Major histocompatiblity complex class II molecules present peptides from an extracellular source of antigens to CD4+ T lymphocytes. The class 11-associated invariant chain affects this role of a and (3 polypeptides by restriction of peptide loading to endocytic vesicles. Up to now no specific portion of the invariant chain has been defined as the class II binding site. We constructed recombinant invariant chain genes and inspected association of the mutant invariant chains with class II polypeptides. Here we demonstrate that an extracytoplasmic sequence of the invariant chain (aa 81-109) that is only 23 residues away from the transmembrane region is essential for contact with class II polypeptides, whereas the remaining C-terminal part is dispensable for binding. The sequence ofinvariantchain-derived peptides that were eluted from class II molecules is contained in this segment and may derme the class II binding site of the invariant chain. The membrane-proximal position of this region suggests that the invariant chain and invariant-chainderived peptides isolated from class II molecules bind to a domain distinct from the class II pocket.Recently, it has been demonstrated that expression of the invariant chain (Ii) facilitates the presentation of various antigens by major histocompatibility complex (MHC) class II molecules (1-5). Ii is a type II membrane protein with the C-terminal domain expressed at the luminal side of the endoplasmic reticulum membrane (6). The following support for two roles of Ii was found. (i) Ii carries a signal sequence on its cytoplasmic domain that is responsible for sorting Ii and associated MHC class II molecules from the secretory to the endocytic route (7-9). (ii) Ii impedes loading of peptides to the MHC class II groove, a process that is postponed until MHC class II molecules enter an endocytic compartment and Ii is degraded (10)(11)(12). Additionally, assembly of Ii with MHC class II polypeptides modulates the shape of a and f3 dimers, which can promote the recognition of the a,3 peptide complex by T cells (13, 14).A soluble 18-kDa fragment of Ii that consists of most of the C-terminal part was found to bind to MHC class II molecules, indicating that association is not mediated by the membranespanning region or the cytoplasmic N terminus (15). Accordingly, a hybrid ofthe luminal domains ofclass II molecules and the transmembrane and cytoplasmic regions of MHC class I molecules associates with Ii (16). In this report we show that a membrane-proximal sequence of the luminal part of Ii is responsible for association with MHC class II molecules. MATERIALS AND METHODSPlasmid Constructions. A 9.4-kb fragment containing the murine Ii gene was subcloned into the plasmid pUC9 (1).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. §1734 solely to indicate this fact.Deletions of coding regions of the Ii gene were obtained by restriction enzyme digestion and reli...

Invariant chain (Ii) is a transmembrane type II protein that forms a complex with the major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum (ER). The membrane proximal luminal region of Ii is responsible for the non-covalent association with MHC class II molecules. Chemical cross-linking in COS cells was used to study the effect of luminal and cytoplasmic deletions on trimerization of Ii. We demonstrate that trimerization of Ii is independent of the cytosolic tail of Ii, whereas residues 162-191 (the sequence encoded by exon 6) in the luminal part of Ii are essential for trimer formation. Immunofluorescence studies of the transfected luminal deletion constructs show that the amino acids encoded by exon 6 of Ii are also essential for the induction of large endosomal vesicles. The data suggest that Ii must be in a trimeric form to modify the endosomal pathway.

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Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.