cells Review Implications of a ‘Third Signal’ in NK Cells Mohamed Khalil 1,2 , Dandan Wang 1,2, Elaheh Hashemi 1,2, Scott S. Terhune 2,* and Subramaniam Malarkannan 1,2,3,4,* 1 Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, Versiti, Milwaukee, WI 53226, USA; [email protected] (M.K.); [email protected] (D.W.); [email protected] (E.H.) 2 Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226, USA 3 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA 4 Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA * Correspondence: [email protected] (S.S.T.); [email protected] (S.M.) Abstract: Innate and adaptive immune systems are evolutionarily divergent. Primary signaling in T and B cells depends on somatically rearranged clonotypic receptors. In contrast, NK cells use germline-encoded non-clonotypic receptors such as NCRs, NKG2D, and Ly49H. Proliferation and effector functions of T and B cells are dictated by unique peptide epitopes presented on MHC or soluble humoral antigens. However, in NK cells, the primary signals are mediated by self or viral proteins. Secondary signaling mediated by various cytokines is involved in metabolic reprogramming, proliferation, terminal maturation, or memory formation in both innate and adaptive lymphocytes. The family of common gamma (γc) cytokine receptors, including IL-2Rα/β/γ, IL- 7Rα/γ, IL-15Rα/β/γ, and IL-21Rα/γ are the prime examples of these secondary signals. A distinct set of cytokine receptors mediate a ‘third’ set of signaling. These include IL-12Rβ1/β2, IL-18Rα/β, β α IL-23R, IL-27R (WSX-1/gp130), IL-35R (IL-12R 2/gp130), and IL-39R (IL-23R /gp130) that can prime, activate, and mediate effector functions in lymphocytes. The existence of the ‘third’ signal is Citation: Khalil, M.; Wang, D.; known in both innate and adaptive lymphocytes. However, the necessity, context, and functional Hashemi, E.; Terhune, S.S.; relevance of this ‘third signal’ in NK cells are elusive. Here, we define the current paradigm of the Malarkannan, S. Implications of a ‘third’ signal in NK cells and enumerate its clinical implications. ‘Third Signal’ in NK Cells. Cells 2021, 10, 1955. https://doi.org/ Keywords: NK cells; Natural Killer cells; NCR—Natural killer cell receptor; IL—interleukin; IFN-γ— 10.3390/cells10081955 interferon gamma; MHC—major histocompatibility complex Academic Editor: Yoshimasa Tanaka Received: 6 July 2021 Accepted: 27 July 2021 1. Introduction Published: 31 July 2021 Natural Killer (NK) cells protect hosts by eliminating pathogens and transformed malignant cells. NK cells are the major subgroup of innate lymphoid cells (ILCs) [1]. The Publisher’s Note: MDPI stays neutral existence of this non-T cell subset was observed in the early 1960s [2,3]. NK cells were with regard to jurisdictional claims in formally characterized as an independent lymphocytic lineage in the mid-1970s [4–7]. published maps and institutional affil- NK cells differentiate ‘self ’ healthy cells against the infected or transformed cells through iations. unique mechanisms. The healthy cells, by virtue of expressing host MHC class I molecules on the cell surface are defined as ‘immunological self ’, which are recognized by inhibitory human killer cell immunoglobulin-like receptors (KIRs) or murine Ly49 receptors initiating negative signaling that trigger membrane-proximal phosphatases to block any activation Copyright: © 2021 by the authors. effectively. Virus-infected cells often down-regulate MHC class I molecules to avoid the Licensee MDPI, Basel, Switzerland. attack from CD8+ T cells [8]. Nevertheless, those cells are particularly vulnerable to NK This article is an open access article cell-mediated clearance and are considered the immunological ‘missing-self ’[9]. Viral distributed under the terms and infections often lead to the expression of ligands that are typically absent on healthy conditions of the Creative Commons cells [10]. These stress-induced ligands serve as the ‘danger signals’ and interact with Attribution (CC BY) license (https:// activating receptors on NK cells to overrule the negative signaling initiated by MHC class I creativecommons.org/licenses/by/ molecules, the immunological ‘induced-self ’[10]. In recent years significant progress has 4.0/). Cells 2021, 10, 1955. https://doi.org/10.3390/cells10081955 https://www.mdpi.com/journal/cells CellsCells2021 2021, 10, 10, 1955, 1955 2 ofof 27 29 beenmade made in understanding in understanding the molecular the molecular basis basisfor these for thesedistinct distinct mechanisms mechanisms operated operated by NK bycells. NK Recent cells. Recent discoveries discoveries have also have started also started to reveal to reveal the critical the critical role of role NK of cells NK cellsin anti in‐ anti-inflammatoryinflammatory responses, responses, tissue tissue repair, repair, and and autoimmunity autoimmunity [11]. [ 11Irrespective]. Irrespective of this of thispro‐ progress,gress, a critical a critical knowledge knowledge gap gap exists exists in defining in defining the basic the basic biology biology of NK of cells NK cellsthat is that es‐ issential essential to realize to realize their their clinical clinical advantages. advantages. One of One these of gaps these relates gaps relates to distinctive to distinctive mecha‐ mechanismsnisms of NK of cell NK activation. cell activation. NKNK cells cells possess possess strong strong functional functional similarities similarities to to their their distant distant CD8 CD8+ +T T cell cell cousins. cousins. NK NK cellscells mediate mediate granzyme/perforin, granzyme/perforin, FasL,FasL, andand antibody-dependentantibody‐dependent cell cell cytotoxicitycytotoxicity (ADCC). (ADCC). TheyThey also also produce produce significant significant quantities quantities of pro-inflammatory of pro‐inflammatory cytokines cytokines or chemokines or chemokines such assuch IFN- asγ ,IFN GM-CSF,‐, GM‐ TNF-CSF, αTNF, CCL3,‐, CCL3, CCL4, CCL4, and CCL5. and CCL5. However, However, unlike unlike CD8+ CD8T cells,+ T cells, NK cellsNK cancells mediate can mediate cytotoxicity cytotoxicity without without prior prior exposure exposure to antigens, to antigens, termed termed ‘priming’ ‘priming’ [4,7]. In[4,7]. addition, In addition, NK cells NK use cells non-clonotypic use non‐clonotypic receptors receptors as their as their primary primary signal signal conduits conduits for activation.for activation. This This inherent inherent ability ability of NK of cells NK tocells utilize to utilize non-clonotypic non‐clonotypic receptors receptors to recognize to rec‐ proteinognize ligandsprotein onligands target on cells target requires cells tightly requires balanced tightly activatingbalanced activating and inhibitory and signalinginhibitory pathways.signaling pathways. However, weHowever, have yet we to have define yet how to define NK cells how diverge NK cells from diverge T cells from in signaling T cells in requirementssignaling requirements in this context. in this Additionally,context. Additionally, NK cells NK communicate cells communicate with dendritic with dendritic cells (DCs),cells (DCs), macrophages, macrophages, neutrophils, neutrophils, and T cells and throughT cells through direct cell-cell direct cell interactions‐cell interactions or soluble or factorssoluble (Figure factors1 ).(Figure 1). FigureFigure 1. 1. Bi-directionalBi‐directional interaction of NK cells with with other other immune immune cells. cells. A A brief brief representation representation of ofNK NK cells’ cells’ significant significant interactions interactions and and the the innate innate (myeloid (myeloid-derived)‐derived) and and adaptive adaptive (T and (T and B cell) B cell) im‐ immunemune system. system. Myeloid Myeloid-derived‐derived cells cells (dendritic (dendritic cells, cells, macrophages, macrophages, and neutrophils) and neutrophils) secrete secretevarious cytokines and chemokines, regulating NK cell effector functions. NK cells secrete cytokines and various cytokines and chemokines, regulating NK cell effector functions. NK cells secrete cytokines chemokines (IFN‐γ, TNF‐a, GM‐CSF, CCL‐3, CCL‐4, and CCL‐5) to regulate lymphoid‐derived cells and chemokines (IFN-γ, TNF-a, GM-CSF, CCL-3, CCL-4, and CCL-5) to regulate lymphoid-derived (T and B cells). cells (T and B cells). ActivationActivation viavia TCR, TCR, BCR, BCR, or or non-clonotypic non‐clonotypic receptors receptors such such as as NCRs, NCRs, NKG2D, NKG2D, and and Ly49HLy49H is is ‘primary ‘primary signaling’ signaling’ that that depends depends on on Src Src kinases kinases and and PLC- PLCγ‐-based‐based pathways.pathways. CostimulatoryCostimulatory signals signals via via cytokine cytokine receptors receptors (IL-2, (IL IL-15)‐2, IL‐15) and and immune-checkpoint immune‐checkpoint proteins pro‐ (CD19,teins (CD19, CD28, CD28, NKG2D) NKG2D) form the form basis the for basis the for ‘second the ‘second signal’, signal’, which is which essential is essential to recruit to and activate PI(3)K-based signaling. Other modes of NK cell activation may involve Cells 2021, 10, 1955 3 of 27 soluble factors such as PDGF-D [12] that binds to NCR2 or cell-free ligands of NKG2D [13]. Activation through the receptors for Type I IFN-α/β, IL-12, IL-18, IL-21, IL-23, IL-27, IL-35, and IL-39 form a distinct set of signaling that constitutes a ‘third signal’. This distinct activation set largely depends on STATs and TRAFs as the conduits of signal transduction [14]. STAT4 downstream