Lymphocyte-derived Cell Lines Research Articles

Abstract 2639: KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers

Abstract MYC is an early-response gene downstream of many receptor complexes, and a component that promotes several pro-growth signal transduction pathways. As a result, MYC expression is highly regulated, but once dysregulated in cancer, MYC promotes tumorigenesis via activation of cell growth, proliferation, and angiogenesis. CDK9 is both an upstream and downstream cofactor of MYC. CDK9 promotes MYC expression through its recruitment to super enhancers at the MYC amplicon and acts as a cofactor to drive gene expression programs. Previously, we profiled pan-cancer sensitivity to KB-0742, a potent, selective, and orally bioavailable small-molecule inhibitor of CDK9, and identified MYC-amplified cancers as being especially sensitive to KB-0742. To better understand the connection between MYC and KB-0742 sensitivity, 11 different tumor types were evaluated preclinically using immortalized cell lines, patient-derived cell lines (PDCs), patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). The 11 indications included head and neck, esophageal, gastric, liver, non-small cell lung cancer (NSCLC), pancreatic, ovarian, triple-negative breast cancer (TNBC), prostate, colon, and lymphoma. For 5 of the indications (head and neck, esophageal, gastric, liver, and colon), little activity was observed in cell lines and in vivo with weak correlations to MYC amplification. TNBC, ovarian cancer, and lymphoma showed good responses to KB-0742 treatment. For TNBC and ovarian, immortalized and patient-derived cell lines indicated lower IC50 values with increased MYC amplification or expression, but the results were not statistically significant. However, in vivo assessments using 15 PDX models showed good correlation of tumor growth inhibition (TGI) and MYC amplification/expression, with some tumor regressions observed in several ovarian models. Two PDO models of TNBC with different treatment histories were also treated with KB-0742 and compared to 4 standard-of-care (SOC) compounds. KB-0742 showed much greater activity in the models, with maximal inhibition rates of 100% and 89%. Cell-line screens of blood cancer-derived cell lines showed that lymphocyte-derived cell lines were the most sensitive to KB-0742 as compared to all other cancer types. We tested several PDX models for response to our compound. Treatment with KB-0742 resulted in TGIs of over 50% in several lymphoma models, including a TGI of 56% in 1 model of double-hit diffuse large B-cell lymphoma. These data support the continual development of KB-0742, which is currently being tested in a phase 1/2 clinical trial (NCT04718675). The dose-escalation phase is opened to relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommend phase 2 dose is determined, we plan to open dedicated expansion arms enrolling patients with MYC over-expression/amplification within the tumor types supported by these studies. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2639.

Auto-antibodies against apolipoprotein A-1 block cancer cells proliferation and induce apoptosis.

Auto-antibodies against apoA-1 (anti-apoA-1 IgGs) have been identified as important actors of atherosclerosis development through pro-inflammatory and pro-atherogenic properties and to also induce apoptosis in tumoral neuronal and lymphocyte derived cell lines through unknown mechanisms. The purpose of this study was to explore the cellular pathways involved in tumoral cell survival modulated by anti-apoA-1 antibodies. We observed that anti-apoA-1 antibodies induce growth arrest (in G2/M phase) and cell apoptosis through caspase 3 activation, accompanied by a selective p53 phosphorylation on serine 15. RNA sequencing indicated that anti-apoA-1 IgGs affect the expression of more than 950 genes belonging to five major groups of genes and respectively involved in i) cell proliferation inhibition, ii) p53 stabilisation and regulation, iii) apoptosis regulation, iv) inflammation regulation, and v) oxidative stress.In conclusion, anti-apoA-1 antibodies seem to have a role in blocking tumoral cell proliferation and survival, by activating a major tumor suppressor protein and by modulating the inflammatory and oxidative stress response. Further investigations are needed to explore a possible anti-cancer therapeutic approach of these antibodies in very specific and circumscribed conditions.

Profiling the B/T cell receptor repertoire of lymphocyte derived cell lines

BackgroundClonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations.MethodsWe systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data.ResultsRearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution.ConclusionsOur analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines.

Molecular mechanisms for Kv1.3 potassium channel current inhibition by CD3/CD28 stimulation in Jurkat T cells

In T lymphocyte, activation of Kv1.3 channel, the major voltage-dependent K+ channel, is an essential step for cell proliferation in immune responses. Here, effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current were examined in three types of human T lymphocyte derived cell lines, Jurkat E6-1, p56lck-kinase deficient mutant JCaM.1, and CD45-phosphatase deficient mutant J45.01. Kv1.3 current was partly reduced by CD3 stimulation and more strongly by addition of anti-CD28 antibody in E6-1. In JCaM.1, Kv1.3 current responses to anti-CD28/CD3 antibodies were similar to those in E6-1. In J45.01, CD3 stimulation partly inhibited Kv1.3 current, but the additive reduction by CD28 stimulation was not significant. The inhibition of tyrosine phosphatase in E6-1 abolished the additional inhibition by anti-CD28 antibody in a similar manner as in J45.01. In conclusion, the stimulation of CD28 in addition to CD3 strongly inhibits Kv1.3 current and this additive inhibition is mediated by CD45 activation.

Possible involvement of post-dopamine D2 receptor signalling components in the pathophysiology of schizophrenia

Par-4 has been suggested to mediate dopamine neurotransmission. Dopamine D2 receptor (DRD2) activation induces a signalling complex of AKT1, PP2A and beta-arrestin2 which dephosphorylates/inactivates AKT1 thereby activating GSK-3beta, transducing dopamine-dependent behaviour. DRD2 activation also results in down-regulation of PKA activity. Among other substrates PKA phosphorylates GSK-3beta. Prolonged DRD2 activation leads to its 'desensitization' which involves GRKs and beta-arrestins. beta-arrestin1 binds to phosphorylated receptors preventing further G-protein stimulation. This study examined whether Par-4, beta-arrestin1, AKT1 and GSK-3beta are involved in the pathophysiology of schizophrenia. Lymphocytes obtained from schizophrenia and bipolar patients and healthy controls recruited from the Beer-Sheva Mental Health Center were transformed by Epstein-Barr virus (EBV) into lymphocyte-derived cell lines (LDCL). Post-mortem brain samples were obtained from the Rebecca L. Cooper Brain Bank, Parkville, Australia. The study was approved by the IRB committees of Beer-Sheva, Israel and Parkville, Australia. Levels of the specific proteins were assayed by Western blotting. beta-arrestin1 protein levels were significantly ~2-fold increased in LDCL from schizophrenia patients while Par-4 protein levels were unaltered. A 63% significant decrease was found in frontal cortex phospho-Ser9-GSK-3beta protein levels in schizophrenia but not in those of AKT1, Par-4 or beta-arrestin1. Elevated beta-arrestin1 protein levels in LDCL and decreased phospho-Ser9-GSK-3beta protein levels in post-mortem frontal cortex of schizophrenia patients vs. control groups support the possible involvement of these proteins in the pathophysiology of schizophrenia. However, since we did not find differences in beta-arrestin1, AKT1 and Par-4 protein levels in post-mortem frontal cortex of schizophrenia patients and although GSK-3beta participates in other signalling cascades we can not rule out the possibility that the differences found reflect deviation in DRD2 signalling.

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

BackgroundHuman T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation.ResultsHerein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes.ConclusionCollectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival.

Human normal T lymphocytes and lymphoid cell lines do express alternative splicing variants of human telomerase reverse transcriptase (hTERT) mRNA

Alternative splicing of telomerase reverse transcriptase (hTERT) mRNA is known to contribute to regulation of telomerase activity in normal and cancerous cells, however, previous studies indicated that normal human T and B cells exhibited constitutive expression of full-length hTERT mRNA without splicing variants and that activation of telomerase upon stimulation of the cells was due to the shuttling of hTERT protein from cytoplasm to nucleus [Proc. Natl. Acad. Sci. USA 96 (1999) 5147; J. Immunol. 166 (2001) 4826]. We found that typical variants of hTERT mRNA were widespread in human lymphocyte-derived cell lines and normal stimulated T cells. In activated T cells, induction of the full-length hTERT mRNA was coupled with increased hTERT protein expression and telomerase activity. Collectively, human normal and malignant lymphocytes, like other human cells, express splicing variants of hTERT mRNA and require transcriptional activation of the hTERT gene to acquire telomerase activity.

Functional nicotinic acetylcholine receptors are expressed in B lymphocyte-derived cell lines.

Nicotine has been shown to affect B lymphocyte immune response. In this study, we have explored the presence of nicotinic receptors in B lymphocyte-derived cell lines, myeloma X63-Ag8 and hybridoma 1D6. We found that myeloma expressed on average 10,170 +/- 1,100 [3H]epibatidine and 6,730 +/- 370 125I-alpha-bungarotoxin binding sites per cell, thus reflecting the presence of both homomeric and heteromeric nicotinic receptors. More specifically, the presence of alpha4- and alpha7-containing nicotinic receptor subunits was demonstrated in both myeloma and hybridoma cells with subunit-specific antibodies. It was significantly higher in dividing than in resting cells. Long-term exposure to nicotine, at physiological concentration found in smokers, resulted in up-regulation of both alpha4 and alpha7 subunits in hybridoma cells. Additionally, nicotine stimulated hybridoma cell proliferation, whereas it decreased antibody production. In contrast, alpha7-specific snake toxins inhibited cell proliferation but increased antibody production. It is concluded that myeloma and hybridoma cells express alpha4- and alpha7-containing nicotinic receptors, which participate in regulating cell proliferation and function.

GSK-3 parameters in lymphocytes of schizophrenic patients

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase that is involved in the signal transduction cascades of multiple cellular processes. GSK-3 has two isoforms, designated α and β. GSK-3β protein levels and GSK-3 enzyme activity have been reported to be reduced by over 40% in postmortem frontal cortex of schizophrenic patients. GSK-3 is also present in peripheral tissue such as lymphocytes. In this study we aimed to find whether the reduction in brain GSK-3β measures is reflected in peripheral tissue of schizophrenic patients. Fresh lymphocytes from schizophrenic patients showed no difference in GSK-3α and GSK-3β mRNA levels, GSK-3β protein levels, or total GSK-3 (α+β) enzyme activity compared with findings in control subjects. In addition, lymphocyte-derived cell lines from schizophrenic patients did not differ in their GSK-3β protein levels from levels in normal control subjects. The results rule out the use of lymphocyte GSK-3 as a marker for central GSK-3 abnormalities in schizophrenia.

Reduced inositol content in lymphocyte-derived cell lines from bipolar patients.

The study aimed to determine whether low inositol content and uptake previously reported in brain and peripheral tissue of bipolar patients are also reflected in lymphocyte-derived cell lines from these patients. Inositol content and uptake were studied in lymphocyte-derived cell lines grown in vitro for at least five generations to eliminate influences of drug treatment. Inositol content was studied gas chromatographically and inositol uptake by following 3H-inositol incorporation at various concentrations. Inositol levels of cell lines derived from bipolar patients were significantly lower than those of cell lines from controls. Low inositol content in lymphocyte-derived cell lines from bipolar patients corroborates previous findings in frontal cortex and in lymphoblastoid cell lines and are consistent with the notion that the phosphatidylinositol signaling system is involved in the pathophysiology of this disorder.

Inositol Monophosphatase Activity in Brain and Lymphocyte-Derived Ceii Lines of Bipolar Patients

Background: Inositol monophosphatase (IMPase) activity was reported to be low in lymphocyte-derived cell lines of bipolar patients.Methods: IMPase activity was measured spectrophotometrically as inorganic phosphate liberated from inositol-1-phosphate.Results: The previously reported reduction was replicated in a new, small group of bipolar patients. The reduction is not present in cell lines of unipolar or schizophrenic patients. IMPase activity in postmortem frontal and occipital cortical samples of unipolar, bipolar and schizophrenic patients was not different from controls.Conclusions: A reduction in lymphocyte-derived IMPase activity without a parallel reduction in cortical IMPase activity could be due to the fact that most leukocyte IMPase activity is the product of the IMPA-2 gene.

Therapeutic ultrasound induces apoptosis in p53 positive and p53 negative malignant hemopoietic cell lines

Abstract None of the modalities currently employed to induce apoptosis involves the use of ultrasound energy. We have demonstrated (Cancer Res. 60: 1014, 2000) that selected physical parameters utilized in therapeutic ultrasound (ULS) application resulting in cavitation process induce an apoptotic cell death. The aim of the present study was to investigate ULS application with rational selected physical parameters to optimize ultrasound cavitation- induced cell death and to determine whether apoptosis is involved. High intensity focussed ULS sonication was delivered with an induction of transient cavitation and intensity of 54 W/cm 2 to malignant T and B lymphocyte-derived cell lines expressing p53 as Raji, Jurkat and NALM-6 as well as myeloid leukemia cell lines p53 negative HL-60, K562, U937. Here we present evidence that much of the cell damage surviving ULS exposure appears to occur through an apoptotic mechanism. Morphological alterations of apoptotic cells involve nuclear fragmentation and apoptotic body formation. Positive identification of apoptotic cells was based also on the detection of nuclear DNA strand break and changes in the surface of treated cells undergoing apaptosis expressed by the breakup of phosphatidylserine from the inner to the outer side of the membrane layer. In summary, after ULS exposure malignant hemopoietic cell lines can be induced to undergo apoptosis by p53-dependent and p53-independent apoptotic pathways.

Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors.

Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID(50) of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.

Oxidative stress and S-nitrosylation of proteins in cells.

The effect of prolonged exposure to nitric oxide on enzymes involved in cell metabolism was investigated in T lymphocyte-derived Jurkat and L929 fibroblast human cell lines using a constant concentration of nitric oxide (1.5 microM) released by the nitric oxide donor DETA-NO (0.5 mM). Nitric oxide inhibited immediately the respiration of the cells acting reversibly at complex IV. With time, the inhibition became progressively persistent, i.e. not reversed by trapping of nitric oxide with oxyhaemoglobin, and was preceded by a decrease in the concentration of the intracellular reduced glutathione. This persistent effect of nitric oxide on respiration was due to inhibition of complex I activity which could be reversed by addition of reduced glutathione or by cold light, suggesting that it was due to S-nitrosylation of thiols necessary for the activity of the enzyme. The activity of other enzymes also known to be susceptible to inhibition by S-nitrosylation, i.e. glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase, was progressively decreased by exposure to nitric oxide with a similar time course to that observed for the inhibition of complex I. Furthermore, inhibition of these enzymes only occurred when the concentrations of reduced glutathione had previously fallen and could be prevented by increasing the intracellular concentrations of reduced glutathione. Our results suggest that S-nitrosylation of different enzymes by nitric oxide may occur only if the reducing potential of the cells is impaired.

Reduced frontal cortex inositol levels in postmortem brain of suicide victims and patients with bipolar disorder.

This study aimed to evaluate aspects of second messenger function in the brain of suicide victims and patients with bipolar disorder. Inositol and its synthetic enzyme, inositol monophosphatase, were measured in postmortem brain samples of 10 suicide victims, eight patients with bipolar affective disorder, and 10 normal comparison subjects. The frontal cortex inositol levels of the suicide victims and the patients with bipolar disorder were significantly less than those of the normal comparison group. No differences in cerebellum or occipital cortex inositol levels were found among the three groups. The groups also showed no differences in inositol monophosphatase activity in any brain area. These results could suggest a deficiency of second messenger precursor in patients with bipolar disorder and suicide victims.

Modulation of Radiation-Induced Apoptosis and G 2 /M Block in Murine T-Lymphoma Cells

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to 137Cs gamma irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of gamma radiation. We studied the effect of several pharmacological agents on the radiation-induced G2/M block and DNA fragmentation. The agents which reduced the radiation-induced G2/M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G2/M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G2/M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G2/M block, and to use this effect in the treatment of patients with malignant disease.

Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme

Toward gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections in AIDS, Moloney murine leukemia virus-derived retroviral vectors were engineered to allow constitutive and tat-inducible expression of an HIV-1 5' leader sequence-specific ribozyme (Rz1). These vectors were used to infect the human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants expressing an HIV-1 RNA-specific ribozyme, under the control of the herpes simplex virus thymidine kinase (tk) promoter, were found to be somewhat resistant to HIV-1 infection as virus production was delayed. In cells allowing ribozyme expression under control of the simian virus 40 or cytomegalovirus promoter, the rate of HIV-1 multiplication was slightly decreased, and virus production was delayed by about 14 days. The highest level of resistance to HIV-1 infection was observed in MT4 cells transformed with a vector containing a fusion tk-TAR (trans activation-responsive) promoter to allow ribozyme expression in a constitutive and tat-inducible manner; no HIV-1 production was observed 22 days after infection of these cells. These results indicate that retroviral vectors expressing HIV-1 RNA-specific ribozymes can be used to confer resistance to HIV-1 infection.

Cell Surface Vitamin D-Binding Protein (GC-Globulin) is Acquired from Plasma*

Vitamin D-binding protein (DBP) is an abundant plasma protein. The observation of immunodetectable, cell-associated DBP on peripheral blood mononuclear cells and placental cytotrophoblasts had presented the question of the origin, function, and precise subcellular localization of cell-associated DBP. Using anti-human DBP F(ab')2 with fluorescence-activated cytometric analysis and immunogold electron microscopy, we detected DBP on the plasmalemma of U937 cells, a monoblastic, histiocytic cell line grown in media supplemented with fetal calf serum (FCS). DBP was then removed from FCS by actin affinity chromatography followed by anti-DBP immunoaffinity chromatography. U937 cells in this DBP-free medium exhibited nearly identical growth rates to cells grown in medium containing native FCS. However, in contrast to cells grown with native FCS, those grown for seven to eight generations with DBP-free FCS exhibited less cell-surface DBP as quantified by fluorescence-activated cytometric analysis (73% decrease) and immunoelectron microscopy (88% decrease). DBP mRNA could not be detected in U937 cells, placental tissues, freshly prepared resting and stimulated B and T lymphocytes, or lymphocyte-derived cell lines by Northern analysis. In addition, using the sensitive reverse transcriptase/polymerase chain reaction assay no DBP fragments were detectable in U937 cells. We conclude that U937 cell-associated DBP is exogenously derived from plasma and is located on the plasmalemma. Based upon this conclusion, we postulate that specific binding sites for DBP may exist on the plasma membranes of certain cell types.

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas.

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.