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M J Bevan

Publications and source records attributed to M J Bevan.

At least 127 records · Page 7Linked to original sources
PubMed1986

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.

Antibodies, Monoclonal↗
PubMed1985

Acquisition of cytotoxic T lymphocyte-specific carbohydrate differentiation antigens.

Cytotoxic T cell (CTL)-specific activation antigens, termed CT determinants, have been detected by monoclonal antibodies (mAb) that inhibit CTL function. At the cell surface, the CT antigens are associated with the T200 glycoproteins and two other proteins of Mr 140,000 and 85,000 and are present on a secreted protein, gp155. Periodate treatment followed by binding analysis and immunoprecipitation experiments using tunicamycin-treated cells indicated that carbohydrate is necessary for CT antigen expression. Furthermore, gp155 is secreted in the presence of tunicamycin while retaining the CT antigens, and the CT determinants are added late in T200 biosynthesis, suggesting that the CT glycans are O-linked. Finally, interleukin 2 was shown to dramatically influence the expression of the CT mAb-reactive oligosaccharides present at the CTL cell surface.

Animals↗
PubMed1985

Antibodies against the transferrin receptor block the induction of cytotoxic T lymphocytes. A new method for antigen-specific negative selection in vitro.

The induction of cytotoxic T lymphocytes (CTL) specific for alloantigen in a primary mixed lymphocyte culture (MLC) was used as a model system to study the potential usefulness of monoclonal antibodies directed against the murine transferrin receptor in eliminating antigen-activated T cells. Two monoclonal antibodies were used which are able to block the transferrin-mediated uptake of iron by growing cells. One of these antibodies is cytotoxic in the presence of complement (C). Treatment of MLC cells from control, uninhibited cultures with the cytotoxic anti-transferrin receptor antibody plus C completely knocked out all CTL effector activity showing that all of the effector cells expressed the receptor. However, surviving cells from such antibody-plus C-treated populations were not depleted of precursors since they were able to mount a strong CTL response to the original alloantigen upon restimulation. Blocking antibody against the transferrin receptor, in the absence of C, when present during the course of a primary MLC, completely inhibited the generation of effector CTL. Conditions were optimized such that antibody present during the course of the MLC could completely and specifically eliminate all antigen-activated T cells including precursors as assessed by secondary restimulation with the original, plus third-party antigens, in the absence of blocking antibody. This method for producing specifically unreactive T cell populations appears to be superior to previously described techniques.

Animals↗
PubMed1985

Cytotoxic T lymphocyte-mediated lysis via the Fc receptor of target cells.

The murine monoclonal antibody F23.1 reacts with an allotypic determinant on the T cell receptors of about 25% of peripheral T lymphocytes in most common mouse strains. In this report we demonstrate that this IgG2a antibody bound to the receptor of a cytotoxic effector T cell can direct that cell to lyse any target cell which expresses receptors for IgG2a. Thus, allotype-positive, Lyt-2+ T lymphocytes from either concanavalin A- or F23.1-activated cell cultures are recruited as cytotoxic effector cells which lyse targets via the Fc receptor. Concanavalin A-induced T cell blasts depleted of Lyt-2+ cells do not mediate cytotoxic effects in this system. The data presented here confirm earlier observations that monoclonal anti-T cell receptor antibodies attached to the surface of a cell can act as target structures for T cell-mediated lysis.

Animals↗
PubMed1985

Rearrangement and transcription of a T-cell receptor beta-chain gene in different T-cell subsets.

The functionally defined sets of T lymphocytes--helper T cells, cytotoxic T cells, and suppressor T cells--were examined for the possible involvement of a recently identified T-cell receptor beta gene locus in receptor formation. Since gene rearrangements are required for functional gene expression, cloned T-cell lines from each of the groups were surveyed for the expression of unique gene rearrangements. In addition, cell lines that showed gene rearrangements were further tested for the expression of the mature 1.2- to 1.3-kilobase mRNA transcribed from a productive gene rearrangement. The results of such experiments show that helper and cytotoxic T cells may use a common beta chain of the receptor, whereas suppressor cells do so rarely, if at all.

Animals↗
PubMed1985

Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor.

Repeated immunization of normal C57L/J (H-2b) mice with peripheral T cells from BALB.B (H-2b) mice results in the production of antibodies which react with the T cell receptor. A monoclonal antibody-producing hybridoma, F23.1, was isolated from immunized C57L/J mice showing this property. This monoclonal antibody recognizes approximately 25% of peripheral T cells in BALB mice. It stains approximately the same fraction of T cells and precipitates the same heterodimer as the rat monoclonal antibody described previously that was made against isolated receptor material. The allotypic determinant recognized by this monoclonal antibody is present in most common laboratory strains (BALB, C57BL, CBA, A, DBA, C3H) and is absent in C57L, C57BR, and SJL mice. Sorting peripheral T cells from BALB.B or (SJL X BALB/c)F1 mice for the F23.1+ and F23.1- subsets revealed that both populations contain approximately the same CTL precursor frequency for alloantigen. Thus, the T cell receptor allotype defined by F23.1 is present on CTL. Furthermore, cytotoxicity mediated by an F23.1+ CTL line could be blocked specifically by the F23.1 monoclonal antibody. Under appropriate conditions, the monoclonal antibody F23.1 bound to Sepharose 4B beads can induce resting peripheral T lymphocytes of allotype-positive strains to proliferate.

Animals↗
PubMed1985

Novel antigenic determinants of the T200 glycoprotein expressed preferentially by activated cytotoxic T lymphocytes.

This report describes the production and characterization of MAb specific for neoantigenic determinants, called CT antigens, present on the T200 glycoprotein of CTL. The CT determinants are expressed at high levels only on activated CTL. Although differences had previously been noted in the cell surface protein profile of distinct T cell subsets, this is the first description of a lineage-specific activation antigen. Furthermore, these determinants appear to have functional significance, because the CT-specific MAb block cytolytic function. Immune precipitation experiments indicated that the appearance of the CT antigens correlated with m.w. changes and the appearance of additional T200 protein species that were CTL specific. MLC cells cultured in the presence of IL 2-containing supernatants undergo a gradual shift from relatively low CT antigen expression to very high CT expression. This shift is accompanied by modifications of T200 eventually leading to a T200 protein profile identical to that of CTL clones. However, MLC cells propagated without the addition of IL 2-containing supernatants do not undergo a shift in CT antigen expression. Thus, the extent to which modifications occur in the T200 molecule is regulated by soluble factors. Competitive binding assays with two CT-specific MAb indicated that at least two distinct but overlapping neo-epitopes appear as the result of the T200 alterations. In sum, we have discovered the presence of CTL-lineage-specific activation antigens whose expression is regulated by lymphokines and is linked to cytolytic function. MAb specific for determinants such as these could be invaluable tools in the enumeration and depletion of specific T cell subsets in in vivo situations.

Animals↗
PubMed1985

Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes.

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.

Animals↗
PubMed1984

Thymic cytotoxic T lymphocytes are primed in vivo to minor histocompatibility antigens.

Potent cytotoxic T lymphocyte (CTL) activity can be derived from cultures of thymocyte responders and minor H different spleen cell stimulators. As is the case of the spleen cell response previously reported, this cytotoxic activity requires in vivo priming. We performed several experiments designed to determine whether the in vivo priming effect is due to the in situ priming of the thymocyte CTL precursors, to contamination of thymus cell preparations with cells of neighboring lymph nodes, or to the appearance in the thymus of antigen-reactive peripheral T cells. We show by depletion of peripheral cells with antilymphocyte serum and part body irradiation that recent thymic immigrants derived from the bone marrow contribute to the primed thymic response. Thymic CTL were primed in animals in which peripheral T cell responses were completely eliminated by repeated treatment in vivo with monoclonal anti-Thy-1 reagents. Primed, antigen-activated lymph node cells were also demonstrated to contribute to the thymus-derived CTL response. Thus, the minor H-specific thymic CTL response is due both to in situ priming and the immigration of activated peripheral T cells. We discuss the possible significance for models of T cell differentiation of the presence within the thymus of antigen and antigen-reactive mature T cells.

Animals↗
PubMed1984

A reexamination of the role of LYT-2-positive T cells in murine skin graft rejection.

We have investigated which T cell subclass defined by cytolysis with monoclonal anti-Lyt-1.2 and anti-Lyt-2.2 antibodies is required to adoptively transfer the ability to reject skin grafts. B6.Thy-1.1 spleen cells immune to graft antigens were fractionated with antibody plus C' and transferred to adult thymectomized, irradiated, bone marrow-reconstituted (ATXBM) B6.Thy-1.2 hosts that were simultaneously grafted with BALB.B skin. We found that when the ATXBM hosts were used 6 wk after irradiation and marrow reconstitution, both Lyt-1-depleted and Lyt-2-depleted immune spleen cells could transfer the ability to promptly reject skin grafts. However, such ATXBM recipients of Lyt-2-depleted cells that had rejected skin grafts were found to contain graft-specific CTL that were largely of host (B6.Thy-1.2) origin. When ATXBM hosts were used for the experiment 1 wk after irradiation and marrow reconstitution, no host-derived graft-specific CTL could be detected. However, graft rejection occurred in recipients of anti-Lyt-1- or anti-Lyt-2 plus C'-treated immune cells and specific CTL were generated from spleen cells of both groups. Thus, in the absence of a host-derived response, adoptively transferred immune Lyt-2+ cells, either resistant to, or that escaped from, antibody plus C' treatment, are able to expand in response to the antigenic stimulus provided by the graft. A more complete elimination of specific T cell subclasses is therefore needed to assess the relative contribution of a particular subset to the graft rejection process.

Animals↗
PubMed1984

Monoclonal antibodies specific for a murine cytotoxic T-lymphocyte clone.

Two antibody-secreting murine hybridomas, F1G3.1 and F2A11.5, have been established from B10.D2 mice immunized with cells from the murine cytotoxic T-lymphocyte clone G4. The two clones used, G4 and B10, were derived from BALB.B (H-2b) mice and the target antigen specificity of both maps to the Dd region of the murine H-2 complex. However, B10 has a lower affinity for the target cells, as shown by its lower specific killing of blasts and its higher susceptibility to blocking by anti-Lyt-2 monoclonal antibody 53-6.75. The monoclonal antibodies, F1G3.1 and F2A11.5, react only with cells from clone G4. Similarly, they block only the specific cytolysis mediated by G4; no effect on cytotoxicity mediated by B10 or by heterogeneous populations of cytotoxic T lymphocytes was found. F1G3.1, especially, is very active in stimulating G4 to secrete immune interferon; B10 in contrast did not show any induction on treatment with these monoclonal antibodies. The structure of the surface antigen on G4 cells recognized by these monoclonal antibodies was revealed by immunoprecipitation studies of radioiodinated cell surface proteins. A protein dimer could be identified with an apparent molecular size of 80,000 daltons consisting of monomers migrating as 42,000-dalton proteins on reduction. So far, electrophoresis in the presence of NaDodSO4 does not indicate any heterogeneity in the size of the monomers. This molecule can be distinguished from the Lyt-2 complex.

Animals↗
PubMed1984

Functional clonal deletion of class I-specific cytotoxic T lymphocytes by veto cells that express antigen.

Intravenous injection of class I incompatible spleen cells into mice results in a drastic reduction of the recipient's cytotoxic T lymphocyte (CTL) response against the injected, but not against third party, class I antigens when measured in bulk cultures initiated 5 to 6 days after the injection. This specific suppressive effect is partly due to T cells but can also be seen when high numbers of anti-Thy-1 and complement-treated spleen cells of nude mice are injected. Such cells suppressing CTL responses against self histocompatibility antigens are called "veto cells." The precursor frequency of CTL specific for the injected class I antigen is found to be reduced greater than 200-fold at days 5 to 6 after the injection, whereas the frequencies of CTL specific for third party class I antigens are not significantly changed. These results indicate that there is a functional clonal deletion of the CTL recognizing class I incompatible veto cells in vivo. The role of such a veto phenomenon in the induction and maintenance of self tolerance and allograft tolerance is discussed.

Animals↗
PubMed1984

Antigen-independent activation of memory cytotoxic T cells by interleukin 2.

Culture supernatants from mitogen- or antigen-activated murine spleen cells are capable of causing reexpression of specific cytolytic activity from inactive memory cytotoxic T lymphocytes (CTL) in the absence of the original priming antigen. We have demonstrated that memory CTL from cytolytically inactive day 14 MLC cells are induced to reexpress high levels of specific cytotoxic activity after incubation with IL 2. Highly purified IL 2 was shown to induce levels of lytic activity comparable with that induced by supernatants from secondary mixed lymphocyte cultures (secondary MLC SN), suggesting that only IL 2 is necessary for the reactivation process. Moreover, only Lyt-2+ cells are necessary for reactivation inasmuch as inactive MLC cells depleted of Lyt-1+ cells by treatment with antibody and complement, followed by FACS selection of Lyt-2+ cells, were efficiently reactivated by IL 2. Because IL 2 is considered a proliferative signal, we examined whether proliferation was requisite for reactivation of memory CTL by IL 2. In the presence of cytosine arabinoside, which effectively inhibited proliferation, IL 2 was capable of reactivating memory CTL as efficiently as antigen, thus implying a differentiative role for IL 2 in secondary CTL activation. Reactivation of CTL by IL 2 and antigen appear to be functionally distinct events, because antigen but not IL 2 could trigger immune interferon release, although either IL 2 or antigen induced high levels of cytotoxicity. We propose that resting, memory CTL retain a heightened level of expression of IL 2 receptors as compared with naive CTL precursors, and thus are able to respond directly to exogenous IL 2. The consequences of this are proliferation and reexpression of specific killing activity, but this signal is not sufficient to induce immune interferon secretion. Rather, it appears that a signal via the antigen receptor is necessary for release of this lymphokine.

Animals↗
PubMed1984

Studies on the mechanism of suppression of primary cytotoxic responses by cloned cytotoxic T lymphocytes.

We have shown in the accompanying companion paper that cloned cytotoxic T lymphocytes (CTL) can serve as veto cells in vitro, suppressing primary cytotoxic activity directed against antigens expressed by those cloned CTL but not against third party antigens. We now explore the mechanism of this antigen-specific suppression by cloned CTL, using as a model system the ability of G4, a BALB.B anti-H-2Dd CTL clone, to specifically suppress a primary in vitro anti-H-2b CTL response. G4 cells do not constitutively secrete a suppressor factor, because suppression cannot be mediated by supernatants removed from G4 cells at a time when they are routinely used as veto cells. Furthermore, medium removed from cultures suppressed by G4 will not suppress, indicating that the veto cell function of G4 is not mediated by soluble factors. Full suppression of primary anti-H-2b CTL responses requires that G4 be present throughout the 5-day mixed lymphocyte culture (MLC). Removal of G4 during the first 3 days of MLC results in a drastic reduction in the level of antigen-specific suppression, with a slight but reproducible loss of suppression after veto cell removal on day 4. The addition of G4 during the course of an ongoing MLC reveals that maximal suppression requires the presence of veto cells during the first 24 to 48 hr of culture. Thus, G4 cells must be present both early and late in an MLC to exert maximal veto cell suppression. Several experiments suggest that G4-induced veto cell activity is unlikely to be due to cytolysis of CTL precursors which are capable of recognizing G4. G4 cannot specifically recognize these CTL precursors, and G4 cells are inefficient at lectin-mediated lysis of non-tumor cell targets. Furthermore, we show that G4 cells cannot lyse CTL which recognize them. Finally, dilutions of anti-clonotypic antibodies which completely block both lectin-mediated and specific cytolysis by G4 do not block (and in fact enhance) G4-mediated veto cell activity.

Animals↗
PubMed1984

Cloned cytolytic T cells can suppress primary cytotoxic responses directed against them.

In vivo and in vitro, murine peripheral T cells can suppress or "veto" the activation of cytotoxic T lymphocytes directed against antigens presented by those T cells. This suppression is antigen-specific and H-2-restricted. The recognition event initiating this suppression appears to be unidirectional; precursors of cytotoxic T lymphocytes recognize the antigen-bearing veto cell and are thereby inactivated--the veto cell need not recognize the CTL precursor. We show here that 3/3 cytolytic T cell clones can exert veto activity in vitro on normal spleen cells which do not bear antigens the T cell clones can recognize. This suppression results in greatly diminished cytotoxic activity generated during a primary 5-day mixed lymphocyte culture against antigens which the veto cell expresses, but not against third-party antigens present in the same culture. In this same system, a noncytolytic T cell clone will not serve as a source of veto cells. Secondary cytotoxic responses are relatively resistant to the veto cell activity of cloned cytolytic T cells. The cloned veto cells do not suppress the generation of cytotoxic activity directed against antigens they recognize (and presumably carry over via antigen-specific receptors). Cold target competition during the cytotoxic assay has been eliminated as a possible mechanism for T cell clone-induced suppression, and suppression cannot be reversed by the addition to the mixed lymphocyte cultures of supernatants from concanavalin A-activated spleen cells. It is suggested that this mechanism of inactivating primary cytotoxic T lymphocyte responses could play an important role in the maintenance of self-tolerance and in the induction and maintenance of tolerance to allografts.

Animals↗