MicroRNA-142 Is Critical for the Homeostasis and Function of Type 1 Innate Lymphoid Cells.

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-ß (TGF-ß) 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.

Memory NK Cells Take Out the (Mitochondrial) Garbage.

The molecular mechanisms important to generate innate natural killer cell "memory" are poorly understood. In this issue of Immunity, O'Sullivan et al. (2015) demonstrate that mitophagy plays a critical role in natural killer cell memory formation following viral infection.

Human Adaptive Natural Killer Cells: Beyond NKG2C.

Paradigm-shifting studies have identified NKG2C(+) adaptive natural killer (NK) cells in individuals infected with cytomegalovirus. Recently in Cell Reports, Liu et al. demonstrate that NKG2C(-/-) HCMV(+) individuals also generate adaptive NK cells, and reveal CD2 as a major co-stimulatory receptor for these NK cells specialized to respond via FcγRIIIa/CD16.

PTEN regulates natural killer cell trafficking in vivo.

Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide-3 kinase pathway, members of which play integral roles in natural killer (NK) cell development and function. However, the functions of PTEN in NK cell biology remain unknown. Here, we used an NK cell-specific PTEN-deletion mouse model to define the ramifications of intrinsic NK cell PTEN loss in vivo. In these mice, there was a significant defect in NK cell numbers in the bone marrow and peripheral organs despite increased proliferation and intact peripheral NK cell maturation. Unexpectedly, we observed a significant expansion of peripheral blood NK cells and the premature egress of NK cells from the bone marrow. The altered trafficking of NK cells from peripheral organs into the blood was due to selective hyperresponsiveness to the blood localizing chemokine S1P. To address the importance of this trafficking defect to NK cell immune responses, we investigated the ability of PTEN-deficient NK cells to traffic to a site of tumor challenge. PTEN-deficient NK cells were defective at migrating to distal tumor sites but were more effective at clearing tumors actively introduced into the peripheral blood. Collectively, these data identify PTEN as an essential regulator of NK cell localization in vivo during both homeostasis and malignancy.

Learning to Fly.

In this narrative medicine essay, an infectious diseases fellow understands while grieving her father that care cannot always be measured in cure but rather in listening and loving.

Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed Natural Killer Cell Antileukemia and FcγRIIIa-Triggered Responses.

Cytokine-induced memory-like natural killer (NK) cells differentiate after short-term preactivation with IL-12, IL-15, and IL-18 and display enhanced effector function in response to cytokines or tumor targets for weeks after the initial preactivation. Conventional NK cell function depends on a licensing signal, classically delivered by an inhibitory receptor engaging its cognate MHC class I ligand. How licensing status integrates with cytokine-induced memory-like NK cell responses is unknown. We investigated this interaction using killer cell immunoglobulin-like receptor- and HLA-genotyped primary human NK cells. Memory-like differentiation resulted in enhanced IFN-γ production triggered by leukemia targets or FcγRIIIa ligation within licensed NK cells, which exhibited the highest functionality of the NK cell subsets interrogated. IFN-γ production by unlicensed memory-like NK cells was also enhanced to a level comparable with that of licensed control NK cells. Mechanistically, differences in responses to FcγRIIIa-based triggering were not explained by alterations in key signaling intermediates, indicating that the underlying biology of memory-like NK cells is distinct from that of adaptive NK cells in human cytomegalovirus-positive individuals. Additionally, memory-like NK cells responded robustly to cytokine receptor restimulation with no impact of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy.

Transcriptional and post-transcriptional regulation of NK cell development and function.

Natural killer (NK) cells are specialized innate lymphoid cells that survey against viral infections and malignancy. Numerous advances have improved our understanding of the molecular mechanisms that control NK cell development and function over the past decade. These include both studies on the regulatory effects of transcription factors and translational repression via microRNAs. In this review, we summarize our current knowledge of DNA-binding transcription factors that regulate gene expression and thereby orchestrate NK cell development and activation, with an emphasis on recent discoveries. Additionally, we highlight our understanding of how RNA-binding microRNAs fine tune the NK cell molecular program. We also underscore the large number of open questions in the field that are now being addressed using new technological approaches and genetically engineered model organisms. Ultimately, a deeper understanding of the basic molecular biology of NK cells will facilitate new strategies to manipulate NK cells for the treatment of human disease.

Ultra-Deep Sequencing Reveals the Mutational Landscape of Classical Hodgkin Lymphoma.

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient.

T-BET and EOMES sustain mature human NK cell identity and antitumor function.

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. Single-cell 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 CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and 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.

A novel fusion protein scaffold 18/12/TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as a cellular immunotherapy for various malignancies. NK cells are particularly dependent on interleukin (IL)-15 for their survival, proliferation, and cytotoxic function. NK cells differentiate into memory-like cells with enhanced effector function after a brief activation with IL-12, IL-15, and IL-18. N-803 is an IL-15 superagonist composed of an IL-15 mutant (IL-15N72D) bound to the sushi domain of IL-15Rα fused to the Fc region of IgG1, which results in physiological trans-presentation of IL-15. Here, we describe the creation of a novel triple-cytokine fusion molecule, 18/12/TxM, using the N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to a heteromeric single-chain IL-12 p70 by the sushi domain of the IL-15Rα. This molecule displays trispecific cytokine activity through its binding and signaling through the individual cytokine receptors. Compared with activation with the individual cytokines, 18/12/TxM induces similar short-term activation and memory-like differentiation of NK cells on both the transcriptional and protein level and identical in vitro and in vivo anti-tumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cellular therapy.

Flow cytometry-based ex vivo murine NK cell cytotoxicity assay.

Direct killing of diseased cells is a hallmark function of NK cells. This protocol describes a flow-based assay to measure in vivo activated murine NK cells' ability to kill target cells ex vivo. Existing published protocols for assaying ex vivo NK cell killing utilized the radioactive chromium release assay or were designed for human NK cells. This protocol details specifically an ex vivo cytotoxicity assay using primary murine NK cells enriched from splenocytes that were activated in vivo with poly(I:C). For complete details on the use and execution of this protocol, please refer to Wagner et al. (2020).

Reliance on Cox10 and oxidative metabolism for antigen-specific NK cell expansion.

Natural killer (NK) cell effector functions are dependent on metabolic regulation of cellular function; however, less is known about in vivo metabolic pathways required for NK cell antiviral function. Mice with an inducible NK-specific deletion of Cox10, which encodes a component of electron transport chain complex IV, were generated to investigate the role of oxidative phosphorylation in NK cells during murine cytomegalovirus (MCMV) infection. Ncr1-Cox10Δ/Δ mice had normal numbers of NK cells but impaired expansion of antigen-specific Ly49H+ NK cells and impaired NK cell memory formation. Proliferation in vitro and homeostatic expansion were intact, indicating a specific metabolic requirement for antigen-driven proliferation. Cox10-deficient NK cells upregulated glycolysis, associated with increased AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) activation, although this was insufficient to protect the host. These data demonstrate that oxidative metabolism is required for NK cell antiviral responses in vivo.

Phase I Trial of N-803, an IL15 Receptor Agonist, with Rituximab in Patients with Indolent Non-Hodgkin Lymphoma.

PURPOSE: N-803 is an IL15 receptor superagonist complex, designed to optimize in vivo persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8+ T cells. Monoclonal antibodies (mAbs) direct Fc receptor-bearing immune cells, including NK cells, to recognize and eliminate cancer targets. The ability of IL15R agonists to enhance tumor-targeting mAbs in patients has not been reported previously. PATIENTS AND METHODS: Relapsed/refractory patients with indolent non-Hodgkin lymphoma were treated with rituximab and intravenous or subcutaneous N-803 on an open-label, dose-escalation phase I study using a 3+3 design (NCT02384954). Primary endpoint was maximum tolerated dose. Immune correlates were performed using multidimensional analysis via mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) which simultaneously measures protein and single-cell RNA expression. RESULTS: This immunotherapy combination was safe and well tolerated and resulted in durable clinical responses including in rituximab-refractory patients. Subcutaneous N-803 plus rituximab induced sustained proliferation, expansion, and activation of peripheral blood NK cells and CD8 T cells, with increased NK cell and T cells present 8 weeks following last N-803 treatment. CITE-seq revealed a therapy-altered NK cell molecular program, including enhancement of AP-1 transcription factor. Furthermore, the monocyte transcriptional program was remodeled with enhanced MHC expression and antigen-presentation genes. CONCLUSIONS: N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.

A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Potently Cytotoxic Natural Killer Cells Initially Emerge from Erythro-Myeloid Progenitors during Mammalian Development.

Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from Hoxaneg/low Kit+CD41+CD16/32+ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXAneg/low CD34+ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA+ CD34+ progenitors, as well as human cord blood CD34+ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.

Stage-Specific Requirement for Eomes in Mature NK Cell Homeostasis and Cytotoxicity.

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages.

Blood natural killer cell deficiency reveals an immunotherapy strategy for atopic dermatitis.

Atopic dermatitis (AD) is a widespread, chronic skin disease associated with aberrant allergic inflammation. Current treatments involve either broad or targeted immunosuppression strategies. However, enhancing the immune system to control disease remains untested. We demonstrate that patients with AD harbor a blood natural killer (NK) cell deficiency that both has diagnostic value and improves with therapy. Multidimensional protein and RNA profiling revealed subset-level changes associated with enhanced NK cell death. Murine NK cell deficiency was associated with enhanced type 2 inflammation in the skin, suggesting that NK cells play a critical immunoregulatory role in this context. On the basis of these findings, we used an NK cell-boosting interleukin-15 (IL-15) superagonist and observed marked improvement in AD-like disease in mice. These findings reveal a previously unrecognized application of IL-15 superagonism, currently in development for cancer immunotherapy, as an immunotherapeutic strategy for AD.

Multidimensional Analyses of Donor Memory-Like NK Cells Reveal New Associations with Response after Adoptive Immunotherapy for Leukemia.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell-treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK-cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. SIGNIFICANCE: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK-cell therapy. These findings identify multiple avenues for optimizing ML NK-cell immunotherapy for cancer and define mechanisms important for ML NK-cell function.This article is highlighted in the In This Issue feature, p. 1775.

CD56bright NK cells exhibit potent antitumor responses following IL-15 priming.

NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56bright NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56bright NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15-based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1-, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56bright compared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.

Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia.

Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell antileukemia potential is unclear. Cytokine-induced memory-like NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for weeks to months after preactivation. We hypothesized that memory-like NK cells exhibit enhanced antileukemia functionality. We demonstrated that human memory-like NK cells have enhanced interferon-γ production and cytotoxicity against leukemia cell lines or primary human AML blasts in vitro. Using mass cytometry, we found that memory-like NK cell functional responses were triggered against primary AML blasts, regardless of killer cell immunoglobulin-like receptor (KIR) to KIR-ligand interactions. In addition, multidimensional analyses identified distinct phenotypes of control and memory-like NK cells from the same individuals. Human memory-like NK cells xenografted into mice substantially reduced AML burden in vivo and improved overall survival. In the context of a first-in-human phase 1 clinical trial, adoptively transferred memory-like NK cells proliferated and expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.