Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1 , HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56 bright and CD56 dim NK cells. Finally, a donor with GATA2 T354M mutation exhibits reduced percentage of CD56 bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells.

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Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-b or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-g-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and highmolecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-b and dsRNA induced messenger RNA (mRNA) expression of BAFF (350-and 452-fold, respectively [n = 3]) and IP-10 (1,081-and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-b or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-b and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P , 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-b. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-b, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6 hours. Our results show that AF extracts suppressed expression of inflammatory cytokines in association with inhibition of the IFN-b signaling pathway and suppression of the formation of pSTAT1. Clinical RelevanceThe components of Aspergillus fumigatus extract that inhibit JAK-signal transducer and activator of transcription 1 (STAT1) signaling could impair antiviral immunity and could also contribute to the skewing of adaptive immune responses toward T helper (Th) 2 observed in Aspergillus-infected individuals by undermining the Th1 responses via inhibition of the activation of STAT1. We believe that the inhibitory effect on STAT1 activation could be exploited for beneficial effects once we better understand the identity of the factor and the molecular mechanism by which STAT1 phosphorylation is inhibited.

The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.