Natural Killer Cell Migration Research Articles

Application of In Vivo Imaging Techniques for Monitoring Natural Killer Cell Migration and Tumor Infiltration.

In recent years, the use of natural killer (NK) cell-based immunotherapy has shown promise against various cancer types. To some extent therapeutic potential of NK cell-based immunotherapy depends on migration of NK cells towards tumors in animal models or human subjects and subsequent infiltration. Constant improvement in the pharmacological and therapeutic properties of NK cells is driving the performance and use of NK cell-based immunotherapies. In this review, we summarize the molecular imaging techniques used in monitoring the migration and infiltration of NK cells in vivo at preclinical and clinical levels. A review of pros and cons of each molecular imaging modality is done. Finally, we provide our perception of the usefulness of molecular imaging approaches for in vivo monitoring of NK cells in preclinical and clinical scenarios.

Cell-based cancer immunotherapy using chimeric antigen receptor (CAR)-engineered natural killer (NK) cells surface-modified with drug-loaded nanoparticles targeting adenosine receptors

Abstract The immunosuppressive tumor microenvironment (TME) plays a key role in failed eradication of solid tumors and tumor recurrence upon cancer treatment, as resident immune cells lose their cancer surveillance abilities. One of the underlying mechanisms responsible for the loss of adoptively transferred immune cell function in the TME is the A2a adenosine receptor (A2aR) inhibitory pathway. Encapsulating SCH-58261 (SCH), a potent antagonists of A2aR, in crosslinked multilamellar liposomal vesicles (cMLVs) was shown to efficiently deliver the drug to adoptively transferred cells in tumors. We genetically modified human NK92 cells to express anti-mesothelin CAR (ameso.CAR.NK) to be used in this study. We hypothesized that the efficacy of NK cell adoptive transfer therapies could be enhanced if CAR.NK cells were used as active carriers to direct SCH-loaded cMLVs to the immunosuppressive TME to block the A2aR pathway. We first covalently attached cMLVs to NK cell surface. We next verified that cMLV conjugation did not have adverse effects on NK cell viability and migration. In addition, surface conjugation of cMLVs did not reduce IFN-γ secretion and cancer cell killing ability of NK cells, suggesting similar levels of cognate antigen-specific activation of ameso.CAR.NK cells by mesothelin-overexpressing ovarian cancer cells. In the future, we will test the in vitro function of irradiated CAR.NK cells, given the requirement of irradiating NK92 cell lines prior to clinical use. Next, we will test our platform in an ovarian cancer xenograft mouse model and examine the effects of our therapy on tumor growth. Finally, we will investigate the potential of this therapy to reverse immune suppression on the TME-resident tumor-infiltrating NK cells.

Immunophenotypic and functional characterisation of Mesodermal Killer (MK) cells: a novel cell type and potential cellular therapy for cancer

Background & Aim We have identified a novel cell type, named mesodermal killer (MK) cells, derived from adult bone marrow under proprietary culture conditions. This study characterized the MK cells by immunophenotype, cytokine profile, immunomodulatory function and cytotoxicity. Methods, Results & Conclusion Methods: We investigated MK cells before and after activation with TNFα or IFNγ for immunophenotype using high throughput-FACS, for secretome and for inflammatory response in the murine air pouch assay. We evaluated MK cell cytotoxicity using the chromium release assay and immunomodulatory function by co-culture with natural killer (NK) cells in transwells. We used immunocytochemistry, flow cytometry and RT-PCR to detect expression of granzyme/perforin genes and proteins and investigated death pathways by siRNA silencing of FasL and TRAIL. Results MK cells displayed a novel immunophenotype (and different from BM-MSCs) including expression of CD16, CD96, CD112, CD137L, FasL, TRAIL and CD277 and some NK cell markers. In contrast to NK cells, MK cells lacked CD56 expression. TNFα and IFNγ activation increased FasL expression. MK cells secreted GROa, IL-12, IL-2Ra, IL-8, TRAIL and IL-6, and GROa and IL-8 secretion was increased by TNFα. MK cell cytotoxicity was observed against leukemia (K562), multiple myeloma (RPMI-8226) and breast cancer (MCF7) cell lines. MK cell conditioned media had no effect on MCF7 and antibody dependent cell mediated cytotoxicity (ADCC) experiments also proved negative. MK cells expressed granzymes and perforin and these were upregulated following exposure to RPMI-8226 (in contrast to BM-MSCs). Pre-treatment of MK cells with EGTA (a non-specific inhibitor of granule exocytosis) demonstrated >50% decrease in cytotoxicity against MCF7 suggesting a partial role of granule exocytosis in MK cell cytotoxicity. Inhibition of TRAIL expression by siRNA led to a significant reduction in MK cell cytotoxicity suggesting TRAIL may also be involved. In contrast to BM-MSCs, MK cells enhanced the cytotoxicity of primary NK cells against K562, RPMI-8226 and U226 (plasma cell myeloma) and IFNγ-activated MK cells enhanced migration of NK cells and monocytes into the murine air pouch suggesting a pro-inflammatory response. Conclusion MK cells exhibit cytotoxicity against a variety of cancer cell lines, are capable of priming NK cells in vitro and are pro-inflammatory in vivo. These results suggest the clinical translation of the MK cells is warranted and animal efficacy studies will begin shortly.

Sesamolin promotes cytolysis and migration activity of natural killer cells via dendritic cells.

The defense mechanism of the immune system is based on the interaction of many kinds of leukocytes. Among them, dendritic cells (DCs) control most immune responses. In our previous study, sesamolin was shown to create an optimal environment for natural killer (NK) cells to kill cancer cells. Here we attempted to demonstrate how sesamolin influences DCs to promote the killing and migration activity of NK cells. We co-cultured DCs and NK cells and analyzed the communication between them. NK cells co-cultured with 5 µg/ml sesamolin-treated mature dendritic cells (mDCs) had better cytolytic activity than did NK cells or mDCs co-cultured NK cells. Moreover, the migration of NK cells toward mDCs was enhanced compared to immature dendritic cells (iDCs). The migration of NK cells stimulated by mDCs was stronger after sesamolin activation of the mDCs. Altogether, this study demonstrated that sesamolin activated NK cells by modulating the differentiation and activation of DCs.

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells' ability to eradicate dysfunctional target cells.

The role of opioids in cancer progression.

The role of opioids in cancer progression.

S1PR5 regulates NK cell responses in preventing graft-versus-host disease while preserving graft-versus-tumour activity in a murine allogeneic haematopoietic stem cell transplantation model.

Graft-versus-host disease (GVHD) remains a major complication following allogeneic haematopoietic stem cell transplantation (allo-HSCT) leading to high transplant-related mortality. Natural killer (NK) cells have been found to mitigate GVHD without attenuating the graft-versus-tumour (GVT) activity in the murine model of haematopoietic stem cell transplantation. Sphingosine-1-phosphate receptor 5 (S1PR5) is a very important chemokine receptor on NK cells that governs NK cell distribution in vivo and trafficking at lesion sites. Our preliminary studies showed that the incidence of GVHD was negatively correlated with S1PR5 expression in the NK cells of patients after allo-HSCT. In the present study, we found that S1PR5 deficiency in murine NK cells blocked the migration of NK cells from the bone marrow to the GVHD target organs and attenuated the inhibitory effects on the alloreactive T cells, especially CD3+ CD8+ T cells, which may be the reason why the loss of S1PR5 in NK cells could aggravate GVHD in recipient mice. Furthermore, we also demonstrated that the absence of S1PR5 expression in NK cells did not interfere with the antitumour effects of NK cells and T cells in vivo. Taken together, our data indicate that S1PR5 plays an essential role in balancing GVHD and GVT activity.

The IRE1-XBP1s Pathway Impairment Underpins NK Cell Dysfunction in Hodgkin Lymphoma, That Is Partly Restored By PD-1 Blockade

Background The frequent loss of Major Histocompatibility Complex I molecule (MHC-I) on Hodgkin/Reed-Sternberg (HRS)-cells renders them susceptible to Natural Killer (NK) cell-mediated lysis in Hodgkin Lymphoma (HL). Optimal NK cell function involves migration from peripheral blood to sites of disease, formation of an immune synapse (NKIS) between NK cells and HRS cells, and release of effector molecules. We recently showed that PD-1+ NK cells are expanded in the circulation of patients with HL (Vari, F Blood 2018), and that PD-1 blockade enhances their anti-HRS capabilities. However, mechanisms behind the functional impairment of NK cells in HL patients and the impact PD-1 blockade has on NK cell function remain to be established. Although the IRE1-XBP1s pathway, part of the unfolded protein response (UPR) system, has established and fundamental roles in macrophage, B, T and dendritic cells homeostatic function, its involvement in NK cells remains unknown. We hypothesized that IRE1-XBP1s dysfunction contributes to NK cell impairment and tested the impact of PD-1 blockade on individual components of NK cell function, including migration, NKIS formation, and cytokine release. Methods Ex-vivo functional assays were performed on blood from 20 participants. Confocal microscopy, time-lapse imaging, trans-well migration, and functional in-vitro immune assays were utilized on a range of NK and HRS cell lines, with and without IRE1-XBP1s small molecule inhibitors (4µ8c and 6-bromo) and/or PD-1 blockade (pembrolizumab). Results Stimulation of both NK cell lines and primary NK cells, with HRS lines resulted in marked and rapid IRE1-XBP1s pathway activation. This occurred independently of the canonical UPR and was associated with increased NK cell effector function. However, IRE1-XBP1s pathway inhibition resulted in aberrant NK cell morphology, reduced motility and migration, deficient NKIS formation and impaired interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) release. Next, we tested the IRE1-XBP1s pathway in the pre-therapy blood of patients with HL and compared this with healthy age/gender matched controls. Strikingly, following co-culture with an HRS-line the pathway was not activated, but this abnormality was restricted to the CD56brightCD16-ve subset that we have previously shown to be expanded and enriched in PD-1 (as well as downregulation of the lymphoid migratory chemokine CCR7) in patients with HL. In subsequent experiments using in-vitro expanded populations of primary NK cells from HL patients, IRE1-XBP1s pathway inhibition impaired the migration, NKIS formation, CD107a degranulation, and secretion of IFNγ and TNFα. Effects were partially but not completely restored by addition of PD-1 blockade (Fig 1). Conclusion Here, we outline a hitherto unrecognized mechanism involving the IRE1-XBP1s pathway that is pivotal to NK cell function, including the relatively poorly understood processes of migration, NKIS formation, and cytokine secretion. Notably, IRE1-XBP1s pathway activation is dysfunctional within the PD-1 enriched CD56brightCD16- NK cell subset. Although PD-1 blockade appears to have a multi-faceted beneficial role on NK cell migrational/NKIS and cytokine release capabilities, it is still only capable of partial restoration of NK cell effector function. Further understanding of the pathways operative in NK cells may result in improved immunotherapeutic strategies to enhance this arm of the immune response in patients with HL. Disclosures Gandhi: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Combined NK Cell Therapy and Radiation Therapy Exhibit Long-Term Therapeutic and Antimetastatic Effects in a Human Triple Negative Breast Cancer Model

We investigated whether adoptive cell therapy with ex vivo-activated natural killer (NK) cells enhances the therapeutic efficacy of local tumor radiation therapy (RT) using a human triple-negative breast cancer xenograft model. NK cells from healthy donors were expanded exvivo. MDA-MB-231/Luc-GFP cells were subcutaneously implanted into the thighs of NSG mice. The animals were divided into 4 experimental groups: control, RT, NK, and RT + NK. On day 17 after tumor implantation, tumors from the RT groups were irradiated. The ex vivo-expanded NK cells were intravenously administered twice, on days 17 and 19. Primary and secondary tumors were evaluated using long-term bioluminescence imaging, and histopathology was performed on resected tumor tissue specimens. The luciferase signals of the primary tumors in the RT + NK group were significantly lower than those of comparably sized primary tumors in the RT group. The long-term migration and infiltration of NK cells into the primary tumor sites were significantly higher in RT + NK than in NK mice. Moreover, lymphatic metastasis to the axillary lymph nodes and liver and lung metastases were highly suppressed in the RT + NK group, as demonstrated by BLI and p53 immunohistochemistry. The long-term survival of the RT + NK group was significantly higher than that of the RT or NK groups. Reduction in tumor burden by combining RT and systemic NK cell therapy improved the suppression of primary tumor growth, with efficient NK cell migration and penetration into the primary tumor site. Administered NK cells were maintained in the primary tissue for a significantly longer time in RT + NK group compared with NK group. Both lymphatic spread and distant metastasis to the lungs and liver were effectively suppressed by the combined therapy.

Senescent hepatocytes enhance natural killer cell activity via the CXCL-10/CXCR3 axis.

Cellular senescence and natural killer (NK) cells play an important role in liver diseases. Chemokines, a component of the senescence-associated secretory phenotype, can recruit NK cells and are involved in the development of various liver diseases. The effect of the C-X-C motif chemokine ligand (CXCL)-9, −10, −11/C-X-C motif chemokine receptor (CXCR)3 axis in senescent hepatocytes remains unknown. The chemokines secreted by senescent hepatocytes, the contribution of the CXCL-9, −10, −11/CXCR3 axis to the migration of NK cells, and the effect of senescent hepatocytes on the function of NK cells were investigated in the present study. The results demonstrated significantly increased levels of C-C motif chemokine ligand 2 and CXCL-1, −2 and −10 in the supernatant of senescent AML12 cells. Despite increased mRNA expression of CXCL-9, −10, and −11 in these cells, western blotting revealed significantly enhanced expression of only CXCL-10. The expression of CXCR3 on the surface of NK cells stimulated by senescent AML12 cells was upregulated (fold change, >3). Following incubation with the supernatant of senescent hepatocytes, both CD107a and interferon γ expression in NK cells increased by >2.5-fold. The cytotoxic effect of NK cells was notably higher stimulated by senescent AML12 cells. Chemotaxis and blocking assays demonstrated that the senescent hepatocytes enhanced the migration of NK cells via the CXCL-10/CXCR3 axis. The present study suggests that senescent hepatocytes secrete various chemokines, including CXCL-10, resulting in the upregulation and activation of CXCR3 in NK cells and the enhancement of NK cell migration via the CXCL-10/CXCR3 axis.

Regulation of CX3CL1 Expression in Human First-Trimester Decidual Cells: Implications for Preeclampsia.

C-X3-C motif ligand 1 (CX3CL1) mediates migration, survival, and adhesion of natural killer (NK) cells, monocytes, and T-cells to endothelial/epithelial cells. Aberrant numbers and/or activation of these decidual immune cells elicit preeclampsia development. Decidual macrophages and NK cells are critical for implantation, while macrophage-derived tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β), and NK cell-derived interferon-γ (IFN-γ) are associated with preeclampsia development. Thus, serum and decidual levels of CX3CL1 from first-trimester pregnancy and preeclampsia-complicated term pregnancy were examined by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The effects of incubating primary human first-trimester decidual cells (FTDCs) with estradiol + medroxyprogesterone acetate + either IL-1β or TNF-α and/or IFN-γ on CX3CL1 expression were also assessed by quantitative reverse transcription-polymerase chain reaction and ELISA. The inhibition of each signaling pathway with each kinase and nuclear factor κB (NFκB) inhibitors was evaluated by ELISA. Chemotaxis of CD56brightCD16- NK cells by various concentrations of CX3CL1 was evaluated. C-X3-C motif ligand 1 is expressed by both cytotrophoblasts and decidual cells in first-trimester decidua. C-X3-C motif ligand 1 expression is increased in term decidua but unchanged in first-trimester and term serum of patients with preeclampsia. Interferon-gamma and either IL-1β or TNF-α synergistically upregulated CX3CL1 expression in FTDCs. Coincubation with IL-1β or TNF-α or IFN-γ, mitogen-activated protein kinase kinase 1 and 2 (MEK1/2), c-JUN N-terminal kinase (JNK), and NFκB inhibitors suppressed CX3CL1 production. C-X3-C motif ligand 1 elicited concentration-dependent enhancement of CD56brightCD16- NK cell migration. In conclusion, the current study suggests that decidual cell-secreted CX3CL1 is involved in the later development of preeclampsia, whereas circulating CX3CL1 levels do not predict preeclampsia. Mitogen-activated protein kinase kinase 1 and 2, JNK, and NFκB signaling mediate IL-1β-, TNF-α-, and IFN-γ-induced CX3CL1 production by FTDCs.

A collagen-based microwell migration assay to study NK-target cell interactions

Natural killer (NK) cell cytotoxicity in tissue is dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Here, we have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix. The assay allows for long-term imaging of NK–target cell interactions within a confined 3D volume. We found marked differences in motility between individual cells with a small fraction of the cells moving slowly and being confined to a small volume within the matrix, while other cells moved more freely. A majority of NK cells also exhibited transient variation in their motility, alternating between periods of migration arrest and movement. The assay could be used as a complement to in vivo imaging to study human NK cell heterogeneity in migration and cytotoxicity.

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4R334X.

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the ex vivo manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by ex vivo expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4. Reduced CXCR4 expression was associated with dampened in vitro NK cell migration toward its cognate ligand stromal-derived factor-1α (SDF-1α). NK cells isolated from patients with WHIM syndrome carry gain-of-function (GOF) mutations in CXCR4 (CXCR4R334X). Compared to healthy donors, we observed that NK cells expanded from WHIM patients have similar surface levels of CXCR4 but have a much stronger propensity to home to BM compartments when adoptively infused into NOD-scid IL2Rgammanull (NSG) mice. Therefore, in order to augment the capacity of adoptively infused NK cells to home to the BM, we genetically engineered ex vivo expanded NK cells to express the naturally occurring GOF CXCR4R334X receptor variant. Transfection of CXCR4R334X-coding mRNA into ex vivo expanded NK cells using a clinically applicable method consistently led to an increase in cell surface CXCR4 without altering NK cell phenotype, cytotoxic function, or compromising NK cell viability. Compared to non-transfected and wild type CXCR4-coding mRNA transfected counterparts, CXCR4R334X-engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro. Importantly, expression of CXCR4R334X on expanded NK cells resulted in significantly greater BM homing following adoptive transfer into NSG mice compared to non-transfected NK cell controls. Collectively, these data suggest up-regulation of cell surface CXCR4R334X on ex vivo expanded NK cells via mRNA transfection represents a novel approach to improve homing and target NK cell-based immunotherapies to BM where hematological malignancies reside.

Role of CXCR3 in natural killer cell migration to melanoma cells

Abstract Natural killer (NK) cell recruitment to solid tumors is essential for direct contact between NK and cancer cells and is regulated by chemokine networks. Tumor-derived CXCL10 increases the infiltration of NK cells into solid tumors. In a xenograft mouse model, adoptively transferred human NK cells efficiently infiltrate CXCL10-transduced tumors and enhance regression of tumor growth compared with mock-transduced tumors. Although this study showed that NK cells homed to solid tumor beds in a CXCL10-depedent manner, several questions remain. How do NK cells find cancer cells after extravasation? Which chemokines do B16F10 cells produce? Which chemokine receptors do NK cells use? How do cancer cell-derived chemokines affect NK cell motility? Do these chemokines directly affect NK cell cytotoxicity? To address these issues, we examined the migration dynamics of NK and cancer cells using time-lapse imaging. Although naïve B16F10 cancer cells produce low levels of chemokines, IFN-g-treated B16F10 cells secreted high levels of CXCL10, low levels of CCL5, but did not secrete CCL2, CCL7, or CXCL12. Wild-type NK cells migrated well toward cancer cells and killed them, whereas NK cells deficient in CXCR3 did not. CXCR3-deficient NK cells also showed slower migration speed than did wild-type NK cells. Taken together, our data show that NK cells find cancer cells, at least in part, by sensing CXCL10 produced by cancer cells and suggest that a strategy to increase CXCL10 secretion by cancer cells may improve the efficacy of NK cell–based immunotherapy.

CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities.

Tissue-resident Natural Killer (NK) cells vary in phenotype according to tissue origin, but are typically CD56bright, CXCR6+, and CD69+. NK cells appear very early in fetal development, but little is known about when markers of tissue residency appear during gestation and whether the expression of these markers, most notably the chemokine receptor CXCR6, are associated with differences in functional capability. Using multi-parametric flow cytometry, we interrogated fetal liver and spleen NK cells for the expression of a multitude of extracellular markers associated with NK cell maturation, differentiation, and migration. We analyzed total NK cells from fetal liver and spleen and compared them to their adult liver and spleen counterparts, and peripheral blood (PB) NK. We found that fetal NK cells resemble each other and their adult counterparts more than PB NK. Maturity markers including CD16, CD57, and KIR are lower in fetal NK cells than PB, and markers associated with an immature phenotype are higher in fetal liver and spleen NK cells (NKG2A, CD94, and CD27). However, T-bet/EOMES transcription factor profiles are similar amongst fetal and adult liver and spleen NK cells (T-bet−/EOMES+) but differ from PB NK cells (T-bet+EOMES−). Further, donor-matched fetal liver and spleen NK cells share similar patterns of expression for most markers as a function of gestational age. We also performed functional studies including degranulation, cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) assays. Fetal liver and spleen NK cells displayed limited cytotoxic effector function in chromium release assays but produced copious amounts of TNFα and IFNγ, and degranulated efficiently in response to stimulation with PMA/ionomycin. Further, CXCR6+ NK cells in fetal liver and spleen produce more cytokines and degranulate more robustly than their CXCR6− counterparts, even though CXCR6+ NK cells in fetal liver and spleen possess an immature phenotype. Major differences between CXCR6− and + NK cell subsets appear to occur later in development, as a distinct CXCR6+ NK cell phenotype is much more clearly defined in PB. In conclusion, fetal liver and spleen NK cells share similar phenotypes, resemble their adult counterparts, and already possess a distinct CXCR6+ NK cell population with discrete functional capabilities.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

NCAM Expression Regulates Integrin and Actin Cytoskeletal Function in Human Natural Killer Cells

Human natural killer (NK) cells are innate immune cells that are broadly defined as being CD56+CD3−, however a functional role for CD56 (neural cell adhesion molecule, NCAM) on NK cells has not been described. NCAM is a member of the Ig superfamily that engages in homotypic and heterotypic interactions in cis and in trans. These include NCAM-NCAM interactions and binding to heparin sulfate proteoglycans. In neuronal cells, NCAM plays a role in neurite outgrowth, cell migration and adhesion, in part through direct interactions with focal adhesion kinase (FAK). We have previously shown that CD56 is required for human NK cell migration on stromal cells, and that deletion of CD56 in primary human NK cells significantly impairs this migration. We deleted CD56 by CRISPR-Cas9 mediated gene editing in a human NK cell line and measured the effect of CD56 deletion on adhesion and migration. Using super-resolution (STED) nanoscopy, we found that activation through NK cell activating receptors and/or αLβ2 integrin leads to an increase in the frequency of actin foci >0.05 um2 in the cortical actin of CD56-knockout (KO) NK cells. This was accompanied by increased colocalization of actin and vinculin in actin asters. In wild-type NK cells, we found that CD56 co-localized with β1 integrin in the uropod of migrating cells. Using live-cell interference reflection microscopy, we showed that while adhesion to integrin ligands was not impaired in CD56-KO NK cells, integrin detachment, specifically in the uropod of migrating cells was dysfunctional. Impaired phosphorylation of the FAK homologue Pyk2 in CD56-KO NK cells in response to activation and integrin ligation further supports a novel role for CD56 in regulating integrin detachment. In summary, we have defined NCAM/CD56 as a novel regulator of human immune cell migration.

The R132H mutation in IDH1 promotes the recruitment of NK cells through CX3CL1/CX3CR1 chemotaxis and is correlated with a better prognosis in gliomas.

Mutations in the isocitrate dehydrogenase (IDH) 1 gene, especially the R132H mutation, have been reported to be associated with a better prognosis in glioma patients. However, the underlying molecular mechanisms are not yet well understood. Many factors may contribute to differences in the survival of IDH1 wild-type and IDH1 mutant glioma patients, in which immune components play a potentially important role. In this study, we analyzed The Cancer Genome Atlas (TCGA), and the Chinese Glioma Genome Atlas (CGGA) databases, as well as glioma patient-derived tumor samples. We found that there was a higher infiltration of natural killer (NK) cells in IDH1 mutant glioma patients, and this was correlated with a better prognosis. We also showed that IDH1-R132 tumor cells had higher expression levels of the chemokine CX3CL1. This arises as a result of the conversion of α-ketoglutarate to R(-)-2-hydroxyglutarate by the IDH1 mutant and the resultant phosphorylation of nuclear factor kappa B. Knockdown of CX3CL1 decreased the migration of NK cells. In addition, the high levels of expression of CX3CL1 were positively correlated with glioma patient survival in the TCGA and CGGA databases, and in the clinical samples. Overall, our data have identified a novel mechanism in which R132H mutation of the IDH1 gene serves as a tumor suppressor by promoting the recruitment of NK cells through CX3CL1/CX3CR1 chemotaxis.

Forced Expression of CXCL10 Prevents Liver Metastasis of Colon Carcinoma Cells by the Recruitment of Natural Killer Cells

CXC chemokine ligand 10 (CXCL10) is a CXC chemokine family protein that transmits signals by binding to its specific receptor, CXCR3. CXCL10 is also known as an interferon-γ-inducible chemokine involved in various biological phenomena, including chemotaxis of natural killer (NK) cells and cytotoxic T lymphocytes, that suppress tumor growth and inhibition of angiogenesis. In this study, we examined the effects of forced expression of CXCL10 in a murine colon carcinoma cell line (CT26) on growth and metastasis in syngeneic mice. We first established CT26 cells that were stably expressing murine CXCL10 (CT26/CXCL10) and compared their growth with their parental CT26 cells in vitro and in vivo. The in vitro growth of the CT26/CXCL10 and CT26 cells was comparable, whereas the in vivo growth of the CT26/CXCL10 cells in the skin was strongly suppressed. Liver metastasis of the CT26/CXCL10 cells was also significantly suppressed after intra-splenic implantation. Removal of NK cells by the administration of anti-asialo GM1 antibody canceled the suppression of subcutaneous growth and liver metastasis of CT26/CXCL10 cells. Immunofluorescence clearly showed that abundant NKp46-positive NK cells were recruited into the liver metastatic lesions of the CT26/CXCL10 cells, consistent with specific NK cell migration towards the culture supernatant from the CT26/CXCL10 cells in the chemotaxis assay using transwells. These findings indicate that CXCL10 prevents in vivo growth and metastasis of colon carcinoma cells by recruiting NK cells, suggesting that forced expression of CXCL10 in the colon tumors by gene delivery should lead to a favorable clinical outcome.

Natural killer cell migration control in microchannels by perturbations and topography.

Natural killer (NK) cells are lymphocytes which play an important role in the immune system by recognizing and killing potentially malignant cells without antigen sensitization, and could be utilized in cancer therapy. NK cell migration is an essential process to find and kill target cells, which is well known to be driven by the chemotaxis effect. NK cells also experience a topographical effect induced by the extracellular matrix (ECM) during their migration. However, topographical effects on NK cell locomotion in three dimensional (3D) environments are not well studied yet. In this work, polydimethylsiloxane based platforms containing microchannels with different types of perturbations and decorated with various surface patterns were fabricated to systematically study the topographical effect on NK cell migration with and without the chemotaxis effect. The results showed that perturbation sites in channels induced pauses and reversals in chemotaxis driven NK cell migration. Surface topography such as gratings in confined environments could introduce directional preference to NK cell movement even without chemoattractants. These findings showed that NK cell migration could be controlled by contact guidance, which provides future possibility to manipulate NK cell migration in controlled in vitro bioengineering systems. Results in this study showed that the complex topography of 3D microenvironments in the ECM could have significant effects on NK cell migration in different tissues and organs, and provided insight for explaining the dynamics of NK cell activities in clinical experiments.