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NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes.

ABSTRACT: Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8⁺ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.

SUBMITTER: Becker JP

PROVIDER: S-EPMC8082087 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes.

Becker Jonas P JP Helm Dominic D Rettel Mandy M Stein Frank F Hernandez-Sanchez Alejandro A Urban Katharina K Gebert Johannes J Kloor Matthias M Neu-Yilik Gabriele G von Knebel Doeberitz Magnus M Hentze Matthias W MW Kulozik Andreas E AE

iScience 20210401 4

Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacyti ...[more]

PMID: 33981976

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Project description:Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.

Project description:BackgroundMesenchymal stromal cells (MSCs) have been extensively used in the clinic due to their exquisite tissue repair capacity. However, they also hold promise in the field of cellular vaccination as they can behave as conditional antigen presenting cells in response to interferon (IFN)-gamma treatment under a specific treatment regimen. This suggests that the immune function of MSCs can be pharmacologically modulated. Given the capacity of the agonist pyrimido-indole derivative UM171a to trigger the expression of various antigen presentation-related genes in human hematopoietic progenitor cells, we explored the potential use of UM171a as a means to pharmacologically instill and/or promote antigen presentation by MSCs.MethodsBesides completing a series of flow-cytometry-based phenotypic analyses, several functional antigen presentation assays were conducted using the SIINFEKL-specific T-cell clone B3Z. Anti-oxidants and electron transport chain inhibitors were also used to decipher UM171a's mode of action in MSCs. Finally, the potency of UM171a-treated MSCs was evaluated in the context of therapeutic vaccination using immunocompetent C57BL/6 mice with pre-established syngeneic EG.7T-cell lymphoma.ResultsTreatment of MSCs with UM171a triggered potent increase in H2-Kb cell surface levels along with the acquisition of antigen cross-presentation abilities. Mechanistically, such effects occurred in response to UM171a-mediated production of mitochondrial-derived reactive oxygen species as their neutralization using anti-oxidants or Antimycin-A mitigated MSCs' ability to cross-present antigens. Processing and presentation of the immunogenic ovalbumin-derived SIINFEKL peptide was caused by de novo expression of the Psmb8 gene in response to UM171a-triggered oxidative stress. When evaluated for their anti-tumoral properties in the context of therapeutic vaccination, UM171a-treated MSC administration to immunocompetent mice with pre-established T-cell lymphoma controlled tumor growth resulting in 40% survival without the need of additional supportive therapy and/or standard-of-care.ConclusionsAltogether, our findings reveal a new immune-related function for UM171a and clearly allude to a direct link between UM171a-mediated ROS induction and antigen cross-presentation by MSCs. The fact that UM171a treatment modulates MSCs to become antigen-presenting cells without the use of IFN-gamma opens-up a new line of investigation to search for additional agents capable of converting immune-suppressive MSCs to a cellular tool easily adaptable to vaccination.

Project description:Rationale: Previous studies have indicated an important role for complement in atherosclerosis, a lipid-driven chronic inflammatory disease associated to oxidative stress in the vessel wall. However, it remains unclear how complement is activated in the process of atherogenesis. An accepted general model for complement activation in the context of ischemia reperfusion injury is that ischemia induces the exposure of neoepitopes that are recognized by natural self-reactive IgM antibodies, and that in turn activate complement. Objective: We investigated whether a similar phenomenon may be involved in the pathogenesis of atherosclerosis, and whether interfering with this activation event, together with inhibition of subsequent amplification of the cascade at the C3 activation step, can provide protection against atherogenesis. Methods and Results: We utilized C2scFv-Crry, a novel construct consisting of a single chain antibody (scFv) linked to Crry, a complement inhibitor that functions at C3 activation. The scFv moiety was derived from C2 IgM mAb that specifically recognizes phospholipid neoepitopes known to be expressed after ischemia. C2scFv-Crry targeted to the atherosclerotic plaque of Apoe -/- mice, demonstrating expression of the C2 neoepitope. C2scFv-Crry administered twice per week significantly attenuated atherosclerotic plaque in the aorta and aortic root of Apoe -/- mice fed with a high-fat diet (HFD) for either 2 or 4 months, and treatment reduced C3 deposition and membrane attack complex formation as compared to vehicle treated mice. C2scFv-Crry also inhibited the uptake of oxidized low-density-lipoprotein (oxLDL) by peritoneal macrophages, which has been shown to play a role in pathogenesis, and C2scFv-Crry-treated mice had decreased lipid content in the lesion with reduced oxLDL levels in serum compared to vehicle-treated mice. Furthermore, C2scFv-Crry reduced the deposition of endogenous total IgM in the plaque, although it did not alter serum IgM levels, further indicating a role for natural IgM in initiating complement activation. Conclusion: Neoepitope targeted complement inhibitors represent a novel therapeutic approach for atherosclerosis.

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NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes.

Publications

NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes.

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