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1.
Front Immunol ; 14: 1256491, 2023.
Article in English | MEDLINE | ID: mdl-38022678

ABSTRACT

Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.


Subject(s)
Melanoma , Receptors, Chimeric Antigen , Humans , T-Lymphocytes , CD28 Antigens , Lymphocytes, Tumor-Infiltrating , Folate Receptor 1 , Receptors, Chimeric Antigen/genetics , CD40 Antigens , Tumor Microenvironment
2.
J Biol Chem ; 299(8): 104981, 2023 08.
Article in English | MEDLINE | ID: mdl-37390984

ABSTRACT

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.


Subject(s)
CD8 Antigens , CD8-Positive T-Lymphocytes , Histocompatibility Antigens Class I , Lymphocyte Activation , Histocompatibility Antigens Class I/metabolism , Peptides/metabolism , Receptors, Antigen, T-Cell/metabolism , Humans
3.
Water Environ Res ; 94(11): e10805, 2022 Oct 18.
Article in English | MEDLINE | ID: mdl-36369990

ABSTRACT

This study evaluates the coagulation performance of kenaf protein fractions (KPFs) comprising of albumin (AlbKP), globulin (GloKP), prolamin (ProKP), and glutenin (GluKP), in the treatment of high (500 NTU), medium (150 NTU), and low (30 NTU) turbidity water. Based on preliminary experimental results, the study focused on GloKP due to it outperforming the other kenaf coagulation products (KCPs) in all water types tested. The influence of GloKP, both as a primary coagulant and coagulant aid to aluminum sulfate (AS) on organic matter removal, was examined. Parametric analysis on turbidity, TSS, pH, dosages, retention time, and KPFs storage time was completed. Results indicated that GloKP could be used both as a primary coagulant and coagulant aid. GloKP had a higher turbidity and solids removal than the AlbKP and other KPFs (ProKP and GluKP). Solution pH greatly influenced the performance of the GloKP, and optimum dosage at pH 2 resulted in the highest organic matter removal. High dosages also resulted in negative mobility of particles and a more stable suspension. When used as a coagulant aid to AS, GloKP was more effective in removing dissolved organic carbon (DOC). Scanning electron microscopy elemental analysis (SEM-EDAX) and Fourier transform infrared (FT-IR) spectra showed the structure of the KPFs. SEM-EDAX indicated the presence of metal cations capable of forming complexes essential for flocs formation. The enhanced floc formation, detailed in this paper, is ascribed to the collective effect of charge neutralization of the AS species and the adsorption and bridging effect of the GloKP, which improves the bonds formed between flocs. The coagulation-flocculation process can be significantly improved using dual coagulants. GloKP was also an excellent alternative to its crude (CrKP) and solvent extract (HxKP) form for removing suspended and dissolved particles from all water types. PRACTITIONER POINTS: Kenaf protein fractionates can destabilize stable particles. The globulin protein fractionate (GloKP) aggregated the most particles and contained least dissolved organic material. GloKP is pH sensitive with pH 2 reported as best working pH. Coagulant dosage and coagulation mechanism were assessed.

4.
Am J Cancer Res ; 12(8): 3967-3984, 2022.
Article in English | MEDLINE | ID: mdl-36119832

ABSTRACT

Nonselected autologous tumor-infiltrating lymphocytes (TILs) may provide advantages over other treatments for solid tumors, including checkpoint inhibitor-refractory melanoma. This retrospective analysis reports a single-center experience of nonselected autologous TILs derived from digested tumors for compassionate use treatment of advanced cutaneous melanoma, including after programmed cell death protein 1 (PD-1) inhibition. Patients with histologically confirmed metastatic cutaneous melanoma and no standard-of-care treatment options underwent tumor resection for TIL product manufacturing. Patients received lymphodepleting chemotherapy with cyclophosphamide for 2 days and fludarabine for 5 days, followed by a single TIL infusion and post-TIL high-dose interleukin (IL)-2. Safety assessments included clinically significant adverse events (AEs). Efficacy assessments included overall response rate (ORR), complete response (CR) rate, disease control rate (DCR), and overall survival. Between October 2011 and August 2019, 21 patients underwent treatment (median follow-up time, 52.2 months from TIL infusion). Among all treated patients, median age was 45 years, median number of disease sites was 4, 100% had M1c or M1d disease, and 90% received prior checkpoint inhibitor. Twelve patients received TILs after prior PD-1 inhibition. The safety profile among all treated patients and the prior PD-1 inhibitor subgroup was generally consistent with lymphodepletion and high-dose IL-2. No treatment-related deaths occurred. Among all patients, the ORR was 67%, CR rate was 19%, and the DCR was 86%, which was consistent with that observed in the prior PD-1 inhibitor subgroup (58%, 8%, and 75%, respectively). Median overall survival in all treated patients and the prior PD-1 inhibitor subgroup was 21.3 months. In total, 5 patients (24%) had durable ongoing responses (>30 months post-TIL infusion) at data cutoff, and all patients who achieved CR remained alive and disease free. To further illustrate how TIL therapy may integrate into established treatment paradigms, several case studies of patients treated in this series were included. Overall, these data demonstrate that manufacturing of nonselected autologous TILs from tumor digests is feasible and resulted in high rates of durable response in poor-risk patient populations, which may address significant unmet medical need.

5.
Environ Technol ; 43(11): 1732-1744, 2022 Apr.
Article in English | MEDLINE | ID: mdl-33180680

ABSTRACT

Although highway runoff has historically been extensively studied, the increasing complexity of stormwater management means that there are still significant gaps regarding the reduction of soluble metals. The work reported in this paper addresses these challenges by analysing the presence and behaviour of iron, copper and zinc in runoff from junction 24 of the M1 motorway in the UK (peak traffic flow: 30,000 vehicles per hour) and comparing it with other urban sources of metals found in the same catchment (a local brook and sewage treatment works). The sampling site included an interceptor and a treatment lagoon and the event monitoring indicated a trend by which the metals did not change their concentration or particulate soluble proportion immediately, hence showing that pre- and post-storm conditions are important factors when analysing the solubility of metals and their behaviour. The data provided further evidence of the important influence of storm characteristics on metal concentrations in highway runoff, in particular the effects of an antecedent dry weather period (ADWP). In addition, this study also helped us to better understand how the release of sodium the application of de-icer for road maintenance in winter affects the availability of zinc.


Subject(s)
Metals, Heavy , Water Pollutants, Chemical , Copper/analysis , Dust/analysis , Environmental Monitoring , Metals/analysis , Metals, Heavy/analysis , Rain , Water Movements , Water Pollutants, Chemical/analysis , Zinc/analysis
6.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Article in English | MEDLINE | ID: mdl-34272276

ABSTRACT

CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.


Subject(s)
CD8 Antigens/immunology , Peptides/agonists , Peptides/immunology , Receptors, Antigen, T-Cell/immunology , Antigens/chemistry , Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Cross Reactions , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Kinetics , Ligands , Lymphocyte Activation , Models, Immunological , Mutation
7.
Water Res ; 188: 116517, 2021 Jan 01.
Article in English | MEDLINE | ID: mdl-33075601

ABSTRACT

The Sustainable Development Goal (SDG) 6.1, established by the United Nations General Assembly in 2015, targets universal and equitable access to safe and affordable drinking water for all by 2030. An essential factor in achieving this goal is the harnessing of "green" coagulants - naturally occurring, environmentally friendly materials which are effective coagulants for use in water treatment, with good availability in developing countries, inherent renewable properties and ease of biodegradation. In order to gain from these benefits, it is essential to fully understand how such coagulants may best be utilised, particularly concerning their practical application in developing countries. In this study, three different plant-based coagulation products (PCPs), namely Hexane (HxKP), saline (StKP) and crude (CrKP) extracts of Kenaf plant seed (Hibiscus cannabinus, a species of the Hibiscus plant), were applied to high (HTW), medium (MTW) and low (LTW) turbidity water in order to determine their performance and coagulation ability. The ability of the three Kenaf coagulant products (KCPs) to remove hydrophobic fractions of natural organic matter (NOM) was measured. The impact of KCPs on the treated water organic matter content (a known disinfection by-product (DBP) precursor) was examined using known surrogates of natural organic matter (NOM) i.e. the dissolved organic carbon (DOC), ultraviolet absorbance at 254 (UV254) and specific ultraviolet absorbance (SUVA254). Results obtained quantify the implications of using these coagulants during the water disinfection process. A parametric study, measuring the effect of different operating parameters, such as untreated water turbidity, pH, dosages, retention time, and KCP storage time, was completed. Turbidity removal performance for HxKP and StKP was very good with > 90% removal recorded for HTW and MTW, respectively, at pH seven within 2 hours retention time. Images obtained from scanning electron microscopy (SEM) analysis revealed a high likelihood of the coagulation mechanism of KCPs to be adsorption-interparticle bridging brought about by their flake-like structures and surfaces charges. Varying pH had no measurable influence on the coagulation performance of the KCPs. Comparing their efficiency with MoringaOleifera (MO, a previously researched PCP) and alum showed that HxKP had a negligibly different particle removal as MO. StKP turbidity removal performance was below HxKP by 1% for HTW and LTW and 2% for MTW but performed higher than the CrKP by 5% and 7% in HTW and MTW, respectively. The optimum dosage of HxKP and StKP reduced DBP surrogate values, indicating that its precursor is also minimized, although a slight shift from this optimum dosage showed a significant rise in their concentration thus signifying a potential increase in DBPs during disinfection.


Subject(s)
Hibiscus , Water Pollutants, Chemical , Water Purification , Adsorption , Disinfection
8.
Ther Adv Vaccines Immunother ; 8: 2515135520933509, 2020.
Article in English | MEDLINE | ID: mdl-32613155

ABSTRACT

With the advent of immunotherapy as a realistic and promising option for cancer treatment, adoptive cellular therapies are gaining significant interest in the clinic. Whilst the recent successes of chimeric antigen receptor T-cell therapies for haematological malignancies are widely known, they have yet to show great success in solid cancers. However, immune cells transduced with T-cell receptors have been shown to traffic to and exert anti-cancer effects on solid tumour cells with some great successes. In this review, we explore the field of transgenic T-cell receptor immunotherapy, highlighting some of the key clinical trials which have paved the way for this type of cellular immunotherapy. Some trials have shown amazing clinical results, including long-term remissions and minimal toxicity, and can be looked at as an exemplar for this adoptive cell therapy. There have also been key trials where unexpected, fatal, off-tumour toxicity has occurred, and these trials have also been instrumental in shaping safer clinical trials, particularly regarding preclinical testing. In addition to previous trials, we analysed the current clinical trial space for T-cell receptor T-cell therapy, showing which trials are dominating in the clinic and which targets are being prioritised by researchers around the world. By looking at both past and current trials, we have been able to identify key drivers in developing transgenic T-cell receptor immunotherapy for the future.

9.
Clin Transl Immunology ; 9(6): e1141, 2020.
Article in English | MEDLINE | ID: mdl-32547743

ABSTRACT

OBJECTIVES: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1-specific CD8+ T cells. METHODS: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or ß-galactosidase (untargeted control). Activation of WT1-specific CD8+ T-cell lines following cross-presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8+ T cells, were used to investigate naïve WT1-specific CD8+ T-cell priming. RESULTS: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines. CONCLUSIONS: Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141+ DCs is sufficient for effective CD8+ T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.

10.
Nat Biotechnol ; 38(5): 609-619, 2020 05.
Article in English | MEDLINE | ID: mdl-32393905

ABSTRACT

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.


Subject(s)
Antigens/analysis , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/cytology , Cell Engineering , Gene Library , Humans , Jurkat Cells , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , T-Lymphocytes/immunology , Viruses/immunology
11.
Nat Commun ; 11(1): 1279, 2020 03 09.
Article in English | MEDLINE | ID: mdl-32152271

ABSTRACT

Climate change and urbanization can increase pressures on groundwater resources, but little is known about how groundwater quality will change. Here, we use a global synthesis (n = 9,404) to reveal the drivers of dissolved organic carbon (DOC), which is an important component of water chemistry and substrate for microorganisms that control biogeochemical reactions. Dissolved inorganic chemistry, local climate and land use explained ~ 31% of observed variability in groundwater DOC, whilst aquifer age explained an additional 16%. We identify a 19% increase in DOC associated with urban land cover. We predict major groundwater DOC increases following changes in precipitation and temperature in key areas relying on groundwater. Climate change and conversion of natural or agricultural areas to urban areas will decrease groundwater quality and increase water treatment costs, compounding existing constraints on groundwater resources.

12.
Sci Total Environ ; 699: 134361, 2020 Jan 10.
Article in English | MEDLINE | ID: mdl-31683216

ABSTRACT

There is a need for an inexpensive, reliable and fast monitoring tool to detect contaminants in a short time, for quick mitigation of pollution sources and site remediation, and for characterization of natural dissolved organic matter (DOM). Fluorescence spectroscopy has proven to be an excellent technique in quantifying aquatic DOM, from autochthonous, allochthonous or anthropogenic sources. This paper reviews the advances in in situ fluorescence measurements of DOM and pollutants in various water environments. Studies have demonstrated, using high temporal-frequency DOM fluorescence data, that marine autochthonous production of DOM is highly complex and that the allochthonous input of DOM from freshwater to marine water can be predicted. Furthermore, river measurement studies found a delayed fluorescence response of DOM following precipitation compared to turbidity and discharge, with various lags, depending on season, site and input of dissolved organic carbon (DOC) concentration. In addition, research has shown that blue light fluorescence (λemission = 430-500 nm) can be a good proxy for DOC, in environments with terrestrial inputs, and ultraviolet fluorescence (λemission = UVA-320-400 nm) for biochemical oxygen demand, and also E. coli in environments with sanitation issues. The correction of raw fluorescence data improves the relationship between fluorescence intensity and these parameters. This review also presents the specific steps and parameters that must be considered before and during in situ fluorescence measurement session for a harmonized qualitative and quantitative protocol. Finally, the strengths and weaknesses of the research on in situ fluorescence are identified.

13.
Sci Rep ; 9(1): 5487, 2019 04 02.
Article in English | MEDLINE | ID: mdl-30940840

ABSTRACT

L-selectin on T-cells is best known as an adhesion molecule that supports recruitment of blood-borne naïve and central memory cells into lymph nodes. Proteolytic shedding of the ectodomain is thought to redirect activated T-cells from lymph nodes to sites of infection. However, we have shown that activated T-cells re-express L-selectin before lymph node egress and use L-selectin to locate to virus-infected tissues. Therefore, we considered other roles for L-selectin proteolysis during T cell activation. In this study, we used T cells expressing cleavable or non-cleavable L-selectin and determined the impact of L-selectin proteolysis on T cell activation in virus-infected mice. We confirm an essential and non-redundant role for ADAM17 in TCR-induced proteolysis of L-selectin in mouse and human T cells and show that L-selectin cleavage does not regulate T cell activation measured by CD69 or TCR internalisation. Following virus infection of mice, L-selectin proteolysis promoted early clonal expansion of cytotoxic T cells resulting in an 8-fold increase over T cells unable to cleave L-selectin. T cells unable to cleave L-selectin showed delayed proliferation in vitro which correlated with lower CD25 expression. Based on these results, we propose that ADAM17-dependent proteolysis of L-selectin should be considered a regulator of T-cell activation at sites of immune activity.


Subject(s)
ADAM17 Protein/metabolism , Clone Cells/immunology , L-Selectin/metabolism , T-Lymphocytes, Cytotoxic/immunology , Virus Diseases/metabolism , ADAM17 Protein/genetics , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Cell Movement , Cells, Cultured , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , L-Selectin/genetics , Lectins, C-Type/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mutation , Proteolysis , Virus Diseases/immunology
14.
Sci Total Environ ; 646: 1-10, 2019 Jan 01.
Article in English | MEDLINE | ID: mdl-30041042

ABSTRACT

This study used fluorescence excitation-emission matrices (EEMs) analysis to investigate the characteristics of natural organic matter (NOM) in treated water using okra crude extract (OCE), sabdariffa crude extract (SCE) and kenaf crude extract (KCE) as coagulants. In addition, an assessment of the impact of purified okra protein (POP), purified sabdariffa protein (PSP) and purified kenaf protein (PKP) was undertaken. The performance evaluation of these coagulants in terms of increase or decrease in dissolved organic carbon (DOC) was compared with Peak T fluorescence intensity observed at excitation wavelength 220-230 nm, and emission wavelength 340-360 nm. Fluorescence analysis of water treated with the crude extracts identified the removal of DOC in peaks A and C region whereas the increase in DOC from the protein was predominantly found in peaks T and B region. Furthermore, it was observed that the purified proteins were noted to be capable of reducing the DOC concentration in raw water where all fluorophores were not detected. The application of OCE, SCE and KCE yielded an increase in DOC of 65, 61 and 55% respectively, corresponding to increases of 65, 29 and 54% in peak T fluorescence intensities, at 100 mg/l dose. Furthermore, DOC concentration was reduced by 25, 24 and 18% using POP, PSP and PKP respectively as coagulants with corresponding decreases in fluorescence intensity of 46%, 44 and 36% in POP, PSP and PKP, at a lower dose of 0.1 mg/l. Therefore, it is clear that Peak T fluorescence intensity could be used to characterise organic matter in treated water using natural extracts to assess final water quality.


Subject(s)
Drinking Water/chemistry , Hibiscus , Water Pollutants/analysis , Water Purification/methods , Humic Substances/analysis , Seeds , Water Quality
15.
Bioresour Technol ; 265: 480-489, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29936352

ABSTRACT

An Euler-Lagrange CFD model is applied to a full-scale, biogas-mixed anaerobic digester to improve mixing efficiency and improve overall performance. Two quantitative mixing criteria previously adopted in anaerobic digestion (viz., uniformity index and dead volume) are critically assessed for the first time. A novel qualitative method is introduced to clarify the output of the quantitative methods. The first-ever quantitative assessment of mixing quality in full-scale, biogas-mixed anaerobic digestion is then proposed, and a strategy to improve mixing, involving the combined use of concentric nozzle manifolds at the base of the digester, is evaluated.


Subject(s)
Biofuels , Bioreactors , Anaerobiosis
16.
Front Immunol ; 8: 1503, 2017.
Article in English | MEDLINE | ID: mdl-29209312

ABSTRACT

Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte epitope produce the SLFNTIAVL triple mutant "ultimate" escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent-exposed residues in the peptide destabilise the peptide-HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate re-evaluation of this exemplar model of HIV TCR escape.

17.
Proc Natl Acad Sci U S A ; 114(51): E10956-E10964, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29158404

ABSTRACT

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells.


Subject(s)
Antigens, CD1/immunology , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Mycolic Acids/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tuberculosis/immunology , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Antigens, CD1/chemistry , Antigens, CD1/genetics , Gene Expression , Granuloma/immunology , Granuloma/metabolism , Granuloma/microbiology , Granuloma/pathology , Humans , Immunohistochemistry , Lymphocyte Activation/immunology , Models, Molecular , Molecular Conformation , Mycolic Acids/chemistry , Mycolic Acids/metabolism , Protein Binding , Receptors, Antigen, T-Cell/metabolism , Tuberculosis/microbiology
18.
Immunol Cell Biol ; 95(7): 620-629, 2017 08.
Article in English | MEDLINE | ID: mdl-28356569

ABSTRACT

The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with non-MHC-restricted γδ T cells and antigen-specific CD8+ T cells. Stable lines with characteristics of breast CSC-like cells were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non-adherent conditions, as well as their potent tumorigenicity, self-renewal and differentiation in xenografted mice. The resistance of CSC-like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS-targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM-1 on CSC-like cells and thereby increased the susceptibility to antigen-specific killing by CD8+ T cells. Alternatively, γδ T-cell responses could be specifically directed against CSC-like cells using the humanised anti-GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T-cell and CD8+ T-cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.


Subject(s)
Breast Neoplasms/immunology , Breast Neoplasms/pathology , CD8-Positive T-Lymphocytes/immunology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Animals , Antibodies/pharmacology , Breast/pathology , Cytotoxicity, Immunologic , Diphosphonates/pharmacology , Epithelial Cells/metabolism , Epitopes/immunology , Female , Humans , Imidazoles/pharmacology , Immunity, Innate , Interferon-gamma/metabolism , Major Histocompatibility Complex , Mice , Phenotype , Zoledronic Acid , ras Proteins/metabolism
19.
Immunol Cell Biol ; 95(1): 68-76, 2017 01.
Article in English | MEDLINE | ID: mdl-27670790

ABSTRACT

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.


Subject(s)
CD8 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class I/immunology , Cell Membrane/metabolism , Humans , Lymphocyte Activation/immunology , Mutation/genetics , Peptides/metabolism
20.
J Water Health ; 14(5): 768-779, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27740543

ABSTRACT

The effects of temperature, storage time and water pH on the coagulation performance of okra seed protein in water treatment were assessed. In a jar test experiment, okra salt extract achieved a notable improvement in treatment efficiency with storage time and showed good performance in quality after thermal treatment at 60, 97 and 140 °C temperatures for 6, 4 and 2 hours, respectively. The performance improvement of more than 8% is considered to be due to the denaturation and subsequent removal of coagulation-hindering proteins in okra seed. Furthermore, the results of a sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis show two distinctive bands of protein responsible for the coagulation process after denaturation. It was further shown that at optimal coagulant dose, the pH of the treated water remained unaffected as a result of the protein's buffering capability during coagulation. Therefore, denatured okra seed exhibited improved performance compared to the native crude extract and offers clear benefits as a water treatment coagulant.


Subject(s)
Abelmoschus/chemistry , Drinking Water/analysis , Plant Proteins/chemistry , Water Purification/methods , Protein Denaturation , Seeds/chemistry , Water Purification/instrumentation
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