Restoring NK cells functionality via cytokine activation enhances cetuximab-mediated NK-cell ADCC: A promising therapeutic tool for HCC patients

Abstract Abstract 2159 Introduction. NK cells are innate immune lymphocytes important for host defense against infection, and also mediate anti-tumor responses. NK cell effector functions are triggered by ligand-mediated engagement of activating receptors and by cytokine stimulation. Human NK cells may be divided into developmental and functional CD56bright and CD56dim subsets with distinct biology. Both NK subsets have constitutive expression of CD122 (IL-2/15Rβ) and CD132 (γc), allowing for responsiveness to IL-15 and high doses (1 nM) of IL-2. The high affinity heterotrimeric IL-2 receptor is formed by CD122, CD132, and CD25 (IL-2Rα), allowing for responses to very low (10 pM) concentrations of IL-2. Previous studies demonstrated that resting CD56bright, but not CD56dim, NK cells constitutively express a functional high-affinity IL-2Rαβγ, as picomolar concentrations of IL-2 result in proliferation and co-stimulate IFN-γ production. We hypothesized that cytokine activation, which occurs at the site of an inflammatory response or interaction with a priming dendritic cell, may induce the expression of CD25 on CD56dimNK cells, allowing for enhanced responsiveness to IL-2. Methods. Purified normal donor NK cells (>95% CD56+CD3-) were cultured for 16h in media containing various cytokines, including IL-15 (100ng/mL) + IL-18 (50ng/mL), IL-15 (100ng/mL) + IL-12 (10ng/mL) or low dose IL-15 (1ng/mL) as a control (to support survival). Following stimulation, cells were washed and analyzed for surface expression of CD25 by antibody staining and flow cytometry. To assess the functional capacity of an induced IL-2Rα chain, NK cells were purified and treated with cytokines as above, washed extensively and replated in media devoid of all cytokines. At 3d after initial stimulation, cultures were briefly (15min) stimulated with IL-2 (10pM, 100pM and 1nM) or IL-15 (100ng/mL) to induce phosphorylation of STAT5. Cells were immediately fixed, permeablized, and assessed for intracellular phosphoSTAT5 by flow cytometry. Results. Pretreatment with IL-15 + IL-18 or IL-12 + IL-18, but no single cytokine, induced marked upregulation of CD25 on both CD56bright as well as CD56dim NK cells, with 93.6 ±1.9% of CD56bright and 97.7 ±1.2% (n=4) of CD56dim NK cells positive for CD25 expression immediately following the initial 16h stimulation with IL-15+18, compared to 20.7 ±6.8 and 3.6 ±1.4% for control treated (low dose IL-15) CD56bright and CD56dim NK. In preliminary experiments, CD25 appears to be markedly upregulated at the mRNA level, following treatment with IL-15+18. While kinetic analyses revealed that the absolute surface receptor expression was maximal at time points early after stimulation, 75.8 ±4.1% of CD56bright and 84.9 ±5.7% (n=4) of CD56dimNK cells pretreated with IL-15+18 retained CD25 surface expression at 7d post-stimulation. Importantly, CD25 induced on both CD56dim and CD56bright NK cells resulted in a signaling-competent high affinity IL-2 receptor, as stimulation with low dose IL-2 at 3d following initial cytokine pre-activation revealed increased production of phosphoSTAT5, versus control treated NK cells. The greatest enhancement was noted in CD56dim NK cells, showing an 8-fold increase in responsiveness to low dose (10pM) IL-2 stimulation (48.2±7.9% pSTAT5+ in IL-15+18 pretreated cells vs. 5.8±2.4% in control treated cells, p<0.02). Cytokine-pretreated CD56bright NK cells demonstrated no enhancement in phosphoSTAT5 following stimulation with low dose IL-2 (10pM) (27.6 ±8.3% in IL-15+18 pretreated vs. 26.0 ±6.4% in control) at this time-point. However, these results are in agreement with published data, which describe expression of CD25 on resting CD56bright but not CD56dimNK cells. Conclusions. Here, we report the induction of CD25, and a signal-competent high-affinity component of the IL-2 receptor, on CD56dim human NK cells, following cytokine pre-activation. These results have implications for the function of both CD56bright and CD56dim NK cells in the context of inflammation and potential for cross-talk with IL-2-producing T cells during an adaptive immune response. In addition, since rhIL-2 is clinically available and used following NK cell adoptive transfer in leukemia patients, these data provides a rationale for low dose IL-2 following allogeneic NK cell pre-activation with combinations of IL-15, IL-12, and IL-18. Disclosures: No relevant conflicts of interest to declare.

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TGFβ drives NK cell metabolic dysfunction in human metastatic breast cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002044 ◽

2021 ◽

Vol 9 (2) ◽

pp. e002044 ◽

Cited By ~ 1

Author(s):

Karen Slattery ◽

Elena Woods ◽

Vanessa Zaiatz-Bittencourt ◽

Sam Marks ◽

Sonya Chew ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Nk Cells ◽

Metabolic Flux ◽

Nk Cell ◽

Metastatic Breast ◽

Metabolic Reprogramming ◽

Metabolic Dysfunction ◽

Cell Therapies ◽

Cytokine Activation

BackgroundNatural killer (NK) cells provide important immune protection from cancer and are a key requirement for particular immunotherapies. There is accumulating evidence that NK cells become dysfunctional during cancer. Overcoming NK cell exhaustion would be an important step to allow them to function optimally in a range of NK cell therapies, including those that depend on autologos circulating NK cells. We have previously demonstrated that NK cells undergo a normal metabolic reprogramming in response to cytokine activation and that this is required for optimal function. The objective of this work was to investigate if cellular metabolism of circulating NK cells is dysregulated in patients with metastatic breast cancer and if so, to gain insights into potential mechanisms underpinning this. Such discoveries would provide important insights into how to unleash the full activity of NK cells for maximum immunotherapy output.MethodsSingle-cell analysis, metabolic flux and confocal analysis of NK cells from patients with metastatic breast cancer and healthy controlsResultsIn addition to reduced interferon-γ production and cytotoxicity, peripheral blood NK cells from patients had clear metabolic deficits including reduced glycolysis and oxidative phosphorylation. There were also distinct morphologically alterations in the mitochondria with increased mitochondrial fragmentation observed. Transforminggrowth factor-β (TGFβ) was identified as a key driver of this phenotype as blocking its activity reversed many metabolic and functional readouts. Expression of glycoprotein-A repetitions predominant (GARP) and latency associated peptide (LAP), which are involved with a novel TGFβ processing pathway, was increased on NK cells from some patients. Blocking the GARP–TGFβ axis recapitulated the effects of TGFβ neutralization, highlighting GARP as a novel NK cell immunotherapy target for the first time.ConclusionsTGFβ contributes to metabolic dysfunction of circulating NK cells in patients with metastatic breast cancer. Blocking TGFβ and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies.

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Cytokine induced natural killer cell training is dependent on cellular metabolism and is defective in obesity

Blood Advances ◽

10.1182/bloodadvances.2021005047 ◽

2021 ◽

Author(s):

Nidhi Kedia-Mehta ◽

Laura Tobin ◽

Vanessa Zaiatz-Bittencourt ◽

Marta Pisarska ◽

Conor De Barra ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Cell Metabolism ◽

Initial Period ◽

Killer Cell ◽

Cellular Metabolism ◽

Cell Responses ◽

Cytokine Activation ◽

Metabolic Signalling

Natural killer (NK) cells are a population of innate immune cells which can rapidily kill cancer cells and produce cytokines such as interferon gamma (IFN-gamma). A key feature of NK cells is their ability to respond without prior sensitation, however it is now well established that NK cells can possess memory-like features. After activation with cytokines, NK cells demonstrate enhanced effector functions upon restimulation days or weeks later. This demonstrates that NK cells may be "trained" to be more effective killers and harnessed as more potent cancer immunotherapy agents. We have previously demonstrated that cellular metabolism is essential for NK cell responses, with NK cells upregulating both glycolysis and oxidative phosphorylation upon cytokine stimulation. Limiting NK cell metabolism results in reduced cytotoxicity and cytokine production. We have also demonstrated that defective NK cell responses in obesity are linked to defective cellular metabolism. In the current study we investigated if cellular metabolism is required during the initial period of NK cell cytokine training, and if NK cells from people with obesity (PWO) can be effectively trained. We show that increased flux through glycolysis and OXPHOS during the initial cytokine activation period is essential for NK cell training, as is the metabolic signalling factor Srepb. We show that NK cells from PWO, which are metabolically defective, display impaired NK cell training, which may have implications for immunotherapy in this particularly vulnerable group.

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Cytokine activation induces human memory-like NK cells

Blood ◽

10.1182/blood-2012-04-419283 ◽

2012 ◽

Vol 120 (24) ◽

pp. 4751-4760 ◽

Cited By ~ 262

Author(s):

Rizwan Romee ◽

Stephanie E. Schneider ◽

Jeffrey W. Leong ◽

Julie M. Chase ◽

Catherine R. Keppel ◽

...

Keyword(s):

Immune Response ◽

Nk Cells ◽

Nk Cell ◽

Human Memory ◽

Leukemia Cells ◽

Recall Response ◽

Cytokine Activation ◽

Cell Immunotherapy ◽

Ifn Γ ◽

Stimulation Of

Abstract Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56dim NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

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Evaluation of Interaction Between NK Cells and Colorectal Carcinoma Cells for Development of NK Cell-Based Immunotherapy in Patients with Refractory Disease

Blood ◽

10.1182/blood.v124.21.5810.5810 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5810-5810

Author(s):

Elisabetta Todisco ◽

Ilaria Turin ◽

Federica Ferulli ◽

Matteo Tanzi ◽

Silvia Brugnatelli ◽

...

Keyword(s):

Gene Expression ◽

Nk Cells ◽

Tumor Cells ◽

Cultured Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Surface Expression ◽

Target Cells ◽

Cytokine Activation ◽

Rate Of Increase

Abstract Background The failure of conventional treatment and target therapies to significantly improve outcomes in metastatic colorectal cancer (mCRC) has prompted the development of immune-based therapies, including natural killer (NK) cells-based strategies. NK cells can kill target cells directly, as well as mediate antibody (Ab)-dependent cellular cytotoxicity (ADCC) via the membrane receptor FcgRIII, which binds to the Fc portion of IgG Ab. This feature makes them of particular importance in the immunotherapy of mCRC patients harboring the KRAS mutation that cannot benefit from administration of anti-epidermal growth factor receptor (EGFR) drugs. In this study we evaluated the capacity of patients derived NK cells, either resting or after cytokine activation, to lyse autologous mCRC cells. mCRC cells were analyzed for expression of ligands for adhesion and triggering NK receptors involved in their recognition and killing. We also evaluated whether KRAS mutated mCRC cells were susceptible to anti-EGFR-induced ADCC mediated by NK cells. Patients and methods. After obtaining informed signed consent, 25 mCRC patients have been enrolled to date. Tumor cells were disaggregated by GentleMACS Dissociator (Miltenyi Biotec,Germany), in vitro expanded and analyzed for the expression of ligands for NK triggering receptors. Ligands expression was evaluated by cytofluorimetric analysis and gene expression by qPCR on mCRC cultured cells and by immunohistochemistry in sections of samples embedded in paraffin. Resting and IL-2- or IL-15-activated NK cells, were analyzed for expression of triggering and inhibitory receptors and for their ability to kill autologous mCRC cells alone or after incubation with anti-EGFR monoclonal antibodies (mAbs) in a 51Cr release cytotoxicity assay. Results. Tumor cells were successfully expanded from 21 of 25 samples. Experiments performed in 10 patients showed the inability of patients resting NK cells to lyse mCRC cells (< 10% at effector:target ratio (E:T) of 20:1. Cytokine overnight (ON) activation resulted in an increased NK cytotoxic activity (IL-2: mean 28%; range:10-71; and IL-15: mean 40%; range 16-76 at E:T ratio of 20:1). Additional days of NK cell activation were able to further enhance their lytic capability. In resting NK cells the mean surface expression of activating receptors DNAM-1 and NKG2D was 79%, (range 75-91) and 39 % (range 29-58), respectively. The latter was up-regulated by ON cytokine activation (IL-2: mean 53%, range 48-70; IL-15: mean 70%, range 63-87). Among activating natural cytotoxicity receptors (NCRs), NKp46 is highly expressed both on resting and activated cells (>90%), while low surface expression of NKp30 and NKp44 was documented on resting NK cells (<10%). Their expression could be slightly up-regulated in particular after IL-15 ON activation (mean: 13%, range 9-30). Molecular analysis demonstrated a sizeable gene expression of NK ligands PVR, Nectin-2 and MICA/B on mCRC cells. Cytofluorimetric analysis showed that PVR and Nectin-2 are expressed in a vast majority of cultured mCRC cells (mean 65%, range 60-76; and mean 78%, range 70-98), respectively, while MICA/B were less expressed (mean 18%, range 15-25). The incubation of mCRC cells with anti-EGFR mAbs increased their susceptibility to NK-mediated lysis irrespective of KRAS status of tumor cells. The average rate of increase was greater using resting NK cells as effectors. The ongoing experiments comparing mCRC ligands expression evaluated on cultured tumor cells versus paraffin embedded sections will clarify whether results obtained in vitrocould be translated to the clinical setting. Conclusions. The evidence that ex vivo activated autologous NK cells are able to lyse patients tumor cells can offer new therapeutic options to a cohort of mCRC patients with a poor prognosis. Disclosures No relevant conflicts of interest to declare.

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