Biology subjects

Dustin, M.

Publications and source records attributed to Dustin, M..

25 records · Page 2Linked to original sources

bioRxiv2020

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

T cells use their T cell receptors (TCRs) to discriminate between peptide MHC (pMHC) ligands that bind with different affinities but precisely how different remains controversial. This is partly because the affinities of physiologically relevant interactions are often too weak to measure. Here, we introduce a surface plasmon resonance protocol to measure ultra-low TCR/pMHC affinities (KD ~ 1000 M). Using naive, memory, and blasted human CD8+ T cells we find that their discrimination power is unexpectedly low, in that they require a large >100-fold decrease in affinity to abolish responses. Interestingly, the discrimination power reduces further when antigen is presented in isolation on artificial surfaces but can be partially restored by adding ligands to CD2 or LFA-1. We were able to fit the kinetic proof-reading model to our data, yielding the first estimates for both the time delay (2.8 s) and number of biochemical steps (2.67). The fractional number of steps suggest that one of the proof-reading steps is not easily reversible.

immunology↗

bioRxiv2020

Intercellular telomere transfer extends T cell lifespan

The common view is that T-lymphocytes activate telomerase, a DNA polymerase that extends telomeres at chromosome ends, to delay senescence. We show that independently of telomerase, T cells elongate telomeres by acquiring telomere vesicles from antigen-presenting cells (APCs). Upon contact with T cells, APCs degraded shelterin to donate telomeres, which were cleaved by TZAP, and then transferred in extracellular vesicles (EVs) at the immunological synapse. Telomere vesicles retained the Rad51 recombination factor that enabled them to fuse with T cell chromosomal ends causing an average lengthening of [~]3000 base pairs. Thus, we identify a previously unknown telomere transfer program that supports T cell lifespan.

immunology↗

bioRxiv2020

Immune responses in pancreatic cancer may be restricted byprevalence of activated regulatory T-cells, dysfunctionalCD8+ T-cells, and senescent T-cells

Pancreatic cancer has the worst prognosis of any human malignancy and leukocyte infiltration is a major prognostic marker of the disease. As current immunotherapies confer negligible survival benefits, there is a need to better characterise leukocytes in pancreatic cancer to identify better therapeutic strategies. In this study, we analysed 32 human pancreatic cancer patients from two independent cohorts. A multi-parameter mass-cytometry analysis was performed on 32,000 T-cells from eight patients. Single-cell RNA sequencing dataset analysis was performed on a cohort of 24 patients. Multiplex immunohistochemistry imaging and spatial analysis were performed to map immune infiltration into the tumour microenvironment. Regulatory T-cell populations demonstrated highly immunosuppressive states with high TIGIT, ICOS and CD39 expression. CD8+ T-cells were found to be either in senescence or an exhausted state. The exhausted CD8 T-cells had low PD-1 expression but high TIGIT and CD39 expression. These findings were corroborated in an independent pancreatic cancer single-cell RNA dataset from additional 24 patients. These data suggest that T-cells are major players in the suppressive microenvironment of pancreatic cancer. Our work identifies novel therapeutic targets that should form the basis for rational design of a new generation of clinical trials in pancreatic ductal adenocarcinoma.

immunology↗

bioRxiv2020

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Protein-protein binding domains are critical in signalling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins have tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signalling, which is a critical requirement of noise filtering mechanisms such as kinetic proofreading. Here, we use modelling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases when tandem domains bind by a kinetic, but not a static, avidity mode. We use surface plasmon resonance to show that ZAP70, a tandem SH2 domain-containing kinase, binds kinetically to biphosphorylated peptides from the T cell antigen receptor (TCR) and that the unbinding rate can be accelerated by the phosphatase CD45. An important functional prediction of regulated unbinding is that the intracellular ZAP70/TCR half-life in T cells will be correlated to the extracellular TCR/antigen half-life and we show that this is the case in both cell lines and primary T cells. The work highlights that binding by kinetic avidity breaks the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discriminated mediated by kinetic proofreading.

immunology↗

bioRxiv2019

IL-15 re-programming compensates for NK cell mitochondrial dysfunction in HIV-1 infection

Dynamic regulation of cellular metabolism is important for maintaining homeostasis and can directly influence immune cell function and differentiation including Natural Killer (NK) cell responses. Persistent HIV-1 infection leads to a state of chronic activation, subset redistribution and progressive NK cell dysregulation. In this study we examined the metabolic processes that characterise NK cell subsets in HIV-1 infection, including adaptive NK cell subpopulations expressing the activating receptor NKG2C, which expand during chronic infection. These adaptive NK cells exhibit an enhanced metabolic profile in human cytomegalovirus (HCMV) infected HIV-1 seronegative individuals. However, the bioenergetic advantage of adaptive CD57+NKG2C+ NK cells is diminished during chronic HIV-1 infection, where NK cells uniformly display reduced oxidative phosphorylation (OXPHOS) and limited fuel flexibility upon CD16 stimulation. Defective OXPHOS was accompanied by increased mitochondrial depolarisation and structural alterations indicative of mitochondrial dysfunction, suggesting that mitochondrial defects are restricting the metabolic plasticity of NK cell subsets in HIV-1 infection. The metabolic requirement for receptor stimulation was alleviated upon IL-15 pre-treatment enhancing mammalian target of rapamycin complex1 (mTORC1) activity and NK cell functionality in HIV-1 infection, representing an effective strategy for pharmacologically boosting NK cell responses.

immunology↗

bioRxiv2018

Cytoskeletal tension actively sustains the T cell immunological synapse

When migratory T cells encounter antigen presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T cell immunity. While the cellular processes underlying synapse formation have been well-characterized, those that maintain the symmetry, and thereby the stability of the synapse remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott -Aldrich syndrome Protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T cell activation, WASP is degraded, leading to cytoskeletal rearrangement and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.

cell biology↗

bioRxiv2017

Immuno-phenotypes of Pancreatic Ductal Adenocarcinoma: Metaanalysis of transcriptional subtypes

Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas and has one of the highest mortality rates of any cancer type with a 5-year survival rate of < 5% and median overall survival of typically six months from diagnosis. Recent transcriptional studies of PDAC have provided several competing stratifications of the disease. However, the development of therapeutic strategies will depend on a unique and coherent classification of PDAC. Here, we use an integrative meta-analysis of four different PDAC gene expression studies to derive the consensus PDAC classification. Despite the fact that immunotherapies have yet to have an impact in treatment of PDAC, the gene expression signatures that stratify PDAC across studies are immunologic. We define these as \"adaptive\", \"innate\" and \"immune-exclusion\" immunologic signatures, which are prognostic across independent cohorts. An appreciation of the immune composition of PDAC with prognostic significance is an opportunity to understand distinct immune escape mechanisms in development of the disease and design novel immune-oncology therapeutic strategies to overcome current barriers.

cancer biology↗