Biology subjects

Polak, M. E.

Publications and source records attributed to Polak, M. E..

8 recordsLinked to original sources

bioRxiv2023

Convergent evolution of monocyte differentiation in adult skin permits repair of the Langerhans cell network

Langerhans cells (LCs) maintain tissue and immunological homeostasis at the epidermal barrier site. They are unique among phagocytes in functioning both as embryo-derived, tissue-resident macrophages that influence skin innervation and repair, and as migrating professional antigen presenting cells, a capability classically assigned to dendritic cells (DCs). Here we report the mechanisms that determine this dual identity. Using ablation of embryo-derived LCs in murine adult skin and tracked differentiation of incoming monocyte-derived replacements, we reveal intrinsic intra-epidermal heterogeneity. We demonstrate that monocyte-dendritic cell progenitor (MDP)-derived monocytes are selected for survival in the skin environment. Within the epidermis, the hair follicle niche subsequently provides an initial site of LC commitment, likely via Notch signaling, prior to metabolic adaptation and survival of differentiated monocyte-derived LCs. In human skin, we show that embryo-derived (e)LCs in newborns retain transcriptional evidence of their macrophage origin, but this is superseded by distinct DC-like immune modules after post-natal expansion of eLCs. Thus, intrinsic and extrinsic adaptations to adult skin niches replicate conditioning of eLC at birth, permitting repair of the unique LC network.

immunology↗

bioRxiv2022

Rank Selection Genetic Algorithm optimises robust parameter estimation for systems biology models

The ability to reliably predict and infer cellular responses to environmental exposures would offer a major advance in the investigation of immune regulation in health and disease. One possible approach is the use of in silico modelling. Design of such a mathematical kinetic model would be based on existing knowledge of a biological system and utilise a partial data set to parameterise. However, the process of parameter estimation, key for the accuracy of the model, is difficult to conduct by hand, and thus a computational alternative is necessary. We report the utility of Genetic Algorithm with Rank Selection (GARS) as a parameter estimation tool on multiple biological models, including heat shock, signal transduction via ERK, circadian rhythm and NF{kappa}B systems, where it showed strong accuracy and superiority to the Extended Kalman Filter method, Algebraic Difference Equations, and MATLAB fminsearch approaches. GARS parameter estimation is a valuable tool for biological data because it reliably infers system behaviour from partial data sets, allowing for the prediction of cellular responses to environmental exposures.

systems biology↗

bioRxiv2021

Dual Dean entrainment with volume ratio modulation for efficient co-encapsulation: Extreme single-cell indexing

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ~1 million cells/hour, while increasing assay signal-to-noise with cell multiplet rates of ~2.5% and capturing ~70% of cells. The method, called Pirouette-seq, extends our capacity to investigate biological systems. TOC AbstractPirouette-seq involves cell and reporter bead inertial ordering for efficient co-encapsulation, achieving a throughput of 1 million cells/hour, a 2.5% multiplet rate and a 70% cell capture efficiency.

cell biology↗

bioRxiv2021

TNF signalling fine-tunes Langerhans cell transcriptional programmes mediating adaptive immunity.

Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.

systems biology↗

bioRxiv2020

A DNA vaccine strategy to activate natural killer cells for therapeutic targeting of XPO1-positive tumors

Natural killer (NK) cells are key components of the immune response to viral infections and cancer. Their functions are controlled by activating and inhibitory killer-cell immunoglobulin-like receptors (KIR) which have MHC class I ligands. KIR2DS2 is an activating KIR, that binds conserved viral peptides in the context of HLA-C and has been associated with protective responses to both cancer and viral infections. We sought to investigate whether NK cells can be specifically activated in a peptide:MHC dependent manner to generate functional immune responses as a potential immunotherapeutic strategy. We developed a peptide-based KIR targeting DNA vaccine. Immunizing KIR-Tg mice with the vaccine construct generated in vivo peptide-specific activation of KIR2DS2-positive NK cells leading to canonical and cross-reactive peptide specific immune responses in vitro, and also in vivo inhibition of tumor growth. Using immunopeptidomics we identified that the nuclear export protein XPO1, which has been associated with a poor prognosis in many different human cancers, furnishes an HLA-C restricted cancer-associated peptide ligand for KIR2DS2-positive NK cells. We thus define a novel strategy to activate KIR in a peptide-specific manner and identify a rationale for its use in cancer immunotherapy. Significance statementNatural killer (NK) cells are known to have important roles in determining the outcomes of viral infections and cancer. The killer cell immunoglobulin-like receptors (KIR), and in particular the activating receptor KIR2DS2, have been associated with the outcome of a number of different human cancers. Specific activation of NK cells through KIR2DS2 is challenging because it shares high (>98%) sequence homology with related inhibitory KIR. We have used a peptide:MHC targeting strategy to activate NK cells through KIR2DS2 and identified a novel cancer-associated ligand for this receptor. The work provides a proof-of-concept for targeting NK cells through activating KIR as a cancer immunotherapy strategy.

immunology↗

bioRxiv2019

Single cell transcriptomic analysis identifies Langerhans cells immunocompetency is critical for IDO1- dependent ability to induce tolerogenic T cells.

Human epidermal Langerhans cells (LCs) can coordinate both immunogenic and tolerogenic immune responses, creating an attractive opportunity for immunomodulation strategies. To investigate transcriptional determinants of human primary LC tolerance we applied single cells RNA-sequencing combined with transcriptional network modelling and functional analysis. Unsupervised clustering of single cell transcriptomes revealed that steady-state LCs exist in immature and immunocompetent states, and become fully immunocompetent on migration. Interestingly, LC migration, which has been shown to result in upregulation of the transcription factor IRF4, led in parallel to increased expression of a tolerogenic gene module including IDO1, LGALS1, LAMTOR1 and IL10RA, which translated to efficient induction of regulatory T cells in co-culture assays by immunocompetent LCs. Using protein expression analysis and perturbation with inhibitors, we confirmed the role of IDO1 as a mediator of LC tolerogenic responses induced during LC migration. Computational analysis of regulons and Partial Information Decomposition analyses identified IRF4 as a key driver for LC tolerogenic programmes. The predicted IRF4-regulated genes were confirmed by analysis of CRISPR-Cas9 edited LCs. These findings suggest that efficient priming of tolerogenic responses by LCs requires upregulation of a migration-coupled maturation program which is superimposed with a tolerance-inducing genomic module.

systems biology↗

bioRxiv2019

Resolving cellular systems by ultra-sensitive and economical single-cell transcriptome filtering

Single-cell transcriptomics has sensitivity limits that restrict low abundance transcript identification, affects clustering and introduce artefact. Here, we describe Constellation DropSeq (C-DropSeq), a molecular transcriptome filter that delivers two orders of magnitude sensitivity gains by maximising read utility while reducing sequencing depth and costs. The simple and powerful method is broadly compatible with library preparation routines and was demonstrated by identifying and characterizing the activation of rare dendritic cell sub-populations.

molecular biology↗

bioRxiv2019

Genomic programming of antigen cross-presentation in IRF4-expressing human Langerhans cells

Langerhans cells (LCs) in the epidermis present MHC I and MHC II-restricted antigens thereby priming either CD8 or CD4 T cell immune responses. The genomic programs and transcription factors regulating antigen presentation in LCs remain to be elucidated. We show human LCs are highly efficient in MHC I-antigen cross-presentation but lack the transcription factor IRF8 that is critical in dendritic cells. LC migration from the epidermis enhances their ability to cross-present antigens and is accompanied by the induction of the transcription factor IRF4, whose expression is correlated by scRNA-seq with genes involved in ubiquitin-dependent protein degradation. Chromatin profiling reveals enrichment of EICE and AICE composite DNA binding motifs in regulatory regions of antigen-presentation genes, which can be recognized by IRF4 in conjunction with PU.1 or BATF3 expressed in LCs. Thus, the genomic programming of human LCs including inducible expression of IRF4 with enhanced cross-presentation distinguishes them from conventional dendritic cells.

immunology↗