scholarly journals Radiotherapy Combined with PD-1 Inhibition Increases NK Cell Cytotoxicity towards Nasopharyngeal Carcinoma Cells

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2458
Author(s):  
Anna Makowska ◽  
Nora Lelabi ◽  
Christina Nothbaum ◽  
Lian Shen ◽  
Pierre Busson ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Nk Cells ◽  
Nk Cell ◽  
Immune Checkpoint Blockade ◽  
Carcinoma Cells ◽  
Checkpoint Blockade ◽  
Therapeutic Modality ◽  
Release Assay ◽  
Nk Cell Cytotoxicity ◽  
Calcein Release

Background: Nasopharyngeal carcinoma (NPC) in endemic regions and younger patients is characterized by a prominent lymphomononuclear infiltration. Radiation is the principal therapeutic modality for patients with NPC. Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade. Here, we investigate the effect of radiotherapy on the killing of NPC cells by Natural Killer (NK) cells. Methods: NPC cell lines and a patient-derived xenograft were exposed to NK cells in the context of radiotherapy. Cytotoxicity was measured using the calcein-release assay. The contribution of the PD-L1/PD-1 checkpoint and signaling pathways to killing were analyzed using specific inhibitors. Results: Radiotherapy sensitized NPC cells to NK cell killing and upregulated expression of PD-1 ligand (PD-L1) in NPC cells and PD-1 receptor (PD-1) in NK cells. Blocking of the PD-L1/PD-1 checkpoint further increased the killing of NPC cells by NK cells in the context of radiotherapy. Conclusion: Radiation boosts the killing of NPC cells by NK cells. Killing can be further augmented by blockade of the PD-L1/PD-1 checkpoint. The combination of radiotherapy with PD-L1/PD-1 checkpoint blockade could therefore increase the efficacy of radiotherapy in NPC tumors.

In-Vitro Culture of Human CD80/IL2 Lentivirus Transduced Acute Myeloid Leukemia Cells (AML) Promote NK Cell Activation and Cytolytic Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2969-2969
Author(s):  
Wendy Ingram ◽  
Lucas Chan ◽  
Hayrettin Guven ◽  
Shahram Kordasti ◽  
Linda Barber ◽  
...  
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Activity ◽  
Cell Cytotoxicity ◽  
Release Assay ◽  
Nk Cell Cytotoxicity

Abstract Natural killer (NK) cells are increasingly recognized as an important component in the graft versus leukemia response following allogeneic hematopoietic stem cell transplantation. Immunotherapeutic strategies aim to promote NK cell activity however, the presence of regulatory T cells (Tregs) which inhibit effector immune responses pose a potential challenge to the efficacy of such regimens. We have previously shown that ‘in-vitro’ culture of AML cells transduced with a self-inactivating lentivirus (LV) encoding CD80 (B7.1) and IL2 enhance allogeneic (allo) and autologous (auto) T cell proliferation and cytotoxicity. The effect on NK cell activity and Tregs has not previously been studied and is of particular importance as IL2 stimulates NK cell and Treg activity. Peripheral blood mononuclear cells (PBMCs) from healthy donors (allo) or AML patients (auto) were cultured for 7 days ‘in-vitro’ with either unmodified or LV-CD80/IL2 AMLs. The number of NK cells (CD3−CD56+) and Tregs (CD3+CD4+CD25highFoxp3+) was examined by multi-color flow cytometry. We observe an increase in the number of NK cells (p<0.001) with an increase in the expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR following allo culture with LV-CD80/IL2 AML compared with unmodified AML. Autologous culture provides a weaker stimulus ‘in-vitro’ however, a higher number of NK cells (p=0.002) and a consistent increased expression of the activation receptors NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, as well as up regulation of the cytolytic marker CD107a was detected following auto stimulation with LV-CD80/IL2 AML compared with unmodified AML. Up regulation of CD107a was also observed in allo cultures stimulated with both unmodified and LV-CD80/IL2 AML cells. In contrast, a consistent increase in the number of Tregs was observed following allo (p=0.043) but not auto (p=0.515) LV-CD80/IL2 AML culture. Foxp3 may be unregulated on activated CD4+ T cells therefore the number of CD3+CD4+CD25highFoxp3+CD27+ Tregs was also examined. An increase in the number of CD27+ Tregs was observed following allo (p=0.017) but not auto (p=0.807) LV-CD80/IL2 AML cell culture. A standard 51Cr release assay was used to examine cytotoxicity against primary unmodified AMLs on days 0 and 7 following LV-CD80/IL2 AML cell culture. Tregs are capable of suppressing CD4+ and CD8+ T cell and NK cell cytotoxicity, therefore lysis of unmodified AMLs was initially examined using whole PBMCs as effectors. Even in the presence of Tregs an increase in lysis of allo unmodified AMLs was observed: 2.2% day 0, 4.6% following culture with unmodified AMLs; 20.4% following LV-CD80/IL2 AML cell culture. Importantly, an increase in lysis of auto AML was also detected: 0% day 0, 2.1% unmodified AML culture, 16% LV-CD80/IL2 AML culture. The ratio of Tregs to effector T cells is important for the suppressive function of Tregs. The number of Tregs in the cytotoxicity assays is likely to be lower than that required for a significant suppressive effect to be observed. We next examined the cytotoxicity of NK cells using K562 and unmodified AMLs as targets. NK cells were negatively isolated on days 0 and 7 following either unmodified AML or LV-CD80/IL2 AML cell culture and used as effectors in a 51Cr release assay. In keeping with the changes in NK cell activation receptor expression, we demonstrate a significant increase in NK cell cytotoxicity against both K562 and primary unmodified AMLs. Lysis of K562 increased from 46.7% on day 0 to 90.4% after LV-CD80/IL2 stimulation. Importantly, an increase in lysis of both allo and auto unmodified AMLs was detected following LV-CD80/IL2 AML cell culture. Lysis of allo AMLs increased from a median of 11.8% on day 0, 8.7% following culture with unmodified AML to 20.1% following LV-CD80/IL2 AML cell culture using a low effector: target ratio of just 5:1. Importantly, an increase in lysis of auto AML from 0.4% on day 0, 2.1% with unmodified AML cells to 21.5% following LV-CD80/IL2 AML stimulation was observed. LV-CD80/IL2 AML cells enhance NK cell activation and cytotoxicity against allo and auto unmodified AMLs. Furthermore, cytotoxicity is enhanced even in the presence of Tregs with an increase in Tregs only observed following allo culture. Vaccination of patients with LV-CD80/IL2 AML cells therefore represents a potential strategy to promote T and NK cell cytotoxicity and enhance anti-leukemia immune responses in patients with AML.

scholarly journals Interferon β and Anti-PD-1/PD-L1 Checkpoint Blockade Cooperate in NK Cell-Mediated Killing of Nasopharyngeal Carcinoma Cells

2019 ◽  
Vol 12 (9) ◽  
pp. 1237-1256 ◽  
Author(s):  
Anna Makowska ◽  
Till Braunschweig ◽  
Bernd Denecke ◽  
Lian Shen ◽  
Valentin Baloche ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Nk Cell ◽  
Carcinoma Cells ◽  
Checkpoint Blockade ◽  
Interferon Β

scholarly journals The effect of IL-2 stimulation and treatment of TRPM3 on channel co-localisation with PIP2 and NK cell function in myalgic encephalomyelitis/chronic fatigue syndrome patients

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Natalie Eaton-Fitch ◽  
Hélène Cabanas ◽  
Stanley du Preez ◽  
Donald Staines ◽  
Sonya Marshall-Gradisnik
Keyword(s):  
Chronic Fatigue Syndrome ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Chronic Fatigue ◽  
Signalling Pathways ◽  
Cell Cytotoxicity ◽  
Fatigue Syndrome ◽  
Intracellular Signalling Pathways ◽  
Nk Cell Cytotoxicity

Abstract Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious multifactorial disorder. The origin remains ambiguous, however reduced natural killer (NK) cell cytotoxicity is a consistent immunological feature of ME/CFS. Impaired transient receptor potential melastatin 3 (TRPM3), a phosphatidylinositol dependent channel, and impaired calcium mobilisation have been implicated in ME/CFS pathology. This investigation aimed to examine the localisation of TRPM3 at the NK cell plasma membrane and co-localisation with phosphatidylinositol 4,5-bisphosphate (PIP2). The effect of IL-2 priming and treatment using pregnenolone sulfate (PregS) and ononetin on TRPM3 co-localisation and NK cell cytotoxicity in ME/CFS patients and healthy controls (HC) was also investigated. Methods NK cells were isolated from 15 ME/CFS patients and 15 age- and sex-matched HC. Immunofluorescent technique was used to determine co-localisation of TRPM3 with the NK cell membrane and with PIP2 of ME/CFS patients and HC. Flow cytometry was used to determine NK cell cytotoxicity. Following IL-2 stimulation and treatment with PregS and ononetin changes in co-localisation and NK cell cytotoxicity were measured. Results Overnight treatment of NK cells with PregS and ononetin resulted in reduced co-localisation of TRPM3 with PIP2 and actin in HC. Co-localisation of TRPM3 with PIP2 in NK cells was significantly reduced in ME/CFS patients compared with HC following priming with IL-2. A significant increase in co-localisation of TRPM3 with PIP2 was reported following overnight treatment with ononetin within ME/CFS patients and between groups. Baseline NK cell cytotoxicity was significantly reduced in ME/CFS patients; however, no changes were observed following overnight incubation with IL-2, PregS and ononetin between HC and ME/CFS patients. IL-2 stimulation significantly enhanced NK cell cytotoxicity in HC and ME/CFS patients. Conclusion Significant changes in co-localisation suggest PIP2-dependent TRPM3 function may be impaired in ME/CFS patients. Stimulation of NK cells with IL-2 significantly enhanced cytotoxic function in ME/CFS patients demonstrating normal function compared with HC. A crosstalk exists between IL-2 and TRPM3 intracellular signalling pathways which are dependent on Ca2+ influx and PIP2. While IL-2R responds to IL-2 binding in vitro, Ca2+ dysregulation and impaired intracellular signalling pathways impede NK cell function in ME/CFS patients.

scholarly journals IMMU-09. CONCURRENT DEXAMETHASONE LIMITS THE CLINICAL BENEFIT OF IMMUNE CHECKPOINT BLOCKADE IN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii106-ii106
Author(s):  
Bryan Iorgulescu ◽  
Prafulla Gokhale ◽  
Maria Speranza ◽  
Benjamin Eschle ◽  
Michael Poitras ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Immune Checkpoint ◽  
Nk Cell ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Innate Immune Responses ◽  
Dose Dependent Fashion ◽  
Dexamethasone Therapy ◽  
Clinical Correlate ◽  
Dose Dependent

Abstract BACKGROUND Dexamethasone, a uniquely potent corticosteroid, is frequently administered to brain tumor patients to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in glioblastoma patients – particularly in the context of immunotherapy. METHODS We evaluated the dose-dependent effects of dexamethasone when administered with anti-PD-1 and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 or CT-2A glioblastoma tumors, including analyses of intracranial tumors, draining lymph nodes, and spleen. Clinically, the effect of dexamethasone on survival was additionally evaluated in 181 consecutive IDH-wildtype glioblastoma patients treated with anti-PD-(L)1, with adjustment for relevant prognostic factors. RESULTS Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy decreased survival in a dose-dependent fashion and decreased survival following anti-PD-1 plus radiotherapy in both GL261 and immunoresistant CT-2A models. Dexamethasone quantitatively decreased T lymphocytes by reducing the proliferation while increasing apoptosis. Dexamethasone also decreased lymphocyte functional capacity. Myeloid and NK cell populations were also generally reduced. Thus, dexamethasone negatively affects both the adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade revealed worse survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone use – regardless of dose – was the strongest predictor of poor survival (reference no dexamethasone; < 2mg HR 2.28, 95%CI=1.41–3.68, p=0.001; ≥2mg HR 1.97, 95%CI=1.27–3.07, p=0.003). CONCLUSIONS Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for glioblastoma patients. Our preclinical analyses also suggest that dexamethasone’s detrimental effects are dose-dependent, suggesting that the lowest possible dose should be used for patients when dexamethasone use is unavoidable. Careful evaluation of dexamethasone use is warranted for neuro-oncology patients undergoing immunotherapy clinical trials.

scholarly journals α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway

2021 ◽  
Vol 22 (2) ◽  
pp. 656
Author(s):  
Hantae Jo ◽  
Byungsun Cha ◽  
Haneul Kim ◽  
Sofia Brito ◽  
Byeong Mun Kwak ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Granzyme B ◽  
Akt Pathway ◽  
Cell Cytotoxicity ◽  
Anticancer Effects ◽  
Nk Cell Cytotoxicity

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

scholarly journals Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e64835 ◽  
Author(s):  
Subhashis Sarkar ◽  
Wilfred T. V. Germeraad ◽  
Kasper M. A. Rouschop ◽  
Elisabeth M. P. Steeghs ◽  
Michel van Gelder ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'>0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

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