Disease-associated mutations in topoisomerase IIß result in defective NK cells.

BACKGROUND: Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. OBJECTIVE: We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. METHODS: Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. RESULTS: Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. CONCLUSIONS: Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.

Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity.

Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.

Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

Concise Review: Human Pluripotent Stem Cells to Produce Cell-Based Cancer Immunotherapy.

Human pluripotent stem cells (PSCs) provide a promising resource to produce immune cells for adoptive cellular immunotherapy to better treat and potentially cure otherwise lethal cancers. Cytotoxic T cells and natural killer (NK) cells can now be routinely produced from human PSCs. These PSC-derived lymphocytes have phenotype and function similar to primary lymphocytes isolated from peripheral blood. PSC-derived T and NK cells have advantages compared with primary immune cells, as they can be precisely engineered to introduce improved anti-tumor activity and produced in essentially unlimited numbers. Stem Cells 2018;36:134-145.

Single Cell Resolution of Human Hematoendothelial Cells Defines Transcriptional Signatures of Hemogenic Endothelium.

Endothelial-to-hematopoietic transition (EHT) is an important stage in definitive hematopoietic development. However, the genetic mechanisms underlying human EHT remain poorly characterized. We performed single cell RNA-seq using 55 hemogenic endothelial cells (HECs: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c+ ), 47 vascular endothelial cells without hematopoietic potential (non-HE: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c- ), and 35 hematopoietic progenitor cells (HPCs: CD34+ CD43+ RUNX1c+ ) derived from human embryonic stem cells (hESCs). HE and HP were enriched in genes implicated in hemogenic endothelial transcriptional networks, such as ERG, GATA2, and FLI. We found transcriptional overlap between individual HECs and HPCs; however, these populations were distinct from non-HE. Further analysis revealed novel biomarkers for human HEC/HPCs, including TIMP3, ESAM, RHOJ, and DLL4. Collectively, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE are more heterogeneous and transcriptionally distinct. Our findings provide a novel strategy to test new genetic targets and optimize the production of definitive hematopoietic cells from human pluripotent stem cells. Stem Cells 2018;36:206-217.

Aryl hydrocarbon receptor inhibition promotes hematolymphoid development from human pluripotent stem cells.

The aryl hydrocarbon receptor (AHR) plays an important physiological role in hematopoiesis. AHR is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and inhibition of AHR results in a marked expansion of human umbilical cord blood-derived HSPCs following cytokine stimulation. It is unknown whether AHR also contributes earlier in human hematopoietic development. To model hematopoiesis, human embryonic stem cells (hESCs) were allowed to differentiate in defined conditions in the presence of the AHR antagonist StemReginin-1 (SR-1) or the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We demonstrate a significant increase in CD34+CD31+ hematoendothelial cells in SR-1-treated hESCs, as well as a twofold expansion of CD34+CD45+ hematopoietic progenitor cells. Hematopoietic progenitor cells were also significantly increased by SR-1 as quantified by standard hematopoietic colony-forming assays. Using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-engineered hESC-RUNX1c-tdTomato reporter cell line with AHR deletion, we further demonstrate a marked enhancement of hematopoietic differentiation relative to wild-type hESCs. We also evaluated whether AHR antagonism could promote innate lymphoid cell differentiation from hESCs. SR-1 increased conventional natural killer (cNK) cell differentiation, whereas TCDD treatment blocked cNK development and supported group 3 innate lymphoid cell (ILC3) differentiation. Collectively, these results demonstrate that AHR regulates early human hematolymphoid cell development and may be targeted to enhance production of specific cell populations derived from human pluripotent stem cells.

Frequent impact of [18F]fluorodeoxyglucose positron emission tomography on the staging and management of patients with indolent non-Hodgkin's lymphoma.

[18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) is useful in staging aggressive non-Hodgkin's lymphoma (NHL). However, its role in indolent NHL has not been established. This retrospective study assessed the sensitivity and clinical impact of PET findings in patients with indolent NHL. Patients with indolent NHL who underwent FDG-PET scanning between May 1997 and August 2001 were identified. Case records were reviewed for FDG-PET and conventional staging/restaging results and compared for concordance. Forty-seven patients were identified. Twelve staging FDG-PET scans and 37 restaging FDG-PET scans were obtained. The FDG-PET case sensitivity rate was 98%. Forty-two percent of staging FDG-PET scans were concordant with conventional staging, with the remaining patients exhibiting more extensive disease on PET. At progression, FDG-PET and conventional assessments were discordant in 46% of cases. Positron emission tomography findings downstaged disease in 30% of these patients and upstaged disease in 16%. Computed tomography (CT) and FDG-PET identified 150 and 146 individual sites of disease, respectively. Among "definite" sites on structural imaging, 74% were also seen on PET. For equivocal lesions, only 19% were seen on both modalities. Clinical management was changed in 34% of patients as a result of FDG-PET findings. Of 22 discordant lesions in which true disease status could be evaluated, the PET findings were confirmed to be correct in 21 (95%; P < 0.0001). These findings demonstrate that FDG-PET has a high sensitivity for indolent NHL and often leads to alteration of disease staging and management. This high accuracy of FDG-PET in assessing discordant lesions suggests a greater diagnostic utility compared with CT.