KIR2DS1 and KIR2DL1-C245 Dominantly Repress NK Cell Degranulation Triggered by Monoclonal or Bispecific Antibodies, whereas Education by Uptuning Inhibitory Killer Ig-related Receptors Exerts No Advantage in Ab-dependent Cellular Cytotoxicity.

NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.

Magnetic targeting of adoptively transferred tumour-specific nanoparticle-loaded CD8+ T cells does not improve their tumour infiltration in a mouse model of cancer but promotes the retention of these cells in tumour-draining lymph nodes.

BACKGROUND: Adoptive T cell-transfer (ATC) therapy is a highly promising cancer-treatment approach. However, in vivo-administered T cells tend to disperse, with only a small proportion reaching the tumour. To remedy this, magnetic targeting of T cells has been recently explored. Magnetic nanoparticles (MNPs) functionalised with antibodies were attached to effector T cells and magnetically recruited to tumour sites under MRI guidance. In this study, we investigated whether 3-aminopropyl-triethoxysilane (APS)-coated MNPs directly attached to CD8+ T cell membranes could also magnetically target and accumulate tumour-specific CD8+ T cells in solid tumours using an external magnetic field (EMF). As it has been shown that T cells associated with APS-coated MNPs are retained in lymph nodes (LNs), and tumour-draining LNs are the most common sites of solid-tumour metastases, we further evaluated whether magnetic targeting of APS-MNP-loaded CD8+ T cells could cause them to accumulate in tumour-draining LNs. RESULTS: First, we show that antigen-specific CD8+ T cells preserve their antitumor activity in vitro when associated with APS-MNPs. Next, we demonstrate that the application of a magnetic field enhanced the retention of APS-MNP-loaded OT-I CD8+ T cells under flow conditions in vitro. Using a syngeneic mouse model, we found similar numbers of APS-MNP-loaded OT-I CD8+ T cells and OT-I CD8+ T cells infiltrating the tumour 14 days after cell transfer. However, when a magnet was placed near the tumour during the transfer of tumour-specific APS-MNP-loaded CD8+ T cells to improve tumour infiltration, a reduced percentage of tumour-specific T cells was found infiltrating the tumour 14 days after cell transfer, which was reflected in a smaller reduction in tumour size compared to tumour-specific CD8+ T cells transferred with or without MNPs in the absence of a magnetic field. Nonetheless, magnet placement near the tumour site during cell transfer induced infiltration of activated tumour-specific CD8+ T cells in tumour-draining LNs, which remained 14 days after cell transfer. CONCLUSIONS: The use of an EMF to improve targeting of tumour-specific T cells modified with APS-MNPs reduced the percentage of these cells infiltrating the tumour, but promoted the retention and the persistence of these cells in the tumour-draining LNs.

T cells loaded with magnetic nanoparticles are retained in peripheral lymph nodes by the application of a magnetic field.

BACKGROUND: T lymphocytes are highly dynamic elements of the immune system with a tightly regulated migration. T cell-based transfer therapies are promising therapeutic approaches which in vivo efficacy is often limited by the small proportion of administered cells that reaches the region of interest. Manipulating T cell localisation to improve specific targeting will increase the effectiveness of these therapies. Nanotechnology has been successfully used for localized release of drugs and biomolecules. In particular, magnetic nanoparticles (MNPs) loaded with biomolecules can be specifically targeted to a location by an external magnetic field (EMF). The present work studies whether MNP-loaded T cells could be targeted and retained in vitro and in vivo at a site of interest with an EMF. RESULTS: T cells were unable to internalize the different MNPs used in this study, which remained in close association with the cell membrane. T cells loaded with an appropriate MNP concentration were attracted to an EMF and retained in an in vitro capillary flow-system. MNP-loaded T cells were also magnetically retained in the lymph nodes after adoptive transfer in in vivo models. This enhanced in vivo retention was in part due to the EMF application and to a reduced circulating cell speed within the organ. This combined use of MNPs and EMFs did not alter T cell viability or function. CONCLUSIONS: These studies reveal a promising approach to favour cell retention that could be implemented to improve cell-based therapy.

Polyethylenimine-coated superparamagnetic iron oxide nanoparticles impair in vitro and in vivo angiogenesis.

Endothelial cells are essential to tumor vascularization and impairing their activity can potentially limit tumor growth. Since polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles (SPIONs) are bioactive nanosystems that modulate inflammatory macrophage responses and limit tumor cell invasion, we evaluated their effects on endothelial cell angiogenesis. PEI-SPION triggered proinflammatory gene profiles in a murine endothelial cell line and in primary human umbilical cord vein endothelial cells (HUVECs). These nanoparticles impaired endothelial cell migration and inhibited HUVEC tube formation. Magnetically tumor-targeted PEI-SPIONs reduced tumor vessel numbers and promoted intratumor macrophage infiltration in a tumor xenograft model. PEI-SPION treatment impaired M2 macrophage-promoted tube formation and affected HUVEC cytoskeleton by limiting Src and Cortactin activation. These mechanisms could contribute to PEI-SPION in vitro and in vivo antiangiogenic potential. These data confirm that PEI-SPION administration and application of a localized magnetic field could offer an affordable anti-angiogenic anti-tumoral targeted treatment that would complement other therapies.

Superparamagnetic iron oxide nanoparticle uptake alters M2 macrophage phenotype, iron metabolism, migration and invasion.

Superparamagnetic iron oxide nanoparticles (SPIONs) have shown promise as contrast agents and nanocarriers for drug delivery. Their impact on M2-polarised macrophages has nonetheless not been well studied. Here we explored the effects of SPIONs coated with dimercaptosuccinic acid, aminopropyl silane or aminodextran in two M2 macrophage models (murine primary IL-4-activated bone marrow-derived macrophages and human M2-like differentiated THP-1 cells). All SPIONs were internalised and no cell toxicity was observed. SPION treatment produced reactive oxygen species and activated the extracellular signal-regulated kinase and AKT pathways. After 24-h SPION incubation, M2 macrophages switched their iron metabolism towards an iron-replete state. SPION treatment in both M2 macrophage models altered their M2 activation profiles, promoted IL-10 production, and stimulated protease-dependent invasion. These results highlight the need to evaluate the interactions between SPIONs and cells to take full advantage of the intrinsic properties of these nanoparticles in biological systems. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxide nanoparticles (SPIONs) have been used as an MRI contrast agent in many experimental studies. The authors here investigated the effects of these nanoparticles on M2 macrophages after cellular uptake. The findings of cell activation further enhanced our current knowledge on the interaction of SPIONS with macrophages.

Harnessing upregulated E-selectin while enhancing SDF-1α sensing redirects infused NK cells to the AML-perturbed bone marrow.

Increased bone marrow (BM) homing of NK cells is associated with positive outcome in patients with acute myeloid leukemia (AML) treated within adoptive NK cell transfer trials. While most efforts to further improve the efficacy focus on augmenting NK cell persistence and cytotoxicity, few address their ability to home to the tumor. Here, we decipher how AML growth alters the BM niche to impair NK cell infiltration and how insights can be utilized to resolve this issue. We show that AML development gradually impairs the BM homing capacity of infused NK cells, which was tightly linked to loss of SDF-1α in this environment. AML development also triggered up-regulation of E-selectin on BM endothelial cells. Given the poor E-selectin-binding capacity of NK cells, introduction of fucosyltransferase-7 (FUT7) to the NK cells per mRNA transfection resulted in potent E-selectin binding and stronger adhesion to E-selectin+ endothelial cells. Co-introduction of FUT7 and gain-of-function CXCR4 (CXCR4R334X) redirected NK cell homing to the BM of AML-bearing mice nearly to the levels in AML-free mice. This work shows how impaired NK cell homing caused by AML-induced microenvironmental changes can be overcome by genetic engineering. We speculate our insights can help further advance future NK cell immunotherapies.

Redirecting NK cells to the lymph nodes to augment their lymphoma-targeting capacity.

CAR-NK cells can induce remission in lymphoma patients. We speculate that the full potential of adoptive NK cell immunotherapy against lymphoma is restricted by their poor lymph node (LN) homing capacity. Here, we have utilized a clinically approved transfection method with the aim of redirecting NK cells to LNs. Electroporation of ex vivo expanded NK cells with mRNAs coding for CCR7, CXCR5, and CD62L resulted in increased in vitro migration towards chemokines and mouse LN-derived supernatant. Following infusion into SCID/Beige mice, modified NK cells showed enhanced LN homing. Importantly, lymphoma patient-derived NK cells were equally well expanded and engineered as healthy donor NK cells, highlighting their translational potential. Additionally, the introduction of high-affinity CD16, together with the homing molecules, also augmented their ADCC capacity against autologous lymphoma cells. Hence, genetic engineering can be utilized to enhance NK cell LN homing. The homing concept may synergize with CAR- or monoclonal/bi-/tri-specific antibody-based approaches.

Iron Oxide Nanoparticle Coatings Dictate Cell Outcomes Despite the Influence of Protein Coronas.

A critical issue in nanomedicine is to understand the complex dynamics that dictate the interactions of nanoparticles (NPs) with their biological milieu. The most exposed part of a nanoparticle is its surface coating, which comes into contact with the biological medium and adsorbs proteins, forming what is known as a protein corona (PC). It is assumed that this PC mainly dictates the nanoparticle-cell interactions. As such, we set out to analyze how different coatings on iron oxide nanoparticles (MNPs) affect the composition of the PC that forms on top of them, and how these newly formed coronas influence the uptake of MNPs by macrophages and tumor cells, their subcellular location upon internalization, and their intracellular degradation. We found that different superficial charges of the coatings did not affect the PC composition, with an enrichment in proteins with affinity for divalent ions regardless of the type of coating. The iron oxide core of the MNP might become exposed to the biological medium, influencing the proteins that constitute the PCs. The presence of enzymes with hydrolase activity in the PC could explain the degradation of the coatings when they come into contact with the biological media. In terms of MNP internalization by cells, coatings mainly determine the endocytic pathways used, especially in terms of receptor-mediated endocytosis. However, the increase in hydrodynamic size provoked by the formation of the associated corona drives uptake mechanisms like macropinocytosis. Once inside the cells, the PC protected the NPs in their intracellular transit to lysosomes, where they were fully degraded. This understanding of how coatings and PCs influence different cellular processes will help design improved NPs for biomedical applications, taking into account the influence of the coating and corona on the biology of the NPs.

Improving Tumor Retention of Effector Cells in Adoptive Cell Transfer Therapies by Magnetic Targeting.

Adoptive cell transfer therapy is a promising anti-tumor immunotherapy in which effector immune cells are transferred to patients to treat tumors. However, one of its main limitations is the inefficient trafficking of inoculated effector cells to the tumor site and the small percentage of effector cells that remain activated when reaching the tumor. Multiple strategies have been attempted to improve the entry of effector cells into the tumor environment, often based on tumor types. It would be, however, interesting to develop a more general approach, to improve and facilitate the migration of specific activated effector lymphoid cells to any tumor type. We and others have recently demonstrated the potential for adoptive cell transfer therapy of the combined use of magnetic nanoparticle-loaded lymphoid effector cells together with the application of an external magnetic field to promote the accumulation and retention of lymphoid cells in specific body locations. The aim of this review is to summarize and highlight the recent findings in the field of magnetic accumulation and retention of effector cells in tumors after adoptive transfer, and to discuss the possibility of using this approach for tumor targeting with chimeric antigen receptor (CAR) T-cells.

Magnetic Nanoparticles Attached to the NK Cell Surface for Tumor Targeting in Adoptive Transfer Therapies Does Not Affect Cellular Effector Functions.

Adoptive cell transfer therapy is currently one of the most promising approaches for cancer treatment. This therapy has some limitations, however, such as the dispersion of in vivo-administered cells, causing only a small proportion to reach the tumor. Nanotechnological approaches could offer a solution for this drawback, as they can increase cell retention and accumulation in a region of interest. In particular, strategies employing magnetic nanoparticles (MNPs) to improve targeting of adoptively transferred T or NK cells have been explored in mice. In vivo magnetic retention is reported using the human NK cell line NK-92MI transfected with MNPs. Primary NK cells are nonetheless highly resistant to transfection, and thus we explore in here the possibility of attaching the MNPs to the NK cell surface to overcome this issue, and examine whether this association would affect NK effector functions. We assessed the attachment of MNPs coated with different polymers to the NK cell surface, and found that APS-MNP attached more efficiently to the NK-92MI cell surface. In association with MNPs, these cells preserved their main functions, exhibiting a continued capacity to degranulate, conjugate with and lyse target cells, produce IFN-γ, and respond to chemotactic signals. MNP-loaded NK-92MI cells were also retained in an in vitro capillary flow system by applying an EMF. A similar analysis was carried out in primary NK cells, isolated from mice, and expanded in vitro. These primary murine NK cells also maintained their functionality intact after MNP treatment and were successfully retained in vitro. This work therefore provides further support for using MNPs in combination with EMFs to favor specific retention of functional NK cells in a region of interest, which may prove beneficial to adoptive cell-therapy protocols.

Time-course assessment of the aggregation and metabolization of magnetic nanoparticles.

To successfully develop biomedical applications for magnetic nanoparticles, it is imperative that these nanoreagents maintain their magnetic properties in vivo and that their by-products are safely metabolized. When placed in biological milieu or internalized into cells, nanoparticle aggregation degree can increase which could affect magnetic properties and metabolization. To evaluate these aggregation effects, we synthesized citric acid-coated iron oxide nanoparticles whose magnetic susceptibility can be modified by aggregation in agar dilutions and dextran-layered counterparts that maintain their magnetic properties unchanged. Macrophage models were used for in vitro uptake and metabolization studies, as these cells control iron homeostasis in the organism. Electron microscopy and magnetic susceptibility studies revealed a cellular mechanism of nanoparticle degradation, in which a small fraction of the particles is rapidly degraded while the remaining ones maintain their size. Both nanoparticle types produced similar iron metabolic profiles but these profiles differed in each macrophage model. Thus, nanoparticles induced iron responses that depended on macrophage programming. In vivo studies showed that nanoparticles susceptible to changes in magnetic properties through aggregation effects had different behavior in lungs, liver and spleen. Liver ferritin levels increased in these animals showing that nanoparticles are degraded and their by-products incorporated into normal metabolic routes. These data show that nanoparticle iron metabolization depends on cell type and highlight the necessity to assess nanoparticle aggregation in complex biological systems to develop effective in vivo biomedical applications. STATEMENT OF SIGNIFICANCE: Magnetic iron oxide nanoparticles have great potential for biomedical applications. It is however imperative that these nanoreagents preserve their magnetic properties once inoculated, and that their degradation products can be eliminated. When placed in a biological milieu nanoparticles can aggregate and this can affect their magnetic properties and their degradation. In this work, we showed that iron oxide nanoparticles trigger the iron metabolism in macrophages, the main cell type involved in iron homeostasis in the organism. We also show that aggregation can affect nanoparticle magnetic properties when inoculated in animal models. This work confirms iron oxide nanoparticle biocompatibility and highlights the necessity to assess in vivo nanoparticle aggregation to successfully develop biomedical applications.

PI3K p85 ß 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 p85ß subunit in NK cells. Using p85ß knockout mice, we found that p85ß deficiency does not alter NK cell differentiation and maturation in spleen or bone marrow. NK cells from p85ß-/- 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 p85ß deficiency impaired NKG2D internalization, which could contribute to the activated phenotype. Decreasing p85ß subunit protein levels might thus constitute a therapeutic target to promote NK cell activity toward NKG2D ligand-expressing cells.