anti-T Monoclonal Antibody Research Articles

Homogeneous time-resolved fluoroimmunoassay of 3,5,3′-triiodo- l-thyronine in human serum by using europium fluorescence energy transfer

A sensitive homogeneous time-resolved fluoroimmunoassay method for 3,5,3′-triiodo- l-thyronine (T3) based on the fluorescence resonance energy transfer (FRET) from a fluorescent Eu 3+ complex, {[(4,6-dichloro-1,3,5-triazin-2-yl)amino-biphenyl-4‴-yl]-2,2′:6′,2″-terpyridine-6,6″-diyl}bis(methylenenitrilo) tetrakis(acetate)-Eu 3+ (DTBTA-Eu 3+) ( λ ex,max = 335 nm, λ em,max = 615 nm), to an organic fluorescence dye Cy5 has been developed. The new assay system combined the use of DTBTA-Eu 3+-labeled T3-bovine serum albumin (BSA) conjugate and Cy5-labeled anti-T3 monoclonal antibody for a competitive-type immunoassay. After the competitive reactions of DTBTA-Eu 3+-labeled T3-BSA and T3 sample with Cy5-labeled anti-T3 antibody, the T3 concentration was measured with a time-resolved mode by monitoring the sensitized emission of Cy5 derived from FRET in a homogeneous format. The method gives the detection limit of 0.26 ng/ml. The coefficient variations of the method are less than 2.0% and the recoveries are in the range of 80–111% for serum sample measurement. The concentrations of T3 in 30 human serum samples were determined, and the results were compared with those of the independently determined by a radio-immunoassay method. A good correlation was obtained with a correlation coefficient of 0.989.

Biotinylated Steroid Derivatives as Ligands for Biospecific Interaction Analysis with Monoclonal Antibodies Using Immunosensor Devices

Systematic ligand-binding studies of the biospecific interaction between steroids and antisteroid antibodies can be performed in real time using biosensor techniques. In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensor systems were applied. Different biotinylated testosterone (T) and 17β-estradiol (E2) derivatives were preincubated with streptavidin and immobilized on the sensor surfaces. We obtained low matrix densities of antigen enabling the investigation of the binding kinetics and position specificities of various anti-E2 and anti-T monoclonal antibodies (mAbs) to these steroidal compounds. The highest immunoreactivity of anti-E2 and anti-T mAbs is not necessarily for the specific modified steroid that was used as a protein-coupled hapten for immunization. The kinetic data confirm that both 3- and 19-specific anti-T mAbs do not discriminate between the 3- and 19-biotinylated T derivatives, whereas the 7α-biotinylated T probe showed no affinity to these two anti-T mAbs. In the case of the 3-specific anti-E2 mAb, comparable interaction data were found for 3- and 6α-biotinylated E2 compounds. The 6-specific anti-E2 mAb showed comparable ligand binding, but a significant higher dissociation rate to the position-specific antigen. The QCM and SPR results correspond well to the data from cross-reactivity studies in solution as well as to enzyme immunoassay equilibrium measurements.

Expression of Sialosyl-T and Disialosyl-T Antigens in Erythroid Cells

Expression of T, sialosyl-T and disialosyl-T antigens on normal blood and bone marrow cells as well as transformed cells was examined using specific monoclonal antibodies and multidimensional flow cytometry. Both anti-sialosyl-T (QSH1) and anti-disialosyl-T (QSH2) monoclonal antibodies aggregated erythrocytes. The anti-disialosyl-T antibody was specific for the erythroid lineage and did not react with neutrophils, monocytes or T-lymphocytes, while the anti-sialosyl-T antibody reacted with erythroid cells and a subset of T-lymphocytes. The developing erythroid cells in bone marrow showed coordinate expression of glycophorin A and the two carbohydrate chains, sialosyl-T and disialosyl-T. Analysis of neoplastic ceils showed that the anti-disialosyl-T antibody only reacted with glycophorin A-positive blasts from erythroleukemia (FAB M6) patients (4/4) and one patient with chronic myeloid leukemia in erythroblastic transformation (CMLET). Leukemic blasts from these patients demonstrated coordinate quantitative expression of glycophorin A and disialosyl-T. The anti-sialosyl-T antibody reacted with glycophorin A-positive blasts from FAB M6 patients (4/4) and one CMLET patient; however, the antibody also reacted with glycophorin A-negative blasts from one FAB M6 and the one CMLET patients and transformed cells from other types of leukemia. The anti-T monoclonal antibody (HH8) did not react with any of the other cells tested. These results indicate that glycophorin A and disialosyl-T expression are tightly linked during normal erythroid development and erythroid leukemogenesis.

Expression of the T antigen on a T-lymphoid cell line, supT1.

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable alpha-N-acetylgalactosaminyl-transferase and beta 1, 3 galactosyltransferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal beta 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with 3H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identified inter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.

Immunological evidence for the association between Simian Virus 40 115-kDa super T antigen and hsp70 proteins in rat, monkey, and human cells

Immunological evidence was provided that in subclone 7 cell line, which is derived from SV40 transformed cells, 115-kDa super T antigen, a transformation-competent, elongated form of large T antigen was physically complexed with hsp70 proteins. This conclusion was first based on the coimmunoprecipitation from unstressed or heat shocked subclone 7 cells of both super T antigen and hsp70 proteins. This was observed with any one of a set of anti-T monoclonal antibodies reacting to determinants located either in the C-terminal region or in the N terminal region. Reciprocally coimmunoprecipitation of both hsp70 and super T was also observed in the anti-hsp70 peptide serum-immunoprecipitate. The formation of complexes between hsp70 proteins and super T antigen in subclone 7 cells was also confirmed by Western blot experiments. Moreover, when expressed in cell lines originating from human (Hela cells) or monkey (CV1 P cells) species following transfection with the relevant plasmid, super T antigen again displayed the ability to associate with hsp70 proteins. Considering that super T antigen was obtained in laboratory experiments as a stable evolutionary variant of SV40 large T antigen, it is suggested that the marked ability of super T antigen to associate with heat shock protein could be selectively advantageous under certain conditions.

Simian virus 40 DNA replication correlates with expression of a particular subclass of T antigen in a human glial cell line

Immunocytochemistry and in situ hybridization were used to identify simian virus 40 (SV40) large T-antigen expression and viral DNA replication in individual cells of infected semipermissive human cell lines. SV40 infection aborts before T-antigen expression in many cells of each of the human cell lines examined. In all but one of the human cell lines, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Since different structural and functional classes of T antigen can be recognized with anti-T monoclonal antibodies, we examined infected A172 cells with a panel of 10 anti-T monoclonal antibodies to determine whether viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal antibody, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentage of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Finally, in situ hybridization was used to show that the percentage of human cells expressing T antigen was similar to the percentage transcribing early SV40 mRNA. Thus, the block to T-antigen expression in human cells is at a stage prior to transcription of early SV40 mRNA.

Stereotactic Biopsy Diagnosis of Primary Non-Hodgkin's Lymphoma of the Central Nervous System: A Histological and Immunohistochemical Study

We report 29 cases of primary non-Hodgkin lymphomas (NHL) of the Central Nervous System (CNS), 26 of which were diagnosed by stereotactic biopsy and 3 by autopsy. In seven cases the patients were affected by AIDS. Histological examination of this series revealed 15 cases of immunoblastic lymphoma, 12 cases of centroblastic lymphoma, 1 case of lymphoplasmacytic immunocytoma and 1 case of unclassified high grade lymphoma. By immunohistochemistry the B-cell origin of lymphoma cells was demonstrated in 28/29 cases. Eight cases were assigned to the B-cell lineage by demonstration of monotypic surface or cytoplasmic immunoglobulin or of the B-cell phenotype CD22+, CD2-, CD3-, CD5-. In twenty cases the B-cell nature of lymphoma was identified by positivity with two or more anti-B monoclonal antibodies (LN1LN2MB2) and negativity by the anti-T monoclonal antibody UCHL1. The histologically unclassified case was a peripheral T-NHL (CD1-, CD2+, CD3-, CD5+, CD22-). We conclude that histological and immunohistological evaluation of stereotactic biopsy specimens provides sufficient information for diagnosis and phenotypic characterization of primary NHL of the CNS. These lymphomas exhibit important predominance of high-grade malignancy histological types and are nearly always B-cell derived. In addition, we provide further evidence that the panel of monoclonal antibodies LN1, LN2, MB2, and UCHL1 is useful for immunophenotypic characterization of brain lymphomas when only paraffin embedded stereotactic biopsy tissue is available.

Triiodothyronine Uptake Measurement in Serum by Time-Resolved Fluorescence Immunofluorometry

A solid phase competition-type fluoroimmunoassay for triiodothyronine (T3) uptake in serum is described. In the assay, exogenous free T3 binds to the unoccupied binding sites on serum thyroxine binding proteins while the remaining unbound T3 competes with immobilized T3 for binding to a soluble biotinylated anti-T3 monoclonal antibody. The bound biotinylated antibody is quantitated by the addition of streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl-1,10 phenanthroline-2,9-bicarboxylic acid (BCPDA) in the presence of excess europium. The fluorescence signal of the final complex, which is directly proportional to the number of unoccupied binding sites on thyroxine binding proteins, is then measured on the dried solid-phase with a pulsed-laser time-resolved fluorometer. The assay requires a 10 microliters serum sample and a total incubation time of 90 minutes. The coefficients of variation for within-run and between-run assays ranged from 2.0 to 5.7%. Results obtained by the present method compared well with those determined by two commercial radioimmunoassays (r greater than 0.9).

Studies on the immunosuppressive properties of 7,25 dihydroxycholesterol-II. Effects on early steps of T-cell activation

The effects of an oxysterol, 7,25-dihydroxycholesterol (7,25-OHC), cyclosporin A (CsA) and dexamethasone (Dex) on the blastogenic response of murine lymphocytes to various stimuli were investigated. 7,25-OHC markedly depressed the rresponse to Con A, anti-T3 monoclonal antibodies and to the combination phorbol myristate acetate (PMA) + IL-2 and PMA + ionomycin. Dexamethasone was also active within the same range of concentrations. However, it did not inhibit the stimulation induced by PMA + ionomycin. On the other hand, Cs A failed to depress the lymphocyte response to IL-2 + PMA. Therefore, the mechanisms of action involved in the blockage of lymphocyte activation are different for these three compounds. Moreover, we noted that 7,25-OHC was less active upon purified T-cells. Taken together, these results suggest that 7,25-OHC may act at the level of signal transduction across the membrane.

Studies on the mechanism of polyclonal B cell stimulation by TH2 cells

Recently, it has been shown that cloned L1/1 T helper cells of type 2 (TH2-cells), when stimulated with antigen, are able to induce polyclonal B cell proliferation. Here we present evidence that this process is dependent on direct cell-cell interaction between T and B cells, which in the effector phase, i.e., during stimulation of the B cells by activated T cells, can be mediated by a mechanism other than cognate interaction. This conclusion is derived from experiments in which highly purified, small B cells of high density were polyclonally stimulated by L1/1 T cells triggered by an anti-T3 monoclonal antibody in the absence of antigen. The triggering process was independent of the presence of the Fc part of the antibody and occurred in cultures devoid of macrophages. Thus, the well-established cognate recognition does not appear to be the only mechanism of B cell induction by T helper cells.

Structural polymorphism of the human platelet Fc gamma receptor.

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.

Antigen-independent activation of T cells mediated by a novel cell surface heterodimer (Tp135-145).

A new heterodimeric structure, Tp135-145, which can mediate interleukin 2 (IL2) production and Ca2+ mobilization by Jurkat cells is described. This structure was identified by a monoclonal antibody, MX24, on the surface of either T3/TcR+ or T3/TcR- human T cell lines as well as on B cell lines. Biochemical studies showed that antibody MX24 precipitated two polypeptide chains of 135 and 145 kDa, respectively, in lysates from 125I-labeled T cells. After reduction the 135-kDa polypeptide chain shifted to 140 kDa, whereas the molecular mass of the other polypeptide remained unchanged. The apparent molecular masses of the desialylated polypeptides differed by 5 kDa. No common peptide fragments between the two polypeptide chains were found after limited proteolysis by Staphylococcus aureus V8 protease. The expression of Tp135-145 was independent of the expression of the T3/TcR molecular complex. Incubation of Jurkat cells with anti-TcR or anti-T3 monoclonal antibody induced complete modulation only of the T3/TcR complex but not of Tp135-145. Conversely complete modulation of Tp135-145 was observed after incubation of these cells with MX24 antibody. Functional studies showed that anti-Tp135-145 antibody MX24 induced high levels of IL2 production in Jurkat cells. In addition, incubation of these cells with MX24 resulted in Ca2+ mobilization from internal stores. In peripheral blood, Tp135-145 was found to be expressed by 39%-76% of resting T cells in individual donors. Two-color flow microfluorimetry showed that the Tp135-145+ cells were equally distributed on the CD4+ and CD8+ subsets. Incubation of peripheral blood T cells with antibody MX24 resulted in IL2 production and cell proliferation. Taken together these results suggest that Tp135-145 is a novel surface molecule involved in antigen-independent pathway of T cell activation.

Theophylline and the immune response: In vitro and in vivo effects

We analyzed the in vitro and in vivo effects of theophylline on various immunological parameters including proliferation of peripheral mononuclear cells (PMNC) in response to phytohemagglutinin (PHA), anti-T3 and anti-T11 monoclonal antibodies (MAb), PHA-induced interleukin-2 (IL-2) and interferon-γ (IFN-γ) production by PMNC, interleukin-1 (IL-1) production by accessory cells, PHA-induced IL-2 production by T-cell clones, and PHA- and anti-T3 MAb-induced DNA synthesis by T cell clones. Results showed that theophylline inhibited PHA- and anti-T3-induced proliferation of both PMNC and T-cell clones, whereas the PMNC proliferation induced by MAb anti-T11 was not affected. The inhibition appeared to be dose-dependent and strictly related to the presence of the drug in the culture. Moreover, PHA-induced IL-2 production by both PMNC and T-cell clones also appeared to be reduced by theophylline. IL-1 production by accessory cells was not affected. These data suggest that the immunological inhibition exerted by theophylline is confined to the T-cell compartment, mainly by acting on structure(s) related to the T3 Ti complex, the primary site for T-cell activation. The alternative pathway of T-cell activation (i.e., via T11 site) seems unaffected. In addition, these results suggest possible clinical relations between the inhibition of the immune response and the plasma levels of the drug reached after a “once daily” or “twice daily” oral ingestion of slow-release theophylline products.

SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene

Monkey cells synthesizing SV40 large T antigen were lysed and the extracts immunoprecipitated with either monoclonal anti-T antibody or monoclonal antibody to p110–114, the product of the retinoblastoma susceptibility gene ( Rb). T and p110–114 coprecipitated in each case, implying that the proteins are complexed with each other. Substitution and internal deletion mutants of T that contain structural alterations in a ten residue, transformation-controlling domain failed to complex with p110–114. In contrast, T mutants bearing structural changes outside of this domain bound to p110–114. These results are consistent with a model for transformation by SV40 which, at least in part, involves T/p110–114 complex formation and the perturbation of Rb protein and/or T function.

Effects of in vivo administration of anti-T3 monoclonal antibody on T cell function in mice. I. Immunosuppression of transplantation responses.

Anti-T3 mAb are being increasingly used clinically in the treatment of organ graft rejection. However, there has not previously been a murine model in which the effects of these mAb on the immune system could be studied in vivo. We have established such a model using the anti-murine-T3 mAb, 145-2C11. Administration of 145-2C11 led to rapid depletion of T cells from peripheral blood and suppression of skin graft rejection. However, depletion of T cells from spleen and lymph node was both delayed and incomplete. Full recovery of T cell number was dependent on the presence of a thymus, but treatment of thymectomized animals revealed that depletion was not the mechanism by which the mAb induced immunosuppression. Rather, alterations in TCR expression may play a more important role. TCR had modulated from T cells in spleen and lymph node early after treatment, and TCR expression remained subnormal for at least 51 days posttreatment. However, subnormal TCR expression alone could not fully explain the observed T cell dysfunction, inasmuch as a period of time existed after TCR re-expression during which T cells appeared to be anergic to CTL and MLR reactivity. These findings implicate T cell dysfunction as an important element in the induction of immunosuppression after anti-T3 administration.

Interleukin 4 (BSF‐1) induces growth in resting murine CD8 T cells triggered via cross‐linking of T3 cell surface structures

To analyze the role of interleukin 4 (IL4, BSF-1) during primary activation of resting (high-density) murine CD8 T cells, a model system was used which bypasses antigen-presenting cells by the use of anti-T3 monoclonal antibodies immobilized on Sepharose beads. In high, but not in low cell density cultures, IL4 alone induced cell growth. In low cell density cultures, further to T3 cross-linking a soluble macrophage product was required as co-stimulator to induce sensitivity to IL4. This co-stimulator activity was unrelated to recombinant (r)IL1, rIL6 and rTNF-alpha (tumor necrosis factor alpha). In primary CD8 T cell responses rIL4-driven growth was about half of that induced by rIL2, and not inhibitable by anti-IL2 receptor antibodies. Higher concentrations of IL4 down-regulated cell proliferation. In the course of IL4-driven growth, the proliferating cells acquired sensitivity to the growth-promoting effect of IL2. Activated CD4 or CD8 T cells were found to be equally sensitive to the IL4 and IL2-driven growth pathway. Taken together, these results define a physiologic role of IL4 as growth factor during primary activation of resting CD8 T cells and thus extend the spectrum of target cells for IL4.

An ELISA method for detecting antibodies to the T cell antigen receptor

An ELISA method for the detection of monoclonal antibodies (MAb) to the T3-T cell antigen receptor (TCR) complex was devised. The T3-TCR complex was solubilised using digitonin and a rat anti-T3 MAb (Campath 3) was used to bind it to an ELISA plate. Normal rat serum was used to block cross-reactivity between the rat MAb and peroxidase-conjugated rabbit anti-mouse immunoglobulins. The assay was tested on four T cell tumour lines and successfully detected MAbs to TCR β chain variable regions, as well as the anti-T3 MAb UCHT1. Other anti-T3 MAbs were not detected because Campath 3 blocked their binding. None of a panel of MAbs reacting with other T cell surface antigens reacted in the assay.

Anti-ovarian carcinoma anti-T3 heteroconjugates or hybrid antibodies induce tumor cell lysis by cytotoxic T-cells.

In the perspective of therapeutic in vivo targeting for T-cell attack, the monoclonal antibody (MAb) MOv18, selected for its restricted reactivity with human ovarian carcinoma, and an anti-T3 MAb were used to produce heteroconjugate or hybrid antibodies derived by fusion of relevant hybridomas. Specificity and activity of bispecific MAbs were analyzed by solid-phase RIA, immunofluorescence and a 51Cr-release assay on the ovarian carcinoma cell line OVCA 432, which expresses the relevant tumor-associated antigen, and on several irrelevant tumor cell lines. Both reagents efficiently promoted, at picomolar concentration, target cell lysis by cytotoxic T-cell (CTL) clones. Although the pattern of tumor cell lines which were lysed was wider than that predicted by binding studies, further studies using a double-determinant immunoradiometric assay confirmed the specificity of MAb targeting. Analysis of reagents indicated that the hybrid MAb was superior to the heteroaggregate as far as purification recovery and storage stability were concerned. Besides CTL clones, peripheral blood lymphocytes could also be used as cytolytic effectors, provided that a suitable in vitro activation scheme was used.

Fenoterol effects on the in vitro immune response

Beta-2-adrenergic agonists are often employed in the treatment of acute bronchostenosis. Following our recent investigations into the influence of some drugs (cromolyn, ketotifen, theophylline) on the immune response, in this study we analyzed the in vitro effects of fenoterol (β-2-adrenergic agonist) on the immune response. The mitogen-(PHA)-induced proliferation of peripheral mononuclear cells (PMNC), the PMNC proliferation induced by anti-T3 and anti-T11 monoclonal antibodies (MAbs), the PHA-induced lymphokine — interleukin 2 (IL-2) and interferon-gamma (IFN-γ) — production were studied in ten healthy volunteers. Since the plasmatic peak of fenoterol following a single inhalation of 200 μg is about 20 ng/ml, in the experiments herein reported the drug was tested in the cultures at concentrations lower, equal and higher than the plasmatic peak: respectively, 2, 20 and 200 ng/ml. Furthermore, for a more detailed study of T-lymphocyte activities, we also evaluated the effect of fenoterol on T-cell clone proliferation. Our results, which reveal no effects of fenoterol on the studied immunological parameters, acquire relevance when related to our previous reports showing a depression of the immunological response exerted by theophylline and ketotifen.

Structure and function of SV40 large-T antigen

The small eukaryotic DNA tumour virus, SV40, has long provided a very useful model for the study of eukaryotic DNA replication and cellular transformation. The viral gene product, large-tumour (large-T) antigen, is essential for the initiation of viral DNA replication and the initiation and maintenance of SV40-virus-mediated cellular transformation. The large-T antigen is a complex multifunctional protein, and to delineate its activity more precisely in viral DNA replication and cellular transformation, small functional domains of the protein have been expressed in Escherichia coli and analysed by using a very extensive library of anti-T monoclonal antibodies.