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M J Bevan

Publications and source records attributed to M J Bevan.

At least 73 records · Page 4Linked to original sources
PubMed1994

Protective cytotoxic T lymphocytes are induced during murine infection with Chlamydia trachomatis.

T cell responses are often an important component in immunity to organisms that replicate intracellularly. Cytotoxic T lymphocyte (CTL) recognition of peptide Ag in the context of MHC class I molecules results in lysis of infected cells and the release of cytokines including IFN-gamma. Members of the genus Chlamydia are obligate intracellular pathogens that cause blindness and sexually transmitted disease worldwide. Even though it replicates within a membrane-bound vacuole, Chlamydia trachomatis may elicit a CTL response if Chlamydia Ags are present in the cytoplasmic compartment where they can be processed for presentation and bound by MHC class I. In this study, we characterized a CTL line derived from mice infected with C. trachomatis. This CTL line is specific for, and able to lyse, Chlamydia-infected cells. The peptide epitope recognized by this CTL line is present on infected cells, and is presented to the CTL by the classical MHC class I molecule H-2 Ld. Adoptive transfer of this CTL line into an infected mouse affords protection, and this protection requires the activity of IFN-gamma.

Animals↗
PubMed1994

Cells infected with Yersinia present an epitope to class I MHC-restricted CTL.

Bacteria of the genus Yersinia cause disease in humans ranging from enteritis and lymphadenitis to plaque. These organisms have the ability to bind to host cells and, in a complex interaction, are able to deliver a specific subset of proteins into the host cell. These proteins alter host structures and functions, and in the case of macrophages, prevent phagocytosis of the organism. In Yersinia enterocolitica, one of these proteins, Yop51, is known to be a protein tyrosine phosphatase. By using a directed approach, we have raised murine CTL that recognize a peptide epitope from Yop51 in the context of the MHC class I molecule H-2 Db. This epitope, Yop51 249-257, seems to be presented both by cells that produce Yop51 endogenously and by epithelial cells infected with virulent Y. enterocolitica. In vivo, Y. enterocolitica is believed to remain mostly in an extracellular niche. Therefore, detection of this organism by cytotoxic T cells and, possibly, resistance to disease may depend on recognition of epitopes from a subset of virulence determinants delivered into the cytoplasm of host cells by surface-bound organisms.

Animals↗
PubMed1994

Specificity and flexibility in thymic selection.

During positive selection, developing thymocytes are rescued from programmed cell death by T-cell receptor (TCR)-mediated recognition of major histocompatibility complex (MHC) molecules. MHC-bound peptides contribute to this process. Recently we identified individual MHC-binding peptides which can induce positive selection of a single TCR. Here we examine peptide fine specificity in positive selection. These data suggest that a direct TCR-peptide interaction occurs during this event, and strengthens the correlation between selecting peptides and TCR antagonists. Certain positively selecting peptides are weakly antigenic. We demonstrate that thymocytes 'educated' on such a peptide are specifically non-responsive to it and have decreased CD8 expression levels. Similar reduction of CD8 expression on mature T cells converts a TCR agonist into a TCR antagonist. These data indicate that thymocytes may maintain self-tolerance towards a positively selecting ligand by regulating co-receptor expression.

Amino Acid Sequence↗
PubMed1994

A positively selecting thymic epithelial cell line lacks costimulatory activity.

The participation of costimulatory molecule interactions in positive selection of T lymphocytes was addressed by assessing the ability of a positively selecting thymic epithelial cell (TEC) line, 427.1, to stimulate allospecific CTL responses. Stimulation of H-2s spleen cells with the H-2b expressing 427.1 line does not result in the generation of cells capable of lysing H-2b target cell lines. The level of expression of MHC class I molecules by 427.1 is lower than that found in other stimulatory TEC lines. However, this finding does not account for the nonstimulatory phenotype. Up-regulation of MHC class I did not result in stimulation, and fusion of 427.1 cells with stimulatory TEC resulted in a line with low MHC class I molecule expression and stimulatory phenotype. The TEC line 427.1 does not express the costimulatory molecule B7/BB1, and transfection of the B7/BB1-encoding DNA results in expression of the molecule and conversion into a stimulatory phenotype, demonstrating directly that the non-stimulatory phenotype is a result of lack of costimulation. However, B7/BB1 expression does not improve the ability of 427.1 TECs to induce positive selection. Intrathymic injection of the B7/BB1 transfected, compared with mock transfected 427.1 cells, rescued fewer CD8+ mature thymocytes in beta 2-microglobulin negative mice. Therefore, unlike the peripheral T cell responses to Ag, positive selection of T cells in the thymus may not depend on the costimulation provided by the presenting cell.

Animals↗
PubMed1994

Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides.

The identification of genes in the class II region of the MHC that are homologous to genes encoding subunits of the proteasome has led to intense interest in the possible role of this enzyme in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase-specific CTL clones. Samples of intact OVA and beta-galactosidase were subjected to digestion in vitro by 20S proteasome purified from bovine red cells and the resulting peptide mixtures were fractionated by reverse-phase HPLC. The fractions were tested for their ability to sensitize appropriate mouse target cells for lysis by specific CTL clones. In both cases, components that under all chromatographic conditions eluted with retention times indistinguishable from synthetic peptides representing known epitopes of the naturally processed proteins were found to be able to sensitize the target cells. Moreover, in the case of OVA, the presence of the expected target peptides was demonstrated directly by amino acid sequence and mass spectrometric analysis. The results demonstrate that the pure 20S proteasome is capable of generating antigenic peptides from two proteins for presentation by class I molecules without the participation of additional components of the protein degradation system. This finding is consistent with the hypothesis of proteasome involvement in Ag processing in vivo.

Amino Acid Sequence↗
PubMed1994

T cell receptor antagonist peptides induce positive selection.

We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic beta 2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These peptide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive selection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, E1, induced positive selection on the beta 2M(-/-) background but negative selection on the beta 2M(+/-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy ligands mediate positive selection.

Amino Acid Sequence↗
PubMed1994

Major histocompatibility complex class I allele-specific peptide libraries: identification of peptides that mimic an H-Y T cell epitope.

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2Kb-restricted octamers and H-2Db-restricted nonamers. Peptides from the Kb-restricted library (KbL: SXIXFXXL) and the Db-restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H-2b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.

Amino Acid Sequence↗
PubMed1994

The ligand for positive selection of T lymphocytes in the thymus.

T cells are spared from programmed cell death in the thymus after an appropriate interaction between the T-cell receptor and a self peptide/MHC complex; this step is referred to as positive selection. Recent work has focused on precise identification of the positively selecting ligand, and the cell that presents it. First, it was shown that bone marrow derived cells or fibroblasts can substitute for epithelial cells in providing the ligand for positive selection. Second, in a T-cell receptor transgenic system, variants of the antigenic peptides were found to induce positive selection. Peptides that served as antagonists or weak agonists for the T-cell receptor efficiently selected immature thymocytes for survival. It appears that the peptide ligands for positive selection of T cells are self peptides, which serve as mimics or look alikes for the universe of pathogen peptides. The challenge remains to identify a naturally occurring thymic self peptide that can cause positive selection and determine the range of reactivities to foreign peptides which it can select.

Animals↗
PubMed1994

A thymic epithelial cell line induces both positive and negative selection in the thymus.

TCR engagement in the thymus results in both survival and elimination signals for developing thymocytes. To examine whether both signals can be provided by the same cell type, we investigated the ability of a thymic epithelial cell (TEC) line 427.1, previously shown to allow positive selection in the thymus, to induce clonal deletion of immature thymocytes. [H-2b/s-->H-2s] bone marrow chimeras are non-responsive to antigens in the context of H-2b. However, chimeras that underwent intrathymic injection of H-2b/s 427.1 cells expressing vesicular stomatitis virus (VSV) nucleocapsid antigen acquired the ability to raise influenza, but not VSV specific H-2b restricted cytotoxic T lymphocyte (CTL) responses. The ability of 427.1 cells to delete CD4+CD8+ thymocytes was determined using mice transgenic for the TCR specific for ovalbumin (OVA) in the context of H-2Kb. OVA transfected, but not mock transfected 427.1 TECs, induced in vitro deletion of CD4+CD8+ TCR transgenic thymocytes manifested as a down-modulation of CD4 and CD8 molecules, a shift in the side versus forward scatter characteristics of thymocytes, and appearance of thymocytes with subdiploid content of DNA indicated the ongoing process of DNA fragmentation. The finding that the same TEC line is capable of inducing both positive and negative selection in the thymus suggests that thymocytes bearing TCRs specific for self peptides expressed by positively selecting thymic epithelium can be deleted. Therefore the expression of a unique set of MHC associated peptides by TECs does not appear to be the basis for the positive outcome of the TCR ligation on immature thymocytes.

Animals↗
PubMed1993

Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes.

The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However, no easily applicable approach for CTL epitope identification has been developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector. Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide. The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2Ld-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131. The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.

Amino Acid Sequence↗
PubMed1993

A conservative mutation in a class I MHC molecule outside the peptide binding groove stimulates responses to self peptides.

A transgenic mouse has been made that expresses a mutant MHC class I H-2Kb molecule with glutamic acid at position 65 (E65) in place of glutamine. The side chain at position 65, on the outward face of the alpha-helix of the alpha 1 domain of the class I molecule, interacts with the TCR, and not with the peptide binding groove. The transgenic mouse, on a DBA/2 background, mounts Kb,E65-restricted Ag-specific responses to conventional Kb-restricted Ag such as OVA and vesicular stomatitis virus, and shows strong alloreactivity to wild-type Kb. The transgenic mouse also mounts a primary in vitro alloreactive response directed to a mutant molecule with aspartic acid at position 65 (D65). This response is relatively weak, probably because of the structural similarities between aspartic and glutamic acid side chains; both have carboxylic termini, and the aspartic acid side chain is shorter by a single secondary carbon. The alloreactive CTL lines elicited by this conservative change are cross-reactive among several position-65 variants of H-2Kb. Individual CTL clones are specific for self peptides that can be extracted from cells expressing Kb,E65, and from purified wild-type Kb molecules, and that are recognized in the context of the D65 residue. Thus, the smallest variance from self in a class I molecule, even outside the peptide binding groove, can be antigenic.

Animals↗
PubMed1993

Clone-specific T cell receptor antagonists of major histocompatibility complex class I-restricted cytotoxic T cells.

A previous report showed that the proliferative response of helper T cells to class II major histocompatibility complex (MHC)-restricted antigens can be inhibited by analogues of the antigen, which act as T cell receptor (TCR) antagonists. Here we define and analyze peptide variants that antagonize various functions of class I MHC-restricted cytotoxic T lymphocyte (CTL) clones. Of 64 variants at individual TCR contact sites of the Kb-restricted octamer peptide ovalbumin257-264 (OVAp), a very high proportion (40%) antagonized lysis by three OVAp-specific CTL clones. This effect was highly clone specific, since many antagonists for one T cell clone have differential effects on another. We show that this inhibition of CTL function is not a result of T cell-T cell interaction, precluding veto-like phenomena as a mechanism for antagonism. Moreover, we present evidence for direct interaction between the TCR and antagonist-MHC complexes. In further analysis of the T cell response, we found that serine esterase release and cytokine production are susceptible to TCR antagonism similarly to lysis. Ca2+ flux, an early event in signaling, is also inhibited by antagonists but may be more resistant to the antagonist effect than downstream responses.

Amino Acid Sequence↗
PubMed1993

Positive selection of CD8+ T cells induced by major histocompatibility complex binding peptides in fetal thymic organ culture.

We have used an in vitro system to study the effects of major histocompatibility complex class I binding peptides on thymic development. Fetal thymus lobes from mice deficient in the class I light chain (beta 2 microglobulin or beta 2 M-/-) were cultured for 10 d in vitro, during which time T cell precursors develop into mature T cells. In these organ cultures, as in the adult or neonatal beta 2 M-/- thymus, CD8+ mature T cells did not develop, demonstrating that the mature T cells seen during early murine thymic development are the result of the positive selection process. To these cultures we added various class I binding peptides with or without a source of exogenous beta 2M. CD8+ T cells developed to various degrees only in the presence of beta 2M and peptides. Using peptide mixtures of differing complexity, we showed that the efficiency of this process is dependent more on peptide complexity than on peptide concentration. These data argue for a specific role for peptides in the process of positive selection. Furthermore, this culture system should be useful in identifying peptides that can promote positive selection of cells expressing a specific T cell receptor (TCR) in TCR transgenic mice.

Amino Acid Sequence↗
PubMed1993

Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule.

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2Kd to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2Kd. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2Kd-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2Kd; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2Kd. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2Kd to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2Kd class I molecule, and only a fraction of these serve as CTL epitopes.

Amino Acid Sequence↗
PubMed1993

Peptide variants reveal how antibodies recognize major histocompatibility complex class I.

The T cell receptor (TcR) on CD8+ T lymphocytes recognizes a complex which consists of a major histocompatibility complex (MHC) heavy chain, beta 2-microglobulin (beta 2M), and peptide on the surface of antigen-presenting cells. Mutational analyses have suggested that the TcR recognizes both the alpha 1 and alpha 2 domains of the heavy chain as well as the peptide. In light of this, it is of interest to know to what extent the heavy chain domains take on distinct conformations when bound to individual peptides. It has recently been shown that antibodies which recognize the Kb MHC complex are sensitive to which peptides are bound in the groove. We have extended this analysis to include eight Kb-specific antibodies, seven of which are peptide sensitive. These antibodies, all of which are allo-antibodies, recognize Kb-bearing cells which, it is now appreciated, have a highly heterogeneous mix of self peptides presented in their grooves. We show that these self peptides also can affect antibody binding. It has been suggested that peptides alter the conformation of the alpha 1 and alpha 2 domains of the heavy chain and that this in turn affects the recognition of Kb by antibody. An alternative hypothesis is that solvent-exposed peptide side chains may prevent the antibody from binding the complex. Using a panel of 128 single-amino acid variants of a Kb-binding antigenic peptide from ovalbumin we show that for most Kb-specific antibodies, the second idea is more likely. Those variants which prevent antibody binding are at solvent exposed positions, and in general, the bulkier the side chain, the greater the inhibition of antibody binding. However, in the case of two antibodies, 100.30 and 34.4.20, the peptide residues which affect antibody recognition are buried, suggesting that these antibodies see an alternate conformation of the peptide/MHC complex.

Amino Acid Sequence↗
PubMed1993

Do nonclassical, class Ib MHC molecules present bacterial antigens to T cells?

Immune responses to bacterial antigens that appear unrestricted by the MHC may involve oligomorphic MHC class Ib molecules. One example is H-2M3, which binds N-formylated peptides and presents a Listeria peptide to cytotoxic T cells from infected mice. Lack of polymorphism makes these molecules a promising target for peptide vaccines.

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