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M J Bevan

Publications and source records attributed to M J Bevan.

17 recordsLinked to original sources
PubMed1979

Properties and applications of monoclonal antibodies directed against determinants of the Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.

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PubMed1978

H-2 antigens of the thymus determine lymphocyte specificity.

After immunization, normal H-2 heterozygous mice (for example H-2(b) x H-2(d)) generate two populations of cytotoxic effector T cells, one specific for target cells expressing H-2(b)-plus-antigen and the other specific for H- 2(d)-plus-antigen. With a multideterminant antigen, these two populations have about the same activity. We show here that the H-2 type of resident cells in the thymus determines the H-2 preference of cytotoxic T lymphocytes. F(1)(B 10 x B 10.D2) (H-2(b) x H-2 (d)) mice were thymectomized, lethally irradiated, and reconstituted with T-cell-depleted syngeneic hematopoietic cells. Groups of such ATXBM mice were grafted subcutaneously with neonatal thymus lobes from parental mice, either B10 (H-2 (b)) or B10.D2 (H-2(d)). 2-3 mo later, the mice were immunized against the minor histocompatibility antigens on F(1)(BALB/c x BALB.B) cells and assayed for cytotoxic T-cell activity. H-2(b) x H-2(d) ATXBM mice with H-2(b) thymus grafts responded to antigen-plus-H-2(b) much better than to antigen-plus-H-2(d), and vice versa for the mice with H-2(d) thymus grafts. As judged by antiserum treatment, the effector cells were of F(1) origin. To explore the possibility that the "thymus preference" may have been due to suppression of T-cell activity, nonimmune spleen and lymph node cells from normal H-2(b) x H-2(d) mice and cells from H-2(b) x H-2(d) mice bearing a homozygous thymus were mixed 1:1 and immunized in adoptive transfer. The mixture responded to antigen-plus-H-2(b) and antigen-plus-H-2(d) equally well, demonstrating that the cells that showed a "thymus preference" could not suppress a response to antigen in association with the nonthymic H-2 type. We conclude from these and other experiments that H-2 antigens present on resident cells of the thymus determine the spectrum of specificity of T cells which mature in that thymus and eventually make up the peripheral T- cell pool.

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PubMed1977

Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes.

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.

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PubMed1977

Killer cells reactive to altered-self antigens can also be alloreactive.

Murine cytotoxic thymus-derived lymphocytes immunized against cells bearing foreign minor histocompatibility antigens are specific for the immunizing minor antigens and for their own major H-2 antigens; they do not lyse target cells that bear the correct minor antigens plus a different H-2 haplotype. These are referred to as "altered-self" or "self-plus-X" killer cells. Alloreactive killer cells are those which respond to allogeneic cells expressing a foreign (non-self) H-2 haplotype. In this study, cytotoxic lymphocytes were immunized against minor histocompatibility differences in vivo and in vitro. These effector cells killed the immunizing altered-self target very well and showed about 1% cross-reactive lysis of an allogeneic target differing from themselves only at H-2. These cross-reactive clones were then selected for by repeated in vitro stimulation with the cells bearing foreign H-2 such that an effector population was obtained which lysed both the altered-self and the alloreactive target with the same efficiency. Cold target competition experiments established that the same killer cell could lyse either target; however, it was not determined if a killer cell uses the same receptor to respond to altered-self antigens as it does respond to foreign H-2 antigens.

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PubMed1977

Priming for a cytotoxic response to minor histocompatibility antigens: antigen specificity and failure to demonstrate a carrier effect.

Spleen cells from normal mice do not give a detectable in vitro cytotoxic T cell (CTL) response to minor H antigens. Spleen cells from animals primed in vivo with minor H antigens give a strong CTL response when boosted in culture with the appropriate stimulating cells. Here I have studied the requirements for priming a CTL response to minor H antigens. It is shown that priming is just as antigen specific as is cytolytic effector function. That is, priming cells have to carry the same minor antigens as the challenge cells. Inducing a graft-vs-host reaction in vivo does not nonspecifically allow spleen cells to respond to minor H antigens in vitro. Using minor H congenic mice (congenic for H-Y and/or H-7) I have also tried, and failed, to demonstrate a carrier effect in priming. Female mice primed to H-Y were challenged in culture with cells bearing H-Y and H-7 antigens in the hope that a helper response to H-Y would augment a CTL response to H-7. This did not happen, however. Such primed and boosted cells gave a strong secondary CTL response to H-Y but none to H-7. It is concluded that in order to prime for a detectable in vitro response to minor antigens it is necessary to expose the CTL precursors to antigen in vivo. This either expands the size of the pool of precursors by cell division or changes them in some qualitative way.

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PubMed1976

H-2 antigen-specific cytotoxic T cells induced by concanavalin A: estimation of their relative frequency.

Specific and nonspecific lysis of DBA/2 (H-2d) mastocytoma cells, P815, by concanavalin A (Con A)-induced cytotoxic T cells was studied. In the assay for nonspecific lysis, phytohemagglutinin (PHA) was present to glue the target and killer cells together. We have presented evidence previously to show that PHA reveals only, and all, cytotoxic T cells. In the assay for specific lysis the only glue present was specific receptors on a fraction of the killer cells and surface antigens of P815. We show that when PHA was present, Con A-induced cells which were syngeneic, semi-syngeneic, or allogeneic, lysed P815 very efficiently in a 4-h 51Cr release assay. However, only Con A-induced T cells which were allogeneic and did not carry H-2d lysed P815 when the assay was carried out in the absence of PHA. In an experiment with two target cells, Con A-induced B10 (H-2b) T cells lysed B10.D2 (H-2d) targets specifically but did not lyse B10 targets, while Con A-induced B10.D2 T cells lysed B10 targets specifically but not B10.D2 targets. Furthermore, Con A-induced B6 (H-2b) T cells from normal mice lysed P815 specifically but Con A-induced B6 T cells from irradiated F1 (B6 x BALB/c) (H-2b/d) mice reconstituted with B6 bone marrow did not lyse P815 specifically. A fraction of Con A-induced T cells therefore appear to bear specific surface receptors for nonself H-2 coded structures. We describe conditions of assay and a new method of plotting the results such that nonspecific (PHA-revealed) and specific (PHA-independent) cytotoxicity can be quantitatively compared. We conclude that 1-4% of the total Con A-induced cytotoxic effector T cells are directed against any particular foreign H-2 haplotype. This is the first estimate of the relative frequency of antigen-reactive cytotoxic T cells.

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PubMed1976

Minor H antigens introduced on H-2 different stimulating cells cross-react at the cytotoxic T cell level during in vivo priming.

The cellular basis of the cross-priming observed with minor histocompatibility antigens on H-2 different cells was investigated. Cytotoxic T cells induced against minor alloantigens show absolute H-2 restriction at the effector level ([51Cr]-release). That is, F1 (BALB/c X BALB.B) (H-2d/b) cytotoxic cells induced by immunization with B10(H-2b) cells are not able to lyse B10.D2(H-2d) targets. But an injection of B10 cells does prime F1 mice for a secondary cytotoxic response to B10.D2. The technique of inducing cytotoxic effector function polyclonally with Con A in the absence of alloantigen was used here to establish that such cross-priming reflects what happens at the cytotoxic cell level. It is shown that an F1 animal previously injected with B10 cells has expanded pools of memory cytotoxic cells reactive with B10 and B10.D2. From this it is concluded that: a) minor H structures on B10.D2 and B10 do cross-react at the cytotoxic T cell level during in vivo priming, and b) because normal cells cross-prime whereas tumor cells do not, then the F1 cytotoxic precursors are probably committed to respond to antigen on cells bearing either the maternal or paternal H-2 haplotype before they encounter antigen. Cross-priming may be explained by foreign minor H antigens being presented to F1 host T cells on the surface of host macrophages. Therefore priming is not restricted to the H-2 type of the injected cells but to both H-2 types of the F1 host.

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PubMed1975

The major histocompatibility complex determines susceptibility to cytotoxic T cells directed against minor histocompatibility antigens.

Cytotoxic cells were generated by immunizing one strain of mouse with cells from an allogeneic strain which carries the same H-2 region. The effector cells assayed in a 4 h 51Cr release assay were shown to be T cells and indistinguishable, except in specificity, from cytotoxic T cells directed at H-2 alloantigens. Although the genetic differences between responder and stimulator cells responsible for the immunization did not code in H-2, the H-2 complex did restrict susceptibility of target cells. For example, BALB.B cytotoxic cells (H-2b) immunized against and capable of lysing C57BL/6 cells (H-2b) would not lyse B6.C/H-2d target cells. C57BL/6 and B6.C/H-2d are congenic and differ in the H-2 region. Two hypotheses are considered to explain the H-2 restriction of susceptibility to cytotoxic T cells generated by an H-2 identical alloimmunization. (a) The dual (self) recognition hypothesis states that the cytotoxic cell has two recognition units, one for H-2-coded structures and another clonally restricted receptor for the minor alloantigen. (b) The interaction antigen hypothesis states that all the surface alloantigenic determinants recognized by cytotoxic T cells are the result of interaction between H-2- and non-H-2-coded gene products. Two lines of evidence, one with F1 effector cells and the other a cold target competition experiment, are presented which argue strongly in favor of the interaction antigen hypothesis. The regions of H-2 required to be histocompatible were mapped to the D region and to the left of IC, probably the K region. These results, and recent work on the response to virus-infected and TNP-modified syngeneic cells, suggest that cytotoxic cells are restricted in specificity to preferentially recognizing alterations in structures that are coded in the major histocompatibility complex.

Animals↗
PubMed1975

Differences in the surface proteins of mouse B and T cells.

We have selectively labeled the surface of mouse thymus-dependent (T) and thymus-independent (B) lymphocytes by means of lactoperoxidase-catalyzed iodination. Examination of the labeled proteins by electrophoresis in the presence of sodium dodecylsulfate followed by autoradiography revealed differences in the cell surface proteins of B and T cells. Proteins from labeled thymocytes, T cell lymphomas, and both normal and activated peripheral T cells give a broad band of radioactivity corresponding to at least two protein components with apparent molecular weights of 170,000 to 190,000. This band is absent from autoradiographs of iodinated B cell proteins, which instead have another band corresponding to a protein with an apparent molecular weight of about 220,000. We have shown that these T and B cell marker proteins are synthesized by the cell and are not serum components selectively bound to the cell surface. We have also established that these proteins are the major iodinated species precipitated from Nonidet P-40 cell lysates by rabbit anti-mouse lymphocyte serum, suggesting that they are the major antigens recognized by rabbit antibodies to mouse lymphocytes.

Animals↗
PubMed1975

Alloimmune cytotoxic T cells: Evidence that they recognize serologically defined antigens and bear clonally restricted receptors.

Murine T cells immunized with allogeneic cells exert a cytotoxic effect on appropriate target cells, as measured by 51-Cr release. Lysis of the 51-Cr-labeled target can be inhibited by addition to the assay of unlabeled cells which bear target antigens. With variants of lymphoid tumors which express different amounts of serologically defined H-2 antigens as competitors in the assay, a positive correlation is shown to exist between the amount of serologically defined antigen and the degree of inhibition of T cell-mediated lysis. The immunized small lymphocytes are shown to be very poor inhibitors of 51-Cr release from target cells which are more susceptible to T cell-mediated lysis. This is interpreted as showing that small lymphocytes are bound very poorly by cytotoxic T cells. With H-2 cross-over strains which carry only the H-2K or H-2D antigens which a cytotoxic population is directed against, it is shown that, 1) approximately equal amounts of cytotoxicity are directed against the K and D antigens, and 2) separate cytotoxic T cells recognize the K and D antigens. Thus, by this criterion, cytotoxic T cells are monospecific and therefore do not acquire their antigen-specific receptors cytophilically.

Animals↗
PubMed1975

Cytotoxic effects of antigen- and mitogen-induced T cells on various targets.

Mitogen-induced cytotoxicity was studied by culturing mouse spleen cells with an optimum mitogenic dose of concanavalin A (Con A) for 2 days and, after washing, assessing their ability to lyse tumor targets. We show that rapid lysis occurs only when the correct concentration of an agglutinating mitogen (phytohemagglutinin P (PHA) or Con A) is present in the assay. PHA is more efficient than Con A in revealing the cytotoxic effect. The Con A-induced effector cytotoxic cell is shown to be sensitive to anti-theta serum and complement. Cytotoxic T cells were also induced by H-2 different allo-immunization. These effector cells specifically lyse targets which bear the immunizing H-2 antigens in the absence of PHA. When PHA is present in the assay, they will also lyse syngeneic tumor targets nonspecifically. Since not all dividing T cells (e.g., T lymphomas, and T blasts induced by M locus different, H-2 similar mixed lymphocyte culture) lyse PHA-P815, we propose that this assay measures only a subset of effector T cells, namely, cytotoxic T cells, regardless of their antigen specificity. The tumor cells, P815 and EL4, are sensitive both to antigen-specific T cell-mediated lysis in the absence of PHA and to nonspecific, PHA-revealed lysis. Small lymphocytes, T cell blasts, and B cell blasts are sensitive to lysis by T cells which are directed against H-2 antigens on their surface, but are not very susceptible to nonspecific T cell lysis in the presence of PHA. The reason for this difference in susceptibility of tumor and normal cells to PHA-revealed nonspecific T cell lysis is not known but may have some relevance to in vivo tumor rejection.

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