Publications
Neoadjuvant combination immunotherapy in MSI/dMMR colorectal cancer
Suarez-Carmona M, Halama N
Trends in cancer
Abbreviations: ICI, immune checkpoint inhibition; MHC, major histocompatibility complex.
Abstract
Neoadjuvant immune checkpoint inhibition (ICI) is a new approach to treat patients with colorectal cancer (CRC). The effects of combined neoadjuvant ICI in locally advanced, DNA mismatch repair (dMMR)-deficient/microsatellite instable (MSI) CRC were recently reported by de Gooyer et al. from the NICHE-3 trial. Further studies will determine whether these impressive pathological responses lead to long-term clinical benefit.
Keywords: MSI; colorectal cancer; dMMR; immune checkpoint inhibition; neoadjuvant therapy.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.
optiPRM: A targeted immunopeptidomics LC-MS workflow with ultra-high sensitivity for the detection of mutation-derived tumor neoepitopes from limited input material
Salek M, Förster J D, Becker J P, Meyer M, Charoentong P, Lyu Y, Lindner K, Lotsch C, Volkmar M, Momburg F, Poschke I, Fröhling S, Schmitz M, Offringa R, Platten M, Jäger D, Zörnig I, Riemer A B.
Abstract
Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor (TCR)-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen (HLA) class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft (PDX) from as little as 2.5×106 cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which makes the identification of actionable neoepitopes possible from sample sizes usually available in the clinic.
© 2024. The Author(s).
Sphinganine recruits TLR4 adaptors in macrophages and promotes inflammation in murine models of sepsis and melanoma
Hering M, Madi A, Sandhoff R, Ma S, Wu J, Mieg A, Richter K, Mohr K, Knabe N. Stichling D, Poschet G, Bestvater F, Frank L, Utikal J, Umansky V, Cui G.
BioRender.com
Abstract
After recognizing its ligand lipopolysaccharide, Toll-like receptor 4 (TLR4) recruits adaptor proteins to the cell membrane, thereby initiating downstream signaling and triggering inflammation. Whether this recruitment of adaptor proteins is dependent solely on protein-protein interactions is unknown. Here, we report that the sphingolipid sphinganine physically interacts with the adaptor proteins MyD88 and TIRAP and promotes MyD88 recruitment in macrophages. Myeloid cell-specific deficiency in serine palmitoyltransferase long chain base subunit 2, which encodes the key enzyme catalyzing sphingolipid biosynthesis, decreases the membrane recruitment of MyD88 and inhibits inflammatory responses in in vitro bone marrow-derived macrophage and in vivo sepsis models. In a melanoma mouse model, serine palmitoyltransferase long chain base subunit 2 deficiency decreases anti-tumor myeloid cell responses and increases tumor growth. Therefore, sphinganine biosynthesis is required for the initiation of TLR4 signal transduction and serves as a checkpoint for macrophage pattern recognition in sepsis and melanoma mouse models.
© 2024. The Author(s).
The immunosuppressive drug cyclosporin A has an immunostimulatory function in CD8+ T cells
Wißfeld J, Hering M, ten Bosch N, Cui G
Abstract
Cyclosporin A is a well-established immunosuppressive drug used to treat or prevent graft-versus-host disease, the rejection of organ transplants, autoimmune disorders, and leukemia. It exerts its immunosuppressive effects by inhibiting calcineurin-mediated dephosphorylation of the nuclear factor of activated T cells (NFAT), thus preventing its nuclear entry and suppressing T cell activation. Here we report an unexpected immunostimulatory effect of cyclosporin A in activating the mammalian target of rapamycin complex 1 (mTORC1), a crucial metabolic hub required for T cell activation. Through screening a panel of tool compounds known to regulate mTORC1 activation, we found that cyclosporin A activated mTORC1 in CD8+ T cells in a 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB/AKT)-dependent manner. Mechanistically, cyclosporin A inhibited the calcineurin-mediated AKT dephosphorylation, thereby stabilizing mTORC1 signaling. Cyclosporin A synergized with mTORC1 pathway inhibitors, leading to potent suppression of proliferation and cytokine production in CD8+ T cells and an increase in the killing of acute T cell leukemia cells. Consequently, relying solely on CsA is insufficient to achieve optimal therapeutic outcomes. It is necessary to simultaneously target both the calcineurin-NFAT pathway and the mTORC1 pathway to maximize therapeutic efficacy.
T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells
Rodriguez Ehrenfried A, Zens S, Steffens LK, Kehm H, Paul A, Lauenstein C, Volkmar M, Poschke I, Meng Z, Offringa R.
Abstract
The advent of single-cell RNA sequencing (scRNAseq) has enabled in-depth gene expression analysis of several thousand cells isolated from tissues. We recently reported the application of scRNAseq toward the dissection of the tumor-infiltrating T-cell repertoire in human pancreatic cancer samples. In this study, we demonstrated that combined whole transcriptome and T-cell receptor (TCR) sequencing provides an effective way to identify tumor-reactive TCR clonotypes on the basis of gene expression signatures. An important aspect in this respect was the experimental validation of TCR-mediated anti-tumor reactivity by means of an in vitro functional assay, which is the subject of the present protocol. This assay involves the transient transfection of mRNA gene constructs encoding TCRα/β pairs into a well-defined human T-cell line, followed by co-cultivation with the tumor cells of interest and detection of T-cell activation by flow cytometry. Due to the high transfectability and the low background reactivity of the mock-transfected T-cell line to a wide variety of tumor cells, this assay offers a highly robust and versatile platform for the functional screening of large numbers of TCR clonotypes as identified in scRNAseq data sets. Whereas the assay was initially developed to test TCRs of human origin, it was more recently also applied successfully for the screening of TCRs of murine origin. Key features • Efficient functional screening of-and discrimination between-TCRs isolated from tumor-reactive vs. bystander T-cell clones. • Applicable to TCRs from CD8+ and CD4+ tumor-infiltrating T-cells originating from patient-derived tumor samples and syngeneic mouse tumor models. • Rapid flow cytometric detection of T-cell activation by means of TNFα and CD107a expression after a 5 h T-cell/tumor cell co-cultivation.
Prediction of tumor-reactive T cell receptors from scRNA-seq data for personalized T cell therapy
Tan CL, Lindner K, Boschert T, Meng Z, Rodriguez Ehrenfried A, De Roia A, Haltenhof G, Faenza A, Imperatore F, Bunse L, Lindner JM, Harbottle RP, Ratliff M, Offringa R, Poschke I, Platten M, Green EW
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Abstract
The identification of patient-derived, tumor-reactive T cell receptors (TCRs) as a basis for personalized transgenic T cell therapies remains a time- and cost-intensive endeavor. Current approaches to identify tumor-reactive TCRs analyze tumor mutations to predict T cell activating (neo)antigens and use these to either enrich tumor infiltrating lymphocyte (TIL) cultures or validate individual TCRs for transgenic autologous therapies. Here we combined high-throughput TCR cloning and reactivity validation to train predicTCR, a machine learning classifier that identifies individual tumor-reactive TILs in an antigen-agnostic manner based on single-TIL RNA sequencing. PredicTCR identifies tumor-reactive TCRs in TILs from diverse cancers better than previous gene set enrichment-based approaches, increasing specificity and sensitivity (geometric mean) from 0.38 to 0.74. By predicting tumor-reactive TCRs in a matter of days, TCR clonotypes can be prioritized to accelerate the manufacture of personalized T cell therapies.
a, An overview of the experimental and computational pipeline underlying the predicTCR classifier: TILs are sorted and subject to scRNA + VDJ-seq, while adjacent resected tumor material is used to establish the BT21 tumor cell line. TCR reactivity data are then integrated with scRNA + VDJ-seq data to train the predicTCR classifier, which is later tested on externally generated TIL datasets from diverse tumor types. b, Unsupervised clustering (UMAP plot) of scRNA-seq data of TILs (n = 5,651) recovered from brain metastasis sample, with key T cell subtypes annotated. c, The percentage frequency of the top 20 TIL TCR clonotypes and their distribution projected onto the UMAP, showing that cells of the same clonotype can occupy diverse phenotypic states. d, T cells transfected with one of the 50 most frequently occurring TIL-derived TCR clonotypes (representing 58 distinct TCR α/ß chain pairs) are cocultured with BT21 cells; the resulting levels of CD107a (as quantified by flow cytometry, gated on mTCRβ+ cells, which express the transgenic TCR as a chimera with the murine constant domain) demonstrate whether a given TCR clonotype recognizes the BT21 cell line. For details of settings per TCR reactivity threshold, see Methods. DMF5 is the HLA mismatched negative control TCR. e, BT21-reactive TCR clonotypes are more frequent than nonreactive clonotypes in the TIL population. f, BT21 reactivity testing results projected onto the UMAP plot (b).
T-FINDER: A highly sensitive, pan-HLA platform for functional T cell receptor and ligand discovery
Miray Cetin, Veronica Pinamonti, Theresa Schmid, Tamara Boschert, Ana Mellado Fuentes, Kristina Kromer, Taga Lerner, Jing Zhang, Yonata Herzig, Christopher Ehlert, Miguel Hernandez-Hernandez, Georgios Samaras, Claudia Maldonado Torres, Laura Fisch, Valeriia Dragan, Arlette Kouwenhoven, Bertrand Van Schoubroeck, Hans Wils, Carl Van Hove, Michael Platten, Edward W. Green, Frederik Stevenaert, Nathan J Felix, John M Lindner
Effective, unbiased, high-throughput methods to functionally identify both class II and class I HLA-presented T cell epitopes and their cognate T cell receptors (TCRs) are essential for and prerequisite to diagnostic and therapeutic applications, yet remain underdeveloped. Here, we present T-FINDER [T cell Functional Identification and (Neo)-antigen Discovery of Epitopes and Receptors], a system to rapidly deconvolute CD4 and CD8 TCRs and targets physiologically processed and presented by an individual's unmanipulated, complete human leukocyte antigen (HLA) haplotype. Combining a highly sensitive TCR signaling reporter with an antigen processing system to overcome previously undescribed limitations to target expression, T-FINDER both robustly identifies unknown peptide:HLA ligands from antigen libraries and rapidly screens and functionally validates the specificity of large TCR libraries against known or predicted targets. To demonstrate its capabilities, we apply the platform to multiple TCR-based applications, including diffuse midline glioma, celiac disease, and rheumatoid arthritis, providing unique biological insights and showcasing T-FINDER's potency and versatility.
H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient
Tamara Boschert, Kristina Kromer, Taga Lerner, Katharina Lindner, Gordon Haltenhof, Chin Leng Tan, Kristine Jähne, Isabel Poschke, Lukas Bunse, Philipp Eisele, Niklas Grassl, Iris Mildenberger, Katharina Sahm, Michael Platten, JohnM Lindner, Edward W Green
H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their complementarity-determining region 3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ and HLA-DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.
Thunder-DDA-PASEF enables high-coverage immunopeptidomics and identifies HLA class-I presented SarsCov-2 spike protein epitopes
David Gomez-Zepeda, Danielle Arnold-Schild, Julian Beyrle, Elena Kumm, Ute Distler, Hansjörg Schild, Stefan Tenzer
Human leukocyte antigen (HLA) class I peptide ligands (HLAIps) are key targets for developing vaccines and immunotherapies against infectious pathogens or cancer cells. Identifying HLAIps is challenging due to their high diversity, low abundance, and patient-specific profiles. Here, we developed a highly sensitive method for identifying HLAIps using liquid chromatography-ion mobility-tandem mass spectrometry (LC-IMS-MS/MS). The optimized method, Thunder-DDA-PASEF, semi-selectively fragments HLAIps based on their IMS and m/z, thus increasing the coverage of immunopeptidomics analyses. Thunder-DDA-PASEF includes singly-charged peptides, which contributes to more than 35% of the HLAIp identifications. Combined with MS2Rescore, Thunder-DDA-PASEF improved ligandome coverage by 150% compared to the original-DDA-PASEF method, and enabled in-depth profiling of HLAIps from two human cell lines, JY and Raji, transfected to express the SARS-CoV-2 spike protein. We identified seventeen spike protein HLAIps, thirteen of which had been reported to elicit immune responses in human patients.
Metabolic regulation of immune responses to cancer
Jannis Wißfeld*, Anke Werner*, Xin Yan*, Nora Ten Bosch*, Guoliang Cui
*authors contributed equally
The tumor microenvironment is an ecosystem composed of multiple types of cells, such as tumor cells, immune cells, and cancer-associated fibroblasts. Cancer cells grow faster than non-cancerous cells and consume larger amounts of nutrients. The rapid growth characteristic of cancer cells fundamentally alters nutrient availability in the tumor microenvironment and results in reprogramming of immune cell metabolic pathways. Accumulating evidence suggests that cellular metabolism of nutrients, such as lipids and amino acids, beyond being essential to meet the bioenergetic and biosynthetic demands of immune cells, also regulates a broad spectrum of cellular signal transduction, and influences immune cell survival, differentiation, and anti-tumor effector function. The cancer immunometabolism research field is rapidly evolving, and exciting new discoveries are reported in high-profile journals nearly weekly. Therefore, all new findings in this field cannot be summarized within this short review. Instead, this review is intended to provide a brief introduction to this rapidly developing research field, with a focus on the metabolism of two classes of important nutrients-lipids and amino acids-in immune cells. We highlight recent research on the roles of lipids and amino acids in regulating the metabolic fitness and immunological functions of T cells, macrophages, and natural killer cells in the tumor microenvironment. Furthermore, we discuss the possibility of "editing" metabolic pathways in immune cells to act synergistically with currently available immunotherapies in enhancing anti-tumor immune responses.
