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E M Shevach

Publications and source records attributed to E M Shevach.

At least 145 records · Page 8Linked to original sources
PubMed1985

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations.

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.

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PubMed1985

Nucleotide sequence and expression of a mouse interleukin 2 receptor cDNA.

We have isolated and sequenced a 1.3 kilobase (kb) cDNA that contains the entire 804 base pair coding region of the murine interleukin 2 (IL 2) receptor. A comparison with the human IL 2 receptor sequence revealed regions of high conservation, including the short (11 amino acid) intracytoplasmic domain, as well as an internally repeated sequence that is consistent with the receptor containing at least two distinct extracytoplasmic domains. When inserted into the expression vector pcEXV-3, this cDNA directs functional membrane expression of the IL 2 receptor on transfected COS 7 cells. An analysis of cytoplasmic RNA from IL 2 receptor-bearing T cells reveals five distinct polyadenylated transcripts ranging in size from 1.7 to 4.7 kb. All five transcripts are evident in both concanavalin A-stimulated splenic T cells and in the IL 2-dependent line CTLL. Some of this size heterogeneity is due to different sites of polyadenylation, resulting in mRNAs that differ in the 3' untranslated region. We cannot exclude the possibility of alternative splicing, however, which may result in distinct forms of the IL 2 receptor on the cell surface.

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PubMed1985

Influence of accessory cell and T cell surface antigens on mitogen-induced IL 2 receptor expression.

We have assessed the inhibitory effects of various monoclonal antibodies on the expression of the IL 2 receptor. Anti-LFA-1, but not anti-Ly-2, markedly inhibited the induction of the IL 2 receptor on the Ly-2+ subset. T-depleted spleen cells, L cells, and B lymphoma cells all functioned as potent accessory cells (AC) for the induction of the IL 2 receptor on L3T4+ T cells. Anti-LFA-1 inhibited the induction of the IL 2 receptor irrespective of the type of AC used. Anti-L3T4 only inhibited the induction of IL 2 receptor expression when L cells were the source of AC. The inhibitory capacity of anti-L3T4 was not related to the expression of Ia on the AC population, because the magnitude of inhibition was comparable in cultures containing either Ia+ or Ia- L cells, whereas no inhibition was seen with either Ia+ or Ia-B lymphoma cells. We conclude from these studies that LFA-1 plays a critical role in mitogen-induced activation of both T cell subsets by promoting both T-AC and T-T interactions. Although anti-L3T4 can inhibit T cell activation in the absence of the recognition of Ia, the mechanism of inhibition and the proposed target molecule for L3T4 on the AC or the T cell have not been determined in our studies. A number of different models for the function of this cell surface antigen are discussed.

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PubMed1985

Characterization and partial purification of a specific interleukin 2 inhibitor.

To investigate the mechanisms that regulate the action of interleukin 2 (IL 2) and possibly limit its activity, we screened supernatants of mouse spleen cell cultures which had been stimulated with concanavalin A (Con A) for their ability to inhibit IL 2-mediated proliferation of a cloned IL 2-dependent line. Inhibitory activities with m.w. of 10,000 to 12,000 and 60,000 to 80,000 daltons could be identified in supernatants of both L3T4+ and Ly-2+ T cells, but not in supernatants of Con A or lipopolysaccharide-stimulated B cells. Maximal inhibitory activity was observed after 3 to 4 days of stimulation, and this inhibitory activity could be overcome by increasing the stimulatory concentration of IL 2. When the factor was further purified by reverse-phase high pressure liquid chromatography, it eluted as a single peak with an m.w. of 11,000 to 12,000 daltons which inhibited IL 2- but not IL 3-dependent proliferation. The mechanisms by which this new lymphokine might play in the control of the clonal expansion of T lymphocytes are discussed.

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PubMed1984

Both interleukin 2 and a second T cell-derived factor in EL-4 supernatant have activity as differentiation factors in IgM synthesis.

B cells cultured with anti-IgM, BSF-p1, and B15-TRF will differentiate into high rate IgM-synthesizing cells in the presence of supernatants from EL-4 cells that have been induced with phorbol myristate acetate. These supernatants contain two molecular species (EL-TRFs) that have differentiative activity. One co-migrates with interleukin 2 (IL-2) and its activity is blocked by antibody to the IL-2 receptor. Furthermore, molecularly cloned IL-2, at concentrations of 100 U/ml or more, expresses such EL-TRF activity. The EL-TRF activity of cloned IL-2 can also be inhibited by antibody to the IL-2 receptor. The other material with EL-TRF activity has a molecular weight of approximately 32,000. This material lacks IL-2 activity. Antibody to the IL-2 receptor does not impair its function. B cells stimulated with anti-IgM and BSF-p1, with or without B15-TRF, express determinants that react with two monoclonal antibodies which recognize distinct epitopes on the T cell IL-2 receptor. These determinants are present at much lower density (approximately 100-fold) on stimulated B cells that on HT-2 cells, an IL-2-dependent T cell line. Very small amounts of [3H]IL-2 (less than 1,000 molecules per cell) bind to activated B cells. These results indicate that IL-2 binds to a receptor on appropriately prepared B cells and causes them to differentiate into high rate IgM-synthesizing cells. The physiologic significance of the B cell differentiative activity of IL-2 remains to be investigated.

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PubMed1984

Mycoplasma contamination: a hazard of screening hybridoma supernatants for inhibition of [3H]thymidine incorporation.

We have utilized a functional screen, inhibition of proliferation of mitogen activated lymphocytes, in an attempt to obtain monoclonal antibodies to soluble mediators of immune responses and to the receptors for such mediators. We have found that contamination of hybridoma cell lines with certain species of mycoplasma can interfere with such a screen. By consuming thymidine, mycoplasma can either mimic the effect of antibodies that inhibit lymphocyte proliferation or obscure the presence of antibodies that stimulate or enhance proliferation.

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PubMed1984

T cell-activating properties of an anti-Thy-1 monoclonal antibody. Possible analogy to OKT3/Leu-4.

We have identified a single rat monoclonal antibody, G7, that is a potent inducer of interleukin (IL-2) production from all functioning T cell hybridomas as well as from normal T cells. G7 is also mitogenic for normal T cells and is a very effective inducer of IL-2 receptor expression. On fluorescence-activated cell sorter analysis, G7 recognized a pan-T cell antigen. Immunoprecipitation studies demonstrated that G7 recognized a cell surface molecule of 28-32 kD that appeared to be identical to Thy-1 in coprecipitation studies. In addition, G7 precipitated a protein of 50 kD. The possible relationship of the putative molecular complex identified by G7 on murine cells to the molecular complex identified on human T cells with anti-T3 reagents is discussed. In addition, G7 should prove to be a very useful reagent for studying the early events of lymphocyte activation as well as an inducer of lymphokine-rich supernatants.

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PubMed1984

Monoclonal antibodies directed against human Ia antigens detect an evolutionary conserved epitope on guinea pig Ia antigens with unique functional properties.

Three murine monoclonal antihuman Ia antibodies were found to cross react with guinea pig Ia antigens. Sequential immunoprecipitation studies demonstrated that these antibodies recognized a subset of guinea pig Ia antigens. In competition binding studies, the antihuman Ia antibodies competed with each other, but not with a number of murine monoclonals raised directly to guinea pig Ia antigens. When the antihuman Ia monoclonals were tested for their ability to inhibit the proliferative response of primed guinea pig T cells to antigen-pulsed macrophages, they completely inhibited the response of strain 2 T cells to the complex antigen ovalbumin while having no effect on the response to the copolymer L-glutamic acid, L-lysine. In contrast, the monoclonals raised against guinea pig Ia had much less dramatic effects on the response of uncloned T cells to these antigens. These results suggest that the cross-reactive monoclonals may recognize an evolutionary conserved epitope on Ia that has a regulatory function in T-cell activation.

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PubMed1984

Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3.

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.

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PubMed1984

Non-activated guinea-pig T cells and thymocytes express Ia antigens: FACS analysis with alloantibodies and monoclonal antibodies.

Conventional alloantisera and monoclonal antibodies to guinea-pig Ia antigens were used for analysis of Ia expression by guinea-pig T cells and thymocytes. Indirect immunofluorescent staining was performed with alloantisera or with ascitic fluid as a source of monoclonal antibody followed by flow microfluorometry analysis on the fluorescence activated cell sorter. About 80% of normal, non-activated peritoneal exudate T cells, lymph node T cells and thymocytes expressed Ia antigens. These data are therefore in contrast to studies with human or murine T cells where Ia antigens were shown to be expressed predominantly on activated but not on non-activated T cells. All the reactivity of the anti-Ia alloantisera for strain 2 T cells could be removed by absorption with an Ia-bearing B cell leukaemia, EN-L2C, but not by its Ia-negative variant, BZ-L2C. Thus, the Ia determinants identified on T and B cells are probably identical. One monoclonal antibody, 25E3, which had previously been shown by serologic analysis to react exclusively with an alloantigenic determinant of strain 2 Ia antigens displayed an unusual pattern of reactivity in that it clearly stained strain 13 thymocytes, but not mature strain 13 T or B lymphocytes. The significance of this possible expression of inappropriate Ia determinants by thymocytes remains unclear. This phenomenon might be associated with differentiation processes in the thymus.

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PubMed1984

The murine IL 2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells.

The properties of three distinct rat monoclonal antibodies, designated 3C7, 7D4, and 2E4, to the murine IL 2 receptor have been compared in binding, biochemical, and functional assays. 3C7 appears to define an epitope near or identical to the IL 2-binding site of the receptor, because 3C7 inhibited the binding of radiolabeled IL 2 to CTL-L cells and because unlabeled IL 2 inhibited the binding of FITC-3C7 to CTL-L cells. 7D4 and 2E4 had no effect on IL 2 binding. Competitive antibody-binding studies confirmed that the epitope seen by 3C7 was distinct from the epitope(s) seen by 7D4 and 2E4. Sequential immunoprecipitation studies demonstrated that all three antibodies were reactive with the same molecular species, and that each precipitated identical components of 20,000 to 25,000 daltons, 50,000 to 60,000 daltons, and 100,000 to 120,000 daltons from the surface of CTL-L. FACS studies demonstrated a quantitatively and qualitatively identical cell distribution for the antigen defined by each antibody. They failed to stain more than 95% of resting lymphocytes, but were strongly reactive with Con A T blasts and substantially less reactive with LPS B blasts. Unlabeled IL 2 was also able to inhibit the binding of FITC-3C7 to LPS B cell blasts, suggesting the presence of IL 2-binding sites on activated B cells. Each antibody inhibited IL 2-driven proliferation of HT2 or CTL-L cells. 3C7 and 7D4 were more potent inhibitors of proliferation than was 2E4, and the combined use of 3C7 and 7D4 resulted in greater levels of inhibition of proliferation than that shown from the use of either antibody alone. Collectively, the results support the hypothesis that these antibodies detect two distinct functional regions of the IL 2 receptor.

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PubMed1984

Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2.

By using as an experimental system the induction of growth of a cloned, antigen:Ia-reactive helper T cell line by an antigen receptor-specific monoclonal antibody, we demonstrated that growth requires two essential co-factors, exogenously produced IL 1 and endogenously produced IL 2. The primary role of the IL 1 is in the expression of receptors on the T cell surface for IL 2, rather than for promoting the synthesis of IL 2. The use of a clone-specific activating monoclonal antibody at nanogram amounts to activate a cloned helper T cell should allow a detailed characterization of T cell activation via antigen receptor cross-linking.

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PubMed1984

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens.

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.

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PubMed1984

The murine IL 2 receptor. II. Monoclonal anti-IL 2 receptor antibodies as specific inhibitors of T cell function in vitro.

We assessed the dependency of a variety of immune responses for IL 2 in vitro by using anti-IL 2 receptor monoclonal antibodies as specific inhibitors of IL 2 function. The generation of allogeneic cytotoxic T lymphocyte (CTL) responses and maximal thymocyte proliferation to phytohemagglutinin (PHA) and IL 1 was readily susceptible to inhibition by these antibodies. Furthermore, the IL 2 receptor-positive, IL 2-responsive cell in the CTL cultures expressed killer cell activity. A greater variability in susceptibility to anti-IL 2 receptor antibody inhibition was noted for proliferation of T cells to concanavalin A, PHA, or allogeneic cells. Under certain conditions, however, each of these responses was almost completely inhibited. In most instances, the failure to block a response could be accounted for by either high levels of endogenous IL 2 production or high density of cell surface IL 2 receptors, which represent two known variables that influence the level of inhibition by these antibodies. Analysis of IL 2 receptor expression by mitogen-stimulated T cells suggested that accessory cells may play a role in the optimal expression of the IL 2 receptor. These experiments demonstrate that IL 2 is the predominant growth factor by which T lymphocytes proliferate, but do not exclude the possibility of an IL 2-independent pathway for growth.

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PubMed1983

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens.

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.

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PubMed1983

Antigen-presenting cells from nonresponder strain 2 guinea pigs are fully competent to present bovine insulin B chain to responder strain 13 T cells. Evidence against a determinant selection model and in favor of a clonal deletion model of immune response gene function.

To test directly the determinant selection hypothesis of immune response gene function, we primed strain 13 T lymphocytes in vitro with allogeneic bovine insulin pulsed strain 2 macrophages. Strain 2 macrophages were found to be fully competent to present bovine insulin B chain to strain 13 T cells despite the fact that strain 2 guinea pigs are normally totally unresponsive to this antigen. These results are incompatible with a strict interpretation of the determinant selection hypothesis, which would have predicted that strain 2 macrophages would have been restricted to the presentation of A chain loop determinants. In addition, a comparison of the reactivity profiles of self-Ia- and allo-Ia-restricted strain 13 T cells to a series of synthetic B chain peptide fragments revealed that the allo-Ia-restricted populations could be activated by autologous guinea pig insulin. Taken together, these observations strongly suggest that the clonal deletion of self-reactive cells is likely to be I region restricted and that nonresponsiveness to any protein antigen may result from a restriction in the T cell repertoire that is generated during ontogeny by a clonal deletion mechanism of tolerance to self.

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PubMed1983

The cellular compartmentalization of macrophage-associated nominal antigen: immunologically relevant macrophage-associated antigen may not require an intracellular phase of macrophage handling.

It has been hypothesized that internalization and "processing" of the nominal antigen by antigen-presenting cells may be a mandatory event in antigen handling prior to cell surface presentation of the immunologically relevant moiety to T lymphocytes. In previous functional and immunochemical analyses of macrophage (M phi)-associated nominal antigen, we obtained data strongly suggesting that an immunologically relevant pool of antigen was detectable on the surface of M phi pulsed with a soluble protein antigen and then aged for 3-24 h. To provide further information about requisite events of M phi antigen handling, we have attempted to this study to determine the origin of the nominal antigen associated with the surface of the pulsed-aged cell using the terpolymer L-glutamic acid, L-lysine, L-tyrosine (GLT) as the antigen. The surface-bound GLT, after a 24-h culture, appeared to be derived from GLT initially surface-associated after the pulse. No evidence was obtained to support the notion that a significant quantity of GLT, initially internalized after the pulse exposure, was recompartmentalized to the cell surface. The turnover of cell-surface GLT resembled the turnover of M phi membrane proteins in general. These results, therefore, imply that the crucial events of M phi antigen handling of GLT may entirely be a cell surface phenomenon.

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