Darren Newton

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Identification of Novel γδT-Cell Subsets following Bacterial Infection in the Absence of Vγ1 + T Cells: Homeostatic Control of γδT-Cell Responses to Pathogen Infection by Vγ1 + T Cells

Infection and Immunity, Feb 1, 2008

FIG. 2. Structural analysis of the CDR3 region of TCR-V␥ chains expressed by Listeria-elicited ␥␦... more FIG. 2. Structural analysis of the CDR3 region of TCR-V␥ chains expressed by Listeria-elicited ␥␦ T cells in wild-type and V␥1 Ϫ/Ϫ mice. (A) TCR CDR3 spectra type analysis of V␥2 and V␥4 transcripts expressed in the spleen. (B) Collated profiles for two or more WT and V␥1 Ϫ/Ϫ mice both before (day 0 [d0]) and 8 days after (d8) Listeria infection (left panels, V␥2; right panels, V␥4).

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CLL-like monoclonal B lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormali... more CLL-like monoclonal B lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5x10 9 /l circulating clonal B cells. However, the clonal size amongst MBL is extremely variable and allows discrimination of two distinct entities (High-and Low

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T Helper 2 IL-4/IL-13 Dual Blockade with Dupilumab Is Linked to Some Emergent T Helper 17‒Type Diseases, Including Seronegative Arthritis and Enthesitis/Enthesopathy, but Not to Humoral Autoimmune Diseases

Journal of Investigative Dermatology, Oct 1, 2022

Dupilumab, an IL-4/IL-13 receptor blocker, has been linked to emergent seronegative inflammatory ... more Dupilumab, an IL-4/IL-13 receptor blocker, has been linked to emergent seronegative inflammatory arthritis and psoriasis that form part of the spondyloarthropathy (SpA) spectrum. We systematically investigated patterns of immune disorders including predominantly Th17- (SpA pattern), Th2-mediated disorders and humoral autoimmune pattern diseases, using VigiBase, the World Health Organization's (WHO) global pharmacovigilance of adverse drug reactions (ADRs). Several bioinformatics databases and repositories were mined to couple Dupilumab-related immune-pharmacovigilance with molecular cascades relevant to reported findings. 37,848 Dupilumab ADR cases were reported, with skin, eye, musculoskeletal systems most affected. Seronegative arthritis (OR 9.61) psoriasis (OR 1.48), enthesitis/enthesopathy (OR 12.65), and iridocyclitis (OR 3.77) were highly associated. However, Ankylosing Spondylitis and IBD were not conclusively associated. Overall, classic polygenic humorally-mediated autoimmune diseases such as RA and SLE were not associated with Dupilumab use. Pathway analysis identified several biological pathways potentially involved in Dupilumab-associated ADRs, including the fibroblast growth factor receptor (FGFR; in particular, FGFR2) pathway. miRNAs analysis revealed the potential involvement of hsa-miR-21-5p and hsa-miR-335-5p. In conclusion, IL-4/IL-13 blockers are not unexpectedly protective against humoral autoimmune diseases but dynamically skew immune responses towards some IL-23/IL-17 cytokine pathway-related diseases. A robust signal potentially towards degenerative related pathology in the eye and vasculature due to loss of IL-4/IL-13 tissue reparative homeostatic mechanisms emerged.

Delineation of the Function of a Major γδ T Cell Subset during Infection

Journal of Immunology, Aug 1, 2005

␥␦ T cells play important but poorly defined roles in pathogen-induced immune responses and in pr... more ␥␦ T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among ␥␦ T cells. Using mice deficient in V␥1 ؉ T cells which are a major component of the ␥␦ T cell response to microbial infection, a specific immunoregulatory role for V␥1 ؉ T cells in macrophage and ␥␦ T cell homeostasis during infection has been established. By contrast, V␥1 ؉ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited V␥1 ؉ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of V␥1 ؉ T cells. These findings, therefore, identify distinct and nonoverlapping roles for ␥␦ T cell subsets in infection and establish the complexity and adaptability of a single population of ␥␦ T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.

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Identification of Novel [gamma][delta] T-Cell Subsets following Bacterial Infection in the Absence of V[gamma]1⁺ T Cells: Homeostatic Control of [gamma][delta] T-Cell Responses to Pathogen Infection by V[gamma]1⁺ T Cells

Infection and Immunity, 2006

Although ␥␦ T cells are a common feature of many pathogen-induced immune responses, the factors t... more Although ␥␦ T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual ␥␦ T-cell subsets to infection is unknown. Here we show that in the absence of V␥1 ؉ T cells, novel subsets of ␥␦ T cells, expressing T-cell receptor (TCR)-V␥ chains that normally define TCR␥␦ ؉ dendritic epidermal T cells (DETCs) (V␥5 ؉), intestinal intraepithelial lymphocytes (iIELs) (V␥7 ؉), and lymphocytes associated with the vaginal epithelia (V␥6 ؉), are recruited to the spleen in response to bacterial infection in TCR-V␥1 ؊/؊ mice. By comparison of phenotype and structure of TCR-V␥ chains and/or-V␦ chains expressed by these novel subsets with those of their epithelium-associated counterparts, the V␥6 ؉ T cells elicited in infected V␥1 ؊/؊ mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of V␥1 ؉ T cells. By contrast, V␥5 ؉ and V␥7 ؉ T cells found in infected V␥1 ؊/؊ mice were distinct from V␥5 ؉ DETCs and V␥7 ؉ iIELs. Functional analyses of the novel ␥␦ T-cell subsets identified for infected V␥1 ؊/؊ mice showed that whereas the V␥5 ؉ and V␥7 ؉ subsets may compensate for the absence of V␥1 ؉ T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of V␥1 ؉ T cells. Collectively, these findings identify novel subsets of ␥␦ T cells, the recruitment and activity of which is under the control of V␥1 ؉ T cells.

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Fibrinogen and Clot Structure Is Affected By Eculizumab in Patients with Paroxysmal Nocturnal Haemoglobinuria

Blood, Dec 7, 2017

Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in w... more Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in which an acquired somatic mutation of the PIGA gene in a haematopoietic stem cell results in the deficiency of key complement regulatory molecules, namely CD59 and CD55, from the surface of blood cells leading to an increased susceptibility of blood cells to complement-mediated activation +/- intravascular haemolysis and thrombosis. Thrombosis has been the leading cause of death in PNH. Multiple mechanisms of thrombosis in PNH have been proposed, including cross talk between the coagulation and complement system. This may lead to denser clots which are more resistant to fibrinolysis. Treatment with eculizumab, which inhibits complement factor C5 and thereby prevents terminal complement activation, reduces thromboembolic events in PNH, indicating that complement activation plays a significant role in the pathophysiology of thrombosis in this disease. Patients with other thrombotic disorders are known to form denser fibrin clots that incur greater resistance to fibrinolysis. It has not previously been explored whether changes to clot architecture contribute to thrombosis in PNH. Furthermore, the role of eculizumab in clot architecture is undocumented. Aims Characterise fibrin clot structure in patients with PNH and determine the effect of eculizumab on clot structure. Methods Informed consent was obtained from all participants. 53 patients from the PNH National Service (Leeds) were recruited. They were grouped according to the proportion of PNH granulocytes, - 90% (n=17) and reanalysed according to treatment with (n=19) or without (n=32) eculizumab. Plasma samples were obtained and ex vivo fibrin clot structure and function was analysed by permeation analysis of clot pore structure, turbidity analysis of clot formation and fibrinolysis, and fibrin ultrastructure by laser scanning confocal microscopy and scanning electron microscopy, according to methodology and protocols well established in our laboratory. The proportion of PNH granulocytes was measured by flow cytometry and fibrinogen levels by Clauss. P values of Results The proportion of PNH granulocytes correlated with fibrinogen (r= -0.35, p=0.048), maximum absorbency (r= -0.34, p=0.046) and clot density (r= -0.36, p=0.04). Seventy per cent of patients with large proportions of PNH granulocytes were on eculizumab. Analysis of patients not on eculizumab showed a trend towards proportion of PNH granulocytes correlating positively with fibrinogen levels (r=0.42, p=ns). Patients on eculizumab had lower fibrinogen levels (3.1±0.2 v 3.6±0.1g/L, P=0.02), maximum absorbance (0.31±0.1 v 0.40±0.1, p=0.028) and fibre density/100µm (18.5±4.4 v 23.9±4.2, p=0.006), indicative of a less dense clot. Summary/Conclusions This is the first study to demonstrate that patients on eculizumab showed reduced fibrinogen, fibre thickness and fibre density. Our data indicate that the beneficial effects of eculizumab in PNH is partly due to reduced complement activation and attenuated inflammatory response, leading to lower fibrinogen levels and a less thrombotic fibrin clot structure. These novel data help to explain the impact of complement inhibition in this disease. Disclosures Arnold: Alexion Pharmaceuticals, Inc.: Honoraria. Griffin: Alexion Pharmaceuticals, Inc.: Honoraria. Munir: Janssen: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; AbbVie: Honoraria; Gilled: Honoraria. Hillmen: Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Richards: Alexion Pharmaceuticals, Inc.: Honoraria. Hill: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees.

THU0013 Normal CD19-Negative Plasma Cells Are Biologically Distinct from Other Normal Plasma Cells and Likely To Be Involved in Lack of Response To B-Cell Depletion for Autoimmune Disorders

Annals of the Rheumatic Diseases, Jun 1, 2016

Background Plasma cells (PCs) are heterogeneous in terms of function and lifespan. Long-lived PCs... more Background Plasma cells (PCs) are heterogeneous in terms of function and lifespan. Long-lived PCs are not depleted by B-cell depletion therapy for autoimmune disorders and therefore likely to be responsible for lack of clinical response in a significant proportion of patients. Until recently [1], the phenotype of long-lived PCs was not known. Understanding the biological features of long-lived PCs could greatly facilitate identification of therapeutic targets. Long-lived PCs contribute to humoral memory, and may represent the cell of origin for myeloma while neoplastic PCs are generally distinguished by lack of CD19 expression. However, polyclonal CD19Neg PCs constitute approximately 15–35% of total PCs in people aged over 50 years. Here we further characterize normal CD19Neg PC populations in the context of normal and therapeutically perturbed bone marrow. Objectives To establish the phenotypic and functional differences between normal CD19Pos and CD19Neg human PCs Methods Extensive phenotyping of surface and intracellular proteins of normal and myeloma PCs were performed by flow cytometry. The regeneration of normal PCs was monitored following bone marrow ablation for myeloma [2] and B-cell depletion with alemtuzumab [3] in a series of clinical trials. The immunoglobulin heavy chain variable (IGHV) region of normal CD19Pos and CD19Neg human PCs were sequenced as described previously [4] to identify phylogenetic relationships. Results CD19Neg PCs are phenotypically distinct from normal CD19Pos plasma cells with weaker CD95 (p<0.0001) and stronger (p<0.0001) BCL2 expression. Normal CD19Neg PCs share some characteristics with neoplastic PCs including variable expression of CD28, CD56, and CD45, but neoplastic PCs are reliably distinguished from normal CD19Neg PCs by differential expression of CD27, CD81 and CD117 as well as CD56 in some cases. After effective myeloablation for myeloma CD19Pos PCs recovered in 74 out of 80 samples, while CD19Neg PCs remained at less than 1% of total PCs in 30 out of 80 samples. Therapeutic B-cell depletion with alemtuzumab for chronic lymphocytic leukaemia results in loss of CD19Pos PCs (p<0.0001) whereas CD19Neg PCs persist. There was little overlap between the IGHV repertoire with only 2.1 to 3.7% of unique IGHV@ rearrangements shared between the CD19Pos and CD19Neg fractions, suggestive of a largely discrete immunoglobulin repertoire in the two fractions at steady state. Conclusions CD19Pos and CD19Neg PCs are biologically distinct with different immunoglobulin repertoires and production kinetics consistent with CD19Neg PCs having greater potential lifespan. Identifying the developmental pathway of normal CD19Neg PC is likely to allow selectively targeting PC compartments for autoimmune disorders. References Mei HE, et al. Blood 2015, 125(11):1739–1748. Rawstron AC, et al. Journal of clinical oncology 2013, 31(20):2540–2547. Rawstron AC, et al. Blood 2004, 103(6):2027–2031. Cocco M, et al. Journal of Immunology 2012, 189(12):5773–5785. Disclosure of Interest None declared

Paneth cells produce Ang4 in response to IL‑22 of which γδ iIELs are a source

PLOS ONE, Dec 17, 2013

Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Cancer immunology research, Apr 19, 2022

Improving the chances of curing patients with cancer who have had surgery to remove metastatic si... more Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec-associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNa secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

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Data from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Improving the chances of curing patients with cancer who have had surgery to remove metastatic si... more Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Presentation clinical, haematological and immunophenotypic features of 1081 patients with GPI‐deficient (paroxysmal nocturnal haemoglobinuria) cells detected by flow cytometry

British Journal of Haematology, Feb 27, 2020

This is a repository copy of Presentation clinical, haematological and immunophenotypic features ... more

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Identification of Novel γδ T-Cell Subsets following Bacterial Infection in the Absence of Vγ1 <sup>+</sup> T Cells: Homeostatic Control of γδ T-Cell Responses to Pathogen Infection by Vγ1 <sup>+</sup> T Cells

Infection and Immunity, Feb 1, 2006

format_quoteV␥1-deficient mice show macrophage accumulation at infection sites, highlighting V␥1's critical role in macrophage homeostasis during Listeria infection.format_quote

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Supplementary Data from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Supplementary Data from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Antican... more

Supplementary Figure from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Supplementary Figure from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Antic... more

Assessment of Eculizumab and C5 Levels in PNH Treatment: Molar Ratio of Eculizumab to C5 Level Effectively Predicts Ongoing Dosage Changes in a Cohort of 50 Patients Treated with Eculizumab

Blood, Dec 7, 2017

Immunophenotypic assessment of PNH clones in major and minor cell lineages in the peripheral blood of patients with paroxysmal nocturnal hemoglobinuria

Cytometry Part B: Clinical Cytometry

BackgroundFlow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturna... more BackgroundFlow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression.MethodsWe studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi‐parameter 7‐color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression.ResultsFor individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest propor...

Extravascular Hemolysis Due to C3-Loading in Patients with PNH Treated with Eculizumab: Defining the Clinical Syndrome

Blood, 2017

Introduction Complement-mediated intravascular hemolysis in patients with paroxysmal nocturnal he... more Introduction Complement-mediated intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria (PNH) is effectively prevented by the terminal-complement inhibitor eculizumab, reducing symptoms and protecting against the clinical consequences of the disease, such as thrombosis and renal failure. Due to the continued activation of the proximal complement cascade upstream of C5, PNH red cells become opsonized with fragments of C3, leading to extravascular hemolysis and a continued transfusion requirement in a proportion of patients. Lactate dehydrogenase (LDH) is utilised as the main indicator of hemolysis in PNH but is particularly a measure of intravascular rather than extravascular hemolysis and therefore is not necessarily elevated in proportion to transfusion requirement due to C3-loading. We therefore conducted data analysis to determine whether other parameters were more indicative of extravascular hemolysis. Methods Data from patients with PNH treated in the Leed...

SAT0361 HEALTHY Human Spinal Processes Peri-Entheseal T-Cells Exhibit a TR1 Rather Than a FOXP3 Regulatory Phenotype

Annals of the Rheumatic Diseases, 2020

Background:We have previously reported that the normal spinal enthesis has populations of convent... more Background:We have previously reported that the normal spinal enthesis has populations of conventional T-cells including CD4+ & CD8+ T-cells that could be induced to produce IL-17A and TNF following anti-CD3/CD28 stimulation. The biology of such cells in health including their normal function and antigen reactivity is completely unknown. The purpose of this work was to define the phenotype, functionality and TCR reactivity of such T-cells in health.Objectives:To investigate whether the T-cells at the normal enthesis were regulatory in nature and to determine the type of regulatory T-cell as Tr1 or FOXP3 regulatory T-cell and to determine T-cell reactivity.Methods:Healthy interspinous ligament and spinous process with matched peripheral blood were harvested from patients undergoing elective spinal surgery (n=20). Entheseal soft tissue (EST) & peri-entheseal bone (PEB) was enzymatically digested and then sorted. Tr1 and Treg phenotypes were investigated using flow cytometry. Analysis ...

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COVID-19 vaccination antibody responses in patients with aplastic anaemia and paroxysmal nocturnal haemoglobinuria

The Lancet Haematology

Effectiveness of SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on DMARDs: as determined by antibody and T cell responses

RMD Open, Mar 1, 2022

Objectives To assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rhe... more Objectives To assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on disease-modifying antirheumatic drugs (DMARDs). Methods This prospective study recruited 100 patients with RA on a variety of DMARDs for antibody and T cell analysis, pre-vaccination and 4 weeks post-vaccination. Positive antibody response was defined as sera IgG binding to ≥1 antigen. Those that remained seronegative after first vaccination were retested 4 weeks after second vaccination; and if still seronegative after vaccination three. A T cell response was defined an ELISpot count of ≥7 interferon (IFN) γ-positive cells when exposed to spike antigens. Type I IFN activity was determined using the luminex multiplex assay IFN score. Results After vaccine one, in patients without prior SARS-CoV-2 exposure, 37/83 (45%) developed vaccine-specific antibody responses, 44/83 (53%) vaccine-specific T cell responses and 64/83 (77%) developed either antibody or T cell responses. Reduced seroconversion was seen with abatacept, rituximab (RTX) and those on concomitant methotrexate (MTX) compared to 100% for healthy controls (p<0.001). Better seroconversion occurred with anti-tumour necrosis factor (TNF) versus RTX (p=0.012) and with age ≤50 (p=0.012). Pre-vaccine SARS-CoV-2 exposure was associated with higher quantitative seroconversion (≥3 antibodies) (p<0.001). In the subgroup of non-seroconverters, a second vaccination produced seroconversion in 54% (19/35), and after a third in 20% (2/10). IFN score analysis showed no change post-vaccine. Conclusion Patients with RA on DMARDs have reduced vaccine responses, particularly on certain DMARDs, with improvement on subsequent vaccinations but with approximately 10% still seronegative after three doses.

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