Search

Sulfur assimilation and the biosynthesis of methionine, cysteine and S-adenosylmethionine (SAM) are critical to life. As a cofactor, SAM is required for the activity of most methyltransferases (MTases) and as such has broad impact on diverse cellular processes. Assigning function to MTases remains a challenge however, as many MTases are partially redundant, they often have multiple cellular roles and these activities can be condition-dependent. To address these challenges, we performed a systematic synthetic genetic analysis of all pairwise MTase double mutations in normal and stress conditions (16°C, 37°C, and LiCl) resulting in an unbiased comprehensive overview of the complexity and plasticity of the methyltransferome. Based on this network, we performed biochemical analysis of members of the histone H3K4 COMPASS complex and the phospholipid methyltransferase OPI3 to reveal a new role for a phospholipid methyltransferase in mediating histone methylation (H3K4) which underscores a potential link between lipid homeostasis and histone methylation. Our findings provide a valuable resource to study methyltransferase function, the dynamics of the methyltransferome, genetic crosstalk between biological processes and the dynamics of the methyltransferome in response to cellular stress.

Background HIV-1-specific CD8+ T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8+ T cells from naive cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8+ T cells from naive cells. Methods HIV-1-specific CD8+ T cells were primed from naive T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3′3′-cGAMP in vitro. We established HIV-1-specific CD8+ T cell lines from primed T cells and then investigated functional properties of these cells. Findings HIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3′3′-cGAMP effectively primed HIV-1-specific CD8+ T cells with strong ability to suppress HIV-1. 3′3′-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3′3′-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3′3′-cGAMP. Interpretation The present study demonstrates the potential of 3′3′-cGAMP to induce HIV-1-specific CD8+ T cells with strong effector function from naive cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.

Toll-like receptor 9 (TLR9) agonists have gained traction in recent years as potential adjuvants for the induction of adaptive immune responses. It has nonetheless remained unclear to what extent such ligands can facilitate the priming events that generate antigen-specific effector and/or memory CD8+ T-cell populations. We used an established in vitro model to prime naive precursors from human peripheral blood mononuclear cells in the presence of various adjuvants, including CpG ODN 2006, a synthetic oligonucleotide TLR9 ligand (TLR9L). Unexpectedly, we found that TLR9L induced a suboptimal inflammatory milieu and promoted the antigen-driven expansion and functional maturation of naive CD8+ T cells ineffectively compared with either ssRNA40 or 2′3′-cGAMP, which activate other pattern recognition receptors (PRRs). TLR9L also inhibited the priming efficacy of 2′3′-cGAMP. Collectively, these results suggest that TLR9L is unlikely to be a good candidate for the optimal induction of de novo CD8+ T-cell responses, in contrast to adjuvants that operate via discrete PRRs.

Toll-like receptor 9 (TLR9) agonists have gained traction in recent years as potential adjuvants for the induction of adaptive immune responses. It has nonetheless remained unclear to what extent such ligands can facilitate the priming events that generate antigen-specific effector and/or memory CD8+ T-cell populations. We used an established in vitro model to prime naive precursors from human peripheral blood mononuclear cells in the presence of various adjuvants, including CpG ODN 2006, a synthetic oligonucleotide TLR9 ligand (TLR9L). Unexpectedly, we found that TLR9L induced a suboptimal inflammatory milieu and promoted the antigen-driven expansion and functional maturation of naive CD8+ T cells ineffectively compared with either ssRNA40 or 2′3′-cGAMP, which activate other pattern recognition receptors (PRRs). TLR9L also inhibited the priming efficacy of 2′3′-cGAMP. Collectively, these results suggest that TLR9L is unlikely to be a good candidate for the optimal induction of de novo CD8+ T-cell responses, in contrast to adjuvants that operate via discrete PRRs.

Additional file 1.

BACKGROUND: HIV-1-specific CD8(+) T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8(+) T cells from naïve cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8(+) T cells from naïve cells. METHODS: HIV-1-specific CD8(+) T cells were primed from naïve T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3′3′-cGAMP in vitro. We established HIV-1-specific CD8(+) T cell lines from primed T cells and then investigated functional properties of these cells. FINDINGS: HIV-1-specific CD8(+) T cells primed with LPS failed to suppress HIV-1. In contrast, 3′3′-cGAMP effectively primed HIV-1-specific CD8(+) T cells with strong ability to suppress HIV-1. 3′3′-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3′3′-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3′3′-cGAMP. INTERPRETATION: The present study demonstrates the potential of 3′3′-cGAMP to induce HIV-1-specific CD8(+) T cells with strong effector function from naïve cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.

Background:Composite measures of disease activity have been developed for use in Psoriatic Arthritis (PsA) to capture the wide spectrum of disease but there is a lack of consensus regarding which to adopt for routine practice. It is recognised that more data is required to understand the measurement properties of existing instruments and consider the impact of modifications that may improve face validity, responsiveness or feasibility. It is important to have an estimate of a measurement instrument’s reliability in the setting of stable disease in order to understand measurement error and responsiveness. To our knowledge no data exists on the stability of composite measures in PsA.Objectives:To measure test re-test reliability of composite measures of disease activity in PsA.Methods:Clinical and patient reported outcomes to enable the calculation of composite measures were administered to 141 patients with PsA at five time points in a UK multicentre observational study. All patients fulfilled the CASPAR criteria. Twenty-nine patients with clinically stable disease and receiving no treatment intervention underwent repeat assessment by the same examiner within 2 weeks. Patients in high and low disease were included. Reliability was evaluated by intra-class correlation coefficient (ICC) and Bland Altman plots.Results:Of the 29 patients included 15 were male, the mean age was 52.4 years (SD 13.39), mean disease duration at T0was 9.2yrs (SD 8.11). The mean swollen joint count was 3.4 (SD 5.1), tender joint count 11.3 (SD 15.03) and PASI 1.0 (SD1.04). The ICC (95% CI) for tender and swollen joint counts were 0.94 (0.87-0.97) and 0.91 (0.80-0.96) respectively. The ICC for PASI was 0.95 (0.90-0.98). All composite measures demonstrated high levels of test-retest reliability with ICC >0.85, table. The most reliable measure was the PADAS ICC 0.98 (95% CI 0.954-0.991). The individual ICC for each composite measures are reported in the table and Bland Altman plots, figure.Conclusion:All composite measures show high levels of test-retest reliability in this cohort. The PASDAS was the most stable measure. Modifications to these instruments can now be tested and the impact compared to the original versions.Table.Test Re-Test reliability of each composite measureIntraclass Correlation Coefficient (95% Confidence Interval)GRACE0.929 (0.842-0.968)*CPDAI0.852 (0.635-0.940)*PASDAS0.978 (0.954-0.991)*DAPSA0.922 (0.831-0.964)*3VAS0.915 (0.815-0.960)*RAPID30.899 (0.782-0.953)*Disease Activity Index for PsA (DAPSA), PsA Disease Activity Score (PASDAS), Composite Psoriatic Disease Activity Index (CPDAI), GRAppa Composite Exercise (GRACE), 3 Visual Analogue Scale (3VAS), Routine Assessment of Patient Index (RAPID3),*PFigure.Bland Altman plots for each composite measureFunding:This report is independent research funded by the National Institute for Health Research, Programme Grants for Applied Research [Early detection to improve outcome in patients with undiagnosed PsA (‘PROMPT’), RP-PG-1212-20007]. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.Disclosure of Interests:William Tillett Grant/research support from: AbbVie, Celgene, Eli Lilly, Janssen, Novartis, Pfizer Inc, UCB, Consultant of: AbbVie, Amgen, Celgene, Lilly, Janssen, Novartis, MSD, Pfizer Inc, UCB, Speakers bureau: AbbVie, Amgen, Celgene, Lilly, Janssen, Novartis, Pfizer Inc, UCB, Philip Helliwell: None declared, Oliver FitzGerald: None declared, Robin Waxman: None declared, Anna Antony: None declared, Laura C Coates: None declared, Deepak Jadon: None declared, Paul Creamer: None declared, Suzanne Lane: None declared, Marco Massarotti: None declared, Charlotte Cavill: None declared, Mel Brooke: None declared, Jonathan Packham: None declared, Eleanor Korendowych: None declared, Anya Lissina: None declared, Neil McHugh: None declared

Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production. We also cover the use of small-scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale-up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.

CD8(+) T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8(+) T-cell receptor (TCR)-V beta(+) populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-V beta(+)) and residual (TCR-V beta(-)) CD8(+) T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8(+) TCR-V beta(+) expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8(+) TCR-V beta(+) expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8(+) TCR-V beta(+) expansions.

International audience; The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8186-194 (ZnT8186-194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186-194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186-194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186-194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186-194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

The immune systems of HIV-infected patients show evidence of exhaustion. Immune exhaustion occurs at three different levels: (i) functional, (ii) clonal, and (iii) systemic. Firstly, HIV-specific cells present functional alterations, largely related to the overexpression of co-inhibitory receptors, upregulated as a result of chronic antigenic stimulation. Secondly, persistently activated HIV-1specific CD8 T-cell clones are driven towards a state of replicative senescence, resulting in the loss of cytotoxic T-cell populations important for the control of viral replication. Lastly, HIV-1-infected individuals are characterized by declining lymphocyte renewal capacities or lymphopoiesis, observed at the level of hematopoietic progenitors and reminiscent of old age. Thus, immune exhaustion in chronic HIV infection is multifaceted and affects the immune response in a number of ways, all of which are likely to contribute towards disease progression.

The human leukocyte antigen (HLA)-A2-restricted zinc transporter (ZnT)8186–194 and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. Here, we show that clonal ZnT8186–194-reactive CD8+ T cells express private T-cell receptors and display equivalent functional properties in T1D and healthy subjects. Ex-vivo analyses further revealed that CD8+ T cells reactive to ZnT8186–194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186–194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children vs. adults, irrespective of disease status. Moreover, some ZnT8186–194-reactive CD8+ T-cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. While ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expressions levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186–194-reactive cells were enriched in the pancreata of T1D donors vs. non-diabetic and type 2 diabetic controls. Thus, islet-reactive CD8+ T cells circulate in most individuals, but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T-cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a pro-inflammatory islet microenvironment.

CD8+ T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8+ T-cell receptor (TCR)-Vβ+ populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-Vβ+) and residual (TCR-Vβ−) CD8+ T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8+ TCR-Vβ+ expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8+ TCR-Vβ+ expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8+ TCR-Vβ+ expansions.

Background: Although it is established that CD8+ T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8+ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8+ T-cell responses against the Gag263–272 KK10 epitope restricted by human leukocyte antigen (HLA)-B27. Methods: To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8+ T-cell clones specific for Nef73–82 QK10 and Gag20–29 RY10, both restricted by HLA-A3, alongside CD8+ T-cell clones specific for Gag263–272 KK10. Results: For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Conclusions: Collectively, these data emphasize the central role of the TCR as a determinant of CD8+ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic “mimics” using subunits that do not exist in the natural world. We developed a platform based on D–amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus–specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.