Florian Erhard

Ribo-seq has provided compelling evidence that there are many translated but unannotated ORFs. Despite a growing body of peptidomic support for cryptic ORFs, the computational discovery of weakly translated, short and mutually overlapping ORFs has remained a major bottleneck. Here we introduce PRICE 2, a method designed to resolve these challenging translation events by adapting concepts from isoform deconvolution. PRICE 2 employs a generative model combined with group LASSO regularized regression to jointly infer active ORFs across multiple sequencing runs. In a comprehensive benchmark, PRICE 2 out-performed thirteen state-of-the-art tools in detecting short and overlapping ORFs. Orthogonal validation via immunopeptidomics demonstrated that ORFs identified by PRICE 2 enable the discovery of ∼40% more cryptic MHC-I peptides than the best-performing competing tool at a fixed false discovery rate. By analyzing public ribo-seq data across 45 distinct cell and tissue types, we constructed ORF-stock , a comprehensive database of non-canonical ORFs for immunopeptidomic studies.

Respiratory syncytial virus (RSV) causes severe lower respiratory disease, yet how it reshapes airway epithelial cells and evades innate immunity remains incompletely understood. We infected adult primary human airway epithelial cultures with RSV and analyzed infected and bystander cells over time using single-cell RNA sequencing and imaging. RSV mainly infected ciliated cells, triggering a virus load–dependent shutdown of genes involved in ciliogenesis, antigen presentation, and innate sensing, including key interferon (IFN) and pattern recognition pathways. Only a subset of infected cells produced type I and III IFNs, while bystander cells exhibited strong IFN-stimulated gene (ISG) signatures. Neither IFN treatment nor ISG induction eliminated infection, but IRF1, an antiviral transcription factor not suppressed by RSV, remained robustly expressed. Ectopic IRF1 expression in vitro reduced viral replication. These findings reveal how RSV evades antiviral defenses and highlight IRF1 as a potential target for therapeutic intervention.

The myeloid growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) exhibits paradoxical pro- and anti-inflammatory functions, but the factors determining these divergent outcomes remain unclear. Here, we report that this functional divergence is controlled by its glycosylation. Murine recombinant fully glycosylated GM-CSF (rgGM-CSF) specifically induces immunosuppressive cell types, whereas its recombinant non-glycosylated counterpart (rngGM-CSF) promotes effector immune cells. Using single-cell ATAC-sequencing and flow cytometry, we show that rgGM-CSF has a previously unrecognized ability to effectively expand IL-10 ⁺ LAG-3 ⁺ PD-L1 ⁺ B-1b plasma cells (PCs) with immunosuppressive properties and self reactive natural IgM secretion. Although rgGM-CSF also promotes the expansion of hematopoietic stem and progenitor cells (HSPCs) and monocytic myeloid-derived suppressor cells (M-MDSCs), adoptive transfer experiments demonstrate that the rgGM-CSF-induced B-1b PCs are responsible for an IL-10-dependent long-term protection in mice from experimental autoimmune-encephalomyelitis (EAE). Our data suggest that glycosylation enhances the systemic bioavailability and activity of GM-CSF and promotes the expansion of immunoregulatory cells rather than pro-inflammatory myeloid effector cells. Together, these results demonstrate that the ‘dual activity’ of GM-CSF is controlled by its glycosylation, resulting in opposing immune functions. These findings support a re-evaluation of human rgGM-CSF (regramostim) as a potential therapeutic strategy for immunosuppression in transplantation and autoimmune diseases. Key points: Glycosylated GM-CSF promotes B-1b cells and B-1b plasma cells expansion and establishes their long-term imprinting as IL-10 ⁺ LAG3 ⁺ PD-L1 ⁺ natural IgM secreting regulatory cells. Albumin binding enhances the systemic activity of glycosylated GM-CSF in generating regulatory B-1b plasma cells. Glycosylated GM-CSF injections into mice expand M-MDSCs, but their suppressive iNOS production is only maintained short-term. Non-glycosylated GM-CSF injections preferentially promote expansion of pro-inflammatory effector monocytes and neutrophils. Graphical

Includes the list of peptides derived from NSM identified at 0.02 FDR and ALC>=30

Includes a large list of all the peptides and details of the HLA-I ligands identified at <=0.01 FDR and >=30 ALC score

Tumor cell line HLA-I typing and tumor type

Supplementary Figure 1. Validation of HLA-I ligand tumor antigen candidates with synthetic peptidesSupplementary Figure 2. Validation of selected HLA-I ligand tumor antigen candidates identified in Mel-1 and Mel-3 with isotope-labeled peptidesSupplementary Figure 3. Frequency of nonC peptides detected in HLA-I.Supplementary Fig. 4. HLA-I allele binding motif of all patients included in the study.Supplementary Figure 5. A fraction of nonC-TL can be detected in publicly available immunopeptidomics datasets from human tumor biopsiesSupplementary Figure 6. Evaluation of pre-existing T-cell responses to candidate tumor antigens in cancer patientsSupplementary Figure 7. Enrichment of antigen-specific T cells from cancer patients by flow cytometry-based sorting.Supplementary Figure 8. Deconvolution of peptide pools containing recognized CGA or melanoma-associated antigens.Supplementary Figure 9. Functional avidity and specificity of neoantigen-specific T cells isolated from cancer patients.Supplementary Figure 10. HLA restriction element and recognition of autologous tumor cell lines by antigen-specific T cells isolated from cancer patients.SupplementaryFigure 11. Genomic location of the three immunogenic nonC-TL identified through IVS.Supplementary Figure 12. Recognition of the predicted ORFs encoding the immunogenic nonC-TL by TCR transduced cells.Supplementary Figure 13. Loss of recognition of TCL transfected with Cas9-sgRNA targeting the predicted 5’U-HOXC13 ORF.Supplementary Figure 14. Assessment of expression and translation of the immunogenic nonC-TL in human healthy cells.Supplementary Figure 15. NonC-TL are the main source of candidate tumor antigens identified by proteogenomics but tumor-reactive T cells preferentially recognize neoantigens derived from NSM.

Show patient characteristics and relevant experimental information

Purpose:Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from noncanonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability.Experimental Design:Peptides presented on HLA-I were identified in 9 patient-derived tumor cell lines from melanoma, gynecologic, and head and neck cancer through proteogenomics. A total of 507 candidate tumor antigens, including nonC-TL, neoantigens, cancer-germline, or melanocyte differentiation antigens, were tested for T-cell recognition of preexisting responses in patients with cancer. Donor peripheral blood lymphocytes (PBL) were in vitro sensitized against 170 selected nonC-TL to isolate antigen-specific T-cell receptors (TCR) and evaluate their therapeutic potential.Results:We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor-reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor PBL against 170 selected nonC-TL, led to the identification of TCRs specific to three nonC-TL, two of which mapped to the 5′ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a noncoding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells.Conclusions:Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.See related commentary by Fox et al., p. 2173

Nsun2 catalyzes 5-methylcytosine (m5C) formation in several types of RNA, including messenger RNAs (mRNAs), transfer RNAs, and other non-coding RNAs. In mRNA, m5C was reported to influence transcript stability. However, it is unclear if it has stabilizing or destabilizing effects. To address the role of Nsun2 in mRNA stability, we characterized the landscape of mRNA turnover dynamics during embryonic stem cell (ESC) differentiation in wild-type and Nsun2-mutant cells. By using an RNA labeling approach combined with thiouridine-to-cytidine-sequencing (TUC-seq), we demonstrate that mRNA synthesis and stability undergo extensive changes during normal cellular differentiation. Remarkably, a large proportion of these changes did not result in altered mRNA abundance, providing evidence for robust transcript buffering during ESC differentiation. Importantly, also the loss of Nsun2 affected mRNA turnover dynamics but not the steady-state levels of transcripts. Furthermore, our data indicate that the effect of Nsun2 on mRNA turnover was not mediated by m5C deposition in mRNA, which is corroborated by catalysis-independent effects of Nsun2 on translation in early ESC differentiation. In conclusion, this study demonstrates that differentiation as well as loss of Nsun2 can induce changes in mRNA turnover dynamics that are independent of mRNA methylation but consistent with a buffering mechanism to maintain constant RNA levels.