IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

CD45 limits early Natural Killer cell development.

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumor cells. To fully harness the tumor killing ability of NK cells, we need to improve NK cell persistence and to overcome suppression of NK cell activation in the tumor microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with the genetic loss of CD45 in mice resulting in increased numbers of mature NK cells. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, we demonstrate here that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development, compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance.

Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells, and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generate mice that specifically lack Hhex in NK cells. NK cell frequency is dramatically reduced, while NK cell differentiation, IL-15 responsiveness, and function at the cellular level remain largely normal in the absence of Hhex. Increased IL-15 availability fails to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches reveal that Hhex regulates NK cell survival by directly binding Bcl2l11 (Bim) and repressing expression of this key apoptotic mediator. These data implicate Hhex as a transcriptional regulator of NK cell homeostasis and immunity.

NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS.

Natural killer (NK) cell activation is controlled by a balance of activating and inhibitory signals and cytokines such as IL-15. We previously identified cytokine-inducible SH2-containing protein (CIS) as a negative regulator of IL-15 signaling in NK cells under inflammatory conditions. While the functional effect of Cish-deficiency in NK cells was obvious by their increased anti-tumor immunity and hyper-proliferative response to IL-15, it remained unclear how CIS regulates NK cell biology in steady-state. Here, we investigated the role of CIS in the homeostatic maintenance of NK cells and found CIS-ablation promoted terminal differentiation of NK cells and increased turnover, suggesting that under steady-state conditions, CIS plays a role in maintaining IL-15 driven regulation of NK cells in vivo. However, hyper-responsiveness to IL-15 did not manifest in NK cell accumulation, even when the essential NK cell apoptosis mediator, Bcl2l11 (BIM) was deleted in addition to Cish. Instead, loss of CIS conferred a lower activation threshold, evidenced by augmented functionality on a per cell basis both in vitro and in vivo without prior priming. We conclude that Cish regulates IL-15 signaling in NK cells in vivo, and through the rewiring of several activation pathways leads to a reduction in activation threshold, decreasing the requirement for priming and improving NK cell anti-tumor function. Furthermore, this study highlights the tight regulation of NK cell homeostasis by several pathways which prevent NK cell accumulation when IL-15 signaling and intrinsic apoptosis are dysregulated.

Hhex regulates murine lymphoid progenitor survival independently of Stat5 and Cdkn2a.

The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development.

Generation of novel Id2 and E2-2, E2A and HEB antibodies reveals novel Id2 binding partners and species-specific expression of E-proteins in NK cells.

NK cells are cytotoxic lymphocytes with a key role in limiting tumour metastases. In mice, the NK cell lineage continually expresses high levels of the Inhibitor of DNA-binding 2 (Id2) protein and loss of Id2 is incongruous with their survival due to aberrant E-protein target gene activity. Using novel Id2 and E-protein antibodies that detect both mouse and human proteins, we have extensively characterised Id2 and E-protein expression in murine and human NK cells. We detected clear expression of E2 A and HEB, and to a lesser extent E2-2 in murine NK cells. In contrast HEB appears to be the major E-protein expressed in human NK cells, with minor E2-2 expression and surprisingly, no E2 A detected in primary NK cells nor human NK cell lines. These novel antibodies are also functional in immunofluorescence and immunoprecipitation. Mass spectrometry analysis of Id2 immuno-precipitated from murine NK cells revealed a number of novel associated proteins including several members of the SWI/SNF-related matrix-associated actin-dependent regulator chromatin (SMARC) and Mediator complex (MED) families. Taken together, these data highlight the utility of novel Id2 and E-protein antibodies and caution against mouse models for understanding Id2/E-protein biology in NK cells given their clearly disparate expression patternbetween species.

Recipient BCL2 inhibition and NK cell ablation form part of a reduced intensity conditioning regime that improves allo-bone marrow transplantation outcomes.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is used to treat over 15,000 patients with acute myeloid leukemia (AML) per year. Donor graft-versus-leukemia (GVL) effect can prevent AML relapse; however, alloSCT is limited by significant toxicity related to conditioning intensity, immunosuppression, opportunistic infections, and graft-versus-host disease (GVHD). Reducing the intensity of conditioning regimens prior to alloSCT has improved their tolerability, but does not alter the pattern of GVHD and has been associated with increased rates of graft rejection and relapse. Here, using a murine pre-clinical model, we describe a novel recipient conditioning approach combining reduced intensity conditioning with either genetic or pharmacological inhibition of NK cell numbers that permits efficient donor engraftment and promotes GVL without inducing GVHD. We show that NK cell-specific deletion of Bcl2 or Mcl1 in mice, or pharmacological inhibition of BCL2 impairs radio-resistant NK cell-mediated rejection of allogeneic engraftment and allows reduction of conditioning intensity below that associated with GVHD priming. The combination of reduced intensity conditioning and NK cell targeting in mice allowed successful donor T cell engraftment and protective immunity against AML while avoiding GVHD. These findings suggest that reduced conditioning in combination with targeted therapies against recipient NK cells may allow the delivery of effective alloSCT against AML while reducing the toxicities associated with more intensive conditioning including GVHD.

Regulation of Murine Natural Killer Cell Development.

Natural killer (NK) cells are effector lymphocytes of the innate immune system that are known for their ability to kill transformed and virus-infected cells. NK cells originate from hematopoietic stem cells in the bone marrow, and studies on mouse models have revealed that NK cell development is a complex, yet tightly regulated process, which is dependent on both intrinsic and extrinsic factors. The development of NK cells can be broadly categorized into two phases: lineage commitment and maturation. Efforts to better define the developmental framework of NK cells have led to the identification of several murine NK progenitor populations and mature NK cell subsets, each defined by a varied set of cell surface markers. Nevertheless, the relationship between some of these NK cell subsets remains to be determined. The classical approach to studying both NK cell development and function is to identify the transcription factors involved and elucidate the mechanistic action of each transcription factor. In this regard, recent studies have provided further insight into the mechanisms by which transcription factors, such as ID2, FOXO1, Kruppel-like factor 2, and GATA-binding protein 3 regulate various aspects of NK cell biology. It is also becoming evident that the biology of NK cells is not only transcriptionally regulated but also determined by epigenetic alterations and posttranscriptional regulation of gene expression by microRNAs. This review summarizes recent progress made in NK development, focusing primarily on transcriptional regulators and their mechanistic actions.

Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival.

Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.