Introduction of NGS into clinical histocompatibility laboratories has greatly increased the frequency of novel HLA allele discovery. We identified 9 DQA1, 7 DQB1, 8 DPA1 and 2 DPB1 novel alleles over the last 2 years. 92% (24 of 26) differed from their closest allele by single nucleotide substitutions detected in coding regions and 84% (22 of 26) contained polymorphism outside of exon 2. Identification of novel alleles in non-coding intronic regions were restricted to splice sites. Two novel alleles arising from nucleotide substitution affecting splicing were identified and named with questionable expression status. Novel alleles with polymorphism detected only in non-coding regions (introns and UTRs) were not reported. 21 additional DQA1, DQB1 and DPA1 alleles detected in multiple individuals as novel at the time of testing, were also detected in other laboratories. The number of novel alleles detected further emphasises the role of NGS in novel allele discovery.

The COVID-19 pandemic, originating in Wuhan in 2019, was caused by SARS-CoV-2, leading to significant global fatalities. Despite the development of vaccines, the virus mutates, creating variants that evade vaccine-induced immunity. To address SARS-CoV-2′s evolving nature, a multiepitope vaccine was developed using immunoinformatics approach, specifically targeting the Omicron variant’s spike protein. This vaccine includes six CD8 + and eleven CD4 + epitopes selected for their immunogenicity, non-toxicity, and significant conservation among former Variants of Concern (VOCs) and Variants of Interest (VOIs), such as Alpha, Beta, Gamma, Delta, Lambda, Mu, R1, and Zeta, as well as current Variants Under Monitoring (VUMs) like XBB.1.5, XBB.1.16, EG.5, BA.2.86, and JN.1. Notably, certain epitopes like ELLHAPATV and PYRVVVLSFELLHAP were fully conserved across all tested variants in the spike protein’s receptor binding domain (RBD). Others, such as NATRFASVYAWNRKR, were fully conserved in all former VOCs and VOIs and 93.33 % in current VUMs, while ERDISTEIYQAGNKP was entirely conserved in current VUMs within the RBD region. The study went on to model, refine, and validate the vaccine prototype’s tertiary structure. Docking experiments and molecular dynamic simulations revealed robust and stable interactions with Toll-like receptor 4. Cloning and codon optimization confirmed successful expression in E. coli. Subsequently, the immunological reaction of the multiepitope vaccine demonstrated that the three-time administration of the prototype significantly enhanced the antibody response while decreasing the number of antigens. The designed vaccine’s epitopes showed significant combined global population coverage of 100 % with 89.75 % for CD8 + and 99.98 % for CD4 + epitopes and conservation across SARS-CoV-2 variants especially in current monitoring omicron subvariants, supporting its broader applicability and potential efficacy. Although, this promising vaccine candidate needs to undergo clinical trials to determine its effectiveness in neutralising SARS-CoV-2.

A Thailand orthohantavirus (THAIV) is endemic in Southeast Asia. This assumption is supported by isolation of THAIV from local small mammals. Also, anti-orthohantavirus antibodies were detected in human serum. However, our understanding of THAIV cross-reactivity with antibodies against other orthohantaviruses remains largely unknown.We used the in-silico approach to identify the cross-reactive immunogenic peptides of THAIV. The immunogenicity of these peptides was tested using convalescent serum from patients infected with Puumala (PUUV), Hantaan (HNTV) and Dobrava (DOBV) orthohantaviruses.We identified three THAIV peptides reacting with orthohantavirus convalescent serum. P1 peptide was reactive with serum from patients infected with PUUV, HNTV and DOBV. These peptides were found to be non-allergenic. Molecular docking and population coverage analysis revealed the potential of selected peptides to interact with diverse HLA alleles worldwide.Our data indicate that THAIV peptides could be used to develop diagnostics for orthohantaviruses circulating in Southeast Asia.

BackgroundThe clinical spectrum of COVID-19 varies considerably, ranging from asymptomatic cases to severe disease and even death. This variability can partly be attributed to genetic differences in genes associated with inflammation and immune responses. Among these genes, Interferon Induced with Helicase C Domain 1 (IFIH1), which codes for a cytoplasmic sensor, plays a crucial role in detecting SARS-CoV-2 viral RNA and initiating the antiviral interferon (IFN) response, thereby constituting a key element of innate immune defense. AimThis study aims to examine the association between genetic variants in the IFIH1 gene and susceptibility to, as well as the severity of, COVID-19 in the Moroccan population. Materials and MethodsWe conducted a case-control study involving 299 COVID-19 positive patients (149 severe, 150 benign) and 145 uninfected-SARS-CoV-2 controls. We determined the genotypes of two functional variants, rs1990760 (Ala946Thr) and rs3747517 (His843Arg), in the IFIH1 gene using predesigned TaqMan real-time allelic discrimination assay. ResultsOur results indicated that the TT genotype of rs1990760 was associated with increased susceptibility to SARS-CoV-2 under a recessive model (odds ratio [OR] = 2.22, 95 % confidence interval [CI] 1.28–3.84, P=0.003). Conversely, the CT genotype appeared to confer protection against SARS-CoV-2 infection (OR=0.58, 95 % CI 0.38–0.91, P=0.016) and COVID-19 severity (OR=0.56, 95 % CI 0.34–0.91, P=0.019). No significant association was found between rs3747517 and the risk of hospitalization or infection susceptibility. ConclusionThese findings underscore the significance of genetic variability in the IFIH1 gene in shaping individual responses to SARS-CoV-2 in the Moroccan population.

Store-operated calcium entry (SOCE) is essential for cellular signaling. Earlier studies of the pyrazole derivative BTP2, an efficient inhibitor SOCE, identified that SOCE blockade suppresses proinflammatory gene expression. The impact of SOCE blockade on gene expression at the whole transcriptome level, however, is unknown. To fill this gap, we performed RNA sequencing (RNA-seq) and investigated at the whole transcriptome level the effect of BTP2 on gene expression in human peripheral blood mononuclear cells signaled with phytohemagglutinin. Our global gene expression analysis identified that SOCE blockade spares activation-induced expression of anti-inflammatory genes (e.g., IL10, TGFB1, FOXP3, and CTLA4) whereas the induced expression of proinflammatory genes such as IFNG and cytopathic genes such as GZMB are inhibited. We validated the differential expression of immunoregulatory genes identified by RNA-seq using preamplification-enhanced RT-qPCR assays. Because IL-2/IL2RA interaction is essential for T cell clonal expansion, we investigated and confirmed that BTP2 inhibits IL2RA expression at the protein level using multiparameter flow cytometry. Our elucidation that SOCE blockade spares activation-induced expression of anti-inflammatory genes while blocking pro-inflammatory gene expression suggests that SOCE blockers may represent a novel class of immunoregulatory drugs of value for treating autoimmune disease states and organ transplantation.

Chimeric antigen receptor (CAR) T cell therapy is a powerful adoptive immunotherapy against blood cancers, but the therapeutic effect was not efficient enough on solid tumors. B cells have been reported to play a critical role in regulating memory T differentiation and cytotoxic T development. However, as of yet the influence of such B cells on CAR T cells has not been discussed. In this study, using ephrin type-A receptor 2 (EphA2) specific CAR T cells, we cultured B cells successfully to stimulate CAR T cells in vitro, and investigated the cell differentiation and anti-tumor efficiency. We observed that EphA2-CAR T cells stimulated by B cells performed increased interferon γ (IFN γ) production and upregulated OX40 expression, as well as the enhanced anti-tumor activity and reduced PD-1 expression. The persistence of CAR T cells was enhanced after B cells stimulation for more than 7 days with the increased subset of central memory T cells (TCM). In addition, next generation sequencing was performed to explore the underlying mechanisms. The up-regulated genes clustered in, immune response activation, chemokine signaling pathway, calcium signaling pathway, cGMP-PKG signaling pathway and et al. which contributed to the upregulated anti-glioblastoma (GBM) activity of CAR T cells stimulated by B cell. Furthermore, MEF2C, CD40, SYK and TNFRSF13B were upregulated in CAR T cells after co-culturing with B cells. These genes functionally enriched in promoting lymphocytes proliferation and may contribute to the enhanced persistence of CAR T cells. In conclusion, these results indicated the critical role of B cells in prolonging CAR T cells longevity and enhancing anti-tumor activity, which paves the way for the therapeutic exploitation of EphA2-CAR T cells against GBM in the future.

Systemic sclerosis (SSc), a rare and lethal autoimmune disorder where patients presents diverse clinical features, therefore unravelling a potential biomarker within a specific cohort is crucial for improving patient care, especially for rare diseases.This study sought to identify potential biomarkers in Tunisian SSc patients. Gene expression analysis of interleukins (IL)-21 and IL-22 in peripheral blood mononuclear cells, using quantitative real-time polymerase chain reaction (qrt-pcr), revealed upregulated IL-21 and downregulated IL-22 in SSc patients compared to healthy controls. Notably, IL-21 overexpression in patients correlated with pulmonary complications, a severe SSc manifestation. Interestingly, flow cytometry analysis displayed no difference in Th17 cells between groups, suggesting that Th17 might not be the primary drivers of cytokine dysregulation. The hypothesis was supported by qRT-PCR, which analysed two key genes: IL-17A and RORγt. Finally, we examined RNA sequencing data to further validate our hypothesis.Collectively, our study provides novel insights into the cytokine landscape of SSc in Tunisian patients, highlighting a dysregulation in IL-21 and IL-22 expression, and suggesting that IL-21 could be a potential biomarker of severity.

BackgroundThe most abundant innate immune cells, neutrophils, contribute significantly to cancer development by stimulating immunosuppression. However, it remains unclear about its function and molecular mechanisms in the immunosuppressive microenvironment of non-small cell lung cancer (NSCLC). MethodsBlood samples were collected from NSCLC patients and healthy volunteers to detect the expression of P2RX1 and PD-L1 in neutrophils using qRT-PCR, western blot (WB), and flow cytometry. Neutrophils were sorted into P2RX1-positive (P2RX1+)/P2RX1-negative (P2RX1−) groups and co-cultured with CD8+ T cells. Changes in the proliferative and cytotoxic capabilities of CD8+ T cells were then detected using flow cytometry and enzyme-linked immunosorbent assay. The content of granzyme B was determined by enzyme-linked immunosorbent assay. The effects of P2RX1-deficient neutrophils on fatty acids, triglycerides, lipid droplet content and FASN expression were detected using kits, Nile red staining and WB, respectively. ResultsThis study revealed a deficiency in P2RX1 expression in peripheral blood neutrophils of NSCLC patients, which was negatively correlated with the expression of PD-L1. P2RX1−neutrophils inhibited T cell proliferation and granzyme B expression and promoted T cell exhaustion. Furthermore, in P2RX1-deficient neutrophils, there was a notable increase in the levels of fatty acids, triglycerides, and lipid droplet accumulation, as well as an upregulation of FASN protein expression. Mechanistically, P2RX1−neutrophils upregulated PD-L1 expression by inducing fatty acid metabolism to improve immunosuppression in NSCLC. ConclusionThe mechanism by which P2RX1-deficient neutrophils contributed to immunosuppressive effects in NSCLC was clarified by our findings, indicating that P2RX1 could be one potential target for counteracting the immunosuppressive effects of neutrophils.

PurposeThe objective of this research is to analyze the clinical progression and identify prognostic factors among elderly patients with sepsis admitted to the emergency intensive care unit (EICU). MethodsA total of 211 patients with sepsis, aged 65 years or above, were selected for inclusion in the study. These patients were admitted to the EICU of the Emergency Department at Harrison International Peace Hospital Affiliated to Hebei Medical University from August 2018 to June 2023. The clinical features, Acute Physiology and Chronic Health Evaluation (APACHE) Π score, Sequential Organ Failure Assessment (SOFA) score, and routine laboratory test indicators were documented. All patients were followed up for 28 days. The factors associated with mortality in both the sepsis group and septic shock group were analyzed by receiver operating characteristic (ROC) curve, MedCalc software, and Kaplan-Meier curve. ResultsAmong the 211 patients, 101 were identified as having septic shock. A significant elevation in blood urea nitrogen-to-albumin ratio (BAR) and inflammatory indicators, APACHE II score, and SOFA score was observed in the septic shock group compared to the sepsis group (P<0.001). Moreover, the sepsis group exhibited a higher proportion of males (P=0.002), while there was no statistically significant difference in age (P=0.467). Further analysis revealed that BAR within 24 h after admission exhibited a positive correlation with infection indicators procalcitonin (PCT) and C-reactive protein (CRP), as well as disease severity scores APACHE Π and SOFA. Additionally, BAR was found to be positively associated with the 28-day mortality rate in patients with sepsis (r = 0.169, P=0.001). The results of the ROC curve analysis showed that BAR exhibited the highest predictive capability for 28-day mortality in elderly patients with sepsis who were admitted to the EICU (AUC=0.614). The Kaplan-Meier survival curve, which identified the optimal cut-off value (≥0.3) of BAR as the most accurate predictor of 28-day mortality in this individual, revealed a significantly higher mortality rate among patients with BAR≥0.3 (χ2 = 12.340, P=0.000). ConclusionThe elderly patients with sepsis in the EICU are generally over the age of 70, with a higher prevalence of males than females, and the albumin level is generally low on admission. Furthermore, BAR is significantly and positively correlated with infectious indexes and has a high predictive value for their mortality outcomes.

BackgroundIt is reported that G protein-coupled receptor 84 (GPR84) can participate in inflammation and immune regulation to repress anti-tumor responses. However, the function of GPR84 in lung cancer (LC) and its potential molecular mechanisms are still largely unknown. MethodsBioinformatics and molecular experiments were employed to assess the expression of GPR84 in LC. The pathways enriched by GPR84 were analyzed by the Kyoto Encyclopedia of Genes and Genomes. Bioinformatics prediction identified the potential upstream regulatory factors of GPR84, which were verified through dual luciferase and chromatin immunoprecipitation experiments. Cell viability was measured by methyl thiazolyl tetrazolium assay. The expression levels of key proteins related to the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway such as JAK2, p-JAK2, p-STAT3, and STAT3 were detected by western blot. Macrophages were co-cultured with LC cells. Flow cytometry was employed to examine the proportion of mannose receptor-positive cells. The expression levels of M2 polarization marker genes chitinase-like protein 3, arginase-1, and found in inflammatory zone 1 were measured by quantitative reverse transcription polymerase chain reaction. We applied an enzyme-linked immunosorbent assay to determine levels of cytokines (interleukin-10 and transforming growth factor beta) to evaluate the M2 macrophage polarization. ResultsGPR84 was highly expressed in LC and substantially enriched in the JAK-STAT pathway. GPR84 facilitated the M2 polarization of macrophages in LC. Adding the JAK-STAT pathway inhibitor weakened the promoting effect of GPR84 overexpression on M2 macrophage polarization. Furthermore, GPR84 also had an upstream regulatory factor lamin B1 (LMNB1). Knocking down LMNB1 blocked the JAK-STAT signaling pathway to repress M2 macrophage polarization in LC, while overexpression of GPR84 reversed the impact of LMNB1 knockdown on macrophage polarization. ConclusionThe project suggested that the LMNB1/GPR84 axis can facilitate M2 polarization of macrophages in LC by triggering the JAK-STAT pathway. Targeting LMNB1/GPR84 or blocking the JAK-STAT pathway may be a novel approach for LC diagnosis and treatment.