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COVID-19 affects multiple organ-systems. Recent studies have indicated perturbations in the circulating metabolome linked to COVID-19 severity. However, several questions pertain with respect to the metabolome in COVID-19. We performed an in-depth assessment of 1129 unique metabolites in 27 hospitalized COVID-19 patients and integrated results with large-scale proteomic and immunology data to capture multi-organ system perturbations. More than half of the detected metabolic alterations in COVID-19 were driven by patient-specific confounding factors ranging from comorbidities to xenobiotic substances. Systematically adjusting for this, a COVID-19 specific metabolic imprint was defined which, over time, underwent a switch in response to SARS-CoV-2 seroconversion. Integration of the COVID-19 metabolome with clinical, cellular, molecular and immunological severity scales further revealed a network of metabolic trajectories aligned with multiple pathways for immune activation, and organ damage including neurological inflammation and damage. Altogether, this resource refines our understanding of the multi organ-system perturbations in severe COVID-19 patients. This article is protected by copyright. All rights reserved.

Background and Aims: Cholangiocarcinoma (CCA) is a malignancy arising from biliary epithelial cells of intra- and extrahepatic bile ducts with dismal prognosis and few nonsurgical treatments available. Despite recent success in the immunotherapy-based treatment of many tumor types, this has not been successfully translated to CCA. Mucosal-associated invariant T (MAIT) cells are cytotoxic innate-like T cells highly enriched in the human liver, where they are located in close proximity to the biliary epithelium. Here, we aimed to comprehensively characterize MAIT cells in intrahepatic (iCCA) and perihilar CCA (pCCA). Approach and Results: Liver tissue from patients with CCA was used to study immune cells, including MAIT cells, in tumor-affected and surrounding tissue by immunohistochemistry, RNA-sequencing, and multicolor flow cytometry. The iCCA and pCCA tumor microenvironment was characterized by the presence of both cytotoxic T cells and high numbers of regulatory T cells. In contrast, MAIT cells were heterogenously lost from tumors compared to the surrounding liver tissue. This loss possibly occurred in response to increased bacterial burden within tumors. The residual intratumoral MAIT cell population exhibited phenotypic and transcriptomic alterations, but a preserved receptor repertoire for interaction with tumor cells. Finally, the high presence of MAIT cells in livers of iCCA patients predicted long-term survival in two independent cohorts and was associated with a favorable antitumor immune signature. Conclusions: MAIT cell tumor infiltration associates with favorable immunological fitness and predicts survival in CCA.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite antigens presented by MR1 and play an important role in antimicrobial immune defense. Staphylococcus aureus is a pathobiont expressing a range of virulence factors including the secreted toxin Leukocidin ED (LukED), which binds to certain chemokine receptors and causes cell death by osmolysis. Here, we investigated the effect of LukED on subsets of human T cells and NK cells that are involved in the early innate response to infection. MAIT cells were strikingly hypersensitive to LukED-mediated lysis and rapidly lost from the peripheral blood T cell pool upon exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid, occurred at lower LukED concentration compared to effects on the overall T cell pool, and coincided with extraordinarily high and uniform expression of CCR5. Furthermore, loss of MAIT cells was efficiently inhibited by the CCR5 inhibitor Maraviroc. Interestingly, pre-activation of MAIT cells with IL-12 and IL-18 also partially rescued these cells from LukED toxicity. Among NK cells, LukED targeted the more mature and cytotoxic CD57+ NK cell subset in a CXCR1-dependent manner. Overall, these results indicate that LukED efficiently eliminates cells of the human immune system that have the capacity to respond rapidly to S. aureus in an innate fashion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED functions as a S. aureus immune evasion mechanism to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Progress in our understanding of MR1-restricted mucosa-associated invariant T (MAIT) cells has raised interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, while preserving their functional response toward their natural ligand and increasing their cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation and expressed a more diverse set of chemokine receptors, potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I–restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy.

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in SARS-CoV-2-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion and migration of granulocytes (e.g. CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers supporting pathophysiologic relevance. Furthermore, clinical features, including multi-organ dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.SignificanceAccumulating evidence shows that granulocytes are key modulators of the immune response to SARS-CoV-2 infection and their dysregulation could significantly impact COVID-19 severity and patient recovery after virus clearance. In the present study, we identify selected immune traits in neutrophil, eosinophil and basophil subsets associated to severity of COVID-19 and to peripheral protein profiles. Moreover, computational modeling indicates that the combined use of phenotypic data and laboratory measurements can effectively predict key clinical outcomes in COVID-19 patients. Finally, patient-matched longitudinal analysis shows phenotypic normalization of granulocyte subsets 4 months after hospitalization. Overall, in this work we extend the current understanding of the distinct contribution of granulocyte subsets to COVID-19 pathogenesis.

International audience; The CD8+ T cell response to an antigen is composed of many T cell clones with unique T cell receptors, together forming a heterogeneous repertoire of effector and memory cells. How individual T cell clones contribute to this heterogeneity throughout immune responses remains largely unknown. In this study, we longitudinally track human CD8+ T cell clones expanding in response to yellow fever virus (YFV) vaccination at the single-cell level. We observed a drop in clonal diversity in blood from the acute to memory phase, suggesting that clonal selection shapes the circulating memory repertoire. Clones in the memory phase display biased differentiation trajectories along a gradient from stem cell to terminally differentiated effector memory fates. In secondary responses, YFV- and influenza-specific CD8+ T cell clones are poised to recapitulate skewed differentiation trajectories. Collectively, we show that the sum of distinct clonal phenotypes results in the multifaceted human T cell response to acute viral infections.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this, we used an integrated approach to characterize tissue-emigrant immune cells in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8(+) T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8(+) T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual antigen-specific clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides a framework of the migratory immune system and defines the nature of tissue-emigrant CD8(+) T cells that recirculate via TDL.

Summary: Bats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.

Antimicrobial resistance is a serious threat to global public health as antibiotics are losing effectiveness due to rapid development of resistance. The human immune system facilitates control and clearance of resistant bacterial populations during the course of antimicrobial therapy. Here we review current knowledge of mucosa-associated invariant T (MAIT) cells, an arm of the immune system on the border between innate and adaptive, and their critical place in human antibacterial immunity. We propose that MAIT cells play important roles against antimicrobial-resistant infections through their capacity to directly clear multidrug-resistant bacteria and overcome mechanisms of antimicrobial resistance. Finally, we discuss outstanding questions pertinent to the possible advancement of host-directed therapy as an alternative intervention strategy for antimicrobial-resistant bacterial infections.

The NK cell activation landscape in acute SARS-CoV-2 infection is associated with COVID-19 disease severity., Activated NK cells in severe COVID-19 Natural killer (NK) cells are cytotoxic lymphocytes that provide innate immune defense against viral infections and cancer, but little is known about their involvement in the host response to COVID-19. Maucourant et al. used high-dimensional flow cytometry to characterize NK cells in patients with moderate or severe COVID-19. SARS-CoV-2 infection was associated with fewer blood NK cells but a higher activation state in circulating NK cells. Severe COVID-19 resulted in an increase in “armed” NK cells containing high levels of cytotoxic proteins such as perforin. The adaptive NK subset was markedly expanded in a subset of severe patients. These findings lay the groundwork for future studies examining the mechanisms of NK cell activation in COVID-19 and their potential roles in host protection and immunopathology., Understanding innate immune responses in coronavirus disease 2019 (COVID-19) is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in single-cell RNA sequencing signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Last, arming of CD56bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.

Understanding innate immune responses in COVID-19 is important for deciphering mechanisms of host responses and interpreting disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections, but might also contribute to immune pathology. Here, using 28-color flow cytometry, we describe a state of strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients, a pattern mirrored in scRNA-seq signatures of lung NK cells. Unsupervised high-dimensional analysis identified distinct immunophenotypes that were linked to disease severity. Hallmarks of these immunophenotypes were high expression of perforin, NKG2C, and Ksp37, reflecting a high presence of adaptive NK cell expansions in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed in course of COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This provides a detailed map of the NK cell activation-landscape in COVID-19 disease.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli., Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans that recognize bacterial metabolites. This study shows that MAIT cells exert potent antimicrobial activity against both cell-associated and extracellular forms of Escherichia coli, including strains that are resistant to the last resort antibiotics carbapenems.

Mucosa-associated invariant T (MAIT) cell loss in chronic HIV-1 infection is a significant insult to antimicrobial immune defenses. Here we investigate the response of MAIT cells during acute HIV-1 infection utilizing the RV217 cohort with paired longitudinal pre- and post-infection samples. MAIT cells are activated and expand in blood and mucosa coincident with peak HIV-1 viremia, in a manner associated with emerging microbial translocation. This is followed by a phase with elevated function as viral replication is controlled to a set-point level, and later by their functional decline at the onset of chronic infection. Interestingly, enhanced innate-like pathways and characteristics develop progressively in MAIT cells during infection, in parallel with TCR repertoire alterations. These findings delineate the dynamic MAIT cell response to acute HIV-1 infection, and show how the MAIT compartment initially responds and expands with enhanced function, followed by progressive reprogramming away from TCR-dependent antibacterial responses towards innate-like functionality., Here, using longitudinal pre- and post-infection samples from the RV217 Early Capture HIV Cohort Study, the authors show that mucosa-associated invariant T (MAIT) cells become activated and expand during the early acute phase of HIV infection, with subsequent reprogramming towards innate-like functionality.

CD8+ T cell exhaustion is a hallmark of many cancers and chronic infections. In mice, T cell factor 1 (TCF-1) maintains exhausted CD8+ T cell responses, whereas thymocyte selection-associated HMG box (TOX) is required for the epigenetic remodeling and survival of exhausted CD8+ T cells. However, it has remained unclear to what extent these transcription factors play analogous roles in humans. In this study, we mapped the expression of TOX and TCF-1 as a function of differentiation and specificity in the human CD8+ T cell landscape. Here, we demonstrate that circulating TOX+ CD8+ T cells exist in most humans, but that TOX is not exclusively associated with exhaustion. Effector memory CD8+ T cells generally expressed TOX, whereas naive and early-differentiated memory CD8+ T cells generally expressed TCF-1. Cytolytic gene and protein expression signatures were also defined by the expression of TOX. In the context of a relentless immune challenge, exhausted HIV-specific CD8+ T cells commonly expressed TOX, often in clusters with various activation markers and inhibitory receptors, and expressed less TCF-1. However, polyfunctional memory CD8+ T cells specific for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) also expressed TOX, either with or without TCF-1. A similar phenotype was observed among HIV-specific CD8+ T cells from individuals who maintained exceptional immune control of viral replication. Collectively, these data demonstrate that TOX is expressed by most circulating effector memory CD8+ T cell subsets and not exclusively linked to exhaustion.

Mucosa-associated invariant T (MAIT) cells are innate-like antimicrobial T cells recognizing a breadth of important pathogens via presentation of microbial riboflavin metabolite Ags by MHC class Ib–related (MR1) molecules. However, the interaction of human MAIT cells with adaptive immune responses and the role they may play in settings of vaccinology remain relatively little explored. In this study we investigated the interplay between MAIT cell–mediated antibacterial effector functions and the humoral immune response. IgG opsonization of the model microbe Escherichia coli with pooled human sera markedly enhanced the capacity of monocytic APC to stimulate MAIT cells. This effect included greater sensitivity of recognition and faster response kinetics, as well as a markedly higher polyfunctionality and magnitude of MAIT cell responses involving a range of effector functions. The boost of MAIT cell responses was dependent on strongly enhanced MR1-mediated Ag presentation via increased FcγR-mediated uptake and signaling primarily mediated by FcγRI. To investigate possible translation of this effect to a vaccine setting, sera from human subjects before and after vaccination with the 13-valent–conjugated Streptococcus pneumoniae vaccine were assessed in a MAIT cell activation assay. Interestingly, vaccine-induced Abs enhanced Ag presentation to MAIT cells, resulting in more potent effector responses. These findings indicate that enhancement of Ag presentation by IgG opsonization allows innate-like MAIT cells to mount a faster, stronger, and qualitatively more complex response and to function as an effector arm of vaccine-induced humoral adaptive antibacterial immunity.

CD8+ T cells play essential roles in immunity to viral and bacterial infections, and to guard against malignant cells. The CD8+ T cell response to an antigen is composed of many T cell clones with unique T cell receptors, together forming a heterogenous repertoire of phenotypically and functionally distinct effector and memory cells. How individual T cell clones contribute to this heterogeneity throughout an immune response is key to understand immunity but remains largely unknown. Here, we longitudinally tracked hundreds of CD8+ T cell clones expanding in response to yellow fever virus vaccination at the single cell level in humans. We show that only a fraction of the clones detected in the acute phase of the response are detected as circulating memory T cells later, indicative of clonal selection shaping the memory repertoire. Clones persisting in the memory phase displayed biased differentiation trajectories along a gradient from stem cell memory to terminally differentiated effector memory fates. Reactivation of single memory CD8+ T cell clones revealed that they were poised to recapitulate skewed differentiation trajectories in secondary responses, and this was generalizable across individuals for both yellow fever and influenza virus. Together, we show that the sum of distinct clonal differentiation repertoires results in the multifaceted T cell response to acute viral infections in humans.

Mucosa‐associated invariant T (MAIT) cells are unconventional T lymphocytes defined by their innate‐like characteristics and broad antimicrobial responsiveness. Whether MAIT cells are part of the tissue‐resident defense in the oral mucosal barrier is unknown. Here, we found MAIT cells present in the buccal mucosa, with a tendency to cluster near the basement membrane, and located in both epithelium and the underlying connective tissue. Overall MAIT cell levels were similar in the mucosa compared to peripheral blood, in contrast to conventional T cells that showed an altered representation of CD4+ and CD8+ subsets. The major mucosal MAIT cell subset displayed a tissue‐resident and activated profile with high expression of CD69, CD103, HLA‐DR, and PD‐1, as well as a skewed subset distribution with higher representation of CD4–/CD8– double‐negative cells and CD8αα+ cells. Interestingly, tissue‐resident MAIT cells had a specialized polyfunctional response profile with higher IL‐17 levels, as assessed by polyclonal stimulus and compared to tissue nonresident and circulating populations. Furthermore, resident buccal MAIT cells were low in perforin. Together, these data indicate that MAIT cells form a part of the oral mucosal T cell compartment, where they exhibit a tissue‐resident‐activated profile biased toward IL‐17 production.

The mucosa-associated invariant T (MAIT) cells are innate-like T cells that recognize microbial vitamin B2 metabolites presented via MR1, a MHC-I-related protein. MAIT cells are abundant in blood and mucosa, where they display a broad range of functions. Spatial distribution of cells and their proximity to other cells, including infected cells and antigen presenting cells, are crucial components of cell-mediated immunity. Here we describe techniques to detect MAIT cells and MR1-expressing cells in situ, which enable the visualization, distribution, and localization of these cells within their histological context. We provide specific protocols and describe potential advantages and limitations for each of the presented methodologies for studying MAIT cells in human tissues.

The mucosa-associated invariant T (MAIT) cells represent the most abundant population of antimicrobial T cells in humans. When encountering cells infected with riboflavin-producing bacteria, this innate-like T cell population rapidly release a plethora of pro-inflammatory cytokines, mediates antimicrobial activity, and kill infected cells. Here, we describe methodological approaches and protocols to measure their cytotoxicity and antimicrobial effector function using multi-color flow cytometry-based and standard microbiological techniques. We provide specific guidance on protocols and describe potential pitfalls for each of the presented methodologies. Finally, we discuss potential applications and current limitations of our approaches to the study of human MAIT cell antimicrobial properties.

NK cells lacking CD56 (CD56neg ) were first identified in chronic HIV-1 infection. However, CD56neg NK cells also exist in healthy individuals, albeit in significantly lower numbers. Here, we provide an extensive proteomic characterisation of human CD56neg peripheral blood NK cells of healthy donors and compare them to their CD56dim and CD56bright counterparts. Unbiased large-scale surface receptor profiling clustered CD56neg cells as part of the main NK cell compartment and indicated an overall CD56dim -like phenotype. Total proteome analyses of CD56neg NK cells further confirmed their similarity with CD56dim NK cells, and revealed a complete cytolytic inventory with high levels of perforin and granzyme H and M. In the present study, twelve proteins discriminated CD56neg NK cells from CD56dim NK cells with nine up-regulated and three down-regulated proteins in the CD56neg NK cell population. Those proteins were functionally related to lytic granule composition and transport, interaction with the extracellular matrix, DNA transcription or repair, and proliferation. Corroborating these results, CD56neg NK cells showed modest cytotoxicity, degranulation, and IFN-ɣ secretion as compared to CD56dim NK cells. In conclusion, CD56neg NK cells constitute functionally competent cells sharing many features of bona fide CD56dim NK cells in healthy individuals, but with some distinct characteristics.

Tick-borne encephalitis virus (TBEV) is a leading cause of viral meningoencephalitis in many parts of Europe and eastwards in Asia, with high morbidity and often long-term neurologic sequelae. With no treatment available, studies of the immune response to TBEV are essential for the understanding of the immunopathogenesis of tick-borne encephalitis and for the development of therapeutics. We have previously demonstrated that CD8+ T cell responses in peripheral blood in patients with acute TBEV peak at around 7 d after hospitalization in the neuroinvasive phase of the disease. In this study, we identified six novel TBEV HLA-A2– and HLA-B7–restricted epitopes, all derived from the nonstructural proteins of TBEV. This identification allowed for a comprehensive phenotypic and temporal analysis of the HLA-A2– and HLA-B7–restricted Ag-specific CD8+ T cell response during the acute stages of human TBEV infection. HLA-A2– and HLA-B7–restricted TBEV epitope–specific effector cells predominantly displayed a CD45RA−CCR7−CD27+CD57− phenotype at day 7, which transitioned into separate distinct phenotypes for HLA-A2– and HLA-B7–restricted TBEV-specific CD8+ T cells, respectively. At day 21, the most prevalent phenotype in the HLA-A2–restricted CD8+ T cell populations was CD45RA−CCR7−CD27+CD57+, whereas the HLA-B7–restricted CD8+ T cell population was predominantly CD45RA+CCR7−CD27+CD57+. Almost all TBEV epitope–specific CD8+ T cells expressed α4 and β1 integrins at days 7 and 21, whereas the bulk CD8+ T cells expressed lower integrin levels. Taken together, human TBEV infection elicits broad responses to multiple epitopes, predominantly derived from the nonstructural part of the virus, establishing distinct maturation patterns for HLA-A2– and HLA-B7–restricted TBEV epitope–specific CD8+ T cells.

Innate lymphocytes are selectively enriched in the liver where they have important roles in liver immunology. Murine studies have shown that type I NKT cells can promote liver inflammation, whereas type II NKT cells have an anti-inflammatory role. In humans, type II NKT cells were found to accumulate in the gut during inflammation and IL13Rα2 was proposed as a marker for these cells. In the human liver, less is known about type I and II NKT cells. Here, we studied the phenotype and function of human liver T cells expressing IL13Rα2. We found that IL13Rα2 was expressed by around 1% of liver-resident memory T cells but not on circulating T cells. In support of their innate-like T-cell character, the IL13Rα2+ T cells had higher expression of promyelocytic leukaemia zinc finger (PLZF) compared to IL13Rα2- T cells and possessed the capacity to produce IL-22. However, only a minority of human liver sulfatide-reactive type II NKT cells expressed IL13Rα2. Collectively, these findings suggest that IL13Rα2 identifies tissue-resident intrahepatic T cells with innate characteristics and the capacity to produce IL-22.

Background & Aims Virus-specific T cell dysfunction is a common feature of HBV-related hepatocellular carcinoma (HBV-HCC). Conventional T (ConT) cells can be redirected towards viral antigens in HBV-HCC when they express an HBV-specific receptor; however, their efficacy can be impaired by liver-specific physical and metabolic features. Mucosal-associated invariant T (MAIT) cells are the most abundant innate-like T cells in the liver and can elicit potent intrahepatic effector functions. Here, we engineered ConT and MAIT cells to kill HBV expressing hepatoma cells and compared their functional properties. Methods Donor-matched ConT and MAIT cells were engineered to express an HBV-specific T cell receptor (TCR). Cytotoxicity and hepatocyte homing potential were investigated using flow cytometry, real-time killing assays, and confocal microscopy in 2D and 3D HBV-HCC cell models. Major histocompatibility complex (MHC) class I-related molecule (MR1)-dependent and MR1-independent activation was evaluated in an Escherichia coli THP-1 cell model and by IL-12/IL-18 stimulation, respectively. Results HBV TCR-MAIT cells demonstrated polyfunctional properties (CD107a, interferon [IFN] γ, tumour necrosis factor [TNF], and IL-17A) with strong HBV target sensitivity and liver-homing chemokine receptor expression when compared with HBV TCR-ConT cells. TCR-mediated lysis of hepatoma cells was comparable between the cell types and augmented in the presence of inflammation. Coculturing with HBV+ target cells in a 3D microdevice mimicking aspects of the liver microenvironment demonstrated that TCR-MAIT cells migrate readily towards hepatoma targets. Expression of an ectopic TCR did not affect the ability of the MAIT cells to be activated via MR1-presented bacterial antigens or IL-12/IL-18 stimulation. Conclusions HBV TCR-MAIT cells demonstrate anti-HBV functions without losing their endogenous antimicrobial mechanisms or hepatotropic features. Our results support future exploitations of MAIT cells for liver-directed immunotherapies. Lay summary Chronic HBV infection is a leading cause of liver cancer. T cell receptor (TCR)-engineered T cells are patients’ immune cells that have been modified to recognise virus-infected and/or cancer cells. Herein, we evaluated whether mucosal-associated invariant T cells, a large population of unconventional T cells in the liver, could recognise and kill HBV infected hepatocytes when engineered with an HBV-specific TCR. We show that their effector functions may exceed those of conventional T cells currently used in the clinic, including antimicrobial properties and chemokine receptor profiles better suited for targeting liver tumours., Graphical abstract, Highlights • Mucosal-associated invariant T (MAIT) cells expanded in vitro can be engineered to kill HBV antigen-presenting hepatoma cells. • MAIT cells produce IL-17A upon encountering their HBV targets, a functional property not observed in conventional T cells currently used in immunotherapy. • HBV-specific MAIT cells migrate readily towards liver targets in a 3D microfluidics system. • Co-expression of an HBV-specific TCR does not affect the ability of MAIT cells to respond to their physiological stimuli.

Common variable immunodeficiency (CVID) is characterized by low levels of Igs leading to increased risk of infections. Mucosal-associated invariant T (MAIT) cells are a recently identified population of innate T cells with potent antibacterial activity. We hypothesized that CVID is associated with alterations in MAIT cells. Cryopreserved PBMC from CVID patients and healthy controls were used to study the frequency, phenotype, and response to Escherichia coli stimulation of MAIT cells by flow cytometry. MAIT cell frequency and absolute counts were depressed in CVID. Residual MAIT presented elevated coexpression of CD38 and HLA-DR, and reduced expression of CCR6, whereas levels of CD127 (IL-7 receptor) were unchanged. CVID patients also had an accumulation of MAIT cells lacking the critical transcription factors eomesodermin and promyelocytic leukemia zinc finger protein. MAIT cell frequency was inversely associated with levels of soluble CD14, with coexpression of CD38 and HLA-DR, and accumulation of MAIT cells lacking eomesodermin or promyelocytic leukemia zinc finger protein expression. None of these changes were normalized by IgG replacement therapy. Finally, MAIT cells from CVID patients displayed poor IFN-γ responses to E. coli stimulation, in part due to defective Ag presentation, and these responses were increased by pretreatment with IL-7. Defective MAIT cell response may contribute to the increased incidence of microbial infections seen in CVID patients on IgG replacement therapy.

Several lines of evidence support the concept that NK cells play an important role in control of hepatitis C virus (HCV) infection via cytokine secretion and cytotoxicity. IL-7 is a homeostatic cytokine with a role in T cell development, activation, proliferation, and cytokine secretion. The IL-7Rα chain [cluster of differentiation (CD)127] is expressed on NK cells, with greatest abundance on the CD56brightCD16dim/− (CD56bright) subset. Here, we measured CD127 expression on CD56bright, CD56dimCD16+ (CD56dim), or CD56negCD16+ (CD56neg) NK cell subsets of 25 uninfected donors (UD); 34 chronic HCV-infected, treatment-naiüve; 25 HIV-infected, virally suppressed on antiretroviral therapy (ART); and 42 HCV–HIV-coinfected subjects on ART. Interestingly, CD127 expression on CD56bright NK cells negatively correlated with HCV plasma levels in HCV monoinfection and HCV–HIV coinfection. IL-7 induced CD69 expression, as well as IFN-γ production, in CD56bright NK cells and also enhanced the IFN-α-induced CD69 expression on these cells. The latter was impaired in HIV infection. Furthermore, IL-7 induced B cell lymphoma 2 (BCL-2) expression and cell cycling of CD56bright NK cells, and this effect was impaired in HCV- and HIV-infected subjects. Whereas IL-7-stimulated CD56bright NK cell degranulation appeared intact in all cohorts, we observed impaired IL-7-activated NK cell cytolytic function in HCV- and HIV-infected subjects. Finally, IL-7-induced phosphorylation of STAT-5 (pSTAT-5) signaling was impaired in NK cells of subjects with chronic viral infection, and this was reversible upon 6 mo of viral suppression with IFN-free HCV therapy. These results implicate that IL-7-dependent NK cell activation and effector function may be other host immune surveillance mechanisms that are impaired in viral infections.

The female genital tract (FGT) mucosa is a critically important site for immune defense against microbes. Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population that recognizes microbial riboflavin metabolite antigens in an MR1-dependent manner. The role of MAIT cells in the FGT mucosa is unknown. Here, we found that MAIT cells and MR1+ antigen-presenting cells were present in the upper and lower FGT, with distinct tissue localization of MAIT cells in endometrium vs. cervix. The MAIT cells from the FGT and blood displayed a distinct phenotype with expression of interleukin (IL)-18Rα, CD127, α4β7, PD-1, as well as the transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, Eomes, and T-bet. Their expression levels of PLZF and Eomes were lower in the FGT compared with blood. When stimulated with Escherichia coli, MAIT cells from the FGT displayed a bias towards IL-17 and IL-22 expression, whereas blood MAIT cells produced primarily IFN-γ, TNF, and Granzyme B. Furthermore, both FGT- and blood-derived MAIT cells were polyfunctional and contributed to the T-cell-mediated response to E. coli. Thus, MAIT cells in the genital mucosa have a distinct IL-17/IL-22 profile and may have an important role in the immunological homeostasis and control of microbes at this site.

Significance Bacterial toxins belonging to the family of superantigens are potent immunostimulatory antigens capable of activating T cells in a nonconventional manner. This results in an overzealous activation of immune cells and release of pathologic levels of pro-inflammatory cytokines, which underlies severe disease manifestations such as streptococcal toxic shock syndrome. Here, we provide evidence that mucosal-associated invariant T (MAIT) cells are major contributors to the overall cytokine response elicited by group A streptococci. Both streptococcal superantigens and surface-attached bacterial factors activate MAIT cells, but through different mechanisms. Furthermore, activated MAIT cells could be detected in patients during the acute phase of streptococcal toxic shock syndrome. Thus, this study identifies an actor and potential target for intervention in streptococcal toxic shock syndrome., Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vβ2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1β, IL-2, and TNFβ. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS.

Objective Activated (CD38HLA-DR) PD-1 CD4 T cells are strongly associated with virus replication and disease progression in untreated HIV-1 infection, and viral persistence in individuals on ART. Few studies have examined cell-associated viral load (CAVL) in different activated CD4 T-cell populations to measure relative contributions to viral reservoirs. Design Longitudinal assessment of HIV-1 chronically infected Ugandans initiating ART, to investigate activated CD4 T-cell populations and their contribution to viral reservoirs. Methods We followed 32 HIV-1 chronically infected individuals from Kampala, Uganda, and determined their CD4 T-cell counts and viral load at baseline, 6, and 12 months after the initiation of ART. T-cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble factors associated with inflammation were measured in plasma using a multiplexed platform. Results Concomitant with viral load decline and CD4 T-cell count rebound, the activated PD-1 CD4 T-cell population contracted upon initiation of ART. Baseline levels of activated PD-1 CD4 T cells correlated with plasma levels of IP-10 and TNFRII. Interestingly, a higher baseline level of activated PD-1 CD4 T cells was associated with poorer CD4 T-cell recovery after 12 months of ART. This population contributed significantly to the cell-associated HIV DNA load at baseline, whereas their contribution declined on ART, indicating high turnover. Conclusion Activated PD-1 CD4 T cells are predictors of poor immunologic recovery on ART and may represent a short-lived component of HIV-1 reservoirs.

The intervillous space of the placenta is a part of the fetal-maternal interface, where maternal blood enters to provide nutrients and gas exchange. Little is known about the maternal immune cells at this site, which are in direct contact with fetal tissues. We have characterized the T cell composition and chemokine profile in paired intervillous and peripheral blood samples from healthy mothers giving birth following term pregnancies. Mucosal-associated invariant T (MAIT) cells and effector memory (EM) T cells were enriched in the intervillous blood compared to peripheral blood, suggesting that MAIT cells and other EM T cells home to the placenta during pregnancy. Furthermore, pregnant women had lower proportions of peripheral blood MAIT cells compared to non-pregnant women. The levels of several chemokines were significantly higher in intervillous compared to peripheral blood, including macrophage migration inhibitory factor (MIF), CXCL10, and CCL25, whereas CCL21, CCL27 and CXCL12 were lower. Migration assays showed that MAIT cells and EM T cells migrated toward conditioned medium from placental explants. A multivariate factor analysis indicated that high levels of MIF and CCL25 were associated with high proportions of MAIT cells in intervillous blood. Blocking of MIF or a combination of MIF, CCL25, and CCL20 in migration assays inhibited MAIT cell migration toward placenta conditioned medium. Finally, MAIT cells showed migratory capacities toward recombinant MIF. Together, these findings indicate that term placental tissues attract MAIT cells, and that this effect is at least partly mediated by MIF.

Opportunistic bacteria in apical periodontitis (AP) may pose a risk for systemic dissemination. Mucosal-associated invariant T (MAIT) cells are innate-like T cells with a broad and potent antimicrobial activity important for gut mucosal integrity. It was recently shown that MAIT cells are present in the oral mucosal tissue, but the involvement of MAIT cells in AP is unknown. Here, comparison of surgically resected AP and gingival tissues demonstrated that AP tissues express significantly higher levels of Vα7.2-Jα33, Vα7.2-Jα20, Vα7.2-Jα12, Cα and tumour necrosis factor (TNF), interferon (IFN)-γ and interleukin (IL)-17A transcripts, resembling a MAIT cell signature. Moreover, in AP tissues the MR1-restricted MAIT cells positive for MR1–5-OP-RU tetramer staining appeared to be of similar levels as in peripheral blood but consisted mainly of CD4+ subset. Unlike gingival tissues, the AP microbiome was quantitatively impacted by factors like fistula and high patient age and had a prominent riboflavin-expressing bacterial feature. When merged in an integrated view, the examined immune and microbiome data in the sparse partial least squares discriminant analysis could identify bacterial relative abundances that negatively correlated with Vα7.2-Jα33, Cα, and IL-17A transcript expressions in AP, implying that MAIT cells could play a role in the local defence at the oral tissue barrier. In conclusion, we describe the presence of MAIT cells at the oral site where translocation of oral microbiota could take place. These findings have implications for understanding the immune sensing of polymicrobial-related oral diseases.

Summary Hantaviruses are zoonotic RNA viruses that cause severe acute disease in humans. Infected individuals have strong inflammatory responses that likely cause immunopathology. Here, we studied the response of mucosal-associated invariant T (MAIT) cells in peripheral blood of individuals with hemorrhagic fever with renal syndrome (HFRS) caused by Puumala orthohantavirus, a hantavirus endemic in Europe. We show that MAIT cell levels decrease in the blood during HFRS and that residual MAIT cells are highly activated. This activation correlates with HFRS severity markers. In vitro activation of MAIT cells by hantavirus-exposed antigen-presenting cells is dependent on type I interferons (IFNs) and independent of interleukin-18 (IL-18). These findings highlight the role of type I IFNs in virus-driven MAIT cell activation and suggest a potential role of MAIT cells in the disease pathogenesis of viral infections., Graphical abstract, Highlights MAIT cells are activated in individuals with hemorrhagic fever with renal syndrome (HFRS) MAIT cell activation correlates with HFRS severity markers during hantavirus infection MAIT cell blood levels decline during acute HFRS Hantavirus-mediated MAIT cell activation is type I IFN dependent, Maleki et al. demonstrate strong activation of mucosal-associated invariant T (MAIT) cells in individuals with hemorrhagic fever with renal syndrome (HFRS) caused by Puumala orthohantavirus. This phenotype is associated with markers of disease severity and is in vitro dependent on type I IFNs.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

Immune exhaustion is a hallmark of chronic viral infections. However, pathogen eradication can result in reinvigorated immune responses. Indeed, this was recently suggested for antigen-specific CD8(+) T cells and NK cells in HCV-infected patients receiving an interferon-free treatment regimen. Mucosal-associated invariant T (MAIT) cells are evolutionarily conserved innate-like effector T cells. Here, we show that MAIT cells are severely diminished in frequency in chronic HCV-infection, and in this regard the most affected immune cell type in peripheral blood of humans with this disease. Residual MAIT cells show an activated phenotype with high expression of granzyme B, HLA-DR, PD-1, and CD69 as well as altered transcription factor expression and suppressed responsiveness to MR1-dependent antigen stimulation. In contrast to other immune cells, MAIT cells are not reinvigorated after successful HCV-clearance using interferon-free therapy. The present results hence demonstrate persistent immune cell-dysfunction in humans despite successful elimination of a chronic pathogen.

Summary SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. We here systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits robust, broad and highly functional memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19., Highlights 1. Acute phase SARS-CoV-2-specific T cells display an activated cytotoxic phenotype 2. Broad and polyfunctional SARS-CoV-2-specific T cell responses in convalescent phase 3. Detection of SARS-CoV-2-specific T cell responses also in seronegative individuals

Current paradigms of CD8 + T cell–mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8 + T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (T RMs ). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8 + T cells in LTs also resemble T RMs . Moreover, high frequencies of HIV-specific CD8 + T RMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific T RMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-T RMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8 + T cell responses resident within LTs.

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid β-peptide (Aβ42), a major constituent of amyloid plaques in Alzheimer’s disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral–host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies., The protein corona around artificial nanoparticles is known to influence activity and biological fate, the formation around viruses is less well understood. Here, the authors observe the formation of protein corona on viruses and study the effects this corona has on viral infectivity and on amyloid protein assembly.

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues., Author summary CD4+ T cells have classically been divided into different subsets based on their different abilities to help and regulate specific parts of the immune system. Recent work in the HIV field has demonstrated that HIV-specific CD4+ T cells with unique effector functions, such as cytolytic activity and β-chemokine production, can play a direct role in control of HIV replication. However, HIV infection is generally considered to be a disease centered in lymphoid tissues, where unique CD4+ T helper cell subsets are present to orchestrate the maturation and priming of adaptive immunity. In this study, we identify that two specific transcription factors, T-bet and Eomes, mark cytolytic and β-chemokine producing CD4+ T cells. While this effector CD4+ T cell population is part of immunosurveillance mechanisms in blood, we find that lymph nodes largely lack this effector population–independent of HIV infection or disease progression status. These results indicate that current effector CD4+ T cell mediated correlates of HIV control are limited to blood and not representative of potential correlates of control in lymphoid tissues.

NK cells play an important role in the defense against viral infections. However, little is known about the regulation of NK cell responses during the first days of acute viral infections in humans. In this study, we used the live attenuated yellow fever virus (YFV) vaccine 17D as a human in vivo model to study the temporal dynamics and regulation of NK cell responses in an acute viral infection. YFV induced a robust NK cell response in vivo, with an early activation and peak in NK cell function at day 6, followed by a delayed peak in Ki67 expression, which was indicative of proliferation, at day 10. The in vivo NK cell response correlated positively with plasma type I/III IFN levels at day 6, as well as with the viral load. YFV induced an increased functional responsiveness to IL-12 and IL-18, as well as to K562 cells, indicating that the NK cells were primed in vivo. The NK cell responses were associated primarily with the stage of differentiation, because the magnitude of induced Ki67 and CD69 expression was distinctly higher in CD57− NK cells. In contrast, NK cells expressing self- and nonself-HLA class I–binding inhibitory killer cell Ig-like receptors contributed, to a similar degree, to the response. Taken together, our results indicate that NK cells are primed by type I/III IFN in vivo early after YFV infection and that their response is governed primarily by the differentiation stage, independently of killer cell Ig-like receptor/HLA class I–mediated inhibition or education.

The bacterium Staphylococcus aureus is an important cause of the life-threatening condition toxic shock syndrome in humans. Bacterial toxins known as superantigens (SAgs) generate this illness by acting as broad activators of a substantial fraction of all T lymphocytes, bypassing the normally highly stringent T-cell receptor antigen specificity to cause a systemic inflammatory cytokine storm in the host. In a new study, Shaler et al. found that immune cells called mucosa-associated invariant T (MAIT) cells make an unexpectedly large contribution to the SAg response in a largely T-cell receptor-independent, cytokine-driven manner. Subsequent to such activation, the MAIT cells remain unresponsive to stimulation with bacterial antigen. Thus, S. aureus hijacks MAIT cells in the cytokine storm and leaves them functionally impaired. This work provides new insight into the role of MAIT cells in antibacterial immunity and opens new avenues of investigation to understand and possibly treat bacterial toxic shock and sepsis.

Summary Persistent inflammatory response in cystic fibrosis (CF) airways is believed to play a central role in the progression of lung damage. Anti-inflammatory treatment may slow lung disease progression, but adverse side effects have limited its use. Vitamin D has immunoregulatory properties. We randomized 16 CF patients to receive vitamin D2, vitamin D3 or to serve as controls, and investigated the effect of vitamin D supplementation on soluble immunological parameters, myeloid dendritic cells (mDCs) and T cell activation. Three months of vitamin D treatment were followed by two washout months. Vitamin D status at baseline was correlated negatively with haptoglobin, erythrocyte sedimentation rate and immunoglobulin A concentration. Total vitamin D dose per kg bodyweight correlated with the down-modulation of the co-stimulatory receptor CD86 on mDCs. Vitamin D treatment was associated with reduced CD279 (PD-1) expression on CD4+ and CD8+ T cells, as well as decreased frequency of CD8+ T cells co-expressing the activation markers CD38 and human leucocyte antigen D-related (HLA-DR) in a dose-dependent manner. There was a trend towards decreased mucosal-associated invariant T cells (MAIT) cell frequency in patients receiving vitamin D and free serum 25-hydroxyvitamin D (free-s25OHD) correlated positively with CD38 expression by these cells. At the end of intervention, the change in free-s25OHD was correlated negatively with the change in CD279 (PD-1) expression on MAIT cells. Collectively, these data indicate that vitamin D has robust pleiotropic immunomodulatory effects in CF. Larger studies are needed to explore the immunomodulatory treatment potential of vitamin D in CF in more detail.

Invariant natural killer T (iNKT) cells are CD1d-restricted immunoregulatory lymphocytes that share characteristics of both the innate and adaptive immune systems. Although it has been reported that iNKT cells are present in the human fetal thymus, it is currently unknown how they distribute, differentiate, and function in fetal peripheral lymphoid and non-lymphoid organs. Here, we show that functional human fetal iNKT cells develop and differentiate in a tissue-specific manner during the second trimester. Fetal iNKT cells accumulated in the small intestine, where they gained a mature phenotype and mounted robust interferon (IFN)-γ responses. In contrast, iNKT cells in the spleen and mesenteric lymph nodes were less frequently detected, less differentiated, mounted poor IFN-γ responses, but proliferated vigorously upon stimulation with α-galactosylceramide. These data demonstrate that fetal iNKT cells can differentiate and acquire potent effector functions in utero before the establishment of the commensal microflora.

Objective: Of the predominant HIV-1 subtypes in Uganda, subtype D infection confers a worse prognosis. HIV-1 infection causes perturbations to natural killer (NK) cells, and yet these cells can exert immune pressure on the virus and influence clinical outcome. Here, we studied NK cell activation and function in Ugandans with chronic untreated HIV-1 subtype D infection in comparison to uninfected community matched controls. Methods: Cryopreserved peripheral blood mononuclear cells (PBMCs) from 42 HIV-infected individuals and 28 HIV-negative controls were analysed using eight-colour flow cytometry. NK cell surface expression of CD16, CD56, CD57, HLA-DR and NKG2A were used to investigate activation, maturation and differentiation status. NK cell function was evaluated by measuring interferon-gamma (IFNγ) production in response to K562 cells, or interleukin (IL)-12 and IL-18. Results: CD56dim NK cells from HIV-infected individuals produced less IFNγ in response to IL-12 and IL-18 than did CD56dim NK cells from uninfected controls. Infected individuals had lower levels of CD56dim NK cells coexpressing the differentiation markers NKG2A and CD57 than controls. In addition, their NKG2A+CD57+ CD56dim NK cells displayed elevated activation levels as assessed by HLA-DR expression. Cytokine-induced IFNγ production correlated directly with coexpression of CD57 and NKG2A on CD56dim NK cells. Conclusion: HIV-1 subtype D infection is associated with impaired NK cell responsiveness to cytokines, decline of the NKG2A+CD57+ CD56dim NK cell subset, as well as elevated activation in this subset. These alterations within the NK cell compartment may contribute to immunopathogenesis of HIV-1 subtype D infection in Ugandans.

Objective Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses to hepatitis delta virus (HDV) are hardly detectable. Thus, the role of other immune responses, including those mediated by natural killer (NK) cells, must be considered in HDV pathogenesis and in treatments with immune-stimulating agents such as interferon (IFN)α. However, the phenotype and function of NK cells in chronic HDV infection, or in HDV-infected individuals undergoing IFNα treatment, have not been extensively studied. Design We performed an extensive analysis of NK cells in chronically HDV-infected patients before and during treatment with IFNα, and compared the results with those for patients with HBV mono-infection as well as healthy controls. Results In untreated HDV-infected patients, a higher than normal frequency of NK cells was observed in peripheral blood with unaltered phenotypic NK cell differentiation status. In contrast, long-term IFNα treatment of HDV-infected patients caused a significant change in NK cell differentiation status, with selective loss of terminally differentiated NK cells and, in parallel, a relative enrichment in immature NK cell subsets. Treatment was associated with marked functional impairment of the NK cells, which was independent of the changes in NK cell differentiation status. Furthermore, treatment polarised NK cell IFN signalling from STAT4 towards STAT1 dependency. Strikingly, a high frequency of CD56 dim NK cells at baseline was positively associated with IFNα treatment outcome in the patients. Conclusions We describe in detail how HDV infection, and IFNα treatment of this infection, affects the NK cell compartment and what consequences this has for the functional capacity of NK cells.

Mucosa-associated Invariant T (MAIT) cells are an evolutionarily conserved innate-like T cell subset that recognizes antigens presented by MR1 molecules. These antigens include vitamin B derivatives shared by many potentially pathogenic microbes, including Mycobacterium tuberculosis and Candida albicans. It was recently discovered that MAIT cells decay numerically and functionally in HIV-1 infection, and that they fail to recover despite several years of effective suppression of viral replication by antiretroviral therapy (ART). Here, we briefly discuss the roles of MAIT cells and their loss in HIV immunopathogenesis. We furthermore propose that the persistence of MAIT cell loss on ART needs to be taken into account when assessing the immunological response to treatment, and when treatment should commence. The importance of this T cell subset in HIV-1 infection needs further study, and interventions to restore the MAIT cell compartment should be considered.

HIV-1 subtype D is associated with faster disease progression compared with subtype A. Immunological correlates of this difference remain undefined. We investigated invariant natural killer T (iNKT) cells and FoxP3⁺ regulatory T cells (Tregs) in Ugandans infected with either subtype. Loss of iNKT cells was pronounced in subtype D, whereas Tregs displayed more profound loss in subtype A infection. The iNKT cell levels were associated with CD4 T-cell interleukin-2 production in subtype A, but not in D, infection. Thus, these viral subtypes are associated with differential loss of iNKT cells and Tregs that may influence the quality of the adaptive immune response.

The live attenuated yellow fever virus (YFV) 17D vaccine provides a good model to study immune responses to an acute viral infection in humans. We studied the temporal dynamics, composition, and character of the primary human T cell response to YFV. The acute YFV-specific effector CD8 T cell response was broad and complex; it was composed of dominant responses that persisted into the memory population, as well as of transient subdominant responses that were not detected at the memory stage. Furthermore, HLA-A2– and HLA-B7–restricted YFV epitope–specific effector cells predominantly displayed a CD45RA−CCR7−PD-1+CD27high phenotype, which transitioned into a CD45RA+CCR7−PD-1−CD27low memory population phenotype. The functional profile of the YFV-specific CD8 T cell response changed in composition as it matured from an effector- to a memory-type response, and it tended to become less polyfunctional during the course of this transition. Interestingly, activation of CD4 T cells, as well as FOXP3+ T regulatory cells, in response to YFV vaccination preceded the kinetics of the CD8 T cell response. The present results contribute to our understanding of how immunodominance patterns develop, as well as the phenotypic and functional characteristics of the primary human T cell response to a viral infection as it evolves and matures into memory.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved antimicrobial MR1-restricted T-cell subset. MAIT cells are CD161(+), express a V7.2 TCR, are primarily CD8(+) and numerous in blood and mucosal tissues. However, their role in HIV-1 infection is unknown. In this study, we found levels of MAIT cells to be severely reduced in circulation in patients with chronic HIV-1 infection. Residual MAIT cells were highly activated and functionally exhausted. Their decline was associated with time since diagnosis, activation levels, and the concomitant expansion of a subset of functionally impaired CD161(+) V7.2(+) T cells. Such cells were generated in vitro by exposure of MAIT cells to Escherichia coli. Notably, whereas the function of residual MAIT cells was at least partly restored by effective antiretroviral therapy, levels of MAIT cells in peripheral blood were not restored. Interestingly, MAIT cells in rectal mucosa were relatively preserved, although some of the changes seen in blood were recapitulated in the mucosa. These findings are consistent with a model in which the MAIT-cell compartment, possibly as a result of persistent exposure to microbial material, is engaged, activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.

A microscopy-based assay to measure iNKT cell responses against rare HIV-1 infected dendritic cells, infected with an eGFP expressing reporter virus., iNKT cells recognize lipid antigens, such as α-GalCer, presented in complex with CD1d expressed by DCs. Exposure of DCs to HIV-1 can lead to productive infection, and it was demonstrated recently that HIV-1 inhibits CD1d surface expression in an apparent mode of immune evasion. However, studies of the interaction between T cells, including iNKT cells and HIV-infected DCs in vitro, are hampered by the low frequency of productive infection in DCs. Here, we demonstrate the utility of full-length HIV-1 modified to express eGFP to address this problem. This virus allowed identification of single, rare productively infected cells in a mixed DC population by fluorescence microscopy and enabled detailed studies of the interaction of such cells with individual iNKT cells. iNKT cell responses to α-GalCer presented by HIV-1-positive and -negative DCs were quantified by intracellular IFN-γ staining in iNKT cells forming conjugates with DCs. Whereas complex formation was observed between iNKT cells and uninfected and infected DCs, only iNKT cells in contact with uninfected DCs produced IFN-γ. This microscopy assay, based on full-length HIV-1 modified to express eGFP, thus allows detailed evaluation of HIV-1 immune-evasion mechanisms in rare virus-infected live DCs.

The mucosa-associated invariant T (MAIT) cells are a large and relatively recently described innate-like antimicrobial T cell subset in humans. The study of human MAIT cells is still in its infancy, and many aspects of MAIT cell immunobiology in health and disease remain unexplored. Here, we describe methodological approaches and protocols to investigate the expression of a broad spectrum of surface receptors on human MAIT cells, and to examine their unique transcription factor profile, as well as their antimicrobial effector function using multicolor flow cytometry-based techniques. We provide specific guidance on protocols and describe potential pitfalls for each of the presented methodologies. Finally, we discuss future prospects and current limitations of multicolor flow cytometry-based approaches to the study of human MAIT cells.