Characterisation of novel MICB alleles by next generation sequencing: MICB*055 and MICB*005:15.

Two novel MICB alleles with coding polymorphisms in exon 3 were detected by next generation sequencing.

Characterisation of the novel MICA*112:02 allele by next generation sequencing.

The novel MICA*112:02 allele was detected by next generation sequencing.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning.

Human leucocyte antigen class I (HLA-I) molecules play a central role for both NK and T-cell responses that prevent serious human cytomegalovirus (HCMV) disease. To create opportunities for viral spread, several HCMV-encoded immunoevasins employ diverse strategies to target HLA-I. Among these, the glycoprotein US10 is so far insufficiently studied. While it was reported that US10 interferes with HLA-G expression, its ability to manipulate classical HLA-I antigen presentation remains unknown. In this study, we demonstrate that US10 recognizes and binds to all HLA-I (HLA-A, -B, -C, -E, -G) heavy chains. Additionally, impaired recruitment of HLA-I to the peptide loading complex was observed. Notably, the associated effects varied significantly dependending on HLA-I genotype and allotype: (i) HLA-A molecules evaded downregulation by US10, (ii) tapasin-dependent HLA-B molecules showed impaired maturation and cell surface expression, and (iii) ß2m-assembled HLA-C, in particular HLA-C*05:01 and -C*12:03, and HLA-G were strongly retained in complex with US10 in the endoplasmic reticulum. These genotype-specific effects on HLA-I were confirmed through unbiased HLA-I ligandome analyses. Furthermore, in HCMV-infected fibroblasts inhibition of overlapping US10 and US11 transcription had little effect on HLA-A, but induced HLA-B antigen presentation. Thus, the US10-mediated impact on HLA-I results in multiple geno- and allotypic effects in a so far unparalleled and multimodal manner.

During a viral infection, the immune system must discriminate between healthy and infected cells to selectively kill infected cells. Healthy cells have different types of molecules known collectively as HLA-I on their surface. These molecules present small fragments of proteins from the cell, called antigens, to patrolling immune cells, known as CTLs or natural killer cells. While CTLs ignore antigens from human proteins (which indicate the cell is healthy), they can bind to and recognize antigens from viral proteins, which triggers them to activate immune responses that kill the infected cell. However, some viruses can prevent infected cells from presenting HLA-I molecules on their surfaces as a strategy to evade the immune system. Natural killer cells have evolved to overcome this challenge. They bind to the HLA-I molecules themselves, which causes them to remain inactive. However, if the HLA-I molecules are missing, the NK cells can more easily switch on and kill the target cell. The human cytomegalovirus is a common virus that causes lifelong infection in humans. Although it rarely causes illness in healthy individuals, it can be life-threatening to newborn babies and for individuals with weakened immune systems. One human cytomegalovirus protein known as US10 was previously found to bind to HLA-I without reducing the levels of these molecules on the surface of the cell. However, its precise role remained unclear. Gerke et al. used several biochemical and cell biology approaches to investigate whether US10 manipulates the quality of the three types of HLA-I, which could impact both CTL and NK cell recognition. The experiments showed that US10 acted differently on the various kinds of HLA-I. To one type, it bound strongly within the cell and prevented it from reaching the surface. US10 also prevented another type of HLA-I from maturing properly and presenting antigens but did not affect the third type of HLA-I. These findings suggest that US10 interferes with the ability of different HLA-I types to present antigens in specific ways. Further research is needed to measure how US10 activity affects immune cells, which may ultimately aid the development of new therapies against human cytomegalovirus and other similar viruses.

Heart immunoengineering by lentiviral vector-mediated genetic modification during normothermic ex vivo perfusion.

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shß2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.

MHC1/LILRB1 axis as an innate immune checkpoint for cancer therapy.

Immune checkpoint blockades (ICBs) have revolutionized cancer therapy through unleashing anti-tumor adaptive immunity. Despite that, they are usually effective only in a small subset of patients and relapse can occur in patients who initially respond to the treatment. Recent breakthroughs in this field have identified innate immune checkpoints harnessed by cancer cells to escape immunosurveillance from innate immunity. MHC1 appears to be such a molecule expressed on cancer cells which can transmit a negative signal to innate immune cells through interaction with leukocyte immunoglobulin like receptor B1 (LILRB1). The review aims to summarize the current understanding of MHC1/LILRB1 axis on mediating cancer immune evasion with an emphasis on the therapeutic potential to block this axis for cancer therapy. Nevertheless, one should note that this field is still in its infancy and more studies are warranted to further verify the effectiveness and safety in clinical as well as the potential to combine with existing immune checkpoints.

NanoHLA: A Method for Human Leukocyte Antigen Class I Genes Typing Without Error Correction Based on Nanopore Sequencing Data.

Human leukocyte antigen (HLA) typing is of great importance in clinical applications such as organ transplantation, blood transfusion, disease diagnosis and treatment, and forensic analysis. In recent years, nanopore sequencing technology has emerged as a rapid and cost-effective option for HLA typing. However, due to the principles and data characteristics of nanopore sequencing, there was a scarcity of robust and generalizable bioinformatics tools for its downstream analysis, posing a significant challenge in deciphering the thousands of HLA alleles present in the human population. To address this challenge, we developed NanoHLA as a tool for high-resolution typing of HLA class I genes without error correction based on nanopore sequencing. The method integrated the concepts of HLA type coverage analysis and the data conversion techniques employed in Nano2NGS, which was characterized by applying nanopore sequencing data to NGS-liked data analysis pipelines. In validation with public nanopore sequencing datasets, NanoHLA showed an overall concordance rate of 84.34% for HLA-A, HLA-B, and HLA-C, and demonstrated superior performance in comparison to existing tools such as HLA-LA. NanoHLA provides tools and solutions for use in HLA typing related fields, and look forward to further expanding the application of nanopore sequencing technology in both research and clinical settings. The code is available at https://github.com/langjidong/NanoHLA .

Designing High Binding Affinity Peptides for MHC Class I Using MAM: An In Silico Approach.

The availability of extensive MHC-peptide binding data has boosted machine learning-based approaches for predicting binding affinity and identifying binding motifs. These computational tools leverage the wealth of binding data to extract essential features and generate a multitude of potential peptides, thereby significantly reducing the cost and time required for experimental procedures. MAM is one such tool for predicting the MHC-I-peptide binding affinity, extracting binding motifs, and generating new peptides with high affinity. This manuscript provides step-by-step guidance on installing, configuring, and executing MAM while also discussing the best practices when using this tool.

In Silico: Predicting Intrinsic Features of HLA Class-I Restricted Neoantigens.

Mutation-containing immunogenic peptides from tumor cells, also named as neoantigens, have various amino acid descriptors and physical-chemical properties characterized intrinsic features, which are useful in prioritizing the immunogenicity potentials of neoantigens and predicting patients' survival. Here, we describe a glioma neoantigen intrinsic feature database, GNIFdb, that hosts computationally predicted HLA-I restricted neoantigens of gliomas, their intrinsic features, and the tools for calculating intrinsic features and predicting overall survival of gliomas. We illustrate the application of GNIFdb in searching for possible neoantigen candidates from ATF6 that plays important roles in tumor growth and resistance to radiotherapy in glioblastoma. We also demonstrate the application of intrinsic feature associated tools in GNIFdb to predict the overall survival of primary IDH wild-type glioblastoma.

MICB Genetic Variants and Its Protein Soluble Level Are Associated with the Risk of Chronic GvHD and CMV Infection after Allogeneic HSCT.

The aim of the present study was to determine the associations between the MICB genetic variability and the expression and the risk of development of post-transplant complications after allogeneic hematopoietic stem cell transplantation (HSCT). HSCT recipients and their donors were genotyped for two MICB polymorphisms (rs1065075, rs3828903). Moreover, the expression of a soluble form of MICB was determined in the recipients' serum samples after transplantation using the Luminex assay. Our results revealed a favorable role of the MICB rs1065075 G allele. Recipients with donors carrying this genetic variant were less prone to developing chronic graft-versus-host disease (cGvHD) when compared to recipients without any symptoms of this disease (41.41% vs. 65.38%, p = 0.046). Moreover, the MICB rs1065075 G allele was associated with a lower incidence of cytomegalovirus (CMV) reactivation, both as a donor (p = 0.015) and as a recipient allele (p = 0.039). The MICB rs1065075 G variant was also found to be associated with decreased serum soluble MICB (sMICB) levels, whereas serum sMICB levels were significantly higher in recipients diagnosed with CMV infection (p = 0.0386) and cGvHD (p = 0.0008) compared to recipients without those complications. A protective role of the G allele was also observed for the rs3828903 polymorphism, as it was more frequently detected among donors of recipients without cGvHD (89.90% vs. 69.23%; p = 0.013). MICB genetic variants, as well as serum levels of sMICB, may serve as prognostic factors for the risk of developing cGvHD and CMV infection after allogeneic HSCT.

Insight into the avidity-affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn.

Monoclonal antibodies (mAbs) as therapeutics necessitate favorable pharmacokinetic properties, including extended serum half-life, achieved through pH-dependent binding to the neonatal Fc receptor (FcRn). While prior research has mainly investigated IgG-FcRn binding kinetics with a focus on single affinity values, it has been shown that each IgG molecule can engage two FcRn molecules throughout an endosomal pH gradient. As such, we present here a more comprehensive analysis of these interactions with an emphasis on both affinity and avidity by taking advantage of switchSENSE technology, a surface-based biosensor where recombinant FcRn was immobilized via short DNA nanolevers, mimicking the membranous orientation of the receptor. The results revealed insight into the avidity-to-affinity relationship, where assessing binding through a pH gradient ranging from pH 5.8 to 7.4 showed that the half-life extended IgG1-YTE has an affinity inflection point at pH 7.2, reflecting its engineering for improved FcRn binding compared with the wild-type counterpart. Furthermore, IgG1-YTE displayed a pH switch for the avidity enhancement factor at pH 6.2, reflecting strong receptor binding to both sides of the YTE-containing Fc, while avidity was abolished at pH 7.4. When compared with classical surface plasmon resonance (SPR) technology and complementary methods, the use of switchSENSE demonstrated superior capabilities in differentiating affinity from avidity within a single measurement. Thus, the methodology provides reliable kinetic rate parameters for both binding modes and their direct relationship as a function of pH. Also, it deciphers the potential effect of the variable Fab arms on FcRn binding, in which SPR has limitations. Our study offers guidance for how FcRn binding properties can be studied for IgG engineering strategies.

Hexamerization: explaining the original sin of IgG-mediated complement activation in acute lung injury.

Although antibody-mediated lung damage is a major factor in transfusion-related acute lung injury (ALI), autoimmune lung disease (for example, coatomer subunit α [COPA] syndrome), and primary graft dysfunction following lung transplantation, the mechanism by which antigen-antibody complexes activate complement to induce lung damage remains unclear. In this issue of the JCI, Cleary and colleagues utilized several approaches to demonstrate that IgG forms hexamers with MHC class I alloantibodies. This hexamerization served as a key pathophysiological mechanism in alloimmune lung injury models and was mediated through the classical pathway of complement activation. Additionally, the authors provided avenues for exploring therapeutics for this currently hard-to-treat clinical entity that has several etiologies but a potentially focused mechanism.

SARS-CoV-2 Selectively Induces the Expression of Unproductive Splicing Isoforms of Interferon, Class I MHC, and Splicing Machinery Genes.

RNA processing is a highly conserved mechanism that serves as a pivotal regulator of gene expression. Alternative processing generates transcripts that can still be translated but lead to potentially nonfunctional proteins. A plethora of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strategically manipulate the host's RNA processing machinery to circumvent antiviral responses. We integrated publicly available omics datasets to systematically analyze isoform-level expression and delineate the nascent peptide landscape of SARS-CoV-2-infected human cells. Our findings explore a suggested but uncharacterized mechanism, whereby SARS-CoV-2 infection induces the predominant expression of unproductive splicing isoforms in key IFN signaling, interferon-stimulated (ISGs), class I MHC, and splicing machinery genes, including IRF7, HLA-B, and HNRNPH1. In stark contrast, cytokine and chemokine genes, such as IL6 and TNF, predominantly express productive (protein-coding) splicing isoforms in response to SARS-CoV-2 infection. We postulate that SARS-CoV-2 employs an unreported tactic of exploiting the host splicing machinery to bolster viral replication and subvert the immune response by selectively upregulating unproductive splicing isoforms from antigen presentation and antiviral response genes. Our study sheds new light on the molecular interplay between SARS-CoV-2 and the host immune system, offering a foundation for the development of novel therapeutic strategies to combat COVID-19.

Extension of the circulatory half-life of recombinant ecallantide via albumin fusion without loss of anti-kallikrein activity.

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (Ki) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.

Proteasome inhibitors FHND6091 enhance the ability of NK cells to kill tumor cells through multiple mechanisms.

The immune system has a strong connection to tumors. When a tumor cell is recognized as an abnormal cell by the immune system, the immune system may initiate an immune response to kill the tumor cell. In this study, RNA sequencing was performed on multiple myeloma (MM) cells treated with the proteasome inhibitor FHND6091. The transcriptional changes induced by FHND6091 in RPMI8226 cells aligned notably with immune response activation and results indicated upregulation of cGAS-STING pathway-related genes in the FHND6091-treated group. In vivo and in vitro experiments had demonstrated that FHND6091 stimulated the immunoreaction of MM cells via activation of the cyclic guanosine monophosphate-adenosine synthase/stimulator of interferon genes (cGAS-STING) pathway. This activation resulted in the generation of type-I interferons and the mobilization of natural killer (NK) cells. Notably, FHND6091 upregulated the levels of calreticulin and the protein ligands UL16-binding protein 2/5/6, MHC class I chain-related A (MICA), and MICB on the surface of MM cells. Subsequently, upon engaging with the surface activation receptors of NK cells, these ligands triggered NK cell activation, leading to the subsequent elimination of tumor cells. Thus, our findings elucidated the mechanism whereby FHND6091 exerted its immunotherapeutic activity as a STING agonist, enhancing the killing ability of NK cells against tumor cells.

Gene and protein sequence features augment HLA class I ligand predictions.

The sensitivity of malignant tissues to T cell-based immunotherapies depends on the presence of targetable human leukocyte antigen (HLA) class I ligands. Peptide-intrinsic factors, such as HLA class I affinity and proteasomal processing, have been established as determinants of HLA ligand presentation. However, the role of gene and protein sequence features as determinants of epitope presentation has not been systematically evaluated. We perform HLA ligandome mass spectrometry to evaluate the contribution of 7,135 gene and protein sequence features to HLA sampling. This analysis reveals that a number of predicted modifiers of mRNA and protein abundance and turnover, including predicted mRNA methylation and protein ubiquitination sites, inform on the presence of HLA ligands. Importantly, integration of such "hard-coded" sequence features into a machine learning approach augments HLA ligand predictions to a comparable degree as experimental measures of gene expression. Our study highlights the value of gene and protein features for HLA ligand predictions.

The Significant Role of PA28αß in CD8+ T Cell-Mediated Graft Rejection Contrasts with Its Negligible Impact on the Generation of MHC-I Ligands.

The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αß). In particular, several immunogenic peptides have been reported to be PA28αß-dependent. In contrast, we did not observe a major impact of PA28αß on the generation of different major histocompatibility complex (MHC) classI ligands. PA28αß-knockout mice infected with the lymphocytic choriomeningitis virus (LCMV) or vaccinia virus showed a normal cluster of differentiation (CD) 8 response and viral clearance. However, we observed that the adoptive transfer of wild-type cells into PA28αß-knockout mice led to graft rejection, but not vice versa. Depletion experiments showed that the observed rejection was mediated by CD8+ cytotoxic T cells. These data indicate that PA28αß might be involved in the development of the CD8+ T cell repertoire in the thymus. Taken together, our data suggest that PA28αß is a crucial factor determining T cell selection and, therefore, impacts graft acceptance.

Non-Classical Swine Leukocyte Antigens SLA-6, -7, and -8, Are Xenoantigens for Some Waitlisted Patients.

Attack of donor tissues by pre-formed anti-pig antibodies is well known to cause graft failure in xenotransplantation. Genetic engineering of porcine donors to eliminate targets of these pre-formed antibodies coupled with advances in immunosuppressive medicines have now made it possible to achieve extended survival in the pre-clinical pig-to-non-human primate model. Despite these improvements, antibodies remain a risk over the lifetime of the transplant, and many patients continue to have pre-formed donor-specific antibodies even to highly engineered pigs. While therapeutics exist that can help mitigate the detrimental effects of antibodies, they act broadly potentially dampening beneficial immunity. Identifying additional xenoantigens may enable more targeted approaches, such as gene editing, to overcome these challenges by further eliminating antibody targets on donor tissue. Because we have found that classical class I swine leukocyte antigens are targets of human antibodies, we now examine whether related pig proteins may also be targeted by human antibodies. We show here that non-classical class I swine leukocyte proteins (SLA-6, -7, -8) can be expressed at the surface of mammalian cells and act as antibody targets.

The impact of MICB mismatches in unrelated haematopoietic stem cell transplantation.

MICA polymorphisms have been associated with increased incidence of acute GvHD and adverse outcome in allogeneic haematopoietic stem cell transplantation (HSCT). MICB is another expressed member of MHC class I-related chain genes and its impact on HSCT outcome is yet to be fully defined. We typed a large cohort of patients and donors for MICB polymorphisms and investigated the impact of MICB matching on outcome after unrelated HSCT. 69.2% of the patients were 10/10 human leukocyte antigen (HLA) matched and 30.8% were 9/10 HLA matched. MICB typing was performed using a short amplicon-based NGS typing assay on the Illumina MiSeq platform. Differences in proteins were considered as mismatches. MICA polymorphisms were identified as possible confounder and were therefore included as parameter in the multivariate analyses. Due to the strong linkage disequilibrium with the classical HLA-genes, sub-stratification for HLA matching status was necessary, and no effect of MICB mismatches was seen in the 10/10 HLA matched group when compared to the MICB matched cases. However, in the 9/10 HLA matched group, MICB mismatched cases showed significantly worse disease free survival (DFS), GvHD and relapse free survival (GRFS) compared to the MICB matched cases (DFS: HR 1.24, p = 0.011; GRFS: HR 1.26, p = 0.002). MICA mismatches had no impact on any outcome parameter. According to our findings, effects previously attributed to MICA differences may have been confounded by MICB polymorphisms. We show that MICB differences contribute a small but relevant effect in 9/10 HLA-matched transplantations, which in turn highlights the possible usefulness of MICB typing in donor selection among similarly suitable 9/10 matched donors, especially when HLA-B mismatches have to be accepted.

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.