In an adaptive immune response, naive T cells proliferate during infection and generate long-lived memory cells that undergo secondary expansion after a repeat encounter with the same pathogen. Although natural killer (NK) cells have traditionally been classified as cells of the innate immune system, they share many similarities with cytotoxic T lymphocytes. We use a mouse model of cytomegalovirus infection to show that, like T cells, NK cells bearing the virus-specific Ly49H receptor proliferate 100-fold in the spleen and 1,000-fold in the liver after infection. After a contraction phase, Ly49H-positive NK cells reside in lymphoid and non-lymphoid organs for several months. These self-renewing 'memory' NK cells rapidly degranulate and produce cytokines on reactivation. Adoptive transfer of these NK cells into naive animals followed by viral challenge results in a robust secondary expansion and protective immunity. These findings reveal properties of NK cells that were previously attributed only to cells of the adaptive immune system.

Adaptive Immune Features of Natural Killer Cells

Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.

/pdf/leveraging-nkg2d-ligands-in-immuno-oncology-2mh6z898et.pdf

Leveraging NKG2D Ligands in Immuno-Oncology

Treatment of cancer with allogeneic natural killer (NK) cell therapies has seen rapid development, especially use against hematologic malignancies. Clinical trials of NK cell-based adoptive transfer to treat relapsed or refractory malignancies have used peripheral blood, umbilical cord blood and pluripotent stem cell-derived NK cells, with each approach undergoing continued clinical development. Improving the potency of these therapies relies on genetic modifications to improve tumor targeting and to enhance expansion and persistence of the NK cells. Induced pluripotent stem cell (iPSC)-derived NK cells allow for routine targeted introduction of genetic modifications and expansion of the resulting NK cells derived from a clonal starting cell population. In this review, we discuss and summarize recent important advances in the development of new iPSC-derived NK cell therapies, with a focus on improved targeting of cancer. We then discuss improvements in methods to expand iPSC-derived NK cells and how persistence of iPSC-NK cells can be enhanced. Finally, we describe how these advances may combine in future NK cell-based therapy products for the treatment of both hematologic malignancies and solid tumors.

/pdf/ipsc-derived-natural-killer-cell-therapies-expansion-and-19rijhlh.pdf

iPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting

EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity.

Frailty is an intermediate status of the human aging process, associated with decompensated homeostasis and death. The immune phenotype of frailty and its underlying cellular and molecular processes remain poorly understood. We profiled 114,467 immune cells from cord blood, young adults and healthy and frail old adults using single-cell RNA and TCR sequencing. Here we show an age-dependent accumulation of transcriptome heterogeneity and variability in immune cells. Characteristic transcription factors were identified in given cell types of specific age groups. Trajectory analysis revealed cells from non-frail and frail old adults often fall into distinct paths. Numerous TCR clonotypes were shared among T-cell subtypes in old adults, indicating differential pluripotency and resilience capabilities of aged T cells. A frailty-specific monocyte subset was identified with exclusively high expression of long noncoding RNAs NEAT1 and MALAT1. Our study discovers human frailty-specific immune cell characteristics based on the comprehensive dimensions in the immune landscape of aging and frailty. 

Multidimensional single-cell analysis of human peripheral blood reveals characteristic features of the immune system landscape in aging and frailty

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy

Immune exhaustion in T cells significantly impacts their ability to control malignancies and infections, and its discovery has led to revolutionary therapies for cancer in the form of checkpoint blockade. NK cells, like T cells, are lymphocytes that recognize virally infected and malignantly transformed cells. However, it remains unclear if NK cells are similarly susceptible to exhaustion. In this review, the aims are to summarize what is currently known and to identify key areas of variability that skew the scientific literature on NK cell exhaustion. A lack of consensus on the defining features of NK cell dysfunctional states such as senescence, suppression, and exhaustion has made a comparison between studies difficult. There are also significant differences in the biology of NK cell subsets with long-lived, adaptive NK cells sharing an epigenetic signature closer to memory CD8+  T cells than to conventional NK cells. Very different checkpoint receptor expression and effector functions have been shown in adaptive versus conventional NK cells chronically exposed to activating signals. Adaptive NK cells develop in individuals with cytomegalovirus (CMV) infection and well over half of the human population worldwide is CMV seropositive by adulthood. Despite this high prevalence, most studies do not account or control for this population. This may contribute to some of the variability reported in the literature on checkpoint receptor expression on NK cells. In this review, the protective role that exhaustion plays in T cells will also be discussed and the evidence for a similar phenomenon in NK cells will be examined.

Unraveling exhaustion in adaptive and conventional NK cells.

Variegated expression of killer Ig-like receptors (KIR) in human NK cells is a stochastic process exclusive to subsets of mature NK cells and CD8+ T cells. Allele-specific KIR expression is maintained by DNA methylation within the proximal promoter regions. Because KIR genes are densely methylated in NK cell progenitors, there is an implied stage of human NK cell development in which DNA demethylation takes place to allow for active transcription. When and how this process occurs is unknown. In this study, we show that KIR proximal promoters are densely methylated in less mature CD56bright NK cells and are progressively demethylated in CD56dim NK cells as they mature and acquire KIR. We hypothesized that ten-eleven translocation (TET) enzymes, which oxidize 5mC on DNA could mediate KIR promoter demethylation. The catalytic efficiency of TET enzymes is known to be enhanced by ascorbic acid. We found that the addition of ascorbic acid to ex vivo culture of sorted CD56bright NK cells increased the frequency of KIR expression in a dose-dependent manner and facilitated demethylation of proximal promoters. A marked enrichment of the transcription factor Runx3 as well as TET2 and TET3 was observed within proximal KIR promoters in CD56bright NK cells cultured with ascorbic acid. Additionally, overexpression of TET3 and Runx3 promoted KIR expression in CD56bright NK cells and NK-92 cells. Our results show that KIR promoter demethylation can be induced in CD56bright, and this process is facilitated by ascorbic acid.

Ascorbic Acid Promotes KIR Demethylation during Early NK Cell Differentiation.

Adaptive natural killer (NK) cells are long-lived and exhibit properties of immunologic memory against cytomegalovirus (CMV). We previously reported that expansion of adaptive NK cells after CMV reactivation in recipients of allogeneic hematopoietic cell transplantation (HCT) was associated with a lower rate of relapse of acute myelogenous leukemia. In the present study, we examined the impact of adaptive NK cell expansion in a cohort of 110 individuals who underwent autologous HCT (AHCT) for a lymphoid malignancy (lymphoma or multiple myeloma [MM]). In this cohort, higher absolute numbers of adaptive NK cells (>1.58/μL) at day 28 post-AHCT were associated with significantly decreased risk of relapse in patients with MM. No significant association was seen in patients with lymphoma. Further stratification of MM patients by CMV serostatus found a strong protective effect of adaptive NK cells only in CMV-seropositive individuals. These findings suggest that strategies to increase adaptive NK cells after AHCT may be a therapeutic option in patients with MM.

Hansol Kim

Papers

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy

Unraveling exhaustion in adaptive and conventional NK cells.

Ascorbic Acid Promotes KIR Demethylation during Early NK Cell Differentiation.

Early Adaptive Natural Killer Cell Expansion Is Associated with Decreased Relapse After Autologous Transplantation for Multiple Myeloma.