Pathology of MHV-A59 infection in beta 2 microglobulin negative mice.
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Animals↗
Biomedical subjects
Y Paterson
Publications and source records attributed to Y Paterson.
Mouse hepatitis virus A59-induced demyelination can occur in the absence of CD8+ T cells.
Mouse hepatitis virus causes a chronic demyelinating disease in C57BL/6 mice. While early studies suggested demyelination is due to direct cytolytic effects of virus on oligodendrocytes, there is increasing evidence for the involvement of the immune system in the mechanism of demyelination. In this study we have asked whether demyelination can occur in the absence of functional MHC class I expression and CD8+ T cells. We infected transgenic mice lacking expression of beta 2 microglobulin (beta 2 M -/- mice) with MHV-A59. In beta 2M-/- mice, virus was much more lethal than in either of the parental strains used to produce the mice; furthermore, while clearance from the CNS did occur in beta 2M-/- mice, it was slower than in C57BL/6 mice. This is consistent with the importance of CD8+ cells in viral clearance. Because of the increased sensitivity of the beta 2M-/- mice to infection, only low levels of virus could be used to evaluate chronic disease. Even at these low levels, demyelination did occur in some animals. To compare infection in beta 2M-/- and C57BL/6 mice we used a higher dose of an attenuated variant of MHV-A59, C12. The attenuated variant induced less demyelination in C57BL/6 mice compared to wild type A59, but the levels observed were not significantly different from those seen in beta 2M-/- mice. Thus, MHV-induced demyelination can occur in some animals in the absence of MHC class I and CD8+ cells.
Animals↗
A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours.
Listeria monocytogenes is an intracellular organism that has the unusual ability to live in the cytoplasm of the cell. It is thus a good vector for targeting protein antigens to the cellular arm of the immune response. Here we use a model system, consisting of colon and renal carcinomas that express the influenza virus nucleoprotein and a recombinant L. monocytogenes that secrets this antigen, to test the potential of this organism as a cancer immunotherapeutic agent. We show that this recombinant organism can not only protect mice against lethal challenge with tumour cells that express the antigen, but can also cause regression of established macroscopic tumours in an antigen-specific T-cell-dependent manner.
Animals↗
T cells from unprimed mice respond to the self antigen heme, in a class II restricted manner, at a frequency similar to alloresponses.
Heme is a non-protein autoantigen which stimulates potent proliferative responses by T cells from unprimed mice of some strains. These studies show that T cells responding to heme in primary responses are predominantly CD4+, classically I-A restricted, and use diverse TCR characterized by the expression of distinct V, D and J gene segments. These characteristics distinguish heme from superantigens and mitogens which exhibit degenerate MHC restriction and, in the case of superantigens, restricted V gene usage. Using limiting dilution analysis these studies also show that the potent primary response of H-2s mice reflects a high frequency (0.26-0.45%) of heme responsive T cells in the periphery, comparable to the frequency of alloresponsive T cells reported by others in primary mixed lymphocyte reactions. In contrast, heme responsive T cells occur at -10-fold lower frequency in unprimed H-2d mice (0.03%). To determine the antigen recognized by heme reactive T cells, the mass spectra of peptides eluted from the high responder haplotype, I-A(s), were examined. These indicated a markedly different molecular weight distribution of peptides isolated from cells grown in the presence of heme, compared with those from cells grown in its absence. This suggests that heme mediates the expansion of diverse T cells in the peripheral repertoire by a mechanism similar to that for allogeneic responses in which the profile of naturally processed peptides bound to the MHC class II molecule is changed.
Amino Acid Sequence↗
Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes.
Listeria monocytogenes is a facultative intracellular pathogen that grows in the cytoplasm of infected host cells. We examined the capacity of L. monocytogenes to introduce influenza nucleoprotein (NP) into the class I pathway of antigen presentation both in vitro and in vivo. Recombinant L. monocytogenes secreting a fusion of listeriolysin O and NP (LLO-NP) targeted infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. Antigen presentation occurred in the context of three different class I haplotypes in vitro. A hemolysin-negative L. monocytogenes strain expressing LLO-NP was able to present in a class II-restricted manner. However, it failed to target infected cells for lysis by CD8+ T cells, indicating that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro. Immunization of mice with a recombinant L. monocytogenes strain that stably expressed and secreted LLO-NP induced NP-specific CD8+ cytotoxic T lymphocytes. These studies have implications for the use of L. monocytogenes to deliver potentially any antigen to the class I pathway in vivo.
Animals↗
Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition.
In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL) induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2bm1 (bm1) mice is identified. An identical sequence, p40-53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2Kb, H-2Db, and H-2Kbm1 class I molecules, although the sequence lacks the usual structural Kb and Db peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2b) targets as well as the L cell transfectants, L+Kb, L+Db, and L+Kbm1. The antigenic peptide with the greatest potency is p41-49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40-53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43-48 peptide from horse cyt c. Residues Pro44 and Thr47, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2Kb, H-2Db, and mutant H-2Kbm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.
Amino Acid Sequence↗
Studies related to the potential antigenicity of the Bowman-Birk inhibitor, an anticarcinogenic protease inhibitor isolated from soybeans.
Soybeans are known to contain certain proteins that are allergenic; therefore the anticarcinogenic Bowman-Birk protease inhibitor (BBI), an 8-kDa protein isolated from soybeans, could be capable of generating an immune response that would contraindicate its use as a cancer chemopreventive agent. In the present investigation, the stimulation of antibody production in response to BBI was measured after the administration of BBI by various routes and treatment regimens. A significant antibody titer to BBI was observed when the animals were treated with BBI by intraperitoneal injection once or three times per week. In these studies, higher antibody titers were observed at earlier time points. When the animals were treated with BBI by oral gavage, little or no antibody production was observed, regardless of the treatment regimen. The doses and treatment regimens used in this study have been previously demonstrated to be effective in preventing tumor formation. The results of this study indicate that antibody production in response to BBI administered orally should not present a complication to BBI treatment.
Animals↗
Syngeneic response to SJL follicular center B cell lymphoma (reticular cell sarcoma) cells is primarily in V beta 16+ CD4+ T cells.
The growth of SJL B cell lymphomas (RCS, reticulum cell sarcoma) in vivo and in vitro is known to depend on cytokine production by RCS-responsive host CD4+ T cells. The high frequency of RCS responsive cells in normal SJL lymph nodes prompted us to prepare a set of 21 RCS-specific T cell hybridomas. Like normal SJL T cells, these hybridoma cells respond to RCS, but not to normal syngeneic B cells; produce IL-2, IL-4, and IL-5; and promote growth of RCS in gamma-irradiated syngeneic hosts. A superantigen-like stimulation by RCS cells was borne out by the fact that all the RCS-specific hybridomas used V beta 16 in their TCR. RCS cells did not stimulate I-As-restricted, V beta 17a+ KLH-specific, or V beta 1+ heme-specific hybridomas, but were excellent Ag presents for these cells. Preincubation of RCS cells with high concentrations of the KM core peptide (high affinity for I-As) did not interfere with the ability of RCS to stimulate RCS-specific hybridomas. The relative representation of mRNA for V beta 1, 4, 10, 15, 16, and 17a was evaluated in RNA extracted from normal SJL lymph node cells responding to Con A or to RCS cells. Only V beta 16 was specifically enriched in the response to RCS. Moreover, the degree of responsiveness to RCS cells in lymph node cells from F1 hybrids of SJL and I-E transgenic SJL mice, corresponds to the relative abundance of V beta 16 in mRNA, but not of V beta 17a mRNA.
Animals↗
Effect of antibody binding on protein motions studied by hydrogen-exchange labeling and two-dimensional NMR.
We have used hydrogen-exchange labeling detected by 2D NMR to study antibody-protein interactions for two monoclonal antibodies raised against horse cytochrome c. The data show that these antibodies bind mainly to the large 37-59 omega-loop of the cytochrome c molecule. In addition, the results provide some suggestive evidence concerning units of local structural flexibility in cytochrome c.
Antibodies, Monoclonal↗
Induction of a cellular immune response to a foreign antigen by a recombinant Listeria monocytogenes vaccine.
A recombinant strain of Listeria monocytogenes that stably and constitutively expresses Escherichia coli beta-galactosidase was used as a live vaccine vector. BALB/c mice were immunized orally or parenterally with the recombinant L. monocytogenes, and their cellular and humoral immune responses to beta-galactosidase were measured. Spleen cells taken 1 week after oral inoculation or 5 weeks after oral or parenteral inoculation (with a boost at 4 weeks) showed beta-galactosidase-specific CTL responses. The CTL line derived from mice immunized i.p. was also shown to be class I restricted and Thy-1.2+, CD8+, and TCR alpha beta+. All mice immunized with the recombinant L. monocytogenes had positive delayed-type hypersensitivity responses to heat-killed L. monocytogenes, but only 15% had a positive delayed-type hypersensitivity reaction to beta-galactosidase. Individual serum samples from mice immunized i.p. or i.v. were tested for antibody to beta-galactosidase. Approximately 11% had low positive titers for beta-galactosidase antibodies. These results demonstrate that both oral and parenteral immunization with recombinant L. monocytogenes results in a cellular immune response to the foreign protein, which is primarily a specific CD8+ CTL response.
Animals↗
Mapping antibody binding sites on protein antigens.
The large surfaces of protein antigens that interact with antibody-combining sites can be determined using deuterium-exchange labelling and two-dimensional 1H nuclear magnetic resonance (NMR). This technique may also be applied to other protein-protein interactions to identify key residues that contribute to the affinity.
Antibodies, Monoclonal↗
Identification of an autologous insulin B chain peptide as a target antigen for H-2Kb-restricted cytotoxic T lymphocytes.
We have examined the CD8+ peripheral T cell repertoire of C57BL/6 (H-2b) mice for cytotoxic T lymphocyte (CTL) reactivities to insulin, using in vitro immunization with a chymotryptic digest of reduced bovine insulin. The results presented in this study demonstrate that potentially autoreactive H-2Kb-restricted cytotoxic T cells specific for an autologous insulin B chain peptide are present in the preimmune splenic T cell repertoire. The immunogenic peptide comprises residues 7-15 from the insulin B chain and has features in common with naturally processed Kb-restricted peptides identified by others. The minimal peptide sequence recognized by these cytotoxic T cells is 10-15, which is highly conserved in mammalian species and constitutes a self-peptide in mice. The presence of class I major histocompatibility complex-restricted CTLs with potentially autoreactive specificities in preimmune animals raises the possibility of a role for such cells in autoimmune disease states. Possible mechanisms for the in vivo expansion of insulin peptide-specific CTLs are discussed.
Amino Acid Sequence↗
Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms.
To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.
Amino Acid Sequence↗
A new mouse T-cell receptor alpha chain variable region family.
Sequence analysis of the rearranged T-cell receptor alpha chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribed V alpha gene family. We have designated this family V alpha 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously described V alpha haplotypes: V alpha a, V alpha b, and V alpha c.
Amino Acid Sequence↗
The structural basis of protein antigenicity.
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Animals↗
An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR.
The interaction of a protein antigen, horse cytochrome c (cyt c), with a monoclonal antibody has been studied by hydrogen-deuterium (H-D) exchange labeling and two-dimensional nuclear magnetic resonance (2D NMR) methods. The H-exchange rate of residues in three discontiguous regions of the cyt c polypeptide backbone was slowed by factors up to 340-fold in the antibody-antigen complex compared with free cyt c. The protected residues, 36 to 38, 59, 60, 64 to 67, 100, and 101, and their hydrogen-bond acceptors, are brought together in the three-dimensional structure to form a contiguous, largely exposed protein surface with an area of about 750 square angstroms. The interaction site determined in this way is consistent with prior epitope mapping studies and includes several residues that were not previously identified. The hydrogen exchange labeling approach can be used to map binding sites on small proteins in antibody-antigen complexes and may be applicable to protein-protein and protein-ligand interactions in general.
Antibodies, Monoclonal↗
T cell receptor junctional regions and the MHC molecule affect the recognition of antigenic peptides by T cell clones.
Nine independent pigeon cytochrome c-specific T cell clones were analyzed by using a panel of antigenic peptide analogs presented in association with three allelic IE-encoded MHC glycoproteins. Eight of the T cell clones expressed a TCR composed of a unique alpha- and beta-chain amino acid sequence, and concordantly, each of these T cell clones exhibited a unique Ag specificity. This was true for several clones which differed only in TCR V-J junctional regions. Interestingly, for a given clone, the response to some of the peptide analogs depended to a large extent on the allelic form of the presenting MHC molecule. A simple interpretation of these data would suggest that certain positions of the peptide Ag are most important for Ag-MHC molecule interactions, and that these specific interactions can influence the antigenic epitope recognized by the TCR. We suggest that an antigenic peptide binds to an MHC glycoprotein in a distinct way, but may retain a measure of flexibility.
Amino Acid Sequence↗
Analysis of peptide binding patterns in different major histocompatibility complex/T cell receptor complexes using pigeon cytochrome c-specific T cell hybridomas. Evidence that a single peptide binds major histocompatibility complex in different conformations.
The interaction of TCR, antigen, and MHC complex has been analyzed using synthetic peptide antigens and a series of single amino acid-substituted analogues. Two similar antigens, mouse cytochrome c (mcyt c) and pigeon cytochrome c (pcyt c), elicit T cell responses in strains of mice bearing MHC class II Ek beta Ek alpha (B10.A), Eb beta Ek alpha [B10.A(5R)], and Es beta Ek alpha [B10.S(9R)]. The immunogenic regions of these antigens are located in the peptide sequence p88-104 for pcyt c and m88-103 for mcyt c. The limited T cell repertoire for these antigens is comprised of four groups of T cell phenotypes that have very few differences in their TCR gene make up. In this paper, we examine the diversity in their fine specificity for each of the antigens, m88-103 and p88-104, complexed with each of the I-Ek haplotypes. Epitopes, i.e., residues that interact with the TCR, and agretopes, i.e., residues in the MHC-binding site, were assigned for the two peptide antigens in the presence of APC bearing E beta kEk alpha, Eb beta Ek alpha, or Eb beta Ek alpha using T cell hybridomas of the phenotypes I, IIIa, and IV. From our results, we conclude that first, the substitution of any residue between 95 and 104 of the cytochrome c peptide changed the antigenic potency of the peptide for at least one of the hybridomas. Second, each T cell type has a different recognition pattern of epitopes and agretopes for a particular antigen-MHC complex, thus, ruling out a static model of T cell recognition, which assigns certain, invariant agretopic residues to the peptide by which it interacts with the MHC molecule independently of the TCR. Third, the same T cell hybridoma responded to the antigens differently when presented on various MHC molecules, implying that overall changes in the MHC groove, as displayed by the three haplotypes, may affect the efficiency in binding the peptide. Fourth, since most of the residues are used as epitopes by at least one of the T cell specificities, the peptide appears to be recognized in a different conformation by each T cell hybridoma phenotype; and, finally, the epitopic and agretopic residues do not segregate, for any one of the T cell specificities, in such a way that suggests they are recognized in a helical conformation. In summary, our results suggest that a single peptide may generate diversity in the T cell response by virtue of its conformational flexibility within the TCR-MHC-antigen complex.
Amino Acid Sequence↗