Human NK cells: From development to effector functions

NK cells are the major lymphocyte subset of the innate immune system that mediates antiviral and anti-tumor responses. It is well established that they develop mechanisms to distinguish self from non-self during the process of NK cell education. Unlike T and B cells, natural killer cells lack clonotypic receptors and are activated after recognizing their target via germline-encoded receptors through natural cytotoxicity, cytokine stimulation, and Ab-dependent cellular cytotoxicity. Subsequently, they utilize cytotoxic granules, death receptor ligands, and cytokines to perform their effector functions. In this review, we provide a general overview of human NK cells, as opposed to murine NK cells, discussing their ontogeny, maturation, receptor diversity, types of responses, and effector functions. Furthermore, we also describe recent advances in human NK cell biology, including tissue-resident NK cell populations, NK cell memory, and novel approaches used to target NK cells in cancer immunotherapy.

CISH targeting in NK cells activates natural cytotoxicity receptor signaling and reduce cell exhaustion to unsilence primary anti-tumor response

Cytokine inducible SH2-containing protein (CISH) is a natural killer (NK) cell negative regulator of cytokine signaling pathway. To further understand-CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cish fl/fl Ncr1Ki/+). We detected no developmental or homeostatic differences in NK cells. However, global gene expression of Cish fl/fl Ncr1Ki/+ NK cells compared to Cish +/+ Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH is not only regulating interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways. Indeed, CISH protein expression level increases upon NCR triggering. Cish fl/fl Ncr1Ki/+ primed-NK cells showed an increased activation upon NCR stimulation. Cish fl/fl Ncr1Ki/+ NK cells display lower activation thresholds and mice are more resistant to tumor metastasis. Remarkably, we found that Cish fl/fl Ncr1Ki/+ mice were also more resistant to primary breast cancer growth in addition to superior control of spontaneous tumor metastasis. CISH deletion favors NK cell accumulation to the tumor burden, optimizes NK cell killing properties and decreases TIGIT expression, an immune checkpoint receptor limiting also NK cell exhaustion. Finally, we argue that specifically enhancing NK cell function is sufficient to boost anti-tumor response to both primary and secondary tumor models, thus validating CISH as a key therapeutic target to enhance NK cell immunotherapy.

Natural cytotoxicity triggering receptor 3 is differentially expressed in lymph node metastasis in human breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). Between the breast and the brain reside the secondary lymphoid organ, the lymph nodes. We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes for the discovery of genes associated with metastasis to the lymph nodes in humans with metastatic breast cancer. We found that natural cytotoxicity triggering receptor 3, NCR3, was among the genes whose expression was most different in the lymph node metastases of patients with metastatic breast cancer as compared to primary tumors of the breast. Analysis of a separate microarray dataset revealed that NCR3 was also differentially expressed in brain metastatic tissues. NCR3 mRNA was present at increased quantities in lymph node metastases as compared to primary tumors of the breast. Importantly, expression of NCR3 in primary tumors of the breast was significantly correlated with patient overall survival, in lymph node negative patients but not in lymph node positive patients. Modulation of NCR3 expression may be relevant to the biology by which tumor cells metastasize from the breast to the lymph nodes and the brain in humans with metastatic breast cancer.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

Influenza A Virus Hemagglutinin and Other Pathogen Glycoprotein Interactions with NK Cell Natural Cytotoxicity Receptors NKp46, NKp44, and NKp30

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions.

The Diverse Functions of the Ubiquitous Fcγ Receptors and Their Unique Constituent, FcRγ Subunit

Fc gamma receptors (FcγRs) are widely expressed on a variety of immune cells and play a myriad of regulatory roles in the immune system because of their structural diversity. Apart from their indispensable role in specific binding to the Fc portion of antibody subsets, FcγRs manifest diverse biological functions upon binding to their putative ligands. Examples of such manifestation include phagocytosis, presentation of antigens, mediation of antibody-dependent cellular cytotoxicity, anaphylactic reactions, and the promotion of apoptosis of T cells and natural killer cells. Functionally, the equilibrium between activating and inhibiting FcγR maintains the balance between afferent and efferent immunity. The γ subunit of the immunoglobulin Fc receptor (FcRγ) is a key component of discrete immune receptors and Fc receptors including the FcγR family. Furthermore, FcγRs exert a key role in terms of crosslinking the innate and adaptive workhorses of immunity. Ablation of one of these receptors might positively or negatively influence the immune response. Very recently, we discovered that FcRγ derived from natural cytotoxicity triggering receptor 1 (NCR1) curtails CD8+ T cell expansion and thereby turns an acute viral infection into a chronic one. Such a finding opens a new avenue for targeting the FcγRs as one of the therapeutic regimens to boost the immune response. This review highlights the structural heterogeneity and functional diversity of the ubiquitous FcγRs along with their featured subunit, FcRγ.