HLA-B2702 (77-83/83-77) peptide binds to beta-tubulin on human NK cells and blocks their cytotoxic capacity

It has been described that peptides derived from a highly conserved region of the alpha1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity blocking both T and NK cell cytotoxicity. In vivo treatment with these peptides prolongs survival of MHC-mismatched allografts. However, the molecular bases of these effects are still unclear. In this study, we further analyze the mechanisms by which the dimeric peptide HLA-B2702 (77-83/83-77) induces suppression of NK cell cytotoxicity. This peptide inhibits natural and redirected lysis mediated by NK cells without significantly affecting effector-target cell binding. We have also isolated and sequenced a protein that binds this inhibitory peptide, which structurally corresponds to beta-tubulin. Tubulin is the major protein of microtubules and is involved in target cell killing. Furthermore, B2702 peptide promotes GTP-independent tubulin assembly, producing aggregates that cannot be depolymerized by cold. Treatment of NK cells with Taxol or demecolcine, which interfere with microtubule organization, also prevents NK cell cytotoxicity. Taken together, these results support the hypothesis that the peptide B2702 (77-83/83-77) exerts its inhibitory effect on NK cell cytotoxicity by inducing polymerization of microtubules and interfering with their normal assembly/disassembly dynamics.     

Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.     

Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity

The present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37 degrees C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromium-release assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to four-fold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (alpha and beta), recombinant interferon gamma, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon gamma for 16-18 h, GM-CSF and interleukin-1 beta for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.     

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri‐implantation period

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1⁺ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single‐cell RNA sequencing (scRNA‐seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue‐resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function‐related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA‐seq and cytotoxicity assays confirmed that CYP26A1⁺ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal‐foetal interface by regulating the activity of NK cells.     

Cutting edge: tumor rejection mediated by NKG2D receptor-ligand interaction is dependent upon perforin

We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) beta. Rae-1beta expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1beta tumor cells were rejected by a combination of NK cells and CD8(+) T cells. Rae-1beta expression stimulated NK cell cytotoxicity and IFN-gamma secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-gamma, was critical for the rejection of Rae-1beta-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.     

Cutting Edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation

The killer cell Ig-like receptor (KIR) gene, KIR3DS1, has been implicated in slowing disease progression in HIV infection; however, little is known about its expression, function, or ligand specificity. Using retrovirally transduced NKL cells and peripheral blood NK cells from KIR3DS1-positive donors we assessed expression of this gene by flow cytometry and its function by in vitro assays measuring KIR3DS1-induced cell-mediated cytotoxicity and cytokine production. In the present study, we demonstrate that KIR3DS1 is expressed on peripheral blood NK cells and triggers both cytotoxicity and IFN-gamma production. Using cotransfection and coimmunoprecipitation, we found that KIR3DS1 associates with the ITAM-bearing adaptor, DAP12. Soluble KIR3DS1-Ig fusion proteins did not bind to EBV-transformed B lymphoid cell lines transfected with HLA-Bw4 80I or 80T allotypes, suggesting that if KIR3DS1 does recognize HLA-Bw4 ligands, this may be peptide dependent.     

A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.     

Age-related variation in the proportion and activity of murine liver natural killer cells and their cytotoxicity against regenerating hepatocytes

We investigated the distribution of liver NK cells in mice of various ages and their cytotoxicity against regenerating hepatocytes. Liver NK cells were identified by asialo GM1 antibody in mononuclear cell suspension from the liver, whereas NK activity was assayed against YAC-1 target cells. Mononuclear cells in the liver consisted of more than 25% NK cells with potent NK activity in C3H/He mice, 8 wk of age. The strain-specific distribution (C3H/He greater than C57BL/6 greater than DBA/2) of liver NK cells was the same as those in the spleen and blood. The proportion of liver NK cells and the level of NK activity in C3H/He mice were further demonstrated to vary depending on age, in that both the proportion and the function were generated at 4 wk of age, reached a maximum between the 6th and 8th wk, and then rapidly decreased around the 9th wk. The appearance of an increased number of NK cells in the liver seemed to coincide with the slowing of the rapid increase of murine liver weight. We then investigated whether liver NK cells mediated their cytotoxicity against regenerating hepatocytes. Both specific 51Cr-release assay and single cell cytotoxicity assay demonstrated that liver NK cells were significantly cytotoxic against regenerating hepatocytes in partially hepatectomized liver, but to a lesser extent against normal hepatocytes in resting liver. Morphologic study revealed that normal liver predominantly consisted of hepatocytes with binuclei (greater than 60%) but that regenerating liver mainly consisted of hepatocytes with a single nucleus (greater than 70%). One-nucleus hepatocytes were more susceptible to the cytotoxicity of liver NK cells. A comparative study of restoration kinetics of the liver weight and the number of liver NK cells after partial hepatectomy also showed a unique relationship. These results raise the possibility that liver NK cells might be responsible for regulating hepatocyte growth.     

TLR7/8-mediated activation of human NK cells results in accessory cell-dependent IFN-gamma production

NK cells express receptors that allow them to recognize pathogens and activate effector functions such as cytotoxicity and cytokine production. Among these receptors are the recently identified TLRs that recognize conserved pathogen structures and initiate innate immune responses. We demonstrate that human NK cells express TLR3, TLR7, and TLR8 and that these receptors are functional. TLR3 is expressed at the cell surface where it functions as a receptor for polyinosinic acid:cytidylic acid (poly(I:C)) in a lysosomal-independent manner. TLR7/8 signaling is sensitive to chloroquine inhibition, indicating a requirement for lysosomal signaling as for other cell types. Both R848, an agonist of human TLR7 and TLR8, and poly(I:C) activate NK cell cytotoxicity against Daudi target cells. However, IFN-gamma production is differentially regulated by these TLR agonists. In contrast to poly(I:C), R848 stimulates significant IFN-gamma production by NK cells. This is accessory cell dependent and is inhibited by addition of a neutralizing anti-IL-12 Ab. Moreover, stimulation of purified monocyte populations with R848 results in IL-12 production, and reconstitution of purified NK cells with monocytes results in increased IFN-gamma production in response to R848. In addition, we demonstrate that while resting NK cells do not transduce signals directly in response to R848, they can be primed to do so by prior exposure to either IL-2 or IFN-alpha. Therefore, although NK cells can be directly activated by TLRs, accessory cells play an important and sometimes essential role in the activation of effector functions such as IFN-gamma production and cytotoxicity.     

CDX2 enhances natural killer cell–mediated immunotherapy against head and neck squamous cell carcinoma through up-regulating CXCL14

(NK) cells are at the first line of defence against tumours, but their anti-tumour mechanisms are not fully understood. We aimed to investigate the mechanism by which NK cells can mediate immunotherapy against head and neck squamous cell carcinoma (HNSCC). We collected fifty-two pairs of HNSCC tissues and corresponding adjacent normal tissues; analysis by RT-qPCR showed underexpression of CXCL14 in HNSCC tissues. Primary NK cells were then isolated from the peripheral blood of HNSCC patients and healthy donors. CXCL14 was found to be consistently under-expressed in the primary NK cells from the HNSCC patients. However, CXCL14 expression was increased in IL2-activated primary NK cells and NK-92 cells. We next evaluated NK cell migration, IFN-γ and TNF-α expression, cytotoxicity and infiltration in response to CXCL14 overexpression or knockdown using gain- and loss-of-function approach. The results exhibited that CXCL14 overexpression promoted NK cell migration, cytotoxicity and infiltration. Subsequent in vivo experiments revealed that CXCL14 suppressed the growth of HNSCC cells via activation of NK cells. ChIP was applied to study the enrichment of H3K27ac, p300, H3K4me1 and CDX2 in the enhancer region of CXCL14, which showed that CDX2/p300 activated the enhancer of CXCL14 to up-regulate its expression. Rescue experiments demonstrated that CDX2 stimulated NK cell migration, cytotoxicity and infiltration through up-regulating CXCL14. In vivo data further revealed that CDX2 suppressed tumorigenicity of HNSCC cells through enhancement of CXCL14. To conclude, CDX2 promotes CXCL14 expression by activating its enhancer, which promotes NK cell–mediated immunotherapy against HNSCC.     

IgE-mediated activation of NK cells through Fc gamma RIII

NK cells express Fc gamma RIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas Fc gamma RIII on NK cells is composed of both Fc gamma RIII alpha and FcR gamma chains, that on mast cells is distinct from NK cells and made of Fc gamma RIII alpha, FcR beta, and FcR gamma. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of Fc gamma RIII as well as Fc epsilon RI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through Fc gamma RIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an Fc gamma RIII-dependent manner. These effects of IgE through Fc gamma RIII were not observed in NK cells from FcR gamma-deficient mice lacking Fc gamma RIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through Fc gamma RIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses.     

In vitro enhancement of natural cytotoxicity by atrial natriuretic peptide fragment 4-28.

The presence of atrial natriuretic peptide (ANP) binding sites in the thymic cortex, medulla, and splenic white pulp suggests that this peptide may have immunoregulatory activity. We examined the effect of ANP on human natural killer (NK) cell activity. ANP significantly augmented NK cell cytotoxicity after twenty-four hours of incubation but had no effect on NK activity after short-term incubations of one hour. In addition, atrial natriuretic peptide did not effect the expression of natural killer or T cell surface markers. This study demonstrates that atrial natriuretic fragment 4-28 enhances natural killer cell activity.     

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented byin vivo priming and subsequentin vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1⁺, asialo GM1⁺, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of¹²⁵I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

Cellular localization and functional role of phosphatidylcholine-specific phospholipase C in NK cells

Although several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (M(r) 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity.     

Synthesis of a prolactin-like peptide by natural killer cells: positive regulation by CD16 and exogenous prolactin.

We have previously shown that Prolactin (PRL) activates the native and the in vitro acquired cytotoxicity and the DNA synthetic activity of Natural Killer (NK) cells. Here we show that the supernatant and the cell lysate of NK cells express a 35S-labelled 50 kDa peptide specifically immunostained by two different PRL-antisera. The supernatant of NK cells was biologically active in a Nb2 assay and the activity could be adsorbed by an anti-PRL antiserum. The production of the PRL-like peptide only occurred when NK cells were isolated through binding to immobilized immunocomplexes, the biological ligand for CD16, and was positively modulated by exogenous PRL. These results indicate that PRL, produced by NK cells following stimulation, may act in an autocrine fashion to maintain and/or activate the NK cell function.     

The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes

The MHC class Ib molecule Qa-1 is the primary ligand for mouse CD94/NKG2A inhibitory receptors expressed on NK cells, in addition to presenting Ags to a subpopulation of T cells. CD94/NKG2A receptors specifically recognize Qa-1 bound to the MHC class Ia leader sequence-derived peptide Qdm. Qdm is the dominant peptide loaded onto Qa-1 under physiological conditions and this peptide has an optimal sequence for binding to Qa-1. Peptide dissociation experiments demonstrated that Qdm dissociates from soluble or cell surface Qa-1(b) molecules with a t(1/2) of approximately 1.5 h at 37 degrees C. In comparison, complexes of an optimal peptide (SIINFEKL) bound to the MHC class Ia molecule H-2K(b) dissociated with a t(1/2) in the range from 11 to 31 h. In contrast to K(b), the stability of cell surface Qa-1(b) molecules was independent of bound peptides, and several observations suggested that empty cell surface Qa-1(b) molecules might be unusually stable. Consistent with the rapid dissociation rate of Qdm from Qa-1(b), cells become susceptible to lysis by CD94/NKG2A(+) NK cells under conditions in which new Qa-1(b)/Qdm complexes cannot be continuously generated at the cell surface. These results support the hypothesis that Qa-1 has been selected as a specialized MHC molecule that is unable to form highly stable peptide complexes. We propose that the CD94/NKG2A-Qa-1/Qdm recognition system has evolved as a rapid sensor of the integrity of the MHC class I biosynthesis and Ag presentation pathway.     

Suppression of natural killer cell cytotoxicity by splenocytes from Corynebacterium parvum-injected, bone marrow-tolerant, and infant mice.

Natural killer (NK) cell cytotoxicity to YAC-1 lymphoma was investigated in mice tolerant to bone marrow grafts (BM-tolerant), Corynebacterium parvum- (C. parvum) treated mice, and infant mice. Also the comparison was made between the NK cell and the hemopoietic-resistance effector (HR-E) cells. It was found that the BM-tolerant mice and C. parvum-treated mice showed either no or markedly decreased NK cell cytotoxicity. These mice were also nonresponders to bone marrow grafts in vivo. The lack of or decreased reactivity was apparently caused by the regulatory cell activities of the suppressor cell since the splenocytes from C. parvum-treated and BM-tolerant mice suppressed significantly the cytotoxic activities of otherwise fully functional NK cells. Similar suppressive effect on NK cells was mounted by splenocytes from infant mice, indicating again the suppressor cell regulation of NK cell cytotoxicity.     

Functional requirements for signaling through the stimulatory and inhibitory mouse NKR-P1 (CD161) NK cell receptors

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56(lck). The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.