C/Ebpg (CCAAT/Enhancer Binding Protein Gamma) Balances Cytotoxic and Secretory Potential of Natural Killer Cells
Authors: Lopes Izabela Aparecida , Kardosova Miroslava , Bianco Thiago Mantello , Arantes Adriana Queiroz , Silva Cleide Araújo , Scheucher Priscila Santos , Tenen Daniel G. , Lee Sanghoon , Rego Eduardo Magalhães , Welner Robert S. , Alberich-Jorda Meritxell , Figueiredo-Pontes Lorena Lobo 
Published: October 2019

Abstract
C/EBPs (CCAAT/enhance-binding proteins) are a family of transcription factors involved in a variety of hematopoietic processes, regulating both terminal differentiation and cellular proliferation. Among these, it was previously reported that C/EBP gamma (C/EBPg) has a role in the development of Natural Killer (NK) cells. However, the mechanisms of such regulation are unknown. NK cells are lymphocytes with effector functions of cytotoxicity and production of cytokines, both dependent on a dynamic equilibrium between the expression of activating and inhibitory receptors as well as cytokine receptors. The two functions (cytotoxic and secretory) make NK cells important components of hematopoiesis, able to eliminate susceptible targets as well as recruit other cells to amplify inflammatory responses. With the aim of studying the regulation of NK cells by C/EBPg, we isolated NK cells from transgenic Cebpg knockout (KO) mice and controls to analyze their function. To characterize NK cells, we analyzed their frequency (Lineage-/CD3-/NK1.1+ cells) and the expression of the receptors NKG2D, Ly49D and NKG2A by flow cytometry of splenocytes. Both analyses showed no difference between control or Cebpg KO NK cells. Although the numbers of NK cells and their receptors were similar between Cebpg WT and KO animals, a functional assay that measured NK cell degranulation by CD107a expression after co-incubation with YAC-1 target cells showed that the expression of this marker was 5-times lower in Cebpg KO splenocytes than in controls (CT = 12.44 ± 2.50%; KO = 2.255 ± 0.67%, p=0.007), suggesting that Cebpg deficient NK cells are not fully activated after target cell recognition. In addition, a cytotoxicity assay by flow cytometry was performed using a fluorescent probe (Cell Tracker Orange) that was incorporated to YAC-1 cells upon exposure to sorted and IL-2 activated NK cells in culture. In the 10:1 NK:target cells ratio, Cebpg KO cells were significantly less cytotoxic than NK control cells (CT = 23.36 ± 8.67%; KO = 10.60 ± 1.66%, p=0.038). The other NK:target cells ratios of 5:1 and 1:1 showed the same tendency. In addition, the functional subtypes of these cells were characterized according to the expression of CD27 and CD11b, which allowed the identification of NK subpopulations as immature secretory, mature secretory, cytotoxic or tolerant. The KO animals showed higher percentages of secretory cells (CT = 10.77 ± 5.38%; KO = 12.98 ± 13.63%, p=0.0002) and a reduction of cytotoxic cells in comparison to the NK control cells (CT = 12.22 ± 11.08%; KO = 10.65 ± 3.82% p=0.013). Cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17α, TNFα and IFNγ, obtained from NK culture supernatants, were measured by flow cytometry, after IL-2 activation. Among these cytokines, the production of IFNγ by Cebpg-deficient NK cells was reduced (CT = 37.68 ± 0.51 pg/mL; KO = 22.34 ± 0.14 pg/mL, p=0.023). Together, these experiments indicate that C/EBPg regulates NK cell cytotoxicity. This may be explained, at least in part, by the reduced frequency of the mature cytotoxic NK subpopulation as compared to the secretory subtypes. Moreover, IFNγ may be an important target for the regulation of NK cell function. Finally, C/EBPg seems to be critical to mediate NK cell functions and not only for their development from the ontogenetic point of view.

Disclosures
No relevant conflicts of interest to declare.