Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 Avian Influenza Virus (AIV).

B2 haplotype MHC has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown and the presenting peptide is rarely identified. Here, we identified the peptide-binding motif of B2 haplotype MHC Ⅰ molecules (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) in vitro using Random Peptide Library-based MHC-Ⅰ LC-MS/MS analysis. To further clarify the structure basis of the peptide binding motif, we determined the crystal structure of the BF2*02:01-PB2552-560 complex at 1.9 Šresolution. We found that BF2*02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets of BF2*02:01 was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2*02:01 than BF2*04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterwards, we explored the H9N2 AIV-induced cellular immune response in B2 haplotype chickens in vivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, IFN-γ, NK lysin, and PARP in PBMCs were significantly increased in defending against H9N2 AIV infection. Especially, we selected 411 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot IFN-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

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.

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.

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

A Prediction Nomogram for Severe Obstructive Sleep Apnea in Snoring Patients: A Retrospective Study.

Purpose: Snoring patients, as a high-risk group for OSA, are prone to the combination of severe OSA and face serious health threats. The aim of our study was to develop and validate a nomogram to predict the occurrence of severe OSA in snorers, in order to improve the diagnosis rate and treatment rate in this population. Patients and Methods: A training cohort of 464 snoring patients treated at our institution from May 2021 to October 2022 was divided into severe OSA and non-severe OSA groups. Univariate and multivariate logistic regression were used to identify potential predictors of severe OSA, and a nomogram model was constructed. An external hospital cohort of 210 patients was utilized as an external validation cohort to test the model. Area under the receiver operating characteristic curve, calibration curve, and decision curve analyses were used to assess the discriminatory power, calibration, and clinical utility of the nomogram, respectively. Results: Multivariate logistic regression demonstrated that body mass index, Epworth Sleepiness Scale total score, smoking history, morning dry mouth, dream recall, and hypertension were independent predictors of severe OSA. The area under the curve (AUC) of the nomogram constructed from the above six factors is 0.820 (95% CI: 0.782-0.857). The Hosmer-Lemeshow test showed that the model had a good fit (P = 0.972). Both the calibration curve and decision curve of the nomogram demonstrated the corresponding dominance. Moreover, external validation further confirmed the reliability of the predicted nomograms (AUC=0.805, 95% CI: 0.748-0.862). Conclusion: A nomogram predicting the occurrence of severe OSA in snoring patients was constructed and validated with external data for the first time, and the findings all confirmed the validity of the model. This may help to improve existing clinical decision making, especially at institutions that do not yet have devices for diagnosing OSA.

Efficient Identification of Tembusu Virus CTL Epitopes in Inbred HBW/B4 Ducks Using a Novel MHC Class I-Restricted Epitope Screening Scheme.

The identification of MHC class I-restricted CTL epitopes in certain species, particularly nonmammals, remains a challenge. In this study, we developed a four-step identification scheme and confirmed its efficiency by identifying the Anpl-UAA*76-restricted CTL epitopes of Tembusu virus (TMUV) in inbred haplotype ducks HBW/B4. First, the peptide binding motif of Anpl-UAA*76 was determined by random peptide library in de novo liquid chromatography-tandem mass spectrometry, a novel nonbiased, data-independent acquisition method that we previously established. Second, a total of 38 TMUV peptides matching the motif were screened from the viral proteome, among which 11 peptides were conserved across the different TMUV strains. Third, the conserved TMUV peptides were refolded in vitro with Anpl-UAA*76 and Anpl-ß2-microglobulin to verify the results from the previous two steps. To clarify the structural basis of the obtained motif, we resolved the crystal structure of Anpl-UAA*76 with the TMUV NS3 peptide LRKRQLTVL and found that Asp34 is critical for the preferential binding of the B pocket to bind the second residue to arginine as an anchor residue. Fourth, the immunogenicity of the conserved TMUV peptides was tested in vivo using specific pathogen-free HBW/B4 ducks immunized with the attenuated TMUV vaccine. All 11 conserved TMUV epitopes could bind stably to Anpl-UAA*76 in vitro and stimulate the secretion of IFN-γ and lymphocyte proliferation, and three conserved and one nonconserved peptides were selected to evaluate the CTL responses in vivo by flow cytometry and their tetramers. We believe that this new scheme could improve the identification of MHC class I-restricted CTL epitopes, and our data provide a foundation for further study on duck anti-TMUV CTL immunity.

Peptidomes and Structures Illustrate How SLA-I Micropolymorphism Influences the Preference of Binding Peptide Length.

Polymorphisms can affect MHC-I binding peptide length preferences, but the mechanism remains unclear. Using a random peptide library combined with LC-MS/MS and de novo sequencing (RPLD-MS) technique, we found that two swine MHC-I molecules with high sequence homology, SLA-1*04:01 and SLA-1*13:01, had significant differences in length preference of the binding peptides. Compared with SLA-1*04:01, SLA-1*13:01 binds fewer short peptides with 8-10 amino acids, but more long peptides. A dodecapeptide peptide (RW12) can bind to both SLA-1*04:01 and SLA-1*13:01, but their crystal structures indicate that the binding modes are significantly different: the entirety of RW12 is embedded in the peptide binding groove of SLA-1*04:01, but it obviously protrudes from the peptide binding groove of SLA-1*13:01. The structural comparative analysis showed that only five differential amino acids of SLA-1*13:01 and SLA-1*04:01 were involved in the binding of RW12, and they determine the different ways of long peptides binding, which makes SLA-1*04:01 more restrictive on long peptides than SLA-1*13:01, and thus binds fewer long peptides. In addition, we found that the N terminus of RW12 extends from the groove of SLA-1*13:01, which is similar to the case previously found in SLA-1*04:01. However, this unusual peptide binding does not affect their preferences of binding peptide length. Our study will be helpful to understand the effect of polymorphisms on the length distribution of MHC-I binding peptides, and to screen SLA-I-restricted epitopes of different lengths and to design effective epitope vaccines.

Structure and Peptidomes of Swine MHC Class I with Long Peptides Reveal the Cross-Species Characteristics of the Novel N-Terminal Extension Presentation Mode.

Antigenic peptide presentation by the MHC is essential for activating T cells. The current view is that the peptide termini are tethered within the closed Ag-binding groove of MHC class I (MHC-I). Recently, the N-terminal extension mode of peptide presentation has been observed in human MHC-I (HLA-I). In this study, we found that the N terminus of the long peptide can extend beyond the groove of swine MHC-I (SLA-1*0401), confirming that this phenomenon can occur across species. Removal of the N-terminal extra (P-1) residue of the RW12 peptide significantly reduced the folding efficiency of the complex, but truncation of the second half of the peptide did not. Consistent with previous reports, the second (P1) residue of the peptide is twisted, and its side chain is inserted into the A pocket to form two hydrogen bonds with polymorphic E63 and conserved Y159. Mutations of E63 disrupt the binding of the peptide, indicating that E63 is necessary for this peptide-binding mode. Compared with W167, which exists in most MHC-Is, SLA-I-specific S167 ensures an open N-terminal groove of SLA-1*0401, enabling the P-1 residue to extend from the groove. In this MHC class II-like peptide-binding mode, the A pocket is restrictive to the P1 residue and is affected by the polymorphic residues. The peptidomes and refolding data indicated that the open N-terminal groove of SLA-1*0401 allows one to three residues to extend out of the Ag-binding groove. These cross-species comparisons can help us better understand the characteristics of this N-terminal extension presentation mode.

Structural Comparison Between MHC Classes I and II; in Evolution, a Class-II-Like Molecule Probably Came First.

Structures of peptide-loaded major histocompatibility complex class I (pMHC-I) and class II (pMHC-II) complexes are similar. However, whereas pMHC-II complexes include similar-sized IIα and IIß chains, pMHC-I complexes include a heavy chain (HC) and a single domain molecule ß2-microglobulin (ß2-m). Recently, we elucidated several pMHC-I and pMHC-II structures of primitive vertebrate species. In the present study, a comprehensive comparison of pMHC-I and pMHC-II structures helps to understand pMHC structural evolution and supports the earlier proposed-though debated-direction of MHC evolution from class II-type to class I. Extant pMHC-II structures share major functional characteristics with a deduced MHC-II-type homodimer ancestor. Evolutionary establishment of pMHC-I presumably involved important new functions such as (i) increased peptide selectivity by binding the peptides in a closed groove (ii), structural amplification of peptide ligand sequence differences by binding in a non-relaxed fashion, and (iii) increased peptide selectivity by syngeneic heterotrimer complex formation between peptide, HC, and ß2-m. These new functions were associated with structures that since their establishment in early pMHC-I have been very well conserved, including a shifted and reorganized P1 pocket (aka A pocket), and insertion of a ß2-m hydrophobic knob into the peptide binding domain ß-sheet floor. A comparison between divergent species indicates better sequence conservation of peptide binding domains among MHC-I than among MHC-II, agreeing with more demanding interactions within pMHC-I complexes. In lungfishes, genes encoding fusions of all MHC-IIα and MHC-IIß extracellular domains were identified, and although these lungfish genes presumably derived from classical MHC-II, they provide an alternative mechanistic hypothesis for how evolution from class II-type to class I may have occurred.

The Structure of a Peptide-Loaded Shark MHC Class I Molecule Reveals Features of the Binding between ß2-Microglobulin and H Chain Conserved in Evolution.

Cartilaginous fish are the most primitive extant species with MHC molecules. Using the nurse shark, the current study is, to the best of our knowledge, the first to present a peptide-loaded MHC class I (pMHC-I) structure for this class of animals. The overall structure was found to be similar between cartilaginous fish and bony animals, showing remarkable conservation of interactions between the three pMHC-I components H chain, ß2-microglobulin (ß2-m), and peptide ligand. In most previous studies, relatively little attention was given to the details of binding between the H chain and ß2-m, and our study provides important new insights. A pronounced conserved feature involves the insertion of a large ß2-m F56+W60 hydrophobic knob into a pleat of the ß-sheet floor of the H chain α1α2 domain, with the knob being surrounded by conserved residues. Another conserved feature is a hydrogen bond between ß2-m Y10 and a proline in the α3 domain of the H chain. By alanine substitution analysis, we found that the conserved ß2-m residues Y10, D53, F56, and W60-each binding the H chain-are required for stable pMHC-I complex formation. For the ß2-m residues Y10 and F56, such observations have not been reported before. The combined data indicate that for stable pMHC-I complex formation ß2-m should not only bind the α1α2 domain but also the α3 domain. Knowing the conserved structural features of pMHC-I should be helpful for future elucidations of the mechanisms of pMHC-I complex formation and peptide editing.

Peptidomes and Structures Illustrate Two Distinguishing Mechanisms of Alternating the Peptide Plasticity Caused by Swine MHC Class I Micropolymorphism.

The micropolymorphism of major histocompatibility complex class I (MHC-I) can greatly alter the plasticity of peptide presentation, but elucidating the underlying mechanism remains a challenge. Here we investigated the impact of the micropolymorphism on peptide presentation of swine MHC-I (termed swine leukocyte antigen class I, SLA-I) molecules via immunopeptidomes that were determined by our newly developed random peptide library combined with the mass spectrometry (MS) de novo sequencing method (termed RPLD-MS) and the corresponding crystal structures. The immunopeptidomes of SLA-1*04:01, SLA-1*13:01, and their mutants showed that mutations of residues 156 and 99 could expand and narrow the ranges of peptides presented by SLA-I molecules, respectively. R156A mutation of SLA-1*04:01 altered the charge properties and enlarged the volume size of pocket D, which eliminated the harsh restriction to accommodate the third (P3) anchor residue of the peptide and expanded the peptide binding scope. Compared with 99Tyr of SLA-1*0401, 99Phe of SLA-1*13:01 could not form a conservative hydrogen bond with the backbone of the P3 residues, leading to fewer changes in the pocket properties but a significant decrease in quantitative of immunopeptidomes. This absent force could be compensated by the salt bridge formed by P1-E and 170Arg. These data illustrate two distinguishing manners that show how micropolymorphism alters the peptide-binding plasticity of SLA-I alleles, verifying the sensitivity and accuracy of the RPLD-MS method for determining the peptide binding characteristics of MHC-I in vitro and helping to more accurately predict and identify MHC-I restricted epitopes.

The Crystal Structure of the MHC Class I (MHC-I) Molecule in the Green Anole Lizard Demonstrates the Unique MHC-I System in Reptiles.

The reptile MHC class I (MCH-I) and MHC class II proteins are the key molecules in the immune system; however, their structure has not been investigated. The crystal structure of green anole lizard peptide-MHC-I-ß2m (pMHC-I or pAnca-UA*0101) was determined in the current study. Subsequently, the features of pAnca-UA*0101 were analyzed and compared with the characteristics of pMHC-I of four classes of vertebrates. The amino acid sequence identities between Anca-UA*0101 and MHC-I from other species are <50%; however, the differences between the species were reflected in the topological structure. Significant characteristics of pAnca-UA*0101 include a specific flip of ∼88° and an upward shift adjacent to the C terminus of the α1- and α2-helical regions, respectively. Additionally, the lizard MHC-I molecule has an insertion of 2 aa (VE) at positions 55 and 56. The pushing force from 55-56VE triggers the flip of the α1 helix. Mutagenesis experiments confirmed that the 55-56VE insertion in the α1 helix enhances the stability of pAnca-UA*0101. The peptide presentation profile and motif of pAnca-UA*0101 were confirmed. Based on these results, the proteins of three reptile lizard viruses were used for the screening and confirmation of the candidate epitopes. These data enhance our understanding of the systematic differences between five classes of vertebrates at the gene and protein levels, the formation of the pMHC-I complex, and the evolution of the MHC-I system.

Does Asthma Increase the Mortality of Patients with COVID-19?: A Systematic Review and Meta-Analysis.

The purpose of this systematic review and meta-analysis was to explore the literature and collate data comparing the mortality of coronavirus disease 2019 (COVID-19) patients with and without asthma. The databases PubMed, Scopus, Embase, Google Scholar, and medRxiv.org were searched for studies comparing the clinical outcomes of asthmatic patients with those of nonasthmatic patients diagnosed with COVID-19. Mortality data were summarized using the Mantel-Haenszel OR with 95% CI in a random-effects model. Five retrospective studies met the inclusion criteria. A meta-analysis of data from 744 asthmatic patients and 8,151 nonasthmatic patients indicated that the presence of asthma had no significant effect on mortality (OR = 0.96; 95% CI 0.70-1.30; I2 = 0%; p = 0.79). Results were stable in a sensitivity analysis. A descriptive analysis of other clinical outcomes indicated no difference in the duration of hospitalization and the risk of intensive care unit (ICU) transfer between asthmatic and nonasthmatic patients. To conclude, preliminary data indicates that asthma as a comorbidity may not increase the mortality of COVID-19. Data on the influence of asthma on the risk of hospitalization, the duration of hospitalization, the requirement of ICU admission, and disease severity is still too limited to draw any strong conclusions. Further studies with a larger sample size are required to establish strong evidence.

LINC00987 Ameliorates COPD by Regulating LPS-Induced Cell Apoptosis, Oxidative Stress, Inflammation and Autophagy Through Let-7b-5p/SIRT1 Axis.

Background: Chronic obstructive pulmonary disease (COPD) is the third cause of disease-related death and brings a heavy burden to human health. Long non-coding RNA (lncRNA) was revealed to participate in COPD pathogenesis. This study aims to establish the effects and regulatory mechanism of lncRNA long intergenic non-coding 00987 (LINC00987) in lipopolysaccharide (LPS)-induced apoptosis, oxidative stress, inflammation and autophagy in BEAS-2B cells. Methods: The expression levels of LINC00987 and let-7b-5p were detected by real-time quantitativepolymerase chain reaction (RT-qPCR). The expression of apoptosis-associated proteins, oxidative stress (ROS)-related proteins, autophagy-related proteins and sirtuin1 (SIRT1) protein was determined by Western blot. Cell viability was illustrated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was investigated by caspase3 activity and apoptosis analysis assays. ROS, inflammation and autophagy were demonstrated by detecting reactive ROS level and superoxide dismutase (SOD) activity, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. The binding sites between let-7b-5p and LINC00987 or SIRT1 were predicted by lncBase or miRWalk online database, and identified by dual-luciferase reporter assay. Results: LINC00987 expression was strikingly downregulated and let-7b-5p expression was obviously upregulated in COPD tissues and LPS-induced BEAS-2B cells compared with control groups. LINC00987 overexpression promoted BEAS-2B cells against LPS-mediated viability, apoptosis, oxidative stress, inflammation and autophagy, whereas these effects were attenuated by let-7b-5p mimic or SIRT1 knockdown. Furthermore, LINC00987 sponged let-7b-5p and let-7b-5p bound to SIRT1. Conclusion: LINC00987 ameliorated COPD through modulating LPS-induced cell apoptosis, oxidative stress, inflammation and autophagy via sponging let-7b-5p to associate with SIRT1. This finding will provide a theoretical basis for the research of LncRNA-mediated treatment in COPD.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of duck MHC class I complexed with a TUMV viral peptide.

The CTL immune response mediated by MHC I plays an important role in duck anti-TMUV infection. This study reports the expression, purification and crystallization of a complex of duck MHC class I molecules Anpl-UAA*SD, duck ß2-microglobulin (Anpl-ß2m) and the polypeptide LRKRQLTVL (LRK9) derived from Tembusu virus (TMUV) NS3. The crystal diffraction resolution is 1.50 Å and belongs to the P62 space group, and the unit cell parameters are a = 82.468, b = 82.468, c = 112.507. The Matthew's constant is calculated to be 2.32 Å3 Da -1, and an asymmetric unit contains a complex molecule with a solvent content of 47%. The research lays the foundation for the structure of immune molecules about duck anti-TMUV research.

The Mechanism of ß2m Molecule-Induced Changes in the Peptide Presentation Profile in a Bony Fish.

Contemporary antigen presentation knowledge is based on the existence of a single ß2m locus, and a classical MHC class I forms a complex with a peptide (i.e., pMHC-I) to trigger CTL immunity. However, two ß2m loci have been found in diploid bony fish; the function of the two ß2m molecules is unclear. Here, we determined the variant peptide profiles originating from different products of the ß2m loci binding to the same MHC-I molecule and further solved the crystal structures of the two pMHC-I molecules (i.e., pCtid-UAA-ß2m-2 and pCtid-UAA-ß2m-1-II). Of note, in pCtid-UAA-ß2m-2, a unique hydrogen bond network formed in the bottom of the peptide-binding groove (PBG) led to α2-helix drift, ultimately leading to structural changes in the PBG. The mechanism of the change in peptide presentation profiles by ß2m molecules is illustrated. The results are also of great significance for antivirus and antitumor functions in cold-blooded vertebrates and even humans.

Structures of the MHC-I molecule BF2*1501 disclose the preferred presentation of an H5N1 virus-derived epitope.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell-mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I-ß2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV-PA123-130 We determined the structure of the BF2*1501-PA123-130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123-130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123-130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV-PA123-130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123-130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

A Glimpse of the Peptide Profile Presentation by Xenopus laevis MHC Class I: Crystal Structure of pXela-UAA Reveals a Distinct Peptide-Binding Groove.

The African clawed frog, Xenopus laevis, is a model species for amphibians. Before metamorphosis, tadpoles do not efficiently express the single classical MHC class I (MHC-I) molecule Xela-UAA, but after metamorphosis, adults express this molecule in abundance. To elucidate the Ag-presenting mechanism of Xela-UAA, in this study, the Xela-UAA structure complex (pXela-UAAg) bound with a peptide from a synthetic random peptide library was determined. The amino acid homology between the Xela-UAA and MHC-I sequences of different species is <45%, and these differences are fully reflected in the three-dimensional structure of pXela-UAAg. Because of polymorphisms and interspecific differences in amino acid sequences, pXela-UAAg forms a distinct peptide-binding groove and presents a unique peptide profile. The most important feature of pXela-UAAg is the two-amino acid insertion in the α2-helical region, which forms a protrusion of ∼3.8 Å that is involved in TCR docking. Comparison of peptide-MHC-I complex (pMHC-I) structures showed that only four amino acids in ß2-microglobulin that were bound to MHC-I are conserved in almost all jawed vertebrates, and the most unique feature in nonmammalian pMHC-I molecules is that the AB loop bound ß2-microglobulin. Additionally, the binding distance between pMHC-I and CD8 molecules in nonmammals is different from that in mammals. These unique features of pXela-UAAg provide enhanced knowledge of T cell immunity and bridge the knowledge gap regarding the coevolutionary progression of the MHC-I complex from aquatic to terrestrial species.

Structure and Peptidome of the Bat MHC Class I Molecule Reveal a Novel Mechanism Leading to High-Affinity Peptide Binding.

Bats are natural reservoir hosts, harboring more than 100 viruses, some of which are lethal to humans. The asymptomatic coexistence with viruses is thought to be connected to the unique immune system of bats. MHC class I (MHC I) presentation is closely related to cytotoxic lymphocyte immunity, which plays an important role in viral resistance. To investigate the characteristics of MHC I presentation in bats, the crystal structures of peptide-MHC I complexes of Pteropus alecto, Ptal-N*01:01/HEV-1 (DFANTFLP) and Ptal-N*01:01/HEV-2 (DYINTNLVP), and two related mutants, Ptal-N*01:01/HEV-1PΩL (DFANTFLL) and Ptal-N*01:01ΔMDL/HEV-1, were determined. Through structural analysis, we found that Ptal-N*01:01 had a multi-Ala-assembled pocket B and a flexible hydrophobic pocket F, which could accommodate variable anchor residues and allow Ptal-N*01:01 to bind numerous peptides. Three sequential amino acids, Met, Asp, and Leu, absent from the α1 domain of the H chain in other mammals, were present in this domain in the bat. Upon deleting these amino acids and determining the structure in p/Ptal-N*01:01ΔMDL/HEV-1, we found they helped form an extra salt-bridge chain between the H chain and the N-terminal aspartic acid of the peptide. By introducing an MHC I random peptide library for de novo liquid chromatography-tandem mass spectrometry analysis, we found that this insertion module, present in all types of bats, can promote MHC I presentation of peptides with high affinity during the peptide exchange process. This study will help us better understand how bat MHC I presents high-affinity peptides from an extensive binding peptidome and provides a foundation to understand the cellular immunity of bats.

Distribution of ancient α1 and α2 domain lineages between two classical MHC class I genes and their alleles in grass carp.

Major histocompatibility complex (MHC) class I molecules play a crucial role in the immune response by binding and presenting pathogen-derived peptides to specific CD8+ T cells. From cDNA of 20 individuals of wild grass carp (Ctenopharyngodon idellus), we could amplify one or two alleles each of classical MHC class I genes Ctid-UAA and Ctid-UBA. In total, 27 and 22 unique alleles of Ctid-UAA and Ctid-UBA were found. The leader, α1, transmembrane and cytoplasmic regions distinguish between Ctid-UAA and Ctid-UBA, and their encoded α1 domain sequences belong to the ancient lineages α1-V and α1-II, respectively, which separated several hundred million years ago. However, Ctid-UAA and Ctid-UBA share allelic lineage variation in their α2 and α3 sequences, in a pattern suggestive of past interlocus recombination events that transferred α2+α3 fragments. The allelic Ctid-UAA and Ctid-UBA variation involves ancient variation between domain lineages α2-I and α2-II, which in the present study was dated back to before the ancestral separation of teleost fish and spotted gar (> 300 million years ago). This is the first report with compelling evidence that recombination events combining different ancient α1 and α2 domain lineages had a major impact on the allelic variation of two different classical MHC class I genes within the same species.