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Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones, collectively called the MHC class I peptide-loading complex (PLC), function in the folding and assembly of MHC class I molecules. The glycan-binding chaperone calreticulin (CRT) and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of CRT and ERp57 to the PLC are codependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance β(2)m and MHC class I heavy (H) chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. In contrast, the conserved MHC class I H chain glycan played a minor role in CRT recruitment into the PLC, but impacted the recruitment of H chains into the PLC, and glycan-deficient H chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of H chain-β(2)m heterodimers, for which tapasin-ERp57 or tapasin-CRT complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate H chain-deficient PLCs.

Within APCs, ubiquitination regulates the trafficking of immune modulators such as MHC class II and CD86 (B7.2) molecules. MARCH1 (membrane-associated RING-CH), a newly identified ubiquitin E3 ligase expressed in APCs, ubiquitinates MHC class II, thereby reducing its surface expression. Following LPS-induced maturation of dendritic cells, MARCH1 mRNA is down-regulated and MHC class II is redistributed to the cell surface from endosomal compartments. Here, we show that MARCH1 expression is also regulated at the posttranscriptional level. In primary dendritic cell and APC cell lines of murine origin, MARCH1 had a half-life of <30 min. MARCH1 degradation appears to occur partly in lysosomes, since inhibiting lysosomal activity stabilized MARCH1. Similar stabilization was observed when MARCH1-expressing cells were treated with cysteine protease inhibitors. Mutational analyses of MARCH1 defined discrete domains required for destabilization, proper localization, and functional interaction with substrates. Taken together, these data suggest that MARCH1 expression is regulated at a posttranscriptional level by trafficking within the endolysosomal pathway where MARCH1 is proteolyzed. The short half-life of MARCH1 permits very rapid changes in the levels of the protein in response to changes in the mRNA, resulting in efficient induction of Ag presentation once APCs receive maturational signals.

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells-particularly when the antigenic peptide has relatively weak affinity for the MHC.

MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with beta(2)-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.

We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-beta(2)-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.

H2-M3 is a class Ib MHC molecule that binds a highly restricted pool of peptides, resulting in its intracellular retention under normal conditions. However, addition of exogenous M3 ligands induces its escape from the endoplasmic reticulum (ER) and, ultimately, its expression at the cell surface. These features of M3 make it a powerful and novel model system to study the potentially interrelated functions of the ER-resident class I chaperone tapasin. The functions ascribed to tapasin include: 1) ER retention of peptide-empty class I molecules, 2) TAP stabilization resulting in increased peptide transport, 3) direct facilitation of peptide binding by class I, and 4) peptide editing. We report in this study that M3 is associated with the peptide-loading complex and that incubation of live cells with M3 ligands dramatically decreased this association. Furthermore, high levels of open conformers of M3 were efficiently retained intracellularly in tapasin-deficient cells, and addition of exogenous M3 ligands resulted in substantial surface induction that was enhanced by coexpression of either membrane-bound or soluble tapasin. Thus, in the case of M3, tapasin directly facilitates intracellular peptide binding, but is not required for intracellular retention of open conformers. As an alternative approach to define unique aspects of M3 biosynthesis, M3 was expressed in human cell lines that lack an M3 ortholog, but support expression of murine class Ia molecules. Unexpectedly, peptide-induced surface expression of M3 was observed in only one of two cell lines. These results demonstrate that M3 expression is dependent on a unique factor compared with class Ia molecules.

H2-M3 is a MHC class Ib molecule with a high propensity to bind N-formylated peptides. Due to the paucity of endogenous Ag, the majority of M3 is retained in the endoplasmic reticulum (ER). Upon addition of exogenous N-formylated peptides, M3 trafficks rapidly to the cell surface. To understand the mechanism underlying Ag presentation by M3, we examined the role of molecular chaperones in M3 assembly, particularly TAP and tapasin. M3-specific CTLs fail to recognize cells isolated from both TAP-deficient (TAP(o)) and tapasin-deficient mice, suggesting that TAP and tapasin are required for M3-restricted Ag presentation. Impaired M3 expression in TAP(o) mice is due to instability of the intracellular pool of M3. Addition of N-formylated peptides to TAP(o) cells stabilizes M3 in the ER and partially restores surface expression. Surprisingly, significant amounts of M3 are retained in the ER in tapasin-deficient mice, even in the presence of N-formylated peptides. Our results define the role of TAP and tapasin in the assembly of M3-peptide complexes. TAP is essential for stabilization of M3 in the ER, whereas tapasin is critical for loading of N-formylated peptides onto the intracellular pool of M3. However, neither TAP nor tapasin is required for ER retention of empty M3.

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-beta(2)-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of L(d) mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient.220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.

The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, L(d), K(d), and K(b). Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.

Successful expression of the TCR beta-chain gene is a multistep process that involves: 1) initial transcription of multiple, unrearranged gene segments, 2) rearrangement of V, D, and J gene segments to form a complete beta-chain gene, and 3) transcription of the fully rearranged beta gene. All of these events have been shown to occur in the thymus, where the majority of T cell development takes place; however, the extent to which any of these events may occur prethymically has not been established. To examine prethymic TCR-beta gene expression, RNA was isolated from a precursor T cell-enriched population (Thy 1low CD3-) of C58/J mouse bone marrow, and analyzed by reverse transcriptase-PCR. A transcript containing TCR-beta constant (C) region sequences but not variable (V) region sequences was amplified, suggesting that an unrearranged TCR-beta gene locus is transcriptionally active in this bone marrow population. The same product was detected in Thy 1+ CD3- bone marrow cells from nude mice, indicating that the thymic microenvironment is not necessary for initiation of TCR-beta gene transcription. This C beta transcript is not confined to pre-B cells, as it was identified in RNA isolated from Thy 1low CD3- B220- bone marrow cells. Germline V beta transcripts were also detected in RNA from this bone marrow population. Furthermore, Sca-1+ Lin- and Sca-1+ Lin+ bone marrow populations from both C58/J mice and nude mice also expressed the C beta transcript. DNA-PCR analyses with D beta-J beta primer sets revealed that partial rearrangement of the beta locus had occurred in all bone marrow populations analyzed. These data suggest that both transcription and partial rearrangement of the TCR-beta locus can initiate in bone marrow cells of adult mice, before exposure of these cells to the thymus.