Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells.

BACKGROUND: Although immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC. METHODS: We assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment. RESULTS: HRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8+ T cells, and cytotoxic CD8+ T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8+ T cell penetration and efficacy, overcoming resistance to ICB therapy. CONCLUSIONS: HRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy.

Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.

Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands.

Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.

Treg-derived TGF-ß1 dampens cGAS-STING signaling to downregulate the expression of class I MHC complex in multiple myeloma.

Multiple myeloma (MM) progression involves diminished tumor antigen presentation and an immunosuppressive microenvironment, characterized by diminished expression of major histocompatibility complexes (MHC) class I molecule and elevated programmed death ligand 1 (PDL1) in MM cells, along with an enriched population of regulatory T cells (Tregs). To investigate Treg's influence on MM cells, we established a co-culture system using Tregs from MM patients and the MM cell lines (MM.1S and SK-MM-1) in vitro and assessed the effects of intervening in the relevant pathways connecting Tregs and MM cells in vivo. In vitro, Tregs induced transforming growth factor beta-1 (TGF-ß1) production, downregulated MHC I members, and increased PDL1 expression in MM cells. Treg-derived TGF-ß1 suppressed the cGAS-STING pathway, contributing to the loss of MHC I molecule expression and PDL1 upregulation. Correspondingly, neutralizing TGF-ß1 or activating the cGAS-STING pathway restored MHC I and PDL1 expression, effectively countering the pro-tumorigenic effect of Tregs on MM cells in vivo. These data elucidated how Tregs influence tumor antigen presentation and immunosuppressive signal in MM cells, potentially providing therapeutic strategies, such as neutralizing TGF-ß1 or activating the cGAS-STING pathway, to address the immune escape and immunosuppressive dynamics in MM.

Amino acid insertion in Bat MHC-I enhances complex stability and augments peptide presentation.

Bats serve as reservoirs for numerous zoonotic viruses, yet they typically remain asymptomatic owing to their unique immune system. Of particular significance is the MHC-I in bats, which plays crucial role in anti-viral response and exhibits polymorphic amino acid (AA) insertions. This study demonstrated that both 5AA and 3AA insertions enhance the thermal stability of the bat MHC-I complex and enrich the diversity of bound peptides in terms of quantity and length distribution, by stabilizing the 310 helix, a region prone to conformational changes during peptide loading. However, the mismatched insertion could diminish the stability of bat pMHC-I. We proposed that a suitable insertion may help bat MHC-I adapt to high body temperatures during flight while enhancing antiviral responses. Moreover, this site-specific insertions may represent a strategy of evolutionary adaptation of MHC-I molecules to fluctuations in body temperature, as similar insertions have been found in other lower vertebrates.

Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

The impact of micropolymorphism in Anpl-UAA on structural stability and peptide presentation.

Micropolymorphism significantly shapes the peptide-binding characteristics of major histocompatibility complex class I (MHC-I) molecules, affecting the host's resistance to pathogens, which is particularly pronounced in avian species displaying the "minimal essential MHC" expression pattern. In this study, we compared two duck MHC-I alleles, Anpl-UAA*77 and Anpl-UAA*78, that exhibit markedly different peptide binding properties despite their high sequence homology. Through mutagenesis experiments and crystallographic analysis of complexes with the influenza virus-derived peptide AEAIIVAMV (AEV9), we identified a critical role for the residue at position 62 in regulating hydrogen-bonding interactions between the peptide backbone and the peptide-binding groove. This modulation affects the characteristics of the B pocket and the stability of the loop region between the 310 helix and the α1 helix, leading to significant changes in the structure and stability of the peptide-MHC-I complex (pMHC-I). Moreover, the proportion of different residues at position 62 among Anpl-UAAs may reflect the correlation between pAnpl-UAA stability and duck body temperature. This research not only advances our understanding of the Anpl-UAA structure but also deepens our insight into the impact of MHC-I micropolymorphism on peptide binding.

Genome-Wide Association Study Identifies IFIH1 and HLA-DQB1*05:02 Loci Associated With Anti-NMDAR Encephalitis.

BACKGROUND AND OBJECTIVES: Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a rare autoimmune neurologic disorder, the genetic etiology of which remains poorly understood. Our study aims to investigate the genetic basis of this disease in the Chinese Han population. METHODS: We performed a genome-wide association study and fine-mapping study within the major histocompatibility complex (MHC) region of 413 Chinese patients with anti-NMDAR encephalitis recruited from 6 large tertiary hospitals and 7,127 healthy controls. RESULTS: Our genome-wide association analysis identified a strong association at the IFIH1 locus on chromosome 2q24.2 (rs3747517, p = 1.06 × 10-8, OR = 1.55, 95% CI, 1.34-1.80), outside of the human leukocyte antigen (HLA) region. Furthermore, through a fine-mapping study of the MHC region, we discovered associations for 3 specific HLA class I and II alleles. Notably, HLA-DQB1*05:02 (p = 1.43 × 10-12; OR, 2.10; 95% CI 1.70-2.59) demonstrates the strongest association among classical HLA alleles, closely followed by HLA-A*11:01 (p = 4.36 × 10-7; OR, 1.52; 95% CI 1.29-1.79) and HLA-A*02:07 (p = 1.28 × 10-8; OR, 1.87; 95% CI 1.50-2.31). In addition, we uncovered 2 main HLA amino acid variation associated with anti-NMDAR encephalitis including HLA-DQß1-126H (p = 1.43 × 10-12; OR, 2.10; 95% CI 1.70-2.59), exhibiting a predisposing effect, and HLA-B-97R (p = 3.40 × 10-8; OR, 0.63; 95% CI 0.53-0.74), conferring a protective effect. Computational docking analysis suggested a close relationship between the NR1 subunit of NMDAR and DQB1*05:02. DISCUSSION: Our findings indicate that genetic variation in IFIH1, involved in the type I interferon signaling pathway and innate immunity, along with variations in the HLA class I and class II genes, has substantial implications for the susceptibility to anti-NMDAR encephalitis in the Chinese Han population.

Characterisation of RAET1E/ULBP4 exon 4 and 3' untranslated region genetic architecture reveals further diversity and allelic polymorphism.

NKG2D is a natural killer cell activating receptor recognising ligands on infected or tumorigenic cells, leading to their cytolysis. There are eight known genes encoding NKG2D ligands: MICA, MICB and ULBP1-6. MICA and MICB are highly polymorphic and well characterised, whilst ULBP ligands are less polymorphic and the functional implication of their diversity is not well understood. Using International HLA and Immunogenetics Workshop (IHIW) cell line DNA, we previously characterised alleles of the RAET1E gene (encoding ULBP4 proteins), including the 5' UTR promoter region and exons 1-3. We found 11 promoter haplotypes associating with alleles based on exons 1-3, revealing 19 alleles overall. The current study extends this analysis using 87 individual DNA samples from IHIW cell lines or cord blood to include RAET1E exon 4 and the 3' UTR, as polymorphism in these regions have not been previously investigated. We found two novel exon 4 polymorphisms encoding amino acid substitutions altering the transmembrane domain. An amino acid substitution at residue 233 was unique to the RAET1E*008 allele whereas the substitution at residue 237 was shared between groups of alleles. Additionally, four haplotypes were found based on 3' UTR sequences, which were unique to certain alleles or shared with allele groups based on exons 1-4 polymorphisms. Furthermore, putative microRNAs were identified that may interact with these polymorphic sites, repressing transcription and potentially affecting expression levels.

Reassessing human MHC-I genetic diversity in T cell studies.

The Major Histocompatibility Complex class I (MHC-I) system plays a vital role in immune responses by presenting antigens to T cells. Allele specific technologies, including recombinant MHC-I technologies, have been extensively used in T cell analyses for COVID-19 patients and are currently used in the development of immunotherapies for cancer. However, the immense diversity of MHC-I alleles presents challenges. The genetic diversity serves as the foundation of personalized medicine, yet it also poses a potential risk of exacerbating healthcare disparities based on MHC-I alleles. To assess potential biases, we analysed (pre)clinical publications focusing on COVID-19 studies and T cell receptor (TCR)-based clinical trials. Our findings reveal an underrepresentation of MHC-I alleles associated with Asian, Australian, and African descent. Ensuring diverse representation is vital for advancing personalized medicine and global healthcare equity, transcending genetic diversity. Addressing this disparity is essential to unlock the full potential of T cells for enhancing diagnosis and treatment across all individuals.

Investigation of HLA susceptibility alleles and genotypes with hematological disease among Chinese Han population.

Several genes involved in the pathogenesis have been identified, with the human leukocyte antigen (HLA) system playing an essential role. However, the relationship between HLA and a cluster of hematological diseases has received little attention in China. Blood samples (n = 123913) from 43568 patients and 80345 individuals without known pathology were genotyped for HLA class I and II using sequencing-based typing. We discovered that HLA-A*11:01, B*40:01, C*01:02, DQB1*03:01, and DRB1*09:01 were prevalent in China. Furthermore, three high-frequency alleles (DQB1*03:01, DQB1*06:02, and DRB1*15:01) were found to be hazardous in malignant hematologic diseases when compared to controls. In addition, for benign hematologic disorders, 7 high-frequency risk alleles (A*01:01, B*46:01, C*01:02, DQB1*03:03, DQB1*05:02, DRB1*09:01, and DRB1*14:54) and 8 high-frequency susceptible genotypes (A*11:01-A*11:01, B*46:01-B*58:01, B*46:01-B*46:01, C*01:02-C*03:04, DQB1*03:01-DQB1*05:02, DQB1*03:03-DQB1*06:01, DRB1*09:01-DRB1*15:01, and DRB1*14:54-DRB1*15:01) were observed. To summarize, our findings indicate the association between HLA alleles/genotypes and a variety of hematological disorders, which is critical for disease surveillance.

The HLA-I landscape confers prognosis and antitumor immunity in breast cancer.

Breast cancer is a highly heterogeneous disease with varied subtypes, prognoses and therapeutic responsiveness. Human leukocyte antigen class I (HLA-I) shapes the immunity and thereby influences the outcome of breast cancer. However, the implications of HLA-I variations in breast cancer remain poorly understood. In this study, we established a multiomics cohort of 1156 Chinese breast cancer patients for HLA-I investigation. We calculated four important HLA-I indicators in each individual, including HLA-I expression level, somatic HLA-I loss of heterozygosity (LOH), HLA-I evolutionary divergence (HED) and peptide-binding promiscuity (Pr). Then, we evaluated their distribution and prognostic significance in breast cancer subtypes. We found that the four breast cancer subtypes had distinct features of HLA-I indicators. Increased expression of HLA-I and LOH were enriched in triple-negative breast cancer (TNBC), while Pr was relatively higher in hot tumors within TNBCs. In particular, a higher Pr indicated a better prognosis in TNBCs by regulating the infiltration of immune cells and the expression of immune molecules. Using the matched genomic and transcriptomic data, we found that mismatch repair deficiency-related mutational signature and pathways were enriched in low-Pr TNBCs, suggesting that targeting mismatch repair deficiency for synthetic lethality might be promising therapy for these patients. In conclusion, we presented an overview of HLA-I indicators in breast cancer and provided hints for precision treatment for low-Pr TNBCs.

Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens.

ABSTRACT: Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

Long-lasting mRNA-encoded interleukin-2 restores CD8+ T cell neoantigen immunity in MHC class I-deficient cancers.

Major histocompatibility complex (MHC) class I antigen presentation deficiency is a common cancer immune escape mechanism, but the mechanistic implications and potential strategies to address this challenge remain poorly understood. Studying ß2-microglobulin (B2M) deficient mouse tumor models, we find that MHC class I loss leads to a substantial immune desertification of the tumor microenvironment (TME) and broad resistance to immune-, chemo-, and radiotherapy. We show that treatment with long-lasting mRNA-encoded interleukin-2 (IL-2) restores an immune cell infiltrated, IFNγ-promoted, highly proinflammatory TME signature, and when combined with a tumor-targeting monoclonal antibody (mAB), can overcome therapeutic resistance. Unexpectedly, the effectiveness of this treatment is driven by IFNγ-releasing CD8+ T cells that recognize neoantigens cross-presented by TME-resident activated macrophages. These macrophages acquire augmented antigen presentation proficiency and other M1-phenotype-associated features under IL-2 treatment. Our findings highlight the importance of restoring neoantigen-specific immune responses in the treatment of cancers with MHC class I deficiencies.

Hippo-signaling-controlled MHC class I antigen processing and presentation pathway potentiates antitumor immunity.

The major histocompatibility complex class I (MHC class I)-mediated tumor antigen processing and presentation (APP) pathway is essential for the recruitment and activation of cytotoxic CD8+ T lymphocytes (CD8+ CTLs). However, this pathway is frequently dysregulated in many cancers, thus leading to a failure of immunotherapy. Here, we report that activation of the tumor-intrinsic Hippo pathway positively correlates with the expression of MHC class I APP genes and the abundance of CD8+ CTLs in mouse tumors and patients. Blocking the Hippo pathway effector Yes-associated protein/transcriptional enhanced associate domain (YAP/TEAD) potently improves antitumor immunity. Mechanistically, the YAP/TEAD complex cooperates with the nucleosome remodeling and deacetylase complex to repress NLRC5 transcription. The upregulation of NLRC5 by YAP/TEAD depletion or pharmacological inhibition increases the expression of MHC class I APP genes and enhances CD8+ CTL-mediated killing of cancer cells. Collectively, our results suggest a crucial tumor-promoting function of YAP depending on NLRC5 to impair the MHC class I APP pathway and provide a rationale for inhibiting YAP activity in immunotherapy for cancer.

Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells.

Pancreatic cancer is a lethal disease, harboring a five-year overall survival rate of only 13%. Current treatment approaches thus require modulation, with attention shifting towards liberating the stalled efficacy of immunotherapies. Select chemotherapy drugs which possess inherent immune-modifying behaviors could revitalize immune activity against pancreatic tumors and potentiate immunotherapeutic success. In this study, we characterized the influence of gemcitabine, a chemotherapy drug approved for the treatment of pancreatic cancer, on tumor antigen presentation by human leukocyte antigen class I (HLA-I). Gemcitabine increased pancreatic cancer cells' HLA-I mRNA transcripts, total protein, surface expression, and surface stability. Temperature-dependent assay results indicated that the increased HLA-I stability may be due to reduced binding of low affinity peptides. Mass spectrometry analysis confirmed changes in the HLA-I-presented peptide pool post-treatment, and computational predictions suggested improved affinity and immunogenicity of peptides displayed solely by gemcitabine-treated cells. Most of the gemcitabine-exclusive peptides were derived from unique source proteins, with a notable overrepresentation of translation-related proteins. Gemcitabine also increased expression of select immunoproteasome subunits, providing a plausible mechanism for its modulation of the HLA-I-bound peptidome. Our work supports continued investigation of immunotherapies, including peptide-based vaccines, to be used with gemcitabine as new combination treatment modalities for pancreatic cancer.

HLA class I NK-epitopes and KIR diversities in patients with multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy caused by the clonal expansion of malignant plasma cells in the bone marrow. Myeloma cells are susceptible to killing by natural killer (NK) cells, but NK cells fail to control disease progression, suggesting immunosuppression. The activation threshold of NK-effector function is regulated by interaction between KIRs and self-HLA class I, during a process called "education" to ensure self-tolerance. NK cells can respond to diseased cells based on the absence of HLA class I expression ("Missing-self" hypothesis). The HLA and KIR repertoire is extremely diverse; thus, the present study aimed to characterize potential variances in genotypic composition of HLA Class I NK-epitopes and KIRs between MM patients and healthy controls. Genotypic expression of KIR and HLA (HLA-C group-C1/C2 and Bw4 motifs (including HLA-A*23, A*24, A*32) were analyzed in 172 MM patients and 195 healthy controls. Compared to healthy controls, we did not observe specific KIR genes or genotypes, or HLA NK-epitopes with higher prevalence among MM patients. The presence of all three HLA NK-epitopes (C1+C2+Bw4+) was not associated with MM occurrence. However, MM patients were more likely to be C1-/C2+/Bw4+ (p = 0.049, OR 1.996). In line with this, there was a trend of increased genetic co-occurrence of Bw4 and KIR3DL1 in MM patients (p = 0.05, OR 1.557). Furthermore, MM patients were more likely to genetically express both C2/KIR2DL1 and Bw4/KIR3DL1 (p = 0.019, OR 2.453). Our results reveal an HLA NK-epitope combination that is associated with the occurrence of MM. No specific KIR genotypes were associated with MM.

HFE genotypes, haemochromatosis diagnosis and clinical outcomes at age 80 years: a prospective cohort study in the UK Biobank.

OBJECTIVES: HFE haemochromatosis genetic variants have an uncertain clinical penetrance, especially to older ages and in undiagnosed groups. We estimated p.C282Y and p.H63D variant cumulative incidence of multiple clinical outcomes in a large community cohort. DESIGN: Prospective cohort study. SETTING: 22 assessment centres across England, Scotland, and Wales in the UK Biobank (2006-2010). PARTICIPANTS: 451 270 participants genetically similar to the 1000 Genomes European reference population, with a mean of 13.3-year follow-up through hospital inpatient, cancer registries and death certificate data. MAIN OUTCOME MEASURES: Cox proportional HRs of incident clinical outcomes and mortality in those with HFE p.C282Y/p.H63D mutations compared with those with no variants, stratified by sex and adjusted for age, assessment centre and genetic stratification. Cumulative incidences were estimated from age 40 years to 80 years. RESULTS: 12.1% of p.C282Y+/+ males had baseline (mean age 57 years) haemochromatosis diagnoses, with a cumulative incidence of 56.4% at age 80 years. 33.1% died vs 25.4% without HFE variants (HR 1.29, 95% CI: 1.12 to 1.48, p=4.7×10-4); 27.9% vs 17.1% had joint replacements, 20.3% vs 8.3% had liver disease, and there were excess delirium, dementia, and Parkinson's disease but not depression. Associations, including excess mortality, were similar in the group undiagnosed with haemochromatosis. 3.4% of women with p.C282Y+/+ had baseline haemochromatosis diagnoses, with a cumulative incidence of 40.5% at age 80 years. There were excess incident liver disease (8.9% vs 6.8%; HR 1.62, 95% CI: 1.27 to 2.05, p=7.8×10-5), joint replacements and delirium, with similar results in the undiagnosed. p.C282Y/p.H63D and p.H63D+/+ men or women had no statistically significant excess fatigue or depression at baseline and no excess incident outcomes. CONCLUSIONS: Male and female p.C282Y homozygotes experienced greater excess morbidity than previously documented, including those undiagnosed with haemochromatosis in the community. As haemochromatosis diagnosis rates were low at baseline despite treatment being considered effective, trials of screening to identify people with p.C282Y homozygosity early appear justified.

Thunder-DDA-PASEF enables high-coverage immunopeptidomics and is boosted by MS2Rescore with MS2PIP timsTOF fragmentation prediction model.

Human leukocyte antigen (HLA) class I peptide ligands (HLAIps) are key targets for developing vaccines and immunotherapies against infectious pathogens or cancer cells. Identifying HLAIps is challenging due to their high diversity, low abundance, and patient individuality. Here, we develop a highly sensitive method for identifying HLAIps using liquid chromatography-ion mobility-tandem mass spectrometry (LC-IMS-MS/MS). In addition, we train a timsTOF-specific peak intensity MS2PIP model for tryptic and non-tryptic peptides and implement it in MS2Rescore (v3) together with the CCS predictor from ionmob. The optimized method, Thunder-DDA-PASEF, semi-selectively fragments singly and multiply charged HLAIps based on their IMS and m/z. Moreover, the method employs the high sensitivity mode and extended IMS resolution with fewer MS/MS frames (300 ms TIMS ramp, 3 MS/MS frames), doubling the coverage of immunopeptidomics analyses, compared to the proteomics-tailored DDA-PASEF (100 ms TIMS ramp, 10 MS/MS frames). Additionally, rescoring boosts the HLAIps identification by 41.7% to 33%, resulting in 5738 HLAIps from as little as one million JY cell equivalents, and 14,516 HLAIps from 20 million. This enables in-depth profiling of HLAIps from diverse human cell lines and human plasma. Finally, profiling JY and Raji cells transfected to express the SARS-CoV-2 spike protein results in 16 spike HLAIps, thirteen of which have been reported to elicit immune responses in human patients.

Hemochromatosis: Ferroptosis, ROS, Gut Microbiome, and Clinical Challenges with Alcohol as Confounding Variable.

Hemochromatosis represents clinically one of the most important genetic storage diseases of the liver caused by iron overload, which is to be differentiated from hepatic iron overload due to excessive iron release from erythrocytes in patients with genetic hemolytic disorders. This disorder is under recent mechanistic discussion regarding ferroptosis, reactive oxygen species (ROS), the gut microbiome, and alcohol abuse as a risk factor, which are all topics of this review article. Triggered by released intracellular free iron from ferritin via the autophagic process of ferritinophagy, ferroptosis is involved in hemochromatosis as a specific form of iron-dependent regulated cell death. This develops in the course of mitochondrial injury associated with additional iron accumulation, followed by excessive production of ROS and lipid peroxidation. A low fecal iron content during therapeutic iron depletion reduces colonic inflammation and oxidative stress. In clinical terms, iron is an essential trace element required for human health. Humans cannot synthesize iron and must take it up from iron-containing foods and beverages. Under physiological conditions, healthy individuals allow for iron homeostasis by restricting the extent of intestinal iron depending on realistic demand, avoiding uptake of iron in excess. For this condition, the human body has no chance to adequately compensate through removal. In patients with hemochromatosis, the molecular finetuning of intestinal iron uptake is set off due to mutations in the high-FE2+ (HFE) genes that lead to a lack of hepcidin or resistance on the part of ferroportin to hepcidin binding. This is the major mechanism for the increased iron stores in the body. Hepcidin is a liver-derived peptide, which impairs the release of iron from enterocytes and macrophages by interacting with ferroportin. As a result, iron accumulates in various organs including the liver, which is severely injured and causes the clinically important hemochromatosis. This diagnosis is difficult to establish due to uncharacteristic features. Among these are asthenia, joint pain, arthritis, chondrocalcinosis, diabetes mellitus, hypopituitarism, hypogonadotropic hypogonadism, and cardiopathy. Diagnosis is initially suspected by increased serum levels of ferritin, a non-specific parameter also elevated in inflammatory diseases that must be excluded to be on the safer diagnostic side. Diagnosis is facilitated if ferritin is combined with elevated fasting transferrin saturation, genetic testing, and family screening. Various diagnostic attempts were published as algorithms. However, none of these were based on evidence or quantitative results derived from scored key features as opposed to other known complex diseases. Among these are autoimmune hepatitis (AIH) or drug-induced liver injury (DILI). For both diseases, the scored diagnostic algorithms are used in line with artificial intelligence (AI) principles to ascertain the diagnosis. The first-line therapy of hemochromatosis involves regular and life-long phlebotomy to remove iron from the blood, which improves the prognosis and may prevent the development of end-stage liver disease such as cirrhosis and hepatocellular carcinoma. Liver transplantation is rarely performed, confined to acute liver failure. In conclusion, ferroptosis, ROS, the gut microbiome, and concomitant alcohol abuse play a major contributing role in the development and clinical course of genetic hemochromatosis, which requires early diagnosis and therapy initiation through phlebotomy as a first-line treatment.