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Exosomes isolated from a murine B cell line present antigen to an MHC class II restricted T cell hybridoma
24 June 1997 - Poster presentations Results and Con&don: We could then demonstrate that HLA-E binds, although poorty. the peptide which binds to Qa-1 and that it also binds with high affinity 9-mer signal sequence-derived psptides from human MHC dass I mofecules. Using alanine and glycfne substitutions, we could define primary anchor residues at positions 2 and 9 and serondaty an&or residues at position 7 and possibly 3.
P.1.05.24 Differential precessing of an influenza nucleoproteln epltope In human and murlne cells V. Braud, Vincenzo Cerundolo, A. McMichael. lnsfifute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Introduotlon: Studies of antigen presentation by MHC class I molecules in different species have so far identified a different selectivity of transport of peptides by the transporter associated with antigen processing (TAP). We investigated whether cytosolic generation of antigenic peptides by the multicatalytic proteinase complex proteasome could also show a functional polymorphism among spedes. We addressed this issue by studying the presentation of an influenza A nucleoprotein epftope in murine cells. This epitope has been shown to sensitize human targets cells for lysis by NP-specific, HLA-A3 restricted CTLs. Materlaband Methods: Cytotoxic assays were performed using L cells transfected with HLA-A3. human 82 microglobulin and LFA3 as targets. These cells were infected with recombinant vaccinia viruses encoding the full length NP, the 9-mer NP peptide, or human TAP complex. Results and Conclusion: We found that the NP epitope was not presented by mutine cells when encoded within the full length NP. The lack of presentation was not due to a defective transport ofthepeptide by murine TAP complex since L-A3-hp2 m-LFA3 cells were lysed when infected by a minlgene expressing the Qmer peptide in the cytosol. The lack of generation of the epitope in murine cells was also confirmed by coinfecting the cells with vaccinia viruses encoding the full length NP and human TAP complex. No presentation was observed in mutine cells whereas presentation was restored in TAP negative cells T2-A3. Altogether, these results suggest a differential processing between murine and human cells.
1P.1.05.251 Processing of bacteria for peptlde presentation on MHC-I occurs by different pathways In dendrltlc cells and macrophages M. Svensson, M.J. Wick. Dept. of Cell and Mok. Biol., Sect for Immunol, Lund Univeniw Box 7031,220 07 Lund, Sweden Introduction: Murine peritoneal macrophages (M0) and bone marrow-derived dendritic cells (DCs) process gram-negative bacteria with no known mechanism of phagosomal escape for peptide presentation on MHC-I. The present study investigates if this antigen processing pathway in DCs requires the transporter assocfated with antigen processing (TAP). Materlab and Methods:DCs from TAP1-I- tics were co-incubated with .E. co/i or S. fyphimurium expressing the Kb-binding OVA(257-264) epitope exoressed in bacteria as a cvtoolasmic fusion protein called Cd-OVA. After 2 hr& antigen processing was &$ped by fixing the cells, the cells were washed and a T hybridoma that produces IL-2 upon recognition of the Kb/OVA(257-264) complex &as added to quantitate antigen pmce&ing and presentation. Fteeuft% Presentation of OVA(257-264) after DCs phagocytosed bacteria expressing Cd-OVA was found to be dependent on TAP. This is in contrast to what was found with M0 where presentation of the same epitope occurred independently of TAP. The in viw significance of bacterial antigen presentation by DCs was also investigated and we show that C57BU6 DCs loaded in vitrowith bactetia expressing the OVA(257-264) epitops elicit OVA(257-264)~specific cytotoxic T cells in viw after injection into C57BU6 mice. Conclusion: DCs and M0s: can both process gram-negative bacteria and present bacterial antigens on MHC-I. However, the mechanism by which this occurs seems to dir between DCs and M0s: DCs process bacteria expressing Cd-OVA and present OVA(257-264) on Kb using a TAP-dependent pathway whereas M0 use a TAP-independent pathway. Furthermore, DC presentation of bacterial antigens on MHC-I may have in viw signfficance in that DCs loaded with bacterial antigens can prime cytotoxic T cells in mice. The ability of DCs and M0 to present bacterial antigens on MHC-I may bs important for priming cytotoxfc T cell immune responses to pathogens capable of replicating within infected host cells.
iUHC molecules: synthesis, assembly and peptide binding
241
I..P 1 05 .26 1 Exosomes
Isolated from a murlna B cell line present antlgen to an MHC class II restrlcted T cell hybrldoma
D.H. Schuurhuis’, M.J. Kleijmeer2, M.L.H. de Bruijn’, H.J. Geuze*, C.J.M. Melief I. 1Dept. of Immunchematol~ Univemity Hospital Leiden, POBox 96w, 2300 RC Leiden, The Netherlands, *Dept. of Cell Biolog)! Facu/iy of Medicine, Utrecht lJniwrsi& POBox 85500, 3508 GA Ukecht, The Netherlands Introduction: MHC class II-enriched compartments (MIIC) are endocytfc structures, which harbor the majority of intracellular MHC class II molecules. They represent the potential binding site of processed internalized antigens to MHC class II. Several morphological subtypes of MllCs can be distinguished, induding those with internal vesicles and membrane sheets, termed multivesicular and multilaminar MIICs, respectively. A possible pathway of peptide-loaded MHC class II molecules to the cell surface has recently been demonstrated. In human B cells multivesicular MllCs have been shown to undergo exccytosis: the MIIC limiting membrane fuses with the plasma membrane and the internal membrane vesicles (exosomes) are secreted. Exosomes are able to induce antigen-specific T cell responses in vitro. To further investigate the functional role of exosomes, an in vivo model system is required. For this, we chose to examine murine exosomes, which we first characterized both biochemically and functionally. Materials and Methods: Exosomes were isolated from the conditioned media of an MHC class II positive murine B cell line (LB27.4) by differential centtifugation. Cell biological analysis included SDS-PAGE and immunoblotting as well as immunoelectmn microscopy. Functional analysis included antigen presentation to an ovafbumin specific, MHC dass II restricted T cell hybrfdoma. Resub lmmunoelectron microscopy showed the primary presence of small vesicles in the exosome preparation, and they were strongly labeled for MHC class II. The exosomes were B220 negative, indicating that these membrane vesicles do not originate from the plasma membrane. Biochemical analysis of pellets after differential centtifugation demonstrated that the cells and the exosome pellet were strongly enriched in both MHC class II a- and p-chain. The enrichment of other proteins, such as costimulatory mofecules, is currently being investigated. Functional analysis showed that exosomss activate an ovalbumin specific, MHC class II restricted T cell hybridoma in a peptide-dependent way. Conclusions: Exosomes have a high expression of MHC class II molecules, which are functional since they present the relevant peptide to an MHC class II restricted T cell hybrfdoma. Further studies may reveal whether exosomes also play a stimulatoty role in vivo, or whether they act in T cell tolerance or maintenance of T cell memory.
P.1.05.27 Proteasome specificity correlates with defective antlgen presentation In Burkltt’s lymphoma cells Teresa Ftisan, Victor Levitsky. Maria G. Masucci. Microbobgy and TumorBiologyCenter Katvlinska Institute, Stockholm, Sweden Introduction: Defects of antigen processing/presentation have been suggested to play a role in the escape of Burkitt’s lymphoma (EL) from cytotoxic T lymphocyte (CTL) mediated rejection. Impaired presentation of an immunodomlnant HLA All restricted epitope from the resident or recombinant vaccinia virus expressed Epstein-Barr virus nuclear antigen (EBNA)4 was demonstrated in the EBV positive E95B-BL28 and its EBV negative parental BL28. Recent evidence suggests that processing of antigens for class I restricted presentation requires both ubiquitination and subsequent degradation by the proteasome. Resuftsz We have analyzed the proteasome activity and specifity in BL cells and compared with lymphoblastoid cell lines (LCLs). Reduced levels of chymotrypsin- and trypsin-like activities against the SucLLVY-MCA and BocLRR-MCA fluorogenic substrates were detected in proteasome preparations from two BL lines, Bl28 and BL72. lmmunoprecipitation and 20 gel fractionation revealed a different subunit composition of the 20s proteasome. The two BL lines coexpressed the p subunits 6 and MB1 and the interferon (IFN)-y inducible subunits Lmp2 and Lmp7 and showed a relative increase of the B subunit MC10.2 and (I subunit MC3 and decrease of the a subunits MC3 and MC6. A synthetic peptide analogue of the immunodominant HLA All-restricted epitope from the EBV nudear antigen (EBNA)-4 was cleaved by the BL proteasomes and by proteasomes purified from the Lmp2I7 deficient T2 cell line while cleavage was not detected with proteasomes from LCLs. Conclurion:The data suggest that the characteristic proteasome subunit compositfon of BL cells results in the generation of a distinct set of endogenous peptides. Failure to produce certain immunogenic peptfdes could favour the escape of the tumor from cytotoxic T lymphocyte (CTL)-mediated rejection.
P.1.05.24 Differential precessing of an influenza nucleoproteln epltope In human and murlne cells V. Braud, Vincenzo Cerundolo, A. McMichael. lnsfifute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Introduotlon: Studies of antigen presentation by MHC class I molecules in different species have so far identified a different selectivity of transport of peptides by the transporter associated with antigen processing (TAP). We investigated whether cytosolic generation of antigenic peptides by the multicatalytic proteinase complex proteasome could also show a functional polymorphism among spedes. We addressed this issue by studying the presentation of an influenza A nucleoprotein epftope in murine cells. This epitope has been shown to sensitize human targets cells for lysis by NP-specific, HLA-A3 restricted CTLs. Materlaband Methods: Cytotoxic assays were performed using L cells transfected with HLA-A3. human 82 microglobulin and LFA3 as targets. These cells were infected with recombinant vaccinia viruses encoding the full length NP, the 9-mer NP peptide, or human TAP complex. Results and Conclusion: We found that the NP epitope was not presented by mutine cells when encoded within the full length NP. The lack of presentation was not due to a defective transport ofthepeptide by murine TAP complex since L-A3-hp2 m-LFA3 cells were lysed when infected by a minlgene expressing the Qmer peptide in the cytosol. The lack of generation of the epitope in murine cells was also confirmed by coinfecting the cells with vaccinia viruses encoding the full length NP and human TAP complex. No presentation was observed in mutine cells whereas presentation was restored in TAP negative cells T2-A3. Altogether, these results suggest a differential processing between murine and human cells.
1P.1.05.251 Processing of bacteria for peptlde presentation on MHC-I occurs by different pathways In dendrltlc cells and macrophages M. Svensson, M.J. Wick. Dept. of Cell and Mok. Biol., Sect for Immunol, Lund Univeniw Box 7031,220 07 Lund, Sweden Introduction: Murine peritoneal macrophages (M0) and bone marrow-derived dendritic cells (DCs) process gram-negative bacteria with no known mechanism of phagosomal escape for peptide presentation on MHC-I. The present study investigates if this antigen processing pathway in DCs requires the transporter assocfated with antigen processing (TAP). Materlab and Methods:DCs from TAP1-I- tics were co-incubated with .E. co/i or S. fyphimurium expressing the Kb-binding OVA(257-264) epitope exoressed in bacteria as a cvtoolasmic fusion protein called Cd-OVA. After 2 hr& antigen processing was &$ped by fixing the cells, the cells were washed and a T hybridoma that produces IL-2 upon recognition of the Kb/OVA(257-264) complex &as added to quantitate antigen pmce&ing and presentation. Fteeuft% Presentation of OVA(257-264) after DCs phagocytosed bacteria expressing Cd-OVA was found to be dependent on TAP. This is in contrast to what was found with M0 where presentation of the same epitope occurred independently of TAP. The in viw significance of bacterial antigen presentation by DCs was also investigated and we show that C57BU6 DCs loaded in vitrowith bactetia expressing the OVA(257-264) epitops elicit OVA(257-264)~specific cytotoxic T cells in viw after injection into C57BU6 mice. Conclusion: DCs and M0s: can both process gram-negative bacteria and present bacterial antigens on MHC-I. However, the mechanism by which this occurs seems to dir between DCs and M0s: DCs process bacteria expressing Cd-OVA and present OVA(257-264) on Kb using a TAP-dependent pathway whereas M0 use a TAP-independent pathway. Furthermore, DC presentation of bacterial antigens on MHC-I may have in viw signfficance in that DCs loaded with bacterial antigens can prime cytotoxic T cells in mice. The ability of DCs and M0 to present bacterial antigens on MHC-I may bs important for priming cytotoxfc T cell immune responses to pathogens capable of replicating within infected host cells.
iUHC molecules: synthesis, assembly and peptide binding
241
I..P 1 05 .26 1 Exosomes
Isolated from a murlna B cell line present antlgen to an MHC class II restrlcted T cell hybrldoma
D.H. Schuurhuis’, M.J. Kleijmeer2, M.L.H. de Bruijn’, H.J. Geuze*, C.J.M. Melief I. 1Dept. of Immunchematol~ Univemity Hospital Leiden, POBox 96w, 2300 RC Leiden, The Netherlands, *Dept. of Cell Biolog)! Facu/iy of Medicine, Utrecht lJniwrsi& POBox 85500, 3508 GA Ukecht, The Netherlands Introduction: MHC class II-enriched compartments (MIIC) are endocytfc structures, which harbor the majority of intracellular MHC class II molecules. They represent the potential binding site of processed internalized antigens to MHC class II. Several morphological subtypes of MllCs can be distinguished, induding those with internal vesicles and membrane sheets, termed multivesicular and multilaminar MIICs, respectively. A possible pathway of peptide-loaded MHC class II molecules to the cell surface has recently been demonstrated. In human B cells multivesicular MllCs have been shown to undergo exccytosis: the MIIC limiting membrane fuses with the plasma membrane and the internal membrane vesicles (exosomes) are secreted. Exosomes are able to induce antigen-specific T cell responses in vitro. To further investigate the functional role of exosomes, an in vivo model system is required. For this, we chose to examine murine exosomes, which we first characterized both biochemically and functionally. Materials and Methods: Exosomes were isolated from the conditioned media of an MHC class II positive murine B cell line (LB27.4) by differential centtifugation. Cell biological analysis included SDS-PAGE and immunoblotting as well as immunoelectmn microscopy. Functional analysis included antigen presentation to an ovafbumin specific, MHC dass II restricted T cell hybrfdoma. Resub lmmunoelectron microscopy showed the primary presence of small vesicles in the exosome preparation, and they were strongly labeled for MHC class II. The exosomes were B220 negative, indicating that these membrane vesicles do not originate from the plasma membrane. Biochemical analysis of pellets after differential centtifugation demonstrated that the cells and the exosome pellet were strongly enriched in both MHC class II a- and p-chain. The enrichment of other proteins, such as costimulatory mofecules, is currently being investigated. Functional analysis showed that exosomss activate an ovalbumin specific, MHC class II restricted T cell hybridoma in a peptide-dependent way. Conclusions: Exosomes have a high expression of MHC class II molecules, which are functional since they present the relevant peptide to an MHC class II restricted T cell hybrfdoma. Further studies may reveal whether exosomes also play a stimulatoty role in vivo, or whether they act in T cell tolerance or maintenance of T cell memory.
P.1.05.27 Proteasome specificity correlates with defective antlgen presentation In Burkltt’s lymphoma cells Teresa Ftisan, Victor Levitsky. Maria G. Masucci. Microbobgy and TumorBiologyCenter Katvlinska Institute, Stockholm, Sweden Introduction: Defects of antigen processing/presentation have been suggested to play a role in the escape of Burkitt’s lymphoma (EL) from cytotoxic T lymphocyte (CTL) mediated rejection. Impaired presentation of an immunodomlnant HLA All restricted epitope from the resident or recombinant vaccinia virus expressed Epstein-Barr virus nuclear antigen (EBNA)4 was demonstrated in the EBV positive E95B-BL28 and its EBV negative parental BL28. Recent evidence suggests that processing of antigens for class I restricted presentation requires both ubiquitination and subsequent degradation by the proteasome. Resuftsz We have analyzed the proteasome activity and specifity in BL cells and compared with lymphoblastoid cell lines (LCLs). Reduced levels of chymotrypsin- and trypsin-like activities against the SucLLVY-MCA and BocLRR-MCA fluorogenic substrates were detected in proteasome preparations from two BL lines, Bl28 and BL72. lmmunoprecipitation and 20 gel fractionation revealed a different subunit composition of the 20s proteasome. The two BL lines coexpressed the p subunits 6 and MB1 and the interferon (IFN)-y inducible subunits Lmp2 and Lmp7 and showed a relative increase of the B subunit MC10.2 and (I subunit MC3 and decrease of the a subunits MC3 and MC6. A synthetic peptide analogue of the immunodominant HLA All-restricted epitope from the EBV nudear antigen (EBNA)-4 was cleaved by the BL proteasomes and by proteasomes purified from the Lmp2I7 deficient T2 cell line while cleavage was not detected with proteasomes from LCLs. Conclurion:The data suggest that the characteristic proteasome subunit compositfon of BL cells results in the generation of a distinct set of endogenous peptides. Failure to produce certain immunogenic peptfdes could favour the escape of the tumor from cytotoxic T lymphocyte (CTL)-mediated rejection.