Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells.

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( B2M ) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M -deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

In vitro models to study natural killer cell dynamics in the tumor microenvironment.

Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.

Generation of tumor spheroids in microwells to study NK cell cytotoxicity, infiltration and phenotype.

The development of new immunotherapeutic drugs and combinatorial strategies requires the implementation of novel methods to test their efficacy in vitro. Here, we present a series of miniaturized in vitro assays to assess immune cell cytotoxic activity, infiltration, and phenotype in renal carcinoma spheroids with the use of a recently developed multichambered microwell chip. We provide protocols for tumor spheroid formation, NK cell culture, fluorescence labelling and imaging of live or fixed cells directly in the chip together with data analysis.

Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires.

The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.

BACKGROUND: Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. METHODS: We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML. RESULTS: ADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes. CONCLUSIONS: These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Targeting myeloid suppressive cells revives cytotoxic anti-tumor responses in pancreatic cancer.

Immunotherapy for cancer that aims to promote T cell anti-tumor activity has changed current clinical practice, where some previously lethal cancers have now become treatable. However, clinical trials with low response rates have been disappointing for pancreatic ductal adenocarcinoma (PDAC). One suggested explanation is the accumulation of dominantly immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells in the tumor microenvironment (TME). Using retrospectively collected tumor specimens and transcriptomic data from PDAC, we demonstrate that expression of the scavenger receptor MARCO correlates with poor prognosis and a lymphocyte-excluding tumor phenotype. PDAC cell lines produce IL-10 and induce high expression of MARCO in myeloid cells, and this was further enhanced during hypoxic conditions. These myeloid cells suppressed effector T and natural killer (NK) cells and blocked NK cell tumor infiltration and tumor killing in a PDAC 3D-spheroid model. Anti-human MARCO (anti-hMARCO) antibody targeting triggered the repolarization of tumor-associated macrophages and activated the inflammasome machinery, resulting in IL-18 production. This in turn enhanced T cell and NK cell functions. The targeting of MARCO thus remodels the TME and represents a rational approach to make immunotherapy more efficient in PDAC patients.

Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip.

Here, we present a methodology based on multiplexed fluorescence screening of two- or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or immune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluorescence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.

A2B adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models.

BACKGROUND: Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A2ARs. While blockade of the A2AARs subtype effectively rescues lymphocyte activity, with four A2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A2AAR/A2BAR dimers in tissues that coexpress the two receptor subtypes, where the A2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy. METHODS: We report the synthesis and functional evaluation of five potent A2BAR antagonists and a dual A2AAR/A2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models. RESULTS: We provide data for six novel small molecules: five A2BAR selective antagonists and a dual A2AAR/A2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A2AAR antagonist AZD-4635. We find that A2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules. CONCLUSIONS: Our results demonstrate that A2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A2BAR blockade. Inhibition of A2BAR signaling restores T cell function and proliferation. Furthermore, A2BAR and dual A2AAR/A2BAR antagonists showed similar or better results than A2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A2BAR in the regulation of the immune system.

Live single cell imaging assays in glass microwells produced by laser-induced deep etching.

Miniaturization of cell culture substrates enables controlled analysis of living cells in confined micro-scale environments. This is particularly suitable for imaging individual cells over time, as they can be monitored without escaping the imaging field-of-view (FoV). Glass materials are ideal for most microscopy applications. However, with current methods used in life sciences, glass microfabrication is limited in terms of either freedom of design, quality, or throughput. In this work, we introduce laser-induced deep etching (LIDE) as a method for producing glass microwell arrays for live single cell imaging assays. We demonstrate novel microwell arrays with deep, high-aspect ratio wells that have rounded, dimpled or flat bottom profiles in either single-layer or double-layer glass chips. The microwells are evaluated for microscopy-based analysis of long-term cell culture, clonal expansion, laterally organized cell seeding, subcellular mechanics during migration and immune cell cytotoxicity assays of both adherent and suspension cells. It is shown that all types of microwells can support viable cell cultures and imaging with single cell resolution, and we highlight specific benefits of each microwell design for different applications. We believe that high-quality glass microwell arrays enabled by LIDE provide a great option for high-content and high-resolution imaging-based live cell assays with a broad range of potential applications within life sciences.

A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo.

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Radiotherapy transiently reduces the sensitivity of cancer cells to lymphocyte cytotoxicity.

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.

Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids.

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

NK cells integrate signals over large areas when building immune synapses but require local stimuli for degranulation.

Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells and for target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process in which the cytoskeleton, cell surface receptors, and intracellular signaling proteins rearrange into supramolecular activation clusters (SMACs). We generated artificial immune synapses (AIS) consisting of synthetic and natural ligands for the NK cell-activating receptors LFA-1 and CD16 by microcontact printing the ligands into circular-shaped SMAC structures. Live-cell imaging and analysis of fixed human NK cells in this reductionist system showed that the spatial distribution of activating ligands influenced the formation, stability, and outcome of NK cell synapses. Whereas engagement of LFA-1 alone promoted synapse initiation, combined engagement of LFA-1 and CD16 was required for the formation of mature synapses and degranulation. Organizing LFA-1 and CD16 ligands into donut-shaped AIS resulted in fewer long-lasting, symmetrical synapses compared to dot-shaped AIS. NK cells spreading evenly over either AIS shape exhibited similar arrangements of the lytic machinery. However, degranulation only occurred in regions containing ligands that therefore induced local signaling, suggesting the existence of a late checkpoint for degranulation. Our results demonstrate that the spatial organization of ligands in the synapse can affect its outcome, which could be exploited by target cells as an escape mechanism.

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells.

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

Ultrasound-Based Scaffold-Free Core-Shell Multicellular Tumor Spheroid Formation.

In cancer research and drug screening, multicellular tumor spheroids (MCTSs) are a popular model to bridge the gap between in vitro and in vivo. However, the current techniques to culture mixed co-culture MCTSs do not mimic the structural architecture and cellular spatial distribution in solid tumors. In this study we present an acoustic trapping-based core-shell MCTSs culture method using sequential seeding of the core and shell cells into microwells coated with a protein repellent coating. Scaffold-free core-shell ovarian cancer OVCAR-8 cell line MCTSs were cultured, stained, cleared and confocally imaged on-chip. Image analysis techniques were used to quantify the shell thickness (23.2 ± 1.8 µm) and shell coverage percentage (91.2 ± 2.8%). We also show that the shell thickness was evenly distributed over the MCTS cores with the exception of being slightly thinner close to the microwell bottom. This scaffold-free core-shell MCTSs formation technique and the analysis tools presented herein could be used as an internal migration assay within the MCTS or to form core-shell MCTS co-cultures to study therapy response or the interaction between tumor and stromal cells.

High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy.

Three-dimensional cell cultures are able to better mimic the physiology and cellular environments found in tissues in vivo compared to cells grown in two dimensions. In order to study the structure and function of cells in 3-D cultures, light microscopy is frequently used. The preparation of 3-D cell cultures for light microscopy is often destructive, including physical sectioning of the samples, which can result in the loss of 3-D information. In order to probe the structure of 3-D cell cultures at high resolution, we have explored the use of expansion microscopy and compared it to a simple immersion clearing protocol. We provide a practical method for the study of spheroids, organoids and tumor-infiltrating immune cells at high resolution without the loss of spatial organization. Expanded samples are highly transparent, enabling high-resolution imaging over extended volumes by significantly reducing light scatter and absorption. In addition, the hydrogel-like nature of expanded samples enables homogenous antibody labeling of dense epitopes throughout the sample volume. The improved labeling and image quality achieved in expanded samples revealed details in the center of the organoid which were previously only observable following serial sectioning. In comparison to chemically cleared spheroids, the improved signal-to-background ratio of expanded samples greatly improved subsequent methods for image segmentation and analysis.

Recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of Vγ1 γδTCRs.

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1-containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate-like αß T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

A Fluorescent Kinase Inhibitor that Exhibits Diagnostic Changes in Emission upon Binding.

The development of a fluorescent LCK inhibitor that exhibits favourable solvatochromic properties upon binding the kinase is described. Fluorescent properties were realised through the inclusion of a prodan-derived fluorophore into the pharmacophore of an ATP-competitive kinase inhibitor. Fluorescence titration experiments demonstrate the solvatochromic properties of the inhibitor, in which dramatic increase in emission intensity and hypsochromic shift in emission maxima are clearly observed upon binding LCK. Microscopy experiments in cellular contexts together with flow cytometry show that the fluorescence intensity of the inhibitor correlates with the LCK concentration. Furthermore, multiphoton microscopy experiments demonstrate both the rapid cellular uptake of the inhibitor and that the two-photon cross section of the inhibitor is amenable for excitation at 700 nm.

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.