miR-17∼92 family clusters control iNKT cell ontogenesis via modulation of TGF-ß signaling.

Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-ß receptor II (TGF-ßRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-ß signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR-17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-ßRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-ß signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.

MicroRNA-133b Regulation of Th-POK Expression and Dendritic Cell Signals Affect NKT17 Cell Differentiation in the Thymus.

NKT17 cells represent a functional subset of Vα14 invariant NKT (iNKT) cells with important effector functions in infections and autoimmune diseases. The mechanisms that drive NKT17 cell differentiation in the thymus are still largely unknown. The percentage of NKT17 cells has a high variability between murine strains due to differential thymic differentiation. For example, the NOD strain carries a high percentage and absolute numbers of NKT17 cells compared with other strains. In this study, we used the NOD mouse model to analyze what regulates NKT17 cell frequency in the thymus and peripheral lymphoid organs. In accordance with previous studies showing that the zinc finger transcription factor Th-POK is a key negative regulator of thymic NKT17 cell differentiation in the thymus, our data indicate that excessive NKT17 cell frequency in NOD mice correlates with defective Th-POK expression by thymic Vα14iNKT cells. Moreover, we found that Th-POK expression is under epigenetic regulation mediated by microRNA-133b whose expression is reduced in Vα14iNKT cells of NOD mice. We also demonstrated in a conditional knockout model of dendritic cell (DC) depletion (CD11cCreXDTA.B6 and CD11cCreRosa26DTA.NOD mice) that DCs play a crucial role in regulating Vα14iNKT cell maturation and their acquisition of an NKT17 cytokine secretion phenotype in the thymus. Overall, our data show that mechanisms regulating NKT17 cell differentiation are unique and completely different from those of Vα14iNKT cells. Specifically, we found that epigenetic regulation through microRNA-133b-regulated Th-POK expression and signals provided by DCs are fundamental for thymic NKT17 cell differentiation.

The circulating microRNome demonstrates distinct lymphocyte subset-dependent signatures.

Upon activation, lymphocytes release vesicles containing microRNAs (miRNAs). However, little is known as to whether this release results in modulation of circulating miRNAs (the miRNome) in the serum. The present work aims to identify lymphocyte subset-specific signatures of miRNAs within the serum circulating miRNome. We therefore assessed serum miRNA expression profiles in wild-type mice; in mice lacking either CD4(+) T cells, CD8(+) T cells, invariant natural killer T (iNKT) cells, or B cells; and, as a control, in mice in which Dicer has been ablated in T lymphocytes. We found that specific serum miRNAs are differentially modulated when different lymphocyte subsets are lacking. In particular, the serum level of miR-181b-5p, previously demonstrated to be fundamental for the development of iNKT cells, is specifically reduced in mice in which iNKT cells are absent. Interestingly, our results indicate a direct link between the biological role of a single miRNA in lymphocyte development and its serum level, and prove that even a population composed of relatively few cells in vivo, such as iNKT lymphocytes, has a measurable effect on the serum circulating miRNome.

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help.

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.

Loss of T cell microRNA provides systemic protection against autoimmune pathology in mice.

With an increasing number of studies demonstrating alterations in T cell microRNA expression during autoimmune disease, modulation of the T cell microRNA network is considered a potential therapeutic strategy. Due to the complex and often opposing interactions of individual microRNA, prioritization of therapeutic targets first requires dissecting the dominant effects of the T cell microRNA network. Initial results utilizing a unidirectional screen suggested that the tolerogenic functions were dominant, with spontaneous colitis resulting from T cell-specific excision of Dicer. Here we performed a bidirectional screen for microRNA function by removing Dicer from the T cells of both wildtype mice and Transforming Growth Factor ß (TGFß) receptor-deficient mice. This allowed the impact of microRNA loss on T cell activation, effector T cell differentiation and autoimmune pathology to be systematically assessed. This bidirectional screen revealed a dominant immunogenic function for T cell microRNA, with potent suppression of T cell activation, IFNγ production and autoimmune pathology in all targeted organs except the colon, where Dicer-dependent microRNA demonstrated a dominant tolerogenic function. These results reverse the original conclusions of microRNA function in T cells by revealing a systemic pro-autoimmune function.

Adoptive Immunotherapy With Engineered iNKT Cells to Target Cancer Cells and the Suppressive Microenvironment.

Invariant Natural Killer T (iNKT) cells are T lymphocytes expressing a conserved semi-invariant TCR specific for lipid antigens (Ags) restricted for the monomorphic MHC class I-related molecule CD1d. iNKT cells infiltrate mouse and human tumors and play an important role in the immune surveillance against solid and hematological malignancies. Because of unique functional features, they are attractive platforms for adoptive cells immunotherapy of cancer compared to conventional T cells. iNKT cells can directly kill CD1d-expressing cancer cells, but also restrict immunosuppressive myelomonocytic populations in the tumor microenvironment (TME) via CD1d-cognate recognition, promoting anti-tumor responses irrespective of the CD1d expression by cancer cells. Moreover, iNKT cells can be adoptively transferred across MHC barriers without risk of alloreaction because CD1d molecules are identical in all individuals, in addition to their ability to suppress graft vs. host disease (GvHD) without impairing the anti-tumor responses. Within this functional framework, iNKT cells are successfully engineered to acquire a second antigen-specificity by expressing recombinant TCRs or Chimeric Antigen Receptor (CAR) specific for tumor-associated antigens, enabling the direct targeting of antigen-expressing cancer cells, while maintaining their CD1d-dependent functions. These new evidences support the exploitation of iNKT cells for donor unrestricted, and possibly off the shelf, adoptive cell therapies enabling the concurrent targeting of cancer cells and suppressive microenvironment.

TCR-engineered iNKT cells induce robust antitumor response by dual targeting cancer and suppressive myeloid cells.

Adoptive immunotherapy with T cells engineered with tumor-specific T cell receptors (TCRs) holds promise for cancer treatment. However, suppressive cues generated in the tumor microenvironment (TME) can hinder the efficacy of these therapies, prompting the search for strategies to overcome these detrimental conditions and improve cellular therapeutic approaches. CD1d-restricted invariant natural killer T (iNKT) cells actively participate in tumor immunosurveillance by restricting suppressive myeloid populations in the TME. Here, we showed that harnessing iNKT cells with a second TCR specific for a tumor-associated peptide generated bispecific effectors for CD1d- and major histocompatibility complex (MHC)-restricted antigens in vitro. Upon in vivo transfer, TCR-engineered iNKT (TCR-iNKT) cells showed the highest efficacy in restraining the progression of multiple tumors that expressed the cognate antigen compared with nontransduced iNKT cells or CD8+ T cells engineered with the same TCR. TCR-iNKT cells achieved robust cancer control by simultaneously modulating intratumoral suppressive myeloid populations and killing malignant cells. This dual antitumor function was further enhanced when the iNKT cell agonist α-galactosyl ceramide (α-GalCer) was administered as a therapeutic booster through a platform that ensured controlled delivery at the tumor site, named multistage vector (MSV). These preclinical results support the combination of tumor-redirected TCR-iNKT cells and local α-GalCer boosting as a potential therapy for patients with cancer.

Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases.

We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (γδT)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues.

Dicer-dependent microRNA pathway controls invariant NKT cell development.

Invariant NK T (iNKT) cells are a separate lineage of T lymphocytes with innate effector functions. They express an invariant TCR specific for lipids presented by CD1d and their development and effector differentiation rely on a unique gene expression program. We asked whether this program includes microRNAs, small noncoding RNAs that regulate gene expression posttranscriptionally and play a key role in the control of cellular differentiation programs. To this aim, we investigated iNKT cell development in mice in which Dicer, the RNase III enzyme that generates functional microRNAs, is deleted in cortical thymocytes. We find that Dicer deletion results in a substantial reduction of iNKT cells in thymus and their disappearance from the periphery, unlike mainstream T cells. Without Dicer, iNKT cells do not complete their innate effector differentiation and display a defective homeostasis due to increased cell death. Differentiation and homeostasis of iNKT cells require Dicer in a cell-autonomous fashion. Furthermore, we identify a miRNA profile specific for iNKT cells, which exhibits features of activated/effector T lymphocytes, consistent with the idea that iNKT cells undergo agonist thymic selection. Together, these results define a critical role of the Dicer-dependent miRNA pathway in the physiology of iNKT cells.

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

miR-21 sustains CD28 signalling and low-affinity T-cell responses at the expense of self-tolerance.

OBJECTIVE: miR-21 is highly expressed in iNKT and activated T cells, but its T-cell autonomous functions are poorly defined. We sought to investigate the role of miR-21 in the development and functions of T and iNKT cells, representing adaptive and innate-like populations, respectively. METHODS: We studied mice with a conditional deletion of miR-21 in all mature T lymphocytes. RESULTS: Thymic and peripheral T and iNKT compartments were normal in miR-21 KO mice. Upon activation in vitro, miR-21 depletion reduced T-cell survival, TH17 polarisation and, remarkably, T- and iNKT cell ability to respond to low-affinity antigens, without altering their response to high-affinity ones. Mechanistically, miR-21 sustained CD28-dependent costimulation pathways required to lower the T-cell activation threshold, inhibiting its repressors in a positive feedback circuit, in turn increasing T-cell sensitivity to antigenic stimulation and survival. Upon immunisation with the low-affinity self-epitope MOG35-55, miR-21 KO mice were indeed less susceptible than WT animals to the induction of experimental autoimmune encephalomyelitis, whereas they mounted normal T-cell responses against high-affinity viral epitopes generated upon lymphocytic choriomeningitis virus infection. CONCLUSION: The induction of T-cell responses to weak antigens (signal 1) depends on CD28 costimulation (signal 2). miR-21 sustains CD28 costimulation, decreasing the T-cell activation threshold and increasing their sensitivity to antigenic stimulation and survival, broadening the immune surveillance range. This occurs at the cost of unleashing autoimmunity, resulting from the recognition of weak self-antigens by autoreactive immune responses. Thus, miR-21 fine-tunes T-cell response and self-/non-self-discrimination.

Mir106b-25 and Mir17-92 Are Crucially Involved in the Development of Experimental Neuroinflammation.

MicroRNAs (miRNAs) are single-stranded RNA that have key roles in the development of the immune system and are involved in the pathogenesis of various autoimmune diseases. We previously demonstrated that two members of the miR106b-25 cluster and the miR17-92 paralog cluster were upregulated in T regulatory cells from multiple sclerosis (MS) patients. The aim of the present work was to clarify the impact of miR106b-25 and miR17-92 clusters in MS pathogenesis. Here, we show that the mice lacking miR17-92 specifically in CD4+ T cells or both total miR106b-25 and miR17-92 in CD4+ T cells (double knockout) are protected from Experimental Autoimmune Encephalomyelitis (EAE) development while depletion of miR106b-25 only does not influence EAE susceptibility. We suggest that the absence of miR106b does not protect mice because of a mechanism of compensation of miR17-92 clusters. Moreover, the decrease of neuroinflammation was found to be associated with a significant downregulation of pro-inflammatory cytokines (GM-CSF, IFNγ, and IL-17) in the spinal cord of double knockout EAE mice and a reduction of Th17 inflammatory cells. These results elucidate the effect of miR106b-25 and miR17-92 deletion in MS pathogenesis and suggest that their targeted inhibition may have therapeutic effect on disease course.

Data on the effects of eIF6 downmodulation on the proportions of innate and adaptive immune system cell subpopulations and on thymocyte maturation.

The data described in this article are related to "High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans" (Manfrini et al., in press) [1]. eIF6 is a translation initiation factor required for ribosomal biogenesis (Sanvito et al., 1999) [2] and for proper translational initiation (Gallo and Manfrini, 2015; Miluzio et al., 2016) [3], [4] whose protein abundance requires tight regulation. Here we analyze by flow cytometry the effects of eIF6 depletion on proportions of specific innate and adaptive immune system subpopulations and on thymocyte maturation in mice.

High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans.

Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that, in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4+ Effector Memory T cells. In human CD4+ T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4+ T cells, eIF6 levels control interferon-γ (IFN-γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery.

Intracellular modulation, extracellular disposal and serum increase of MiR-150 mark lymphocyte activation.

Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.

Inhibition of human pancreatic cell line MIA PaCa2 proliferation by HA-But, a hyaluronic butyric ester: a preliminary report.

OBJECTIVES: Inhibition of histone deacetylase activity is one of the epigenetic mechanisms in the regulation of the cellular gene expression. We investigated the antitumor effect of HA-But, a new histone deacetylase inhibitor, in which hyaluronic acid is esterified with butyric acid residues and selectively bind to CD44, overexpressed in most human cancers, including pancreatic cancer. METHODS: We analyzed the effect of HA-But on the expression level of some cell cycle (p21 waf1/cip1, p27 kip1, p53, and cyclin D1), apoptosis (BAX, caspase-7, Bcl-2, and survivin), and angiogenesis-related (vascular endothelial growth factor [VEGF] A165, VEGF-C, and VEGF-D) proteins on MIA PaCa-2, a pancreas carcinoma cell line that expressed CD44 in a high percentage (99%) of cells. RESULTS: HA-But was 7-fold more effective than sodium butyrate in inhibiting cell proliferation; it induced p21 waf1/cip1, p27 kip1, p53, and cyclin D1 modulation, resulting in a block of the cell cycle at G0/G1 and G2/M phases. Moreover, Ha-But induced apoptosis, affecting the expression level of either proapoptotic or antiapoptotic factors, reduced the expression level of VEGF-A165 and VEGF-D, and inhibited the angiogenesis process in vitro. CONCLUSIONS: On the basis of these results, which demonstrated an interesting antiproliferative, proapoptotic, and antiangiogenic activity, Ha-But could be a promising candidate for the treatment of pancreatic cancer.