Gene editing in T-cells and T-cell targets

Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost. As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct. To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clones from the A549 lung carcinoma and THP-1 monocytic cell lines in order to study MR1 biology. Mucosal-associated invariant T-cell (MAIT) clones were not activated by MR1 deficient A549 or THP-1 clones infected with bacteria

‘Inappropriate’ attenders to the Adult Emergency Department – A Critical Review

Background: The Department of Health (DoH) emphasise the increased attendance rates to the Emergency Department (ED) over the last decade from 14,044,018 in 2001- 2 (DoH, 2002) up to 21,342,543 in 2010-11 (DoH, 2010-11). Ainsworth (2008) identified that annually up to 14 million attenders could have been treated by their General Practitioner which coincides with Lee, Hazlett, Chow, Lau, Kam, Wong & Wong’s (2003) suggestion that ‘inappropriate’ attenders are the cause of the increased attendance rates. The literature is heavily criticised for the lack of definition and this is respective in society illustrated through the increased attendance rates. Aims: This study aims to critically review literature on ‘inappropriate’ adult attenders to the Emergency Department. Method: A critical review was used, reviewing 24 literature articles from ScienceDirect, CINAHL, Medline and Embase using the keywords: “Emergency Service, Hospital/”, “Emergency Medical Services/”, “Emergency Department.mp”, “Inappropriate.mp”, “Primary Care.mp or Primary Health Care/”. Findings: A lack of consistency between definitions of ‘inappropriateness’ to the ED was found from the literature, leading to a vast discrepancy between definitions generated by healthcare professional’s opinions and patients’. The role of the ED was found to relate to functionality through name, suggesting that society is unaware of the role of the ED. The reasons patients attend ED are variable, complex and consider health seeking behaviour from a psychology approach. VIII Conclusion: The review found that the definition of ‘inappropriate attendance’ is elusive and therefore open for interpretation by staff and patients. The use of personal opinions as a definition has created a vast discrepancy between staff and patients’, leading to ‘blame’ and ‘labelling’ of patients

Ice tectonic deformation during the rapid in situ drainage of a supraglacial lake on the Greenland Ice Sheet.

We present detailed records of lake discharge, ice motion and passive seismicity capturing the behaviour and processes preceding, during and following the rapid drainage of a 4 km<sup>2</sup> supraglacial lake through 1.1-km-thick ice on the western margin of the Greenland Ice Sheet. Peak discharge of 3300 m<sup>3</sup> s<sup>−1</sup> coincident with maximal rates of vertical uplift indicates that surface water accessed the ice–bed interface causing widespread hydraulic separation and enhanced basal motion. The differential motion of four global positioning system (GPS) receivers located around the lake record the opening and closure of the fractures through which the lake drained. We hypothesise that the majority of discharge occurred through a 3-km-long fracture with a peak width averaged across its wetted length of 0.4 m. We argue that the fracture's kilometre-scale length allowed rapid discharge to be achieved by combining reasonable water velocities with sub-metre fracture widths. These observations add to the currently limited knowledge of in situ supraglacial lake drainage events, which rapidly deliver large volumes of water to the ice–bed interface

T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer

Thermal stability of heterotrimeric pMHC proteins as determined by circular dichroism spectroscopy

T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity

Peptide super-agonist enhances T-cell responses to melanoma

Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.

Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies

Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses

Structural mechanism underpinning cross-reactivity of a CD8(+) T-cell clone that recognizes a peptide derived from human telomerase reverse transcriptase

T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8(+) T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents