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P M Allen

Publications and source records attributed to P M Allen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Structural basis for T cell recognition of altered peptide ligands: a single T cell receptor can productively recognize a large continuum of related ligands.

T cells recognize short linear peptides bound to major histocompatibility complex (MHC)-encoded molecules. Subtle molecular changes in peptide antigens produce altered peptide ligands (APLs), which induce different T cell responses from those induced by the antigenic ligand. A molecular basis for how these slight molecular variations lead to such different consequences for the T cell has not been described. To address this issue, we have made amino acid substitutions at the primary T cell receptor (TCR) contact residue of the murine hemoglobin determinant, Hb(64-76)/I-Ek and produced 12 peptides that interact with the TCR of the T cell clone 3.L2. The 3.L2 T cell responds to these peptides, which vary 1 million-fold in their activity, and enables them to be ranked according to their relative ability to signal through the 3.L2 TCR. Such a ranking reveals that the ability of the 3.L2 T cell to respond to these peptides depends on how well the structure of the side chain at the primary TCR contact site mimics that of the Asn residue present in the antigenic ligand. The reactivity of the 3.L2 T cell also depends on an MHC contact residue that is next to the primary TCR contact residue, suggesting that conformation of the Asn side chain is also important. By using nonnatural amino acids at a TCR contact residue, we have demonstrated that APLs can be rationally designed based on structure. These data are consistent with a model in which the affinity of a peptide-MHC complex for the TCR determines how the T cell will respond.

Animals↗
PubMed1996

Selective activation of the calcium signaling pathway by altered peptide ligands.

We previously demonstrated that altered peptide ligands (APL) can partially activate T cells, resulting in multiple distinct functional phenotypes, including the induction of anergy. Such APL stimulate a unique pattern of T cell receptor (TCR) phospho-zeta species, and lack associated ZAP-70 kinase activity. While these data suggested that selective signaling pathways downstream of the TCR/CD3 molecules are activated upon APL stimulation, they did not directly demonstrate this. Thus, we pursued intracellular signaling events successfully stimulated by APL. Because our previous studies showed that cyclosporin A (CsA) completely inhibited anergy induction, we assessed whether TCR ligation by APL cause a rise in cytosolic calcium (Ca+2). Our results show that these ligands can induce Ca+2 transients, in contrast to data generated using analogue peptides in other antigen systems. These opposing results may reflect differences in the intracellular signaling pathways utilized by different APL, or may be due to the exquisite sensitivity of the assay used here. Importantly, the APL-stimulated Ca+2 induction is both initiated and sustained at lower levels than that stimulated by a strong agonist signal, but resembles that stimulated by a weaker agonist stimulus. Alone, the less than optimal Ca+2 induction does not cause anergy, because ionomycin treatment together with the APL does not result in a proliferative signal. Instead, we propose that a combination of this and other signaling pathways induces T cell anergy. Overall, these data support the concept of differential signaling in T cells, as a direct consequence of the phosphotyrosine status of the TCR/CD3 molecules.

Animals↗
PubMed1996

TCR recognition of the Hb(64-76)/I-Ek determinant: single conservative amino acid changes in the complementarity-determining region 3 dramatically alter antigen fine specificity.

To identify the structural basis of Ag fine specificity, TCR sequences from a panel of Hb(64-76)/I-Ek-specific T cells were compared and found to be restricted in variable (V) gene usage, predominantly using BV1 or BV15 and AV4 or AV10 genes. TCRA and TCRB junctional sequences were extremely diverse. No conservation of length or position was found, which distinguishes this response from others, but correlates with the range of fine specificities that these T cells display. A remarkable subtlety in the recognition of Hb(64-76) was revealed from the study of the response to the D73 variant of Hb(64-76), which contains a conservative change in an MHC anchor residue not affecting the binding affinity to I-Ek. To one group of T cells this determinant was non-cross-reacting with Hb(64-76), whereas another recognized both Ags. Interesting, they all used a different constellation of TCRBV genes than that found in Hb(64-76) recognition. To limit the variability in the anti-Hb(64-76) TCR repertoire, transgenic mice expressing a fixed TCRB rearrangement from a Hb(64-76)-specific T cell were used. In Hb(64-76)-specific TCR from these mice, the endogenous alpha-chains pairing with the transgenic beta-chain were highly restricted in their AV gene usage. A comparison of two pairs of closely related T cells of these endogenous TCR variants, one differing by a single, conservative substitution in the complementarity-determining region 3 and the other containing a positional switch of two amino acids, revealed dramatically different fine specificities. Overall, these findings highlight the exquisite sensitivity of the TCR- peptide/MHC interaction to subtle alterations in any of the components.

Amino Acid Sequence↗
PubMed1996

Essential flexibility in the T-cell recognition of antigen.

alpha beta T cells specifically recognize a ligand composed of a peptide bound to a self-major-histocompatibility-complex molecule, but the recognition of slightly altered ligands by T cells can lead to a partial activation. This flexibility is crucial for T-cell development and can have both beneficial and harmful effects on peripheral T cells.

Antigens↗
PubMed1996

Endogenous altered peptide ligands can affect peripheral T cell responses.

T cells potentially encounter a large number of endogenous self-peptide/MHC ligands in the thymus and the periphery. These endogenous ligands are critical to both positive and negative selection in the thymus; however, their effect on peripheral T cells has not been directly ascertained. Using the murine allelic Hbd (64-76)/I-Ek self-antigen model, we have previously identified altered peptide ligands (APLs) which are able to stimulate some but not all TCR-mediated effector functions. To determine directly the effect of endogenously synthesized APL/MHC complexes on peripheral T cells, we used a TCR transgenic mouse which had reversed our normal antigen system, with Ser69 peptide now being the agonist and Hbd(64-76) being the APL. In this report, we show that the constitutive level of endogenous Hbd(64-76)/I-Ek complexes presented by APCs in vivo is too low to affect the response of Ser69 reactive T cells. However, by increasing the number of Hbd(64-76)/I-Ek complexes expressed by the APCs, TCR antagonism is observed for both primary T cells and T cell hybridomas. In addition, the level of the CD4 coreceptor expressed on T cells and T cell hybridomas. In addition, the level of the CD4 coreceptor expressed on T cells changes the response pattern to endogenously presented Hbd(64-76)/I-Ek ligand. These findings demonstrate that T cells are selected to ignore the constitutive levels of endogenous complexes they encounter in the periphery. T cell responses can be affected by endogenous APLs in the periphery under limited but attainable circumstances which change the efficacy of the TCR/ligand interaction. Thus, endogenous APLs play a role in both the selection of T cells in the thymus and the responses of peripheral T cells.

Animals↗
PubMed1996

Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.

The highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes.

14-3-3 Proteins↗
PubMed1996

The effect of endogenous altered peptide ligands on peripheral T-cell responses.

Numerous studies have shown that a T cell can productively interact through its TCR with less-than-optimal ligands resulting in partial T-cell activation. These ligands, that we called 'altered peptide ligands' (APLs), can act as partial agonists, antagonists or weak agonists for the T cells. Here we discuss the self-antigen system that we used to provide evidence that endogenous APLs exist in vivo and affect T-cell development. We also report the ability of endogenous APLs to induce partial T-cell activation of peripheral T cells, and describe in which circumstances this could occur in vivo.

Animals↗
PubMed1996

Altered T cell receptor ligands trigger a subset of early T cell signals.

TCR ligands are complexes of peptides and MHC proteins on the surfaces of APCs. Some of these ligands cause T cell proliferation (agonists), while others block it (antagonists). We compared the acid release, calcium flux, and proliferation response of helper T cells to a variety of ligands. We found that all agonist ligands but not most antagonist ligands trigger acid release, a general indicator of early cellular activation. Only a subset of ligands triggering acid release cause sustained calcium flux, and only a subset of these ligands cause T cell proliferation. Antagonist ligands and anti-CD4 antibodies both effectively block T cell proliferation. However, significantly greater antagonist ligand or antibody concentrations are required to block acid release and initial calcium influx. These data demonstrate a hierarchy of early T cell signaling steps and show that altered TCR ligands can initiate some steps while blocking the completion of others.

Amino Acid Sequence↗
PubMed1996

Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.

AIDS-Related Opportunistic Infections↗
PubMed1996

Processing and presentation of an antigen of Mycobacterium avium require access to an acidified compartment with active proteases.

We have generated a murine T-cell hybridoma, 1C9, which recognizes an antigen expressed by a virulent clinical isolate of Mycobacterium avium. Both peritoneal exudate macrophages and bone marrow-derived macrophages infected in vitro with M. avium process and present the antigen to the T-cell hybridoma. Gel filtration chromatography of a sonicate of M. avium followed by T-cell Western blotting (immunoblotting) demonstrated that the antigen recognized by hybridoma 1C9 is approximately 50 kDa. In addition, treatment of macrophages with the lysosomotropic agent chloroquine or with inhibitors of acid proteases inhibits processing and presentation of the antigen. These results indicate that the antigen must encounter an acidic compartment with active proteases for processing and presentation to occur. Our results are discussed in the context of our current understanding of how mycobacterial antigens are processed and presented by infected macrophages to T cells.

Animals↗
PubMed1996

Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology.

The elucidation of the phenomena of T cell antagonism and partial activation by altered peptide ligands has necessitated a revision in the traditional concepts of TCR recognition of antigen and subsequent signal transduction. Whereas previous models supported a single ligand specificity for any particular T cell, many studies using analogs of immunogenic peptides have now demonstrated a flexibility in this recognition. Moreover, interaction with such altered peptide ligands can result in dramatically different phenotypes of the T cells, ranging from inducing selective stimulatory functions to completely turning off their functional capacity. Investigations of the biochemical basis leading to these phenotypes have shown that altered peptide ligands can induce a qualitatively different pattern of signal transduction events than does any concentration of the native ligand. Such observations imply that several signaling modules are directly linked to the TCR/CD3 complex and that they can be dissociated from each other as a direct result of the nature of the ligand bound. Interestingly, many in vivo models of T cell activation are compatible with a selective signaling model, and several studies have shown that peptide analogs can play a role in various T cell biologic phenomena. These data strongly suggest that naturally occurring altered peptide ligands for any TCR exist in the repertoire of self-peptides or, in nature, derived from pathogens, and recent reports provide compelling evidence that this is indeed the case. The concept of altered peptide ligands, their effects on T cell signaling, the hypothesized mechanisms by which they exert their effects, and their possible roles in shaping the T cell immune response are the scope of this review.

Animals↗
PubMed1995

Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.

Immune interactions in the heart were studied using a murine model of myosin-induced autoimmune myocarditis. A T cell hybridoma specific for mouse cardiac myosin was generated from A/J mice and used to demonstrate that endogenous myosin/I-Ak complexes are constitutively expressed on antigen-presenting cells in the heart. This T cell hybridoma, Seu.5, was used as a functional probe to identify a myocarditis-inducing epitope of cardiac myosin. Overlapping peptides based on the cardiac myosin heavy chain alpha (myhc alpha) sequences were synthesized and tested for their ability to stimulate Seu.5 T cells. One peptide, myhc alpha (325-357) strongly stimulated the Seu.5 T cells, localizing the epitope to this region of the myhc alpha molecule. Using truncated peptides, the epitope was further localized to residues 334-352. The myhc alpha (334-352) peptide strongly induced myocarditis when administered to A/J mice, which was histologically indistinguishable from that induced by myosin. The myhc alpha (334-352) epitope was present in cardiac myosin and not skeletal muscle myosins, providing a biochemical basis for the cardiac specificity of this autoimmune disease. Induction of myocarditis by this epitope was restricted to the myhc alpha isoform and not the myhc beta isoform, suggesting there may be a difference in the efficiency of generating tolerance to these isoforms of cardiac myosin, which are differentially developmentally regulated. The myhc alpha (334-352) epitope bound to purified I-Ak molecules in a similar manner to other I-Ak-restricted immunogenic epitopes, HEL(48-61) and RNase(43-56). Importantly, the myhc alpha (334-352) epitope was able to bind to I-Ak molecules on the surface of antigen-presenting cells in a stable manner. These findings demonstrate that autoantigenic epitopes can behave in a dominant manner and constitutively bind to class II molecules in the target organ in a similar manner to foreign immunogenic epitopes.

Amino Acid Sequence↗
PubMed1995

Intramolecular mimicry. Identification and analysis of two cross-reactive T cell epitopes within a single protein.

The recognition of peptide Ags by T cells through the TCR has exquisite specificity. Cross-reactive T cell responses have been described; however, the structural basis for these responses is not known. We show that two peptides derived from the same protein can exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive T cell responses. In addition, we explore the structural basis for cross-reactivity. T cell hybridomas recognizing PiM and PiZ allelic forms of human alpha 1-antitrypsin (hAAT) each recognized both PiM 205-220 and PiM 335-350. These two peptides possessed primary sequence identity at only two of 16 amino acid residues. Cross-reactive peptides also exhibited homology at the bulk T cell level because lymph node T cells primed with one peptide proliferated to the other peptide in vitro. Critical amino acids for the responding T cells were determined, and the core was transferred into the less reactive peptide in an attempt to increase homology by increasing sequence identity. Interestingly, as identity increased, homology decreased: peptides with the least primary sequence identity appeared most homologous to the T cells. These results have important implications for understanding the development of autoimmune diseases, and imply that minimal obvious primary sequence identity may be sufficient to initiate cross-reactive T cell responses. The ability of structurally dissimilar peptides to mimic each other when bound to a class II MHC molecule may also be important to the understanding of T development and autoimmunity.

Alleles↗
PubMed1995

Modulation of T cell development by an endogenous altered peptide ligand.

T cells potentially encounter numerous endogenous peptides during selection in the thymus and in the periphery. We examined the impact of an endogenous peptide on in vivo T cell development, using a TCR transgenic mouse model based on a hemoglobin-specific T cell clone. In these mice, the transgenic beta chains paired with endogenous alpha chains. This led to a serendipitous primary reactivity to Ser69 peptide, an altered peptide ligand of the Hbd (64-76) epitope of the parent clone. Two Ser69-reactive T cell populations were identified. A smaller population of the Ser69-reactive T cells responded both to Ser69 and Hbd (64-76). A majority reacted only to Ser69, and not to Hbd(64-76); in fact, Hbd(64-76) was a specific TCR antagonist for these Ser69-only-reactive T cells. Thus, in this unique experimental system, Ser69 became an agonist, and Hbd (64-76) was an antagonist. Endogenous presentation of the antagonist ligand in the thymus selectively eliminated the high-avidity cells, while sparing low-avidity cells in the Ser69-reactive T cell repertoire. These results highlight how specificity guides developing T cells through a network of ligands and indicate that the endogenous peptide pool has a profound effect on T cell development and repertoire.

Amino Acid Sequence↗
PubMed1995

Signalling events in the anergy induction of T helper 1 cells.

T cells can interact productively with altered peptide ligands (APLs) resulting in different phenotypic outcomes. Stimulation of T helper 1 cells with an APL on live antigen-presenting cells results in the induction of anergy. We investigated the intracellular signalling events involved in generating this anergy by comparing protein tyrosine phosphorylation patterns after stimulation with the anergy-inducing APL or the immunogenic peptide. Stimulation by an APL resulted in a unique pattern of T cell receptor (TCR) phospho-zeta species, which was not observed with any dose of immunogenic peptide. This altered phospho-zeta pattern had a profound functional significance, in that the tyrosine kinase ZAP-70 was not activated. Thus, anergy can be induced by changing the constellation of intracellular signalling events in a T cell. These findings demonstrate that the TCR-CD3 complex can engage selective intracellular biochemical signalling pathways as a direct consequence of the nature of the ligand recognized and the initial phosphotyrosine pattern of the TCR-CD3 proteins. This then leads to different phenotypes.

Clonal Anergy↗
PubMed1995

Significance of T-cell stimulation by altered peptide ligands in T cell biology.

Investigations of T-cell responses to altered peptide ligands have provided functional evidence that a T-cell receptor can interpret subtle structural changes in its ligand, highlighting the complexity of this antigen receptor signaling system. Over the past year, observations from many studies have suggested several roles for such analog peptides in various aspects of immune responses. Collectively, these data strongly suggest the existence of naturally occurring altered peptide ligands in the endogenous peptide repertoire, that can actively participate in the development and shaping of T-cell immunity.

Animals↗