Abstract
T-box transcription factors (TF) Eomes and T-bet are vital for NK cell development. However, their role in mature NK cell homeostasis and function remains unclear. Recently we reported that ...Eomes regulates mouse NK cell cytotoxicity and stage-specific survival using an NK-specific, tamoxifen-inducible Eomes KO model. However, the role of Eomes and T-bet have not been investigated in primary human NK (hNK) cell biology using genetic loss-of-function approaches. We hypothesized that these TFs play a critical role in maintaining NK cell functional molecular programs.
Here we used CRISPR-Cas9 to genetically delete Eomes and T-bet expression in primary hNK cells. In vitro stimulation assays revealed that IFNγ response is impaired in CD56bright NK cells only when Eomes is deleted (p<0.01) but not T-bet. This impairment is even more striking when both TFs are deleted, resulting in both CD56bright and CD56dim NK cells having reduced IFNγ (p<0.01 and p<0.05, respectively).
Using NSG xenografts, we found that Eomes/T-bet double CRISPR-edited (DKO) hNK cells have impaired persistence/expansion in vivo. After 3 weeks, we recovered in the spleen on average 3E5 control compared to only 6E4 DKO hNK cells. A similar ~80% decrease was also observed in the blood. To determine the mechanism of this, we assessed proliferation and apoptosis of control compared to DKO cells. Ki-67 and dye dilution revealed impaired proliferation of DKO cells in vivo, and we also observed increased apoptosis measured by Annexin V/7-AAD staining (p<0.05).
Our data suggest that Eomes and T-bet are critical for the function and homeostasis of mature hNK cells. Understanding their function in mature NK cells will provide insights to improve NK cell therapy for diseases such as cancer.
Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular ...programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Singlecell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56.sup.bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORCand AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.
Abstract
Adoptive cell therapy (ACT) using NK cells is a promising armament in the fight against cancer. Cytokine induced memory like (CIML) NK cells have been shown in clinical studies to have ...potent antitumor activity with superior in vivo persistence. Currently, the expansion of NK cells for clinical development is mainly based on feeder cells, which imposes significant regulatory hurdles and increases the costs for manufacturing. We have developed fusion proteins, HCW9201 and HCW9206 comprising of IL-15/IL-18/IL-12 and IL15/IL-7/IL-21, respectively, capable of priming memory-like differentiation and expanding CIML NK cell products without using feeder cells. This “Kick and Expand” strategy allows greater than 100x expansion of CIML NK cells from donor PBMCs in as little as 14 days without the use of exogenous feeder cells. Continued expansion can yield sufficient CIML NK cells for cryopreservation and multiple ACT infusions. The NK cells generated have bona fide memory-like properties: enhanced antitumor activity across multiple cancer cell lines, higher metabolic capacity, stable epigenetic demethylation of the IFN-γ promoter and increased persistence in NSG mice, when compared to conventional NK cells. In conclusion, this “Kick and Expand” process supports generation of abundant CIML NK cells for multiple ACT infusions and provides simpler, more regulatory friendly, off-the-shelf platform for generating NK cell products, including those with chimeric antigen receptor (CAR) constructs.
Purpose
– The purpose of this case study is to discuss the creation of robust preservation functionality within PURR. The study seeks to discuss the customization of the HUBzero platform, composition ...of digital preservation policies, and the creation of a novel, machine-actionable metadata model for PURR's unique digital content. Additionally, the study will trace the implementation of the Open Archival Information System (OAIS) model and track PURR's progress towards Trustworthy Digital Repository certification.
Design/methodology/approach
– This case study discusses the use of the Center for Research Libraries Trusted Repository Audit Checklist (TRAC) certification process and ISO 16363 as a rubric to build an OAIS institutional repository for the publication, preservation, and description of unique datasets.
Findings
– ISO 16363 continues to serve as a rubric, barometer and set of goals for PURR as development continues. To become a trustworthy repository, the PURR project team has consistently worked to build a robust, secure, and long-term home for collaborative research. In order to fulfill its mandate, the project team constructed policies, strategies, and activities designed to guide a systematic digital preservation environment. PURR expects to undertake the full ISO 16363 audit process at a future date in expectation of being certified as a Trustworthy Digital Repository. Through its efforts in digital preservation, the Purdue University Research Repository expects to better serve Purdue researchers, their collaborators, and move scholarly research efforts forward world-wide.
Originality/value
– PURR is a customized instance of HUBzero®, an open source software platform that supports scientific discovery, learning, and collaboration. HUBzero was a research project funded by the United States National Science Foundation (NSF) and is a product of the Network for Computation Nanotechnology (NCN), a multi-university initiative of eight member institutions. PURR is only one instance of a HUBzero's customization; versions have been implemented in many disciplines nation-wide. PURR maintains the core functionality of HUBzero, but has been modified to publish datasets and to support their preservation. Long-term access to published data are an essential component of PURR services and Purdue University Libraries' mission. Preservation in PURR is not only vital to the Purdue University research community, but to the larger digital preservation issues surrounding dynamic datasets and their long-term usability.
The Purdue University Research Repository C. Dearborn, Carly; J. Barton, Amy; A. Harmeyer, Neal
OCLC systems & services,
02/2014, Letnik:
30, Številka:
1
Journal Article
Purpose
– The purpose of this case study is to discuss the creation of robust preservation functionality within PURR. The study seeks to discuss the customization of the HUBzero platform, composition ...of digital preservation policies, and the creation of a novel, machine-actionable metadata model for PURR's unique digital content. Additionally, the study will trace the implementation of the Open Archival Information System (OAIS) model and track PURR's progress towards Trustworthy Digital Repository certification.
Design/methodology/approach
– This case study discusses the use of the Center for Research Libraries Trusted Repository Audit Checklist (TRAC) certification process and ISO 16363 as a rubric to build an OAIS institutional repository for the publication, preservation, and description of unique datasets.
Findings
– ISO 16363 continues to serve as a rubric, barometer and set of goals for PURR as development continues. To become a trustworthy repository, the PURR project team has consistently worked to build a robust, secure, and long-term home for collaborative research. In order to fulfill its mandate, the project team constructed policies, strategies, and activities designed to guide a systematic digital preservation environment. PURR expects to undertake the full ISO 16363 audit process at a future date in expectation of being certified as a Trustworthy Digital Repository. Through its efforts in digital preservation, the Purdue University Research Repository expects to better serve Purdue researchers, their collaborators, and move scholarly research efforts forward world-wide.
Originality/value
– PURR is a customized instance of HUBzero®, an open source software platform that supports scientific discovery, learning, and collaboration. HUBzero was a research project funded by the United States National Science Foundation (NSF) and is a product of the Network for Computation Nanotechnology (NCN), a multi-university initiative of eight member institutions. PURR is only one instance of a HUBzero's customization; versions have been implemented in many disciplines nation-wide. PURR maintains the core functionality of HUBzero, but has been modified to publish datasets and to support their preservation. Long-term access to published data are an essential component of PURR services and Purdue University Libraries' mission. Preservation in PURR is not only vital to the Purdue University research community, but to the larger digital preservation issues surrounding dynamic datasets and their long-term usability.
Accurate clinical assessment of a patient's response to treatment for glioblastoma multiforme (GBM), the most malignant type of primary brain tumor, is undermined by the wide patient-to-patient ...variability in GBM dynamics and responsiveness to therapy. Using computational models that account for the unique geometry and kinetics of individual patients' tumors, we developed a method for assessing treatment response that discriminates progression-free and overall survival following therapy for GBM. Applying these models as untreated virtual controls, we generate a patient-specific "Days Gained" response metric that estimates the number of days a therapy delayed imageable tumor progression. We assessed treatment response in terms of Days Gained scores for 33 patients at the time of their first MRI scan following first-line radiation therapy. Based on Kaplan-Meier analyses, patients with Days Gained scores of 100 or more had improved progression-free survival, and patients with scores of 117 or more had improved overall survival. Our results demonstrate that the Days Gained response metric calculated at the routinely acquired first post-radiation treatment time point provides prognostic information regarding progression and survival outcomes. Applied prospectively, our model-based approach has the potential to improve GBM treatment by accounting for patient-to-patient heterogeneity in GBM dynamics and responses to therapy.
Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function ...remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142−/− mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-β (TGF-β) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.
Display omitted
•miR-142 is induced by IL-15 and is required for type 1 ILC development and homeostasis•miR-142 targets Socs1 thereby promotes IL-15 receptor signaling in type 1 ILCs•αV integrin, a miR142-3p target, promotes IL-15-independent survival in type 1 ILCs•miR-142 is critical for cytokine production and NK-cell-mediated viral control
IL-15 and TGF-β support the homeostasis and molecular programs of distinct innate lymphoid cell (ILC) types. Berrien-Elliott and colleagues identify that microRNA-142 is required for IL-15 receptor signaling and NK cell survival, whereas loss of microRNA-142 results in distinct TGF-β- and/or integrin-supported type 1 ILCs.
Background
Glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than gliomas with wild-type IDH1.
Methods
Here we compare the IDH1 mutational ...status in 172 contrast-enhancing glioma patients with the invasion profile generated by a patient-specific mathematical model we developed based on MR imaging.
Results
We show that IDH1-mutated contrast-enhancing gliomas were relatively more invasive than wild-type IDH1 for all 172 contrast-enhancing gliomas as well as the subset of 158 histologically confirmed glioblastomas. The appearance of this relatively increased, model-predicted invasive profile appears to be determined more by a lower model-predicted net proliferation rate rather than an increased model-predicted dispersal rate of the glioma cells. Receiver operator curve analysis of the model-predicted MRI-based invasion profile revealed an area under the curve of 0.91, indicative of a predictive relationship. The robustness of this relationship was tested by cross-validation analysis of the invasion profile as a predictive metric for IDH1 status.
Conclusions
The strong correlation between IDH1 mutation status and the MRI-based invasion profile suggests that use of our tumor growth model may lead to noninvasive clinical detection of IDH1 mutation status and thus lead to better treatment planning, particularly prior to surgical resection, for contrast-enhancing gliomas.
PDF
