Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8 ؉ ... more Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8 ؉ T cell response, but the development of similar reagents for detection of CD4 ؉ T cells based on class II MHC proteins has been more difficult. We evaluated fluorescent streptavidin-based oligomers of HLA-DR1 for use as reagents to analyze Ag-specific human CD4 ؉ T cells. Staining was blocked at low temperatures and by drugs that disrupt microfilament formation and endocytosis. Cellassociated MHC oligomers were resistant to a surface stripping protocol and were observed by microscopy in intracellular compartments. This behavior indicates that detection of CD4 ؉ T cells using class II MHC oligomers can depend on an active cellular process in which T cells cluster and/or endocytose their Ag receptors. T cells of identical specificity but in different activation states varied greatly in their ability to be detected by class II MHC oligomers.
Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its con... more Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5–8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this “effector checkpoint” dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus–infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced b...
The impact of initial peptide antigen affinity for TCR in driving memory fate has been studied pr... more The impact of initial peptide antigen affinity for TCR in driving memory fate has been studied previously, however its contributions when effectors contract to memory is unclear. To become memory, effector CD4 T cells must recognize antigen at 5-8 days post-infection, at what we call the “effector checkpoint.” We examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We made an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP-peptide variants that bind FluNP TCR with a broad range of avidity. To vary avidityin vivo, we primed naïve donor FluNP in IAV-infected hosts, purified 6d FluNP effectors and co-transferred them with peptide-pulsed APC into uninfected second hosts. Higher affinity peptides yielded higher numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN, and drove better protection against lethal influenza infection...
The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu fr... more The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph and its role in immunological recognition is still missing and is the focus of this investigation. Using advanced proteomics and biochemical approaches; we sequenced the proteome/degradome/peptidome of the human lymph and compared it with the MHC II peptidome displayed by HLA-DR1+ dendritic cells. Analysis of more than 3000 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration. P...
Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mat... more Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in pre-formed MHCI–peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA–B*58, HLA–B*08 and HLA–A*02. For all MHCI–peptide combinations, peptide binding onto MHCI protected against ERAP1–mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08–bound 12mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic and computational analyses suggested that this 12mer is highly labile and that apparent on–MHC trimming rates are alwa...
ERAP1 is an endoplasmic reticulum-resident zinc aminopeptidase that plays an important role in th... more ERAP1 is an endoplasmic reticulum-resident zinc aminopeptidase that plays an important role in the immune system by trimming peptides for loading onto major histocompatibility complex (MHC-I) proteins. Here we report discovery of the first inhibitors selective for ERAP1 over its paralogs ERAP2 and IRAP. Compound 1 (N-(N-(2-(1H-indol-3-yl)ethyl)carbamimidoyl)-2,5-difluorobenzenesulfonamide) and compound 2 (1-(1-(4-acetylpiperazine-1-carbonyl)cyclohexyl)-3-(p-tolyl)urea) are competitive inhibitors of ERAP1 aminopeptidase activity. Compound 3 (4-methoxy-3-(N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)sulfamoyl)benzoic acid) allosterically activates ERAP1's hydrolysis of fluorogenic and chromogenic amino acid substrates but competitively inhibits its activity towards a nonamer peptide representative of physiological substrates. Compounds 2 and 3 inhibit antigen presentation in a cellular assay. Compound 3 displays higher potency for an ERAP1 variant associated with increased risk of autoimmune disease. These inhibitors provide mechanistic insight into the determinants of specificity for ERAP1, ERAP2 and IRAP, and offer a new therapeutic approach of specifically inhibiting ERAP1 activity in vivo.
Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can generate or de... more Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can generate or destroy potential peptide ligands for MHC class I molecules. ERAP1 activity influences the cell-surface immunopeptidome and epitope immunodominance patterns but in complex and poorly understood manners. Two main distinct pathways have been proposed to account for ERAP1's effects on the nature and quantity of MHCI-bound peptides: i) ERAP1 trims peptides in solution, generating the correct length for binding to MHCI or overtrimming peptides so that they are too short to bind, and ii) ERAP1 trims peptides while they are partially bound onto MHCI in manner that leaves the peptide amino terminus accessible. For both pathways, once an appropriate length peptide is generated it could bind conventionally to MHCI, competing with further trimming by ERAP1. The two pathways, although not necessarily mutually exclusive, provide distinct vantage points for understanding of the rules behind the gene...
Shigella is unique among enteric pathogens, as it invades colonic epithelia through the basolater... more Shigella is unique among enteric pathogens, as it invades colonic epithelia through the basolateral pole. Therefore, it has evolved the ability to breach the intestinal epithelial barrier to deploy an arsenal of effector proteins, which permits bacterial invasion and leads to a severe inflammatory response. However, the mechanisms used by Shigella to regulate epithelial barrier permeability remain unknown. To address this question, we used both an intestinal polarized model and a human ex-vivo model to further characterize the early events of host-bacteria interactions. Our results showed that secreted Serine Protease A (SepA), which belongs to the serine protease autotransporter of Enterobacteriaceae family, is responsible for critically disrupting the intestinal epithelial barrier. Such disruption facilitates bacterial transit to the basolateral pole of the epithelium, ultimately fostering the hallmarks of the disease pathology. SepA was found to cause a decrease in active LIM Kin...
Recent advances in mass spectrometry technology have facilitated detailed examination of MHC-II i... more Recent advances in mass spectrometry technology have facilitated detailed examination of MHC-II immunopeptidomes, i.e. the repertoires of peptides bound to MHC-II molecules expressed in antigen presenting cells. These studies have deepened our view of MHC-II presentation. Other studies have broadened our view of pathways leading up to peptide loading. Here we review these recent studies in the context of earlier work on conventional and non-conventional MHC-II processing. The message that emerges is that sources of antigen beyond conventional endosomal processing of endocytosed proteins are important for generation of cellular immune responses to pathogens and maintenance of central and peripheral tolerance. The multiplicity of pathways results in a broad MHC II immunopeptidome that conveys the sampled environment to patrolling T cells.
Patients with obesity, metabolic syndrome, or type-2 diabetes (T2D) often exhibit hyperglycemia a... more Patients with obesity, metabolic syndrome, or type-2 diabetes (T2D) often exhibit hyperglycemia and hyperlipidemia. A consequence of the dysmetabolism is non-enzymatic coupling of reactive sugars and lipids with proteins and DNA inducing glycation and glycoxidation. Biochemically, these modifications disrupt the cellular proteome by changing the protein chemical structure and inducing protein unfolding, cross-linking and aggregation. We investigated protein post-translational modifications (PTM) induced by glycation and glycoxidation in PBMC from non-diabetic, pre-diabetic and diabetic individuals as well as CD11c+ dendritic cells purified from mice fed a high-fat diet, obese (Ob/Ob) mice and relative controls. By employing label-free bottom-up proteomic and bioinformatics analysis we mapped several cellular pathways affected by the oxidative PTMs, including the antigen processing and presentation machinery. Functional assays of endosomal processing, using transthyretin and insulin ...
Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in seve... more Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in several metabolic diseases including diabetes. These highly reactive moieties bind to the cellular proteome to form advanced glycation end-products (AGEs), which can alter the structure and function of targeted proteins. To address whether the AGE-mediated modification of immunologically relevant proteins could have functional consequences we incubated soluble recombinant HLA- DR1 (HLA-DRA*01:01/DRB1*01:01) in presence of glucose at concentration commonly observed in diabetic subjects (15–20 mM). Analysis of the AGE adducts was achieved by using bottom-up proteomics and tandem mass spectrometry analysis. Major observed modifications were glyoxal-derived hydroimiadazolone, argpyrimidine, dihydroxy and methylglyoxal adducts on arginine residues, pyralline and glyceraldehyde-derived pyridinium compound adducts of lysine and carboxy ethylation and methylation of both arginine and lysine residues. These AGE-related modifications were comparable to what observed on MHCII, immunoprecipitated from the PBMC of Diabetic patients. AGE PTMs mapped to both the binding groove and HLA-DM binding domains. AGE-modified and unmodified HLA-DR1 were then assessed for their binding affinity of several relevant peptides in a fluorescence polarization assay in the presence or absence of HLA-DM. Changes in binding affinity for the selected peptides was observed in AGE-HLA-DR1 as compared to the unmodified HLA-DR1. Altogether our data indicate that protein PTM, as commonly observed in conditions of metabolic and oxidative stress can change HLA-DR1/peptide binding affinity thus compromising MHC-II restricted immune responses.
Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8 ؉ ... more Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8 ؉ T cell response, but the development of similar reagents for detection of CD4 ؉ T cells based on class II MHC proteins has been more difficult. We evaluated fluorescent streptavidin-based oligomers of HLA-DR1 for use as reagents to analyze Ag-specific human CD4 ؉ T cells. Staining was blocked at low temperatures and by drugs that disrupt microfilament formation and endocytosis. Cellassociated MHC oligomers were resistant to a surface stripping protocol and were observed by microscopy in intracellular compartments. This behavior indicates that detection of CD4 ؉ T cells using class II MHC oligomers can depend on an active cellular process in which T cells cluster and/or endocytose their Ag receptors. T cells of identical specificity but in different activation states varied greatly in their ability to be detected by class II MHC oligomers.
Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its con... more Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5–8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this “effector checkpoint” dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus–infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced b...
The impact of initial peptide antigen affinity for TCR in driving memory fate has been studied pr... more The impact of initial peptide antigen affinity for TCR in driving memory fate has been studied previously, however its contributions when effectors contract to memory is unclear. To become memory, effector CD4 T cells must recognize antigen at 5-8 days post-infection, at what we call the “effector checkpoint.” We examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We made an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP-peptide variants that bind FluNP TCR with a broad range of avidity. To vary avidityin vivo, we primed naïve donor FluNP in IAV-infected hosts, purified 6d FluNP effectors and co-transferred them with peptide-pulsed APC into uninfected second hosts. Higher affinity peptides yielded higher numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN, and drove better protection against lethal influenza infection...
The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu fr... more The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph and its role in immunological recognition is still missing and is the focus of this investigation. Using advanced proteomics and biochemical approaches; we sequenced the proteome/degradome/peptidome of the human lymph and compared it with the MHC II peptidome displayed by HLA-DR1+ dendritic cells. Analysis of more than 3000 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration. P...
Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mat... more Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in pre-formed MHCI–peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA–B*58, HLA–B*08 and HLA–A*02. For all MHCI–peptide combinations, peptide binding onto MHCI protected against ERAP1–mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08–bound 12mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic and computational analyses suggested that this 12mer is highly labile and that apparent on–MHC trimming rates are alwa...
ERAP1 is an endoplasmic reticulum-resident zinc aminopeptidase that plays an important role in th... more ERAP1 is an endoplasmic reticulum-resident zinc aminopeptidase that plays an important role in the immune system by trimming peptides for loading onto major histocompatibility complex (MHC-I) proteins. Here we report discovery of the first inhibitors selective for ERAP1 over its paralogs ERAP2 and IRAP. Compound 1 (N-(N-(2-(1H-indol-3-yl)ethyl)carbamimidoyl)-2,5-difluorobenzenesulfonamide) and compound 2 (1-(1-(4-acetylpiperazine-1-carbonyl)cyclohexyl)-3-(p-tolyl)urea) are competitive inhibitors of ERAP1 aminopeptidase activity. Compound 3 (4-methoxy-3-(N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)sulfamoyl)benzoic acid) allosterically activates ERAP1's hydrolysis of fluorogenic and chromogenic amino acid substrates but competitively inhibits its activity towards a nonamer peptide representative of physiological substrates. Compounds 2 and 3 inhibit antigen presentation in a cellular assay. Compound 3 displays higher potency for an ERAP1 variant associated with increased risk of autoimmune disease. These inhibitors provide mechanistic insight into the determinants of specificity for ERAP1, ERAP2 and IRAP, and offer a new therapeutic approach of specifically inhibiting ERAP1 activity in vivo.
Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can generate or de... more Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can generate or destroy potential peptide ligands for MHC class I molecules. ERAP1 activity influences the cell-surface immunopeptidome and epitope immunodominance patterns but in complex and poorly understood manners. Two main distinct pathways have been proposed to account for ERAP1's effects on the nature and quantity of MHCI-bound peptides: i) ERAP1 trims peptides in solution, generating the correct length for binding to MHCI or overtrimming peptides so that they are too short to bind, and ii) ERAP1 trims peptides while they are partially bound onto MHCI in manner that leaves the peptide amino terminus accessible. For both pathways, once an appropriate length peptide is generated it could bind conventionally to MHCI, competing with further trimming by ERAP1. The two pathways, although not necessarily mutually exclusive, provide distinct vantage points for understanding of the rules behind the gene...
Shigella is unique among enteric pathogens, as it invades colonic epithelia through the basolater... more Shigella is unique among enteric pathogens, as it invades colonic epithelia through the basolateral pole. Therefore, it has evolved the ability to breach the intestinal epithelial barrier to deploy an arsenal of effector proteins, which permits bacterial invasion and leads to a severe inflammatory response. However, the mechanisms used by Shigella to regulate epithelial barrier permeability remain unknown. To address this question, we used both an intestinal polarized model and a human ex-vivo model to further characterize the early events of host-bacteria interactions. Our results showed that secreted Serine Protease A (SepA), which belongs to the serine protease autotransporter of Enterobacteriaceae family, is responsible for critically disrupting the intestinal epithelial barrier. Such disruption facilitates bacterial transit to the basolateral pole of the epithelium, ultimately fostering the hallmarks of the disease pathology. SepA was found to cause a decrease in active LIM Kin...
Recent advances in mass spectrometry technology have facilitated detailed examination of MHC-II i... more Recent advances in mass spectrometry technology have facilitated detailed examination of MHC-II immunopeptidomes, i.e. the repertoires of peptides bound to MHC-II molecules expressed in antigen presenting cells. These studies have deepened our view of MHC-II presentation. Other studies have broadened our view of pathways leading up to peptide loading. Here we review these recent studies in the context of earlier work on conventional and non-conventional MHC-II processing. The message that emerges is that sources of antigen beyond conventional endosomal processing of endocytosed proteins are important for generation of cellular immune responses to pathogens and maintenance of central and peripheral tolerance. The multiplicity of pathways results in a broad MHC II immunopeptidome that conveys the sampled environment to patrolling T cells.
Patients with obesity, metabolic syndrome, or type-2 diabetes (T2D) often exhibit hyperglycemia a... more Patients with obesity, metabolic syndrome, or type-2 diabetes (T2D) often exhibit hyperglycemia and hyperlipidemia. A consequence of the dysmetabolism is non-enzymatic coupling of reactive sugars and lipids with proteins and DNA inducing glycation and glycoxidation. Biochemically, these modifications disrupt the cellular proteome by changing the protein chemical structure and inducing protein unfolding, cross-linking and aggregation. We investigated protein post-translational modifications (PTM) induced by glycation and glycoxidation in PBMC from non-diabetic, pre-diabetic and diabetic individuals as well as CD11c+ dendritic cells purified from mice fed a high-fat diet, obese (Ob/Ob) mice and relative controls. By employing label-free bottom-up proteomic and bioinformatics analysis we mapped several cellular pathways affected by the oxidative PTMs, including the antigen processing and presentation machinery. Functional assays of endosomal processing, using transthyretin and insulin ...
Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in seve... more Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in several metabolic diseases including diabetes. These highly reactive moieties bind to the cellular proteome to form advanced glycation end-products (AGEs), which can alter the structure and function of targeted proteins. To address whether the AGE-mediated modification of immunologically relevant proteins could have functional consequences we incubated soluble recombinant HLA- DR1 (HLA-DRA*01:01/DRB1*01:01) in presence of glucose at concentration commonly observed in diabetic subjects (15–20 mM). Analysis of the AGE adducts was achieved by using bottom-up proteomics and tandem mass spectrometry analysis. Major observed modifications were glyoxal-derived hydroimiadazolone, argpyrimidine, dihydroxy and methylglyoxal adducts on arginine residues, pyralline and glyceraldehyde-derived pyridinium compound adducts of lysine and carboxy ethylation and methylation of both arginine and lysine residues. These AGE-related modifications were comparable to what observed on MHCII, immunoprecipitated from the PBMC of Diabetic patients. AGE PTMs mapped to both the binding groove and HLA-DM binding domains. AGE-modified and unmodified HLA-DR1 were then assessed for their binding affinity of several relevant peptides in a fluorescence polarization assay in the presence or absence of HLA-DM. Changes in binding affinity for the selected peptides was observed in AGE-HLA-DR1 as compared to the unmodified HLA-DR1. Altogether our data indicate that protein PTM, as commonly observed in conditions of metabolic and oxidative stress can change HLA-DR1/peptide binding affinity thus compromising MHC-II restricted immune responses.
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Papers by Lawrence Stern