Search

Although trajectories of youth volunteering were disrupted by the COVID-19 pandemic, nevertheless some youth persisted in volunteering, and others emerged as volunteers. To understand volunteering trajectories, the present mixed method study proposed a model adapted from prior literature and examined volunteer trajectories during the pandemic. Youths' volunteer trajectories were categorized (as persistent, emergent, or former volunteer, or persistent non-volunteer), and their justifications for their volunteer decisions were classified using social domain theory (personal, social, moral, and prudential). A sample of 461 youth (M age = 19.26; 68.8% female; 41.6% European or North American) from a large Canadian university completed a retrospective survey on pandemic volunteering and volunteer decisions. Volunteer decisions were coded using conventional and directed qualitative content analysis. Although the pandemic disrupted the volunteering trajectories of former volunteers, overall, more youth persisted or emerged as volunteers during the pandemic, a finding framed in both the trajectory and emergency and disaster literature. Volunteers were more likely to use moral justifications, whereas prudential justifications were more frequent among non-volunteers. The present study offers insight into the impact of the pandemic on youth volunteering and is one of the first studies to find a substantive role for prudential reasoning in youth decision making. [ABSTRACT FROM AUTHOR]

Objectives: Seasonal influenza viruses cause roughly 650 000 deaths annually despite available vaccines. CD8+ T cells typically recognise influenza‐derived peptides from internal structural and non‐structural influenza proteins and are an attractive avenue for future vaccine design as they could reduce the severity of disease following infection with diverse influenza strains. CD8+ T cells recognise peptides presented by the highly polymorphic Human Leukocyte Antigens class I molecules (HLA‐I). Each HLA‐I variant has distinct peptide binding preferences, representing a significant obstacle for designing vaccines that elicit CD8+ T cell responses across broad populations. Consequently, the rational design of a CD8+ T cell‐mediated vaccine would require the identification of highly immunogenic peptides restricted to a range of different HLA molecules. Methods: Here, we assessed the immunogenicity of six recently published novel influenza‐derived peptides identified by mass‐spectrometry and predicted to bind to the prevalent HLA‐B*18:01 molecule. Results: Using CD8+ T cell activation assays and protein biochemistry, we showed that 3/6 of the novel peptides were immunogenic in several HLA‐B*18:01+ individuals and confirmed their HLA‐B*18:01 restriction. We subsequently compared CD8+ T cell responses towards the previously identified highly immunogenic HLA‐B*18:01‐restricted NP219 peptide. Using X‐ray crystallography, we solved the first crystal structures of HLA‐B*18:01 presenting immunogenic influenza‐derived peptides. Finally, we dissected the first TCR repertoires specific for HLA‐B*18:01 restricted pathogen‐derived peptides, identifying private and restricted repertoires against each of the four peptides. Conclusion: Overall the characterisation of these novel immunogenic peptides provides additional HLA‐B*18:01‐restricted vaccine targets derived from the Matrix protein 1 and potentially the non‐structural protein and the RNA polymerase catalytic subunit of influenza viruses. [ABSTRACT FROM AUTHOR]

Seasonal influenza viruses continue to cause severe medical and financial complications annually. Although there are many licenced influenza vaccines, there are billions of cases of influenza infection every year, resulting in the death of over half a million individuals. Furthermore, these figures can rise in the event of a pandemic, as seen throughout history, like the 1918 Spanish influenza pandemic (50 million deaths) and the 1968 Hong Kong influenza pandemic (~4 million deaths). In this review, we have summarised many of the currently licenced influenza vaccines available across the world and current vaccines in clinical trials. We then briefly discuss the important role of CD8+ T cells during influenza infection and why future influenza vaccines should consider targeting CD8+ T cells. Finally, we assess the current landscape of known immunogenic CD8+ T‐cell epitopes and highlight the knowledge gaps required to be filled for the design of rational future influenza vaccines that incorporate CD8+ T cells. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is on the rise globally and is a known susceptibility factor for severe influenza virus infections. However, the mechanisms by which diabetes increases the severity of an influenza virus infection are yet to be fully defined. Diabetes mellitus is hallmarked by high glucose concentrations in the blood. We hypothesized that these high glucose concentrations affect the functionality of CD8+ T cells, which play a key role eliminating virus-infected cells and have been shown to decrease influenza disease severity. To study the effect of hyperglycemia on CD8+ T cell function, we stimulated peripheral blood mononuclear cells (PBMCs) from donors with and without diabetes with influenza A virus, anti-CD3/anti-CD28-coated beads, PMA and ionomycin (PMA/I), or an influenza viral peptide pool. After stimulation, cells were assessed for functionality [as defined by expression of IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1β, and lysosomal-associated membrane protein-1 (CD107a)] using flow cytometry. Our results showed that increasing HbA1c correlated with a reduction in TNF-α production by CD8+ T cells in response to influenza stimulation in a TCR-specific manner. This was not associated with any changes to CD8+ T cell subsets. We conclude that hyperglycemia impairs CD8+ T cell function to influenza virus infection, which may be linked with the increased risk of severe influenza in patients with diabetes. [ABSTRACT FROM AUTHOR]

Objective: Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally, infecting humans and causing widespread morbidity and mortality. Here, we investigate the T cell response towards an immunodominant IAV epitope, NP265‐273, and its IBV and ICV homologues, presented by HLA‐A*03:01 molecule expressed in ~ 4% of the global population (~ 300 million people). Methods: We assessed the magnitude (tetramer staining) and quality of the CD8+ T cell response (intracellular cytokine staining) towards NP265‐IAV and described the T cell receptor (TCR) repertoire used to recognise this immunodominant epitope. We next assessed the immunogenicity of NP265‐IAV homologue peptides from IBV and ICV and the ability of CD8+ T cells to cross‐react towards these homologous peptides. Furthermore, we determined the structures of NP265‐IAV and NP323‐IBV peptides in complex with HLA‐A*03:01 by X‐ray crystallography. Results: Our study provides a detailed characterisation of the CD8+ T cell response towards NP265‐IAV and its IBV and ICV homologues. The data revealed a diverse repertoire for NP265‐IAV that is associated with superior anti‐viral protection. Evidence of cross‐reactivity between the three different influenza virus strain‐derived epitopes was observed, indicating the discovery of a potential vaccination target that is broad enough to cover all three influenza strains. Conclusion: We show that while there is a potential to cross‐protect against distinct influenza virus lineages, the T cell response was stronger against the IAV peptide than IBV or ICV, which is an important consideration when choosing targets for future vaccine design. [ABSTRACT FROM AUTHOR]

Mental ill health forms an increasingly significant part of the burden of disease in developing countries. The growing interest in social risk factors for mental health coincides with the development of social capital research which may further inform the social model of mental health. The objective of the study reported here was to discover if there is an independent association between social capital and mental health when taking into account an array of demographic and violence variables. A total of 1168 youth (15-25 years) in a low income community in Cali, Colombia were surveyed. Mental health was measured by a 20 item self-report questionnaire. The instrument used to measure social capital covered structural and cognitive social capital. Twenty-four per cent of the sample were probable cases of mental ill health. Females had a prevalence rate three times higher than males. Using a model which considered demographic and social capital measures as potential risk factors for mental ill health, the significant risk factors emerged as being female, having limited schooling, working in the informal sector, being a migrant, and having low trust in people. The 'classic' poverty type variables (poor education and employment) were more important than social capital, as was the commonly dominant risk factor for mental ill health--being a woman. When violence factors were added to the model, the 'trust' factor fell out and the most important risk factors became (in descending order of importance): being female; no schooling/incomplete primary; and being a victim of violence. The dominance of poverty related factors, as opposed to social capital, prompts renewed attention to the explanatory mechanisms that link income inequality and poor mental health. Keywords: Mental health; Social capital; Youth; Colombia: Violence

Chemoresistance poses a great barrier to breast cancer treatment and is thought to correlate with increased matrix stiffness. We developed two-dimensional (2D) polyacrylamide (PAA) and three-dimensional (3D) alginate in vitro models of tissue stiffness that mimic the stiffness of normal breast and breast cancer. We then used these to compare cell viability in response to chemotherapeutic treatment. In both 2D and 3D we observed that breast cancer cell growth and size was increased at a higher stiffness corresponding to tumours compared to normal tissue. When chemotherapeutic response was measured, a specific differential response in cell viability was observed for gemcitabine in 2 of the 7 breast cancer cell lines investigated. MCF7 and T-47D cell lines showed gemcitabine resistance at 4 kPa compared to 500 Pa. These cell lines share a common phenotype of progesterone receptor (PGR) expression and, indeed, pre-treatment with the selective progesterone receptor modulator (SPRM) mifepristone abolished resistance to gemcitabine at high stiffness. Our data reveals that combined treatment with SPRMs may therefore help in reducing resistance to gemcitabine in stiffer breast tumours which are PGR positive. [ABSTRACT FROM AUTHOR]

As part of a drug discovery program, 4-pyrazin-2-yl-1 H -pyrrole-2-carboxamides were accessed along with a number of bicyclic analogues. Routes to these compounds were largely absent from the scientific literature. The synthesis of a 4-(pyrazin-2-yl)-1 H -pyrrole-2-carboxamide and several fused bicyclic analogues all using standard procedures (SN Ar, borylation, C–C cross couplings, hydrolysis, amide bond formation, cyclisation, halogenation, and alkylation) from readily available starting materials is reported. The synthetic sequences range from 4–12 steps per final compound, with yields of isolated intermediates ranging from 20 to ∼100%. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic has highlighted the vulnerability of people with diabetes mellitus (DM) to respiratory viral infections. Despite the short history of COVID‐19, various studies have shown that patients with DM are more likely to have increased hospitalisation and mortality rates as compared to patients without. At present, the mechanisms underlying this susceptibility are unclear. However, prior studies show that the course of COVID‐19 disease is linked to the efficacy of the host's T‐cell responses. Healthy individuals who can elicit a robust T‐cell response are more likely to limit the severity of COVID‐19. Here, we investigate the hypothesis that an impaired T‐cell response in patients with type 2 diabetes mellitus (T2DM) drives the severity of COVID‐19 in this patient population. While there is currently a limited amount of information that specifically addresses T‐cell responses in COVID‐19 patients with T2DM, there is a wealth of evidence from other infectious diseases that T‐cell immunity is impaired in patients with T2DM. The reasons for this are likely multifactorial, including the presence of hyperglycaemia, glycaemic variability and metformin use. This review emphasises the need for further research into T‐cell responses of COVID‐19 patients with T2DM in order to better inform our response to COVID‐19 and future disease outbreaks. [ABSTRACT FROM AUTHOR]

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease‐modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment‐screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment–protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics. [ABSTRACT FROM AUTHOR]

The city of Hawassa is growing fast, driven by construction of a flagship industrial park that is expected to attract up to 60,000 workers by 2021, mostly young women, arriving without families or dependents, and living off very low wages. Along with these young women, female-headed households; divorced, separated and widowed women; elderly women; and women with disabilities all face severe/acute shelter vulnerabilities. These groups are most likely to struggle to access both formal and informal shelter, related to their below-average income levels but also to other forms of bias and discrimination. This paper draws out key findings on gender and housing from a collaborative study investigating shelter provision in Hawassa, part of a wider research study on inclusive cities in East Africa led by the International Institute for Environment and Development (IIED). It aims to highlight specific constraints faced by women in accessing shelter, and around such issues as informality, safety and security, and infrastructure provision. [ABSTRACT FROM AUTHOR]

Seasonal influenza virus infections cause 290,000–650,000 deaths annually and severe morbidity in 3–5 million people. CD8+ T-cell responses towards virus-derived peptide/human leukocyte antigen (HLA) complexes provide the broadest cross-reactive immunity against human influenza viruses. Several universally-conserved CD8+ T-cell specificities that elicit prominent responses against human influenza A viruses (IAVs) have been identified. These include HLA-A*02:01-M158-66 (A2/M158), HLA-A*03:01-NP265-273, HLA-B*08:01-NP225-233, HLA-B*18:01-NP219-226, HLA-B*27:05-NP383-391 and HLA-B*57:01-NP199-207. The immunodominance hierarchies across these universal CD8+ T-cell epitopes were however unknown. Here, we probed immunodominance status of influenza-specific universal CD8+ T-cells in HLA-I heterozygote individuals expressing two or more universal HLAs for IAV. We found that while CD8+ T-cell responses directed towards A2/M158 were generally immunodominant, A2/M158+CD8+ T-cells were markedly diminished (subdominant) in HLA-A*02:01/B*27:05-expressing donors following ex vivo and in vitro analyses. A2/M158+CD8+ T-cells in non-HLA-B*27:05 individuals were immunodominant, contained optimal public TRBV19/TRAV27 TCRαβ clonotypes and displayed highly polyfunctional and proliferative capacity, while A2/M158+CD8+ T cells in HLA-B*27:05-expressing donors were subdominant, with largely distinct TCRαβ clonotypes and consequently markedly reduced avidity, proliferative and polyfunctional efficacy. Our data illustrate altered immunodominance patterns and immunodomination within human influenza-specific CD8+ T-cells. Accordingly, our work highlights the importance of understanding immunodominance hierarchies within individual donors across a spectrum of prominent virus-specific CD8+ T-cell specificities prior to designing T cell-directed vaccines and immunotherapies, for influenza and other infectious diseases. Author summary: Annual influenza infections cause significant morbidity and morbidity globally. Established T-cell immunity directed at conserved viral regions provides some protection against influenza viruses and promotes rapid recovery, leading to better clinical outcomes. Killer CD8+ T-cells recognising viral peptides in a context of HLA-I glycoproteins, provide the broadest ever reported immunity across distinct influenza strains and subtypes. We asked whether the expression of certain HLA-I alleles affects CD8+ T cells responses. Our study clearly illustrates altered immunodominance hierarchies and immunodomination within broadly-cross-reactive influenza-specific CD8+ T-cells in individuals expressing two or more universal HLA-I alleles, key for T cell-directed vaccines and immunotherapies. [ABSTRACT FROM AUTHOR]

The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probe‐based competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest. [ABSTRACT FROM AUTHOR]

Background Our Integrated Care Hub has been operating since mid-2017 providing patient-centred multidisciplinary team (MDT) care operating via a number of specialised care pathways. There has been an early emphasis on the response to crisis presentations and with the introduction of the Hub we now have an extensive and organised response to such presentations. We wanted to assess does this system result in earlier referral of the 'Crisis' patient to an Integrated Care Hub. Methods We performed a retrospective quantitative analysis of data comparing two time-periods January to June 2018 with January to April 2019 (year-to-date). The number and type of MDT contacts were classified into 4 categories: Immediate Crisis (would present to the Emergency Department within 48 hrs if not reviewed), Emerging Crisis (would present to the Emergency Department within 2 weeks if not reviewed), Stable Situation (needs optimisation), Stable Situation (no intervention). Data were extracted from File-Maker Pro. Results There were 424 MDT contacts in January-June 2018 compared with 658 MDT contacts in the year-to-date 2019. The number of patients in these time-periods presenting in 'Immediate Crisis' was reduced in the year-to-date 2019 - 4.41% when compared to the same time-period in 2018 – 11.79%. There was a similar reduction in 'Emerging' Crisis presentations in these time-periods: 15.65% in 2019 vs. 24.29% in 2018. While the patients attending that were classified as Stable Situation (needs optimisation) increased in 2019 compared to 2018 – 66.57% vs. 47.88%. Conclusion We can see from our data that the creation of our Integrated Care Hub has resulted in an increased demand for our service, however, interestingly we have found that the crisis patient load has, in fact, decreased. This suggests that we are influencing referral patterns by both recognising and intervening at an earlier stage to manage, stabilise and reverse crisis in these patients in an organised manner within the MDT. [ABSTRACT FROM AUTHOR]

Background The Occupational Therapy (OT) service in the integrated care HUB team was established in January 2018. Prior to this, there was an established memory clinic, however with inconsistent access to OT. The streamlining and structuring of memory clinics further highlighted the need for post-diagnostic support for example individual sessions focusing on memory education and practical strategies to enable independence in daily activities. The integration of OT enabled person-centred strategies to be provided to individuals experiencing memory loss. Methods A retrospective analysis was completed comparing the number of OT memory patient contacts between January to June 2018 and November 2018 to April 2019 also reflecting the memory care pathway developed during this period. OT assessed all new patients experiencing memory difficulties that impacted on functional activities to initiate referral process. In April 2018, the Memory Technology Resource Room (MTRR) opened and the OT HUB using to facilitate patient contacts. The design/implementation of cognitive rehabilitation groups in May 2018 added another step to pathway. Dementia cafés were established in public cafés in the city and county area, sponsored by local care provider, also supported by HUB team. Feedback was gathered from surveys given to individuals, carers and HUB team in the Café and MTRR. Results From January to June 2018 there were 50 memory streamed patient contacts completed by the OT. From November 2018 to April 2019, 206 patient contacts were completed. These include both individual and group cognition focused sessions. Positive qualitative feedback was retrieved from attendees to MTRR and Café all indicating at least one positive outcome from post-diagnostic supports and services. Conclusion A multi-domain cognitive OT service when initiated by an integrated care HUB proves to be an effective and acceptable memory care pathway. The development of this holistic pathway enabled the person to be seen in the right place, at the right stage of their journey with memory difficulties. [ABSTRACT FROM AUTHOR]

Background The development of an Integrated Care Hub has resulted in a complete review of the triage system for older people referred to our service. Our aim was to develop a multidisciplinary triage process (MTP) to ensure patients are seen by the right person at the right time for a First Contact Assessment (FCA). Methods A quantitative retrospective analysis of referrals and frequency of multidisciplinary team input from January 2018 – June 2018, highlighted the majority of patients were seen for FCA by the staff nurse. This subsequently resulted in onward referrals to Allied Health Professionals within the 'Hub', and patients attended for multiple visits. A working group was formed to address these issues. This resulted in the development of the MTP where patients are triaged by the Multidisciplinary team (MDT) and allocated to the most appropriate team member based on the referring information. Following the introduction of this triage process, data analysed from November 2018 - April 2019 highlighted its impact on our service. Results In the first reference period the proportion of patients seen for FCA by each discipline was as follows; Staff nurse (SN) 53%, Clinical Nurse Specialist (CNS) 18%, Occupational Therapist (OT) 16% and Physiotherapist 9%. In the second reference period the proportions changed to the following; SN 12%, CNS 31%, OT 33%, Physiotherapist 20% and Dietitian 4%. It has reduced the number of patient visits to the 'Hub' and the waiting times to see the right person within the team. Conclusion Empowering supported MDT members to triage and the use of this new MTP has resulted in patients having rapid access to the most appropriate team member for FCA. It has resulted in a change in the distribution of the caseload to ensure patients are seen by the right person within the team, and at the right time for early patient centred intervention. [ABSTRACT FROM AUTHOR]

Abstract: Influenza epidemics lead to severe illness, life‐threatening complications, and deaths, especially in the elderly. As CD8+ T cells are associated with rapid recovery from influenza, we investigated the effects of aging on antigen‐specific CD8+ T cells across the universal influenza epitopes in humans. We show that aging is characterized by altered frequencies in T cell subsets, with naive T cells being partially replaced by activated effector/memory populations. Although we observed no striking differences in TCR signaling capacity, T cells in the elderly had increased expression of transcription factors Eomes and T‐bet, and such changes were most apparent in CD8+ T cells. Strikingly, the numbers of antigen‐specific CD8+ T cells across universal influenza epitopes were reduced in the elderly, although their effector/memory phenotypes remained stable. To understand whether diminished numbers of influenza‐specific CD8+ T cells in the elderly resulted from alteration in TCR clonotypes, we dissected the TCRαβ repertoire specific for the prominent HLA‐A*02:01‐restricted‐M158–66 (A2/M158) influenza epitope. We provide the first ex vivo data on paired antigen‐specific TCRαβ clonotypes in the elderly, showing that influenza‐specific A2/M158+ TCRαβ repertoires in the elderly adults varied from those in younger adults, with the main features being a reduction in the frequency of the public TRAV27–TRBV19 TCRαβ clonotype, increased proportion of private TCRαβ signatures, broader use of TRAV and TRBV gene segments, and large clonal expansion of private TCRαβ clonotypes with longer CDR3 loops. Our study supports the development of T cell‐targeted influenza vaccines that would boost the T cell compartment during life and maintain the numbers and optimal TCRαβ signatures in the elderly. [ABSTRACT FROM AUTHOR]

It has widely been accepted that major histocompatibility complex class I molecules (MHC-I) are limited to binding small peptides of 8-10 residues in length. However, this consensus has recently been challenged with the identification of longer peptides (= 11 residues) that can also elicit cytotoxic CD8 + T cell responses. Indeed, a growing number of studies demonstrate that these noncanonical epitopes are important targets for the immune system. As long epitopes represent up to 10% of the peptide repertoire bound to MHC-I molecules, here we review their impact on antigen presentation by MHC-I, TCR recognition, and T cell immunity. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair. [ABSTRACT FROM AUTHOR]

CD27 is a co-stimulatory immune-checkpoint receptor, constitutively expressed on a broad range of T-cells (αβ and γδ), NK-cells and B-cells. Ligation of CD27 with CD70 results in potent co-stimulatory effects. In mice, co-stimulation of CD8 + T-cells through CD27 promotes immune activation and enhances primary, secondary, memory and recall responses towards viral infections. Limited in vitro human studies support mouse experiments and show that CD27 co-stimulation enhances antiviral T-cell immunity. Given the potent co-stimulatory effects of CD27, manipulating CD27 signalling is of interest for viral, autoimmune and anti-tumour immunotherapies. This review focuses on the role of CD27 co-stimulation in anti-viral T-cell immunity and discusses clinical studies utilising the CD27 co-stimulation pathway for anti-viral, anti-tumour and autoimmune immunotherapy. [ABSTRACT FROM AUTHOR]

Epstein‐Barr virus (EBV) is one of the most common viruses in humans, capable of causing life‐threatening infections and cancers in immunocompromised individuals. Although CD8+ T cells provide key protection against EBV, the persistence and dynamics of specific T‐cell receptor (TCR) clones during immunosuppression in transplant patients is largely unknown. For the first time, we used a novel single‐cell TCRαβ multiplex‐nested reverse transcriptase PCR to dissect TCRαβ clonal diversity within GLCTLVAML (GLC)‐specific CD8+ T cells in healthy individuals and immunocompromised lung transplant recipients. The GLC peptide presented by HLA‐A*02:01 is one of the most immunogenic T‐cell targets from the EBV proteome. We found that the GLC‐specific TCRαβ repertoire was heavily biased toward TRAV5 and encompassed five classes of public TCRαβs, suggesting that these clonotypes are preferentially utilized following infection. We identified that a common TRAV5 was diversely paired with different TRAJ and TRBV/TRBJ genes, in both immunocompetent and immunocompromised individuals, with an average of 12 different TCRαβ clonotypes/donor. Moreover, pre‐transplant GLC‐specific TCRαβ repertoires were relatively stable over 1 year post transplant under immunosuppression in the absence or presence of EBV reactivation. In addition, we provide the first evidence of early GLC‐specific CD8+ T cells at 87 days post transplant, which preceded clinical EBV detection at 242 days in an EBV‐seronegative patient receiving a lung allograft from an EBV‐seropositive donor. This was associated with a relatively stable TCRαβ repertoire after CD8+ T‐cell expansion. Our findings provide insights into the composition and temporal dynamics of the EBV‐specific TCRαβ repertoire in immunocompromised transplant patients and suggest that the early detection of EBV‐specific T cells might be a predictor of ensuing EBV blood viremia. [ABSTRACT FROM AUTHOR]

αβT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized.Toprovide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR crossreactivity remain unclear, with conflicting results on anti-viralT cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M158, respectively) or with the hepatitis C and influenza viruses (NS31073 and NA231, respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell crossreactivity. We combined ex vivo and in vitro functional assays, single-cell αβTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αβTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell crossreactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific. [ABSTRACT FROM AUTHOR]

Memory CD8+ T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158 and the hypervariable HLA-B*3501-NP418 antigens. The TCRαβs for HLA-B*3501-NP418 + CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19+ TCRαβ was selected in HLA-A*0201+ donors responding to M158. This public TCR cross-recognized naturally occurring M158 variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19+ TCR specificity for the WT and mutant M158 peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201+ individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158. Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) cause significant morbidity and mortality worldwide, despite new strain-specific vaccines being available annually. As IAV-specific CD8 + T cells promote viral control in the absence of neutralizing antibodies, and can mediate cross-reactive immunity toward distinct IAVs to drive rapid recovery from both mild and severe influenza disease, there is great interest in developing a universal T cell vaccine. However, despite detailed studies in mouse models of influenza virus infection, there is still a paucity of data on human epitope-specific CD8 + T cell responses to IAVs. This review focuses on our current understanding of human CD8 + T cell immunity against distinct IAVs and discusses the possibility of achieving a CD8 + T cell mediated-vaccine that protects against multiple, distinct IAV strains across diverse human populations. We also review the importance of CD8 + T cell immunity in individuals highly susceptible to severe influenza infection, including those hospitalised with influenza, the elderly and Indigenous populations. [ABSTRACT FROM AUTHOR]

The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus–specific CD8+ T lymphocytes (CTLs) can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16–57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701, B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development. [ABSTRACT FROM AUTHOR]

To understand the interactions between innate and adaptive immunity, and specifically how virally infected macrophages impact T cell function, novel assays examining the ability of macrophages to present antigen to CD8+ T cells are needed. In the present study, we have developed a robust in vitro assay to measure how antigen presentation by human monocyte-derived macrophages (MDMs) affects the functional capacity of autologous CD8+ T cells. The assay is based on the polyfunctional characteristics of antigen-specific CD8+ T cells, and is thus called a Mac-CD8 Polyfunctionality Assay. Following purification of monocytes and their maturation to MDMs, MDMs were pulsed with an antigenic peptide to be presented to CD8+ T cells. Peptide-pulsed MDMs were then incubated with antigen-specific CD8+ T cells in order to assess the efficacy of antigen presentation to T cells. CD8+ T cell polyfunctionality was assessed by staining with mAbs to IFN-γ, TNF-α, and CD107a in a multi-color intracellular cytokine staining assay. To highlight the utility of the Mac-CD8 Polyfunctionality Assay, we assessed the effects of influenza infection on the ability of human macrophages to present antigen to CD8+ T cells. We found that influenza infection of human MDMs can alter the effector efficacy of MDMs to activate more CD8+ T cells with cytotoxic capacity. This has important implications for understanding how the virus-infected macrophages affect adaptive immunity at the site of infection. [ABSTRACT FROM AUTHOR]

Influenza A virus causes annual epidemics and sporadic pandemics, resulting in significant morbidity and mortality worldwide. Vaccines are currently available; however, they induce a non-strain-cross protective humoral immune response directed against the rapidly mutating surface glycoproteins, and thus need to be updated annually. As T cells are directed against more conserved internal influenza proteins, a T-cell-based vaccine has the potential to induce long-lasting and cross-strain protective CD8+ T-cell immunity, and in that way minimize the severity of influenza infection. However, to rationally design such vaccines, we need to identify immunogenic T-cell regions within the most antigenic viral proteins. In this study, we have used a systematic approach to identify immunodominant peptides in HLA-A2-negative donors. A broad range of CD8+ T-cell responses were observed and 6/7 donors had an immunodominant response against the relatively conserved internal nucleoprotein (NP). Dissecting the minimal epitope regions within the immunogenic NP led to the identification of six novel immunodominant epitopes, which include a 12-mer and an 8-mer peptides. The majority of immunodominant epitopes was clustered within the carboxyl terminal 2/3 of the NP protein and were highly conserved. We also subjected NP to three common computer algorithms for epitope prediction and found that most of the novel epitopes would not have been predicted. Our study emphasizes the importance of using a systematic approach to identify immunodominant CD8+ T-cell responses and suggests that the epitope-rich regions within NP present a promising target for the T-cell-mediated multi-strain influenza vaccine. [ABSTRACT FROM AUTHOR]

The effects of combined treatment with a glucocorticoid receptor (GR) antagonist, Org 34850, and a selective serotonin reuptake inhibitor (SSRI), fluoxetine, were investigated on pre- and postsynaptic aspects of 5-HT neurotransmission. Rats were treated for 14 days with Org 34850 (15 mg per kg per day subcutaneously), fluoxetine (10 mg per kg per day intraperitoneally), or a combination of both drugs. [3H]-citalopram binding (an index of 5-HT transporter (5-HTT) expression) was only slightly affected by Org 34850 alone: decreased in cortex and midbrain and increased in hippocampus. In contrast, chronic fluoxetine markedly decreased 5-HTT levels in all regions. Importantly, this decrease was significantly enhanced by combined Org 34850/fluoxetine treatment. There were no changes in the expression of 5-HTT mRNA, suggesting these effects were not due to changes in gene transcription. Expression of tryptophan hydroxylase mRNA and both 5-HT1A autoreceptor mRNA and protein (assessed using [3H]-8-OH-DPAT binding) were unchanged by any treatment. The expression of postsynaptic 5-HT1A receptor protein in the forebrain was unaltered by fluoxetine, Org 34850 or the combined Org 34850/fluoxetine treatment. This downregulation of 5-HTT by fluoxetine and its enhancement by Org 34850 can explain our recent observation that GR antagonists augment the SSRI-induced increase in extracellular 5-HT. In addition, these data suggest that the augmentation of forebrain 5-HT does not result in downregulation of forebrain 5-HT1A receptor expression. Given the importance of 5-HT1A receptor-mediated transmission in the forebrain to the antidepressant response, these data indicate that co-administration of GR antagonists may be effective in augmenting the antidepressant response to SSRI treatment.Neuropsychopharmacology (2009) 34, 399–409; doi:10.1038/npp.2008.70; published online 21 May 2008 [ABSTRACT FROM AUTHOR]