Kinetic, imaging based assay to measure NK cell cytotoxicity against adherent cells

Croom-Perez, Tayler J.; Robles-Carillo, Liza D.; Oyer, Jeremiah; Dieffenthaller, Thomas; Hasan, Md Faqrul; Copik, Alicja J.

doi:10.1016/bs.mcb.2022.07.012

Cited by 3 publications

(3 citation statements)

References 32 publications

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“…Kinetic live-cell imaging cytotoxicity assays were performed as previously described [58]. Lung cancer cell lines A549-NLR, NCI-H358-NLR, NCI-H1975-NLR, and NCI-H1299-NLR, stably expressing nuclear red fluorescent protein (Nuclight Red; NLR) for tracking were used as target cells.…”

Section: Kinetic Live-cell Imaging Cytotoxicity Assaysmentioning

confidence: 99%

TIGIT Expression on Activated NK Cells Correlates with Greater Anti-Tumor Activity but Promotes Functional Decline upon Lung Cancer Exposure: Implications for Adoptive Cell Therapy and TIGIT-Targeted Therapies

Hasan

Croom-Perez

Oyer

et al. 2023

Cancers

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Treatments targeting TIGIT have gained a lot of attention due to strong preclinical and early clinical results, particularly with anti-PD-(L)1 therapeutics. However, this combination has failed to meet progression-free survival endpoints in phase III trials. Most of our understanding of TIGIT comes from studies of T cell function. Yet, this inhibitory receptor is often upregulated to the same, or higher, extent on NK cells in cancers. Studies in murine models have demonstrated that TIGIT inhibits NK cells and promotes exhaustion, with its effects on tumor control also being dependent on NK cells. However, there are limited studies assessing the role of TIGIT on the function of human NK cells (hNK), particularly in lung cancer. Most studies used NK cell lines or tested TIGIT blockade to reactivate exhausted cells obtained from cancer patients. For therapeutic advancement, a better understanding of TIGIT in the context of activated hNK cells is crucial, which is different than exhausted NK cells, and critical in the context of adoptive NK cell therapeutics that may be combined with TIGIT blockade. In this study, the effect of TIGIT blockade on the anti-tumor activities of human ex vivo-expanded NK cells was evaluated in vitro in the context of lung cancer. TIGIT expression was higher on activated and/or expanded NK cells compared to resting NK cells. More TIGIT+ NK cells expressed major activating receptors and exerted anti-tumor response as compared to TIGIT− cells, indicating that NK cells with greater anti-tumor function express more TIGIT. However, long-term TIGIT engagement upon exposure to PVR+ tumors downregulated the cytotoxic function of expanded NK cells while the inclusion of TIGIT blockade increased cytotoxicity, restored the effector functions against PVR-positive targets, and upregulated immune inflammation-related gene sets. These combined results indicate that TIGIT blockade can preserve the activation state of NK cells during exposure to PVR+ tumors. These results support the notion that a functional NK cell compartment is critical for anti-tumor response and anti-TIGIT/adoptive NK cell combinations have the potential to improve outcomes.

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Section: Kinetic Live-cell Imaging Cytotoxicity Assaysmentioning

confidence: 99%

TIGIT Expression on Activated NK Cells Correlates with Greater Anti-Tumor Activity but Promotes Functional Decline upon Lung Cancer Exposure: Implications for Adoptive Cell Therapy and TIGIT-Targeted Therapies

Hasan

Croom-Perez

Oyer

et al. 2023

Cancers

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“…Given that CAP-NK cells had higher proliferation and expression of functional markers, the next question was if CAP-NK cells had further enhanced anti-tumor function compared to PM21-NK cells. To test this, live-cell imaging cytotoxicity assays were performed as previously described ( 24 , 43 ). Tumor growth in the absence or presence of increasing amounts of NK cells was monitored over time ( Supplementary Figure 4 ).…”

Section: Resultsmentioning

confidence: 99%

“…Cancer cell lines stably expressing nuclear red fluorescent protein (NucLight Red; NLR) for tracking were used as target cells and live-cell imaging cytotoxicity assays were performed as previously described ( 43 ). Cancer cell monolayers were co-cultured with NK cells at the indicated effector-to-target (E:T) ratios and imaged for 45 h with an IncuCyte ® S3 Live-Cell Analysis System (Sartorius, Göttingen, Germany).…”

Section: Methodsmentioning

confidence: 99%

PM21-particle stimulation augmented with cytokines enhances NK cell expansion and confers memory-like characteristics with enhanced survival

Oyer,

Croom-Perez,

Hasan

et al. 2024

Front. Immunol.

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NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both in vitro and in vivo. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications.

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Knockout of the inhibitory receptor TIGIT enhances the antitumor response of ex vivo expanded NK cells and prevents fratricide with therapeutic Fc-active TIGIT antibodies

Hasan,

Campbell,

Croom-Perez

et al. 2023

J Immunother Cancer

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BackgroundInhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in the development of cancer immunotherapeutics. Although early results of anti-TIGIT and its combinations with antiprogrammed death-ligand 1 were highly exciting, results from an interim analysis of phase III trials are disappointing. With mixed results, there is a need to understand the effects of therapeutic anti-TIGIT on the TIGIT+immune cells to support its clinical use. Most of the TIGIT antibodies in development have an Fc-active domain, which binds to Fc receptors on effector cells. In mouse models, Fc-active anti-TIGIT induced superior immunity, while Fc receptor engagement was required for its efficacy. NK-cell depletion compromised the antitumor immunity of anti-TIGIT indicating the essential role of NK cells in the efficacy of anti-TIGIT. Since NK cells express TIGIT and Fc-receptor CD16, Fc-active anti-TIGIT may deplete NK cells via fratricide, which has not been studied.MethodsCRISPR-Cas9-based TIGIT knockout (KO) was performed in expanded NK cells. Phenotypic and transcriptomic properties of TIGIT KO and wild-type (WT) NK cells were compared with flow cytometry, CyTOF, and RNA sequencing. The effect of TIGIT KO on NK-cell cytotoxicity was determined by calcein-AM release and live cell imaging-based cytotoxicity assays. The metabolic properties of TIGIT KO and WT NK cells were compared with a Seahorse analyzer. The effect of the Fc-component of anti-TIGIT on NK-cell fratricide was determined by co-culturing WT and TIGIT KO NK cells with Fc-active and Fc-inactive anti-TIGIT.ResultsTIGIT KO increased the cytotoxicity of NK cells against multiple cancer cell lines including spheroids. TIGIT KO NK cells upregulated mTOR complex 1 (mTORC1) signaling and had better metabolic fitness with an increased basal glycolytic rate when co-cultured with cancer cells compared with WT NK cells. Importantly, TIGIT KO prevented NK-cell fratricide when combined with Fc-active anti-TIGIT.ConclusionsTIGIT KO in ex vivo expanded NK cells increased their cytotoxicity and metabolic fitness and prevented NK-cell fratricide when combined with Fc-active anti-TIGIT antibodies. These fratricide-resistant TIGIT KO NK cells have therapeutic potential alone or in combination with Fc-active anti-TIGIT antibodies to enhance their efficacy.

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Kinetic, imaging based assay to measure NK cell cytotoxicity against adherent cells

Cited by 3 publications

References 32 publications

TIGIT Expression on Activated NK Cells Correlates with Greater Anti-Tumor Activity but Promotes Functional Decline upon Lung Cancer Exposure: Implications for Adoptive Cell Therapy and TIGIT-Targeted Therapies

TIGIT Expression on Activated NK Cells Correlates with Greater Anti-Tumor Activity but Promotes Functional Decline upon Lung Cancer Exposure: Implications for Adoptive Cell Therapy and TIGIT-Targeted Therapies

PM21-particle stimulation augmented with cytokines enhances NK cell expansion and confers memory-like characteristics with enhanced survival

Knockout of the inhibitory receptor TIGIT enhances the antitumor response of ex vivo expanded NK cells and prevents fratricide with therapeutic Fc-active TIGIT antibodies

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