well as SU-DUL-1, have been devoid of Epstein-Barr virus (EBV) genomes by the EBNA test, whereas two of the three American Burkitt's lymphoma cell lines were positive. Spontaneous production of a C-type RNA virus was first detected in post-mitochondrial cytoplasmic fractions and culture fluids of the SU-DHL-1 cell line. Screening assays for the detection of reverse transcriptase-positive particles in the culture fluids of the other cell lines indicate that eight of the fifteen cell lines tested to date have spontaneously initiated C-type RNA virus production. After partial purification by ion-exchange and affinity chromatography, the reverse transcriptases of the virus isolated from SU-DHL-1 cells is partially inhibited by antibodies to the reverse transcriptases of C-type viruses of subhuman primate and endogenous feline, but not of murine, origin. Conversely, antibody prepared against the purified SU-DHL-1 viral reverse transcriptase, at concentrations which maximally inhibit the homologous enzyme, partially inhibits the reverse transcriptases of subhuman primate C-type viruses, but has little or no inhibitory activity against the reverse transcriptases of feline or murine leukemia viruses. The viruses produced by the SU-DHL-1 and SU-AmB-3 cell lines have been shown to be infectious for normal human peripheral blood mononuclear cells, normal human bone marrow cells, and certain human lymphoblastoid cell lines. After infection by these viruses, normal human peripheral blood mononuclear cells and human bone marrow cells have exhibited striking changes in growth behavior and morphology which, though not permanently sustained, have many of the features of abortive transformation.