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We recently conducted an exploratory analysis of murine T cell receptor (TCR) repertoire data (biorxiv doi:10.1101/2022.11.23.517563).Specifically, we analyzed the complement-determining region 3 (CDR3) of the TCRβ chain from naïve CD4+T cell populations that express high or low levels of the surface marker CD5 (CD5loand CD5hicells). This document describes in advance a series of analyses we will conduct on a held-out validation dataset from 10 mice to confirm the hypotheses derived from our exploratory analysis. We are providing this document in the interest of transparency. We are also publishing the exploration and validation datasets and the code used to perform the planned analyses alongsidethis document.

SummaryThe T cell receptor (TCR) determines the specificity and affinity for both foreign and self-peptides presented by MHC. It is established that self-pMHC reactivity impacts T cell function, but it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naïve CD4+T cells with low versus high self-pMHC reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that predicts self-reactivity directly from TCRβ sequences. This approach revealed that n-nucleotide additions and acidic amino acids weaken self-reactivity. We tested our ML predictions of TCRβ sequence self-reactivity using retrogenic mice. Extrapolating our analyses to independent datasets, we found high predicted self-reactivity for regulatory CD4+T cells and low predicted self-reactivity for T cells responding to chronic infection. Our analyses suggest a potential trade-off between repertoire diversity and self-reactivity intrinsic to the architecture of a TCR repertoire.

Listeria monocytogenes (Lm) is a food-borne pathogen that causes severe bacterial gastroenteritis, with high rates of hospitalization and mortality. Lm is ubiquitous in soil, water and livestock, and can survive and proliferate at low temperatures. Following oral ingestion of contaminated food, Lm crosses the epithelial through intestinal goblet cells in a mechanism depending on Lm InlA and host E-cadherin. Importantly, human infections typically occur with Lm growing at or below room temperature, which are flagellated and motile. Even though many important human bacterial pathogens are flagellated, little is known regarding the effect of bacterial motility on invasion and immune evasion.Here, we used complementary imaging and computer modeling approaches to test the hypothesis that bacterial motility helps Lm locate and engage target cells permissive for invasion. Imaging explanted mouse and human intestine, we confirmed that Lm grown at room temperature uses motility to scan the epithelial surface and preferentially attach to target cells. Furthermore, we integrated quantitative parameters from our imaging experiments to construct a versatile “layered” cellular Potts model (L-CPM) that simulates host-pathogen dynamics. Simulated data are consistent with the hypothesis that bacterial motility enhances invasion by allowing bacteria to search the epithelial surface for their preferred invasion targets. Indeed, our model consistently predicts that motile bacteria have invaded ∼2-fold more at the 1-hour mark. This invasion advantage persists even in the presence of host phagocytes, with the balance between invasion and phagocytosis governed almost entirely by bacterial motility.In conclusion, our simulations provide insight into host pathogen interactions and challenge fundamental assumptions regarding how phagocytes might limit bacterial invasion early during infection.

BackgroundImmunotherapy is currently part of the standard of care for patients with advanced-stage non-small cell lung cancer (NSCLC). However, many patients do not respond to this treatment, therefore combination strategies are being explored to increase clinical benefit. The PEMBRO-RT trial combined the therapeutic programmed cell death 1 (PD-1) antibody pembrolizumab with stereotactic body radiation therapy (SBRT) to increase the overall response rate and study the effects on the tumor microenvironment (TME).MethodsHere, immune infiltrates in the TME of patients included in the PEMBRO-RT trial were investigated. Tumor biopsies of patients treated with pembrolizumab alone or combined with SBRT (a biopsy of the non-irradiated site) at baseline and during treatment were stained with multiplex immunofluorescence for CD3, CD8, CD20, CD103 and FoxP3 for lymphocytes, pan-cytokeratin for tumors, and HLA-ABC expression was determined.ResultsThe total number of lymphocytes increased significantly after 6 weeks of treatment in the anti-PD-1 group (fold change: 1.87, 95% CI: 1.06 to 3.29) and the anti-PD-1+SBRT group (fold change: 2.29, 95% CI: 1.46 to 3.60). The combination of SBRT and anti-PD-1 induced a 4.87-fold increase (95% CI: 2.45 to 9.68) in CD103+cytotoxic T-cells 6 weeks on treatment and a 2.56-fold increase (95% CI: 1.03 to 6.36) after anti-PD-1 therapy alone. Responders had a significantly higher number of lymphocytes at baseline than non-responders (fold difference 1.85, 95% CI: 1.04 to 3.29 for anti-PD-1 and fold change 1.93, 95% CI: 1.08 to 3.44 for anti-PD-1+SBRT).ConclusionThis explorative study shows that that lymphocyte infiltration in general, instead of the infiltration of a specific lymphocyte subset, is associated with response to therapy in patients with NSCLC.Furthermore, anti-PD-1+SBRT combination therapy induces an immunological abscopal effect in the TME represented by a superior infiltration of cytotoxic T cells as compared with anti-PD-1 monotherapy.

Visualization of cell migration via time-lapse microscopy has greatly advanced our understanding of the immune system. However, subtle differences in migration dynamics are easily obscured by biases and imaging artifacts. While several analysis methods have been suggested to address these issues, an integrated tool implementing them is currently lacking. Here, we present celltrackR, an R package containing a diverse set of state-of-the-art analysis methods for (immune) cell tracks. CelltrackR supports the complete pipeline for track analysis by providing methods for data management, quality control, extracting and visualizing migration statistics, clustering tracks, and simulating cell migration. CelltrackR supports the analysis of both 2D and 3D cell tracks. CelltrackR is an open-source package released under the GPL-2 license, and is freely available on both GitHub and CRAN. Although the package was designed specifically for immune cell migration data, many of its methods will also be of use in other research areas dealing with moving objects.

Contains fulltext : 244706.pdf (Publisher’s version ) (Open Access) Causal diagrams such as directed acyclic graphs (DAGs) are used in several scientific fields to help design and analyze studies that aim to infer causal effects from observational data; for example, DAGs can help identify suitable strategies to reduce confounding bias. However, DAGs can be difficult to design, and the validity of any DAG-derived strategy hinges on the validity of the postulated DAG itself. Researchers should therefore check whether the assumptions encoded in the DAG are consistent with the data before proceeding with the analysis. Here, we explain how the R package 'dagitty', based on the web tool dagitty.net, can be used to test the statistical implications of the assumptions encoded in a given DAG. We hope that this will help researchers discover model specification errors, avoid erroneous conclusions, and build better models. (c) 2021 The Authors. Basic Protocol 1: Constructing and importing DAG models from the dagitty web interface Support Protocol 1: Installing R, RStudio, and the dagitty package Basic Protocol 2: Testing DAGs against categorical data Basic Protocol 3: Testing DAGs against continuous data Support Protocol 2: Testing DAGs against continuous data with non-linearities Basic Protocol 4: Testing DAGs against a combination of categorical and continuous data.

SummaryThe Cellular Potts Model (CPM) is a powerful in silico method for simulating diverse biological processes at tissue scale. Because of its inherently graphical nature, this model should in theory be accessible to a large audience of biologists – without requiring extensive mathematical expertise. But in practice, CPMs are mostly implemented in specialized frameworks that users need to master before they can run and modify the simulation. We here present Artistoo (Artificial Tissue Toolbox), a JavaScript library for building “explorable” CPM simulations where users can change model parameters and see their effects in real time. Artistoo simulations run directly in the web browser and do not require any third-party software, plugins, or back-end servers. Although implemented in JavaScript, Artistoo does not suffer from a major performance loss compared to frameworks written in C++; it remains sufficiently fast to let users interact with simulations in real time. Artistoo provides an opportunity to unlock CPM models for a broader audience: interactive simulations can be shared through a simple URL in a zero-install setting. We discuss how such model sharing may benefit modelling research, science dissemination, open science, and education.Availability and ImplementationArtistoo is an open-source library released under the MIT license, and is freely available on GitHub at https://github.com/ingewortel/artistoo.ContactJohannes.Textor@radboudumc.nl

Activating transcription factor 4 (ATF4) is a stress-induced transcription factor that is frequently upregulated in cancer cells. ATF4 controls the expression of a wide range of adaptive genes that allow cells to endure periods of stress, such as hypoxia or amino acid limitation. However, under persistent stress conditions, ATF4 promotes the induction of apoptosis. Recent advances point to a role for post-translational modifications (PTMs) and epigenetic mechanisms in balancing these pro- and anti-survival effects of ATF4. We review here how PTMs and epigenetic modifiers associated with ATF4 may be exploited by cancer cells to cope with cellular stress conditions that are intrinsically associated with tumor growth. Identification of mechanisms that modulate ATF4-mediated transcription and its effects on cellular metabolism may uncover new targets for cancer treatment.

SummaryVisualization of cell migration via time-lapse microscopy has greatly advanced our understanding of the immune system. However, subtle differences in migration dynamics are easily obscured by biases and imaging artifacts. While several analysis methods have been suggested to address these issues, an integrated tool implementing them is currently lacking. Here, we present CelltrackR, an R package containing a diverse set of state-of-the-art analysis methods for (immune) cell tracks. CelltrackR supports the complete pipeline for track analysis by providing methods for data management, quality control, extracting and visualizing migration statistics, clustering tracks, and simulating cell migration.Availability and ImplementationCelltrackR is an open-source package released under the GPL-2 license, and is freely available on GitHub at https://github.com/ingewortel/celltrackR.Contactmmiller23@wustl.edu, Johannes.Textor@radboudumc.nl

Contains fulltext : 190315.pdf (Publisher’s version ) (Open Access) Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.

Contains fulltext : 172708.pdf (Publisher’s version ) (Open Access) Chemotherapeutics, including the platinum compounds oxaliplatin (OXP) and cisplatin (CDDP), are standard care of treatment for cancer. Although chemotherapy has long been considered immunosuppressive, evidence now suggests that certain cytotoxic agents can efficiently stimulate antitumor responses, through the induction of a form of apoptosis, called immunogenic cell death (ICD). ICD is characterized by exposure of calreticulin and heat shock proteins (HSPs), secretion of ATP and release of high-mobility group box 1 (HMGB1). Proper activation of the immune system relies on the integration of these signals by dendritic cells (DCs). Studies on the crucial role of DCs, in the context of ICD, have been performed using mouse models or human in vitro-generated monocyte-derived DCs (moDCs), which do not fully recapitulate the in vivo situation. Here, we explore the effect of platinum-induced ICD on phenotype and function of human blood circulating DCs. Tumor cells were treated with OXP or CDDP and induction of ICD was investigated. We show that both platinum drugs triggered translocation of calreticulin and HSP70, as well as the release of ATP and HMGB1. Platinum treatment increased phagocytosis of tumor fragments by human blood DCs and enhanced phenotypic maturation of blood myeloid and plasmacytoid DCs. Moreover, upon interaction with platinum-treated tumor cells, CD1c(+) DCs efficiently stimulated allogeneic proliferation of T lymphocytes. Together, our observations indicate that platinum-treated tumor cells may exert an active stimulatory effect on human blood DCs. In particular, these data suggest that CD1c(+) DCs are critical mediators of immune responses induced by ICD. 15 p.