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Biomedical subjects

E G Pamer

Publications and source records attributed to E G Pamer.

At least 37 records · Page 2Linked to original sources
PubMed1999

T cell responses to bacterial infection.

Many exciting advances in our understanding of T cell mediated immunity to bacterial infection have occurred in the past several years. T cell responses have been more fully characterized, due in part to the development of MHC class I tetramers. The importance of cytokines and various effector molecules in defense against infection has come to light. Finally, intracellular bacteria are being exploited to deliver antigens and DNA in an effort to induce immunity to pathogens.

Animals↗
PubMed1999

Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors.

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

Alleles↗
PubMed1999

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

Amino Acid Sequence↗
PubMed1999

Antigen presentation in the immune response to infectious diseases.

T lymphocytes mediate immune defenses against infectious diseases. The major histocompatibility complex class I and class II antigen-processing pathways play an essential role in the activation of pathogen-specific T lymphocytes by presenting peptide fragments derived from pathogen-encoded proteins. The past 5 years have seen remarkable advances in our understanding of both of these antigen-processing pathways. Many new proteins that are critical to T-cell-mediated defenses against infectious pathogens have been identified and characterized. Although the direct effects of aging on antigen processing and presentation are relatively unexplored, it is likely that immunosenescence affects and is affected by the antigen-processing pathways.

Aged↗
PubMed1999

Processing of Listeria monocytogenes antigens and the in vivo T-cell response to bacterial infection.

Presentation of antigens to T lymphocytes is a critical step in the clearance of pathogens from their hosts and in the establishment of protective immunity. Several animal models have been developed to study this process, but few have been as informative as the murine immune response to Listeria monocytogenes infection. Herein we review the presentation of L. monocytogenes proteins by the MHC class I antigen-processing pathway and the in vivo T-cell response to these bacterial antigens. These studies demonstrate the following: 1) The size of a peptide-specific T-cell response does not correlate with the amount of epitope presented by infected cells; 2) T cells specific for dominant epitopes do not, in the case of L. monocytogenes infection, inhibit responses to subdominant epitopes; 3) T cells responding to different epitopes presented by MHC class Ia molecules expand, contract and enter the memory pool synchronously; 4) Repeated in vivo expansion of antigen-specific T-cell populations results in a narrowing of their T-cell receptor repertoire and in an increase in their affinity for antigen; and 5) T cells restricted by H2-M3 MHC class Ib molecules constitute a major part of the primary response to bacterial infection, but appear to play a relatively smaller role in memory responses. These studies have provided a novel glimpse of the relationship between antigen processing and in vivo T-cell responses to infection, and provide a foundation for more detailed analyses of T-cell mediated adaptive immunity.

Amino Acid Sequence↗
PubMed1999

Noncompetitive expansion of cytotoxic T lymphocytes specific for different antigens during bacterial infection.

Listeria monocytogenes is an intracellular bacterium that elicits complex cytotoxic T-lymphocyte (CTL) responses in infected mice. The responses of CTL populations that differ in antigen specificity range in magnitude from large, dominant responses to small, subdominant responses. To test the hypothesis that dominant T-cell responses inhibit subdominant responses, we eliminated the two dominant epitopes of L. monocytogenes by anchor residue mutagenesis and measured the T-cell responses to the remaining subdominant epitopes. Surprisingly, the loss of dominant T-cell responses did not enhance subdominant responses. While mice immunized with bacteria lacking dominant epitopes developed L. monocytogenes-specific immunity, their ability to respond to dominant epitopes upon rechallenge with wild-type bacteria was markedly diminished. Recall responses in mice immunized with wild-type or epitope-deficient L. monocytogenes showed that antigen presentation during recall infection is sufficient for activating memory cells yet insufficient for optimal priming of naive T lymphocytes. Our findings suggest that T-cell priming to different epitopes during L. monocytogenes infection is not competitive. Rather, T-cell populations specific for different antigens but the same pathogen expand independently.

Animals↗
PubMed1998

Evolution of a complex T cell receptor repertoire during primary and recall bacterial infection.

The mechanisms underlying the genesis and maintenance of T cell memory remain unclear. In this study, we examined the evolution of a complex, antigen-specific T cell population during the transition from primary effector to memory T cells after Listeria monocytogenes infection. T cell populations specific for listeriolysin O (LLO)91-99, the immunodominant epitope recognized by H2-Kd-restricted T lymphocytes, were directly identified in immune spleens using tetrameric H2-Kd-epitope complexes. The T cell receptor (TCR) Vbeta repertoire of specific T cells was determined by direct, ex vivo staining with a panel of mAbs. We demonstrate that LLO91-99-specific, primary effector T cell populations have a diverse TCR Vbeta repertoire. Analyses of memory T cell populations demonstrated similar TCR diversity. Furthermore, experiments with individual mice demonstrated that primary effector and memory T cells have indistinguishable TCR repertoires. Remarkably, after reinfection with L. monocytogenes, LLO91-99-specific T cells have a narrower TCR repertoire than do primary effector or memory T cells. Thus, our studies show that the TCR repertoire of primary effector T lymphocytes is uniformly transmitted to memory T cells, whereas expansion of memory T cells is selective.

Animals↗
PubMed1998

MHC class I/peptide stability: implications for immunodominance, in vitro proliferation, and diversity of responding CTL.

Infection of BALB/c mice with Listeria monocytogenes primes CD8+ cytotoxic T cells specific for four different H2-Kd-restricted peptides. In vitro restimulation of L. monocytogenes immune splenocytes with each of these peptides resulted in larger T cell responses to p60 217-225 and mpl 84-92 than to LLO 91-99 and p60 449-457. Direct frequency analyses of immune splenocytes, however, revealed that LLO 91-99 and p60 217-225 elicit dominant T cell responses, while p60 449-457 and mpl 84-92 elicit minor, subdominant responses. Restimulation of immune splenocytes with a range of peptide concentrations revealed that T cells with dominant specificities respond optimally to low peptide concentrations, while T cells specific for subdominant epitopes expand maximally to high peptide concentrations. This disparity correlates with the stability of H2-Kd/epitope complexes: the two dominant epitopes form stable complexes, while the subdominant epitopes form less stable complexes with H2-Kd. Interestingly, T cells specific for LLO 91-99 and p60 217-225 express more complex TCR-Vbeta repertoires than p60 449-457- and mpl 84-92-specific T cells. Thus, in our system, dominant T cell responses have relatively diverse TCR repertoires and are specific for peptides that form stable complexes with MHC class I molecules. Determining the precise roles of epitope/MHC class I stability and TCR repertoire in the generation of dominant T cell responses will require further investigation.

Animals↗
PubMed1998

Effect of antigen-processing efficiency on in vivo T cell response magnitudes.

T lymphocytes eradicate and provide long-term immunity to infections caused by intracellular pathogens. The mechanisms that determine in vivo T cell response sizes are poorly understood. Although it is speculated that the relative processing efficiency of different epitopes determines the hierarchy of T cell responses following immunization, this hypothesis has not been rigorously tested. We therefore mutagenized the secreted p60 Ag of Listeria monocytogenes to alter the efficiency of T cell epitope generation. Ag-processing efficiencies in cells infected with the different L. monocytogenes mutants ranged from one H2-Kd-associated p60 217-225 epitope generated per 15 intracellularly degraded p60 molecules (1/15) to one epitope per 350 degraded p60 molecules (1/350), i.e., a spectrum encompassing a 20-fold range of efficiencies. Mice infected with L. monocytogenes secreting inefficiently processed p60 (1/350) did not mount p60 217-225-specific T cell responses. However, increasing the efficiency of Ag processing by a factor of 5 to 1/70 restored the T cell response size to normal, while further increases in the efficiency of p60 217-225 generation to 1/50, 1/35, and 1/17 did not further augment specific T cell responses. Our studies demonstrate an Ag-processing threshold for in vivo T cell activation. Surprisingly, once this threshold is achieved, further enhancement of Ag-processing efficiency does not enhance the size of T cell responses.

Amino Acid Sequence↗
PubMed1998

Coordinate regulation of complex T cell populations responding to bacterial infection.

Bacterial infections activate complex T cell populations that differ in size and antigen specificity. We used tetramerized MHC class I molecules complexed with Listeria monocytogenes-derived epitopes to characterize four distinct CD8+ T lymphocyte populations during bacterial infection. Surprisingly, T cell populations differing in antigen specificity expand, contract, and enter the T cell memory compartment synchronously. Because the four L. monocytogenes epitopes are presented with different efficiencies and have distinct stabilities in infected cells, our findings suggest that these factors do not determine in vivo T cell dynamics. While T cell activation requires antigen presentation, the timing and extent of T cell expansion appear to be regulated in a coordinated fashion independent of antigen quantity and stability.

Animals↗
PubMed1997

The Listeria monocytogenes-secreted p60 protein is an N-end rule substrate in the cytosol of infected cells. Implications for major histocompatibility complex class I antigen processing of bacterial proteins.

Cytosolic antigen degradation is an initial step in the generation of major histocompatibility complex (MHC) class I-associated cytolytic T lymphocyte epitopes. Intracellular Listeria monocytogenes secretes p60, a murein hydrolase, into the host cell cytosol, where it is degraded by proteasomes. Roughly 3% of degraded p60 gives rise to p60 217-225, a nonamer peptide that is bound by H-2Kd MHC class I molecules. Herein, we introduce targeted deletions throughout the p60 gene to identify potential proteolytic signals within p60. Degradation of mutant forms of p60 was investigated in macrophages infected with recombinant L. monocytogenes. We found that deletions within the amino-terminal two-thirds of p60 enhanced cytosolic degradation. In contrast, truncation of the C terminus resulted in modest stabilization of p60 in the host cell cytosol. Because a protein's N-terminal amino acid can determine its rate of degradation, we mutagenized this residue in p60 into known stabilizing and destabilizing residues. Valine substitution dramatically stabilized cytosolic p60 molecules, while substitution with aspartic acid resulted in rapid degradation. The number of p60 217-225 epitopes isolated from infected cells directly correlated with the rates of p60 degradation. Our data, therefore, indicate that the N-terminal amino acid and multiple internal regions of p60 influence its stability in the cytosol of infected cells. Antigen degradation and epitope generation are linked, and different degradation signals can channel bacterial proteins into the MHC class I antigen processing pathway.

Antigen Presentation↗
PubMed1997

Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes.

Association of antigenic peptides with newly synthesized major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum and is a critical early step for the initiation of cytotoxic T lymphocyte (CTL)-mediated immune defenses. Pathogen-derived peptides compete with a plethora of endogenous peptides for MHC class I grooves. We find that two H2-K(d)-restricted peptides, which derive from the Listeria monocytogenes p60 antigen, accumulate in infected cells with different kinetics. Although competition assays suggest that both epitopes are bound with equivalent affinity, they dissociate from MHC class I molecules at markedly different rates. p60 217-225 forms complexes with H2-K(d) with a half-life >6 h, while p60 449-457 dissociates from H2-K(d) with a half-life of approximately 1 h. We find that p60 449-457-H2-K(d) complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes. While peptide dissociation from retained MHC class I molecules is enhanced, retained H2-K(d) molecules maintain a remarkable capacity to bind new T cell epitopes. We find that intracellular H2-K(d) molecules can bind new CTL epitopes for up to 3 h after their synthesis. Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells. We propose that the increased intracellular lability of peptide-MHC class I complexes may function to optimize the spectrum of peptides presented to T lymphocytes during cellular infection.

Animals↗
PubMed1997

Immunodominant and subdominant CTL responses to Listeria monocytogenes infection.

Protective immunity to infection by intracellular pathogens begins with expansion of Ag-specific, effector T lymphocytes and is followed by persistence of pathogen-specific memory T cells. Infection by Listeria monocytogenes, an intracellular bacterium, induces cytolytic T lymphocytes that mediate systemic sterilization and long term immunity. In cells infected with L. monocytogenes, H2-Kd class I molecules present three nonamer peptides, listeriolysin (LLO) 91-99, p60 217-225, and p60 449-457, to CTL. Herein we show that during the peak CTL response to L. monocytogenes infection, the ratio of T cells specific for LLO 91-99, p60 217-225, and p60 449-457 is approximately 20:10:1, respectively. While the number of Ag-specific T lymphocytes decreases in the weeks after infection, the proportion of T lymphocytes specific for the three epitopes is maintained. Repertoire analysis of a subset of L. monocytogenes-specific T cells, using alanine-substituted variants of p60 217-225, indicates that the range of T cell specificities is maintained by memory cells. These results indicate that the breadth and relative magnitude of T cell specificities initially elicited by an infection are transmitted to the memory compartment. Our results suggest that T lymphocytes with different gross and fine Ag specificities are equally likely to become memory T cells.

Acute Disease↗
PubMed1997

The CD40-CD154 system in anti-infective host defense.

Research in the past few years has documented significant advances in our understanding of the CD40-CD40 ligand (CD154) system in diverse immune functions. This system influences many T cell mediated inflammatory immune responses and effector functions, unmasking a previously unexpected role for CD40-CD154 in cell mediated immunity. Manipulation of CD154 in animal models of infection by the use of CD154-deficient mice or anti-CD154 antibodies has shown the importance of this system in the initiation of the inflammatory response, in the activation of antigen-presenting cells and in resistance to infections.

Animals↗
PubMed1997

MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses.

Listeria monocytogenes (L. monocytogenes) secretes proteins associated with its virulence into the cytosol of infected cells. These secreted proteins are degraded by host cell proteasomes and processed into peptides that are bound by MHC class I molecules in the endoplasmic reticulum. We have found that the MHC class I antigen-processing pathway is very efficient at generating the epitopes that are presented to cytolytic T lymphocytes (CTL). Depending on which antigen is investigated, from 3 to 30% of degraded antigens are processed into nonamer peptides that are bound by MHC class I molecules. Surprisingly, neither the efficiency of epitope generation nor the absolute number of epitopes per infected cell determines the magnitude of the in vivo CTL response. One of the least prevalent epitopes, derived from an antigen that is virtually undetectable in infected cells, primes the immunodominant CTL response in L. monocytogenes-infected mice. Our studies suggest that immunodominant and subdominant T-cell responses cannot be predicted by the prevalence of antigens or epitopes alone, and that additional factors, yet to be determined, are involved.

Animals↗
PubMed1997

A nonamer peptide derived from Listeria monocytogenes metalloprotease is presented to cytolytic T lymphocytes.

Listeria monocytogenes is an intracellular bacterium that secretes proteins into the cytosol of infected macrophages. Major histocompatibility complex (MHC) class I molecules bind peptides that are generated by the degradation of bacterial proteins and present them to cytolytic T lymphocytes (CTL). In this study we have investigated CTL responses in L. monocytogenes-immunized mice to peptides that (i) derive from the L. monocytogenes proteins phosphatidylinositol-specific phospholipase C, lecithinase (most active on phosphatidylcholine), metalloprotease (Mpl), PrfA, and the ORF-A product and (ii) conform to the binding motif of the H2-Kd MHC class I molecule. We identified a nonamer peptide, Mpl 84-92, that is presented to L. monocytogenes-specific CTL by H2-Kd MHC class I molecules. Unlike other motif-conforming peptides derived from the secreted Mpl of L. monocytogenes, Mpl 84-92 is bound with high affinity by H2-Kd. Mpl 84-92 is the fourth L. monocytogenes-derived peptide found to be presented to CTL by the H2-Kd molecule during infection and demonstrates the importance of high-affinity interactions between antigenic peptides and MHC class I molecules for CTL priming.

Animals↗