Biology subjects

Volkmar, M.

Publications and source records attributed to Volkmar, M..

5 recordsLinked to original sources

bioRxiv2025

Single-cell chromatin landscape and DNA methylation patterns reveal shared molecular programs in human tumor and non-tumor tissue CCR8+ Treg cells

Regulatory T (Treg) cells, a subset of CD4+ T cells, play a crucial role in immunoregulation. Notably, CCR8-expressing Treg cells in tissues also contribute to organ homeostasis and repair. To determine whether these tissue-regenerative programs are active in the tumor microenvironment, we employed single-cell chromatin accessibility and genome-wide DNA methylation analyses to investigate CCR8+ tissue Treg cells isolated from human tumor and adjacent tumor-free tissues. Our findings indicate that CCR8+ tissue Treg cells from tumor and corresponding tumor-free tissues exhibit a high degree of similarity, suggesting that the tumor microenvironment may harbor highly activated tissue Treg cells. This observation was consistent across various tumor types and origins, including primary tumors and metastases. Using quantitative proteomics, we identified several candidate factors associated with the regenerative and suppressive programs of Treg cells, which may serve as potential reservoir of druggable targets for future therapeutic interventions.

immunology↗

bioRxiv2023

optiPRM: A targeted immunopeptidomics LC-MS workflow with ultra-high sensitivity for the detection of mutation-derived tumor neoepitopes from limited input material

Personalized cancer immunotherapies such as vaccines and 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. For their detection by liquid chromatography mass spectrometry (LC-MS), 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 2.5x106 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, we here present optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which allowed for the identification of actionable neoepitopes from sample sizes usually available in the clinic.

immunology↗

bioRxiv2023

Incorporation of TRDV segments into TCR alpha chains

In human tumor models we tried to identify and clone the TCR of tumor-reactive T cells enriched in mixed lymphocyte tumor-cell cultures (MLTC). In a particular MLTC, we identified a predominant TCR beta chain using the Beta Mark Vbeta Kit, but a corresponding alpha chain could not be amplified via RT-PCR using TRAV-specific forward primers. Therefore, we applied 5RACE to obtain the TCR alpha chain sequences. The 5RACE product revealed an alpha chain that encompassed 89bp of the TRDV1 5UTR, followed by the TRDV1 coding sequence joined in frame to TRAJ24. The ORF reaching from the TRDV1 start codon to the TRAC segment was intact, suggesting a functional TCR. To analyze this MLTC population in greater depth we conducted 10X VDJ sequencing. CellRanger identified the beta chain known from the Beta Mark analysis, but no corresponding alpha chain in the filtered results. The corresponding TRDV-containing TCR alpha chain could, however, be detected in the "all_contig_annotations" files. In a separate project, we performed TCR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Also here, a predominant clonotype contained a TCR alpha chain joining Trdv2-2 in frame to Traj49. Transfection of both TCR cDNAs resulted in cell surface localization of TCR and CD3 as validated by FACS. Tumor recognition of the human, TRDV1-containing TCR could be demonstrated by IFNgamma ELISpot whereas the murine TCR did not recognize a tumor-derived cell line. TRDV-containing alpha chains have been reported in the literature for two HLA I-restricted TCRs against HIV peptides (Ueno et al, Eur J Immunol, 2003). To determine whether such TDRV-containing TCRs are unique events or whether Vdelta segments are commonly incorporated into TCR alpha chains, we queried the NCBI Sequence Read Archive (SRA) for 10X VDJ data and analyzed 21 human and 23 murine datasets. We found that especially TRDV1, Trdv1 and to some extent Trdv2-2 are more commonly incorporated into TCR alpha chains than some TRAV genes, making the TRDV segments a relevant contribution to TCR alpha diversity. For apparently solitary beta chains in 10X VDJ datasets, we suggest to scrutinize the "all_contig_annotations" files as these may contain an accompanying, TRDV-containing alpha chain.

immunology↗

bioRxiv2021

Deep molecular characterization linked to drug response profiling of pancreatic ductal adenocarcinoma using patient-derived organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by high drug resistance and poor prognosis. Novel therapeutic and stratification strategies are urgently needed. Here, we present an integration of in-depth genomic and transcriptomic characterization with drug screening and clinical outcome based on a catalogue of 51 patient-derived tumor organoids (PDOs) from resected PDAC. Known PDAC molecular subtypes and their prognostic value are conserved in organoids. Integration of transcriptomic and drug response profiles suggest a metabolism-mediated modulations of drug resistance. Copy number alterations on chromosome 13q and wild-type status of TP53 emerged as potential novel genomic biomarkers for sensitivity to 5-FU and oxaliplatin treatment, respectively. Functional testing of targeted drugs in PDOs revealed its additional value for genome-driven personalized oncology. Co-deletion of TP53/POLR2A increased vulnerability to RNA polymerase II inhibition, pointing to a promising target for personalized treatment in PDAC. SignificancePatient-derived PDAC organoids hold great promise as surrogate tumor models for personalized oncology. By integrating highly granular molecular, drug sensitivity and clinical data, we demonstrate that PDOs are valid models for molecular characterization and response prediction that also enable identification of novel drug sensitivity biomarkers and resistance mechanisms in PDAC.

cancer biology↗

bioRxiv2019

Phosphoproteomics of CD2 signaling reveals an AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells

The in-depth analysis of costimulatory signaling enhancing the activity of cytotoxic T cells (CTLs) represents a major approach towards immunotherapy development. Here we report that CD2 costimulation plays a critical role in killing by freshly isolated human CTLs, which represent a challenging but valuable study model to gain insight into CTL biology. We show that CD2 triggering critically aids signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules towards the microtubule-organizing center (MTOC). To gain insight into the underlying elusive mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeleton organization, autophagy and metabolism. Strikingly, signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) represents a functionally critical node of the CD2 network which regulates granule polarization towards the MTOC in CTLs. Granule trafficking is driven by active AMPK enriched on adjacent lysosomes, illustrating a novel signaling cross-talk between vesicular compartments in CTLs. Our results thus establish CD2 signaling as key for regulating cytotoxic killing and granule polarization in freshly isolated CTLs and strengthens the rationale to choose CD2 and AMPK as therapeutic targets to boost CTL activity.

immunology↗