Biology subjects

Wagner, A.

Publications and source records attributed to Wagner, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Dose-dependent regulation of immune memory responses against HIV by saponin monophosphoryl lipid A nanoparticle adjuvant

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play an important role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (TMem and BMem) and long-lived plasma cells in non-human primates. Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L+OX40+ CD4 TMem (11-fold), IFN{gamma}+ CD4 TMem (15-fold), IL21+ CD4 TMem (9-fold), circulating TFH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in non-human primates, which are relevant for human use and next-generation vaccines.

immunology↗

bioRxiv2024

Tree diversity increases carbon stocks and fluxes above- but not belowground in a tropical forest experiment

International commitments advocate large-scale forest restoration as a nature-based solution to climate change mitigation through carbon (C) sequestration. Mounting evidence suggests that mixed compared to monospecific planted forests may sequester more C, exhibit lower susceptibility to climate extremes and offer a broader range of ecosystem services. However, experimental studies comprehensively examining the control of tree diversity on multiple C stocks and fluxes above- and belowground are lacking. To address this gap, we leverage data from the Sardinilla experiment in Panama, the oldest tropical tree diversity experiment which features a gradient of one-, two-, three- and five-species mixtures of native tree species. Over 16 years, we measured multiple above- and belowground C stocks and fluxes, ranging from tree aboveground C, over leaf litter C production, to soil organic carbon (SOC). We show that tree diversity significantly increased aboveground C stocks and fluxes, with a 57% higher gain in aboveground tree C in five-species mixtures compared to monocultures (35.7{+/-}1.8 vs 22.8{+/-}3.4 Mg C ha-1) 16 years after planting. In contrast, we observed a net reduction in SOC (on average -11.2{+/-}1.1 Mg C ha-1) and no significant difference in SOC3 stocks (the predominantly tree-derived, i.e., C3 plant-derived SOC fraction) between five-species mixtures and monocultures (13.0{+/-}0.9 vs 15.1{+/-}1.3 Mg C ha-1). Positive tree diversity effects persisted despite repeated climate extremes and strengthened over time for aboveground tree growth. Structural equation models showed that higher tree growth in mixtures enhanced leaf litter and coarse woody debris C fluxes to the soil, resulting in a tightly linked C cycle aboveground. However, we did not observe significant links between above- and belowground C stocks and fluxes. Our study elucidates the mechanisms through which higher tree diversity bolsters the climate mitigation potential of tropical forest restoration. Restoration schemes should prioritize mixed over monospecific planted forests.

ecology↗

bioRxiv2024

Engineered Ubiquitin Variants Mitigate Pathogenic Bacterial Ubiquitin Ligase Function

During infection some pathogenic gram-negative bacteria, such as Salmonella, manipulate the host ubiquitination system through the delivery of secreted effectors known as novel E3 ubiquitin ligases (NELs). Despite the presence of NELs amongst these well-studied bacterial species, their unique structure has limited the tools that are available to probe their molecular mechanisms and explore their therapeutic potential. In this work, we report the identification of two high affinity engineered ubiquitin variants that can modulate the activity of the Salmonella enterica serovar Typhimurium encoded NEL, SspH1. We show that these ubiquitin variants suppress SspH1-mediated toxicity phenotypes in Saccharomyces cerevisiae. Additionally, we provide microscopic and flow cytometric evidence that SspH1-mediated toxicity is caused by interference with S. cerevisiae cell cycle progression that can be suppressed in the presence of ubiquitin variants. In vitro ubiquitination assays revealed that these ubiquitin variants increased the amount of SspH1-mediated ubiquitin chain formation. Interestingly, despite the increase in ubiquitin chains, we observe a relative decrease in the formation of SspH1-mediated K48-linked ubiquitin chains on its substrate, PKN1. Taken together our findings suggest that SspH1 toxicity in S. cerevisiae occurs through cell cycle interference and that an engineered ubiquitin variant approach can be used to identify modulators of bacterially encoded ubiquitin ligases. Author SummaryNovel E3 ligases (NELs) are a family of secreted effectors found in various pathogenic gram- negative bacteria. During infection these effectors hijack vital host ubiquitin signaling pathways to aid bacterial invasion and persistence. Despite interacting with a protein as highly conserved as ubiquitin, they have a distinct architecture relative to the eukaryotic E3 enzymes. This unique architecture combined with the indispensable role ubiquitin signaling plays in host cell survival has made hindering the contribution of NELs to bacterial infections a difficult task. Here, we applied protein engineering technology to identify two ubiquitin variants (Ubvs) with high affinity for SspH1, a Salmonella-encoded NEL. We provide evidence that these high affinity Ubvs suppress a known SspH1-meidated toxicity phenotype in the eukaryotic model system Saccharomyces cerevisiae. We also show that this suppression occurs without interfering with host ubiquitin signaling. Furthermore, we demonstrate the ability of a Ubv to modulate the activity of SspH1 in vitro, ultimately altering the lysine linkages found in SspH1-mediated ubiquitination. To our knowledge, this is the first evidence that an engineered ubiquitin variant approach can be implemented to modulate the activity of a family of previously untargetable bacterial-encoded E3 ligases.

microbiology↗

bioRxiv2024

Full-length structure of the host targeted bacterial effector Bep1 reveals a novel structural domain conserved in FIC effector proteins from Bartonella

Bacterial effector proteins translocated via a type-IV secretion system (T4SS) typically harbor a C-terminal segment required for recognition by the type-IV secretion coupling protein 1. In the -proteobacterial pathogen Bartonella, the signal is bipartite being composed of a BID (Bep intracellular delivery) domain and a positively charged C-terminal tail 2. Here, we show the crystal structure of full length Bartonella effector protein 1 (Bep1), which shows a novel FIC - OB - BAS(BID) domain arrangement conserved in the majority of Beps with the BID domain inserted into the newly discovered BAS parent domain. We propose that the BAS domain is necessary for the overall "boomerang"-like shape of Bep1 and that it plays a role during translocation through the T4SS.

microbiology↗

bioRxiv2024

5'-untranslated region sequences enhance plasmid-based protein production in Sulfolobus acidocaldarius

Sulfolobus acidocaldarius, a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of the saci_2122 promoter (Para), providing low basal activity and high pentose-dependent induction. However, available expression vectors lack a 5-terminal untranslated region (5-UTR), which is a typical element in bacterial expression vectors, usually significantly enhancing protein production in bacteria. To establish S. acidocaldarius as a production strain in biotechnology in the long-term, it is intrinsically relevant to optimize its tools and capacities to increase production efficiencies. Here we show that protein production is increased by the integration of S. acidocaldarius 5-UTRs into Para expression plasmids. Using the esterase Saci_1116 as a reporter protein, we observed a fourfold increase in soluble and active protein yield upon insertion of the saci_1322 (alba) 5-UTR. Screening of four additional 5-UTRs from other highly abundant proteins (th, slaA, slaB, saci_0330) revealed a consistent enhancement in target protein production. Additionally, site-directed mutagenesis of the Shine-Dalgarno (SD) motif within the alba 5-UTR revealed its significance for protein synthesis. Ultimately, the alba 5-UTR optimized expression vector demonstrated successful applicability in expressing various proteins, exemplified by its utilization for archaeal glycosyltransferases. Our results demonstrate that the integration of SD-motif containing 5-UTRs significantly boosted plasmid-based protein production in S. acidocaldarius. This advancement in recombinant expression not only broadens the utility of S. acidocaldarius as an archaeal expression platform but also marks a significant step toward potential biotechnological applications.

molecular biology↗

bioRxiv2024

Promoter motifs facilitate as well as impede the emergence and evolution of gene expression

Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called -10 and -35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 "promoter islands", DNA sequences enriched with -10 and -35 boxes. We mutagenize these starting "parent" sequences, and measure gene expression driven by 240000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new -10 and -35 boxes at unique positions in the library, but only [~]0.3% of these resulted in de-novo promoter activity. Only [~]13% of all -10 and -35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations create new -10 and -35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that -10 and -35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de-novo genes, and phenotypic evolution.

evolutionary biology↗

bioRxiv2024

Genotype sampling for deep-learning assisted experimental mapping of fitness landscapes

MotivationExperimental characterization of fitness landscapes, which map genotypes onto fitness, is important for both evolutionary biology and protein engineering. It faces a fundamental obstacle in the astronomical number of genotypes whose fitness needs to be measured for any one protein. Deep learning may help to predict the fitness of many genotypes from a smaller neural network training sample of genotypes with experimentally measured fitness. Here I use a recently published experimentally mapped fitness landscape of more than 260,000 protein genotypes to ask how such sampling is best performed. ResultsI show that multilayer perceptrons, recurrent neural networks (RNNs), convolutional networks, and transformers, can explain more than 90 percent of fitness variance in the data. In addition, 90 percent of this performance is reached with a training sample comprising merely {approx}103 sequences. Generalization to unseen test data is best when training data is sampled randomly and uniformly, or sampled to minimize the number of synonymous sequences. In contrast, sampling to maximize sequence diversity or codon usage bias reduces performance substantially. These observations hold for more than one network architecture. Simple sampling strategies may perform best when training deep learning neural networks to map fitness landscapes from experimental data.

bioinformatics↗

bioRxiv2024

Multimodal profiling reveals tissue-directed signatures of human immune cells altered with age

The immune system comprises multiple cell lineages and heterogeneous subsets found in blood and tissues throughout the body. While human immune responses differ between sites and over age, the underlying sources of variation remain unclear as most studies are limited to peripheral blood. Here, we took a systems approach to comprehensively profile RNA and surface protein expression of over 1.25 million immune cells isolated from blood, lymphoid organs, and mucosal tissues of 24 organ donors aged 20-75 years. We applied a multimodal classifier to annotate the major immune cell lineages (T cells, B cells, innate lymphoid cells, and myeloid cells) and their corresponding subsets across the body, leveraging probabilistic modeling to define bases for immune variations across donors, tissue, and age. We identified dominant tissue-specific effects on immune cell composition and function across lineages for lymphoid sites, intestines, and blood-rich tissues. Age-associated effects were intrinsic to both lineage and site as manifested by macrophages in mucosal sites, B cells in lymphoid organs, and T and NK cells in blood-rich sites. Our results reveal tissue-specific signatures of immune homeostasis throughout the body and across different ages. This information provides a basis for defining the transcriptional underpinnings of immune variation and potential associations with disease-associated immune pathologies across the human lifespan.

immunology↗

bioRxiv2023

Oligomerisation mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 [A]2 and a smaller one of 415 [A]2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

biochemistry↗

bioRxiv2023

Mobile DNA is replete with hotspots for the de novo emergence of gene regulation

Transposable elements can alter gene regulation in their host genome, either when they integrate into a genome, or when they accrue mutations after integration. However, the extent to which transposons can alter gene expression, as well as the necessary mutational steps, are not well characterized. Here we study the gene regulatory potential of the prominent IS3 family of transposable elements in E.coli. We started with 10 sequences from the ends of 5 IS3 sequences, created 18,537 random mutations in them, and measured their promoter activity using a massively parallel reporter assay. All 10 sequences could evolve de-novo promoter activity from single point mutations. De-novo promoters mostly emerge from existing proto-promoter sequences when mutations create new -10 boxes downstream of preexisting -35 boxes. The ends of IS3s harbor ~1.5 times as many such proto-promoter sequences than the E. coli genome. We also estimate that at least 26% of the 706 characterized IS3s already encode promoters. Our study shows that transposable elements can have a high latent cis-regulatory potential. Our observations can help to explain why mobile DNA may persist in prokaryotic genomes. They also underline the potential use of transposable elements as a substrate for evolving new gene expression.

evolutionary biology↗

bioRxiv2023

Horizontal gene transfer of a key translation protein has shaped the polyproline proteome

Prolines take longer than other amino acids to be incorporated into nascent proteins and cause ribosomes to stall during translation. This phenomenon occurs in all domains of life and is exacerbated at polyproline motifs. Such stalling can be eased by elongation factor P (EFP) in bacteria. We discovered a potential connection between horizontally transferred EFP variants and genomic signs of EFP dysfunction. Horizontal transfer of the efp gene has occurred several times throughout the bacterial tree of life, and such transfer events are associated with the loss of otherwise highly conserved polyproline motifs. In this study, we pinpoint cases of horizontal EFP transfer among a diverse set of bacterial genomes and examine the consequences of these events on genome evolution in the phyla Thermotogota and Planctomycetes. In these phyla, horizontal EFP transfer is not only associated with the loss of conserved polyproline motifs, but also with the loss of entire polyproline motif containing proteins, whose expression is likely dependent on EFP. In particular, three proteases (Lon, ClpC, and FtsH) and three tRNA synthetases (ValS, IleS1, IleS2) appear highly sensitive to EFP transfer. The conserved polyproline motifs within these proteins all reside within, or in close proximity to ATP binding regions, some of which have been shown to be crucial to their function. Our work shows that the horizontal transfer of EFP has left genomic traces that persist to this day. It also implies that the process of domesticating a horizontally transferred efp gene can perturb the overall function of EFP.

evolutionary biology↗

bioRxiv2023

Activation-induced shift in nutrient preference and function-specific nutrient dependence in human neutrophils

Neutrophils - the first responders in innate immunity - perform a variety of effector functions associated with specific metabolic demand. To maintain fitness and support functions, neutrophils have been found to utilize extracellular glucose, intracellular glycogen, and other alternative substrates. However, the quantitative contribution of these nutrients under specific conditions and the relative dependence of various cell functions on specific nutrients remain unclear. Here, using ex vivo and in vivo isotopic tracing, we reveal that under resting condition, human peripheral blood neutrophils, in contrast to in vitro cultured human neutrophil-like cell lines, rely on glycogen as a major direct source of glycolysis and pentose phosphate pathway. Upon activation with a diversity of stimuli, neutrophils undergo a significant and often rapid nutrient preference shift, with glucose becoming the dominant metabolic source thanks to a multi-fold increase in glucose uptake mechanistically mediated by the phosphorylation and translocation of GLUT1. At the same time, cycling between gross glycogenesis and glycogenolysis is also substantially increased, while the net flux favors sustained or increased glycogen storage. The shift in nutrient utilization impacts neutrophil functions in a function-specific manner. The activation of oxidative burst specifically depends on the utilization of extracellular glucose rather than glycogen. In contrast, the release of neutrophil traps can be flexibly supported by either glucose or glycogen. Neutrophil migration and fungal control is promoted by the shift away from glycogen utilization. Together, these results quantitatively characterize fundamental features of neutrophil metabolism and elucidate how metabolic remodeling shapes neutrophil functions upon activation.

biochemistry↗

bioRxiv2023

A direct experimental test of Ohno's hypothesis

Gene duplication drives evolution by providing raw material for proteins with novel functions. The oldest and historically most influential hypothesis about the evolutionary fate and potential of duplicated genes has been proposed by Susumu Ohno in 1970. This hypothesis essentially posits that gene duplication can help genes tolerate new mutations and thus facilitates the evolution of new phenotypes. Competing hypotheses argue that deleterious mutations will usually inactivate gene duplicates too rapidly for Ohnos hypothesis to work. Here, we provide a first direct experimental test of Ohnos hypothesis. Specifically, we evolved one or exactly two copies of a gene encoding a fluorescent protein in Escherichia coli through multiple rounds of mutagenesis and selection. We then analyzed the genotypic and phenotypic evolutionary dynamics of the evolving populations through high-throughput DNA sequencing, biochemical assays, and engineering of selected variants. In support of Ohnos hypothesis, populations carrying two gene copies displayed higher mutational robustness than those carrying a single gene copy. As a consequence, the double-copy populations experienced relaxed purifying selection, evolved higher phenotypic and genetic diversity, carried more mutations and accumulated combinations of key beneficial mutations earlier. However, their phenotypic evolution was not accelerated, possibly because one gene copy rapidly became inactivated by deleterious mutations. Our work provides an experimental platform to test models of evolution by gene duplication, and it supports alternatives to Ohnos hypothesis that point to the importance of gene dosage.

evolutionary biology↗

bioRxiv2023

The highly rugged yet navigable regulatory landscape of the bacterial transcription factor TetR

Transcription factor binding sites (TFBSs) are important sources of evolutionary innovations. Understanding how evolution navigates the sequence space of such sites can be achieved by mapping TFBS adaptive landscapes. In such a landscape, an individual location corresponds to a TFBS bound by a transcription factor. The elevation at that location corresponds to the strength of transcriptional regulation conveyed by the sequence. We developed an in vivo massively parallel reporter assay to map the landscape of bacterial TFBSs. We applied this assay to the TetR repressor, for which few TFBSs are known. We quantify the strength of transcriptional repression for 17,765 TFBSs and show that the resulting landscape is highly rugged, with 2,092 peaks. Only a few peaks convey stronger repression than the wild type. Non-additive (epistatic) interactions between mutations are frequent. Despite these hallmarks of ruggedness, most high peaks are evolutionarily accessible. They have large basins of attraction and are reached by around 20% of populations evolving on the landscape. Which high peak is reached during evolution is unpredictable and contingent on the mutational path taken. This first in-depth analysis of a prokaryotic gene regulator reveals a landscape that is navigable but much more rugged than the landscapes of eukaryotic regulators. SignificanceUnderstanding how evolution explores the vast space of genotypic possibilities is a fundamental question in evolutionary biology. The mapping of genotypes to quantitative traits (such as phenotypes and fitness) allows us to delineate adaptive landscapes and their topological properties, shedding light on how evolution can navigate such vast spaces. In this study, we focused on mapping a transcription factor binding site (TFBS) landscape to gene expression levels, as changes in gene expression patterns play a crucial role in biological innovation. We developed a massively parallel reporter assay and mapped the first comprehensive in vivo gene regulatory landscape for a bacterial transcriptional regulator, TetR. Surprisingly, this landscape is way more rugged than those observed in eukaryotic regulators. Despite its ruggedness, the landscape remains highly navigable through adaptive evolution. Our study presents the first high-resolution landscape for a bacterial TFBS, offering valuable insights into the evolution of TFBS in vivo. Moreover, it holds promise as a framework for discovering new genetic components for synthetic biological systems.

evolutionary biology↗

bioRxiv2023

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend towards high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time, and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass resolving quadrupole, the Orbitrap and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared to state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with high dynamic range and resolution, synchronized with Astral analyzers acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/543408v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1eedfdeorg.highwire.dtl.DTLVardef@1ab7939org.highwire.dtl.DTLVardef@a3b2f8org.highwire.dtl.DTLVardef@13ac46b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A longitudinal single-cell therapeutic atlas of anti-tumour necrosis factor treatment in inflammatory bowel disease

Precision medicine in immune-mediated inflammatory diseases (IMIDs) requires an understanding of how cellular networks change following therapy. We describe a therapeutic atlas for Crohns disease (CD) and ulcerative colitis (UC) following anti-tumour necrosis factor (TNF) therapy. We generated ~1 million single-cell transcriptomes, organised into 109 cell states, from 216 gut biopsies from 38 patients and three controls, revealing disease- and therapy-specific differences. A systems-biology analysis identified distinct spatially-resolved cellular microenvironments: granuloma signatures in CD and interferon (IFN)-response signatures localising to T-cell aggregates and epithelial damage in CD and UC. Longitudinal comparisons demonstrated that disease progression in non-responders associated with myeloid and stromal cell perturbations in CD and increased multi-cellular IFN signalling in UC. IFN signalling was also observed in rheumatoid arthritis (RA) synovium with a lymphoid pathotype. Our therapeutic atlas informs drug positioning across IMIDs, and suggests a rationale for the use of janus kinase (JAK) inhibition following anti-TNF resistance.

immunology↗

bioRxiv2023

Clonal associations of lymphocyte subsets and functional states revealed by single cell antigen receptor profiling of T and B cells in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is an autoimmune disease initiated by antigen-specific T cells and B cells, which promote synovial inflammation through a complex set of interactions with innate immune and stromal cells. To better understand the phenotypes and clonal relationships of synovial T and B cells, we performed single-cell RNA and repertoire sequencing on paired synovial tissue and peripheral blood samples from 12 donors with seropositive RA ranging from early to chronic disease. Paired transcriptomic-repertoire analyses highlighted 3 clonally distinct CD4 T cells populations that were enriched in RA synovium: T peripheral helper (Tph) and T follicular helper (Tfh) cells, CCL5+ T cells, and T regulatory cells (Tregs). Among these cells, Tph cells showed a unique transcriptomic signature of recent T cell receptor (TCR) activation, and clonally expanded Tph cells expressed an elevated transcriptomic effector signature compared to non-expanded Tph cells. CD8 T cells showed higher oligoclonality than CD4 T cells, and the largest CD8 T cell clones in synovium were highly enriched in GZMK+ cells. TCR analyses revealed CD8 T cells with likely viral-reactive TCRs distributed across transcriptomic clusters and definitively identified MAIT cells in synovium, which showed transcriptomic features of TCR activation. Among B cells, non-naive B cells including age-associated B cells (ABC), NR4A1+ activated B cells, and plasma cells, were enriched in synovium and had higher somatic hypermutation rates compared to blood B cells. Synovial B cells demonstrated substantial clonal expansion, with ABC, memory, and activated B cells clonally linked to synovial plasma cells. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate RA synovium.

immunology↗

bioRxiv2023

A phase-field model for non-small cell lung cancer under the effects of immunotherapy

Formulating tumor models that predict growth under therapy is vital for improving patient-specific treatment plans. In this context, we present our recent work on simulating non-small-scale cell lung cancer (NSCLC) in a simple, deterministic setting for two different patients receiving an immunotherapeutic treatment. At its core, our model consists of a Cahn-Hilliard-based phase-field model describing the evolution of proliferative and necrotic tumor cells. These are coupled to a simplified nutrient model that drives the growth of the proliferative cells and their decay into necrotic cells. The applied immunotherapy decreases the proliferative cell concentration. Here, we model the immunotherapeutic agent concentration in the entire lung over time by an ordinary differential equation (ODE). Finally, reaction terms provide a coupling between all these equations. By assuming spherical, symmetric tumor growth and constant nutrient inflow, we simplify this full 3D cancer simulation model to a reduced 1D model. We can then resort to patient data gathered from computed tomography (CT) scans over several years to calibrate our model. For the reduced 1D model, we show that our model can qualitatively describe observations during immunotherapy by fitting our model parameters to existing patient data. Our model covers cases in which the immunotherapy is successful and limits the tumor size, as well as cases predicting a sudden relapse, leading to exponential tumor growth. Finally, we move from the reduced model back to the full 3D cancer simulation in the lung tissue. Thereby, we show the predictive benefits a more detailed patient-specific simulation including spatial information could yield in the future. Author summaryLung cancer is one of the deadliest diseases, with low long-term survival rates. Its treatment is still very heuristic since patients respond to the same treatment plans differs significantly. Therefore, patient-specific models for predicting tumor growth and the treatment response are necessary for clinicians to make informed decisions about the patients therapy and avoid a trial and error based approach. We made a first small step in that direction by introducing a model for simulating cancer growth and its treatment inside a 3D lung geometry. In this model, we represented tumor cells by a volume fraction field that varies over space and time. We described their evolution by a system of partial differential equations, which include patient- and treatment-specific parameters capturing the different responses of patients to the therapies. Our simulation results were compared to clinical data and showed that we can quantitatively describe the tumors behavior with a suitable parameter set. This enabled us to change therapies in simulation runs and analyze how these changes could have impacted the patients health.

cell biology↗