Tapasin−/− and TAP1−/− Macrophages Are Deficient in Vacuolar Alternate Class I MHC (MHC-I) Processing due to Decreased MHC-I Stability at Phagolysosomal pH This information is current as of September 29, 2021. Peter J. Chefalo, Andres G. Grandea III, Luc Van Kaer and Clifford V. Harding J Immunol 2003; 170:5825-5833; ; doi: 10.4049/jimmunol.170.12.5825 http://www.jimmunol.org/content/170/12/5825 Downloaded from References This article cites 55 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/170/12/5825.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Tapasin؊/؊ and TAP1؊/؊ Macrophages Are Deficient in Vacuolar Alternate Class I MHC (MHC-I) Processing due to Decreased MHC-I Stability at Phagolysosomal pH1 Peter J. Chefalo,* Andres G. Grandea III,2† Luc Van Kaer,† and Clifford V. Harding3* Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin؊/؊ cells and experiments with tapasin؊/؊ and TAP1؊/؊ macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin؊/؊ cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin؊/؊ cells might be Downloaded from peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both ta- pasin؊/؊ and TAP1؊/؊ macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin؊/؊ and TAP1؊/؊ macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin؊/؊ or TAP1؊/؊ cells at 26°C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed http://www.jimmunol.org/ for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments. The Journal of Immunology, 2003, 170: 5825–5833. onventional class I MHC (MHC-I)4 Ag processing in- vacuolar compartments to the cytosol, where they enter the con- volves Ags expressed in the cytosol of APC and does not ventional MHC-I processing and peptide loading pathway (3, 10– deal with exogenous Ags. Professional APCs, however, 12). This mechanism is sensitive to proteasome inhibitors, brefel- C by guest on September 29, 2021 do process exogenous Ags for presentation by MHC-I molecules din A (BFA; which blocks anterograde transport of nascent MHC-I ϩ to CD8 T cells. This mechanism has been termed alternate through the Golgi complex and therefore its delivery to the cell MHC-I Ag processing and forms the basis for cross-presentation surface) and deficiencies in TAP (3). The vacuolar alternate of Ags and cross-priming of CD8 T cells (1–3). Alternate MHC-I MHC-I pathway involves processing of Ags in vacuolar compart- ϩ processing plays an important role in the initiation of CD8 T cell ments (e.g., phagosomes or endosomes, without access to the cy- responses (4, 5). A more complete understanding of alternate tosol) and binding of peptides to MHC-I molecules in post-Golgi MHC-I processing is important to better understand host defense vacuolar compartments (e.g., within phagosomes or possibly at the for infectious disease and to develop new vaccine strategies. cell surface after recycling and regurgitation of peptide) (7, 13, Alternate MHC-I processing has been reported in many systems 14). This pathway is resistant to proteasome inhibitors and short and may proceed by distinct pathways (1, 2, 4, 6–9), including term treatments with BFA. In some experimental systems vacuolar cytosolic and vacuolar processing mechanisms, with processing in alternate MHC-I processing is TAP-independent (5, 15–17), but in different subcellular compartments. The cytosolic alternate MHC-I others it is partially inhibited by TAP deficiency due to a conse- pathway involves transfer of exogenous antigenic proteins from quent decrease in expression of post-Golgi peptide-receptive MHC-I molecules (8, 9, 18). Mutant cell lines and mouse strains that have mutations in com- *Department of Pathology, Case Western Reserve University, Cleveland, OH 44106; and †Department of Microbiology and Immunology, Vanderbilt University School of ponents of the conventional MHC-I pathway have been instrumen- Medicine, Nashville, TN 37232 tal for studying alternate MHC-I Ag processing. TAP is necessary Received for publication January 7, 2003. Accepted for publication March 19, 2003. for transport of peptides from the cytosol into the endoplasmic The costs of publication of this article were defrayed in part by the payment of page reticulum (ER) to bind MHC-I molecules (19–22). TAP-deficient charges. This article must therefore be hereby marked advertisement in accordance Ϫ/Ϫ with 18 U.S.C. Section 1734 solely to indicate this fact. cell lines and TAP1 mice are well characterized (19, 23), and Ϫ/Ϫ 1 This work was supported by National Institutes of Health Grants AI34343, AI35726, cells from TAP1 mice have been used to study the influence of and AI47255 (to C.V.H.). TAP on the expression of peptide-receptive MHC-I molecules that 2 Current address: Celltech R&D, Inc., 1631 220th Street, SE, Bothell, WA 98021. contribute to vacuolar alternate MHC-I processing (8, 9, 18). Ta- 3 ␤ Address correspondence and reprint requests to Dr. Clifford V. Harding, Department pasin is complexed with calreticulin, MHC-I- 2-microglobulin, of Pathology, BRB 925, Case Western Reserve University, 10900 Euclid Avenue, and TAP, and is necessary for association of TAP with the other Cleveland, OH 44106-4943. E-mail address: [email protected] components of this peptide loading complex (24, 25). In addition 4 Abbreviations used in this paper: MHC-I, class I MHC; BFA, brefeldin A; BMM, bone marrow-derived macrophages; CBS, citrate-buffered saline; ER, endoplasmic to promoting assembly of the MHC-I peptide loading complex, reticulum; PEM, peritoneal exudate macrophages; PeM, peritoneal macrophages. tapasin has been suggested to help retain MHC-I molecules in the Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 5826 TAPASIN AND TAP DEFICITS IMPACT PHAGOSOMAL MHC-I Ag PROCESSING ER until they are loaded with high affinity peptides (25–32). Re- phagosome, where acidic pH facilitates peptide exchange to form cently, tapasinϪ/Ϫ mice were described (33, 34). The present stud- complexes of MHC-I with antigenic peptides from exogenous Ag. ies provide the first examination of alternate MHC-I Ag processing in tapasinϪ/Ϫ cells. Materials and Methods Macrophages from both TAP1Ϫ/Ϫ and tapasinϪ/Ϫ mice have Cells and Ags MHC-I molecules that are poorly loaded, i.e., contain peptides that Ϫ/Ϫ Ϫ/Ϫ Ϫ Ϫ Tapasin mice (33) on C57BL/6 genetic background, TAP1 mice are not bound with high affinity. In TAP1 / cells this is due to the (23) on C57BL/6 genetic background, and wild-type C57BL/6 mice (The absence of peptide transport into the ER, greatly reducing the num- Jackson Laboratory, Bar Harbor, ME) were bred and housed under specific ber and diversity of peptides available for MHC-I binding. In ta- pathogen-free conditions. Cells were cultured in standard medium consist- Ϫ/Ϫ ing of DMEM (Life Technologies, Grand Island, NY) supplemented with pasin cells this may result from the absence of the complete 10% heat-inactivated FCS (HyClone, Logan, UT), 5 ϫ 10Ϫ5 M 2-ME, 1 peptide loading complex, and insufficient ER retention and peptide mM sodium pyruvate, HEPES buffer, and penicillin/streptomycin (Life exchange to optimize peptide loading of MHC-I. Consequently, Technologies). Cells were incubated in a 5% CO2 atmosphere and, unless MHC-I molecules on TAP- or tapasin-deficient cells have a short otherwise indicated, at 37°C. Activated peritoneal macrophages (PeM) were harvested by peritoneal lavage 4 days after i.p. inoculation