Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.     

IRX-2, a novel immunotherapeutic, enhances and protects NK-cell functions in cancer patients

Background

IRX-2 is a primary biologic which has been used for the therapy of head and neck squamous cell cancer (HNSCC) with promising clinical results. Since NK-cell function is compromised in HNSCC patients, we tested the effects of IRX-2 on the restoration of human NK-cell functions in vitro.

Methods

Peripheral blood mononuclear cells (PBMC) were isolated from 23 HNSCC patients and 10 normal controls (NC). The NK-cell phenotype and functions were compared before and after culture?±?IRX-2 or?±?50?IU/ml rhIL-2. Flow cytometry was used to study the NK-cell phenotype, cytotoxic activity and cytokine expression.

Results

Impaired NK-cell cytotoxicity in HNSCC patients was related to lower expression of NKG2D, NKp30 and NKp46 receptors (P?P?P?P?P?Conclusions IRX-2 was more effective than IL-2 in enhancing NK-cell cytotoxicity and protecting NK-cell function of HNSCC patients in vitro, emphasizing the potential advantage of IRX-2 as a component of future therapies for HNSCC.     

Developmental pathways that generate natural-killer-cell diversity in mice and humans

Natural killer (NK) cells are large granular lymphocytes capable of producing inflammatory cytokines and spontaneously killing malignant, infected or 'stressed' cells. These NK-cell functions are controlled by cell-surface receptors that titrate stimulatory and inhibitory signals. However, we remain puzzled about where and when NK cells develop and differentiate, and this has fuelled the debate over the diversification of the peripheral NK-cell pool: are NK cells functionally homogeneous or are there subsets with specialized effector functions? In this Review, we consider the developmental relationships and biological significance of the diverse NK-cell subsets in mice and humans, and discuss how new humanized mouse models may help to characterize them further.     

CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation

Natural killer (NK) cells are part of the first line defense against tumors, parasites and virus-infected cells. Therefore, factors that control NK-cell numbers and their function are important. CD27 is constitutively expressed on NK cells and its expression correlates with sequential phases in NK-cell development, discriminating phenotypically and functionally different subsets within the NK-cell population. Although CD27 has been described to have an important regulatory role in effector and memory T and B lymphocytes, its role in NK-cell biology remains to be addressed. In this study, we used CD27(-/-) mice to investigate the role of CD27 in NK-cell development and function, both during the resting state and upon stimulation. The results show that NK-cell numbers are not impaired in CD27(-/-) mice. Moreover, CD27(-/-) NK cells reach full phenotypic maturity, evidenced by normal expression of CD49b, CD43 and CD11b. Expression of activating receptors is unaltered, whereas expression of several inhibitory receptors is increased. Cytotoxicity and interferon-γ production by NK cells from CD27(-/-) mice in the resting state are normal. However, upon in vivo anti-CD40- or poly-I:C-mediated activation, or in vitro interleukin-15 priming plus anti-NKp46 stimulation, the absence of CD27 results in decreased cytolytic activity and cytokine production by spleen and liver NK cells. In conclusion, this study demonstrates that CD27 is dispensable for the development of functional NK cells. However, upon stimulation of NK cells, CD27 displays an important role in their activation and functionality.     

Self-tolerance of natural killer cells

Natural killer (NK) cells, similar to other lymphocytes, acquire tolerance to self. This means that NK cells have the potential to attack normal self cells but that there are mechanisms to ensure that this does not usually occur. Self-tolerance is acquired by NK cells during their development, but the underlying molecular and cellular mechanisms remain poorly understood. Recent studies have produced important new information about NK-cell self-tolerance. Here, we review the evidence for and against possible mechanisms of NK-cell self-tolerance, with an emphasis on the role of MHC-specific receptors.     

Human-specific evolution of killer cell immunoglobulin-like receptor recognition of major histocompatibility complex class I molecules

In placental mammals, natural killer (NK) cells are a population of lymphocytes that make unique contributions to immune defence and reproduction, functions essential for survival of individuals, populations and species. Modulating these functions are conserved and variable NK-cell receptors that recognize epitopes of major histocompatibility complex (MHC) class I molecules. In humans, for example, recognition of human leucocyte antigen (HLA)-E by the CD94:NKG2A receptor is conserved, whereas recognition of HLA-A, B and C by the killer cell immunoglobulin-like receptors (KIRs) is diversified. Competing demands of the immune and reproductive systems, and of T-cell and NK-cell immunity-combined with the segregation on different chromosomes of variable NK-cell receptors and their MHC class I ligands-drive an unusually rapid evolution that has resulted in unprecedented levels of species specificity, as first appreciated from comparison of mice and humans. Counterparts to human KIR are present only in simian primates. Observed in these species is the coevolution of KIR and the four MHC class I epitopes to which human KIR recognition is restricted. Unique to hominids is the emergence of the MHC-C locus as a supplier of specialized and superior ligands for KIR. This evolutionary trend is most highly elaborated in the chimpanzee. Unique to the human KIR locus are two groups of KIR haplotypes that are present in all human populations and subject to balancing selection. Group A KIR haplotypes resemble chimpanzee KIR haplotypes and are enriched for genes encoding KIR that bind HLA class I, whereas group B KIR haplotypes are enriched for genes encoding receptors with diminished capacity to bind HLA class I. Correlating with their balance in human populations, B haplotypes favour reproductive success, whereas A haplotypes favour successful immune defence. Evolution of the B KIR haplotypes is thus unique to the human species.     

Activation or tolerance of natural killer cells is modulated by ligand quality in a nonmonotonic manner

Natural killer (NK) cells extend important immune resistance in vertebrates by lysing infected and tumor cells. A fine balance between opposing signals generated by a diverse set of stimulatory and inhibitory NK-cell receptors determines the fate of target cells interacting with the NK cells. We have developed a mathematical model involving membrane proximal initial signaling events that provides novel mechanistic insights into how activation of NK cells is modulated by the half-life of receptor-ligand interaction and ligand concentrations. We show that strong stimulatory ligands produce digital activation, whereas weaker stimulatory ligands can mediate inhibition by strengthening the signals generated by inhibitory ligands, as indicated in experiments in knockout mice. We find under certain conditions, counterintuitively, inhibitory receptors can help mediate activation instead of inhibition. Mechanistic insights gained from NK-cell signaling can facilitate understanding of complex signaling responses that occur due to cross talk between dueling signaling pathways in other cell types.     

A new self: MHC-class-I-independent natural-killer-cell self-tolerance

A fundamental tenet of the immune system is the requirement for lymphocytes to respond to transformed or infected cells while remaining tolerant of normal cells. Natural killer (NK) cells discriminate between self and non-self by monitoring the expression of MHC class I molecules. According to the 'missing-self' hypothesis, cells that express self-MHC class I molecules are protected from NK cells, but those that lack this self-marker are eliminated by NK cells. Recent work has revealed that there is another system of NK-cell inhibition, which is independent of MHC class I molecules. Newly discovered NK-cell inhibitory receptors that have non-MHC-molecule ligands broaden the definition of self as seen by NK cells.     

HIV-1 Vpu disarms natural killer cells

Natural killer (NK)-cell killing of virus-infected cells is regulated in part by the engagement of activation and coactivation receptors. In this issue of Cell Host & Microbe, Shah et?al. (2010) demonstrate that HIV-1 protects infected cells from NK-cell-mediated killing by hindering NK-cell degranulation through downmodulation of NTB-A coactivation receptor ligands by the Vpu accessory protein.     

Hitting the complexity of the TIGIT-CD96-CD112R-CD226 axis for next-generation cancer immunotherapy

Antibody-based therapeutics targeting the inhibitory receptors PD-1, PD-L1, or CTLA-4 have shown remarkable clinical progress on several cancers. However, most patients do not benefit from these therapies. Thus, many efforts are being made to identify new immune checkpoint receptor-ligand pathways that are alternative targets for cancer immunotherapies. Nectin and nectin-like molecules are widely expressed on several types of tumor cells and play regulatory roles in T- and NK-cell functions. TIGIT, CD226, CD96 and CD112R on lymphoid cells are a group of immunoglobulin superfamily receptors that interact with Nectin and nectin-like molecules with different affinities. These receptors transmit activating or inhibitory signals upon binding their cognate ligands to the immune cells. The integrated signals formed by their complex interactions contribute to regu-lating immune-cell functions. Several clinical trials are currently evaluating the efficacy of anti-TIGIT and anti-CD112R blockades for treating patients with solid tumors. However, many questions still need to be answered in order to fully understand the dynamics and functions of these receptor networks. This review addresses the rationale behind targeting TIGIT, CD226, CD96, and CD112R to regulate T- and NK-cell functions and discusses their potential application in cancer immunotherapy.     

Whatever turns you on: accessory-cell-dependent activation of NK cells by pathogens

Natural killer (NK) cells have a crucial role in combating infections and cancers and their surface receptors can directly recognize and respond to damaged, transformed or non-self cells. Whereas some virus-infected cells are recognized by this same route, NK-cell responses to many pathogens are triggered by a different mechanism. Activation of NK cells by these pathogens requires the presence of accessory cells such as monocytes, macrophages and dendritic cells. Recent studies have identified numerous pathogen-recognition receptors that enable accessory cells to recognize different pathogens and subsequently transmit signals--both soluble and contact-dependent--to NK cells, which respond by upregulating their cytotoxic potential and the production of inflammatory cytokines.     

Reactive phenotypes after acute and chronic NK-cell activation

Several phenotypic changes have been shown to occur after NK-cell stimulation, involving molecules that have been proved to regulate NK-cell migration into tissues and NK-cell activation and proliferation as well as target cell recognition and killing. Here, we review the reactive phenotypes observed in vivo after acute and chronic NK-cell activation.     

NK and K cell activity in children with leukemias and aplastic anemias before and after allogeneic bone marrow transplantation

Mononuclear leukocytes from the peripheral blood and bone-marrow of children affected with aplastic anemia and leukemia were investigated for K-cell activity (antibody-dependent cellular cytotoxicity) and NK-cell activity before and after allogenous bone-marrow transplantation. 51Cr liberation test against murine Graffi erythroblast leukemic cells covered with xenoantibodies and K-562 cells were used for identification. Strongly lowered NK- and K-cell activities could be found in aplastic anemia prior to bone-marrow transplantation. However, NK-cell activity was only lowered significantly in leukemic patients with indication of bone-marrow transplantation. K-cell and NK-cell activities normalised after bone-marrow transplantation. K-cell and NK-cell activities could be observed to be reconstituted very early after bone-marrow transplantation.     

Peripheral natural killer cell maturation depends on the transcription factor Aiolos

Natural killer (NK) cells are an innate lymphoid cell lineage characterized by their capacity to provide rapid effector functions, including cytokine production and cytotoxicity. Here, we identify the Ikaros family member, Aiolos, as a regulator of NK-cell maturation. Aiolos expression is initiated at the point of lineage commitment and maintained throughout NK-cell ontogeny. Analysis of cell surface markers representative of distinct stages of peripheral NK-cell maturation revealed that Aiolos was required for the maturation in the spleen of CD11b^highCD27⁻ NK cells. The differentiation block was intrinsic to the NK-cell lineage and resembled that found in mice lacking either T-bet or Blimp1; however, genetic analysis revealed that Aiolos acted independently of all other known regulators of NK-cell differentiation. NK cells lacking Aiolos were strongly hyper-reactive to a variety of NK-cell-mediated tumor models, yet impaired in controlling viral infection, suggesting a regulatory function for CD27⁻ NK cells in balancing these two arms of the immune response. These data place Aiolos in the emerging gene regulatory network controlling NK-cell maturation and function.     

Mutations in mice that influence natural killer (NK) cell activity

A series of mutations in mice was tested for splenic NK-cell activity against YAC-1 target cells. Mutations at six loci that reduce NK-cell activity in the homozygous state were identified, including beige (bg), hairless (hr), motheaten (me), obese (ob), steel (Sl) and, to a lesser extent, dominant spotting (W). Motheaten mice displayed the most profound NK-cell deficiency, with NK-cell activity virtually absent. Two mutations, nude (nu) and lymphoproliferation (Ipr), produced elevated NK-cell-mediated lysis. The double homozygous recessivenu/nu bg/bg nude-beige mouse was viable and NK-cell-deficient, with activity slightly higher than that of +/?bg/bg beige littermate controls. Pigmentation mutants related to beige, including pale ears (ep), pearl (pe), and ruby eyes (ru ^2J ) did not dramatically influence NK-cell levels. Unlike the obese gene, other mutations leading to obesity, diabetes (db) and yellow (Asuy), did not impair NK-cell function. The possible site of gene action of these mutants in the NK-cell pathway is discussed.     

NK cytotoxic activity and relative distribution of NK cells in 3-acetylpyridine influenced mice

NK-cell cytotoxic activity and their relative distributions were studied in the spleen of female Lurcher mice with spontaneous olivopontocerebellar degeneration (C3H) and female athymic nu/nu mice (BALB/c) influenced by 3-acetylpyridine (the neurotoxin causing selective degeneration of cerebellar and inferior olive neurons in some rodent species). The congenital olivopontocerebellar degeneration in Lurcher mice is followed by only an insignificant increase of NK-cell cytotoxic activity (1.2 times). On the other hand, the congenital thymic dysgenesis in nu/nu mice is compensated by a substantial increase in cytotoxic activity (19.4-fold). The administration of 3-acetylpyridine (including prevalent neuronal destruction particularly in Lurcher mutants) caused a decrease of NK-cell cytotoxic activities in all groups of mice (in Lurcher and C3H controls to 60 and 50%, respectively, and in nu/nu and BALB/c controls to 25 and 60%). Relative distributions of NK-cells in spleens of non-influenced and influenced animals were not significantly changed. Some fundamental immune mechanisms, such as the NK-cell cytotoxic activity, were demonstrated to be controlled by congenitally determined or artificially induced changes in both the nervous and the immune systems.     

Natural killer cell cytotoxic potential of patients with ovarian carcinoma and its modulation with virus-modified tumor cell extract

Summary We have demonstrated that cancer patients with ovarian carcinoma display deficient peripheral blood NK-cell cytotoxic potential against the K-562 target cell line. Furthermore, no NK-cell activity against the same tumor was detected in ascitic fluids of these patients. The inferior peripheral blood NK-cell cytotoxicity of ovarian carcinoma patients was significantly augmented after ID inoculation with virus-modified tumor cell extract. Similarly, NK-cell activity in the ascitic fluids was dramatically increased after IP in vivo therapy with the same tumor extract preparation. Interestingly, in some of the cancer patients the augmentation of NK-cell activity in ascitic fluids after IP injection of virus-modified tumor cells extract was associated with a clinical response of the patients, as demonstrated by regression of ascitic tumors. These studies indicate, first, that virus-modified tumor extract displays immunopotentiating activity, as reflected by its marked NK cell-augmenting potential, and secondly, that regional activation of NK cells could underlie the mechanism of regression of ascitic tumors.