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Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding. [ABSTRACT FROM AUTHOR]

• Study on rpMHC-I thermostability shows a two-step mechanism of thermal denaturation. • High-affinity rpMHC-I are pH-stable, but buffer substances influence Tm. • NanoDSF is a robust and sensitive method for measuring stability and folding energies of rpMHC-I. • NanoDSF allows buffer formula optimization and quality control of rpMHC-I. • NanoDSF is suitable for estimating the binding affinity of peptides to MHC-I. Recombinant major histocompatibility complex class I molecules are used in diagnostic and therapeutic approaches in cancer immunotherapy, with many studies exploring their binding to antigenic peptides. Current techniques for kinetic peptide binding studies are hampered by high sample consumption, low throughput, interference with protein stability, and/or high background signal. Here, we validate nanoscale differential scanning fluorimetry (nanoDSF), a method using the tryptophan fluorescence of class I molecules, for class I/peptide binding, and we use it to determine the molecular mechanism of the thermal denaturation of HLA-A*02:01. [ABSTRACT FROM AUTHOR]

Adaptive immunity vitally depends on major histocompatibility complex class I (MHC I) molecules loaded with peptides. Selective loading of peptides onto MHC I, referred to as peptide editing, is catalyzed by tapasin and the tapasin-related TAPBPR. An important catalytic role has been ascribed to a structural feature in TAPBPR called the scoop loop, but the exact function of the scoop loop remains elusive. Here, using a reconstituted system of defined peptide-exchange components including human TAPBPR variants, we uncover a substantial contribution of the scoop loop to the stability of the MHC I-chaperone complex and to peptide editing. We reveal that the scoop loop of TAPBPR functions as an internal peptide surrogate in peptide-depleted environments stabilizing empty MHC I and impeding peptide rebinding. The scoop loop thereby acts as an additional selectivity filter in shaping the repertoire of presented peptide epitopes and the formation of a hierarchical immune response.

Abstract Background Currently, mismatch repair-deficient (dMMR) status is a promising candidate for targeted immune checkpoint inhibition therapy in colorectal cancer (CRC) patients, however, the potential immunological mechanism has not yet been well clarified and some other predictors need to be excavated as well. Methods We collected 330 CRC patients by the match of mismatch repair-proficient (167) and dMMR (163), explored the relationship between MMR status and some important immune molecules including MHC class I, CD3, CD4, CD8, CD56, programmed death-1 and programmed death ligand-1, and investigated the risk factors for dMMR status as well as low MHC class I expression. The Pearson Chi square test was used for analyzing the associations between clinicopathological and immune characteristics and MMR status, and two categories logistic regression model was used for univariate and multivariate analysis to predict the odds ratio of risk factors for dMMR status and low MHC class I expression. Results Multivariate logistic regression analysis showed that low MHC class I and CD4 expression and high CD8 expression were significant risk factors for dMMR status [odds ratio (OR) = 24.66, 2.94 and 2.97, respectively; all p

Herein, we demonstrate that HCMV miR-UL112-5p targets ERAP1, thereby inhibiting the processing and presentation of the HCMV pp65495-503 peptide to specific CTLs. In addition, we show that the rs17481334 G variant, naturally occurring in the ERAP1 3′ UTR, preserves ERAP1 from miR-UL112-5p-mediated degradation. Specifically, HCMV miR-UL112-5p binds the 3′ UTR of ERAP1 A variant, but not the 3′ UTR of ERAP1 G variant, and, accordingly, ERAP1 expression is reduced both at RNA and protein levels only in human fibroblasts homozygous for the A variant. Consistently, HCMV-infected GG fibroblasts were more efficient in trimming viral antigens and being lysed by HCMV-peptide-specific CTLs. Notably, a significantly decreased HCMV seropositivity was detected among GG individuals suffering from multiple sclerosis, a disease model in which HCMV is negatively associated with adult-onset disorder. Overall, our results identify a resistance mechanism to HCMV miR-UL112-5p-based immune evasion strategy with potential implications for individual susceptibility to infection and other diseases.

Neuronal MHC class I proteins have been previously reported to regulate synaptic plasticity. Several reports indicate MHC class I proteins are expressed early during development of the nervous system, suggesting they may also play a role in neuronal development. Using cultured cortical neurons, we show MHC class I proteins aggregate at specific sites in neuronal cell bodies, which overlap with the actin cytoskeleton. Knockout of MHC class I in cultured neurons increases total dendritic length and the number of branch points. These effects are abolished by reintroducing MHC class I expression. Similarly, blocking of MHC class I proteins or PirB by an MHCI antibody or a soluble PirB ectodomain respectively, mimics the knock out phenotype of increased dendritic branching. This effect is correlated with decreased phosphorylation of both LIMK and cofilin, suggesting it may be mediated by an induction of cofilin activity. Finally, layer II and III cortical neurons in the sensorimotor region of an MHC class I deficiency mouse model show increased dendritic growth and branching. Altogether, our results suggest MHC class I plays a role in inhibiting or limiting the degree of dendrite arborization during the development of cortical neurons. [ABSTRACT FROM AUTHOR]

Autophagy-related (Atg) gene-encoded proteins were originally described for their crucial role in macroautophagy, a catabolic pathway for cytoplasmic constituent degradation in lysosomes. Recently it has become clear that modules of this machinery can also be used to influence endo- and exocytosis. This mini review discusses how these alternative Atg functions support virus replication and viral antigen presentation on major histocompatibility (MHC) class I and II molecules. A better understanding of the modular use of the macroautophagy machinery might enable us to manipulate these alternative functions of Atg proteins during anti-viral therapies and to attenuate virus-induced immune pathologies. [ABSTRACT FROM AUTHOR]

Pluripotent parthenogenetic stem cells (pSCs) can be derived by pharmacological activation of unfertilized oocytes. Homozygosity of the major histocompatibility complex (MHC) in pSCs makes them an attractive cell source for applications in allogeneic tissue repair. This was recently demonstrated for pSC-based tissue-engineered heart repair. A detailed analysis of immunological properties of pSC-derived cardiomyocytes and engineered heart muscle (EHM) thereof is, however, lacking. The aim of this study was to determine baseline and cytokine-inducible MHC class I and MHC class II as well as programmed death ligand-1 (PDL-1) and co-stimulatory protein (CD40, CD80, CD86) expression in pSC-derived cardiomyocytes and pSC-EHM in vitro and in vivo. Cardiomyocytes from an MHC-homologous (H2d/d) pSC-line were enriched to ~90% by making use of a recently developed cardiomyocyte-specific genetic selection protocol. MHC class I and MHC class II expression in cardiomyocytes could only be observed after stimulation with interferon gamma (IFN-γ). PDL-1 was markedly upregulated under IFN-γ. CD40, CD80, and CD86 were expressed at low levels and not upregulated by IFN-γ. EHM constructed from H2d/d cardiomyocytes expressed similarly low levels of MHC class I, MHC class II, and costimulatory molecules under basal conditions. However, in EHM only MHC class I, but not MHC class II, molecules were upregulated after IFN-γ-stimulation. We next employed a cocultivation system with MHC-matched and MHC-mismatched splenocytes and T-cells to analyze the immune stimulatory properties of EHMs. Despite MHC-mismatched conditions, EHM did not induce splenocyte or T-cell proliferation in vitro. To evaluate the immunogenicity of pSC-derived cardiomyocytes in vivo, we implanted pSC-derived embryoid bodies after elimination of non-cardiomyocytes (cardiac bodies) under the kidney capsules of MHC-matched and - mismatched mice. Spontaneous beating of cardiac bodies could be observed for 28 days in the matched and for 7 days in the mismatched conditions. Teratomas formed after 28 days only in the MHC-matched conditions. Immunohistochemistry revealed single clusters of CD3-positive cells in the border zone of the implant in the mismatched conditions with few CD3-positive cells infiltrating the implant. Taken together, MHC-matched pSC-cardiomyocyte allografts show little immune cell activation, offering an explanation for the observed longterm retention of pSC-EHM allografts in the absence of immunosuppression. [ABSTRACT FROM AUTHOR]

Summary Herein, we demonstrate that HCMV miR-UL112-5p targets ERAP1, thereby inhibiting the processing and presentation of the HCMV pp65 495-503 peptide to specific CTLs. In addition, we show that the rs17481334 G variant, naturally occurring in the ERAP1 3′ UTR, preserves ERAP1 from miR-UL112-5p-mediated degradation. Specifically, HCMV miR-UL112-5p binds the 3′ UTR of ERAP1 A variant, but not the 3′ UTR of ERAP1 G variant, and, accordingly, ERAP1 expression is reduced both at RNA and protein levels only in human fibroblasts homozygous for the A variant. Consistently, HCMV-infected GG fibroblasts were more efficient in trimming viral antigens and being lysed by HCMV-peptide-specific CTLs. Notably, a significantly decreased HCMV seropositivity was detected among GG individuals suffering from multiple sclerosis, a disease model in which HCMV is negatively associated with adult-onset disorder. Overall, our results identify a resistance mechanism to HCMV miR-UL112-5p-based immune evasion strategy with potential implications for individual susceptibility to infection and other diseases. [ABSTRACT FROM AUTHOR]

Cross-presentation, in which exogenous antigens are presented via MHC I complexes, is involved both in the generation of anti-infectious and anti-tumoral cytotoxic CD8+ T cells and in the maintenance of immune tolerance. While cross-presentation was described almost four decades ago and while it is now established that some dendritic cell subsets are better than others in processing and cross-presenting internalized antigens, the involved molecular mechanisms remain only partially understood. Some of the least explored molecular mechanisms in cross-presentation concern the origin of cross-presenting MHC I molecules and the cellular compartments where antigenic peptide loading occurs. This review focuses on MHC I molecules and their intracellular trafficking. We discuss the source of cross-presenting MHC I in dendritic cells as well as the role of the endocytic pathway in their recycling from the cell surface. Next, we describe the importance of the TAP peptide transporter for delivering peptides to MHC I during cross-presentation. Finally, we highlight the impact of innate immunity mechanisms on specific antigen cross-presentation mechanisms in which TLR activation modulates MHC I trafficking and TAP localization.

Abstract: Autophagy-related (Atg) gene-encoded proteins were originally described for their crucial role in macroautophagy, a catabolic pathway for cytoplasmic constituent degradation in lysosomes. Recently it has become clear that modules of this machinery can also be used to influence endo- and exocytosis. This mini review discusses how these alternative Atg functions support virus replication and viral antigen presentation on major histocompatibility (MHC) class I and II molecules. A better understanding of the modular use of the macroautophagy machinery might enable us to manipulate these alternative functions of Atg proteins during anti-viral therapies and to attenuate virus-induced immune pathologies.

Cross-presentation, in which exogenous antigens are presented via MHC I complexes, is involved both in the generation of anti-infectious and anti-tumoral cytotoxic CD8+ T cells and in the maintenance of immune tolerance. While cross-presentation was described almost four decades ago and while it is now established that some dendritic cell (DC) subsets are better than others in processing and cross-presenting internalized antigens, the involved molecular mechanisms remain only partially understood. Some of the least explored molecular mechanisms in cross-presentation concern the origin of cross-presenting MHC I molecules and the cellular compartments where antigenic peptide loading occurs. This review focuses on MHC I molecules and their intracellular trafficking. We discuss the source of cross-presenting MHC I in DCs as well as the role of the endocytic pathway in their recycling from the cell surface. Next, we describe the importance of the TAP peptide transporter for delivering peptides to MHC I during cross-presentation. Finally, we highlight the impact of innate immunity mechanisms on specific antigen cross-presentation mechanisms in which TLR activation modulates MHC I trafficking and TAP localization. [ABSTRACT FROM AUTHOR]

The use of MHC multimers allows precise and direct detecting and analyzing of antigen-specific T-cell populations and provides new opportunities to characterize T-cell responses in humans and animals. MHC-multimers enable us to enumerate specific T-cells targeting to viral, tumor and vaccine antigens with exceptional sensitivity and specificity. In the field of HIV/SIV immunology, this technique provides valuable information about the frequencies of HIV- and SIV-specific CD8+ cytotoxic T lymphocytes (CTLs) in different tissues and sites of infection, AIDS progression, and pathogenesis. Peptide-MHC multimer technology remains a very sensitive tool in detecting virus-specific T -cells for evaluation of the immunogenicity of vaccines against HIV-1 in preclinical trials. Moreover, it helps to understand how immune responses are formed following vaccination in the dynamics from priming point until T-cell memory is matured. Here we review a diversity of peptide-MHC class I multimer applications for fundamental immunological studies in different aspects of HIV/SIV infection and vaccine development. [ABSTRACT FROM AUTHOR]

RV144 clinical trial was modestly effective in preventing HIV infection. New alternative approaches are needed to design improved HIV-1 vaccines and their delivery strategies. One of these approaches is construction of synthetic polyepitope HIV-1 immunogen using protective T- and B-cell epitopes that can induce broadly neutralizing antibodies and responses of cytotoxic (CD8+ CTL) and helpers (CD4+ Th) T-lymphocytes. This approach seems to be promising for designing of new generation of vaccines against HIV-1, enables in theory to cope with HIV-1 antigenic variability, focuses immune responses on protective determinants and enables to exclude from the vaccine compound that can induce autoantibodies or antibodies enhancing HIV-1 infectivity. Herein, the authors will focus on construction and rational design of polyepitope T-cell HIV-1 immunogens and their delivery, including: advantages and disadvantages of existing T-cell epitope prediction methods; features of organization of polyepitope immunogens, which can generate high-level CD8+ and CD4+ T-lymphocyte responses; the strategies to optimize efficient processing, presentation and immunogenicity of polyepitope constructs; original software to design polyepitope immunogens; and delivery vectors as well as mucosal strategies of vaccination. This new knowledge may bring us a one step closer to developing an effective T-cell vaccine against HIV-1, other chronic viral infections and cancer. [ABSTRACT FROM AUTHOR]

BET inhibition has been shown to have a promising antitumor effect in multiple tumors. However, the impact of BET inhibition on antitumor immunity was still not well documented in HNSCC. In this study, we aim to assess the functional role of BET inhibition in antitumor immunity and clarify its mechanism. We show that BRD4 is highly expressed in HNSCC and inversely correlated with the infiltration of CD8 + T cells. BET inhibition potentiates CD8 + T cell-based antitumor immunity in vitro and in vivo. Mechanistically, BRD4 acts as a transcriptional suppressor and represses the expression of MHC class I molecules by recruiting G9a. Pharmacological inhibition or genetic depletion of BRD4 potently increases the expression of MHC class I molecules in the absence and presence of IFN-γ. Moreover, compared to PD-1 blocking antibody treatment or JQ1 treatment individually, the combination of BET inhibition with anti-PD-1 antibody treatment significantly enhances the antitumor response in HNSCC. Taken together, our data unveil a novel mechanism by which BET inhibition potentiates antitumor immunity via promoting the expression of MHC class I molecules and provides a rationale for the combination of ICBs with BET inhibitors for HNSCC treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Highlights: [•] The peptide-loading complex (PLC) has an essential function in adaptive immunity directed against infected or tumor cells. [•] This multitasking machinery involves a set of specialized chaperones, transporters, and receptors. [•] Current knowledge concerning the molecular architecture, function, and dynamics of the PLC is summarized. [•] The fully assembled PLC comprises one TAP heterodimer, two Tsn-ERp57, and up to two MHC I and Crt molecules. [Copyright &y& Elsevier]

Abstract: Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection. [Copyright &y& Elsevier]

In order to illustrate the structure of the swine MHC class I (SLA-I) molecule and to evaluate the cytotoxic T lymphocyte (CTL) response against porcine reproductive and respiratory syndrome virus (PRRSV), the ternary complex of the SLA-I molecule termed SLA-1*1502 with β2-microglobulin and the CTL epitope TMPPGFELY (PRRSV-NSP9TY9) derived from PRRSV nonstructural protein 9 (residues 198-206) was assembled and crystallized. The crystal diffracted X-rays to 2.2 Å resolution and belonged to space group P212121, with unit-cell parameters a = 66.1, b = 74.1, c = 98.6 Å; it contained one molecule in the asymmetric unit. The Matthews coefficient and the solvent content were calculated to be 2.74 Å3 Da−1 and 55.17%, respectively. The results will be helpful in obtaining insight into the structural basis of the presentation of viral epitopes by SLA-I. [ABSTRACT FROM AUTHOR]

Abstract: The E3-19K immunomodulatory protein from adenoviruses (Ads) inhibits antigen presentation by major histocompatibility complex (MHC) class I molecules. As a result, the ability of Ad-specific cytotoxic T lymphocytes (CTLs) to lyse infected cells is suppressed. The ER-lumenal domain of E3-19K is subdivided into a variable (residues 1 to ∼78/81) and conserved (residues ∼79/82 to 98) region followed by a linker (residues 99–107). Using molecular and cellular approaches, we characterized in detail the properties of the ER-lumenal domain of E3-19K that enable it to target MHC class I molecules. Proteolysis of recombinant serotype 2 E3-19K (residues 1–100) (with six His residues) generated a large N-terminal (residues 1–88) and a small C-terminal fragment (residues 94–100) in solution. Neither of these fragments associates with HLA-A*1101 as shown by a native gel band-shift assay. In contrast, the N-terminal 1-93 residues of Ad2 E3-19K exhibited the same binding affinity to HLA-A*1101 as E3-19K. Using a site-directed mutational analysis and flow cytometry, we show that Tyr93, but not Tyr88, critically modulates the cell-surface expression of MHC class I molecules. Taken together, these results indicate that the sequence comprising residues 89–93 (M89SKQY93), and in particular Tyr93, in the conserved region of E3-19K is critical for its immunomodulatory function. Residues 89–93 likely form a linker or loop in E3-19K. Overall, our data provide novel insights into the structure of E3-19K and identify key determinants for association with and ER-retention of its cellular target protein. This knowledge is important for our understanding of the molecular basis of Ad pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: MHC class I molecules present short peptides, usually 8–10 amino acids in length, to CD8+ T cells. These peptides are typically generated from full-length endogenously synthesized proteins degraded by the antigen processing machinery of the target cell. However, exogenous proteins, whether originating from intracellular bacteria or parasites or via phagocytosis during cross-presentation, can also be processed for presentation by MHC class I molecules. It is currently not known whether endogenously synthesized proteins and proteins acquired from exogenous sources follow the same presentation pathway. One clue that the processing pathways followed by endogenous and exogenous proteins may not be identical is the vastly different presentation efficiencies reported for viral versus bacterial antigens. Because class I antigen processing involves multiple steps, we sought to determine where in the processing pathway these differences in efficiency occur. To accomplish this, we expressed identical minimal peptide determinants from viral and bacterial vectors using a minigene expression system and determined the rate of peptide-MHC generation per molecule of minigene product synthesized. We found that peptides expressed from either the viral or bacterial vector were presented with virtually identical efficiencies. These results suggest that differences in the processing pathways followed by endogenous versus exogenous proteins most likely occur at a point prior to where free peptide is liberated from full-length protein. [ABSTRACT FROM AUTHOR]

Abstract: The complex interaction between natural killer (NK) cells and cytomegalovirus is a paradigm of the co-evolution between genomes of large DNA viruses and their host immune systems. Both human and mouse cytomegalovirus posses numerous mechanisms to avoid NK cell detection. Linkage studies, positional cloning and functional studies in mice and cells, have led to the identification of key genes governing resistance to cytomegalovirus, including various NK cell activating receptors of major histocompatibility complex (MHC) class I. These receptors, however, seem to require either viral or host MHC class I molecules to operate recognition and elimination of the cytomegalovirus-infected cell leading to host resistance. Here we will review the genes and molecules involved in these mechanisms while contrasting their function with that of other NK cell receptors. Activating receptors of MHC class I may represent a window of therapeutic intervention during human infection with viruses, of which cytomegalovirus remains an important health threat. [Copyright &y& Elsevier]

Background/Aims: Hepatitis C virus leads to chronic hepatitis in the majority of infected individuals. The mechanism of viral persistence is not completely understood. Hepatitis C virus core protein is produced within hepatocytes and is secreted during HCV infection. Our study characterizes the effects of core protein on T cell priming in mice. Methods: We used a system of antigen-specific in vitro priming of CD4+ and CD8+ T cells by myeloid dendritic cells, hepatoma cells or primary hepatocytes. Core protein was either added to the cultures or expressed by antigen-presenting cells. Results: Antigen-presenting cells treated with core protein showed reduced surface expression of major histocompatibility molecules. Myeloid dendritic cells showed also reduced expression of costimulatory molecules. CD4+ and CD8+ T cells primed by these cells showed defects in activation, proliferation, and cytokine production. Importantly, CD4+ and also CD8+ T cells primed in the presence of core protein showed an increase in interleukin-10 production resembling the phenotype of regulatory T cells. Conclusions: Hepatitis C virus core protein inhibits priming of antigen-specific CD4+ and CD8+ T cell responses by downregulation of major histocompatibility molecules and costimulatory molecules on antigen-presenting cells and induces development of IL-10-producing T cells. [Copyright &y& Elsevier]

Abstract: Immune cells are usually considered non-attached blood cells, which would exclude the formation of gap junctions. This is a misconception since many immune cells express connexin 43 (Cx43) and other connexins and are often residing in tissue. The role of gap junctions is largely ignored by immunologists as is the immune system in the field of gap junction research. Here, the current knowledge of the distribution of connexins and the function of gap junctions in the immune system is discussed. Gap junctions appear to play many roles in antibody productions and specific immune responses and may be important in sensing danger in tissue by the immune system. Gap junctions not only transfer electrical and metabolical but also immunological information in the form of peptides for a process called cross-presentation. This is essential for proper immune responses to viruses and possibly tumours. Until now only 40 research papers on gap junctions in the immune system appeared and this will almost certainly expand with the increased mutual interest between the fields of immunology and gap junction research. [Copyright &y& Elsevier]

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MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposi's sarcoma-associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3-associated class I by lysine-63-linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine-63-linked polyubiquitination requires both UbcH5b/c and Ubc13-conjugating enzymes for initiating mono- and subsequent polyubiquitination of class I, and the clathrin-dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine-63-linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors. [ABSTRACT FROM AUTHOR]

The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHCI-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCI recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell–cell recognition receptors. Thermodynamic analyses showed that LILRB1–MHCI interactions are entropically driven (−TΔS=−9.4∼−6.6kcalmol−1) with low heat capacity changes (ΔC p=−0.22∼−0.10kcalmol−1 K−1). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility. Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1–MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from “induced-fit” binding, which is associated with large reductions in conformational flexibility, and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells. [Copyright &y& Elsevier]

Abstract: The human cytomegalovirus (HCMV) gene product US11 dislocates MHC I heavy chains from the endoplasmic reticulum (ER) and targets them for proteasomal degradation in the cytosol. To identify the structural and functional domains of US11 that mediate MHC class I molecule degradation, we constructed truncated mutants and chimeric proteins, and analyzed these to determine their intracellular localization and their ability to degrade MHC class I molecules. We found that only the luminal domain of US11 was essential to confer ER localization to the protein but that the ability to degrade MHC class I molecules required both the transmembrane domain and the luminal domain of US11. By analyzing a series of point mutants of the transmembrane domain, we were also able to identify Gln192 and Gly196 as being crucial for the functioning of US11, suggesting that these residues may play a critical role in interacting with the components of the protein degradation machinery. [Copyright &y& Elsevier]

The MHC class I molecule plays an important role in immune response, pathogen recognition and response against vaccines and self- versus non-self-recognition. Studying MHC class I characteristics thus became a priority when dealing withAotusto ensure its use as an animal model for biomedical research. Isolation, cloning and sequencing of exons 1-8 from 27 MHC class I alleles obtained from 13 individuals classified as belonging to three owl monkey species (A. nancymaae,A. nigricepsandA. vociferans) were carried out to establish similarities betweenAotusMHC class I genes and those expressed by other New and Old World primates. SixAotusMHC class I sequence groups (Ao-g1,Ao-g2,Ao-g3,Ao-g4,Ao-g5andAo-g6) weakly related to non-classical Catarrhini MHC were identified. An allelic lineage was also identified in oneA. nancymaaeand twoA. vociferansmonkeys, exhibiting a high degree of conservation, negative selection along the molecule and premature termination of the open reading frame at exon 5 (Ao-g5). These sequences’ high conservation suggests that they more likely correspond to a soluble form ofAotusMHC class I molecules than to a new group of processed pseudogenes. Another group, namedAo-g6, exhibited a strong relationship with Catarrhini’s classicalMHC-B-Cloci. Sequence evolution and variability analysis indicated thatAotusMHC class I molecules experience inter-locus gene conversion phenomena, contributing towards their high variability. [ABSTRACT FROM AUTHOR]

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals. [ABSTRACT FROM AUTHOR]

MHC class I molecules are loaded with peptides that mostly originate from the degradation of cytosolic protein antigens and that are translocated across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). The ER-resident molecule tapasin (Tpn) is uniquely dedicated to tether class I molecules jointly with the chaperone calreticulin (Crt) and the oxidoreductase ERp57 to TAP. As learned from the study of a Tpn-deficient cell line and from mice harboring a disrupted Tpn gene, the transient association of class I molecules with Tpn and TAP is critically important for the stabilization of class I molecules and the optimization of the peptide cargo presented to cytotoxic T cells. The different functions of molecular domains of Tpn and the highly coordinated formation of the TAP-associated peptide loading complex will also be discussed in this review. [Copyright &y& Elsevier]

Major histocompatibility complex (MHC) class I molecules usually present endogenous peptides at the cell surface. This is the result of a cascade of events involving various dedicated proteins like the peptide transporter associated with antigen processing (TAP) and the ER chaperone tapasin. However, alternative ways for class I peptide loading exist which may be highly relevant in a process called cross-priming. Both pathways are described here in detail. One major difference between these pathways is that the proteases involved in the generation of peptides are different. How proteases and peptidases influence peptide generation and degradation will be discussed. These processes determine the amount of peptides available for TAP translocation and class I binding and ultimately the immune response. [Copyright &y& Elsevier]

Similar to other herpesviruses, human cytomegalovirus remains in the infected host following resolution of the primary infection. The ability to persist in the host after primary infection is believed to be strongly influenced by the ability of HCMV to down-regulate immune recognition of infected cells. One of the genes contributing to immune evasion is the US3 gene. The US3 gene has been shown to retain major histocompatibility complex type I molecules in the endoplasmic reticulum. The US3 gene gives rise to three alternatively spliced RNAs which encode distinct but related proteins. Each of the alternatively spliced transcripts is present early in viral infection, suggesting that the encoded proteins play a role in the viral life cycle. We demonstrate that only the protein encoded by the unspliced US3 transcript is able to retain MHC class I heavy chains in the endoplasmic reticulum. The protein encoded by the singly spliced US3 transcript appears to be processed through the secretory pathway while the protein encoded by the doubly spliced transcript becomes localized to the Golgi apparatus. These experiments raise interesting questions about the functions of the smaller US3 proteins during viral infection in the host. [Copyright &y& Elsevier]

A query learning algorithm based on hidden Markov models (HMMs) isdeveloped to design experiments for string analysis and prediction of MHCclass I binding peptides. Query learning is introduced to aim at reducingthe number of peptide binding data for training of HMMs. A multiple numberof HMMs, which will collectively serve as a committee, are trained withbinding data and used for prediction in real-number values. The universeof peptides is randomly sampled and subjected to judgement by the HMMs.Peptides whose prediction is least consistent among committee HMMs aretested by experiment. By iterating the feedback cycle of computationalanalysis and experiment the most wanted information is effectivelyextracted. After 7 rounds of active learning with 181 peptides in all,predictive performance of the algorithm surpassed the so far bestperforming matrix based prediction. Moreover, by combining the bothmethods binder peptides (log Kd < -6) could be predicted with84% accuracy. Parameter distribution of the HMMs that can be inspectedvisually after training further offers a glimpse of dynamic specificity ofthe MHC molecules. [ABSTRACT FROM AUTHOR]

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acidtreatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells. [ABSTRACT FROM AUTHOR]

βMicroglobulin(βm)deficient non-obese diabetic (NOD) mice were established by crossing βm-deficient 129/Sv mice with NOD mice, and used to examine the possible involvement of MHC class I molecules and CD8+ T cells in the development of insulitis and diabetes. In these mice, MHC class I molecules were not expressed, resulting In no generation of CD8+ T cells. None of eight lines of βm-deficient NOD mice (−/−) established developed overt diabetes by 32 weeks, while control littermates (+/+) became diabetic by 22 weeks, histological studies showed no significant lymphocyte infiltration of the islets (insulitis score: 0.03 ± 0.03) in any of the βm-deficient NOD mice (−/−) compared with littermate NOD mice (+/+) with overt insulitis (1.42 ± 0.28). These findings support the notion that the expression of MHC class I molecules and/or CD8+T cells plays an essential role in the infiltration of CD4+ T cells in islets as well as the development of diabetes in NOD mice. [ABSTRACT FROM PUBLISHER]

Minor histocompatibility (H) antigens are T cell recognized self proteins which can cause graft versus host disease or organ transplant rejection. We have analysed the number of peptide epitopes involved in cytotoxic T lymphocyte (CTL) responses to single or multiple minor H antigens. Bulk CTL responses were generated in H-2 mice differing in one (H-1), two (H-1 and H-25), or multiple (>29) minor H loci, and HPLC separation was used to analyse the complexity of CTL recognized peptides. Anti-H-1 CTL recognize one out of 50 HPLC peptide fractions and recognition is H-2K restricted. The same peptide fraction is also recognized by anti-H-1/H-25 CTL and no additional epitopes are detected, indicating that the H-25 locus does not stimulate CTL when combined with H-1. CTL generated to multiple minor H loci (including H-1 and H-25) recognize two HPLC peptide fractions which are presented by H-2D and H-2K class I molecules, respectively. The H-2k presented fraction is the same as that recognized by anti-H-1 and anti-H-1/H-25 CTL, and it is shown to contain a H-1-derived peptide. Subfractionatlon of the CTL recognized HPLC fractions is consistent with the presence of only one peptide epitope. Thus, in the responses analysed here one minor H locus encodes probably only one CTL epitope. The study provides a molecular explanation for immunodominance among minor H antigens, suggesting that dominant loci encode single peptide epltopes which are presented efficiently by MHC class I molecules enabling them to readily stimulate CTL responses. [ABSTRACT FROM PUBLISHER]

Human renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to cytokines, were examined for their susceptibility to lymphokine-activated killer (LAK) cells. Flow-cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by interferon α (IFNα), IFNγ and tumor necrosis factor α (TNFα). A 4-hCr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFNγ or IFNα, but not with TNFα, decreased their susceptibility to LAK cells. IFNγ also decreased susceptibility to natural killer cells in a 16-hCr-release cytotoxicity assay. IFNγ treatment decreased the susceptibility of ACHN cells in a dose-dependent manner. 'Cold'-target competition assay clearly showed that IFNγ- but not TNFα-pretreated cells compete less effectively than do untreated target cells. Pretreatment with IFNγ, however, increased expression of intercellular adhesion molecule-1 (ICAM-1) to a degree comparable to that with TNFα. Northern blot analyses using a 520-base-pair ICAM-1 cDNA as a probe demonstrated that more 3.3-kb mRNA is expressed in IFNγ- and TNFα-pretreated cells. These results suggest that IFNγ-treated RCC cell lines may reduce their ability to be recognized by LAK cells, and that IFN-induced protection of RCC cell lines against LAK cells may depend upon a mechanism independent of the expression of class I antigens or ICAM-1 on tumor cells. [ABSTRACT FROM AUTHOR]

Adaptive immunity vitally depends on major histocompatibility complex class I (MHC I) molecules loaded with peptides. Selective loading of peptides onto MHC I, referred to as peptide editing, is catalyzed by tapasin and the tapasin-related TAPBPR. An important catalytic role has been ascribed to a structural feature in TAPBPR called the scoop loop, but the exact function of the scoop loop remains elusive. Here, using a reconstituted system of defined peptide-exchange components including human TAPBPR variants, we uncover a substantial contribution of the scoop loop to the stability of the MHC I-chaperone complex and to peptide editing. We reveal that the scoop loop of TAPBPR functions as an internal peptide surrogate in peptide-depleted environments stabilizing empty MHC I and impeding peptide rebinding. The scoop loop thereby acts as an additional selectivity filter in shaping the repertoire of presented peptide epitopes and the formation of a hierarchical immune response., Competing Interests: LS, FH, CT, RT No competing interests declared, (© 2020, Sagert et al.)

Major histocompatibility complex (MHC) class I molecules function to present pathogen derived peptides to cytotoxic T cells and act as ligands for Natural Killer cells, thus alerting the immune system to the presence of invading pathogens. However, some MHC class I molecules, most notably HLA-B27, can be strongly associated with autoimmune diseases. In addition, the MHC class I pathway is a target for numerous viral evasion strategies Understanding not only the antigen presenting functions, but also the biosynthesis and the degradation pathways of MHC class I molecules has therefore become important in determining their role in pathogen and autoimmune related diseases. Here, we describe how using epitope tagged MHC class I molecules can aid in the analysis of MHC class I molecule biosynthesis and degradation as well as complementary studies using conventional conformationally specific antibodies. Coupled together with pharmacological manipulation which can target both biosynthetic and degradative pathways, this offers a powerful tool in analyzing MHC class I molecules.