Human CD8+ T cell cross-reactivity across influenza A, B and C viruses.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people.

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8+ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8+ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8+ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8+ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8+ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2320-331 and A11/PB2323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M41-49, A11/NS1186-195 and A11/NP511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8+ T cell epitopes has implications for understanding how CD8+ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.

The allopurinol metabolite, oxypurinol, drives oligoclonal expansions of drug-reactive T cells in resolved hypersensitivity cases and drug-naïve healthy donors.

Allopurinol (ALP) is a successful drug used in the treatment of gout. However, this drug has been implicated in hypersensitivity reactions that can cause severe to life-threatening reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Individuals who carry the human leukocyte antigen (HLA)-B*58:01 allotype are at higher risk of experiencing a hypersensitivity reaction (odds ratios ranging from 5.62 to 580.3 for mild to severe reactions, respectively). In addition to the parent drug, the metabolite oxypurinol (OXP) is implicated in triggering T cell-mediated immunopathology via a labile interaction with HLA-B*58:01. To date, there has been limited information regarding the T-cell receptor (TCR) repertoire usage of reactive T cells in patients with ALP-induced SJS or TEN and, in particular, there are no reports examining paired αßTCRs. Here, using in vitro drug-treated PBMCs isolated from both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors, we show that OXP is the driver of CD8+ T cell-mediated responses and that drug-exposed memory T cells can exhibit a proinflammatory immunophenotype similar to T cells described during active disease. Furthermore, this response supported the pharmacological interaction with immune receptors (p-i) concept by showcasing (i) the labile metabolite interaction with peptide/HLA complexes, (ii) immunogenic complex formation at the cell surface, and (iii) lack of requirement for antigen processing to elicit drug-induced T cell responsiveness. Examination of paired OXP-induced αßTCR repertoires highlighted an oligoclonal and private clonotypic profile in both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors.

A Shared TCR Bias toward an Immunogenic EBV Epitope Dominates in HLA-B*07:02-Expressing Individuals.

EBV is one of the most common viruses found in humans and is prototypic of a persistent viral infection characterized by periods of latency. Across many HLA class I molecules, the latent-specific CD8+ T cell response is focused on epitopes derived from the EBNA-3 protein family. In the case of HLA-B*07:02 restriction, a highly frequent class I allele, the T cell response is dominated by an epitope spanning residues 379-387 of EBNA-3 (RPPIFIRRL [EBVRPP]). However, little is known about either the TCR repertoire specific for this epitope or the molecular basis for this observed immunodominance. The EBVRPP CD8+ T cell response was common among both EBV-seropositive HLA-B*07:02+ healthy and immunocompromised individuals. Similar TCRs were identified in EBVRPP-specific CD8+ T cell repertoires across multiple HLA-B7+ individuals, indicating a shared Ag-driven bias in TCR usage. In particular, TRBV4-1 and TRAV38 usage was observed in five out of six individuals studied. In this study, we report the crystal structure of a TRBV4-1+ TCR-HLA-B*07:02/EBVRPP complex, which provides a molecular basis for the observed TRBV4-1 bias. These findings enhance our understanding of the CD8+ T cell response toward a common EBV determinant in HLA-B*07:02+ individuals.

Resourcing, annotating, and analysing synthetic peptides of SARS-CoV-2 for immunopeptidomics and other immunological studies.

SARS-CoV-2 has caused a significant ongoing pandemic worldwide. A number of studies have examined the T cell mediated immune responses against SARS-CoV-2, identifying potential T cell epitopes derived from the SARS-CoV-2 proteome. Such studies will aid in identifying targets for vaccination and immune monitoring. In this study, we applied tandem mass spectrometry and proteomic techniques to a library of ∼40,000 synthetic peptides, in order to generate a large dataset of SARS-CoV-2 derived peptide MS/MS spectra. On this basis, we built an online knowledgebase, termed virusMS (https://virusms.erc.monash.edu/), to document, annotate and analyse these synthetic peptides and their spectral information. VirusMS incorporates a user-friendly interface to facilitate searching, browsing and downloading the database content. Detailed annotations of the peptides, including experimental information, peptide modifications, predicted peptide-HLA (human leukocyte antigen) binding affinities, and peptide MS/MS spectral data, are provided in virusMS.

The complexity of T cell-mediated penicillin hypersensitivity reactions.

Penicillin refers to a group of beta-lactam antibiotics that are the first-line treatment for a range of infections. However, they also possess the ability to form novel antigens, or neoantigens, through haptenation of proteins and can stimulate a range of immune-mediated adverse reactions-collectively known as drug hypersensitivity reactions (DHRs). IgE-mediated reactions towards these neoantigens are well studied; however, IgE-independent reactions are less well understood. These reactions usually manifest in a delayed manner as different forms of cutaneous eruptions or liver injury consistent with priming of an immune response. Ex vivo studies have confirmed the infiltration of T cells into the site of inflammation, and the subsets of T cells involved appear dependent on the nature of the reaction. Here, we review the evidence that has led to our current understanding of these immune-mediated reactions, discussing the nature of the lesional T cells, the characterization of drug-responsive T cells isolated from patient blood, and the potential mechanisms by which penicillins enter the antigen processing and presentation pathway to stimulate these deleterious responses. Thus, we highlight the need for a more comprehensive understanding of the underlying genetic and molecular basis of penicillin-induced DHRs.

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames.

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.

Inability To Detect Cross-Reactive Memory T Cells Challenges the Frequency of Heterologous Immunity among Common Viruses.

Human memory T cells that cross-react with epitopes from unrelated viruses can potentially modulate immune responses to subsequent infections by a phenomenon termed heterologous immunity. However, it is unclear whether similarities in structure rather than sequence underpin heterologous T cell cross-reactivity. In this study, we aimed to explore the mechanism of heterologous immunity involving immunodominant epitopes derived from common viruses restricted to high-frequency HLA allotypes (HLA-A*02:01, -B*07:02, and -B*08:01). We examined EBV-specific memory T cells for their ability to cross-react with CMV or influenza A virus-derived epitopes. Following T cell immunoassays to determine phenotype and function, complemented with biophysical and structural investigations of peptide/HLA complexes, we did not detect cross-reactivity of EBV-specific memory T cells toward either CMV or influenza A virus epitopes presented by any of the selected HLA allomorphs. Thus, despite the ubiquitous nature of these human viruses and the dominant immune response directed toward the selected epitopes, heterologous virus-specific T cell cross-reactivity was not detected. This suggests that either heterologous immunity is not as common as previously reported, or that it requires a very specific biological context to develop and be clinically relevant.

Identification of Native and Posttranslationally Modified HLA-B*57:01-Restricted HIV Envelope Derived Epitopes Using Immunoproteomics.

The recognition of pathogen-derived peptides by T lymphocytes is the cornerstone of adaptive immunity, whereby intracellular antigens are degraded in the cytosol and short peptides assemble with class I human leukocyte antigen (HLA) molecules in the ER. These peptide-HLA complexes egress to the cell surface and are scrutinized by cytotoxic CD8+ T-cells leading to the eradication of the infected cell. Here, naturally presented HLA-B*57:01 bound peptides derived from the envelope protein of the human immunodeficiency virus (HIVenv) are identified. HIVenv peptides are present at a very small percentage of the overall HLA-B*57:01 peptidome (<0.1%) and both native and posttranslationally modified forms of two distinct HIV peptides are identified. Notably, a peptide bearing a natively encoded C-terminal tryptophan residue is also present in a modified form containing a kynurenine residue. Kynurenine is a major product of tryptophan catabolism and is abundant during inflammation and infection. Binding of these peptides at a molecular level and their immunogenicity in preliminary functional studies are examined. Modest immune responses are observed to the modified HIVenv peptide, highlighting a potential role for kynurenine-modified peptides in the immune response to HIV and other viral infections.

Maintenance of the EBV-specific CD8+ TCRαß repertoire in immunosuppressed lung transplant recipients.

Epstein-Barr virus (EBV) is one of the most common viruses in humans, capable of causing life-threatening infections and cancers in immunocompromised individuals. Although CD8+ T cells provide key protection against EBV, the persistence and dynamics of specific T-cell receptor (TCR) clones during immunosuppression in transplant patients is largely unknown. For the first time, we used a novel single-cell TCRαß multiplex-nested reverse transcriptase PCR to dissect TCRαß clonal diversity within GLCTLVAML (GLC)-specific CD8+ T cells in healthy individuals and immunocompromised lung transplant recipients. The GLC peptide presented by HLA-A*02:01 is one of the most immunogenic T-cell targets from the EBV proteome. We found that the GLC-specific TCRαß repertoire was heavily biased toward TRAV5 and encompassed five classes of public TCRαßs, suggesting that these clonotypes are preferentially utilized following infection. We identified that a common TRAV5 was diversely paired with different TRAJ and TRBV/TRBJ genes, in both immunocompetent and immunocompromised individuals, with an average of 12 different TCRαß clonotypes/donor. Moreover, pre-transplant GLC-specific TCRαß repertoires were relatively stable over 1 year post transplant under immunosuppression in the absence or presence of EBV reactivation. In addition, we provide the first evidence of early GLC-specific CD8+ T cells at 87 days post transplant, which preceded clinical EBV detection at 242 days in an EBV-seronegative patient receiving a lung allograft from an EBV-seropositive donor. This was associated with a relatively stable TCRαß repertoire after CD8+ T-cell expansion. Our findings provide insights into the composition and temporal dynamics of the EBV-specific TCRαß repertoire in immunocompromised transplant patients and suggest that the early detection of EBV-specific T cells might be a predictor of ensuing EBV blood viremia.

Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity.

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

Recognition of distinct cross-reactive virus-specific CD8+ T cells reveals a unique TCR signature in a clinical setting.

Human CMV still remains problematic in immunocompromised patients, particularly after solid organ transplantation. CMV primary disease and reactivation greatly increase the risks associated with incidences of chronic allograft rejection and decreased survival in transplant recipients. But whether this is due to direct viral effects, indirect viral effects including cross-reactive antiviral T cell immunopathology, or a combination of both remains undetermined. In this article, we report the novel TCR signature of cross-reactive HLA-A*02:01 (A2) CMV (NLVPMVATV [NLV])-specific CD8(+) T cells recognizing a specific array of HLA-B27 alleles using technical advancements that combine both IFN-γ secretion and multiplex nested RT-PCR for determining paired CDR3α/ß sequences from a single cell. This study represents the first evidence, to our knowledge, of the same A2-restricted cross-reactive NLV-specific TCR-α/ß signature (TRAV3TRAJ31_TRBV12-4TRBJ1-1) in two genetically distinct individuals. Longitudinal posttransplant monitoring of a lung transplant recipient (A2, CMV seropositive) who received a HLA-B27 bilateral lung allograft showed a dynamic expansion of the cross-reactive NLV-specific TCR repertoire before CMV reactivation. After resolution of the active viral infection, the frequency of cross-reactive NLV-specific CD8(+) T cells reduced to previremia levels, thereby demonstrating immune modulation of the T cell repertoire due to antigenic pressure. The dynamic changes in TCR repertoire, at a time when CMV reactivation was subclinical, illustrates that prospective monitoring in susceptible patients can reveal nuances in immune profiles that may be clinically relevant.

Deciphering the clinical relevance of allo-human leukocyte antigen cross-reactivity in mediating alloimmunity following transplantation.

PURPOSE OF REVIEW: Despite a growing awareness regarding the potential of cross-reactive virus-specific memory T cells to mediate alloimmunity, there has been limited clinical evaluation on allograft immunopathology. This review will explore published models of human T-cell cross-reactivity and discuss criteria required to drive this mechanism as a contributing cause of allograft dysfunction in transplantation. RECENT FINDINGS: Published models of human allogeneic (allo)-human leukocyte antigen (HLA) cross-reactivity have enabled dissection of the cross-reactive T cell receptor/peptide/major histocompatibility complex (TCR/peptide/MHC) interaction. In many of the models, the cross-reactive T cells express a unique TCR, although the relevance of a public cross-reactive TCR repertoire has yet to be determined. Equally, allopeptide identity, a vital component driving cross-recognition, remains unknown in the majority of models thereby prompting further characterization utilizing novel technologies. Although clinical studies examining the presence and impact of specific cross-reactive virus-specific T cells have been minimally explored, the existing data suggest that there may be a marginal set of requirements that need to be satisfied before the potentially damaging effects of allo-HLA cross-reactivity can be realized. SUMMARY: Our understanding of allo-HLA cross-reactivity continues to evolve as improved technology and novel strategies allow us to better question the contribution of allo-HLA cross-reactivity in clinically relevant allograft dysfunction.

The oral iron chelator deferasirox inhibits NF-κB mediated gene expression without impacting on proximal activation: implications for myelodysplasia and aplastic anaemia.

The myelodysplastic syndromes (MDS) are a group of disorders characterized by ineffective haematopoiesis, bone marrow dysplasia and cytopenias. Failure of red cell production often results in transfusion dependency with subsequent iron loading requiring iron chelation in lower risk patients. Consistent with previous reports, we have observed haematopoietic improvement in a cohort of patients treated with the oral iron chelator deferasirox (DFX). It has been postulated that MDS patients have a pro-inflammatory bone marrow environment with increased numbers of activated T cells producing elevated levels of tumour necrosis factor (TNF), which is detrimental to normal haematopoiesis. We demonstrate that DFX inhibits nuclear factor (NF)-κB dependent transcription without affecting its proximal activation, resulting in reduced TNF production from T cells stimulated in vitro. These results suggest that the haematopoietic improvement observed in DFX-treated patients may reflect an anti-inflammatory effect, mediated through inhibition of the transcription factor NF-κB and support the therapeutic targeting of this pathway, which is aberrantly activated in a large proportion of haematological malignancies.

The activin A antagonist follistatin inhibits cystic fibrosis-like lung inflammation and pathology.

Cystic fibrosis (CF) is the most common life-limiting genetically acquired respiratory disorder. Patients with CF have thick mucus obstructing the airways leading to recurrent infections, bronchiectasis and neutrophilic airway inflammation culminating in deteriorating lung function. Current management targets airway infection and mucus clearance, but despite recent advances in care, life expectancy is still only 40 years. We investigated whether activin A is elevated in CF lung disease and whether inhibiting activin A with its natural antagonist follistatin retards lung disease progression. We measured serum activin A levels, lung function and nutritional status in CF patients. We studied the effect of activin A on CF lung pathogenesis by treating newborn CF transgenic mice (ß-ENaC) intranasally with the natural activin A antagonist follistatin. Activin A levels were elevated in the serum of adult CF patients, and correlated inversely with lung function and body mass index. Follistatin treatment of newborn ß-ENaC mice, noted for respiratory pathology mimicking human CF, decreased the airway activin A levels and key features of CF lung disease including mucus hypersecretion, airway neutrophilia and levels of mediators that regulate inflammation and chemotaxis. Follistatin treatment also increased body weight and survival of ß-ENaC mice, with no evidence of local or systemic toxicity. Our findings demonstrate that activin A levels are elevated in CF and provide proof-of-concept for the use of the activin A antagonist, follistatin, as a therapeutic in the long-term management of lung disease in CF patients.

Mapping the immunopeptidome of seven SARS-CoV-2 antigens across common HLA haplotypes.

Most COVID-19 vaccines elicit immunity against the SARS-CoV-2 Spike protein. However, Spike protein mutations in emerging strains and immune evasion by the SARS-CoV-2 virus demonstrates the need to develop more broadly targeting vaccines. To facilitate this, we use mass spectrometry to identify immunopeptides derived from seven relatively conserved structural and non-structural SARS-CoV-2 proteins (N, E, Nsp1/4/5/8/9). We use two different B-lymphoblastoid cell lines to map Human Leukocyte Antigen (HLA) class I and class II immunopeptidomes covering some of the prevalent HLA types across the global human population. We employ DNA plasmid transfection and direct antigen delivery approaches to sample different antigens and find 248 unique HLA class I and HLA class II bound peptides with 71 derived from N, 12 from E, 28 from Nsp1, 19 from Nsp4, 73 from Nsp8 and 45 peptides derived from Nsp9. Over half of the viral peptides are unpublished. T cell reactivity tested against 56 of the detected peptides shows CD8+ and CD4+ T cell responses against several peptides from the N, E, and Nsp9 proteins. Results from this study will aid the development of next-generation COVID vaccines targeting epitopes from across a number of SARS-CoV-2 proteins.

CD8+ T-cell responses towards conserved influenza B virus epitopes across anatomical sites and age.

Influenza B viruses (IBVs) cause substantive morbidity and mortality, and yet immunity towards IBVs remains understudied. CD8+ T-cells provide broadly cross-reactive immunity and alleviate disease severity by recognizing conserved epitopes. Despite the IBV burden, only 18 IBV-specific T-cell epitopes restricted by 5 HLAs have been identified currently. A broader array of conserved IBV T-cell epitopes is needed to develop effective cross-reactive T-cell based IBV vaccines. Here we identify 9 highly conserved IBV CD8+ T-cell epitopes restricted to HLA-B*07:02, HLA-B*08:01 and HLA-B*35:01. Memory IBV-specific tetramer+CD8+ T-cells are present within blood and tissues. Frequencies of IBV-specific CD8+ T-cells decline with age, but maintain a central memory phenotype. HLA-B*07:02 and HLA-B*08:01-restricted NP30-38 epitope-specific T-cells have distinct T-cell receptor repertoires. We provide structural basis for the IBV HLA-B*07:02-restricted NS1196-206 (11-mer) and HLA-B*07:02-restricted NP30-38 epitope presentation. Our study increases the number of IBV CD8+ T-cell epitopes, and defines IBV-specific CD8+ T-cells at cellular and molecular levels, across tissues and age.

Screening self-peptides for recognition by mouse alloreactive CD8+ T cells using direct ex vivo multimer staining.

Here, we present a protocol to identify immunogenic self-peptide/allogeneic major histocompatibility complex (MHC) epitopes. We describe the generation of enriched alloreactive CD8+ T cells by priming mice with a skin graft expressing the allogeneic MHC class I molecule of interest, followed by boosting with a liver-specific AAV vector encoding the heavy chain of that donor MHC allomorph. We then use a peptide-exchange approach to assemble a range of peptide-MHC (pMHC) multimers for measuring recognition of the various epitopes by these alloreactive T cells. For complete details on the use and execution of this protocol, please refer to Son et al. (2021).1.

Endogenous H3.3K27M derived peptide restricted to HLA-A∗02:01 is insufficient for immune-targeting in diffuse midline glioma.

Diffuse midline glioma (DMG) is a childhood brain tumor with an extremely poor prognosis. Chimeric antigen receptor (CAR) T cell therapy has recently demonstrated some success in DMG, but there may a need to target multiple tumor-specific targets to avoid antigen escape. We developed a second-generation CAR targeting an HLA-A∗02:01 restricted histone 3K27M epitope in DMG, the target of previous peptide vaccination and T cell receptor-mimics. These CAR T cells demonstrated specific, titratable, binding to cells pulsed with the H3.3K27M peptide. However, we were unable to observe scFv binding, CAR T cell activation, or cytotoxic function against H3.3K27M+ patient-derived models. Despite using sensitive immunopeptidomics, we could not detect the H3.3K27M26-35-HLA-A∗02:01 peptide on these patient-derived models. Interestingly, other non-mutated peptides from DMG were detected bound to HLA-A∗02:01 and other class I molecules, including a novel HLA-A3-restricted peptide encompassing the K27M mutation and overlapping with the H3 K27M26-35-HLA-A∗02:01 peptide. These results suggest that targeting the H3 K27M26-35 mutation in context of HLA-A∗02:01 may not be a feasible immunotherapy strategy because of its lack of presentation. These findings should inform future investigations and clinical trials in DMG.