Molecular insight into interactions between the Taf14, Yng1 and Sas3 subunits of the NuA3 complex.

The NuA3 complex is a major regulator of gene transcription and the cell cycle in yeast. Five core subunits are required for complex assembly and function, but it remains unclear how these subunits interact to form the complex. Here, we report that the Taf14 subunit of the NuA3 complex binds to two other subunits of the complex, Yng1 and Sas3, and describe the molecular mechanism by which the extra-terminal domain of Taf14 recognizes the conserved motif present in Yng1 and Sas3. Structural, biochemical, and mutational analyses show that two motifs are sandwiched between the two extra-terminal domains of Taf14. The head-to-toe dimeric complex enhances the DNA binding activity of Taf14, and the formation of the hetero-dimer involving the motifs of Yng1 and Sas3 is driven by sequence complementarity. In vivo assays in yeast demonstrate that the interactions of Taf14 with both Sas3 and Yng1 are required for proper function of the NuA3 complex in gene transcription and DNA repair. Our findings suggest a potential basis for the assembly of three core subunits of the NuA3 complex, Taf14, Yng1 and Sas3.

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment.

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment.

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we developed an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrated that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacted nucleosome diffusive properties in a manner that was dependent on local chromatin density and supportive of a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Our results reveal that nuclear heterogeneity arises from both active and passive process and highlights the need to account for different organizational principals when modeling different chromatin environments.

MicroRNA-124 Enhances T Cells Functions by Manipulating the Lactic Acid Metabolism of Tumor Cells.

High production of lactic acid is a common feature of various tumors. Lactic acid is an immunosuppressive molecule with crucial roles in tumor cells' immune escape, which could largely be attributed to its negative effects on the T cells present in the tumor microenvironment (TME). Strategies that decrease the glycolysis rate of tumor cells could enhance immunosurveillance and limit tumor growth. Pyruvate kinase M2 (PKM2) is a key enzyme in the glycolysis pathway, and it plays a vital role in lactic acid buildup in the TME. MicroRNA (miR)-124 has been shown to be able to decrease tumor cell lactic acid synthesis indirectly by reducing PKM2 levels. In this study, we first overexpressed miR-124 in the tumor cells and evaluated its effects on the PKM2 expression and lactic acid production of the tumor cells using quantitative real-time polymerase chain reaction (qRT-PCR) and spectrophotometry, respectively. Then, we cocultured miR-124-treated tumor cells with T cells to investigate the effects of miR-124 overexpression on T cell proliferation, cytokine production, and apoptosis. Our results demonstrated that miR-124 overexpression could significantly reduce the amount of lactic acid produced by tumor cells by manipulating their glucose metabolism, which led to the augmented proliferation and IFN-γ production of T cells. Moreover, it rescued T cells from lactic acid-induced apoptosis. Our data suggest that lactic acid is a hindering factor for T-cell-based immunotherapies; however, manipulating tumor cells' metabolism via miR-124 could be a promising way to improve antitumor responses of T cells.

Prevalence and specificity of red blood cell alloantibodies and autoantibodies in transfused Iranian ß-thalassemia patients: A systematic review and meta-analysis.

BACKGROUND: Repeated allogeneic blood transfusions in thalassemia major patients stimulate the patient's immune system to generate antibodies against foreign erythrocyte antigens. This study was carried out to systematically review the findings of available studies about the prevalence of alloantibodies and autoantibodies, as well as the type of causative antigens among transfusion-dependent thalassemia patients in Iran. METHODS: Electronic search was conducted on Medline, PubMed, Cochrane, EMBASE, ScienceDirect, and Persians databases. All relevant articles published from January 1990 to July 2018 were included. Abstracts of conference booklets which that been published in the last 5 years were also included in the meta-analysis. The search language was restricted to English and Persian. The quality of studies was evaluated according to a checklist developed by authors, and Cochrane Risk of Bias Assessment Tool was used to evaluate the risk of bias. RESULTS: Twenty-three relevant articles met all the inclusion criteria. The prevalence of alloimmunization was 13%. Our study showed that anti-D (25%) and anti-K (25%) were most prevalent among Iranian ß-thalassemia patients. Data analysis shows the autoantibody prevalence to be 1% among 3787 patients. Meta-regression revealed that the prevalence of alloantibodies increases with each year as the average age of the study population increases. CONCLUSION: The prevalence of red blood cell (RBC) alloantibodies in transfused Iranian ß-thalassemia patients was high. Appropriate preventive strategies such as RBC phenotyping for patients before beginning transfusion and using extended RBC donor-recipient matching, specifically for Rh and Kell system, could be implemented to avoid complications in thalassemia patients.

Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

Mitochondria as Playmakers of CAR T-cell Fate and Longevity.

The development of chimeric antigen receptor (CAR) T-cell therapy has led to a paradigm shift in cancer treatment. However, patients often do not benefit from CAR T-cell therapy due to poor persistence of the adoptively transferred cells. Development of strategies based on the generation and maintenance of long-lasting memory T cells may expand the therapeutic effects of CAR T cells. Mitochondrial metabolic pathways play crucial roles in regulating the fate, function, and longevity of T cells. Here, we discuss how reprogramming of mitochondrial metabolic pathways influences function, persistence, and determination of CAR T-cell fate toward a memory phenotype. Moreover, we explore how mitochondrial activity determines persistence and the clinical outcome of CAR T-cell therapy. In addition, we review some strategies for manipulating CAR T-cell mitochondria to improve the survival of CAR T cells.

Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells.

T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells. To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling.

A metabolic switch to memory CAR T cells: Implications for cancer treatment.

Therapeutic efficacy of chimeric antigen receptor (CAR) T cells is associated with their expansion, persistence and effector function. Although CAR T cell therapy has shown remarkable therapeutic effects in hematological malignancies, its therapeutic efficacy has been limited in some types of cancers - in particular, solid tumors - partially due to the cells' inability to persist and the acquisition of T cell dysfunction within a harsh immunosuppressive tumor microenvironment. Therefore, it would be expected that generation of CAR T cells with intrinsic properties for functional longevity, such as the cells with early-memory phenotypes, could beneficially enhance antitumor immunity. Furthermore, because the metabolic pathways of CAR T cells help determine cellular differentiation and lifespan, therapies targeting such pathways like glycolysis and oxidative phosphorylation, can alter CAR T cell fate and durability within tumors. Here we discuss how reprogramming of CAR T cell metabolism and metabolic switch to memory CAR T cells influences their antitumor activity. We also offer potential strategies for targeting these metabolic circuits in the setting of adoptive CAR T cell therapy, aiming to better unleash the potential of adoptive CAR T cell therapy in the clinic.

Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell.

Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3ζ and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) α, and Interferon-(IFN) γ production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting.

Pharmacological targeting of immune checkpoint A2aR improves function of anti-CD19 CAR T cells in vitro.

In spite of impressive results in the treatment of acute lymphoblastic B cell leukemia (B-ALL) with chimeric antigen receptor (CAR) T cells, the clinical outcome of some hematological cancers like follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL) has not been very promising likely due to immunosuppressive networks within tumor microenvironment. Hypoxia in the microenvironment of hematological malignancies and consequently generation of adenosine molecule is appeared to be correlated with immunosuppression, tumor progression, and relapse. Herein, we hypothesized that whether pharmacological targeting of adenosine 2a receptor (A2aR) can enhance antitumor activity of anti-CD19 CAR T cells in vitro. Prior to functional assays, A2aR expression was assessed in CAR-expressing T cells. Our results showed that A2aR was not only up-regulated in the fully human anti-CD19 CAR T cells (hereafter referred to as huCAR19 T cells) but also was further overexpressed following re-stimulation with target cells. Although pharmacological inhibition of A2aR could significantly increase proliferation capacity and cytokine production of huCAR19 T cells following treatment with an adenosine analog, cytotoxic activity of huCAR19 T cells was not significantly improved. Considering A2aR overexpression in huCAR19 T cells in the tumor microenvironment, our results indicated that pharmacological targeting of A2aR could not only improve huCAR19 T cells functionality in a hostile tumor microenvironment but also could have a therapeutic advantage, and sought to assess the possibility in a pre-clinical setting.

Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells.

BACKGROUND: CAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). METHODS: In this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software. RESULTS: Our experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells. CONCLUSION: Altogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

Role of microRNAs in Chronic Lymphocytic Leukemia Pathogenesis.

MicroRNAs (miRNAs) are a group of small endogenous non-coding RNAs involved in many cancers and various cellular processes such as cellular growth, DNA methylation, apoptosis, and differentiation. 13q14.3 chromosomal region contains miR-15 and miR-16 and deletion of this region is a commonly reported aberration in Chronic Lymphoblastic Leukemia (CLL), suggesting miRNAs involvement in CLL pathogenesis. MicroRNAs are known as oncogenes and tumor suppressors in CLL which may also serve as markers of onset and progression of the disease. The most prevalent form of leukemia diagnosed in adults in the western world, chronic lymphocytic leukemia, accounts for one-third of all leukemias. CLL is characterized by the presence of B Cell Malignant Clones in secondary lymphoid tissues, peripheral blood and bone marrow. The precise etiology of CLL is remained to be known, however, a number of Chromosomal Abnormalities such as deletions of 13q14.3, 11q and 17p and trisomy 12 have been detected. In this review, we offer our prospect on how miRNAs are involved in the CLL pathogenesis and disease progression. Further understanding of the underlying mechanisms and regulation of CLL pathogenesis has underscored the need for further research regarding their role in this disease.