Nucleobase adducts bind MR1 and stimulate MR1-restricted T cells.

MR1T cells are a recently found class of T cells that recognize antigens presented by the major histocompatibility complex-I-related molecule MR1 in the absence of microbial infection. The nature of the self-antigens that stimulate MR1T cells remains unclear, hampering our understanding of their physiological role and therapeutic potential. By combining genetic, pharmacological, and biochemical approaches, we found that carbonyl stress and changes in nucleobase metabolism in target cells promote MR1T cell activation. Stimulatory compounds formed by carbonyl adducts of nucleobases were detected within MR1 molecules produced by tumor cells, and their abundance and antigenicity were enhanced by drugs that induce carbonyl accumulation. Our data reveal carbonyl-nucleobase adducts as MR1T cell antigens. Recognizing cells under carbonyl stress allows MR1T cells to monitor cellular metabolic changes with physiological and therapeutic implications.

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.

Shared graft-versus-leukemia minor histocompatibility antigens in DISCOVeRY-BMT.

T-cell responses to minor histocompatibility antigens (mHAs) mediate graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation. Therapies that boost T-cell responses improve allogeneic hematopoietic cell transplant (alloHCT) efficacy but are limited by concurrent increases in the incidence and severity of GVHD. mHAs with expression restricted to hematopoietic tissue (GVL mHAs) are attractive targets for driving GVL without causing GVHD. Prior work to identify mHAs has focused on a small set of mHAs or population-level single-nucleotide polymorphism-association studies. We report the discovery of a large set of novel GVL mHAs based on predicted immunogenicity, tissue expression, and degree of sharing among donor-recipient pairs (DRPs) in the DISCOVeRY-BMT data set of 3231 alloHCT DRPs. The total number of predicted mHAs varied by HLA allele, and the total number and number of each class of mHA significantly differed by recipient genomic ancestry group. From the pool of predicted mHAs, we identified the smallest sets of GVL mHAs needed to cover 100% of DRPs with a given HLA allele. We used mass spectrometry to search for high-population frequency mHAs for 3 common HLA alleles. We validated 24 predicted novel GVL mHAs that are found cumulatively within 98.8%, 60.7%, and 78.9% of DRPs within DISCOVeRY-BMT that express HLA-A∗02:01, HLA-B∗35:01, and HLA-C∗07:02, respectively. We confirmed the immunogenicity of an example novel mHA via T-cell coculture with peptide-pulsed dendritic cells. This work demonstrates that the identification of shared mHAs is a feasible and promising technique for expanding mHA-targeting immunotherapeutics.

Construction of three-gene-based prognostic signature and analysis of immune cells infiltration in children and young adults with B-acute lymphoblastic leukemia.

BACKGROUND: Although B-acute lymphoblastic leukemia (B-ALL) patients' survival has been improved dramatically, some cases still relapse. This study aimed to explore the prognosis-related novel differentially expressed genes (DEGs) for predicting the overall survival (OS) of children and young adults (CAYAs) with B-ALL and analyze the immune-related factors contributing to poor prognosis. METHODS: GSE48558 and GSE79533 from Gene Expression Omnibus (GEO) and clinical sample information and mRNA-seq from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database were retrieved. Prognosis-related key genes were enrolled to build a Cox proportional model using multivariate Cox regression. Five-year OS of patients, clinical characteristic relevance and clinical independence were assessed based on the model. The mRNA levels of prognosis-related genes were validated in our samples and the difference of immune cells composition between high-risk and low-risk patients were compared. RESULTS: One hundred and twelve DEGs between normal B cells and B-ALL cells were identified based on GSE datasets. They were mainly participated in protein binding and HIF-1 signaling pathway. One hundred and eighty-nine clinical samples were enrolled in the study, both Kaplan-Meier (KM) analysis and univariate Cox regression analysis showed that CYBB, BCL2A1, IFI30, and EFNB1 were associated with prognosis, CYBB, BCL2A1, and EFNB1 were used to construct prognostic risk model. Moreover, compared to clinical indicators, the three-gene signature was an independent prognostic factor for CAYAs with B-ALL. Finally, the mRNA levels of CYBB, BCL2A1, and EFNB1 were significantly lower in B-ALL group as compared to controls. The high-risk group had a significantly higher percentage of infiltrated immune cells. CONCLUSION: We constructed a novel three-gene signature with independent prognostic factor for predicting 5-year OS of CAYAs with B-ALL. Additionally, we discovered the difference of immune cells composition between high-risk and low-risk groups. This study may help to customize individual treatment and improve prognosis of CAYAs with B-ALL.

The P5-type ATPase ATP13A1 modulates major histocompatibility complex I-related protein 1 (MR1)-mediated antigen presentation.

The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included ß2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.

A Model of Minor Histocompatibility Antigens in Allogeneic Hematopoietic Cell Transplantation.

Minor histocompatibility antigens (mHAg) composed of peptides presented by HLA molecules can cause immune responses involved in graft-versus-host disease (GVHD) and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (HCT). The current study was designed to identify individual graft-versus-host genomic mismatches associated with altered risks of acute or chronic GVHD or relapse after HCT between HLA-genotypically identical siblings. Our results demonstrate that in allogeneic HCT between a pair of HLA-identical siblings, a mHAg manifests as a set of peptides originating from annotated proteins and non-annotated open reading frames, which i) are encoded by a group of highly associated recipient genomic mismatches, ii) bind to HLA allotypes in the recipient, and iii) evoke a donor immune response. Attribution of the immune response and consequent clinical outcomes to individual peptide components within this set will likely differ from patient to patient according to their HLA types.

Mouse Models of Antigen Presentation in Hematopoietic Stem Cell Transplantation.

Allogeneic stem cell transplantation (alloSCT) is a curative therapy for hematopoietic malignancies. The therapeutic effect relies on donor T cells and NK cells to recognize and eliminate malignant cells, known as the graft-versus-leukemia (GVL) effect. However, off target immune pathology, known as graft-versus-host disease (GVHD) remains a major complication of alloSCT that limits the broad application of this therapy. The presentation of recipient-origin alloantigen to donor T cells is the primary process initiating GVHD and GVL. Therefore, the understanding of spatial and temporal characteristics of alloantigen presentation is pivotal to attempts to separate beneficial GVL effects from detrimental GVHD. In this review, we discuss mouse models and the tools therein, that permit the quantification of alloantigen presentation after alloSCT.

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein.

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.

Consequence of Histoincompatibility beyond GvH-Reaction in Cytomegalovirus Disease Associated with Allogeneic Hematopoietic Cell Transplantation: Change of Paradigm.

Hematopoietic cell (HC) transplantation (HCT) is the last resort to cure hematopoietic malignancies that are refractory to standard therapies. Hematoablative treatment aims at wiping out tumor cells as completely as possible to avoid leukemia/lymphoma relapse. This treatment inevitably co-depletes cells of hematopoietic cell lineages, including differentiated cells that constitute the immune system. HCT reconstitutes hematopoiesis and thus, eventually, also antiviral effector cells. In cases of an unrelated donor, that is, in allogeneic HCT, HLA-matching is performed to minimize the risk of graft-versus-host reaction and disease (GvHR/D), but a mismatch in minor histocompatibility antigens (minor HAg) is unavoidable. The transient immunodeficiency in the period between hematoablative treatment and reconstitution by HCT gives latent cytomegalovirus (CMV) the chance to reactivate from latently infected donor HC or from latently infected organs of the recipient, or from both. Clinical experience shows that HLA and/or minor-HAg mismatches increase the risk of complications from CMV. Recent results challenge the widespread, though never proven, view of a mechanistic link between GvHR/D and CMV. Instead, new evidence suggests that histoincompatibility promotes CMV disease by inducing non-cognate transplantation tolerance that inhibits an efficient reconstitution of high-avidity CD8+ T cells capable of recognizing and resolving cytopathogenic tissue infection.

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

60 Years Young: The Evolving Role of Allogeneic Hematopoietic Stem Cell Transplantation in Cancer Immunotherapy.

The year 2020 marked the 30th anniversary of the Nobel Prize in Medicine awarded to E. Donnall Thomas for the development of allogeneic hematopoietic stem cell transplantation (allo-HSCT) to treat hematologic malignancies and other blood disorders. Dr. Thomas, "father of bone marrow transplantation," first developed and reported this technique in 1957, and in the ensuing decades, this seminal study has impacted fundamental work in hematology and cancer research, including advances in hematopoiesis, stem cell biology, tumor immunology, and T-cell biology. As the first example of cancer immunotherapy, understanding the mechanisms of antitumor biology associated with allo-HSCT has given rise to many of the principles used today in the development and implementation of novel transformative immunotherapies. Here we review the historical basis underpinning the development of allo-HSCT as well as advances in knowledge obtained by defining mechanisms of allo-HSCT activity. We review how these principles have been translated to novel immunotherapies currently utilized in clinical practice and describe potential future applications for allo-HSCT in cancer research and development of novel therapeutic strategies.

DNA and modified vaccinia Ankara prime-boost vaccination generates strong CD8+ T cell responses against minor histocompatibility antigen HA-1.

Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

LMP7 polymorphism may modify the presentation and clinical impact of minor histocompatibility antigens in matched related hematopoietic stem cell transplantation.

Differential expression of minor histocompatibility antigens between the recipient and donor determines their disparity and can be modified by immunoproteasomes that regulate their processing and presentation. We examined the impact of HA-1 and HA-8 disparity, and immunoproteasome LMP7 polymorphism in 130 pairs. In multivariate analysis, HA-1 disparity showed a statistically significant association with an increased incidence of acute graft-versus-host disease (aGVHD) II-IV (p = 0.043, HR: 3.71, 95%CI = 1.04-13.26), while LMP7-Q/Q showed a trend toward increased incidence of aGVHD compared to LMP7-Q/K and K/K genotypes (p = 0.087, HR: 2.36, 95%CI = 0.88-6.31). All HA-1 and HA-8 disparate patients who developed aGVHD had the LMP7-Q/Q genotype. No significant association could be detected between HA-1, HA-8, or LMP7 and chronic GVHD, relapse-free survival (RFS), overall survival (OS), or transplant-related mortality (TRM). In conclusion, we suggested an association between the HA-1 disparity and the risk of developing aGVHD with a possible modifying effect of LMP7.

Laser-assisted epicutaneous immunization to target human skin dendritic cells.

Dendritic cells (DC) are promising targets for immunotherapy of cancer. Clinically, immunization against cancer antigens by means of the most potent antigen-presenting cells, that is DC, remains an important treatment option in combination with the modern immune checkpoint approaches. Instead of adoptively transferring in vitro monocyte-derived DC, they can also be loaded in situ by antibody-mediated targeting of antigen. Conventionally, these vaccines are delivered by classical intradermal injections. Here, we tested an alternative approach, namely laser-assisted epicutaneous immunization. With an infrared laser ("Precise Laser Epidermal System"/P.L.E.A.S.E.® Laser System), we created micropores in human skin and applied monoclonal antibodies (mAbs) against C-type lectins, for example DEC-205/CD205 and Langerin/CD207. Optimal parameters for formation of pores in epidermis and dermis were determined. We could induce pores of defined depths without enhanced apoptosis around them. Antibodies applied epicutaneously to the laser-porated skin could be detected both in Langerhans cells (LC) in situ in the epidermis and in migratory skin DC subsets from short term human skin explant culture, demonstrating uptake and transport of Langerin and DEC-205 mAbs. Efficacy of targeting was similar between the different laser treatments and pore depths. Thus, laser-assisted epicutaneous immunization may be a valuable alternative to intradermal injection, yet the loading efficacy of DC needs to be further improved.

Myo1g is required for efficient adhesion and migration of activated B lymphocytes to inguinal lymph nodes.

Cell migration is a dynamic process that involves adhesion molecules and the deformation of the moving cell that depends on cytoskeletal remodeling and actin-modulating proteins such as myosins. In this work, we analyzed the role of the class I Myosin-1 g (Myo1g) in migratory processes of LPS + IL-4 activated B lymphocytes in vivo and in vitro. In vivo, the absence of Myo1g reduced homing of activated B lymphocytes into the inguinal lymph node. Using microchannel chambers and morphology analysis, we found that the lack of Myo1g caused adhesion and chemotaxis defects. Additionally, deficiency in Myo1g causes flaws in adopting a migratory morphology. Our results highlight the importance of Myo1g during B cell migration.

Tripartite Motif 22 (TRIM22) protein restricts herpes simplex virus 1 by epigenetic silencing of viral immediate-early genes.

Intrinsic resistance is a crucial line of defense against virus infections, and members of the Tripartite Ring Interaction Motif (TRIM) family of proteins are major players in this system, such as cytoplasmic TRIM5α or nuclear promyelocytic leukemia (PML/TRIM19) protein. Previous reports on the antiviral function of another TRIM protein, TRIM22, emphasized its innate immune role as a Type I and Type II interferon-stimulated gene against RNA viruses. This study shows that TRIM22 has an additional intrinsic role against DNA viruses. Here, we report that TRIM22 is a novel restriction factor of HSV-1 and limits ICP0-null virus replication by increasing histone occupancy and heterochromatin, thereby reducing immediate-early viral gene expression. The corresponding wild-type equivalent of the virus evades the TRIM22-specific restriction by a mechanism independent of ICP0-mediated degradation. We also demonstrate that TRIM22 inhibits other DNA viruses, including representative members of the ß- and γ- herpesviruses. Allelic variants in TRIM22 showed different degrees of anti-herpesviral activity; thus, TRIM22 genetic variability may contribute to the varying susceptibility to HSV-1 infection in humans. Collectively, these results argue that TRIM22 is a novel restriction factor and expand the list of restriction factors functioning in the infected cell nucleus to counter DNA virus infection.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Common allotypes of ER aminopeptidase 1 have substrate-dependent and highly variable enzymatic properties.

Polymorphic variation of immune system proteins can drive variability of individual immune responses. Endoplasmic reticulum aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by major histocompatibility complex class I molecules. Coding SNPs in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes has not been thoroughly explored. We used phased genotype data to estimate ERAP1 allotype frequency in 2504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the ten most common ERAP1 allotypes, and systematically characterized their in vitro enzymatic properties. We find that ERAP1 allotypes possess a wide range of enzymatic activities, up to 60-fold, whose ranking is substrate dependent. Strikingly, allotype 10, previously associated with Behçet's disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a subactive ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multistep trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site suggest that allotypic variation influences the communication between the regulatory and the active site. Our work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with major histocompatibility complex haplotypes, to immune response variability and predisposition to chronic inflammatory conditions.

T cell receptor diversity, specificity and promiscuity of functionally heterogeneous human MR1-restricted T cells.

The monomorphic MHC-class I-like molecule, MR1, presents small metabolites to T cells. MR1 is the restriction element for microbe-reactive mucosal-associated invariant T (MAIT) cells. MAIT cells have limited TCR usage, including a semi-invariant TCR alpha chain and express high levels of CD161 and CD26. In addition to microbial lumazine metabolites, recent studies have demonstrated that MR1 is able to capture a variety of diverse chemical entities including folate-derivatives, a number of drug-like and other synthetic small molecules, and as yet undefined compounds of self-origin. This capacity of MR1 to bind distinct ligands likely accounts for the recent identification of additional, non-canonical, subsets of MR1-restricted T (MR1T) cells. These subsets can be defined based on their ability to recognize diverse microbes as well as their reactivity to non-microbial cell-endogenous ligands, including tumor-associated antigens. Herein, we will discuss our current understanding of MR1T cell diversity in terms of TCR usage, ligand recognition and functional attributes.