Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Circulating miRNAs can serve as potential diagnostic biomarkers in chronic myelogenous leukemia patients.

INTRODUCTION: Chronic Myelogenous Leukemia (CML) is a myeloproliferative disorder described as a malignant blood disorder by accounts for 15-20% of all adult leukemia. MicroRNAs (miRNAs) play an important role in post-transcriptional regulation of gene expressions. Expression level of tumor suppressor-miRNAs, described as miRNAs that target the oncogens, can contribute to diagnosis and prognosis of some malignant disorders including CML. We theorized that according to the excessive proliferation and alteration in miRNA expressions, there could be a change in the expression of miRNAs in plasma carried by exosomes. METHODS: We consequently decided to detect the differences between normal and aberrant miRNA expression in human plasma sample to find out the possibility of diagnosis by these alterations. The expression of candidate miRNAs were compared using RNA extracted from the plasma of 50 patients, as well as 30 healthy individuals. We analysed the plasma miR-16-1, miR-20, miR-106, miR-126, miR-155, miR-222, and miR-451 expression levels in CML patients by individual real-time quantitative RT-PCR. RESULTS: All selected miRNAs were found to be upregulated in newly diagnosed CML patients compared to the control, while upregulation of only three (miR-20, 106 and 222) were significant (17.4, 19 and 74.95 fold change, respectively; p<0.0001). IN CONCLUSION: microRNAs have a potential use in treatment of CML, as they can target the genes involved in cell cycle, MAPK, growth inhibition, TGF beta, and p53 signaling pathways. Therefore, these miRNA signatures provide the basis for their utilization as biomarkers in CML.

Chronic HIV infection induces transcriptional and functional reprogramming of innate immune cells.

Chronic inflammation and immune dysfunction play a key role in the development of non-AIDS-related comorbidities. The aim of our study was to characterize the functional phenotype of immune cells in people living with HIV (PLHIV). We enrolled a cross-sectional cohort study of PLHIV on stable antiretroviral therapy and healthy controls. We assessed ex vivo cytokine production capacity and transcriptomics of monocytes and T cells upon bacterial, fungal, and viral stimulation. PLHIV exhibited an exacerbated proinflammatory profile in monocyte-derived cytokines, but not in lymphocyte-derived cytokines. Particularly, the production of the IL-1ß to imiquimod, E. coli LPS, and Mycobacterium tuberculosis was increased, and this production correlated with plasma concentrations of high-sensitivity C-reactive protein and soluble CD14. This increase in monocyte responsiveness remained stable over time in subsequent blood sampling after more than 1 year. Transcriptome analyses confirmed priming of the monocyte IL-1ß pathway, consistent with a monocyte-trained immunity phenotype. Increased plasma concentrations of ß-glucan, a well-known inducer of trained immunity, were associated with increased innate cytokine responses. Monocytes of PLHIV exhibited a sustained proinflammatory immune phenotype with priming of the IL-1ß pathway. Training of the innate immune system in PLHIV likely plays a role in long-term HIV complications and provides a promising therapeutic target for inflammation-related comorbidities.

Controlled Human Malaria Infection Induces Long-Term Functional Changes in Monocytes.

Innate immune memory responses (also termed "trained immunity") have been described in monocytes after BCG vaccination and after stimulation in vitro with microbial and endogenous ligands such as LPS, ß-glucan, oxidized LDL, and monosodium urate crystals. However, whether clinical infections are also capable of inducing a trained immunity phenotype remained uncertain. We evaluated whether Plasmodium falciparum infection can induce innate immune memory by measuring monocyte-derived cytokine production from five volunteers undergoing Controlled Human Malaria Infection. Monocyte responses followed a biphasic pattern: during acute infection, monocytes produced lower amounts of inflammatory cytokines upon secondary stimulation, but 36 days after malaria infection they produced significantly more IL-6 and TNF-α in response to various stimuli. Furthermore, transcriptomic and epigenomic data analysis revealed a clear reprogramming of monocytes at both timepoints, with long-term changes of H3K4me3 at the promoter regions of inflammatory genes that remain present for several weeks after parasite clearance. These findings demonstrate an epigenetic basis of trained immunity induced by human malaria in vivo.

Outcomes of controlled human malaria infection after BCG vaccination.

Recent evidence suggests that certain vaccines, including Bacillus-Calmette Guérin (BCG), can induce changes in the innate immune system with non-specific memory characteristics, termed 'trained immunity'. Here we present the results of a randomised, controlled phase 1 clinical trial in 20 healthy male and female volunteers to evaluate the induction of immunity and protective efficacy of the anti-tuberculosis BCG vaccine against a controlled human malaria infection. After malaria challenge infection, BCG vaccinated volunteers present with earlier and more severe clinical adverse events, and have significantly earlier expression of NK cell activation markers and a trend towards earlier phenotypic monocyte activation. Furthermore, parasitemia in BCG vaccinated volunteers is inversely correlated with increased phenotypic NK cell and monocyte activation. The combined data demonstrate that BCG vaccination alters the clinical and immunological response to malaria, and form an impetus to further explore its potential in strategies for clinical malaria vaccine development.

Network of three specific microRNAs influence type 2 diabetes through inducing insulin resistance in muscle cell lines.

Insulin resistance has been implicated as one of the best predictors for type 2 diabetes. Growing evidence propose the involvement of microRNAs (miRNAs) as short regulatory molecules in modulating and inducing resistance. In this regard, we have investigated the role of three selected miRNAs in insulin resistance development (miR-135, miR-202, and miR-214), via assessing glucose uptake levels in C2C12 and L6 muscle cell lines. Interestingly, miRNA-transfected cells demonstrated a significantly different glucose uptake compared to the positive control cells. In addition, we evaluated the expression levels of three putative miRNA target genes (Rho-associated coiled-coil containing protein kinase 1, serine/threonine kinase 2, and vesicle-associated membrane protein 2) in transfected cells, recruiting luciferase assay. Our results indicated the targeting and downregulation of Rho-associated coiled-coil containing protein kinase 1 and serine/threonine kinase 2 genes in all miR-transfected cell lines ( P ≤ 0.05), but not for vesicle-associated membrane protein 2. MiRNA upregulation led to the poor stimulation of glucose uptake through insulin and developed insulin-resistant phenotype in both muscle cell lines. Our study showed the role of three miRNAs in the induction of insulin resistance in cell lines and making them prone to type 2 diabetes development.

Tissue engineering; strategies, tissues, and biomaterials.

Current tissue regenerative strategies rely mainly on tissue repair by transplantation of the synthetic/natural implants. However, limitations of the existing strategies have increased the demand for tissue engineering approaches. Appropriate cell source, effective cell modification, and proper supportive matrices are three bases of tissue engineering. Selection of appropriate methods for cell stimulation, scaffold synthesis, and tissue transplantation play a definitive role in successful tissue engineering. Although the variety of the players are available, but proper combination and functional synergism determine the practical efficacy. Hence, in this review, a comprehensive view of tissue engineering and its different aspects are investigated.

ß-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance.

Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, ß-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo ß-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT.

MicroRNA-129-1 acts as tumour suppressor and induces cell cycle arrest of GBM cancer cells through targeting IGF2BP3 and MAPK1.

BACKGROUND: MicroRNA-129-1 (miR-129-1) seems to behave as a tumour suppressor since its decreased expression is associated with different tumours such as glioblastoma multiforme (GBM). GBM is the most common form of brain tumours originating from glial cells. The impact of miR-129-1 downregulation on GBM pathogenesis has yet to be elucidated. METHODS: MiR-129-1 was overexpressed in GBM cells, and its effect on proliferation was investigated by cell cycle assay. MiR-129-1 predicted targets (CDK6, IGF1, HDAC2, IGF2BP3 and MAPK1) were also evaluated by western blot and luciferase assay. RESULTS: Restoration of miR-129-1 reduced cell proliferation and induced G1 accumulation, significantly. Several functional assays confirmed IGF2BP3, MAPK1 and CDK6 as targets of miR-129-1. Despite the fact that IGF1 expression can be suppressed by miR-129-1, through 3'-untranslated region complementary sequence, we could not find any association between IGF1 expression and GBM. MiR-129-1 expression inversely correlates with CDK6, IGF2BP3 and MAPK1 in primary clinical samples. CONCLUSION: This is the first study to propose miR129-1 as a negative regulator of IGF2BP3 and MAPK1 and also a cell cycle arrest inducer in GBM cells. Our data suggests miR-129-1 as a potential tumour suppressor and presents a rationale for the use of miR-129-1 as a novel strategy to improve treatment response in GBM.

MiR-371-373 cluster acts as a tumor-suppressor-miR and promotes cell cycle arrest in unrestricted somatic stem cells.

Recent advances in small RNA research have implicated microRNAs (miRNAs) as important regulators of proliferation and development. The miR-371-373 cluster is prominently expressed in human embryonic stem cells (ESCs) and rapidly decreases after cell differentiation. MiR-371-373 cluster was investigated as one of the key factors of stem cell maintenance and pluripotency in unrestricted somatic stem cells (USSCs) using a lentivirus system. Gene expression showed a dual effect on proliferation, which revealed a transient cell cycle progression and consequent repression in pluripotency factors and cell cycle genes. Cell proliferation analysis with CFU, MTT, and DNA content assays further confirmed the dual effect of cluster after prolonged exposure. Analyzing the course of action, it seems that miR-371-373 cluster acts as an onco/tumor suppressor-miR. MiR371-373 cluster acts by modulating the function of these factors and limiting the excessive cell cycle propagation upon oncogenic stimuli to protect cells from replicative stress, but also activate CDK inhibitors and transcriptional repressors of the retinoblastoma family to cause cell cycle arrest. In contrast to the previous studies, we believe that miR-371-373 cluster functions as a self-renewal miRNA to induce and maintain the pluripotent state but also to potentially inhibit dysregulated proliferation through cell cycle arrest. It seems that miR-371-373 cluster presents with a dual effect in this cellular context which may possess different actions in various cells. This not only expands the basic knowledge of the cluster but may offer a great chance for therapeutic interventions.

7SK small nuclear RNA inhibits cancer cell proliferation through apoptosis induction.

7SK small nuclear RNA (snRNA) is a 331-333-bp non-coding RNA, which recruits HEXIM 1/2 protein to inhibit positive elongation factor b (P-TEFb) activity. P-TEFb is an essential factor in alleviating promoter-proximal paused RNA polymerase II (Pol II) and initiating the productive elongation phase of gene transcription. Without this protein, Pol II will remain in its hypophosphorylated state, and no transcription occurs. In this study, we inhibited P-TEFb activity by over-expressing 7SK snRNA in human embryonic kidney (HEK) 293T cancer cell line. This inhibition led to a significant decrease in cell viability, which can be due to the transcription inhibition. Moreover, 7SK snRNA over-expression promoted apoptosis in cancerous cells. Our results suggest 7SK snRNA as a potential endogenous anti-cancer agent, and to the best of our knowledge, this is the first study that uses a long non-coding RNA's over-expression against cancer cell growth and proliferation.