Intrinsic factors and CD1d1 but not CD1d2 expression levels control invariant natural killer T cell subset differentiation.

Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

Identification of Rare Thymic NKT Cell Precursors by Multiparameter Flow Cytometry.

Mouse invariant natural killer T (NKT) cells are a subset of T lymphocytes which have been shown to play a significant role in innate and adaptive immune responses. Features of innate responses are attributed to these cells because they can be stimulated simultaneously with the same ligand to produce quickly and in large amount cytokines without prior immunization. Because these characteristics could be exploited for clinical applications, NKT cells have attracted considerable interest. Many studies have investigated the molecular mechanisms through which they are selected and differentiate. These studies are based on developmental models that serve as a scaffold to understand the specific roles played by various factors and to identify checkpoints during cellular development. Analysis of NKT cell precursors at the HSAhigh stage, stage 0, can reveal potential selection defects, whereas analysis of NKT cells at the HSAlow stage can shed light on defects in the maturation/differentiation of the different NKT cell subsets (NKT1, 2, and 17). Unlike HSAlow NKT cell subsets, HSAhigh NKT cell precursors are not accurately identified by flow cytometry because of their extreme rarity. Here, we describe an NKT cell enrichment strategy to identify unambiguously NKT cell precursors at the HSAhigh stage that can be used to assess their distribution and characteristics by multicolor flow cytometry.

PLZF Acetylation Levels Regulate NKT Cell Differentiation.

The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.

Cortical Thymocytes Along With Their Selecting Ligands Are Required for the Further Thymic Maturation of NKT Cells in Mice.

Following positive selection, NKT cell precursors enter an "NK-like" program and progress from an NK- to an NK+ maturational stage to give rise to NKT1 cells. Maturation takes place in the thymus or after emigration of NK- NKT cells to the periphery. In this study, we followed the fate of injected NKT cells at the NK- stage of their development in the thymus of a series of mice with differential CD1d expression. Our results indicate that CD1d-expressing cortical thymocytes, and not epithelial cells, macrophages, or dendritic cells, are necessary and sufficient to promote the maturation of thymic NKT1 cells. Migration out of the thymus of NK- NKT cells occurred in the absence of CD1d expression, however, CD1d expression is required for maturation in peripheral organs. We also found that the natural ligand Isoglobotriosylceramide (iGb3), and the cysteine protease Cathepsin L, both localizing with CD1d in the endosomal compartment and crucial for NKT cell positive selection, are also required for NK- to NK+ NKT cell transition. Overall, our study indicates that the maturational transition of NKT cells require continuous TCR/CD1d interactions and suggest that these interactions occur in the thymic cortex where DP cortical thymocytes are located. We thus concluded that key components necessary for positive selection of NKT cells are also required for subsequent maturation.

A focus on natural killer T-cell subset characterization and developmental stages.

Almost 20 years ago, CD1d tetramers were developed to track invariant natural killer T (NKT) cells based on their specificity, and to define developmental steps during which differentiation markers and functional features are progressively acquired from early NKT cell precursor to fully mature NKT cell subsets. Based on these findings, a linear developmental model was proposed and subsequently used by all studies investigating the specific role of factors that control NKT cell development. More recently, based on intracellular staining patterns of lineage-specific transcription factors such as T-bet, GATA-3, promyelocytic leukemia zinc finger and RORγt, a lineage differentiation model was proposed for NKT cell development. Currently, studies on NKT cells development present lineage differentiation model data in addition to the linear maturation model. In the perspective presented here, we discuss current knowledge relating to NKT cell developmental models and particularly focus on the approaches and strategies, some of which appear nebulous, used to define NKT cell developmental stages and subsets.

Characterization of the developmental landscape of murine RORγt+ iNKT cells.

Invariant natural killer T (iNKT) cells expressing the retinoic acid receptor-related orphan receptor γt (RORγt) and producing IL-17 represent a minor subset of CD1d-restricted iNKT cells (iNKT17) in C57BL/6J (B6) mice. We aimed in this study to define the reasons for their low distribution and the sequence of events accompanying their normal thymic development. We found that RORγt+ iNKT cells have higher proliferation potential and a greater propensity to apoptosis than RORγt- iNKT cells. These cells do not likely reside in the thymus indicating that thymus emigration, and higher apoptosis potential, could contribute to RORγt+ iNKT cell reduced thymic distribution. Ontogeny studies suggest that mature HSAlow RORγt+ iNKT cells might develop through developmental stages defined by a differential expression of CCR6 and CD138 during which RORγt expression and IL-17 production capabilities are progressively acquired. Finally, we found that RORγt+ iNKT cells perceive a strong TCR signal that could contribute to their entry into a specific 'Th17 like' developmental program influencing their survival and migration. Overall, our study proposes a hypothetical thymic developmental sequence for iNKT17 cells, which could be of great use to study molecular mechanisms regulating this developmental program.

TCR density in early iNKT cell precursors regulates agonist selection and subset differentiation in mice.

It is established that iNKT cells are a cell type that require strong TCR signal for their proper development and represent a model for thymic agonist selection. The nature of the signal perceived by iNKT cells promoting their specification is not well understood. To address this question, we analyzed iNKT cell development in relevant TCR Vα14-Jα18 alpha chain transgenic mice (Vα14Tg). In CD4-Vα14Tg mice, where the transgene is driven by CD4 promoter, we identified a block in iNKT cell development at early developmental stages due to a reduced expression of key transcription factors accompanied with a reduced TCR expression levels. This indicates that TCR signal strength control iNKT cell differentiation. Importantly, we found in WT mice that early precursors of iNKT cells express higher TCR levels compared to positively selected precursors of mainstream T cells showing that TCR levels could contribute to the strength of iNKT cell TCR signaling. Overall, our study highlights TCR signal strength associated with a higher TCR density as an important regulator of iNKT cell lineage specification.

Critical Contribution of NK Group 2 Member D Expressed on Invariant Natural Killer T Cells in Concanavalin A-Induced Liver Hepatitis in Mice.

Natural killer group 2D (NKG2D) is a well-characterized activating receptor expressed on many immune cells, including invariant natural killer T (iNKT) cells. These cells were shown to be responsible of liver injury in the model of concanavalin A (Con A)-induced hepatitis, considered to be an experimental model of human autoimmune hepatitis. In this study, we investigated whether NKG2D plays a role in the hepatitis induced by iNKT cell-mediated immune response to Con A. By using killer cell lectin-like receptor subfamily K, member 1 deficient (Klrk1-/-) mice, we found that the absence of NKG2D reduced the hepatic injury upon Con A administration. This was not due to an intrinsic functional defect of NKG2D-deficient iNKT cells as mice missing NKG2D have normal distribution and function of iNKT cells. Furthermore, increased resistance to Con A-induced hepatitis was confirmed using neutralizing anti-NKG2D antibodies. The reduced pathogenic effect of Con A in the absence of NKG2D correlates with a reduction in pathogenic cytokine production and FAS-Ligand (FAS-L) expression by iNKT cells. We also found that Con A administration led to an increase in the retinoic acid early inducible (RAE-1) surface expression on wild-type hepatocytes. Finally, we found that Con A has no direct action on FAS-L expression or cytokine production by iNKT cells and thus propose that NKG2D-L expression on stressed hepatocytes promote cytotoxic activity of iNKT cells via its interaction with NKG2D contributing to hepatic injury. In conclusion, our results highlight NKG2D as an essential receptor required for the activation of iNKT cells in Con A-induced hepatitis and indicate that it represents a potential drug target for prevention of autoimmune hepatitis.

Potential role of IL-17-producing iNKT cells in type 1 diabetes.

We explored in this study the status and potential role of IL-17-producing iNKT cells (iNKT17) in type 1 diabetes (T1D) by analyzing these cells in patients with T1D, and in NOD mice, a mouse model for T1D. Our analysis in mice showed an increase of iNKT17 cells in NOD vs control C57BL/6 mice, partly due to a better survival of these cells in the periphery. We also found a higher frequency of these cells in autoimmune-targeted organs with the occurrence of diabetes, suggesting their implication in the disease development. In humans, though absent in fresh PMBCs, iNKT17 cells are detected in vitro with a higher frequency in T1D patients compared to control subjects in the presence of the proinflammatory cytokine IL-1ß, known to contribute to diabetes occurrence. These IL-1ß-stimulated iNKT cells from T1D patients keep their potential to produce IFN-γ, a cytokine that drives islet ß-cell destruction, but not IL-4, with a reverse picture observed in healthy volunteers. On the whole, our results argue in favour of a potential role of IL-17-producing iNKT cells in T1D and suggest that inflammation in T1D patients could induce a Th1/Th17 cytokine secretion profile in iNKT cells promoting disease development.

Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

Smad and NFAT pathways cooperate to induce CD103 expression in human CD8 T lymphocytes.

The interaction of integrin αE(CD103)ß7, often expressed on tumor-infiltrating T lymphocytes, with its cognate ligand, the epithelial cell marker E-cadherin on tumor cells, plays a major role in antitumor CTL responses. CD103 is induced on CD8 T cells upon TCR engagement and exposure to TGF-ß1, abundant within the tumor microenvironment. However, the transcriptional mechanisms underlying the cooperative role of these two signaling pathways in inducing CD103 expression in CD8 T lymphocytes remain unknown. Using a human CTL system model based on a CD8(+)/CD103(-) T cell clone specific of a lung tumor-associated Ag, we demonstrated that the transcription factors Smad2/3 and NFAT-1 are two critical regulators of this process. We also identified promoter and enhancer elements of the human ITGAE gene, encoding CD103, involved in its induction by these transcriptional regulators. Overall, our results explain how TGF-ß1 can participate in CD103 expression on locally TCR-engaged Ag-specific CD8 T cells, thus contributing to antitumor CTL responses and cancer cell destruction.

Consistent high concentration of the viral microRNA BART17 in plasma samples from nasopharyngeal carcinoma patients--evidence of non-exosomal transport.

BACKGROUND: Because latent Epstein Barr (EBV)-infection is a specific characteristic of malignant nasopharyngeal carcinoma (NPC), various molecules of viral origin are obvious candidate biomarkers in this disease. In a previous study, we could show in a few clinical samples that it was possible to detect a category of EBV microRNAs called miR-BARTs in the plasma of at least a fraction of NPC patients. The first aim of the present study was to investigate the status of circulating miR-BART17-5p (one of the miR-BARTs hereafter called miR-BART17) and EBV DNA in a larger series of NPC plasma samples. The second aim was to determine whether or not circulating miR-BART17 was carried by plasma exosomes. PATIENTS AND METHODS: Plasma samples were collected from 26 NPC patients and 10 control donors, including 9 patients with non-NPC Head and Neck squamous cell carcinoma and one healthy EBV carrier. Concentrations of miR-BART17 and two cellular microRNAs (hsa-miR-16 and -146a) were assessed by real-time quantitative PCR with spike-in normalization and absolute quantification. In addition, for 2 patients, exosome distributions of miR-BART17 and miR-16 were investigated following plasma lipoprotein fractionation by isopycnic density gradient ultrcentrifugation. RESULTS: The miR-BART17 was significantly more abundant in plasma samples from NPC patients compared to non-NPC donors. Above a threshold of 506 copies/mL, detection of miR-BART17 was highly specific for NPC patients (ROC curve analysis: AUC=0.87 with true positive rate = 0.77, false positive rate = 0.10). In this relatively small series, the concentration of plasma miR-BART17 and the plasma EBV DNA load were not correlated. When plasma samples were fractionated, miR-BART17 co-purified with a protein-rich fraction but not with exosomes. CONCLUSIONS: Detection of high concentrations of plasma miR-BART17 is consistent in NPC patients. This parameter is, at least in part, independent of the viral DNA load. Circulating miR-BART17 does not co-purify with exosomes.

Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a human epithelial malignancy consistently associated with the Epstein-Barr virus. The viral genome is contained in the nuclei of all malignant cells with abundant transcription of a family of viral microRNAs called BART miRNAs. MicroRNAs are well known intra-cellular regulatory elements of gene expression. In addition, they are often exported in the extra-cellular space and sometimes transferred in recipient cells distinct from the producer cells. Extra-cellular transport of the microRNAs is facilitated by various processes including association with protective proteins and packaging in secreted nanovesicles called exosomes. Presence of microRNAS produced by malignant cells has been reported in the blood and saliva of tumor-bearing patients, especially patients diagnosed with glioblastoma or ovarian carcinoma. In this context, it was decided to investigate extra-cellular release of BART miRNAs by NPC cells and their possible detection in the blood of NPC patients. To address this question, we investigated by quantitative RT-PCR the status of 5 microRNAs from the BART family in exosomes released by NPC cells in vitro as well as in plasma samples from NPC xenografted nude mice and NPC patients. RESULTS: We report that the BART miRNAs are released in the extra-cellular space by NPC cells being associated, at least to a large extent, with secreted exosomes. They are detected with a good selectivity in plasma samples from NPC xenografted nude mice as well as NPC patients. CONCLUSIONS: Viral BART miRNAs are secreted by NPC cells in vitro and in vivo. They have enough stability to diffuse from the tumor site to the peripheral blood. This study provides a basis to explore their potential as a source of novel tumor biomarkers and their possible role in communications between malignant and non-malignant cells.

Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is the third most frequent virus-associated human malignancy. How this tumor escapes immune recognition despite the expression of several viral antigens has remained poorly understood. Our previous in vitro studies have shown that NPC cells release exosomes containing high amounts of galectin-9, a ligand of the membrane receptor Tim-3, which is able to induce apoptosis in mature Th1 lymphocytes. Here, we sought to determine whether galectin-9-carrying exosomes were produced in NPC patients and whether such exosomes might play a role in the immune evasion of NPC cells. We report that galectin-9-containing exosomes are selectively detected in plasma samples from NPC patients and mice xenografted with NPC tumors. The incorporation into exosomes protects galectin-9 against proteolytic cleavage but retains its Tim-3-binding capacity. Importantly, NPC exosomes induce massive apoptosis in EBV-specific CD4(+) cells used as a model of target T cells. This effect is inhibited by both anti-Tim-3 and antigalectin-9 blocking antibodies. These results indicate that blocking galectin-9/Tim-3 interaction in vivo might alleviate the Th1-suppressive effect of NPC exosomes and sustain antitumoral T-cell responses and thereby improve clinical efficacy of immunotherapeutic approaches against NPC.