Alter NK Cell Research Articles

Methodological Approaches for Increasing the Retroviral Transduction Efficiency of Primary NK Cells.

The growing attention to NK cells for cancer cell therapy is associated with the need to establish highly efficient protocols for their genetic modification, particularly by retroviral transduction. In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets. Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells. We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts. Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.

Tumor Cells Alter NK Cell Surface Topology for Immune Evasion.

Cancer cells alter NK cell surface topology to block immune synapse formation and cytotoxicity.

Janus kinase inhibitors alter NK cell phenotypes and inhibit their antitumour capacity.

Janus kinase inhibitors (JAKi) are efficacious in RA but concerns regarding the risk of cancer associated with their exposure have recently emerged. Given the role of NK cells in antitumour response, we investigated the impact of JAKi [tofacitinib (TOFA), baricitinib (BARI), upadacitinib (UPA) and filgotinib (FIL)] on NK cells. We first performed an ex vivo phenotype of NK cells in RA patients treated with TOFA, BARI or MTX. We next phenotyped sorted NK cells from healthy donors cultured with four JAKi or dimethyl sulphoxide (DMSO) at three concentrations, including the licensed dose (therapeutic concentration). Third, we assessed NK cell function using anti-NKp30 cross-linking and co-cultures with two different tumour cell lines: A549 and SU-DHL-4. Twenty-eight RA patients were included. Patients treated with TOFA had reduced expression of CD69 on NK cells compared with MTX (P < 0.05). We confirmed in vitro the negative impact of JAKi on NK cell maturation (CD57), activation (CD69) and activating receptor (NKp30), these latter two being specifically altered with TOFA and UPA. When NK cells were stimulated by NKp30, we observed reduced CD107a (P < 0.01) and IFN-γ/TNF expression (P < 0.05) with TOFA. Lastly, NK cells exposed to TOFA showed reduced CD107a (P < 0.05) and altered cytotoxicity (P < 0.05) when co-cultured with the two cell lines. JAKi have a phenotypic and functional impact on NK cell activation and impair their antitumour activity, with a variable impact depending on the JAKi. It remains an open question whether this mechanism can explain the increased tumour risk observed with TOFA.

SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway.

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells’ exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.

Immunometabolic Dysfunction of Natural Killer Cells Mediated by the Hypoxia-CD73 Axis in Solid Tumors.

NK cell infiltration into solid tumors is often low and is largely represented by the poorly-cytotoxic CD56bright subset. Numerous studies have demonstrated that CD73, overexpressed under conditions of hypoxia, is involved in a variety of physiological processes, while its overexpression has been correlated with tumor invasiveness, metastasis and poorer patient survival in many cancers. Hypoxia itself favors aggressive glycolytic fueling of cancer cells, in turn driving reprogramming of NK cell metabolism. In addition, the hypoxia-driven activity of CD73 immunometabolically impairs NK cells in tumors, due to its catalytic role in the generation of the highly immunosuppressive metabolite adenosine. Adenosinergic signaling was shown to alter NK cell metabolic programs, leading to tumor-promoting environments characterized by NK cell dysfunction. Despite the demonstrated role of NK cell responses in the context of CD73 targeting, the engagement of NK cells in the setting of hypoxia/CD73 signaling has not been extensively studied or exploited. Here, we discuss available evidence on the role of hypoxic signaling on CD73-mediated activity, and how this relates to the immunometabolic responses of NK cells, with a particular focus on the therapeutic targeting of these pathways.

Natural selection for killer receptors and their MHC class I ligands: In pursuit of gene pairs that fit well in tandem.

Our understanding of the genetic basis of host resistance to viral infection and disease has progressed significantly over the last century. Numerous genes coding for modifiers of immune functions have been identified, which impact a variety of critical cellular processes, including signaling via lymphocyte receptors and their ligands, signal transduction, cytokine signaling, production and release of cytotoxic effectors, transcriptional regulation, and proliferation. Genome-wide association studies implicate an important role for both highly polymorphic NK cell receptors and their MHC class I ligands in modifying host resistance. These findings indicate NK cells are critical mediators of viral control with considerable potential to affect morbidity and mortality outcomes. They further suggest that both stimulatory and inhibitory NK receptor polymorphisms alter NK cell sensing of MHC I ligands on viral targets, which influences how NK cells respond to infection. In many cases, however, the underlying causes associated with host outcomes remain elusive. Herein, we discuss several modes of NK cell sensing of MHC I and MHC I-like molecules on viral targets, and the role of genetic diversity in this evolutionarily dynamic process. We further suggest that natural selection for paired NK receptors with opposing function, but shared MHC I ligands may give rise to rare, but highly effective MHC I-dependent modes of NK cell sensing of viral targets.

M2-polarized and tumor-associated macrophages alter NK cell phenotype and function in a contact-dependent manner

The crosstalk between NK cells and M1 macrophages has a vital role in the protection against infections and tumor development. However, macrophages in the tumor resemble an M2 phenotype, and, at present, their effect on NK cells is less clear. This study investigated whether tumor-associated macrophages (TAMs) have a role in altering NK cell function and phenotype using in vitro cocultures of murine NK cells with peritoneal or bone marrow-derived, M2-polarized macrophages or TAMs isolated from spontaneous mouse breast tumors. We report here that both peritoneal and bone marrow-derived M2 macrophages, as well as TAMs, substantially inhibit NK cell activation and concordant cytotoxicity against tumor cells. The mechanism for this inhibition was found to require contact between the respective cell types. Both M2 macrophages and TAMs are producers of the immunosuppressive cytokine TGF-β. The inhibition of TGF-β restored the cytotoxicity of NK cells in contact with M2 macrophages, implicating TGF-β in the mechanism for NK cell inhibition. In addition to affecting NK cell function, TAMs also induced a CD27lowCD11bhigh-exhausted NK cell phenotype, which corresponds with the reduced activation and cytotoxicity observed. This study reveals a novel implication of TAMs in the tumor-associated inhibition of NK cell function by demonstrating their capacity to directly alter NK cell cytotoxicity and phenotype in a contact-dependent mechanism involving TGF-β. These findings identify the interaction between NK cells and TAMs as a prospective therapeutic target to enhance NK cell effector function for effective NK cell cancer therapies.

PI3K p85 β regulatory subunit deficiency does not affect NK cell differentiation and increases NKG2D-mediated activation.

Activation of NK cells depends on a balance between activating and inhibitory signals. Class Ia PI3K are heterodimeric proteins with a catalytic and a regulatory subunit and have a central role in cell signaling by associating with tyrosine kinase receptors to trigger signaling cascades. The regulatory p85 subunit participates in signaling through NKG2D, one of the main activating receptors on NK cells, via its interaction with the adaptor protein DAP10. Although the effects of inhibiting catalytic subunits or deleting the regulatory p85α subunit have been studied, little attention has focused on the role of the p85β subunit in NK cells. Using p85β knockout mice, we found that p85β deficiency does not alter NK cell differentiation and maturation in spleen or bone marrow. NK cells from p85β-/- mice nonetheless produced more IFN-γ and degranulated more effectively when stimulated with anti-NKG2D antibody. These cells also degranulated and killed NKG2D ligand-expressing target cells more efficiently. We show that p85β deficiency impaired NKG2D internalization, which could contribute to the activated phenotype. Decreasing p85β subunit protein levels might thus constitute a therapeutic target to promote NK cell activity toward NKG2D ligand-expressing cells.

PTEN is a negative regulator of NK cell cytolytic function.

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56(dim) NK cell efficiently kills malignant targets at rest, whereas the less mature CD56(bright) NK cells cannot. In this study, we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells.

Activation-specific metabolic regulation of NK cell IFN-gamma production (INC6P.334)

Abstract Many factors that stimulate NK cell functional responses have been described; however metabolic fuels that drive NK cell function are unknown. Metabolism is significantly altered in disease states and metabolic regulation of T cell function is well-described. We hypothesize that NK cells require distinct metabolic fuels for survival, proliferation, and functional responses. NK cell metabolic phenotype was determined with an extracellular flux analyzer to determine basal oxidative phosphorylation (oxphos) and glycolysis of resting and activated NK cells. NK cells were then cultured under a variety of conditions using inhibitors and nutrient deprivation to determine cellular requirements for glucose, fatty acids, and oxphos for NK cell survival, proliferation, and function. NK cells primarily utilize oxphos at baseline and, in contrast to T cells, upon proliferation. However, there is significant plasticity in the metabolic requirements for NK cell IFN-gamma production. NK cell IFN-gamma production in response to cytokine stimulation is not dependent on either glycolysis or oxphos. By contrast, NK cell IFN-gamma production in response to receptor stimulation requires oxphos and is partially dependent on glycolysis. The metabolic requirements for NK cell IFN-gamma production are not altered by priming in vitro or in vivo, but do change with proliferation. These findings suggest that the metabolic microenvironment in vivo may significantly alter NK cell function.

Functional NK Cell Repertoires Are Determined By Survival Mechanisms Through IL-2Ra and FasL

NK cell based immunotherapy can be used to treat advanced acute myelogenous leukemia and data shows that success depends on the function of NK cells and how long the cells are maintained in the recipient. An important determinant of NK cell function is expression of Killer-cell immunoglobulin-like receptors (KIRs), which are involved in the maintenance of self tolerance and acquisition of NK cell functional competence, a process termed education. Little is known about how NK cell education influences NK cell survival. Utilizing a serum starvation assay we found that KIR+ and educated NK cells survived better when compared to those that did not have a KIR or KIR matching cognate HLA-ligand respectively (Figure 1A and 1B). Under basal conditions both KIR+ and educated NK cells had more anti-apoptotic proteins (Bcl-2 and Bcl-xL) and expressed less Fas. When NK cells were incubated with serum and IL-15 (10 ng/ml) followed by IL-15 withdrawal, a substantial increase of pro-apoptotic Bim was found on uneducated NK cells (1.4 fold increase; P = 0.01). Under this same condition educated NK cells expressed more FasL (1.5 fold increase; P = 0.0003), indicating that they could be driving cell death on neighboring NK cells when cytokine is limiting. Since both NK cell survival and homeostasis are mediated by cytokine signaling, we studied expression of IL-15 and IL-2 signaling components. The most significant change was IL-2Ra, which was expressed at higher levels on uneducated NK cells (3 fold increase; P = 0.0004) when the cells were stimulated with serum and IL-15 (1 ng/ml) for 72 hrs. Higher IL-2Ra expression correlated well with cell death and Bim expression so we decided to test if modulating its expression would alter NK cell survival in an IL-15 withdrawal setting. Compared to the control, transient overexpression of IL-2Ra lead to a 1.55 fold decrease in NK cell numbers (P = 0.01) while siRNA knockdown of IL-2Ra lead to a 1.5 fold increase in NK cell numbers (P = 0.07). Importantly, at the time of harvest there was a 1.4 fold decrease in cell death in the IL-2Ra knockdown condition when compared to the control (P = 0.0007). Given that no IL-2 or crosslinking signals were present, the mechanism must differ from the well-described role of IL-2 on activation induced cell death. Since FasL is upregulated on educated NK cells when IL-15 is limiting and IL-2Ra renders NK cells more sensitive to cell death, we tested if educated cells could drive cell death of IL-2Rahi NK cells when survival signals are scarce using a co-culturing assay and FasL blocking antibodies. IL-2Rahi NK cells were more sensitive to apoptosis when co-cultured with KIR+ NK cells (presumably enriched for educated subsets) than with KIR- NK cells from the same donor that were subjected to IL-15 stimulation and withdrawal (34±4.8% vs. 20.6±4.7%; P = 0.002). The effect was reduced when FasL was blocked (P = 0.003), and no differences in killing were seen on the IL-2Ralo NK cells regardless of the treatment. Taken together these findings indicate that educated NK cells outlive their counterparts through two mechanisms: decreased expression of proteins involved in cell death and by killing competing NK cells when IL-15 is limiting. Finally, since we have previously reported on expansion of educated NK cells on transplant patients post CMV reactivation and since CMV reactivation can be associated with decreased relapse, we wanted to investigate if CMV reactivation could alter NK cell survival. There was increased survival on the NK cells from adult donor HCT (2 fold increase at 6 months; P = 0.03) and umbilical cord blood (3 fold increase at 100 days; P = 0.01) allogeneic transplant patients that had undergone CMV reactivation supporting the physiologic role of NK cell survival in vivo from a pathologic challenge. Taken together, these findings show that NK cell functional repertoires are determined by class I interactions, infection, and NK-NK interactions through IL-2Ra, Bim, and FasL to mediate clonal dominance that might be exploited in order to enhance NK cell survival and function after adoptive transfer of allogeneic NK cells for therapeutic use in cancer. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms.

Azacytidine impairs NK cell reactivity while decitabine augments NK cell responsiveness toward stimulation

Azacytidine and decitabine are approved for treatment of acute myeloid leukemias and myelodysplastic syndromes. While clinical responses are attributed to epigenetic effects and induction of apoptosis in malignant cells, these azanucleosides also affect antitumor immune responses. NK cells as components of innate immunity may confine development and progression of cancer. Numerous therapeutic strategies presently aim to reinforce NK reactivity against hematopoietic malignancies. We here comparatively analyzed the effect of the two clinically available azanucleosides and report that NK cytotoxicity and IFN-γ production are significantly impaired by pharmacological concentrations of azacytidine but enhanced by decitabine. This was not due to alterations in the target cells but caused by direct effects on NK cells depending on the chemical modifications by which azanucleosides differ from their physiological analogues. Although azacytidine impaired mRNA synthesis and induced apoptosis in NK cells, decitabine did not per se alter NK cell viability or reactivity but enhanced responsiveness to activating stimuli by inducing transcription of genes involved in NK reactivity. Tantalizingly, these effects were independent of incorporation of the azanucleosides into DNA during cell division. While azacytidine impairs NK antitumor immunity, decitabine augments NK reactivity by yet unidentified mechanisms and may thus serve well in therapeutic strategies combining its effects on malignant cells with its ability to enhance NK functions.

Unidirectional signaling triggered through 2B4 (CD244), not CD48, in murine NK cells

Engagement of 2B4 (CD244) with CD48 results in activation, costimulation, or inhibition of NK cell activities, depending on the cell types and the stage of differentiation. In vivo, 2B4+ NK cells can interact with CD48+ NK cells and also with surrounding CD48+ hematopoietic cells. Similarly, CD48+ NK cells may be triggered by adjacent 2B4+ NK cells or other hematopoietic cells expressing 2B4, e.g., monocytes, basophils, γδ T cells, etc. As CD48 was also shown to function as an activating receptor, 2B4/CD48 binding in the settings of NK-to-NK or NK-to-non-NK cell interactions may generate bidirectional signals. To address this question, we examined the consequence of CD48 or 2B4 ligation using two experimental settings: one with target (syngeneic EL4 and allogeneic P815) cells, ectopically expressing surface 2B4 or CD48, and the other with direct cross-linking with plate-bound mAb. Here, we report that ligation of CD48 with 2B4+ EL4 or 2B4+ P815 targets, in the absence of other receptor engagement, did not alter NK cell cytotoxicity or proliferation significantly. Similarly, cross-linking of NK cells with plate-bound anti-CD48 mAb in the absence or presence of a suboptimal dose of IL-2 did not modulate NK proliferation, cytotoxicity, or cytokine production. Nonetheless, 2B4 cross-linking promoted NK cell proliferation and effector functions consistently in both settings. Therefore, our results demonstrate unequivocally that CD48 on surrounding NK or non-NK cells serves primarily as a ligand to stimulate 2B4 on the adjacent NK cells in mice.

Active Hexose Correlated Compound (AHCC) improves immune cell populations after influenza vaccination of healthy subjects.

Active Hexose Correlated Compound (AHCC), an extract of the Basidiomycetes mushroom, has immunomodulatory effects in animal and human studies. We performed a randomized, double‐blind placebo‐controlled trial to evaluate the effects of AHCC supplementation on the immune response of healthy adults after immunization with 2009–10 seasonal influenza vaccine. A total of 29 subjects were enrolled in this study: 14 were supplemented with AHCC (total of 3000 mg/day) and 15 were given a placebo on the day of vaccination throughout two weeks post‐vaccination. Blood was drawn at immunization and two weeks later for phenotypic analysis of lymphocytes using flow cytometry. The fold increases in percentages of T cells, CD8+ T (cytotoxic) cells, CD56+ (NK) cells, but not CD4/CD8 ratios, were significantly higher than at vaccination for AHCC‐supplemented subjects. Although AHCC supplementation did not alter NK cell percentages post‐vaccination, CD56 bright cytotoxic NK cells were higher in AHCC than the placebo group. AHCC supplementation had a more dramatic effect on immune cell phenotypes after vaccination of subjects over 60 years old. This suggests that short‐term AHCC supplementation may be a good therapeutic intervention to sustain, or increase, the immune response to influenza vaccination in healthy subjects. Supported by Amino Up Chemical Company, Sapporo, Japan.

A Fc-Engineered RANK-Fc Fusion Protein Neutralizes Soluble RANK Ligand and Targets Malignant Hematopoietic Cells for NK Cell Reactivity.

Abstract 411Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NFκB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) as key regulators of bone resorption. Multiple myeloma (MM) disrupts the balance within this molecule system, and severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of this disease. Besides MM cells, other malignant hematopoietic cells have also been found to express RANKL at the cell surface and to release this molecule in soluble form (sRANKL). Neutralization of RANKL using RANK-Fc fusion protein or monoclonal antibody (Denosumab/AMG162), which mimics the RANKL-neutralizing endogenous effects of osteoprotegerin, decreases osteolysis in multiple in vivo models and is presently being evaluated as a means to treat both non-malignant and malignant osteolysis. We here confirmed and extended previously published data and report that all investigated MM cell lines (n = 5) as well as primary leukemic cells of CLL patients (n = 12) displayed substantial levels of RANKL mRNA and surface expression. Moreover, we report that substantial levels of sRANKL can be detected in culture supernatants of MM and primary CLL cells, but not in supernatants of healthy PBMC. Next we engineered RANK-Fc fusion proteins with modified affinity to FcγR by mutating amino acids in the Fc portion as previously described (Lazar et al., PNAS 2006; Armour et al., Eur J Immunol 1999). Compared to wildtype RANK-Fc (RANK-Fc-wt), our mutants (S239D/I332E and E233P/L234V/L235A/DeltaG236/A327G/A330S) displayed highly enhanced and abrogated (RANK-Fc-ADCC+ and RANK-Fc-KO, respectively) affinity to FcγRIIIa expressed on NK cells, which play an important role in anti-tumor immunity due to their ability to lyse target cells directly and to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of therapeutic antibodies. The RANK-Fc-ADCC+ displayed similar capacitiy to neutralize sRANKL compared to the RANK-Fc-KO and the RANK-Fc-wt as revealed by binding competition assays. Next we cultured NK cells with L cells or P815 cells transfected to express RANKL and the parental, RANKL-negative controls in the presence or absence of the different RANK-Fc constructs. Addition of RANK-Fc-KO or RANK-Fc-wt did not substantially alter NK cell reactivity against the target cells. However, presence of the RANK-Fc-ADCC+ dramatically enhanced NK cell cytotoxicity and cytokine production in cultures with the RANKL-expressing target cells (increase from 20% to 89%, E:T ratio 30:1 and 12 pg/ml to 290 pg/ml respectively; both p<0.01, Student's t-test). Neither of the three RANK-Fc proteins altered NK cell cytotoxicity and cytokine production in cultures with the RANKL negative controls demonstrating that the RANK-Fc-ADCC+ specifically induced NK cell reactivity against RANKL-expressing malignant cells. Moreover, treatment with RANK-Fc-ADCC+ also significantly augmented NK cell anti-tumor reactivity in cultures with RANKL-expressing primary CLL cells of patients, and this was observed both in settings using allogenic NK cells and analyzing autologous NK cells among PBMC of the leukemia patients (both p<0.01, Student's t-test). Taken together, our Fc-engineered RANK-Fc-ADCC+ fusion protein may neutralize detrimental effects of sRANKL, can target RANKL-expressing malignant cells for NK cell anti-tumor reactivity and may thus constitute an attractive immunotherapeutic means for treatment of hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

Four Weeks Of Acupuncture Treatments Did Not Alter NK Cell Number Or Activity Following 60-min Of Cycling

Four Weeks Of Acupuncture Treatments Did Not Alter NK Cell Number Or Activity Following 60-min Of Cycling

LNFPIII/LeX-Stimulated Macrophages Activate Natural Killer Cells via CD40-CD40L Interaction

Lacto-N-fucopentaose III (LNFPIII) is a human milk sugar containing the biologically active Lewis X (LeX) trisaccharide. LNFPIII/LeX is also expressed by immunosuppressive helminth parasites, by bacteria, and on a number of tumor/cancer cells. In this report, we first demonstrate that LNFPIII activates macrophages in vitro as indicated by upregulation of Gr-1 expression on F4/80(+) cells. Further, we investigated the effect of LNFPIII-activated macrophages on NK cell activity. We found that LNFPIII-stimulated F4/80(+) cells were able to activate NK cells, inducing upregulation of CD69 expression and gamma interferon (IFN-gamma) production. The experiments show that NK cell activation is macrophage dependent, since NK cells alone did not secrete IFN-gamma in response to LNFPIII. Furthermore, we found that activation of NK cells by glycan-stimulated macrophages required cell-cell contact. As part of the cell-cell contact mechanism, we determined that CD40-CD40L interaction was critical for IFN-gamma secretion by NK cells, as the addition of anti-CD40L antibodies to the coculture blocked IFN-gamma production. We also demonstrated that LNFPIII-stimulated macrophages secrete prostaglandin E(2), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) but a very low level of IL-12. Interestingly, addition of anti-TNF-alpha, anti-IL-10, or anti-IL-12 monoclonal antibodies did not significantly alter NK cell activity. Our data show that these soluble mediators are not critical for LNFPIII-stimulated macrophage activation of NK cells and provide further evidence for the importance of cell-cell contact and CD40-CD40L interactions between macrophages and NK cells.

Reduced splenic natural killer cell activity in rats with a hyperreactive dopaminergic system

Interactions between the nervous system and the immune system have been recognized as important regulatory processes in determining the activity of the immune response. We have previously shown that rats, which differ in the reactivity of the dopaminergic system (APO-SUS and APO-UNSUS rats), also differ in experimental metastasis formation and in susceptibility to autoimmunity. APO-SUS rats have a high response to administration of apomorphine and can be characterized as hyperdopaminergic, whereas their APO-UNSUS counterparts show low susceptibility to apomorphine and have a hypodopaminergic phenotype. In this study we investigated whether the decreased experimental metastasis formation of APO-SUS rats compared to APO-UNSUS rats is associated with higher natural killer cell activity in APO-SUS rats. Surprisingly, splenic NK cell activity of hyperdopaminergic APO-SUS female as well as male rats is significantly lower than NK cell activity of their hypodopaminergic APO-UNSUS counterparts. The reduced splenic NK activity of female APO-SUS rats is associated with lower percentages of NK cells in the spleen cell population. In contrast, male APO-SUS and APO-UNSUS rats show similar numbers of NK cells in the spleen. There was no difference in plasma dopamine levels between APO-SUS and APO-UNSUS rats and i.p. treatment of rats with the dopaminergic agonist quinpirole did not alter NK cell activity. In conclusion, our data demonstrate that differences in the reactivity of the dopaminergic system are associated with differences in splenic NK cell activity. Moreover, our data demonstrate that in this model lower splenic NK cell activity is not related to increased experimental lung metastasis formation.

IL-2 and IL-12 Alter NK Cell Responsiveness to IFN-γ-Inducible Protein 10 by Down-Regulating CXCR3 Expression

Cytokine treatment of NK cells results in alterations in multiple cellular responses that include cytotoxicity, cytokine production, proliferation, and chemotaxis. To understand the molecular mechanisms underlying these responses, microarray analysis was performed and the resulting gene expression patterns were compared between unstimulated, IL-2, IL-2 plus IL-12, and IL-2 plus IL-18-stimulated NK92 cells. RNase protection assays and RT-PCR confirmed microarray predictions for changes in mRNA expression for nine genes involved in cell cycle progression, signal transduction, transcriptional activation, and chemotaxis. Multiprobe RNase protection assay also detected changes in the expression of CCR2 mRNA, a gene that was not imprinted on the microarray. We subsequently expanded our search for other chemokine receptor genes absent from the microarray and found an IL-2- and IL-12-dependent decrease in CXCR3 receptor mRNA expression in NK92 cells. A detailed analysis of CXCR3 expression in primary NK cells revealed that an IL-2 and an IL-12 together significantly decreased the CXCR3 receptor mRNA and receptor surface expression by 6 and 24 h of treatment, respectively. This decrease in receptor expression was associated with a significant reduction in chemotaxis in the presence of IFN-gamma-inducible protein-10. The decline in CXCR3 mRNA was due to transcriptional and posttranscriptional mechanisms as the addition of actinomycin D to IL-2- and IL-12-treated NK92 slightly altered the half-life of the CXCR3 mRNA. Collectively, these data suggest that IL-2 and IL-12 directly affect NK cell migratory ability by rapid and direct down-regulation of chemokine receptor mRNA expression.