Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

Distinct functions for the glycans of tapasin and heavy chains in the assembly of MHC class I molecules.

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 ß(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-ß(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.

Ubiquitin-mediated regulation of CD86 protein expression by the ubiquitin ligase membrane-associated RING-CH-1 (MARCH1).

The activation of naïve T cells requires antigen presentation by dendritic cells (DCs), and the process of antigen presentation is regulated over the course of DC maturation. One key aspect of this regulation is the cell surface up-regulation upon DC maturation of peptide·MHC-II complexes and the costimulatory molecule CD86. It is now clear that these critical induction events involve changes in ubiquitin-dependent trafficking of MHC-II and CD86 by the E3 ligase membrane-associated RING-CH-1 (MARCH1). Although ubiquitin-dependent trafficking of MHC-II has been well characterized, much less is known regarding the post-transcriptional regulation of CD86 expression. Here, we examined the physical and functional interaction between CD86 and MARCH1. We observed that CD86 is rapidly endocytosed in the presence of MARCH1 followed by lysosome-dependent degradation. Furthermore, we found that the association between CD86 and MARCH1 was conferred primarily by the transmembrane domains of the respective proteins. In contrast to MHC-II, which has a single, conserved ubiquitin acceptor site in the cytosolic domain, we found that multiple lysine residues in the cytosolic tail of CD86 could support ubiquitination consistent with the relative lack of sequence conservation across species within the CD86 cytosolic domain. These findings suggest that MARCH1 recruits multiple substrates via transmembrane domain-mediated interactions to permit substrate ubiquitination in the face of diverse cytosolic domain sequences.

Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3.

The mechanism by which substrates for endoplasmic reticulum-associated degradation are retrotranslocated to the cytosol remains largely unknown, although ubiquitination is known to play a key role. The mouse gamma-herpesvirus protein mK3 is a viral RING-CH-type E3 ligase that specifically targets nascent major histocompatibility complex I heavy chain (HC) for degradation, thus blocking the immune detection of virus-infected cells. To address the question of how HC is retrotranslocated and what role mK3 ligase plays in this action, we investigated ubiquitin conjugation sites on HC using mutagenesis and biochemistry approaches. In total, our data demonstrate that mK3-mediated ubiquitination can occur via serine, threonine, or lysine residues on the HC tail, each of which is sufficient to induce the rapid degradation of HC. Given that mK3 has numerous cellular and viral homologues, it will be of considerable interest to determine the pervasiveness of this novel mechanism of ubiquitination.

Discrete domains of MARCH1 mediate its localization, functional interactions, and posttranscriptional control of expression.

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.

Licensing of natural killer cells by host major histocompatibility complex class I molecules.

Self versus non-self discrimination is a central theme in biology from plants to vertebrates, and is particularly relevant for lymphocytes that express receptors capable of recognizing self-tissues and foreign invaders. Comprising the third largest lymphocyte population, natural killer (NK) cells recognize and kill cellular targets and produce pro-inflammatory cytokines. These potentially self-destructive effector functions can be controlled by inhibitory receptors for the polymorphic major histocompatibility complex (MHC) class I molecules that are ubiquitously expressed on target cells. However, inhibitory receptors are not uniformly expressed on NK cells, and are germline-encoded by a set of polymorphic genes that segregate independently from MHC genes. Therefore, how NK-cell self-tolerance arises in vivo is poorly understood. Here we demonstrate that NK cells acquire functional competence through 'licensing' by self-MHC molecules. Licensing involves a positive role for MHC-specific inhibitory receptors and requires the cytoplasmic inhibitory motif originally identified in effector responses. This process results in two types of self-tolerant NK cells--licensed or unlicensed--and may provide new insights for exploiting NK cells in immunotherapy. This self-tolerance mechanism may be more broadly applicable within the vertebrate immune system because related germline-encoded inhibitory receptors are widely expressed on other immune cells.

DC-expressed MHC class I single-chain trimer-based vaccines prime cytotoxic T lymphocytes against exogenous but not endogenous antigens.

The poor immunogenicity of many tumors can be partly explained by the inefficiency of the MHC class I peptide presentation pathway. MHC-I-based single-chain trimers (SCT) represent a new class of molecules with the potential to overcome this limitation. We here evaluated the ability of SCT presenting a melanoma antigen peptide (TRP-2) to prime cytotoxic T lymphocyte (CTL) responses in mice when given as DNA vaccines via Gene Gun or when expressed by dendritic cells. The SCT was unable to induce detectable priming or significant anti-tumor activity of CTL using either vaccination strategy, whereas control SCT (with an exogenous peptide) primed strong responses. This study thus provides the first data related to the use of SCT in combination with DC and their application toward self antigens and suggest this potent technology, alone, is insufficient to overcome self tolerance.

Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways.

The CD8+ cytotoxic-T-cell response is a potent mechanism that controls intracellular pathogens, including many viruses. To facilitate transmission, viruses often counter this response by inhibiting the cell surface display of virus-derived peptides on MHC class I molecules. More specifically, recent studies have demonstrated that viruses have evolved remarkable mechanisms to inhibit MHC class I expression by interfering with the function of the MHC class I assembly machinery (the peptide-loading complex) in the endoplasmic reticulum and/or by exploiting endoplasmic-reticulum-associated degradation pathways. These viral molecules are proving invaluable as research tools to illuminate the novel features of physiological pathways that are central to normal cell biology. Furthermore, the detailed characterization of such pathways has yielded significant new insights into host-pathogen interplay.

Structural engineering of pMHC reagents for T cell vaccines and diagnostics.

MHC class I peptide complexes (pMHC) are routinely used to enumerate T cell populations and are currently being evaluated as vaccines to tumors and specific pathogens. Herein, we describe the structures of three generations of single-chain pMHC progressively designed for the optimal presentation of covalently associated epitopes. Our ultimate design employs a versatile disulfide trap between an invariant MHC residue and a short C-terminal peptide extension. This general strategy is nondisruptive of native pMHC conformation and T cell receptor engagement. Indeed, cell-surface-expressed MHC complexes with disulfide-trapped epitopes are refractory to peptide exchange, suggesting they will make safe and effective vaccines. Furthermore, we find that disulfide-trap stabilized, recombinant pMHC reagents reliably detect polyclonal CD8 T cell populations as proficiently as conventional reagents and are thus well suited to monitor or modulate immune responses during pathogenesis.

The E3 ubiquitin ligase MARCH1 regulates glucose-tolerance and lipid storage in a sex-specific manner.

Type 2 diabetes is typified by insulin-resistance in adipose tissue, skeletal muscle, and liver, leading to chronic hyperglycemia. Additionally, obesity and type 2 diabetes are characterized by chronic low-grade inflammation. Membrane-associated RING-CH-1 (MARCH1) is an E3 ubiquitin ligase best known for suppression of antigen presentation by dendritic and B cells. MARCH1 was recently found to negatively regulate the cell surface levels of the insulin receptor via ubiquitination. This, in turn, impaired insulin sensitivity in mouse models. Here, we report that MARCH1-deficient (knockout; KO) female mice exhibit excessive weight gain and excessive visceral adiposity when reared on standard chow diet, without increased inflammatory cell infiltration of adipose tissue. By contrast, male MARCH1 KO mice had similar weight gain and visceral adiposity to wildtype (WT) male mice. MARCH1 KO mice of both sexes were more glucose tolerant than WT mice. The levels of insulin receptor were generally higher in insulin-responsive tissues (especially the liver) from female MARCH1 KO mice compared to males, with the potential to account in part for the differences between male and female MARCH1 KO mice. We also explored a potential role for MARCH1 in human type 2 diabetes risk through genetic association testing in publicly-available datasets, and found evidence suggestive of association. Collectively, our data indicate an additional link between immune function and diabetes, specifically implicating MARCH1 as a regulator of lipid metabolism and glucose tolerance, whose function is modified by sex-specific factors.

TCRbeta enhancer activation in early and late lymphoid progenitors.

An earlier report from our laboratory indicates that the activation of the T cell receptor (TCR) beta enhancer (Ebeta) is not always an indicator of T lineage potential in bone marrow-resident pre-lymphocytes. In order to more precisely investigate the consequences of Ebeta activation in lymphopoiesis, a genetic reporter animal, in which the expression of green fluorescent protein (GFP) is controlled by Ebeta, was used to examine two well-defined lymphopotent populations. Adoptive transfer experiments suggest that primitive lymphoid precursor populations (specifically, hematopoietic stem cells) consist of two discrete-populations discernible by Ebeta-GFP activation, although the two populations display no overt differences in lineage potential. In contrast, subsets of more differentiated pre-lymphocytes (specifically, common lymphoid progenitors), while also discernible by Ebeta-GFP activation, display different capacities for reconstituting lymphoid compartments. Interestingly, late lymphoid progenitors containing inactive Ebeta elements generated both T and B cells in vivo, in accord with the original description of this population; however, progenitors containing active Ebeta elements displayed an unexpected bias toward the B lineage. Our findings suggest that Ebeta activation is an indicator of B lineage specification in late, but not early lymphoid precursors.

Applications of major histocompatibility complex class I molecules expressed as single chains.

Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.

Low GILT Expression is Associated with Poor Patient Survival in Diffuse Large B-Cell Lymphoma.

The major histocompatibility complex (MHC) class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4(+) T cells. Multiple cancers express MHC class II, which may influence the anti-tumor immune response and patient outcome. Low MHC class II expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL), the most common form of aggressive non-Hodgkin lymphoma. Therefore, we investigated whether gamma-interferon-inducible lysosomal thiol reductase (GILT), an upstream component of the MHC class II-restricted antigen processing pathway that is not regulated by the transcription factor class II transactivator, may be important in DLBCL biology. GILT reduces protein disulfide bonds in the endocytic compartment, exposing additional epitopes for binding to MHC class II and facilitating antigen presentation. In each of four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of a classical MHC class II gene, HLA-DRA, was associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival was independent of established clinical and molecular prognostic factors, the International Prognostic Index and the cell of origin classification, respectively. Immunohistochemical analysis of GILT expression in 96 DLBCL cases demonstrated variation in GILT protein expression within tumor cells which correlated strongly with GILT mRNA expression. These studies identify a novel association between GILT expression and clinical outcome in lymphoma. Our findings underscore the role of antigen processing in DLBCL and suggest that molecules targeting this pathway warrant investigation as potential therapeutics.

Adapter-mediated substrate selection for endoplasmic reticulum-associated degradation.

During endoplasmic reticulum (ER)-associated degradation (ERAD), a relatively small number of ubiquitin ligases (E3) must be capable of ubiquitinating an assortment of substrates diverse in both structure and location (ER lumen, membrane, and/or cytosol). Therefore, mechanisms that operate independently of primary sequence determinants must exist to ensure specificity during this process. Here we provide direct evidence for adapter-mediated substrate recruitment for a virus-encoded ERAD E3 ligase, mK3. Members of an ER membrane protein complex that normally functions during major histocompatibility complex class I biogenesis in the immune system are required for mK3 substrate selection. We demonstrate that heterologous substrates could be ubiquitinated by mK3 if they were recruited by these ER accessory molecules to the proper position relative to the ligase domain of mK3. This mechanism of substrate recruitment by adapter proteins may explain the ability of some E3 ligases, including cellular ERAD E3 ligases, to specifically target the ubiquitination of multiple substrates that are unrelated in sequence.

A single peptide-MHC complex positively selects a diverse and specific CD8 T cell repertoire.

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

[Expression of a single-chain trimer of MHC restricted HBsAg CTL epitope using adenovirus vector containing GFP-report gene].

OBJECTIVE: To generate a recombinant Adenovirus encoding a GFP (green fluorescent protein)-report gene and a single-chain trimer of MHC restricted HBsAg CTL epitope. METHODS: An oligonucleotide encoding H-2L(d) restricted HBsAg CTL epitope was synthesized and fused with H-2L(d) DNA molecule to construct the eukaryotic expression vector carrying the HBsAg-SCT gene. The HBsAg-SCT gene was subcloned into a GFP adenovirus expression vector,which was transfected into Ad293 cells for packaging and amplification of recombinant adenovirus encoding HBsAg-SCT. RESULTS: HBsAg-SCT has been cloned into an adenovirus vector encoding GFP report gene successfully as confirmed by double enzyme digestion and direct sequencing. HBsAg-SCT was expressed by infected Ad293 cells demonstrated by western blot assay. CONCLUSION: A recombinant adenovirus expressing HBsAg-SCT and green fluorescent protein report gene has been generated.

Human major histocompatibility complex (MHC) class I molecules with disulfide traps secure disease-related antigenic peptides and exclude competitor peptides.

The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, beta(2)m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.

Disulfide bond engineering to trap peptides in the MHC class I binding groove.

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.

Recognition of open conformers of classical MHC by chaperones and monoclonal antibodies.

There is considerable evidence that the conformation and stability of class I and class II major histocompatibility complex (MHC) proteins is dependent upon high-affinity peptide ligation, but structural data for an empty MHC protein unfortunately is lacking. However, several monoclonal antibodies (mAbs) that specifically detect open MHC conformers have been characterized, and they provide insights into the changes associated with peptide loading and unloading. Here, the structural changes make the argument that certain of these open conformer-specific mAbs recognize analogous MHC segments as the molecular chaperones tapasin and DM. MHC residues located in regions flanking the peptide-terminal anchoring pockets have been implicated in both chaperone and monoclonal antibody binding. Indeed, we propose these regions serve as peptide-binding hinges that are uniquely accessible in open MHC.

Requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class I proteins by the viral immune evasion molecule mK3.

Recent studies suggest that certain viral proteins co-opt endoplasmic reticulum (ER) degradation pathways to prevent the surface display of major histocompatibility complex class I molecules to the immune system. A novel example of such a molecule is the mK3 protein of gammaherpesvirus 68. mK3 belongs to an extensive family of structurally similar viral and cellular proteins that function as ubiquitin ligases using a conserved RING-CH domain. In the specific case of mK3, it selectively targets the rapid degradation of nascent class I heavy chains in the ER while they are associated with the class I peptide-loading complex (PLC). We present here evidence that the PLC imposes a relative proximity and/or orientation on the RING-CH domain of mK3 that is required for it to specifically target class I molecules for degradation. Furthermore, we demonstrate that full assembly of class I molecules with peptide is not a prerequisite for mK3-mediated degradation. Surprisingly, although the cytosolic tail of class I is required for rapid mK3-mediated degradation, we observed that a class I mutant lacking lysine residues in its cytosolic tail was ubiquitinated and degraded in the presence of mK3 in a manner indistinguishable from wild-type class I molecules. These findings are consistent with a "partial dislocation" model for turnover of ER proteins and define some common features of ER degradation pathways initiated by structurally distinct herpesvirus proteins.