Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality.

Successful translation of chimeric antigen receptor (CAR) T cell therapy for the treatment of solid tumors has proved to be troublesome, mainly due to the complex tumor microenvironment promoting T cell dysfunction and antigen heterogeneity. Mesothelin (MSLN) has emerged as an attractive target for CAR T cell therapy of several solid malignancies, including ovarian cancer. To improve clinical response rates with MSLN-CAR T cells, a better understanding of the mechanisms impacting CAR T cell functionality in vitro is crucial. Here, we demonstrated superior cytolytic capacity of CD28-costimulated MSLN-CAR T cells (M28z) relative to 4-1BB-costimulated MSLN-CAR T cells (MBBz). Furthermore, CD28-costimulated MSLN CAR T cells displayed enhanced cytolytic capacity against tumor spheroids with heterogeneous MSLN expression compared to MBBz CAR T cells. In this study, we identified CAR-mediated trogocytosis as a potential impeding factor for successful MSLN-CAR T cell therapy due to fratricide killing and contributing to tumor antigen heterogeneity. Moreover, we link antigen-dependent upregulation of LAG-3 with reduced CAR T cell functionality. Taken together, our study highlights the therapeutic potential and bottlenecks of MSLN-CAR T cells, providing a rationale for combinatorial treatment strategies.

Targeting of Nrf2 improves antitumoral responses by human NK cells, TIL and CAR T cells during oxidative stress.

BACKGROUND: Adoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair NK cell and T cell function. Auranofin, a gold (I)-containing phosphine compound, is a strong activator of the transcription factor Nrf2. Nrf2 controls a wide range of downstream targets important for the cells to obtain increased resistance to ROS. In this study, we present a strategy using auranofin to render human cytotoxic lymphocytes resistant toward oxidative stress. METHODS: Melanoma patient-derived tumor infiltrating lymphocytes (TIL) and healthy donor-derived NK cells and CD19-directed CAR T cells were pretreated with a low dose of auranofin. Their resistance toward oxidative stress was assessed by measuring antitumoral responses (killing-assay, degranulation/CD107a, cytokine production) and intracellular ROS levels (flow cytometry) in conditions of oxidative stress. To confirm that the effects were Nrf2 dependent, the transcription level of Nrf2-driven target genes was analyzed by qPCR. RESULTS: Pretreatment of human TIL and NK cells ex vivo with a low-dose auranofin significantly lowered their accumulation of intracellular ROS and preserved their antitumoral activity despite high H2O2 levels or monocyte-derived ROS. Furthermore, auranofin pretreatment of CD19 CAR-T cells or TIL increased their elimination of CD19 +tumor cells or autologous tumor spheroids, respectively, especially during ROS exposure. Analysis of Nrf2-driven target genes revealed that the increased resistance against ROS was Nrf2 dependent. CONCLUSION: These novel findings suggest that Nrf2 activation in human cytotoxic lymphocytes could be used to enhance the efficacy of adoptive cell therapy.

Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads.

For the past decade, adoptive cell therapy including tumor-infiltrating lymphocytes, genetically modified cytotoxic lymphocytes expressing a chimeric antigen receptor, or a novel T-cell receptor has revolutionized the treatment of many cancers. Progress within exome sequencing and neoantigen prediction technologies provides opportunities for further development of personalized immunotherapies. In this study, we present a novel strategy to deliver in silico predicted neoantigens to autologous dendritic cells (DCs) using paramagnetic beads (EpiTCer beads). DCs pulsed with EpiTCer beads are superior in enriching for healthy donor and patient blood-derived tumor-specific CD8+ T cells compared to DC loaded with whole-tumor lysate or 9mer neoantigen peptides. A dose-dependent effect was observed, with higher EpiTCer bead per DC being favorable. We concluded that CD8+ T cells enriched by DC loaded with EpiTCer beads are tumor specific with limited tumor cross-reactivity and low recognition of autologous non-activated monocytes or CD8+ T cells. Furthermore, tumor specificity and recognition were improved and preserved after additional expansion using our Good Manufacturing Process (GMP)-compatible rapid expansion protocol. Phenotypic analysis of patient-derived EpiTCer DC expanded CD8+ T cells revealed efficient maturation, with high frequencies of central memory and effector memory T cells, similar to those observed in autologous expanded tumor-infiltrating lymphocytes. These results indicate that DC pulsed with EpiTCer beads enrich for a T-cell population with high capacity of tumor recognition and elimination, which are features needed for a T-cell product to be used for personalized adoptive cell therapy.

Interleukin-33 is a Novel Immunosuppressor that Protects Cancer Cells from TIL Killing by a Macrophage-Mediated Shedding Mechanism.

Recognition of specific antigens expressed in cancer cells is the initial process of cytolytic T cell-mediated cancer killing. However, this process can be affected by other non-cancerous cellular components in the tumor microenvironment. Here, it is shown that interleukin-33 (IL-33)-activated macrophages protect melanoma cells from tumor-infiltrating lymphocyte-mediated killing. Mechanistically, IL-33 markedly upregulates metalloprotease 9 (MMP-9) expression in macrophages, which acts as a sheddase to trim NKG2D, an activating receptor expressed on the surface of natural killer (NK) cells, CD8+ T cells, subsets of CD4+ T cells, iNKT cells, and γδ T cells. Further, MMP-9 also cleaves the MHC class I molecule, cell surface antigen-presenting complex molecules, expressed in melanoma cells. Consequently, IL-33-induced macrophage MMP-9 robustly mitigates the tumor killing-effect by T cells. Genetic and pharmacological loss-of-function of MMP-9 sheddase restore T cell-mediated cancer killing. Together, these data provide compelling in vitro and in vivo evidence showing novel mechanisms underlying the IL-33-macrophage-MMP-9 axis-mediated immune tolerance against cancer cells. Targeting each of these signaling components, including IL-33 and MMP-9 provides a new therapeutic paradigm for improving anticancer efficacy by immune therapy.

Cisplatin inhibits frequency and suppressive activity of monocytic myeloid-derived suppressor cells in cancer patients.

Cancer immunotherapies have induced long-lasting responses in cancer patients including those with melanoma and head and neck squamous cell carcinoma (HNSCC). However, the majority of treated patients does not achieve clinical benefit from immunotherapy because of systemic tumor-induced immunosuppression. Monocytic myeloid-derived suppressor cells (M-MDSCs) are implicated as key players in inhibiting anti-tumor immune responses and their frequencies are closely associated with tumor progression. Tumor-derived signals, including signaling via STAT3-COX-2, induce the transformation of monocytic precursors into suppressive M-MDSCs. In a retrospective assessment, we observed that survival of melanoma patients undergoing dendritic cell vaccination was negatively associated with blood M-MDSC levels. Previously, it was shown that platinum-based chemotherapeutics inhibit STAT signaling. Here, we show that cisplatin and oxaliplatin treatment interfere with the development of M-MDSCs, potentially synergizing with cancer immunotherapy. In vitro, subclinical doses of platinum-based drugs prevented the generation of COX-2+ M-MDSCs induced by tumor cells from melanoma patients. This was confirmed in HNSCC patients where intravenous cisplatin treatment drastically lowered M-MDSC frequency while monocyte levels remained stable. In treated patients, expression of COX-2 and arginase-1 in M-MDSCs was significantly decreased after two rounds of cisplatin, indicating inhibition of STAT3 signaling. In line, the capacity of M-MDSCs to inhibit activated T cell responses ex vivo was significantly decreased after patients received cisplatin. These results show that platinum-based chemotherapeutics inhibit the expansion and suppressive activity of M-MDSCs in vitro and in cancer patients. Therefore, platinum-based drugs have the potential to enhance response rates of immunotherapy by overcoming M-MDSC-mediated immunosuppression.

Predicting anti-PD-1 responders in malignant melanoma from the frequency of S100A9+ monocytes in the blood.

BACKGROUND: While programmed cell death receptor 1 (PD-1) blockade treatment has revolutionized treatment of patients with melanoma, clinical outcomes are highly variable, and only a fraction of patients show durable responses. Therefore, there is a clear need for predictive biomarkers to select patients who will benefit from the treatment. METHOD: To identify potential predictive markers for response to PD-1 checkpoint blockade immunotherapy, we conducted single-cell RNA sequencing analyses of peripheral blood mononuclear cells (PBMC) (n=8), as well as an in-depth immune monitoring study (n=20) by flow cytometry in patients with advanced melanoma undergoing treatment with nivolumab at Karolinska University Hospital. Blood samples were collected before the start of treatment and at the time of the second dose. RESULTS: Unbiased single-cell RNA sequencing of PBMC in patients with melanoma uncovered that a higher frequency of monocytes and a lower ratio of CD4+ T cells to monocyte were inversely associated with overall survival. Similarly, S100A9 expression in the monocytic subset was correlated inversely with overall survival. These results were confirmed by a flow cytometry-based analysis in an independent patient cohort. CONCLUSION: Our results suggest that monocytic cell populations can critically determine the outcome of PD-1 blockade, particularly the subset expressing S100A9, which should be further explored as a possible predictive biomarker. Detailed knowledge of the biological role of S100A9+ monocytes is of high translational relevance.

Counteracting CAR T cell dysfunction.

In spite of high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, the efficacy of this approach is limited by generation of dysfunctional CAR T cells in vivo, conceivably induced by immunosuppressive tumor microenvironment (TME) and excessive antigen exposure. Exhaustion and senescence are two critical dysfunctional states that impose a pivotal hurdle for successful CAR T cell therapies. Recently, modified CAR T cells with an "exhaustion-resistant" phenotype have shown superior antitumor functions and prolonged lifespan. In addition, several studies have indicated the feasibility of senescence delay in CAR T cells. Here, we review the latest reports regarding blockade of CAR T cell exhaustion and senescence with a particular focus on the exhaustion-inducing pathways. Subsequently, we describe what potential these latest insights offer for boosting the potency of adoptive cell transfer (ACT) therapies involving CAR T cells. Furthermore, we discuss how induction of costimulation, cytokine exposure, and TME modulation can impact on CAR T cell efficacy and persistence, while potential safety issues associated with reinvigorated CAR T cells will also be addressed.

Targeting a scavenger receptor on tumor-associated macrophages activates tumor cell killing by natural killer cells.

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.

PD-1 checkpoint blockade in advanced melanoma patients: NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates.

Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival (OS) and a dramatic improvement in patient quality of life. Despite the success of this approach, the number of benefitting patients is limited and there is a need for predictive biomarkers as well as a deeper mechanistic analysis of the cellular populations involved in clinical responses. With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients receiving pembrolizumab or nivolumab treatment at Karolinska University Hospital. Blood samples were collected before treatment and before administration of the second and fourth doses. Peripheral blood mononuclear cells were isolated and stained for flow cytometric analysis within 2 h of sample collection. Overall survival and progression-free survival (PFS) were inversely correlated with CD69 expression NK cells. In the myeloid compartment, high frequencies of non-classical monocytes and low frequencies of monocytic myeloid derived suppressor cells (MoMDSCs) correlated with response rates and OS. A deeper characterization of monocytic subsets showed that PD-L1 expression in MDSCs, non-classical and intermediate monocytes was significantly increased in patients with shorter PFS in addition to correlating inversely with OS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of activated NK cells and monocytic subsets are inversely correlated with survival and clinical benefit, suggesting that their role as predictive biomarkers should be further evaluated.

Complete and long-lasting clinical responses in immune checkpoint inhibitor-resistant, metastasized melanoma treated with adoptive T cell transfer combined with DC vaccination.

Development of T cell-directed immune checkpoint inhibitors (ICI) has revolutionized metastatic melanoma (MM) therapy, but <50% of treated patients experience durable responses. This phase I trial (NCT01946373) investigates the safety/feasibility of tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) combined with dendritic cell (DC) vaccination in MM patients progressing on ICI. An initial cohort (5 patients) received TIL therapy alone to evaluate safety and allow for optimization of TIL expansion protocols. A second cohort (first-in-man, 5 patients) received TIL combined with autologous tumor lysate-loaded DC vaccination. All patients received cyclophosphamide/fludarabine preconditioning prior to, and intravenous (i.v.) IL-2 after, TIL transfer. The DC vaccine was given as five intradermal injections after TIL and IL-2 administration. [18F]-FDG PET/CT radiology was performed to evaluate clinical response, according to RECIST 1.1 (on the CT part). Immunological monitoring was performed by flow cytometry and T-cell receptor (TCR) sequencing. In the safety/optimization cohort, all patients had a mixed response or stable disease, but none durable. In the combination cohort, two patients experienced complete responses (CR) that are still ongoing (>36 and >18 months, respectively). In addition, two patients had partial responses (PR), one still ongoing (>42 months) with only a small bone-lesion remaining, and one of short duration (<4 months). One patient died early during treatment and did not receive DC. Long-lasting persistency of the injected TILs was demonstrated in blood. In summary, we report clinical responses by TIL therapy combined with DC vaccination in 4 out of 4 treated MM patients who previously failed ICI.

Visualization of human T lymphocyte-mediated eradication of cancer cells in vivo.

Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.

Caveolin-1-Mediated Tumor Suppression Is Linked to Reduced HIF1α S-Nitrosylation and Transcriptional Activity in Hypoxia.

Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.

Genetically modified immune cells targeting tumor antigens.

Immunotherapy approaches consisting of genetically modified immune cells have become a promising platform for cancer treatment. Such 'living' therapies targeting tumor antigens have shown success in many cancer patients in the form of durable responses in a growing number of clinical studies. Besides, a large number of ongoing studies have been designed to introduce reliable methods for identification of tumor antigens. In addition, technical and biotechnological developments are being applied to the generation and expansion of genetically modified immune cells. In this review, we summarize and discuss the latest progress and current challenges in the tumor antigen landscape and in the generation of genetically modified immune cells in view of their clinical efficacy, either as monotherapy or combinational therapy.

The Outcome of Ex Vivo TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic In Vitro Growth Capacity between T-Cell Clones.

PURPOSE: During our efforts to develop tumor-infiltrating lymphocyte (TIL) therapy to counter the devastating recurrence rate in patients with primary resectable pancreatic ductal adenocarcinoma (PDA), we found that PDA TILs can readily be expanded in vitro and that the majority of resulting TIL cultures show reactivity against the autologous tumor. However, the fraction of tumor-reactive T cells is low. We investigated to which extent this was related to the in vitro expansion. EXPERIMENTAL DESIGN: We compared the clonal composition of TIL preparations before and after in vitro expansion using T-cell receptor (TCR) deep sequencing. Our findings for PDA were benchmarked to experiments with melanoma TILs. RESULTS: We found that the TIL TCR repertoire changes dramatically during in vitro expansion, leading to loss of tumor- dominant T-cell clones and overgrowth by newly emerging T-cell clones that are barely detectable in the tumor. These changes are primarily driven by differences in the intrinsic in vitro expansion capacity of T-cell clones. Single-cell experiments showed an association between poor proliferative capacity and expression of markers related to antigen experience and dysfunction. Furthermore, we found that spatial heterogeneity of the TIL repertoire resulted in TCR repertoires that are greatly divergent between TIL cultures derived from distant tumor samples of the same patient. CONCLUSIONS: Culture-induced changes in clonal composition are likely to affect tumor reactivity of TIL preparations. TCR deep sequencing provides important insights into the factors that govern the outcome of in vitro TIL expansion and thereby a path toward optimization of the production of TIL preparations with high therapeutic efficacy.See related commentary by Lozano-Rabella and Gros, p. 4177.

High expression of ID1 in monocytes is strongly associated with phenotypic and functional MDSC markers in advanced melanoma.

The efficacy of immunotherapies for malignant melanoma is severely hampered by local and systemic immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Inhibitor of differentiation 1 (ID1) is a transcriptional regulator that was shown to be centrally involved in the induction of immunosuppressive properties in myeloid cells in mice, while it was overexpressed in CD11b+ cells in the blood of late-stage melanoma patients. Therefore, we comprehensively assessed ID1 expression in PBMC from stage III and IV melanoma patients, and studied ID1 regulation in models for human monocyte differentiation towards monocyte-derived dendritic cells. A highly significant elevation of ID1 was observed in CD33+CD11b+CD14+HLA-DRlow monocytic MDSC in the blood of melanoma patients compared to their HLA-DRhigh counterparts, while expression of ID1 correlated positively with established MDSC markers S100A8/9 and iNOS. Moreover, expression of ID1 in monocytes significantly decreased in PBMC samples taken after surgical removal of melanoma metastases, compared to those taken before surgery. Finally, maturation of monocyte-derived DC coincided with a significant downregulation of ID1. Together, these data indicate that increased ID1 expression is strongly associated with expression of phenotypic and immunosuppressive markers of monocytic MDSC, while downregulation is associated with a more immunogenic myeloid phenotype. As such, ID1 may be an additional phenotypic marker for monocytic MDSC. Investigation of ID1 as a pharmacodynamic biomarker or its use as a target for modulating MDSC is warranted.

Assessment of Antitumor T-Cell Responses by Flow Cytometry After Coculture of Tumor Cells with Autologous Tumor-Infiltrating Lymphocytes.

As a consequence of the ever-increasing use of immunotherapy for the treatment of cancer, interest in the direct interaction between T cells and tumors has surged tremendously. In vitro coculture of tumor cells with autologous tumor-infiltrating lymphocytes (TIL) is a highly physiological and clinically relevant model to study functional T-cell responses that result from the array of activatory and inhibitory signals that naturally occur on the patient's own tumor cells. This chapter describes a detailed protocol to set up a tumor-TIL coculture and assess ensuing functional T-cell responses, in order to establish the strength of tumor recognition by T cells and identify key determinants that govern this.

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Chromium Release Assay.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Flow Cytometry.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.