Antigenic Repertoire Research Articles

ERAP2 Increases the Abundance of a Peptide Submotif Highly Selective for the Birdshot Uveitis-Associated HLA-A29

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.

Abstract PR012: Adoptive transfer of T cells surface-tethered with IL-12 activates a natural killer/dendritic cell axis to promote antigen spreading for enhanced anti-tumor efficacy

Abstract Despite the promise of adoptive cell therapy (ACT) in the treatment of some cancer types, success against solid tumors remains elusive due to multiple immune escape pathways. T cell therapies that affect tumor microenvironment or provide adjuvant activity in addition to tumor cell lysis hold strong potential for overcoming immune escape in solid tumors. Interleukin-12 (IL-12) is a potent stimulator of innate and adaptive immune cells that holds strong potential for cancer immunotherapy, but its clinical utility has been limited by high systemic toxicities. We have previously shown that tethering an IL-12 immunocytokine to the surface of T cells prior to adoptive cell transfer (ACT) safely improves anti-tumor efficacy by promoting T cell function specifically in the tumor. Here, we demonstrate that tethered IL-12 further delivers adjuvant activity by initiating an immune cascade involving natural killer cell-mediated activation of cross-priming dendritic cells to resulting in broader anti-tumor T cell responses. In a preclinical cell therapy model tumor-specific T cells tethered with IL-12, but not the T cells alone, triggered proliferation of tumor-specific endogenous tumor infiltrating lymphocytes (TIL). This was accompanied by increased activation and infiltration of cross-presenting dendritic cells (cDC1) in tumor-draining lymph nodes (tdLN). Analyses of cDC1 subsets revealed upregulation of CD86 expression on both migratory and resident cDC1s within the tdLN, two cell types that are critical for both tumor antigen transport to tdLN and activation of naïve CD8 T cells, respectively. This activity was specific to cDC1s, as increased activation or accumulation of type 2 dendritic cells (cDC2) was not observed. Mechanistically, the IL-12 tethered T cells upregulated expression of FLT3 ligand on NK cells, a key cytokine for cDC1 recruitment and expansion. NK cell depletion ablated cDC1 tdLN infiltration and endogenous tumor infiltrating T cell expansion, indicating that recruitment and activation of endogenous tumor-specific T cells is at least partially driven by tethered IL-12-mediated activation of NK cells to recruit and expand the cross-priming dendritic cells. Together, this demonstrates that our surface tethered cytokine technology provides powerful adjuvant activity to adoptively transferred T cells and promotes activation of endogenous anti-tumor adaptive immune response, thus promoting a broad antigenic repertoire. This approach thus holds potential to help overcome challenges of antigen escape and tumor heterogeneity that limit efficacy of T cell therapies against solid tumors. This abstract is also being presented as PO078. Citation Format: Kate L. Stokes, Ditte E. Jaehger, Alvin Pratama, Holmfridur R. Halldorsdottir, Gulzar Ahmad, Jon D. Nardozzi, Katharine L. Sackton, Thomas L. Andresen, Douglas S. Jones. Adoptive transfer of T cells surface-tethered with IL-12 activates a natural killer/dendritic cell axis to promote antigen spreading for enhanced anti-tumor efficacy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR012.

Cross-reactivity of 4CMenB vaccine-induced antibodies against meningococci belonging to non-B serogroups in Italy

ABSTRACT The four-component meningococcal serogroup B vaccine (4CMenB) contains antigens present in the majority of meningococci causing invasive meningococcal disease (IMD) and may potentially offer protection against strains belonging to non-B serogroups. This study aimed to evaluate the ability of 4CMenB-induced antibodies to kill, in a human serum bactericidal assay (hSBA), non-B meningococci belonging to the main genotypes responsible for IMD in Italy. Meningococci, collected between 2015 and 2017, was characterized for PorA, FetA and sequence type, and for clonal complex. Twenty non-B isolates, representative of the most frequent genotypes, were molecularly characterized for 4CMenB antigens and tested in hSBA with sera from 4CMenB-vaccinated infants and adolescents. Among twenty isolates, eleven were serogroup C, five were Y, two W and two X. All isolates contained genes encoding for fHbp and NHBA antigens and four harbored the NadA full-length encoding gene. Positive hSBA titers were obtained against all serogroup W, X and Y isolates and against five serogroup C isolates. These data show that the 4CMenB vaccine can induce bactericidal antibodies against genetically representative meningococcal W, Y and X strains from Italy. For serogroup C, different susceptibilities to killing were observed for strains with similar antigenic repertoires.

Interaction between ERAP Alleles and HLA Class I Types Support a Role of Antigen Presentation in Hodgkin Lymphoma Development.

Simple SummaryHodgkin lymphoma (HL) is a common lymphoma in young adults derived from B cells. Emerging evidence suggests that antigen presentation by the malignant B cells is critically involved in HL pathogenesis. In fact, genetic variants of the antigen presenting Human Leukocyte Antigens (HLA) are strongly associated with HL susceptibility. Interestingly, the endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP2 genes, that code for enzymes that process antigens, also appear to be associated. In this study, we show that genetic variants of ERAP genes strongly affect expression levels of ERAP1 and ERAP2. In addition, we find that certain ERAP variants interact with specific HLA class I types in HL patients. This suggests that mechanisms that determine the repertoire of antigens that are presented to the immune system, affect the chance of developing HL. Our findings therefore support a prominent role of antigen presentation in HL susceptibility.Genetic variants in the HLA region are the strongest risk factors for developing Hodgkin lymphoma (HL), suggesting an important role for antigen presentation. This is supported by another HL-associated genomic region which contains the loci of two enzymes that process endogenous proteins to peptides to be presented by HLA class I, i.e., endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2. We hypothesized that ERAP and HLA class I type interact in HL susceptibility, as shown previously for several autoimmune diseases. We detected ERAP1 and ERAP2 expression in tumor cells and cells in the microenvironment in primary HL tissue samples. Seven ERAP SNPs and ERAP1 haplotypes showed strong associations with RNA and protein levels of ERAP1 and ERAP2 in LCLs and HL cell lines. Analysis of HLA class I types, ERAP SNPs and ERAP haplotypes by direct genotyping or imputation from genome-wide association data in 390 HL patients revealed significant interactions between HLA-A11, rs27038 and the rs27038 associated ERAP haplotype, as well as between HLA-Cw2 and rs26618. In conclusion, our results show that ERAP and HLA class I interact in genetic susceptibility to HL, providing further evidence that antigen presentation is an important process in HL susceptibility and pathogenesis.

Copper Sulfide Nanoparticle‐Redirected Macrophages for Adoptive Transfer Therapy of Melanoma

AbstractAdoptive cell therapy (ACT) has achieved landmark advances in treating cancer in clinic. Recent advances in ACT of macrophages engineered to express chimeric antigen receptors (CARs) have shown effectiveness in treating solid tumors. However, the CAR‐macrophage therapy is dependent on tumor antigen recognition and gene editing methods. Herein, an adoptive macrophage therapy is presented through copper sulfide nanoparticle‐regulation that exhibits substantial antitumor effect in melanoma‐bearing mice, without the need for tumor antigen repertoire. Bone marrow derived macrophages (BMDMs) incubated with the nanoparticles promote the cellular production of reactive oxygen species (ROS) through dynamin‐related protein 1 (Drp1)‐mediated mitochodrial fission. The high intracellular ROS level directs BMDMs polarization toward M1 phenotype by classical IKK‐dependent NF‐κB activation. Moreover, the copper sulfide nanoparticle‐stimulated BMDMs (CuS‐MΦ) reduce the expression of programmed death‐1 (PD‐1) and exhibit enhanced phagocytic and digestive ability. Intratumoral transfer of CuS‐MΦ significantly prolongs the median survival time of the tumor‐bearing mice, remodels the tumor microenvironment, and elicits systemic antitumor immunity. These results suggest a cancer therapeutic approach of adoptively transferred macrophages through the induction of intracellular ROS with nanomaterials.

Unraveling the Regulation of Cancer/Testis Antigens in Tumorigenesis Through an Analysis of Normal Germ Cell Development in Rodents.

Cancer/testis (CT) antigens are proteins aberrantly overexpressed in various tumorigenic cells, but they can also be normally expressed in the mammalian germline. Most CT antigens are highly immunogenic and known to be involved in cancer cell proliferation and tumor metastasis. A recent genome-wide analysis systematically identified CT antigen expression in 19 cancer types, significantly expanding the repertoire of CT antigens by 5-fold,from over 200 to approximately 1000. However, their function and regulation in tumorigenesis remain poorly understood. The shared functional characteristics between germ cells and cancer cells, if methodically defined, offer a unique gateway to understanding the regulation of CT antigens in cancers by studying gametogenesis. Nonetheless, such studies also provide insightful information on the role of CT antigens in spermatogenesis. Herein, we analyzed publicly available next generation sequencing datasets generated from normal adult testes in rodents, primordial germ cells and cancer samples across a series of published studies and databases. Based on these analyses, we report that a subset of CT antigens belonged to the core fitness gene family. Furthermore, super-enhancers both in normal testes and various cancers controlled specific CT antigens. We found that DNA methylation of CT antigens, such as TEX101 and TAF7L, was inversely correlated with their expression in both normal primordial germ cells and various cancers, which was mediated at least partly by DNA methyltransferase1 (DNMT1). By analyzing data from a testis knockout model, we showed that TAF7L could further influence the expression of additional CT antigens, which also held true in tumors. These findings not only confirmed the previous notion that CT antigens regulate cancer dynamics, but also showed that understanding the regulation of CT antigens during gametogenesis can offer new insights for cancer research.

CIITA-Transduced Glioblastoma Cells Uncover a Rich Repertoire of Clinically Relevant Tumor-Associated HLA-II Antigens

AbstractCD4+ T cell responses are crucial for inducing and maintaining effective anticancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry–based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17, and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared with interferon gamma (IFNɣ) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

Recent Advances in the Development of Protein- and Peptide-Based Subunit Vaccines against Tuberculosis.

The World Health Organization (WHO) herald of the “End TB Strategy” has defined goals and targets for tuberculosis prevention, care, and control to end the global tuberculosis endemic. The emergence of drug resistance and the relative dreadful consequences in treatment outcome has led to increased awareness on immunization against Mycobacterium tuberculosis (Mtb). However, the proven limited efficacy of Bacillus Calmette-Guérin (BCG), the only licensed vaccine against Mtb, has highlighted the need for alternative vaccines. In this review, we seek to give an overview of Mtb infection and failure of BCG to control it. Afterward, we focus on the protein- and peptide-based subunit vaccine subtype, examining the advantages and drawbacks of using this design approach. Finally, we explore the features of subunit vaccine candidates currently in pre-clinical and clinical evaluation, including the antigen repertoire, the exploited adjuvanted delivery systems, as well as the spawned immune response.

Opportunities for Antigen Discovery in Metastatic Breast Cancer.

Immune checkpoint inhibitor-based immunotherapy (ICI) of breast cancer is currently efficacious in a fraction of triple negative breast cancers (TNBC) as these cancers generally carry high tumor mutation burden (TMB) and show increased tumor infiltration by CD8+ T cells. However, most estrogen receptor positive breast cancers (ERBC) have low TMB and/or are infiltrated with immunosuppressive regulatory T cells (Tregs) and thus fail to induce a significant anti-tumor immune response. Our understanding of the immune underpinning of the anti-tumor effects of CDK4/6 inhibitor (CDKi) treatment coupled with new knowledge about the mechanisms of tolerance to self-antigens suggests a way forward, specifically via characterizing and exploiting the repertoire of tumor antigens expressed by metastatic ERBC. These treatment-associated tumor antigens (TATA) may include the conventional tumor neoantigens (TNA) encoded by single nucleotide mutations, TNA encoded by tumor specific aberrant RNA transcription, splicing and DNA replication induced frameshift (FS) events as well as the shared tumor antigens. The latter may include the conventional tumor associated antigens (TAA), cancer-testis antigens (CTA) and antigens encoded by the endogenous retroviral (ERV) like sequences and repetitive DNA sequences induced by ET and CDKi treatment. An approach to identifying these antigens is outlined as this will support the development of a multi-antigen-based immunotherapy strategy for improved targeting of metastatic disease with potential for minimal autoimmune toxicity against normal tissues.

Unveiling the Hidden Treasury: CIITA-Driven MHC Class II Expression in Tumor Cells to Dig up the Relevant Repertoire of Tumor Antigens for Optimal Stimulation of Tumor Specific CD4+ T Helper Cells.

Simple SummaryIt is becoming clear that combined approaches are required to fight and succeed against cancer. As far as immune-based strategies, new generation anti-tumor vaccines will certainly play a crucial role. A key aspect is to identify tumor antigens which optimally stimulate CD4+ T helper cells, as these cells are crucial to triggering and maintaining all effector mechanisms of the adaptive immune response. Our approach is to render tumor cells surrogate antigen presenting cells for their own tumor antigen by forcing them to express de novo MHC class II molecules after the genetic transfer of CIITA, the major transcriptional controller of MHC class II gene expression. The unprecedented strong stimulation of tumor-specific CD4+ T cells that were obtained with our approach let us hope that this will help in identifying a constellation of MHC class II-restricted tumor antigens for more potent next generation anti-tumor vaccines.Despite the recent enthusiasm generated by novel immunotherapeutic approaches against cancer based on immune checkpoint inhibitors, it becomes increasingly clear that single immune-based strategies are not sufficient to defeat the various forms and types of tumors. Within this frame, novel vaccination strategies that are based on optimal stimulation of the key cell governing adaptive immunity, the CD4+ T helper cell, will certainly help in constructing more efficient treatments. In this review, we will focus on this aspect, mainly describing our past and recent contributions that, starting with a rather unorthodox approach, have ended up with the proposition of a new idea for making available an unprecedented extended repertoire of tumor antigens, both in quantitative and qualitative terms, to tumor-specific CD4+ T helper cells. Our approach is based on rendering the very same tumor cells antigen presenting cells for their own tumor antigens by gene transfer of CIITA, the major transcriptional coordinator of MHC class II expression discovered in our laboratory. CIITA-driven MHC class II-expressing tumor cells optimally stimulate in vivo tumor specific MHC class II-restricted CD4 T cells generating specific and long lasting protective immunity against the tumor. We will discuss the mechanism underlying protection and elaborate not only on the applicability of this approach for novel vaccination strategies amenable to clinical setting, but also on the consequence of our discoveries on sedimented immunological dogmas that are related to antigen presentation.

Engineered Nanoparticles for Cancer Vaccination and Immunotherapy.

The immune system has evolved over time to protect the host from foreign microorganisms. Activation of the immune system is predicated on a distinction between self and nonself. Unfortunately, cancer is characterized by genetic alterations in the host's cells, leading to uncontrolled cellular proliferation and evasion of immune surveillance. Cancer immunotherapy aims to educate the host's immune system to not only recognize but also attack and kill mutated cancer cells. While immune checkpoint blockers have been proven to be effective against multiple types of advanced cancer, the overall patient response rate still remains below 30%. Therefore, there is an urgent need to improve current cancer immunotherapies. In this Account, we present an overview of our recent progress on nanoparticle-based strategies for improving cancer vaccines and immunotherapies. We also present other complementary strategies to give a well-rounded snapshot of the field of combination cancer immunotherapy. The versatility and tunability of nanoparticles make them promising platforms for addressing individual challenges posed by various cancers. For example, nanoparticles can deliver cargo materials to specific cells, such as vaccines delivered to antigen-presenting cells for strong immune activation. Nanoparticles also allow for stimuli-responsive delivery of various therapeutics to cancer cells, thus forming the basis for combination cancer immunotherapy. Here, we focus on nanoparticle platforms engineered to deliver tumor antigens, whole tumor cells, and chemotherapeutic or phototherapeutic agents in a manner to effectively and safely trigger the host's immune system against tumor cells. For each work, we discuss the nanoparticle platform developed, synthesis chemistry, and in vivo applications. Nanovaccines offer a unique platform for codelivery of personalized tumor neoantigens and adjuvants and elicitation of robust immune responses against aggressive tumors. Nanovaccines either delivering whole tumor cell lysate or formed from tumor cell lysate may increase the repertoire of tumor antigens as immune targets while exploiting immunogenic cell death to prime antitumor immune responses. We also discuss how antigen- and whole tumor cell-based approaches may open the door for personalized cancer vaccination and immunotherapy. On the other hand, chemotherapy, phototherapy, and radiotherapy are more standardized cancer therapies, and nanoparticle-based approaches may promote their ability to initiate T cell activation against tumor cells and improve antitumor efficacy with minimal toxicity. Finally, building on the recent progress in nanoparticle-based cancer immunotherapy, the field should set the ultimate goal to be clinical translation and clinical efficacy. We will discuss regulatory, analytical, and manufacturing hurdles that should be addressed to expedite the clinical translation of nanomedicine-based cancer immunotherapy.

Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

Immunomolecular characterization of Ehrlichia chaffeensis (E. ch.) and E. canis (E. ca.) has defined protein orthologs, including tandem repeat proteins (TRPs) that have immunodominant linear antibody epitopes. In this study, we combined bioinformatic analysis and cell-free protein expression to identify undiscovered immunoreactive E. ch. and E. ca. hypothetical proteins. Antigenicity of the E. ch. and E. ca. ORFeomes (n = 1105 and n = 925, respectively) was analyzed by the sequence-based prediction model ANTIGENpro, and we identified ~250 ORFs in each respective ORFeome as highly antigenic. The hypothetical proteins (E. ch. n = 93 and E. ca. n = 98) present in the top 250 antigenic ORFs were further investigated in this study. By ELISA, 46 E. ch. and 30 E. ca. IVTT-expressed hypothetical proteins reacted with antibodies in sera from naturally E. ch.-infected patients or E. ca.-infected dogs. Moreover, 15 E. ch. and 16 E. ca. proteins consistently reacted with a panel of sera from patients or dogs, including many that revealed the immunoreactivity of “gold standard” TRPs. Antibody epitopes in most (>70%) of these proteins exhibited partial or complete conformation-dependence. The majority (23/31; 74%) of the major immunoreactive proteins identified were small (≤250 aa), and 20/31 (65%) were predicted to be secreted effectors. Unlike the strong linear antibody epitopes previously identified in TRP and OMP orthologs, there were contrasting differences in the E. ch. and E. ca. antigenic repertoires, epitopes and ortholog immunoreactivity. This study reveals numerous previously undefined immunodominant and subdominant antigens, and illustrates the breadth, complexity, and diversity of immunoreactive proteins/epitopes in Ehrlichia.

A Weakened Immune Response to Synthetic Exo-Peptides Predicts a Potential Biosecurity Risk in the Retrieval of Exo-Microorganisms.

Simple SummaryWe tested the immune response of T cells of the mammalian immune system towards protein antigens that includes the unusual amino acids isovaline and α-aminoisobutyric. Those amino acids have been found in high abundance on carbonaceous meteorites but are extremely rare in proteomes of earth organisms. We hypothesised that proteins of non-terrestrial alien life forms might contain such amino acids and tested whether chemically synthesised “exopeptides” that contain these amino acids could be detected by the immune system. Our assays, based on the responses of CD8+ T cells to these exopeptides, indicated that antigen cleavage, processing, and subsequent T cell activation still occurred, but were less efficient than the response to control peptides that lacked these amino acids. We therefore speculate that the encounter of putative exo-microorganisms of an unusual antigenic repertoire might pose an immunological risk for space missions aiming to retrieve potentially biotic samples from exoplanets and moons.The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth’s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and α-aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and α-aminoisobutyric acid residues and demonstrated that these peptides can promote naïve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

Defining the Helicobacter pylori Disease-Specific Antigenic Repertoire.

The analysis of the interaction between Helicobacter pylori (HP) and the host in vivo is an extremely informative way to enlighten the molecular mechanisms behind the persistency/latency of the bacterium as well as in the progression of the infection. An important source of information is represented by circulating antibodies targeting the bacteria that define a specific “disease signature” with prospective diagnostic implications. The diagnosis of some of the HP induced diseases such as gastric cancer (GC), MALT lymphoma (MALT), and autoimmune gastritis (AIG) is not easy because patients do not show symptoms of illness in early-onset stages, at the same time they progress rapidly. The possibility of identifying markers able to provide an early diagnosis would be extremely beneficial since a late diagnosis results in a delay in undergoing active therapy and reduces the survival rate of patients. With the aim to identify the HP antigens recognized during the host immune-response to the infection and possibly disease progression, we applied a discovery-driven approach, that combines “phage display” and deep sequencing. The procedure is based on the selection of ORF phage libraries, specifically generated from the pathogen’s genome, with sera antibodies from patients with different HP-related diseases. To this end two phage display libraries have been constructed starting from genomic DNA from the reference HP 26695 and the pathogenic HP B128 strains; libraries were filtered for ORFs by using an ORF selection vector developed by our group (Di Niro et al., 2005; Soluri et al., 2018), selected with antibodies from patients affected by GC, MALT, and AIG and putative HP antigens/epitopes were identified after Sequencing and ranking. The results show that individual selection significantly reduced the library diversity and comparison of individual ranks for each condition allowed us to highlight a pattern of putative antigens specific for the different pathological outcomes or common for all of them. Within the putative antigens enriched after selection, we have validated protein CagY/Cag7 by ELISA assay as a marker of HP infection and progression. Overall, we have defined HP antigenic repertoire and identified a panel of putative specific antigens/epitopes for three different HP infection pathological outcomes that could be validated in the next future.

Engineered T cells Flt around their targets.

Antigen escape by solid tumors has limited the efficacy of genetically modified T cells. T cells engineered to secrete the cytokine Flt3L induce the activation of endogenous T cells, enabling a broader repertoire of tumor antigens to be targeted via the expansion of intratumoral antigen presenting cells, significantly improving tumor responses.

Post-translational modifications contribute to neoepitopes in Type-1 diabetes: Challenges for inducing antigen-specific tolerance

Type-1 Diabetes (T1D) is the major autoimmune disease affecting the juvenile population in which insulin-producing pancreatic β-cells are destroyed by self-reactive T-cells and B-cells. Emerging studies have identified the presence of autoantibodies and altered T-cell reactivity against several autoantigens in individuals who are at risk of developing T1D even before the clinical onset of diabetes. Whilst these findings could lead to the development of predictive biomarkers for early diagnosis, growing evidence on the generation of neoepitopes, epitope spreading and diverse antigen repertoire in T1D poses a major challenge for developing approaches to induce antigen-specific tolerance. Mechanisms of neoepitope generation include post-translational modifications of existing epitopes, aberrant translational products, peptide fusion, and differences in MHC binding registers. Here, we focus our discussion on how post-translational modifications can give rise to immunogenic neoepitopes in T1D and present our perspective on how it could affect the development of therapeutic approaches to induce antigen-specific tolerance.

Towards new horizons: characterization, classification and implications of the tumour antigenic repertoire.

Immune-checkpoint inhibition provides an unmatched level of durable clinical efficacy in various malignancies. Such therapies promote the activation of antigen-specific T cells, although the precise targets of these T cells remain unknown. Exploiting these targets holds great potential to amplify responses to treatment, such as by combining immune-checkpoint inhibition with therapeutic vaccination or other antigen-directed treatments. In this scenario, the pivotal hurdle remains the definition of valid HLA-restricted tumour antigens, which requires several levels of evidence before targets can be established with sufficient confidence. Suitable antigens might include tumour-specific antigens with alternative or wild-type sequences, tumour-associated antigens and cryptic antigens that exceed exome boundaries. Comprehensive antigen classification is required to enable future clinical development and the definition of innovative treatment strategies. Furthermore, clinical development remains challenging with regard to drug manufacturing and regulation, as well as treatment feasibility. Despite these challenges, treatments based on diligently curated antigens combined with a suitable therapeutic platform have the potential to enable optimal antitumour efficacy in patients, either as monotherapies or in combination with other established immunotherapies. In this Review, we summarize the current state-of-the-art approaches for the identification of candidate tumour antigens and provide a structured terminology based on their underlying characteristics.

Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by proteome-wide PhIP-Seq.

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to more common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from a cohort of people with APS1 and discovered multiple common antibody targets. These novel APS1 autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells, pineal gland, and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including a link between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for extensively interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.

Overcoming Heterogeneity of Antigen Expression for Effective CAR T Cell Targeting of Cancers.

Chimeric antigen receptor (CAR) gene-modified T cells (CAR T cells) can eradicate B cell malignancies via recognition of surface-expressed B lineage antigens. Antigen escape remains a major mechanism of relapse and is a key barrier for expanding the use of CAR T cells towards solid cancers with their more diverse surface antigen repertoires. In this review we discuss strategies by which cancers become amenable to effective CAR T cell therapy despite heterogeneous phenotypes. Pharmaceutical approaches have been reported that selectively upregulate individual target antigens on the cancer cell surface to sensitize antigen-negative subclones for recognition by CARs. In addition, advanced T cell engineering strategies now enable CAR T cells to interact with more than a single antigen simultaneously. Still, the choice of adequate targets reliably and selectively expressed on the cell surface of tumor cells but not normal cells, ideally by driving tumor growth, is limited, and even dual or triple antigen targeting is unlikely to cure most solid tumors. Innovative receptor designs and combination strategies now aim to recruit bystander cells and alternative cytolytic mechanisms that broaden the activity of CAR-engineered T cells beyond CAR antigen-dependent tumor cell recognition.

Optimized Whole Genome Association Scanning for Discovery of HLA Class I-Restricted Minor Histocompatibility Antigens

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A∗01:01, A∗02:01, A∗03:01, B∗07:02, B∗08:01, C∗07:01, and C∗07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A∗24:02 and HLA-A∗23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation.