Biology subjects

Winter, P.

Publications and source records attributed to Winter, P..

5 recordsLinked to original sources

bioRxiv2025

Hierarchical cross-entropy loss improves atlas-scale single-cell annotation models

Accurately annotating cell types is essential for extracting biological insight from single-cell RNA-seq data. Although cell types are naturally organized into hierarchical ontologies, most computational models do not explicitly incorporate this structure into their training objectives. We introduce a hierarchical cross-entropy loss that aligns model objectives with biological structure. Applied to architectures ranging from linear models to transformers, this simple modification significantly improves out-of-distribution performance (12-15%) without added computational cost.

cell biology↗

bioRxiv2022

Cancer tissue of origin constrains the growth and metabolism of metastases

Metastases arise from a subset of cancer cells that disseminate from the primary tumor; however, the factors that contribute to proliferation of cancer cells in a secondary site are incompletely understood. The ability of cancer cells to thrive in a new tissue site is influenced by genetic and epigenetic changes that are important for disease initiation and progression, but these factors alone do not predict if and where cancers metastasize. Specific cancer types metastasize to consistent subsets of tissues, suggesting that factors associated with the primary tumor influence the tissue environments where cancers can grow. Using pancreatic cancer as a model, we find that primary and metastatic tumors share many metabolic similarities and that the tumor initiating capacity and proliferation of both primary- and metastasis-derived cells is favored in the primary site relative to the metastatic site. Moreover, propagating lung or liver metastatic cells in vivo to enrich for tumor cells adapted to grow in the lung or the liver does not enhance their relative ability to form large tumors in those sites, change their preference to grow in the primary site, nor stably alter some aspects of their metabolism relative to primary tumors. We also analyzed primary liver and lung cancer cells and find that these cells also exhibit a preference to grow in their primary site relative to metastatic sites. Together, these data suggest that the cancer tissue-of-origin influences the metabolism of both primary and metastatic tumors and may impact whether cancer cells can thrive in a metastatic site.

cancer biology↗

bioRxiv2020

Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

Advanced analytical strategies including top-down and middle-up HPLC-MS approaches have become powerful alternatives to classical bottom-up analysis for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up analysis of polyclonal IgGs posing additional challenges due to extensive sequence variability. The presented workflow is based on Fc/2 portions as conserved subunits of IgGs and enables global profiling of subclasses and their glycosylation patterns, both of which influence IgG effector functions. To obtain subunits of murine IgGs, we established digestion with the bacterial protease SpeB. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry allowing relative quantification of IgG subclasses and their N-glycosylation variants. In order to assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. This analysis simultaneously revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine as demonstrated for monoclonal IgGs, which may be implemented for quality control of functional antibodies.

biochemistry↗

bioRxiv2020

In silico design of Phl p 6 variants with altered folding stability significantly impacts antigen processing, immunogenicity and immune polarization

AbstactO_ST_ABSIntroductionC_ST_ABSProtein fold stability has been proposed to represent an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Using in silico prediction, we introduced point mutations in proteins that either increase or decrease their fold-stability without altering immunodominant epitopes or changing the overall structure of the protein. Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. MethodsUsing the MAESTRO software tool, stabilizing or destabilizing mutations were selected and verified by molecular dynamics simulations. The mutants were expressed in E. coli, purified tag-free, and analyzed for thermal stability and resistance to endolysosomal proteases. The resulting peptides were analysed by degradome assay and mass spectrometry. The structure of the most stable mutant protein was obtained by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. ResultsFour stabilizing and two destabilizing mutations were identified by MAESTRO. Experimentally determined changes in thermal stability compared to the wild type protein ranged from -5 to +14 {degrees}C. Destabilization led to faster proteolytic processing in vitro, whereas highly stabilized mutants were degraded very slowly. However, the overall pattern of identified peptides remained very similar. This was confirmed in bone marrow derived dendritic cells that processed and presented the immune dominant epitope from a destabilized mutant more efficiently. In vivo, stabilization resulted in a shift in immune polarization as indicated by higher levels of IgG2a and increased secretion of TH1/TH17 cytokines. ConclusionMAESTRO was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with susceptibility to protease resistance and presentation of pMHCII on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

immunology↗

bioRxiv2019

Mast cells and γδ T cells are largely dispensable for adaptive immune responses after laser-mediated epicutaneous immunization

BackgroundThe skin resembles an attractive target for vaccination due to its accessibility and abundance of resident immune cells. Cells like {gamma}{delta} T cells and mast cells (MCs) are part of the first line of defence against exogenous threats. Despite being important mediators for eliciting TH2 immune responses after epithelial stress, {gamma}{delta} T cell and MC function still remains to be completely understood. Here, we aimed to characterize their roles in shaping adaptive immune responses after laser-mediated epicutaneous immunization (EPI).\n\nMethods{gamma}{delta} T cell knock out, MC depleted, and wildtype control mice were immunized with mannan-conjugated grass pollen allergen Phl p 5 (P5-MN) by laser-mediated EPI. After 2-3 immunizations, cytokine expression, T helper polarization, and antigen-specific IgG1/IgE levels were analysed. The local cytokine/chemokine milieu after laser microporation was determined.\n\nResultsWhile the majority of inflammatory chemokines and cytokines induced by laser treatment was not affected by the presence of {gamma}{delta} T cells or MCs, RANTES, was elevated in {gamma}{delta} T cell knock out mice, and GRO and TSLP, were significantly decreased after MC depletion. However, absence of {gamma}{delta} T cells or depletion of MC had no substantial effect on adaptive humoral or cellular immune responses after laser-mediated EPI, except for slightly reduced IgG1 and effector T cell levels in MC depleted mice.\n\nConclusions{gamma}{delta} T cells did not play a pivotal role in shaping the humoral and cellular adaptive immune response after laser-mediated EPI, whereas MC depletion decreased numbers of effector T cells, indicating a potential role of MCs in the activation and maturation of T cells after EPI.\n\nHighlightsO_LILaser microporation induces an inflammatory chemokine milieu at the site of immunization\nC_LIO_LI{gamma}{delta} T cells and mast cells contribute to the steady-state or damage-induced cytokine milieu in the skin\nC_LIO_LI{gamma}{delta} T cells and mast cells are dispensable for adaptive immunity after laser-mediated immunization\nC_LI

immunology↗