NFKB1 regulates human NK cell maturation and effector functions

doi:10.1016/j.clim.2016.11.012

Clinical Immunology

Volume 175, February 2017, Pages 99-108

https://doi.org/10.1016/j.clim.2016.11.012 Get rights and content

Highlights

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NK cells from patients with NFKB1 mutations show impaired peripheral maturation.
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NK cells from NFKB1 mutated patients show profound defects in key effector functions, ie cytotoxicity and IFN-γ production.
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NFKB1 mutated NK cells show a trend towards impaired IL-2 induced proliferation and NKp44 up-regulation.
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NFKB1 haploinsufficient patients show a high prevalence of viral infections.

Abstract

NFKB1, a component of the canonical NF-κB pathway, was recently reported to be mutated in a limited number of CVID patients. CVID-associated mutations in NFKB2 (non-canonical pathway) have previously been shown to impair NK cell cytotoxic activity. Although a biological function of NFKB1 in non-human NK cells has been reported, the role of NFKB1 mutations for human NK cell biology and disease has not been investigated yet. We decided therefore to evaluate the role of monoallelic NFKB1 mutations in human NK cell maturation and functions. We show that NFKB1 mutated NK cells present impaired maturation, defective cytotoxicity and reduced IFN-γ production upon in vitro stimulation. Furthermore, human IL-2 activated NFKB1 mutated NK cells fail to up-regulate the expression of the activating marker NKp44 and show reduced proliferative capacity. These data suggest that NFKB1 plays an essential novel role for human NK cell maturation and effector functions.

Introduction

The NF-κB (NF-kappaB; nuclear factor of kappa light polypeptide gene enhancer in B cells) signaling pathway plays an important role both in the innate and the adaptive immune system [1], [2], [3]. The NF-κB transcription factor family consists of five members: NF-κB1, NF-κB2, RelA, RelB and c-Rel. NFKB1 encodes the precursor p105 which is processed to the mature p50. NFKB2 encodes the precursor p100 and the mature p52. The canonical pathway, which includes NFKB1, mediates numerous immunological and inflammatory cellular responses, and may be activated upon stimulation with a broad range of stimuli, including proinflammatory cytokines, activation of innate immune receptors, T-cell receptor (TCR) and B-cell receptor (BCR) signaling, and others [1], [2], [3]. The non-canonical pathway, which involves NFKB2, has more restricted immunological functions mainly focusing on B cell homeostasis and is activated upon engagement of a limited set of members of the TNF receptor superfamily, including BAFF receptor, CD40 and the lymphotoxin receptor [2], [3], [4].

Data on the NF-κB involvement in NK cell function and maturation are limited. The first indirect description of the biological role of NF-κB in NK cells was derived from in vitro pharmacological NF-κB inhibition that led to impaired NK cell cytotoxicity [5]. Studies on Interleukin-2 (IL-2) induced NK cell functional stimulation have implicated the activation of NF-κB in both, cytotoxic activity and production of Interferon-gamma (IFN-γ) [6]. However, most of these studies did not investigate the role of individual components of the NF-κB signaling pathway. Observations in Nfkb1-deficient mice suggested that p50 is a negative regulator of NK cell proliferation and IFN-γ production [7].

Primary immunodeficiencies provide unique opportunities for a better understanding of the human immune system. For instance, congenital mutations in NIK [8] or NEMO [9], both of which encode upstream components of the NF-κB pathways, have demonstrated their importance for NK cell function. Monoallelic mutations in NFKB2, causing a functional haploinsufficiency due to expression of unprocessable p100 precursors have been reported in CVID patients [10], [11] and were shown to impair NK cell cytotoxic activity [12]. Recently, monoallelic mutations in NFKB1 leading to p50 haploinsufficiency have also been reported in three CVID families [13]. However, the impact of these NFKB1 mutations in human NK cell biology has not been investigated. In the present study, we analyzed NK cell maturation and effector functions in patients with NFKB1 mutations affecting the canonical NF-κB pathway.

Section snippets

Monoclonal antibodies

The fine characterization of surface markers of resting and IL-2 activated NK cells was performed using the following monoclonal antibodies (mAbs) generated in our laboratory (Department of Molecular and Translational Medicine, University of Brescia), or in the laboratory directed by A. Moretta (Laboratory of Molecular Immunology, DIMES, University of Genoa): BAB281 (IgG1, anti-NKp46); AZ20 (IgG1, anti-NKp30); ON72 (IgG1, anti-NKG2D); C127 and SUS142 (IgG1 and IgG2b respectively, anti-CD16);

Patients and novel NFKB1 mutations

Seven adult patients affected with CVID carrying monoallelic NFKB1 mutations were included in this study. Table 1 summarizes their clinical and immunological features. All patients presented with recurrent respiratory infections, both of the upper and the lower respiratory tract. None of the patients presented a history of mycobacterial disease. Lymphadenopathy and splenomegaly is a frequent feature of this cohort of patients (4/7 and 4/7 patients, respectively). Autoimmune manifestations

Discussion

The two NF-κB pathways, canonical and non-canonical, play an important role in immune cell biology. Although diverse in terms of components and type of activating stimuli, both pathways have been shown to play crucial roles in distinct biological functions of immune cells [1], [2], [3]. This diversity has been studied mainly in mouse models and mainly for B and T lymphocytes [1], [4]. Data on the involvement of NF-κB in NK cell function and maturation are limited. Regarding the NFKB1-dependent

Conclusions

In this study, we provide evidence that monoallelic NFKB1 mutations affect human NK cell homeostasis in terms of maturation and effector functions. NFKB1 mutated NK cells showed impaired peripheral maturation with a significant reduction of the CD56^dimCD57^hi, CD56^dimCD62L⁺ NK cell subsets, as well as altered expression pattern of NKp46, KIRs, CXCR1, CCCR7 and CD16. Furthermore, NFKB1 mutated NK cells showed functional impairments in terms of cytotoxicity, IFN-γ production, NKp44 up-regulation

Funding

The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7/2007–2013 under grant agreement no 201549 (EURO-PADnet HEALTH-F2-2008-201549) and from the Italian Ministerial Grant GR-2010-2315762. The research leading to these results also received funding from the “Fondazione C. Golgi”, Brescia, Italy and Associazione Immunodeficienze Primitive (A.I.P.). This study was supported by the German Federal Ministry of Education and Research (

Acknowledgments

We would like to thank Fondazione Camillo Golgi, Brescia, Italy and Associazione Immunodeficienze Primitive (A.I.P.). We would also like to thank Alessandro Moretta for providing monoclonal antibodies anti-NK cells receptors, produced in the Laboratory of Molecular Immunology, DIMES, University of Genoa, Italy. We would also like to thank the patients, the patients' families and the nurses for all their efforts. We thank Katrin Hübscher and Jessica Rojas-Restrepo for excellent technical

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Hematopoietic stem cell transplantation is currently planned for 4 patients with EBV-lymphoproliferative disease, refractory cytopenia with lymphoproliferation, and mycobacterial disease (AB.II.1, S.I.1, AP.I.1, and BL.II.1). Heterozygous NFKB1 mutations causing p50 haploinsufficiency have previously been reported to be associated with various phenotypes ranging from mere antibody deficiency to multiorgan autoinflammatory conditions.4-10 However, a comprehensive clinical description of the extended phenotype of the NF-κB1–related phenotype has been lacking.
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View full text

NFKB1 regulates human NK cell maturation and effector functions

Highlights

Abstract

Introduction

Section snippets

Monoclonal antibodies

Patients and novel NFKB1 mutations

Discussion

Conclusions

Funding

Acknowledgments

Blood

Am. J. Hum. Genet.

Blood

Trends Immunol.

Blood

Immunol. Lett.

Trends Immunol.

Blood

Unravelling the complexities of the NF-kappaB signalling pathway using mouse knockout and transgenic models

Oncogene

NF-kB, the first quarter century: remarkable progress and oustanding questions

Genes Dev.

NF-kB Iin immunobiology

Cell Res.

Functions of NF-kappaB1 and NF-kappaB2 in immune cell biology

Biochem. J.

Cellular redox influences both cytotoxic and NF-kappa B activation in natural killer cells

Immunology