Involvement of autophagy in NK cell development and function

ABSTRACT

Natural killer (NK) cells are the prototypical members of the recently identified family of innate lymphoid cells (ILCs). Thanks to their cytotoxic and secretory functions, NK cells play a key role in the immune response to cells experiencing various forms of stress, including viral infection and malignant transformation. Autophagy is a highly conserved network of degradative pathways that participate in the maintenance of cellular and organismal homeostasis as they promote adaptation to adverse microenvironmental conditions. The relevance of autophagy in the development and functionality of cellular components of the adaptive immune system is well established. Conversely, whether autophagy also plays an important role in the biology of ILC populations such as NK cells has long remained elusive. Recent experimental evidence shows that ablating Atg5 (autophagy-related 5, an essential component of the autophagic machinery) in NK cells and other specific ILC populations results in progressive mitochondrial damage, reactive oxygen species (ROS) overgeneration, and regulated cell death, hence interrupting ILC development. Moreover, disrupting the interaction of ATG7 with phosphorylated FOXO1 (forkhead box O1) in the cytosol of immature NK cells prevents autophagic responses that are essential for NK cell maturation. These findings suggest that activating autophagy may support the maturation of NK cells and other ILCs that manifest antiviral and anticancer activity.

Natural killer (NK) cells are large lymphocytes of the innate immune system endowed with the ability to recognize and destroy infected and transformed cells.^1,2 The robust cytotoxic activity of NK cells is governed by an array of surface receptors that deliver stimulatory or inhibitory signals. Whereas inhibitory NK cell receptors are mainly engaged by MHC class I molecules, the ligands of their stimulatory counterparts encompass a wide array of molecules that are specifically exposed on the surface of cells experiencing stressful conditions, including viral infection and malignant transformation.³ Upon activation, NK cells release cytotoxic granules containing—among various effectors—PRF1 (perforin 1), GZMA (granzyme A) and GZMB (granzyme B), which ultimately promote the demise of target cells. In addition, NK cells secrete a plethora of cytokines, including IFNG (interferon gamma) and TNF (tumor necrosis factor), as well as chemokines, such as CCL3 (chemokine [C-C motif] ligand 3), CCL4 and CCL5. These soluble mediators allow NK cells to modulate the activity of other components of the innate and adaptive immune system, including dendritic cells and CD8⁺ T lymphocytes.⁴ In humans, NK cells are typically defined as CD3⁻NCAM1/CD56⁺ cells, whereas in mice the traditional NK cell surface phenotypes are CD3⁻NCR1/NKp46⁺ or CD3⁻KLRB1C/NK1.1⁺, depending on the strain.⁵ Of note, NK cells are the main representatives of so-called innate lymphoid cells (ILCs), a recently identified component of the innate immune system with emerging key roles in tissue homeostasis and defense against viral and malignant threats.^6,7 Similar to other ILCs, NK cells originate from a common lymphoid progenitor owing to the activity of specific transcription factors that dictate cell lineage commitment. As they develop, ILCs express or downregulate several receptors that endow them with the ability to respond to prosurvival and differentiating factors. In particular, 3 main stages are observed during NK cell development: (1) NK cell precursors (NKPs), which upon stimulation with IL15/IL-15 (interleukin 15) differentiate toward (2) immature NK (iNK) and (3) mature NK (mNK) cells.⁸

Previous work has demonstrated that multiple lymphoid cell populations of the adaptive immune system, including T and B lymphocytes, depend on macroautophagy (herein referred to as autophagy) for differentiation, survival, proliferation and effector functions.^9-12 This is not surprising given the central position occupied by autophagy in the maintenance of homeostasis in physiologic conditions, as well as in the cellular adaptation to potentially harmful microenvironmental perturbations.¹³ However, the potential impact of autophagy on the biology of NK cells and other ILCs has long remained elusive. Recent experimental evidence uncovers a crucial function of autophagy not only in the regulation of NK cell development and survival, but also in the NK cell responses against infected cells.

Bone marrow-derived murine iNK cells exhibit several biomarkers of an ongoing autophagic response, including MAP1LC3B/LCB3 (microtubule-associated protein 1 light chain 3 β) lipidation and SQSTM1/p62 (sequestosome 1) degradation in baseline conditions, ¹⁴ as do rapidly proliferating murine mNK cells and group 1 ILCs during homeostatic expansion upon adoptive transfer into immunodeficient Rag2^−/−Il2rg^−/− mice.¹⁵ These observations suggest that autophagy may be involved in different steps of NK cell maturation and homeostatic proliferation. To obtain further insights into this possibility, mice with an NK cell-specific deletion of Atg5 were generated (Ncr1^creAtg5^flox/flox mice). These mice show a notable reduction in the numbers of circulating iNK and mNK cells as compared with their wild-type (WT) counterparts, yet exhibit normal amounts of peripheral T and B cells, as well as a normal number of lymphocytes.^14,15 Accordingly, NKPs isolated from Ncr1^creAtg5^flox/flox mice are unable to differentiate into iNK or mNK cells upon exposure to IL15, an effect that can be reproduced by silencing Atg3 or Atg7 with short-hairpin RNAs in WT NKPs.¹⁴ Moreover, adoptively transferred Atg5-deficient common lymphoid progenitors fail to generate mature type 2 and type 3 ILCs in the small intestine of recipient mice.¹⁵ Altogether, these observations demonstrate that the proper differentiation of NK cells and other ILCs critically depends on autophagy, at least in mice.

Autophagy-deficient NK cells accumulate damaged mitochondria and overproduce reactive oxygen species (ROS), which favors their demise via regulated cell death.^14,16 The cell-permeant ROS scavenger Tiron limits the death of autophagy-incompetent NK cells, providing a mechanistic link between Atg5 deletion and cell death. Furthermore, systemic induction of autophagy with the antidiabetic drug metformin increases the amount of CD8⁺ T cells, NKT cells, mNK cells and type 1 ILCs in a mouse model of lymphopenia, and such an effect critically depends on the presence of Atg5.¹⁵ Finally, IFNG secretion and cytotoxic activity are markedly impaired in NK cells from Ncr1^creAtg5^flox/flox mice exposed to IL12/IL-12 or IL18/IL-18 in vitro, as is the control of murine cytomegalovirus (MCMV) infection in vivo.^14,15 Thus, impaired NK cell development imposed by the Ncr1^cre Atg5^flox/flox genotype also affects the functional competence of mature NK cells.¹⁷ In a previous work, the inducible deletion of Atg3 in half of adoptively transferred mature Ly49H⁺ NK cells did not alter IFNG expression and early NK cell expansion in response to MCMV, but considerably affected the percentage of circulating NK cells at later time points (during the contraction phase).¹⁷ Taken together, these observations suggest that proficient autophagic responses may influence several aspects of NK cell biology (Fig. 1).

Figure 1.

Autophagy is activated at different stages of NK cell and ILC development. When autophagy is disabled through genetic deletion of Atg5 (autophagy related 5) or Atg7, or by disrupting the interaction of cytosolic FOXO1 (forkhead box 1) with ATG7, immature natural killer (iNK) cells are not able to eliminate damaged and dysfunctional mitochondria, which are a major source of reactive oxygen species (ROS). The consequent accumulation of ROS severely compromises iNK cell homeostasis as iNK cells succumb to ROS-driven regulated cell death and/or fail to develop and mature properly. Globally, this results in a numerically and functionally impaired mature NK cell compartment. N, nucleus.

To gain insights into the molecular mechanisms underpinning the regulation of autophagy during NK cell development, the status of MTOR (mechanistic target of rapamycin [serine/threonine kinase]—a protein kinase that controls proliferation and autophagy)¹⁸—was investigated. These studies revealed that MTOR and many of its substrates are hyperphosphorylated in iNK cells as compared with NKPs,¹⁴ pointing to a possible involvement of MTOR in NK cell development. Depletion of RPTOR (regulatory associated protein of MTOR, complex 1) or RICTOR (RPTOR independent companion of MTOR, complex 2), which results in the destabilization of 2 distinct MTOR-containing complexes (i.e., MTORC1 and MTORC2, respectively), as well as administration of the MTOR inhibitor rapamycin arrest NKP differentiation, but surprisingly no induction of autophagy is observed.¹⁴ Therefore, MTOR appears to participate in the development of NK cells but fails to directly regulate autophagy in this specific setting. These observations are in line with previous work demonstrating that NK cell development and effector functions critically rely on MTOR.¹⁹

Based on these findings, FOXO transcription factors (which regulate autophagy independent of MTOR signaling) were interrogated. Considerable levels of phosphorylated FOXO1 (forkhead box O1) are detected in the cytosol of iNK cells, whereas NKPs and mNK cells express low levels of this transcription factor.¹⁴ FOXO1 phosphorylation, which promotes its retention in the cytoplasm, modulates the expression of several FOXO1 target genes including Atg7 (which is normally repressed by FOXO1). Notably, the NK cell-specific deletion of Foxo1 (obtained in Ncr1^creFoxo1^flox/flox mice) abrogates NK cell development in vivo, as does the reconstitution of these mice with a non-excisable gene encoding a mutant form of FOXO1 in which 3 phosphorylation sites are rendered nonphosphorylatable (FOXO1^AAA).¹⁴ Both of these genetic interventions inhibit autophagy in iNK cells, hence favoring ROS overproduction and regulated cell death, a phenotype that can be rescued by the reintroduction of FOXO1. Corroborating the importance of autophagy for the development of a fully functional mature NK cell compartment, Ncr1^creFoxo1^flox/flox mice as well as mice with a knock-in for FOXO1^AAA are more susceptible to MCMV infection than their WT counterparts.¹⁴

Further experiments revealed that phosphorylated FOXO1 interacts with ATG7 on phagophores (the precursors to autophagosomes), the latter being the only protein that, among several autophagy regulators, scores as highly expressed in iNK cells as compared with NKPs.¹⁴ Remarkably, abolishing the association between FOXO1 and ATG7 upon deletion of a specific FOXO1 domain or Atg7 silencing impairs the activation of autophagy in iNK cells as well as the development of functional NK cells in mice.¹⁴ These findings suggest that the interaction of FOXO1 with ATG7 is required for proficient autophagic responses in iNK cells, and hence for proper NK cell maturation. In cancer cells exposed to stress conditions, FOXO1 is acetylated and thus binds ATG7 in the cytoplasm, an interaction that is disrupted by the deacetylation of FOXO1 by SIRT2 (sirtuin 2).²⁰ Thus, it will be interesting to elucidate the role of FOXO1 acetylating/deacetylating enzymes in the regulation of autophagy in the context of NK cell development and function.

Collectively, the observations discussed here above strongly support the contention that autophagy is crucial for the proper maturation of NK cells and other ILCs with antiviral and anticancer activity (Fig. 1). Although whether autophagy should be activated or inhibited in the context of cancer therapy is highly debated,²¹ it will be important to investigate the role of autophagic responses in NK cell-dependent anticancer immunosurveillance.

Abbreviations

FOXO1^AAA

nonphosphorylatable FOXO1

ILCs

innate lymphoid cells

iNK

immature NK

MCMV

murine cytomegalovirus

mNK

mature NK

NK

natural killer

NKPs

NK cell precursors

ROS

reactive oxygen species

WT

wild type

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.