Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes

Mellins, Elizabeth; Smith, Lauren M.; Arp, Benjamin; Cotner, Tom; Celis, Esteban; Pious, Donald

doi:10.1038/343071a0

Cited by 224 publications

(153 citation statements)

References 28 publications

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“…Samples were normalized for protein and cell number before electrophoretic analysis and immunoblotting (10,12). Class II a3 dimers were detected on immunoblots by using antibody L243 and sample buffer with 0.2% SDS (13 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.…”

Section: Methodsmentioning

confidence: 99%

“…Fragments of the I-chain accumulate in B-lymphoblastoid cell lines (B-LCLs) exposed to inhibitors such as leupeptin, suggesting that a cysteine proteinase catalyzes the final stages of I-chain dissociation (2). Intermediates in I-chain processing include the 21-kDa leupeptininduced protein (LIP) fragment and small leupeptin-induced peptides (SLIP) of [11][12][13][14] Cysteine and acidic aspartic proteinases colocalize in endosomes with class II a,8 and I-chain (7). These proteinases have been implicated in antigen processing, yet their role in I-chain processing has not been established.…”

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confidence: 99%

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Endosomal aspartic proteinases are required for invariant-chain processing.

Marić

Taylor

Blum

1994

Proc. Natl. Acad. Sci. U.S.A.

163

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Immunogenic peptides are displayed in the context of class II histocompatibility proteins on the surface of antigen-presenting cells. Class H a and 13 subunits bind the invariant chain (I-chain), a transmembrane glycoprotein which must dissocite prior to peptide presentation. Proteolytic release of I-chain in an acidic compartment is followed by class II afi surface expression. Two distinct proteinases sequentially catalyze I-chain dissociation in B-lymphoblastoid cell lines. An aspartic proteinase initiates processing whereas a cysteine proteinase catalyzes the rmal stages of I-chain release. Inactivation of these enzymes prevents class II a1 maturation, demonstrating that acidic proteinases are essential for the generation of functional class II complexes. MATERIALS AND METHODSCells and Proteinase Inhibitors. The B-LCL Sweig (DR5) was cultured in Iscove's modification ofDulbecco's modified Eagle's medium plus 5% calf serum. Leupeptin (stock solution, 25 mg/ml in water) was used at a finial concentration of 5-50 jmg/ml. The aspartic proteinase inhibitors PD136517, PD136809, and PD134678 inactivate cathepsins D and E (8, 9). These inhibitors (50 mM stock solutions in dimethyl sulfoxide) were used at 25-50 ,uM, concentrations which reduced intracellular aspartic proteinase activity >75% after 24 hr.Antibodies. The antibody DA6.147 recognizes class II DR a chain, and L243 recognizes an epitope on mature DR5(wll) af3 (2, 10, 11). I-chain was detected with PIN1.1, an antibody specific for residues 12-28 (10).Immunoblotting and Metabolic Radiolabeling. Samples were normalized for protein and cell number before electrophoretic analysis and immunoblotting (10, 12). Class II a3 dimers were detected on immunoblots by using antibody L243 and sample buffer with 0.2% SDS (13). For metabolic radiolabeling, cells were pretreated with proteinase inhibitors for 1-2 hr, incubated with [35S]methionine for 30 min, and cultured in complete medium with or without inhibitors for 3.5 hr or 8 hr. In some experiments cells were radiolabeled for 4 hr. Cell lysates were immunoprecipitated for SDS/PAGE, and autoradiographs were quantitated by densitometry with the integrated area expressed in total intensity units. Resolution of glycosylated I-chain (Ip) from DR a was difficult on SDS/polyacrylamide gels. To calculate the accumulation of intact I-chain (It = I + Ip) with class II antigens, the formula (It + a)/, was used, assuming a constant a/l3 ratio. Subcellular Fractionation. Sweig cells (3 x 108) cultured with or without proteinase inhibitors were lysed in 0.5 mM CaCl2/1 mM NaHCO3/0.25 M sucrose/0.2 mM phenylmethanesulfonyl fluoride/0. 1 mM 7-amino-1-chloro-3-tosylamido-2-heptanone ("tosyllysine chloromethyl ketone") at pH 7.2 in a glass tissue homogenizer at 40C. The cell lysate was centrifuged at 900 x g for 10 min to pellet nuclei, and the supernatant was layered above 100 bil of 1.58 M sucrose and spun at 2000 x g for 15 min to remove mitochondria. The resulting supernatant was layered above continuous sucrose gradients (...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Endosomal aspartic proteinases are required for invariant-chain processing.

Marić

Taylor

Blum

1994

Proc. Natl. Acad. Sci. U.S.A.

163

View full text Add to dashboard Cite

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“…1B). The class II molecules expressed by the murine M12.C3 transfectants were peptide loaded, as demonstrated by the detection of SDS-stable dimers by immunoblots (37) and staining with the peptide-dependent, DR-specific mAb 16.23 (29,38) (Fig. 1B and data not shown).…”

Section: Regulated Expression Of Hla Dr and Dq Gene Products In Yac-tmentioning

confidence: 97%

A 320-Kilobase Artificial Chromosome Encoding the Human HLA DR3-DQ2 MHC Haplotype Confers HLA Restriction in Transgenic Mice

Chen

Dudek

Wijburg

et al. 2002

The Journal of Immunology

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MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ∼320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Aβnull/null) and expressing only murine CD4. A broad range of TCR Vβ families was used in the peripheral T cell repertoire, which was also purged of Vβ5-, Vβ11-, and Vβ12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Aβnull/null background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.

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“…Selective loss of class II conformational epitopes in human somatic variants (38) and mice lacking DM function (23,24,39) closely correlates with CLIP occupancy. In contrast, considerable evidence suggests DM activities are nonessential for conformational maturation of other class II allelic products, but there are also contradictory results in the literature (18 -21, 30).…”

Section: Partially Disrupted Class II Conformational Maturationmentioning

confidence: 99%

Relaxed DM Requirements During Class II Peptide Loading and CD4+ T Cell Maturation in BALB/c Mice

Bikoff

Wutz

Kenty

et al. 2001

The Journal of Immunology

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Current ideas about DM actions have been strongly influenced by studies of mutant strains expressing the H-2b haplotype. To evaluate DM contributions to class II activities in BALB/c mice, we generated a novel mutation at the DMa locus via embryonic stem cell technology. Unlike long-lived Ab/class II-associated invariant chain-derived peptide (CLIP) complexes, mature Ad and Ed molecules are loosely occupied by class II-associated invariant chain-derived peptide and are SDS unstable. BALB/c DM mutants weakly express BP107 conformational epitopes and toxic shock syndrome toxin-1 superantigen-binding capabilities, consistent with partial occupancy by wild-type ligands. Near normal numbers of mature CD4+ T cells fail to undergo superantigen-mediated negative selection, as judged by TCR Vβ usage. Ag presentation assays reveal consistent differences for Ad- and Ed-restricted T cells. Indeed, the mutation leads to decreased peptide capture by Ad molecules, and in striking contrast causes enhanced peptide loading by Ed molecules. Thus, DM requirements differ for class II structural variants coexpressed under physiological conditions in the intact animal.

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Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes

Cited by 224 publications

References 28 publications

Endosomal aspartic proteinases are required for invariant-chain processing.

Endosomal aspartic proteinases are required for invariant-chain processing.

A 320-Kilobase Artificial Chromosome Encoding the Human HLA DR3-DQ2 MHC Haplotype Confers HLA Restriction in Transgenic Mice

Relaxed DM Requirements During Class II Peptide Loading and CD4+ T Cell Maturation in BALB/c Mice

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