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Invariant chain as a tool to load antigenic peptides on MHC class I
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Abstracts / Molecular Immunology 51 (2012) 5–41
Invariant chain as a tool to load antigenic peptides on MHC class I Sébastien Wälchli a , Ole Landsverk b , Lars-Egil Fallang a , Johanna Olweus a , Oddmund Bakke b , Tone Gregers b,∗ a
Department of Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Norway b Department of Molecular Biosciences and Centre for Immune Regulation, University of Oslo, Norway CD4+ T cells recognize exogenously derived peptides presented on the cell surface of antigen presenting cells in the MHC class II (MHC II) context. The biosynthesis and transport of MHC II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. Several studies have shown that by genetically exchanging the CLIP region with antigenic peptides, MHC II molecules are efficiently loaded with the peptide and presented to peptide specific CD4+ T cells. Unlike MHC II, MHC I binds mainly endogenously derived peptides generated by the proteasome and targeted to the endoplasmic reticulum (ER) prior to MHC I pepitde loading and presentation for specific CD8+ T cells. MHC I is independent on Ii, however, a few studies have shown that Ii interacts with MHC I probably via the CLIP region. We therefore asked if it was possible to load a class I peptide onto MHC-I using a modified Ii as a vehicle. We here show that Ii and MHC-I co-localize in the endosomal pathway when co-expressed in model cell lines. We present biochemical evidence that MHC-I can interact with Iiwt, and that the interaction is increased when the CLIP peptide is exchanged by known MHC-I peptides, indicating that the interaction is mediated via the CLIP region. Finally, we use these constructs in a CD8+ Tcell functional assay. Taken together these data demonstrate that Ii-peptide can efficiently load MHC-I and that the loaded complex can be presented to T-cells. doi:10.1016/j.molimm.2012.02.039 Why the structure but not the activity of an immunoproteasome subunit rescues antigen presentation Marcus Groettrup a,∗ , Christoph Lauer a , Jacqueline Moebius a , Reinhold Weber b , Michael Przybylski b , Alexei Kisselev c , d e Christopher Tsu , Michael Basler a
Division of Immunology, Department of Biology, Constance University b Laboratory of Analytical Chemistry, Department of Chemistry, Constance University c Department of Pharmacology & Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School d Millenium Pharamceuticals Inc e Biotechnology Institute Thurgau at the University of Constance The replacement of the catalytically active proteasome subunits beta1, beta2, and beta5 by the immunoproteasome subunits LMP2 (beta1i), MECL-1 (beta2i), and LMP7 (beta5i) is required for the production of numerous class-I-ligands. The structural features rather than the proteolytic activity of an immunoproteasome subunit are needed for the generation of some epitopes but the underlying mechanisms have remained elusive. Experiments with LMP2-deficient splenocytes revealed that the generation of the male HY-derived CTL-epitope UTY246-254 was dependent on LMP2. Unexpectedly, treatment of male splenocytes with an LMP2-selective inhibitor did not reduce UTY246-254-presentation,
whereas inhibition or genetic silencing of beta1-activity, increased presentation of UTY246-254. In vitro degradation experiments showed that the caspase-like activity of beta1 was responsible for destruction of this CTL-epitope, while it was preserved when LMP2 replaced beta1. Moreover, inhibition of the beta5 subunit rescued the presentation of the HLA-A0201 influenza Matrix M1 58-66 epitope previously described as dependent on the “structure” rather than the activity of LMP7, thus indicating that a similar mechanism can apply to the exchange of beta5 by LMP7. It therefore appears that an important function of immunoproteasome subunits is that they preserve epitopes by avoiding destructive cleavages by replacing their constitutive homologues. This insight will be discussed in ˚ X-ray crystallographic structure light of the high resolution (2.9 A) of the mouse constitutive proteasome and immunoproteasome which we have recently obtained. Taken together, our data provide a rationale why the structural property of an immunoproteasome subunit rather than its activity is required for the generation of a CTL-epitope. doi:10.1016/j.molimm.2012.02.040 The tapasin isoform NeTT (new tapasin transcript) encoded by an alternatively spliced transcript lacking exon 3 impairs PLC (peptide loading complex) conferred stabilization of MHC class I molecules Nele Beutler a , Sebastian Hauka a , Julia Uhlmann a , Alexandra Niepel a , Esther Ghanem b , Heiner Schaal a , Frank Momburg c , Sebastian Springer b , Hartmut Hengel a , Anne Halenius a,∗ a
Institute for Virology, HHU Duesseldorf Biochemistry and Cell Biology, Jacobs University Bremen c Division of Translational Immunology (D015), German Cancer Research Center b
CD8 T-cell recognition of a foreign peptide presented on MHC class I results in elimination of the target cell, thereby preventing spread of virus infections or tumor growth. To assure efficient MHC I peptide loading the peptide loading complex (PLC) recruits the peptide receptive form of MHC I in the ER. In this process the chaperone tapasin plays a crucial role, connecting MHC I to the peptide transporter TAP. A stable disulfide bond formed between tapasin and ERp57 is important for selection of high-affinity peptides that stabilize the MHC I complex. In cells infected with human cytomegalovirus (HCMV), an alternatively spliced tapasin transcript lacking exon 3 was observed, here called NeTT (new tapasin transcript). Recognition of exon 3 was found to be regulated via Gruns downstream of its 5 splice site suggesting that members of the hnRNP family regulate NeTT expression. Since exon 3 encodes cysteine 95, responsible for the disulfide bond formation with ERp57, NeTT interaction with ERp57 was strongly impaired While in tapasin-deficient cells NeTT specifically stabilized TAP expression, but not MHC I, in tapasin-proficient cells, NeTT reduced the cell surface expression of the tapasin-dependent HLA-B*44:02 but not of the tapasin-independent HLA-B*44:05. Detailed analysis of the PLC composition in NeTT expressing cells suggest that NeTT and tapasin can bind to TAP simultaneously. Thus, NeTT expression decreases the number of functional interaction sites for MHC I on the PLC, which implies that the quality control of MHC I peptide loading by tapasin is reduced. doi:10.1016/j.molimm.2012.02.041
Abstracts / Molecular Immunology 51 (2012) 5–41
Invariant chain as a tool to load antigenic peptides on MHC class I Sébastien Wälchli a , Ole Landsverk b , Lars-Egil Fallang a , Johanna Olweus a , Oddmund Bakke b , Tone Gregers b,∗ a
Department of Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Norway b Department of Molecular Biosciences and Centre for Immune Regulation, University of Oslo, Norway CD4+ T cells recognize exogenously derived peptides presented on the cell surface of antigen presenting cells in the MHC class II (MHC II) context. The biosynthesis and transport of MHC II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. Several studies have shown that by genetically exchanging the CLIP region with antigenic peptides, MHC II molecules are efficiently loaded with the peptide and presented to peptide specific CD4+ T cells. Unlike MHC II, MHC I binds mainly endogenously derived peptides generated by the proteasome and targeted to the endoplasmic reticulum (ER) prior to MHC I pepitde loading and presentation for specific CD8+ T cells. MHC I is independent on Ii, however, a few studies have shown that Ii interacts with MHC I probably via the CLIP region. We therefore asked if it was possible to load a class I peptide onto MHC-I using a modified Ii as a vehicle. We here show that Ii and MHC-I co-localize in the endosomal pathway when co-expressed in model cell lines. We present biochemical evidence that MHC-I can interact with Iiwt, and that the interaction is increased when the CLIP peptide is exchanged by known MHC-I peptides, indicating that the interaction is mediated via the CLIP region. Finally, we use these constructs in a CD8+ Tcell functional assay. Taken together these data demonstrate that Ii-peptide can efficiently load MHC-I and that the loaded complex can be presented to T-cells. doi:10.1016/j.molimm.2012.02.039 Why the structure but not the activity of an immunoproteasome subunit rescues antigen presentation Marcus Groettrup a,∗ , Christoph Lauer a , Jacqueline Moebius a , Reinhold Weber b , Michael Przybylski b , Alexei Kisselev c , d e Christopher Tsu , Michael Basler a
Division of Immunology, Department of Biology, Constance University b Laboratory of Analytical Chemistry, Department of Chemistry, Constance University c Department of Pharmacology & Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School d Millenium Pharamceuticals Inc e Biotechnology Institute Thurgau at the University of Constance The replacement of the catalytically active proteasome subunits beta1, beta2, and beta5 by the immunoproteasome subunits LMP2 (beta1i), MECL-1 (beta2i), and LMP7 (beta5i) is required for the production of numerous class-I-ligands. The structural features rather than the proteolytic activity of an immunoproteasome subunit are needed for the generation of some epitopes but the underlying mechanisms have remained elusive. Experiments with LMP2-deficient splenocytes revealed that the generation of the male HY-derived CTL-epitope UTY246-254 was dependent on LMP2. Unexpectedly, treatment of male splenocytes with an LMP2-selective inhibitor did not reduce UTY246-254-presentation,
whereas inhibition or genetic silencing of beta1-activity, increased presentation of UTY246-254. In vitro degradation experiments showed that the caspase-like activity of beta1 was responsible for destruction of this CTL-epitope, while it was preserved when LMP2 replaced beta1. Moreover, inhibition of the beta5 subunit rescued the presentation of the HLA-A0201 influenza Matrix M1 58-66 epitope previously described as dependent on the “structure” rather than the activity of LMP7, thus indicating that a similar mechanism can apply to the exchange of beta5 by LMP7. It therefore appears that an important function of immunoproteasome subunits is that they preserve epitopes by avoiding destructive cleavages by replacing their constitutive homologues. This insight will be discussed in ˚ X-ray crystallographic structure light of the high resolution (2.9 A) of the mouse constitutive proteasome and immunoproteasome which we have recently obtained. Taken together, our data provide a rationale why the structural property of an immunoproteasome subunit rather than its activity is required for the generation of a CTL-epitope. doi:10.1016/j.molimm.2012.02.040 The tapasin isoform NeTT (new tapasin transcript) encoded by an alternatively spliced transcript lacking exon 3 impairs PLC (peptide loading complex) conferred stabilization of MHC class I molecules Nele Beutler a , Sebastian Hauka a , Julia Uhlmann a , Alexandra Niepel a , Esther Ghanem b , Heiner Schaal a , Frank Momburg c , Sebastian Springer b , Hartmut Hengel a , Anne Halenius a,∗ a
Institute for Virology, HHU Duesseldorf Biochemistry and Cell Biology, Jacobs University Bremen c Division of Translational Immunology (D015), German Cancer Research Center b
CD8 T-cell recognition of a foreign peptide presented on MHC class I results in elimination of the target cell, thereby preventing spread of virus infections or tumor growth. To assure efficient MHC I peptide loading the peptide loading complex (PLC) recruits the peptide receptive form of MHC I in the ER. In this process the chaperone tapasin plays a crucial role, connecting MHC I to the peptide transporter TAP. A stable disulfide bond formed between tapasin and ERp57 is important for selection of high-affinity peptides that stabilize the MHC I complex. In cells infected with human cytomegalovirus (HCMV), an alternatively spliced tapasin transcript lacking exon 3 was observed, here called NeTT (new tapasin transcript). Recognition of exon 3 was found to be regulated via Gruns downstream of its 5 splice site suggesting that members of the hnRNP family regulate NeTT expression. Since exon 3 encodes cysteine 95, responsible for the disulfide bond formation with ERp57, NeTT interaction with ERp57 was strongly impaired While in tapasin-deficient cells NeTT specifically stabilized TAP expression, but not MHC I, in tapasin-proficient cells, NeTT reduced the cell surface expression of the tapasin-dependent HLA-B*44:02 but not of the tapasin-independent HLA-B*44:05. Detailed analysis of the PLC composition in NeTT expressing cells suggest that NeTT and tapasin can bind to TAP simultaneously. Thus, NeTT expression decreases the number of functional interaction sites for MHC I on the PLC, which implies that the quality control of MHC I peptide loading by tapasin is reduced. doi:10.1016/j.molimm.2012.02.041