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Towards understanding HLA-E expression and function
Abstracts
0494
91
0495
TOWARDS UNDERSTANDING HLA-EEXPRESSION ANDFUNCTION Ni Lee, Hans Marquardt, and Daniel E. Geraghty Fred Hutchinson Cancer Research Center, Seattle WA, USA To date, no clear understanding 01 HLA-E expression has been elucidated and previous work had shown that HLA-E was expressed intracellularly but was not expressed on the surface of the class I deficient cell line .221. In order to gain insight into the signals controlling surface expression of HLA-E, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner. we identified a hybrid construct containing the' A2 signal sequence which was expressed on the cell surface of .221. Biochemical analysis showed that both the mature E and AEH proteins were identical in their primary structure and quantitative analysis showed both proteins were expressed at essentially similar levels. These results demonstrated the different signal sequences did not affect the position of signal peptide cleavage from the mature proteins. Further analysis showed that AEH was complexed with hound peptide while the HLA-E protein lacked substantial bound peptide. These results led us to explore the antigen presentation of HLA-E in normal cells. To do this we developed an HLA-E specific antibody useful for analysis of HLA-E expression in the background of other HLA class I proteins. Using FACS analysis. biochemical analysis. and analysis of peptides eluted from HLA-E expressed in normal LCL. we were able to decipher important new information about the expression of HLA-E and about the specificity of peptide binding by HLA-E. These findings indicate a unique functional role for HLA-E in the immune process, both relating to classical class I peptide binding as well as to a direct functional interaction of HLA-E to the immune response.
A STRATEGY FOR PREDICTION OF PEPTIDE BINDING AFFINITY FOR THE HUMAN PEPTIDE TRANSPORTER Van Endert PM, Daniel S, Hammer J*, Sinigaglia F*, Riganelli D#, Petrovsky Nt. Bach JF, Caillat-Zucman S. INSERM U25, Paris, France; *Roche Milano Ricerche, Milan, and #Institut for Int. Med., Perugia, Italy; '(WEHI, Melbourne, Australia.
0496
0497
RESIDUES IN TAP PEPTIDE TRANSPORTERS CONTROLLING SUBSTRATE SPECIFICITY
Selection of epitopes for presentation by HI..A class I molecules depends on their affinity for HI..A and the efficiency of peptide generation in antigen processing which is governed by so far unknown rules. Peptide transport into the ER by the TAP complex may account for part or all of this selectivity. In order to analyze TAP affinities of a large number of epitopes, and potentially to predict those peptides with high HI..Aaffinities likely to be generated by processing, we are trying to develop a method for prediction of TAP affinities (measured as IC50s in a binding assay using human TAP expressed in insect cells). As a first step, we have measured IC50s of a library of 171 poly-Ala peptides with 19 single substitutions in each of the 9 positions. The results of this analysis allow limited prediction of efficient TAP binders. We hope to increase predictive power by a complex chemometric analysis of variables determining the affinities of 9-mer (6-parameter analysis) and 12-mer (4-parameters) peptides, and by training a neural network with a large set of peptide IC50s.
EVIDENCE FOR lILA CLASS II SUPERDIMERS ON ANTIGEN PRESENTING CELLS
Frank Momburg, Elena A. Arrnandola, Jens-O. Koopmann, Markus Post, Gunter J.
Hammerling Department of Molecular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the endoplasmic reticulum (ER) where they associate with major histocompatibility complex (MHC) class I molecules. Using an in vitro assay on permeabilized
cells two specificity patterns with regard to the C-tenninal residue of transported peptides have been previously shown, While the u allele of rat TAP and the mouse TAP preferentially transport peptides with hydrophobic C-terminal residues, no such selection was reported for the a allele of rat TAP and for the human TAP. We constructed several chimeric molecules containing parts of rat lAna and TAP2u and expressed them together
with rat TAPI in TAP-deficient T2 cells. With thesehybrids we wereableto maptwo short stretches in rat TAP2, with two polymorphic residues each, that essentially control
thedifferential peptide transport ohserved fortheratalleles. Thecritical residues are located in putative cytoplasmic loops close to the membrane. Using hybrid TAP molecules consisting efTAPl and TAP2 subunits derived from different species a subtle influence of the TAPl subunit on the specificity of transport could be demonstrated.The specificity of transport with regard to the length of the peptide substrate was analyzed with series of peptides that contained an Asnl X/Thr glycosylation consensus signal and an iodinated Tyr residua at opposite ends. Peptide transporters from different species preferentially transported peptides of8-12 amino acids, a length that fits with the requirements of class I molecules, but also shorter peptides (6-mers) and much longer peptides of up to 40 residues were found to be translocated and glycosylated. The length selection observed for the ATP-dependent peptide translocation was confirmed using an assay that measures the ATP-independent binding of peptides to TAP.
0498 INTRACELLULAR PATHWAY OF MHC CLASS II MOLECULES IN HUMAN DENDRITIC CELLS, 1. Salamero, C. Saudrais, D. Spehner*, H. de la Salle*, A. Bohbot", J-P Cazenave**, B. Goud, D. Ranau* UMR 144 CNRS-Institut Curie, 75005 Paris, *ClF INSERM 94-03, Laboratoire d'Histocompatibilite, and **INSERM U.311, ETS and "Service d'Onco-Hematologie, Hopital de Hautepierre, 67200 Strasbourg, France. From previous studies in B lympbocytes, it is currently believed that binding of antigenic peptides on MHC-II molecules occurs in the endocytic pathway following direct targeting of newly synthesized MHC-II molecules from the trans Golgi network to endosomal compartments. By using pulse chase metabolic labeling followed by cell surface biotinylation, we show here that in human dendritie cells, more than 50% of MHC-1I are first routed to the plasma membrane. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC II to the endosomal lysosomal system. These complexes rapidly internalize and allow the delivery of anti-Ii antibodies to MHC-II rich compartments as assessed by double immunolabeling in electron microscopy. Following degradation of Ii, these apIi complexes are rapidly converted to MHC Il-peptide complexes as shown here hy their sodium dodecyl sulfate stability, Ultimately, these complexes are delivered to the cell surface. If removal of Ii from the apli complexes is a sufficient prerequisite for peptide binding in DC, rapid dissociation of Ii from internalized apli complexes suggests that this pathway may allow newly liberated class II ap heterodimers to bind peptides in different compartments along the endocyticroute in these cell type.
Roucard Corinne, Ericson Mats, Garban Frederic, Mooney Nuala, Charron Dominique. INSERM U396, ] 5, rue de l'Ecole de Medecine, 75006 Paris, France. The conformation of lILA class II molecules on peripheral blood lymphocytes and on monocytes, freshly isolated or activated with IFNy, was studied. A Dk-speciflc mAb (DU2) was shown to preferentially precipitate dimers of a~ heterodimers, or « superdimers » from surface-labelled cell lysates. The 120 kD superdimers were stable in SDS at room temperature but dissociated into 60 kD heterodimers when heated to 50°C and into free a- and ~ monomers when boiled. After in vivo labelling of B cells, the superdimers were first detectable after Ih of chase which coincided with the appearance of SDS-stable a~-dimers and of proteolytic fragments of the invariant chain. This finding indicated that the superdimers form intracellularly before transport to the cell surface. A Western blot performed on whole cell lysates and subsequently quantified by densitometry indicated that the superdimers may represent some 15% of the total number of SDS-stable molecules in monocytes. A role of superdimers in the generation of multimeric coaggregates with CD4 molecules can be hypothesized.
0499
Ii CHAIN DETERMINES MHC CLASS II TRANSPORT INTO AND OUT OF LYSOSOMES S. Amigorena, V. Brachet and I. Mellman. Institut Curie, Section de Recherche, 12, rue Lhomond, 75005, Paris, France We have identified and isolated an endocytic compartment in B lymphocytes, different from endosomes and lysosomes, where MHC class II molecules transiently accumulate before arriving to the cell surface. Endosomal, but not lysosomal, markers where found by electron rnicroscocopy in isolated CIIV, indicating that these vesicles are not related to lysosornes. To question the relationship between conventional and specialized endocytic compartments, we analyzed the effect of leupeptin, an inhibitor of lysosomal proteases which interferes with Ii degradation. on the intracellular distributions of class II molecules. In the presence of leupeplin, a 10 kDa Ii-fragment remained associated to all dimers and retained class II molecules intracellularly. The Ii-plOa~ complexes were found in both CIIV and conventional Iysosomes. MHC class II molecules in these lysosomal compartments were not destined for degradation, since upon removal of leupeplin Ii-p I0 was degraded and peptide-loaded dimers were efficiently transported to the cell surface.
a~
0494
91
0495
TOWARDS UNDERSTANDING HLA-EEXPRESSION ANDFUNCTION Ni Lee, Hans Marquardt, and Daniel E. Geraghty Fred Hutchinson Cancer Research Center, Seattle WA, USA To date, no clear understanding 01 HLA-E expression has been elucidated and previous work had shown that HLA-E was expressed intracellularly but was not expressed on the surface of the class I deficient cell line .221. In order to gain insight into the signals controlling surface expression of HLA-E, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner. we identified a hybrid construct containing the' A2 signal sequence which was expressed on the cell surface of .221. Biochemical analysis showed that both the mature E and AEH proteins were identical in their primary structure and quantitative analysis showed both proteins were expressed at essentially similar levels. These results demonstrated the different signal sequences did not affect the position of signal peptide cleavage from the mature proteins. Further analysis showed that AEH was complexed with hound peptide while the HLA-E protein lacked substantial bound peptide. These results led us to explore the antigen presentation of HLA-E in normal cells. To do this we developed an HLA-E specific antibody useful for analysis of HLA-E expression in the background of other HLA class I proteins. Using FACS analysis. biochemical analysis. and analysis of peptides eluted from HLA-E expressed in normal LCL. we were able to decipher important new information about the expression of HLA-E and about the specificity of peptide binding by HLA-E. These findings indicate a unique functional role for HLA-E in the immune process, both relating to classical class I peptide binding as well as to a direct functional interaction of HLA-E to the immune response.
A STRATEGY FOR PREDICTION OF PEPTIDE BINDING AFFINITY FOR THE HUMAN PEPTIDE TRANSPORTER Van Endert PM, Daniel S, Hammer J*, Sinigaglia F*, Riganelli D#, Petrovsky Nt. Bach JF, Caillat-Zucman S. INSERM U25, Paris, France; *Roche Milano Ricerche, Milan, and #Institut for Int. Med., Perugia, Italy; '(WEHI, Melbourne, Australia.
0496
0497
RESIDUES IN TAP PEPTIDE TRANSPORTERS CONTROLLING SUBSTRATE SPECIFICITY
Selection of epitopes for presentation by HI..A class I molecules depends on their affinity for HI..A and the efficiency of peptide generation in antigen processing which is governed by so far unknown rules. Peptide transport into the ER by the TAP complex may account for part or all of this selectivity. In order to analyze TAP affinities of a large number of epitopes, and potentially to predict those peptides with high HI..Aaffinities likely to be generated by processing, we are trying to develop a method for prediction of TAP affinities (measured as IC50s in a binding assay using human TAP expressed in insect cells). As a first step, we have measured IC50s of a library of 171 poly-Ala peptides with 19 single substitutions in each of the 9 positions. The results of this analysis allow limited prediction of efficient TAP binders. We hope to increase predictive power by a complex chemometric analysis of variables determining the affinities of 9-mer (6-parameter analysis) and 12-mer (4-parameters) peptides, and by training a neural network with a large set of peptide IC50s.
EVIDENCE FOR lILA CLASS II SUPERDIMERS ON ANTIGEN PRESENTING CELLS
Frank Momburg, Elena A. Arrnandola, Jens-O. Koopmann, Markus Post, Gunter J.
Hammerling Department of Molecular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the endoplasmic reticulum (ER) where they associate with major histocompatibility complex (MHC) class I molecules. Using an in vitro assay on permeabilized
cells two specificity patterns with regard to the C-tenninal residue of transported peptides have been previously shown, While the u allele of rat TAP and the mouse TAP preferentially transport peptides with hydrophobic C-terminal residues, no such selection was reported for the a allele of rat TAP and for the human TAP. We constructed several chimeric molecules containing parts of rat lAna and TAP2u and expressed them together
with rat TAPI in TAP-deficient T2 cells. With thesehybrids we wereableto maptwo short stretches in rat TAP2, with two polymorphic residues each, that essentially control
thedifferential peptide transport ohserved fortheratalleles. Thecritical residues are located in putative cytoplasmic loops close to the membrane. Using hybrid TAP molecules consisting efTAPl and TAP2 subunits derived from different species a subtle influence of the TAPl subunit on the specificity of transport could be demonstrated.The specificity of transport with regard to the length of the peptide substrate was analyzed with series of peptides that contained an Asnl X/Thr glycosylation consensus signal and an iodinated Tyr residua at opposite ends. Peptide transporters from different species preferentially transported peptides of8-12 amino acids, a length that fits with the requirements of class I molecules, but also shorter peptides (6-mers) and much longer peptides of up to 40 residues were found to be translocated and glycosylated. The length selection observed for the ATP-dependent peptide translocation was confirmed using an assay that measures the ATP-independent binding of peptides to TAP.
0498 INTRACELLULAR PATHWAY OF MHC CLASS II MOLECULES IN HUMAN DENDRITIC CELLS, 1. Salamero, C. Saudrais, D. Spehner*, H. de la Salle*, A. Bohbot", J-P Cazenave**, B. Goud, D. Ranau* UMR 144 CNRS-Institut Curie, 75005 Paris, *ClF INSERM 94-03, Laboratoire d'Histocompatibilite, and **INSERM U.311, ETS and "Service d'Onco-Hematologie, Hopital de Hautepierre, 67200 Strasbourg, France. From previous studies in B lympbocytes, it is currently believed that binding of antigenic peptides on MHC-II molecules occurs in the endocytic pathway following direct targeting of newly synthesized MHC-II molecules from the trans Golgi network to endosomal compartments. By using pulse chase metabolic labeling followed by cell surface biotinylation, we show here that in human dendritie cells, more than 50% of MHC-1I are first routed to the plasma membrane. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC II to the endosomal lysosomal system. These complexes rapidly internalize and allow the delivery of anti-Ii antibodies to MHC-II rich compartments as assessed by double immunolabeling in electron microscopy. Following degradation of Ii, these apIi complexes are rapidly converted to MHC Il-peptide complexes as shown here hy their sodium dodecyl sulfate stability, Ultimately, these complexes are delivered to the cell surface. If removal of Ii from the apli complexes is a sufficient prerequisite for peptide binding in DC, rapid dissociation of Ii from internalized apli complexes suggests that this pathway may allow newly liberated class II ap heterodimers to bind peptides in different compartments along the endocyticroute in these cell type.
Roucard Corinne, Ericson Mats, Garban Frederic, Mooney Nuala, Charron Dominique. INSERM U396, ] 5, rue de l'Ecole de Medecine, 75006 Paris, France. The conformation of lILA class II molecules on peripheral blood lymphocytes and on monocytes, freshly isolated or activated with IFNy, was studied. A Dk-speciflc mAb (DU2) was shown to preferentially precipitate dimers of a~ heterodimers, or « superdimers » from surface-labelled cell lysates. The 120 kD superdimers were stable in SDS at room temperature but dissociated into 60 kD heterodimers when heated to 50°C and into free a- and ~ monomers when boiled. After in vivo labelling of B cells, the superdimers were first detectable after Ih of chase which coincided with the appearance of SDS-stable a~-dimers and of proteolytic fragments of the invariant chain. This finding indicated that the superdimers form intracellularly before transport to the cell surface. A Western blot performed on whole cell lysates and subsequently quantified by densitometry indicated that the superdimers may represent some 15% of the total number of SDS-stable molecules in monocytes. A role of superdimers in the generation of multimeric coaggregates with CD4 molecules can be hypothesized.
0499
Ii CHAIN DETERMINES MHC CLASS II TRANSPORT INTO AND OUT OF LYSOSOMES S. Amigorena, V. Brachet and I. Mellman. Institut Curie, Section de Recherche, 12, rue Lhomond, 75005, Paris, France We have identified and isolated an endocytic compartment in B lymphocytes, different from endosomes and lysosomes, where MHC class II molecules transiently accumulate before arriving to the cell surface. Endosomal, but not lysosomal, markers where found by electron rnicroscocopy in isolated CIIV, indicating that these vesicles are not related to lysosornes. To question the relationship between conventional and specialized endocytic compartments, we analyzed the effect of leupeptin, an inhibitor of lysosomal proteases which interferes with Ii degradation. on the intracellular distributions of class II molecules. In the presence of leupeplin, a 10 kDa Ii-fragment remained associated to all dimers and retained class II molecules intracellularly. The Ii-plOa~ complexes were found in both CIIV and conventional Iysosomes. MHC class II molecules in these lysosomal compartments were not destined for degradation, since upon removal of leupeplin Ii-p I0 was degraded and peptide-loaded dimers were efficiently transported to the cell surface.
a~