The C-type lectin-like receptor CD161 is expressed on lymphocytes found in human gut and liver, as well as blood, especially Natural Killer cells, T helper 17 cells and a population of unconventional T cells known as Mucosal Associated Invariant T (MAIT) cells. The association of high CD161 expression with innate T cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including anti-viral populations, which display a memory phenotype. These memory CD161int CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterised by enhanced polyfunctionality, increased levels of cytotoxic mediators and high expression of the transcription factors T-bet and Eomesodermin. Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against Hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T cell populations in humans. Since induction of such responses represents a major aim of T cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

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T-cell biomarkers for diagnosis of tuberculosis: candidate evaluation by a simple whole blood assay for clinical translation To the Editor: Diagnosis of pulmonary tuberculosis (TB) disease, the most common cause of death due to infection globally [1], depends on direct detection of Mycobacterium tuberculosis, typically from a sputum specimen. However, up to half of individuals with microbiologically proven TB do not have a prolonged productive cough [2], required to produce sputum. To address this challenge, investigators have identified candidate blood-based host biomarkers for TB diagnosis, based on M. tuberculosis-specific T-cell memory phenotypes, activation or cytokine expression profiles [3-9]. Such biomarkers have also shown potential as surrogates to monitor TB treatment response [3, 10]. Here, we aimed to: 1) compare the diagnostic performance of several blood-based T-cell biomarkers in TB patients and controls; 2) determine if combinations of these biomarkers improve diagnostic performance; 3) identify the minimal number of flow cytometry parameters required to distinguish active TB patients from persons with latent M. tuberculosis infection (LTBI); and 4) translate measurement of these biomarkers from peripheral blood mononuclear cells to whole blood to enable a simplified application in field studies. We enrolled 25 healthy adults with LTBI, defined by QuantiFERON-TB Gold In-Tube assay (QFT; Qiagen, Hilden, Germany), and 25 HIV-negative adults with TB disease (XpertMTB/RIF+) (Cepheid, Sunnyvale, CA, USA) from a region endemic for TB in South Africa. Blood was collected prior to treatment initiation in persons with active TB and 12-18 months later, after they were declared cured (n=19). In controls, blood was also collected at 12-18 months after enrolment (n=20). Whole blood was stimulated with whole mycobacteria (BCG, Connaught), ESAT-6/CFP-10 peptides from QFT tubes, and TB122 peptide pool [11] using a standardised protocol [12]. Cells were stained with the following antibodies: anti-CD3 (Beckman Coulter, Krefeld, Germany; clone UCHT1; ECD), anti-CD4

ObjectiveRecently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA.DesignWe developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case–control study comparing results with those obtained with the Abbott RealTime HCV test.ResultsThe PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country.ConclusionWe report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions.Trial registration numberNCT02992184.

Germinal centers (GC) are microanatomical structures critical for the development of high-affinity antibodies and B-cell memory. They are organised into two zones, light and dark, with coordinated roles, controlled by local signalling. The innate Lectin-like transcript 1 (LLT1) is known to be expressed on B-cells, but its functional role in the GC reaction has not been explored. Here we report high expression of LLT1 on GC-associated B-cells, early plasmablasts and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40 and CpG stimulation on B-cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on Follicular dendritic cells (FDCs). Triggering of LLT1 supported B-cell activation, CD83 upregulation and CXCR4 downregulation. Overall, these data suggest that LLT1-CD161 interactions play a novel and important role in B-cell maturation within the GC in humans.

Lactate is the main product generated at the end of anaerobic glycolysis or during the Warburg effect and its role as an active signalling molecule is increasingly recognised. Lactate can be released and used by host cells, by pathogens and commensal organisms, thus being essential for the homeostasis of host–microbe interactions. Infection can alter this intricate balance, and the presence of lactate transporters in most human cells including immune cells, as well as in a variety of pathogens (including bacteria, fungi and complex parasites) demonstrates the importance of this metabolite in regulating host–pathogen interactions. This review will cover lactate secretion and sensing in humans and microbes, and will discuss the existing evidence supporting a role for lactate in pathogen growth and persistence, together with lactate's ability to impact the orchestration of effective immune responses. The ubiquitous presence of lactate in the context of infection and the ability of both host cells and pathogens to sense and respond to it, makes manipulation of lactate a potential novel therapeutic strategy. Here, we will discuss the preliminary research that has been carried out in the context of cancer, autoimmunity and inflammation.

Aims/hypothesis Self-antigen-specific T cell responses drive type 1 diabetes pathogenesis, but alterations in innate immune responses are also critical and not as well understood. Innate immunity in human type 1 diabetes has primarily been assessed via gene-expression analysis of unstimulated peripheral blood mononuclear cells, without the immune activation that could amplify disease-associated signals. Increased responsiveness in each of the two main innate immune pathways, driven by either type 1 IFN (IFN-1) or IL-1, have been detected in type 1 diabetes, but the dominant innate pathway is still unclear. This study aimed to determine the key innate pathway in type 1 diabetes and assess the whole blood immune stimulation assay as a tool to investigate this. Methods The TruCulture whole blood ex vivo stimulation assay, paired with gene expression and cytokine measurements, was used to characterise changes in the stimulated innate immune response in type 1 diabetes. We applied specific cytokine-induced signatures to our data, pre-defined from the same assays measured in a separate cohort of healthy individuals. In addition, NOD mice were stimulated with CpG and monocyte gene expression was measured. Results Monocytes from NOD mice showed lower baseline vs diabetes-resistant B6.g7 mice, but higher induced IFN-1associated gene expression. In human participants, ex vivo whole blood stimulation revealed higher induced IFN-1 responses in type 1 diabetes, as compared with healthy control participants. In contrast, neither the IL-1-induced gene signature nor response to the adaptive immune stimulant Staphylococcal enterotoxin B were significantly altered in type 1 diabetes samples vs healthy control participants. Targeted gene-expression analysis showed that this enhanced IFN response was specific to IFN-1, as IFN-γ-driven responses were not significantly different. Conclusions/interpretation Our study identifies increased responsiveness to IFN-1 as a feature of both the NOD mouse model of autoimmune diabetes and human established type 1 diabetes. A stimulated IFN-1 gene signature may be a potential biomarker for type 1 diabetes and used to evaluate the effects of therapies targeting this pathway. Kameron B. Rodrigues and Matthew J. Dufort contributed equally to this study.

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