Major Histocompatibility Complex Class I Alleles Research Articles

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.

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.

The electrostatic landscape of MHC-peptide binding revealed using inception networks

Predictive modeling of macromolecular recognition and protein-protein complementarity represents one of the cornerstones of biophysical sciences. However, such models are often hindered by the combinatorial complexity of interactions at the molecular interfaces. Exemplary of this problem is peptide presentation by the highly polymorphic major histocompatibility complex class I (MHC-I) molecule, a principal component of immune recognition. We developed human leukocyte antigen (HLA)-Inception, a deep biophysical convolutional neural network, which integrates molecular electrostatics to capture non-bonded interactions for predicting peptide binding motifs across 5,821 MHC-I alleles. These predictions of generated motifs correlate strongly with experimental peptide binding and presentation data. Beyond molecular interactions, the study demonstrates the application of predicted motifs in analyzing MHC-I allele associations with HIV disease progression and patient response to immune checkpoint inhibitors. A record of this paper's transparent peer review process is included in the supplemental information.

MHC-I alleles mediate clearance and antibody response to the zoonotic Lassa virus in Mastomys rodent reservoirs.

West African Mastomys rodents are the primary reservoir of the zoonotic Lassa virus (LASV). The virus causes haemorrhagic Lassa fever and considerable mortality in humans. To date, the role of Mastomys immunogenetics in resistance to, and persistence of, LASV infections is largely unknown. Here, we investigated the role of Major Histocompatibility Complex class I (MHC-I) on LASV infection status (i.e., active vs. cleared infection, determined via PCR and an immunofluorescence assay on IgG antibodies, respectively) in Mastomys natalensis and M. erythroleucus sampled within southwestern Nigeria. We identified more than 190 and 90 MHC-I alleles by Illumina high throughput-sequencing in M. natalensis and M. erythroleucus, respectively, with different MHC allele compositions and frequencies between LASV endemic and non-endemic sites. In M. natalensis, the MHC allele ManaMHC-I*006 was negatively associated with active infections (PCR-positive) and positively associated with cleared infections (IgG-positive) simultaneously, suggesting efficient immune responses that facilitate LASV clearance in animals carrying this allele. Contrarily, alleles ManaMHC-I*008 and ManaMHC-I*021 in M. natalensis, and MaerMHC-I*008 in M. erythroleucus, were positively associated with active infection, implying susceptibility. Alleles associated with susceptibility shared a glutamic acid at the positively selected codon 57, while ManaMHC-I*006 featured an arginine. There was no link between number of MHC alleles per Mastomys individual and LASV prevalence. Thus, specific alleles, but not MHC diversity per se, seem to mediate antibody responses to viremia. We conclude that co-evolution with LASV likely shaped the MHC-I diversity of the main LASV reservoirs in southwestern Nigeria, and that information on reservoir immunogenetics may hold insights into transmission dynamics and zoonotic spillover risks.

Only rare classical MHC-I alleles are highly expressed in the European house sparrow.

The exceptional polymorphism observed within genes of the major histocompatibility complex (MHC), a core component of the vertebrate immune system, has long fascinated biologists. The highly polymorphic classical MHC class-I (MHC-I) genes are maintained by pathogen-mediated balancing selection (PMBS), as shown by many sites subject to positive selection, while the more monomorphic non-classical MHC-I genes show signatures of purifying selection. In line with PMBS, at any point in time, rare classical MHC alleles are more likely than common classical MHC alleles to confer a selective advantage in host-pathogen interactions. Combining genomic and expression data from the blood of wild house sparrows Passer domesticus, we found that only rare classical MHC-I alleles were highly expressed, while common classical MHC-I alleles were lowly expressed or not expressed. Moreover, highly expressed rare classical MHC-I alleles had more positively selected sites, indicating exposure to stronger PMBS, compared with lowly expressed classical alleles. As predicted, the level of expression was unrelated to allele frequency in the monomorphic non-classical MHC-I alleles. Going beyond previous studies, we offer a fine-scale view of selection on classical MHC-I genes in a wild population by revealing differences in the strength of PMBS according to allele frequency and expression level.

Improved haplotype resolution of highly duplicated MHC genes in a long-read genome assembly using MiSeq amplicons.

Long-read sequencing offers a great improvement in the assembly of complex genomic regions, such as the major histocompatibility complex (MHC) region, which can contain both tandemly duplicated MHC genes (paralogs) and high repeat content. The MHC genes have expanded in passerine birds, resulting in numerous MHC paralogs, with relatively high sequence similarity, making the assembly of the MHC region challenging even with long-read sequencing. In addition, MHC genes show rather high sequence divergence between alleles, making diploid-aware assemblers incorrectly classify haplotypes from the same locus as sequences originating from different genomic regions. Consequently, the number of MHC paralogs can easily be over- or underestimated in long-read assemblies. We therefore set out to verify the MHC diversity in an original and a haplotype-purged long-read assembly of one great reed warbler Acrocephalus arundinaceus individual (the focal individual) by using Illumina MiSeq amplicon sequencing. Single exons, representing MHC class I (MHC-I) and class IIB (MHC-IIB) alleles, were sequenced in the focal individual and mapped to the annotated MHC alleles in the original long-read genome assembly. Eighty-four percent of the annotated MHC-I alleles in the original long-read genome assembly were detected using 55% of the amplicon alleles and likewise, 78% of the annotated MHC-IIB alleles were detected using 61% of the amplicon alleles, indicating an incomplete annotation of MHC genes. In the haploid genome assembly, each MHC-IIB gene should be represented by one allele. The parental origin of the MHC-IIB amplicon alleles in the focal individual was determined by sequencing MHC-IIB in its parents. Two of five larger scaffolds, containing 6-19 MHC-IIB paralogs, had a maternal and paternal origin, respectively, as well as a high nucleotide similarity, which suggests that these scaffolds had been incorrectly assigned as belonging to different loci in the genome rather than as alternate haplotypes of the same locus. Therefore, the number of MHC-IIB paralogs was overestimated in the haploid genome assembly. Based on our findings we propose amplicon sequencing as a suitable complement to long-read sequencing for independent validation of the number of paralogs in general and for haplotype inference in multigene families in particular.

Abstract 5376: Protein structure-based modeling to improve MHC class I epitope predictions

Abstract The ability to accurately identify peptide ligands for a given major histocompatibility complex class I (MHC-I) molecule has immense value for targeted anticancer therapeutics. However, the highly polymorphic nature of the MHC-I protein makes universal prediction of peptide ligands challenging due to lack of experimental data describing most MHC-I variants. To address this challenge, we have developed a deep convolutional neural network, HLA-Inception, capable of predicting MHC-I peptide binding motifs using electrostatic properties of the MHC-I binding pocket. By approaching this immunological issue using molecular biophysics, we measure the impact of sidechain arrangement and topology on peptide binding, feature not captured by sequence-based MHC-I prediction methods. Through a combination of molecular modeling and simulation, 5821 MHC-I alleles were modeled, providing extensive coverage across human populations. Predicted peptide binding motifs fell into distinct clusters, each defined with different degrees of submotif heterogeneity. Peptide binding scores generated by HLA-Inception are strongly correlated with quantitative MHC-I binding data, indicating predicted peptides can be ranked, both within and between alleles. HLA-inception also showed high precision when predicting naturally presented peptides and can be used for rapid proteome-scale MHC-I peptide binding predictions. Finally, we show that the binding pocket diversity measured by HLA inception predicts response to checkpoint blockade. Citation Format: Eric A. Wilson, John Kevin Cava, Diego Chowell, Abhishek Singharoy, Karen S. Anderson. Protein structure-based modeling to improve MHC class I epitope predictions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5376.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein β2-microglobulin (β2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their invivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of β2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.

Abstract 1379: Discovery of tumor-associated, immunogenic peptides presented in a patient-derived, mutant calreticulin-driven myeloproliferative neoplasm cell line

Abstract Background: Myeloproliferative neoplasms (MPNs) are a type of chronic blood cancers. A subset are driven by frameshift mutations in the calreticulin gene (CALR). The CALR protein is a lectin chaperone and primarily assists in glycoprotein folding in the endoplasmic reticulum. It is also part of the peptide loading complex, a set of proteins involved in antigen processing and peptide presentation by major histocompatibility complex class I (MHC-I) molecules. All MPN-associated frameshift mutations in CALR lead to a similar change in its C-terminal, altering its protein-protein interactions. Examples include impaired chaperone activity of mutant CALR that impacts protein homeostasis and compromised activity of the MHC-I peptide loading complex. We hypothesized that these impaired functions will alter the repertoire of peptides presented by MHC-I in mutant CALR expressing cells, which we characterized in a patient-derived, mutant-CALR-driven myeloid tumor cell line, Marimo. Methods: We carried out an immunopeptidome analysis on Marimo cell line derivates expressing either wildtype or mutant CALR to identify potential MPN-associated peptides presented by MHC-I, and validated their immunogenicity in human samples. MHC-I-peptide complexes were immunoprecipitated from these Marimo cell line derivates, followed by peptide elution and analysis by mass spectrometry. A subset of peptides that were found in the mass spectrometry analysis of mutant CALR cells but not wildtype CALR cells were tested for their immunogenicity by assaying the in vitro T cell responses they evoked. Naïve T cells from healthy donors, carrying at least one MHC-I allele that matched the Marimo cells, were primed and expanded with the selected peptides and peptide-specific T cell responses were measured by intracellular staining detecting effector cytokines IFN-γ and TNF-α. Results: 1. The expression of mutant CALR disrupts the function of the peptide loading complex, leading to reduced recruitment of MHC-I and reducing its cell surface expression by almost 40% compared to cells expressing wildtype CALR. 2. Peptide repertoire of MHC-I is significantly altered by the expression of mutant CALR. Immunopeptidomics revealed a qualitative and quantitative difference in peptides presented by cells expressing wildtype CALR vs mutant CALR 3. The immunogenicity of a curated list of 24 peptides was tested in healthy donors, and at least 6 peptides activated T cell responses. Similar testing in MPN patients is planned. Conclusions: This is the first study to investigate the impact of mutant CALR on the repertoire of peptides presented by MHC-I in myeloid cells. We discovered tumor-associated, immunogenic peptides that may serve as potential targets of immunotherapy allowing the specific elimination of mutant CALR-expressing cells, which has been a challenge in the treatment of MPNs. Citation Format: Najla Arshad, Nathalie Vigneron, Cansu Cimen Bozkus, Vincent Stroobant, Stefan Naulaerts, Nina Bhardwaj, Benoît Van den Eynde, Peter Cresswell. Discovery of tumor-associated, immunogenic peptides presented in a patient-derived, mutant calreticulin-driven myeloproliferative neoplasm cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1379.

HLA-Inception: A structure-based deep learning framework for MHC-I binding motif prediction

Abstract The ability to accurately identify peptide ligands for a given major histocompatibility complex class I (MHC-I) molecule has immense value for targeted anticancer and antiviral therapeutics. However, the highly polymorphic nature of the MHC-I protein makes universal prediction of peptide ligands challenging due to lack of experimental data describing most MHC-I variants, and the vast number of protein variants precludes comprehensive experimental determination. Therefore, there is a need for a framework to cluster MHC-I alleles to prioritize for experimental validation as well as identify alleles with potential disease associations. To address this challenge, we have developed a deep convolutional neural network, HLA-Inception, capable of predicting MHC-I peptide binding motif using data derived from the structure of the MHC-I binding pocket. By approaching this problem from a 3-dimensional perspective, we can fully consider the impact of sidechain arrangement and topology of the MHC-I binding pocket on peptide binding motif, which is not inherently captured by the popular protein sequence-based approaches. Through a combination of homology modeling and biophysical simulations, we created protein structure models for all full-length HLA-ABC alleles. The topology and interaction forces within the MHC-I binding pocket were accounted for by solving the 3-dimensional electrostatic potential near the surface of the protein. HLA-Inception was then trained on all MHC-I alleles with known MHC-I binding motifs and applied to the full set of MHC-I models. We found that predicted peptide binding motifs fell into distinct and well-defined clusters which maintained known peptide binding and disease associations.

Immunogenetic variation shapes the gut microbiome in a natural vertebrate population

BackgroundThe gut microbiome (GM) can influence many biological processes in the host, impacting its health and survival, but the GM can also be influenced by the host’s traits. In vertebrates, Major Histocompatibility Complex (MHC) genes play a pivotal role in combatting pathogens and are thought to shape the host’s GM. Despite this—and the documented importance of both GM and MHC variation to individual fitness—few studies have investigated the association between the GM and MHC in the wild.ResultsWe characterised MHC class I (MHC-I), MHC class II (MHC-II) and GM variation in individuals within a natural population of the Seychelles warbler (Acrocephalus sechellensis). We determined how the diversity and composition of the GM varied with MHC characteristics, in addition to environmental factors and other host traits. Our results show that the presence of specific MHC alleles, but not MHC diversity, influences both the diversity and composition of the GM in this population. MHC-I alleles, rather than MHC-II alleles, had the greatest impact on the GM. GM diversity was negatively associated with the presence of three MHC-I alleles (Ase-ua3, Ase-ua4, Ase-ua5), and one MHC-II allele (Ase-dab4), while changes in GM composition were associated with the presence of four different MHC-I alleles (Ase-ua1, Ase-ua7, Ase-ua10, Ase-ua11). There were no associations between GM diversity and TLR3 genotype, but GM diversity was positively correlated with genome-wide heterozygosity and varied with host age and field period.ConclusionsThese results suggest that components of the host’s immune system play a role in shaping the GM of wild animals. Host genotype—specifically MHC-I and to a lesser degree MHC-II variation—can modulate the GM, although whether this occurs directly, or indirectly through effects on host health, is unclear. Importantly, if immune genes can regulate host health through modulation of the microbiome, then it is plausible that the microbiome could also influence selection on immune genes. As such, host–microbiome coevolution may play a role in maintaining functional immunogenetic variation within natural vertebrate populations.6pCJo_ud_f-NPN_tGFHK9CVideo abstract

NK-Cell Alloreactivity Is Associated with Acute Gvhd and Decreased Relapse Incidence after T-Replete Haplo-Identical Allotransplant with High-Dose Post-Transplant Cyclophosphamide

Background: NK cells express activating (act) and inhibitory (inh) receptors, which recognize MHC class-I alleles, termed “Killer cell Immunoglobulin-like Receptors” (KIRs). Previous clinical data from haplo-identical T-cell-depleted allotransplant have demonstrated that donor vs recipient NK cell alloreactivity (or KIR/HLA incompatibility, KIR/HLA inc) plays a major role as effectors against acute myeloid leukemia (AML). Recently, T-replete haplo-transplant using high-dose post-transplant cyclophosphamide (PTCY) has been a revolution in the field as it is associated with good rate of engraftment and very low incidence of severe acute and chronic GVHD.Methods: The main objective of this prospective study was to study the impact of NK alloreactivity after T-replete haplo-transplant using PTCY. The Baltimore conditioning regimen (Luznik, 2008) was used for lymphoid malignancies while fludarabine as part of this regimen was replaced by clofarabine (Clo-Baltimore) for patients with myeloid malignancies.KIR/HLA inc were studied between donor and recipient considering various act and inh KIR receptors. We then studied the correlation between the presence of KIR/HLA inc and inh KIR/HLA inc and the occurrence of GVHD and relapse post-transplant. Finally, we compared reconstitution of various NK cell subsets (KIR2DL3, KIR2DL1, NKp46+2B4-, NKp46-2B4+, NKG2A+) between cases with or without acute GVHD, KIR/HLA inc, and between Baltimore vs Clo-Baltimore patients. For this purpose, peripheral blood samples were collected at days+10, +20, +30, +60 and +90/100 post-transplant. All patients gave informed consent.Results: Between August 2014 and March 2017, 34 patients (males n=23, median age: 62.5 years) were included in the study. Twelve cases received a Baltimore regimen vs 22 a Clo-Baltimore regimen. All patients except one received peripheral blood stem cell as source of graft. 56% and 14.7% of patients developed grade 2-4 and 3-4 acute GVHD, respectively, at a median of 35 days post-transplant. In July 2017, 9 patients had relapsed and 10 are dead. No differences were observed in terms of acute GVHD incidence, relapse or deaths between Baltimore and Clo-Baltimore sub-groups.KIR/HLA inc was documented in 17 cases of whom 13 presented with acute grade 2-4 while it was only 6/17 cases among patients with no KIR/HLA inc (p=0.001). The same was observed when considering only inhKIR/HLA inc (n=11/13 vs n=8/21, p=0.006). Conversely, relapse was less frequent in patients with KIR/HLA inc (n=2/17 vs n=7/17, p=0.05). However, incidence of relapse was similar between patients developing GvHD (n 5/19) vs no (n=4/15), suggesting that decreased incidence of relapse was not due to GVHD but was related to KIR/HLA inc.The proportions of NK cells at each kinetic point considered after transplant were similar regardless of GVHD, KIR/HLA inc, or conditioning. Phenotypic analyses of NK cell subsets showed that patients developing GVHD had higher mature/activated NKp46+2B4- and NKG2A+NK cells at day +20 post-transplant. The proportion of KIR2DL3+ NK cells was also significantly higher in absence of KIR/HLA inc at day+30, in contrast to KIR2DL1+ NK cells that were similar between each sub-groups at any time considered.Conclusion: KIR/HLA inc are associated with acute GVHD and decreased relapse incidence after T-repleted haplo-identical allotransplantation using PTCY. NKp46+2B4- and NKG2A+NK cells were significantly higher in cases developing GVHD at day+20, suggesting a role of these activated NK cells in the occurrence of GVHD. Also, KIR/HLA inc impact the phenotypic NK cell reconstitution and may favor NK cell expansion with better leukemic effect. These results may be of crucial importance regarding the selection of haplo-donor. DisclosuresNo relevant conflicts of interest to declare.

Analyses of the interactions of tapasin and ERp57-tapasin proteins with PaSTa 1 and PaSTa 2 antibodies and MHC-I molecules

Abstract The intracellular loading of major histocompatibility complex class I (MHC-I) molecules with high affinity peptides is a pre-requisite for their cell surface display as ligands for T and NK cells. Peptide acquisition occurs in the ER within the peptide loading complex (PLC) and is accomplished through the concerted action of several proteins, most notably tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. In order to gain a mechanistic understanding of tapasin function as well as its apparent allelomorph specificity we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSTa 1 and PaSTa 2, and investigated their binding to human recombinant soluble tapasin and ERp57/tapasin by SPR methods. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. Based on these results we developed an MHC-I binding assay employing PaSTa 1-captured tapasin or ERp57/tapasin which reveals affinities in the micromolar range for selected MHC-I alleles. These affinities are notably weaker than those obtained for the structurally related but PLC-independent chaperone, TAPBPR (TAP-binding protein, related), and may reflect distinct evolutionarily derived functions for the two related molecules.

Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity.

Simple SummaryMany cancers down-regulate the expression of major histocompatibility complex class I (MHC-I) to avoid recognition and elimination by the adaptive immune system. Natural Killer (NK) cells, as part of innate immunity, complement tumor recognition by their ability to sense foreign and malignant cells by the lack of major MHC-I expression. During development, NK cells learn to perceive physiological levels of self MHC-I as healthy in a process termed NK cell education. In the current study, we assessed whether the stimulation of natural killer T (NKT) cells could impact the tumor-killing ability of NK cells. We can show that stimulation with a well-established activator of NKT cells induces the preferential expansion of educated NK cells, and improves NK-cell-mediated tumor-killing of both MHC-I+ and MHC-I− tumor cells. Furthermore, this increased tumor-killing capacity of NK cells can be achieved by nanovesicle-mediated delivery, which is known to improve adaptive immunity as well. Our approach thus holds great potential for future cancer immunotherapy.Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

Immunoinformatic approach to assess SARS-CoV-2 protein S epitopes recognised by the most frequent MHC-I alleles in the Brazilian population

AimsBrazil is nowadays one of the epicentres of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and new therapies are needed to face it. In the context of specific...

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in preformed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08, and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12-mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic, and computational analyses suggested that this 12-mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from preformed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1/MHCI/peptide complex. Similarly, no interactions between ERAP1 and purified peptide-loading complex were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution along with the dynamic nature of peptide binding to MHCI are sufficient to explain ERAP1 processing of antigenic peptide precursors.

Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques

Cumulative studies on human immunodeficiency virus (HIV)-infected individuals have shown association of major histocompatibility complex class I (MHC-I) polymorphisms with lower viral load and delayed AIDS progression, suggesting that HIV replication can be controlled by potent CD8+ T-cell responses. We have previously established an AIDS model of simian immunodeficiency virus (SIV) infection in Burmese rhesus macaques and found a potent CD8+ T cell targeting the Mamu-A1*065:01-restricted Gag241-249 epitope, which is located in a region corresponding to the HIV Gag240-249 TW10 epitope restricted by a protective MHC-I allele, HLA-B*57. In the present study, we determined a T cell receptor (TCR) of this Gag241-249 epitope-specific CD8+ T cell. cDNA clones encoding TCR-α and TCR-β chains were obtained from a Gag241-249-specific CD8+ T-cell clone. Coexpression of these TCR-α and TCR-β cDNAs resulted in reconstitution of a functional TCR specifically detected by Gag241-249 epitope-Mamu-A1*065:01 tetramer. Two of three previously-reported CD8+ T-cell escape mutations reduced binding affinity of Gag241-249 peptide to Mamu-A1*065:01 but the remaining one not. This is consistent with the data obtained by molecular modeling of the epitope-MHC-I complex and TCR. These results would contribute to understanding how viral CD8+ T-cell escape mutations are selected under structural constraint of viral proteins.

CD8+ T cell-based strong selective pressure on multiple simian immunodeficiency virus targets in macaques possessing a protective MHC class I haplotype

In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host major histocompatibility complex class I (MHC-I) genotypes have a great impact on viral replication and MHC-I-associated viral genome mutations are selected under CD8+ T-cell pressure. Association of MHC-I genotypes with HIV/SIV control has been investigated at MHC-I allele levels but not fully at haplotype levels. We previously established groups of rhesus macaques sharing individual MHC-I haplotypes. In the present study, we compared viral genome diversification after SIV infection in macaques possessing a protective MHC-I haplotype, 90-010-Id, with those possessing a non-protective MHC-I haplotype, 90-010-Ie. These two MHC-I haplotypes are associated with immunodominant CD8+ T-cell responses targeting similar regions of viral Nef antigen. Analyses of viral genome sequences and antigen-specific T-cell responses showed four and two candidates of viral CD8+ T-cell targets associated with 90-010-Id and 90-010-Ie, respectively, in addition to the Nef targets. In these CD8+ T-cell target regions, higher numbers of mutations were detected at the setpoint after SIV infection in macaques possessing 90-010-Id than those possessing 90-010-Ie. These results indicate higher selective pressure on overall CD8+ T-cell targets associated with the protective MHC-I haplotype, suggesting a pattern of HIV/SIV control by multiple target-specific CD8+ T-cell responses.

The Frequency of Vaccine-Induced T-Cell Responses Does Not Predict the Rate of Acquisition after Repeated Intrarectal SIVmac239 Challenges in Mamu-B*08 + Rhesus Macaques

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8+ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+ ) RMs. Here we evaluated if robust vaccine-elicited CD8+ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08+ RMs following mucosal SIV challenges. Ten Mamu-B*08+ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8+ T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8+ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8+ T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.

How ERAP1 and ERAP2 Shape the Peptidomes of Disease-Associated MHC-I Proteins.

Four inflammatory diseases are strongly associated with Major Histocompatibility Complex class I (MHC-I) molecules: birdshot chorioretinopathy (HLA-A*29:02), ankylosing spondylitis (HLA-B*27), Behçet's disease (HLA-B*51), and psoriasis (HLA-C*06:02). The endoplasmic reticulum aminopeptidases (ERAP) 1 and 2 are also risk factors for these diseases. Since both enzymes are involved in the final processing steps of MHC-I ligands it is reasonable to assume that MHC-I-bound peptides play a significant pathogenetic role. This review will mainly focus on recent studies concerning the effects of ERAP1 and ERAP2 polymorphism and expression on shaping the peptidome of disease-associated MHC-I molecules in live cells. These studies will be discussed in the context of the distinct mechanisms and substrate preferences of both enzymes, their different patterns of genetic association with various diseases, the role of polymorphisms determining changes in enzymatic activity or expression levels, and the distinct peptidomes of disease-associated MHC-I allotypes. ERAP1 and ERAP2 polymorphism and expression induce significant changes in multiple MHC-I-bound peptidomes. These changes are MHC allotype-specific and, without excluding a degree of functional inter-dependence between both enzymes, reflect largely separate roles in their processing of MHC-I ligands. The studies reviewed here provide a molecular basis for the distinct patterns of genetic association of ERAP1 and ERAP2 with disease and for the pathogenetic role of peptides. The allotype-dependent alterations induced on distinct peptidomes may explain that the joint association of both enzymes and unrelated MHC-I alleles influence different pathological outcomes.