NKG2D Gene Polymorphism Is Associated with Disease Control of Chronic Myeloid Leukemia By Dasatinib

Abstract Dasatinib (DA) is a tyrosine kinase inhibitor (TKI), and is approved for the treatment of naïve and relapsed/refractory Philadelphia chromosome positive leukemia. DA is a potent and multi-target TKI, and a part of patients treated with DA are reported to exhibit lymphocytosis and that is caused by a proliferation of NK cells or CD8 T cells. Nowadays, a lot of reports about the stop TKI trials suggested that treatment free remission of the CML after TKI discontinuation was significantly associated with NK cell proliferation in peripheral blood of the patients. So far, little is known about the individual differences in NK cell response among patients treated with Dasatinib. NKG2D is an activating receptor expressed on NK cells. Recently, functional SNP were identified between LNK1 and HNK1 haplotypes of the NKG2D gene. We analyzed the association between patient's NKG2D polymorphism and achievement of the deep molecular response by Dasatinib. We retrospectively investigated thirty-one patients who were treated with Dasatinib for the first-line treatment of CML at Tokai university school of medicine between 2011 and 2015. Patients with HNK1 haplotype tended to achieve MR4.5 more frequently (not significant), and these patients accomplished MR4.5significantly faster than others. Next, we harvested NK cells from healthy volunteers, and investigated NK cell responses by Dasatinib in vitro. There were differences in the degree of NK cell proliferation capacity among subjects after DA treatment in vitro. We found that NK cells with NKG2D LNK1/LNK1 haplotype exhibited significantly lower proliferation capacity than others in the population. NK cells of higher proliferation capacity exhibited Vav-1 Tyr174 phosphorylation, but they didn't show any differences in cytotoxicity capacity. In this study, our data suggest that DA may stimulate NK cells with NKG2D HNK1 haplotype more strongly than LNK1 haplotype, and therefore patients with HNK1 haplotype can achieve deep molecular response faster than others. So, personalized medicine according to their NKG2D haplotype may be useful to the treatment of CML, especially when they are considered to discontinue DA in stop TKI trials. Disclosures No relevant conflicts of interest to declare.

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Cyclosporine A (CSA) Significantly Reduces the Cytotoxic Effects of In Vitro Expanded NK Cells: Implications for Adoptive NK Cell Therapy in the Setting of Allogeneic Hematopoietic Stem Cell Transplantation (HCT).

Blood ◽

10.1182/blood.v110.11.4899.4899 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4899-4899

Author(s):

Hisayuki Yokoyama ◽

Maria Berg ◽

Andreas Lundqvist ◽

J. Philip McCoy ◽

Shivani Srivastava ◽

...

Keyword(s):

Cell Proliferation ◽

Nk Cells ◽

Nk Cell ◽

Immunofluorescent Staining ◽

Hematopoietic Stem ◽

Trail Expression ◽

And Function ◽

The Impact ◽

Cells Cultured

Abstract The ability to expand NK cells in vitro has led to the recent initiation of protocols incorporating adoptive NK cell infusions after HCT. Calcineurin inhibitors such as CSA are commonly used to prevent graft versus host disease (GVHD) in HCT recipients. Recently, Hong et al found the phenotype and function of fresh NK cells cultured in vitro with CSA was altered, with CSA treated NK cell cultures having enhanced cytotoxicity against tumor targets. However, the impact of CSA on in vitro expanded NK cell function and phenotype has not been explored. We analyzed cell proliferation, IFN-gamma production, cell surface immunofluorescent staining and cytotoxicity against K562 and renal cell carcinoma cell lines by in vitro expanded vs freshly isolated NK cells cultured in physiological doses of CSA (40ng/ml, 200ng/ml, 1000ng/ml for 18hrs). Fresh NK cells were obtained from the PBMC of healthy donors using immunomagnetic beads to isolate CD56+/ CD3− cells. NK cells were expanded in vitro using irradiated EBV transformed B cells as feeder cells in media containing IL-2 [500U/ml] for 12–14 days. Comparing CSA containing cultures to controls, there was a significant reduction in IL-2 stimulated fresh NK cell proliferation (stimulation index 0.51± 0.1) and TRAIL expression (MFI 10.4 vs 3.01). Furthermore, an ELISA assay showed fresh NK cells treated with CSA had a significant reduction in IL-2 induced IFN-g production compared to controls (median 231 vs 57 pg/ml, p=0.025). In contrast, in vitro expanded NK cells cultured in CSA showed no significant reduction of proliferation or TRAIL expression. At the highest doses of CSA (1000ng/ml), minimal inhibition of K562 killing of freshly isolated NK cells was observed. In contrast, expanded NK cells cultured in CSA for 18 hours compared to controls had a significant reduction in the killing of K562 cells (E:T=10:1, median 66 vs 43% lysis, p=0.011) and RCC tumor cells (E:T=20:1, 14.8 vs 8.8%, p=0.043). Figure Figure These data confirm CSA alters the phenotype and function of CD3−/CD56 + NK cells. Importantly, CSA appears to have a deleterious effect on expanded NK cell tumor cytotoxicity that was not observed with fresh NK cells. These finding suggest the anti-tumor effects of in vitro expanded NK cells could be hindered when adoptively infused in HCT patients receiving CSA.

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Tumor-Experienced Human NK Cells Express High Levels of PD-L1 and Inhibit CD8+ T Cell Proliferation

Frontiers in Immunology ◽

10.3389/fimmu.2021.745939 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jessica M. Sierra ◽

Florencia Secchiari ◽

Sol Y. Nuñez ◽

Ximena L. Raffo Iraolagoitia ◽

Andrea Ziblat ◽

...

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Nk Cells ◽

Cancer Patients ◽

Nk Cell ◽

The Cancer Genome Atlas ◽

T Cell Proliferation ◽

Dependent Manner ◽

Effector Functions

Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.

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Adoptively-Infused NK Cells Maintain Their Antitumor Effects in Vivo in the Presence of CyclosporineA (CSA)

Blood ◽

10.1182/blood.v112.11.2563.2563 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2563-2563

Author(s):

Hisayuki Yokoyama ◽

Andreas Lundqvist ◽

Maria Berg ◽

Muthalagu Ramanathan ◽

Rebecca Lopez ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Median Survival ◽

Nk Cell ◽

Stimulation Index ◽

Surface Expression ◽

Inhibition Of Proliferation

Abstract Animal models show infusions of donor NK cells given after allogeneic HCT can prevent GVHD while simultaneously mediating a graft-vs-tumor effect. However, it is unclear whether adoptively infused NK cells can mediate these beneficial effects in the presence of CSA, which is commonly given after HCT to prevent GVHD. In this study, we analyzed the in vitro effects of pharmacological concentrations of CSA on NK cell phenotype, cell proliferation, and tumor cytotoxicity. We also evaluated in vivo whether CSA administration would reduce the anti-tumor effects of adoptively infused NK cells in tumor bearing mice. PBMCs collected from healthy donors were labeled with CFSE then were stimulated in vitro with IL-2 for 7 days in the presence or absence of CSA (1000ng/ml). CFSE proliferation assays on fresh PBMC showed CSA inhibited IL-2 stimulated CD3+ T-cell proliferation more than CD3−/CD56+ NK cell proliferation (mean percentage inhibition of proliferation 49.4% vs. 22.2% for T cells and NK cells respectively; p<0.05). CD3−/CD56+ NK cells were then isolated from PBMCs of healthy donors and expanded in vitro with irradiated EBV-LCL and IL-2 for 10 days. In contrast to T-cells, CSA only minimally inhibited IL-2 induced proliferation of expanded NK cells (mean 9.5% inhibition of proliferation by CFSE staining). T cells and NK cells were next isolated from PBMCs and stimulated with either OKT3, PMA-Ionomycin (PI), or IL-2. A [3H] TdR uptake assay showed T cell proliferation was inhibited at a substantially higher level by CSA (mean stimulation index for OKT3: 0.03, for PI: 0.35, for IL-2: 0.55) compared to that of expanded NK cells (mean stimulation index for IL-2: 0.82, p<0.05). Furthermore, an ELISA assay showed CSA treatment reduced IL-2 induced secretion of INF-g by T cells more than expanded NK cells (mean reduction in INF-g secretion in T cells of 94.7 % vs. 36.5 % in NK cells, p<0.05). Compared to controls, culturing in vitro expanded NK cells in CSA did not alter surface expression of the activating receptors NKp30, NKp42, and NKG2D but did reduce surface expression of NKp44 and TRAIL (mean reduction in surface expression 36% and 36.3% respectively). Cytotoxic granule release assessed by CD107a staining was inhibited by CSA in CD8+ melanoma specific T cells co-cultured with melanoma cells (mean 12.2 % inhibition) in contrast to NK cells co-cultured with K562 cells where CSA increased CD107a expression a mean 29.7% (p<0.05). Furthermore, at a 20:1 E:T ratio, 51Cr cytotoxity assays showed CSA did not reduce the cytotoxicity of in vitro expanded NK cells against renal cell carcinoma (RCC) cells (58% mean lysis) compared to NK cells cultured in control media (55% mean; p=n.s.). In contrast, melanoma specific T-cell killing of tumor targets was significantly lower in CTL cultures containing CSA compared to control media (38.0% vs. 50.2% respectively P<0.05). Next, we assessed the impact of CSA administration on the anti-tumor effects of adoptive NK cell infusions in tumor bearing animals where syngeneic NK cell infusions following bortezomib treatment have been shown to delay tumor progression and prolong survival. BALB/c mice injected with 100,000 luciferase transfected RENCA tumor cells i.v. received 3 weekly treatments with the combination of bortezomib (5ug/mouse i.v.) and 2×106 syngeneic NK cells i.v. with or without daily administration of CSA (15mg/kg sc). Bioluminescence imaging in controls that did not receive CSA showed tumor growth was slower and survival was prolonged in mice receiving adoptive NK cell infusions (median survival 53 days) compared to mice that did not receive NK cells (median survival 30.2 days; p<0.05). CSA administration did not impair the anti-tumor effects of adoptive NK cell infusions; mice receiving CSA and adoptive NK cell infusions had similar tumor growth and survival as recipients of NK cells without CSA (median survival 47 vs. 53 days; p=n.s.). These results show that CSA is significantly more immunosuppressive to T cells compared to NK cells and provide evidence that the anti-tumor effects of adoptively infused in vitro expanded NK cells are maintained even in the presence CSA.

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Costimulatory Effects of Interleukin-18 (IL-18) on Human Natural Killer (NK) Cells Activated through Fc Receptors for Immunoglobulin (IgG)

Blood ◽

10.1182/blood.v116.21.2780.2780 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2780-2780

Author(s):

Shivani Srivastava ◽

Hailin Feng ◽

Menggang Yu ◽

David Pelloso ◽

Michael Robertson

Keyword(s):

Monoclonal Antibodies ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fc Receptors ◽

Cytolytic Activity ◽

Mitogen Activated Protein Kinase ◽

Target Cells

Abstract Abstract 2780 NK cells play an important role in innate and adaptive immune responses. Most human NK cells express CD16, an Fc receptor for IgG that mediates lysis of antibody-coated target cells and costimulates interferon (IFN)-g production in response to cytokines. IL-18 is an immunostimulatory cytokine with antitumor activity in preclinical animal models. The effects of IL-18 on human NK cell function were examined. Here we show that NK cells stimulated with immobilized IgG in vitro secreted IFN-g; such IFN-g production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-g production by NK cells stimulated with immobilized IgG or CD16 antibodies (Figure 1). NK cell IFN-g production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk, extracellular signal-related kinases (ERK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Stimulation with IL-18 or immobilized IgG could augment IL-12-induced IFN-g production by STAT4-deficient lymphocytes obtained from lymphoma patients after autologous stem cell transplantation (Figure 2). IL-18 also augmented the in vitro lysis of rituximab-coated Raji cells by human NK cells (Figure 3). These observations that IL-18 can co stimulate IFN-g production and cytolytic activity of NK cells activated through Fc receptors makes it an attractive cytokine to combine with monoclonal antibodies for treatment of cancer. Disclosure: No relevant conflicts of interest to declare.

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The Role of Her2/Neu in ALL: Long Term Clinical Follow-up of Surface-Positive Patients and Induction of NK Cell ADCC by Trastuzumab in Vitro

Blood ◽

10.1182/blood.v120.21.1421.1421 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1421-1421

Author(s):

Sebastian P. Haen ◽

Benjamin J Schmiedel ◽

Nora Roth ◽

Christoph Faul ◽

Robert Möhle ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Additional Risk Factor ◽

Cytokine Release ◽

Breast Cancer Patients ◽

Time To Relapse

Abstract Abstract 1421 In about one third of patients, acute lymphoblastic leukemia (ALL) cells express Her2/neu (p185Her-2). In epithelial malignancies Her2/neu contributes to oncogenic transformation and metastatic potential, and overexpression often is associated with poor prognosis. Previous reports also suggested an inferior prognosis in patients with Her2/neu-positive ALL, but as of now no study has performed survival analyses. In breast cancer patients, targeting of Her2/neu with the monoclonal antibody Trastuzumab results in improved disease outcome, and induction of antibody dependent cellular cytotoxicity (ADCC) and cytokine release of NK cells contributes to the same. Most recently, application of Trastuzumab in patients with relapsed or refractory ALL was shown to yield promising results, but the mechanisms by which Trastuzumab mediates its efficacy have not been studied so far. Here, we studied patient records and flow cytometry data on Her2/neu expression of patients treated for ALL at our hospital between 2000 and 2011 and determined the effect of Trastuzumab and/or Rituximab on NK cell reactivity against ALL blasts in vitro. In 57 patients (37 men, 20 women, median age at diagnosis 42 years, range 17–83 years) data on Her2/neu expression were available. Her2/neu expression was detected in 15/57 patients (26%). Clinical follow up was assessed for all 57 patients. Median follow-up was 44 months (range 0.5–356 months) until death or last follow-up visit. Overall, 60% of the patients had died at the end of follow-up (25 of 42 Her2/neu-negative and 9 of 15 Her2/neu-positive patients, respectively). Median survival times were 43 (Her2/neu-negative, range 0.5–356 months) and 41 months (Her2/neu-positive, range 3.5–138 months; p = 0.81. Kaplan Meyer regression analysis). Also, median time to relapse was comparable in both groups (22.5 months vs. 15.5 months; p = 0.77). In functional analyses with NK cells, incubation of Her2/neu-positive CD20-positive ALL blasts with Trastuzumab or Rituximab comparably induced ADCC and cytokine release of NK cells. Only minor additional effects were observed upon combined application of both antibodies. When blasts from Her2/neu-negative ALL patients were employed (phentotype CD20-positive Her2/neu-negative or CD20-negative Her2/neu-negative), no effect of Trastuzumab was observed, and Rituximab exerted effects solely with blasts from CD20-positive ALL patients. Taken together, in our patient cohort Her2/neu expression on ALL blasts was not associated with time to relapse or overall survival and thus did not appear to be an additional risk factor. Rather, Her2/neu is a promising target for antibody therapy, especially in patients with Her2/neu-positive CD20-negative ALL, as treatment with Trastuzumab is suitable to induce ADCC and cytokine production of NK cells also against Her2/neu-positive CD20-negative ALL blasts. Disclosures: No relevant conflicts of interest to declare.

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Dasatinib Expands Pre-Existing, CMV-Associated, Highly Differentiated NK Cells in Ph+ Leukemia

Blood ◽

10.1182/blood.v124.21.3149.3149 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3149-3149

Author(s):

Ken-ichi Ishiyama ◽

Toshio Kitawaki ◽

Akifumi Takaori ◽

Norimitsu Kadowaki

Keyword(s):

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Strong Predictor ◽

Philadelphia Chromosome ◽

Principal Component ◽

Leukemic Cells ◽

Cmv Reactivation ◽

Therapeutic Responses

Abstract [Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Among these TKIs, only dasatinib expands large granular lymphocytes (LGLs) including NK cells in some patients. This dasatinib-induced lymphocytosis is associated with enhanced therapeutic responses. Recently, it has been suggested that dasatinib-associated LGL expansion is linked to cytomegalovirus (CMV) reactivation. CMV infection induces substantial changes in phenotypic and functional profiles of NK cells. However, characteristics of NK cells in dasatinib-treated Ph+ leukemia patients (DA) and mechanisms by which dasatinib expands NK cells remain unknown. [Method] NK-cell marker expression and cytotoxic activity of NK cells were examined in 31 DA, 15 patients treated with other TKIs (OT) and 15 healthy donors (HD). We mapped NK-cell profiles, using principal component analysis (PCA) of 19 NK-cell markers. [Result] NK cells increased in 18 of 26 CMV-seropositive (CMV+) DA, in contrast to no increase in 5 CMV- DA and 13 CMV+ OT. Expression of an activating receptor NKG2C was enhanced on the increased NK cells. PCA mapped NK-cell profiles according to the degree of differentiation, which correlated with higher expression of NKG2C, CD57 and LIR-1, and lower expression of NKp30 and NKp46, revealing that NK cells in CMV+ DA were highly differentiated compared to those in CMV- DA and HD (Figure 1). NK cells in CMV+ OT were mapped between CMV+ DA and HD. In 10 CMV+ DA, recent CMV reactivation was detected by CMV IgM and/or quantitative PCR. Of these, CMV reactivation was symptomatic only in 4 patients, indicating that dasatinib induces subclinical CMV reactivation in the majority of patients. CD107a degranulation assay showed higher cytotoxic activity of NKG2C+ NK cells, compared to NKG2C- NK cells. Interestingly, some of the CMV+ patients with Ph+ leukemia had NKG2C+ highly differentiated NK cells already at diagnosis, suggesting that CMV has been reactivated before starting TKIs at least in some patients. In fact, we detected CMV genome by nested PCR in PBMCs from some of the pretreatment patients. Moreover, the CMV+ patients with highly differentiated NK cells developed NK-LGL expansion earlier after starting dasatinib, compared to those with less differentiated NK cells. [Discussion] Our data indicate that CMV seropositivity underlies the development of NK-LGL expansion in DA patients. Intriguingly, it appears that CMV has been subclinically reactivated at least in some Ph+ leukemia patients even before starting dasatinib, leading to expansion of "ready-to-go" NKG2C+ highly differentiated NK cells. It is likely that dasatinib further expands such NK cells presumably due to chronic stimulation by reactivated CMV. The NKG2C+ NK cells have high cytotoxic activity against leukemic cells, which might contribute to enhanced therapeutic responses in DA patients. The phenotypic profile of NK cells at diagnosis may be a strong predictor of NK-LGL expansion during dasatinib therapy. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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T/NK Lymphocytosis in CML Ph+ Patients During Dasatinib Therapy.

Blood ◽

10.1182/blood.v114.22.3279.3279 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3279-3279

Author(s):

Antonella Russo Rossi ◽

Alessandra Ricco ◽

Paola Carluccio ◽

Mario Delia ◽

Manuela Leo ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Nk Cell ◽

Conflicts Of Interest ◽

Cytotoxic T Cell ◽

Molecular Response ◽

Imatinib Treatment ◽

Long Term Outcome ◽

Significant Difference ◽

The Impact

Abstract Abstract 3279 Poster Board III-1 Background. Some authors reported that Natural Killer (NK) cells from CML patients are defective in NK cell activity and NK cell number decrease as the disease progresses to the advanced phase and probably the abnormal BCR/ABL gene causes abnormal NK cell differentiation.TK inhibitors reduce BCR/ABL transcription and could restore NK cell numbers and/or function.Moreover Dasatinib,by the blockade of SRC Kinases,could affect the development of NK cells as well as T-lymphocytes.Our aim was to verify the impact of Dasatinib treatment on T CD8+ and NK cells modulation. Methods. We evaluated 24 patients with CML resistant/intollerant to Imatinib and treated with Dasatinib at a starting dose of 70 mg/BID or 100 mg/QD.Blood count were monitored; lymphocytosis has been definited by an increased number of peripheral blood lymphocyte counts ≥ 3.0×10(e)9/L and by the predominance of LGLs in peripheral blood smear. Immunophenotyping was done with flow-cytometry using antibodies against the following antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD16, and CD56. Results. With a median of 19 mo. of Dasatinib therapy (range 3–43), 15/24 cases (62.5%) developed peripheral blood lymphocytosis. Median onset of lymphocytosis was 3 months after the initiation of Dasatinib therapy (range 1–12) and duration was 14 months (range 6–40).Lymphocytosis was CD3+/CD8+/Cytotoxic T Cell in 9 patients (60%) and CD3-/CD16+/CD56+/NK Cell in 6 patients (40%).In all 15 patients no symptoms or signs suggestive of LGL leukemia or viral infections were documented.There was no significant difference in terms of the frequency of severe adverse events, including pleural effusion between patients with and without lymphocytosis. 11(73%) of the 15 patients who developed lymphocytosis achieved MMolR and 4/11 presented Bcr/Abl mutation at the time of imatinib treatment (F317L, E255K, F359V, E255K),whereas only 3(33%) of the 9 patients without lymphocytosis achieved MMolR and 1/3 presented Bcr/Abl mutation (F359V). Moreover molecular response was earlier in the group of patients with lymphocytosis (8vs12 mo.). Conclusions. The development of lymphocytosis in our patients seems to be associated to an improved response to dasatinib in terms of molecular response and time to response. The assessment of higher frequency lymphocytosis requires further analysis; a larger patients'cohort should be needed to explore the biological role of lymphocytosis and the impact on the long term outcome in patients treated with dasatinib. Disclosures: No relevant conflicts of interest to declare.

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Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation

Frontiers in Immunology ◽

10.3389/fimmu.2021.711621 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hemant K. Mishra ◽

Kate J. Dixon ◽

Nabendu Pore ◽

Martin Felices ◽

Jeffrey S. Miller ◽

...

Keyword(s):

Cell Proliferation ◽

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Feedback System ◽

Cytotoxic Lymphocytes ◽

Cell Therapies ◽

Surface Receptors

Natural killer (NK) cells are innate cytotoxic lymphocytes that can recognize assorted determinants on tumor cells and rapidly kill these cells. Due to their anti-tumor effector functions and potential for allogeneic use, various NK cell platforms are being examined for adoptive cell therapies. However, their limited in vivo persistence is a current challenge. Cytokine-mediated activation of these cells is under extensive investigation and interleukin-15 (IL-15) is a particular focus since it drives their activation and proliferation. IL-15 efficacy though is limited in part by its induction of regulatory checkpoints. A disintegrin and metalloproteinase-17 (ADAM17) is broadly expressed by leukocytes, including NK cells, and it plays a central role in cleaving cell surface receptors, a process that regulates cell activation and cell-cell interactions. We report that ADAM17 blockade with a monoclonal antibody markedly increased human NK cell proliferation by IL-15 both in vitro and in a xenograft mouse model. Blocking ADAM17 resulted in a significant increase in surface levels of the homing receptor CD62L on proliferating NK cells. We show that NK cell proliferation in vivo by IL-15 and the augmentation of this process upon blocking ADAM17 are dependent on CD62L. Hence, our findings reveal for the first time that ADAM17 activation in NK cells by IL-15 limits their proliferation, presumably functioning as a feedback system, and that its substrate CD62L has a key role in this process in vivo. ADAM17 blockade in combination with IL-15 may provide a new approach to improve NK cell persistence and function in cancer patients.

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KIR-LIGAND Mismatched Natural Killer Cells Effectively Kill Primary Myeloma and Myeloma Cell Lines.

Blood ◽

10.1182/blood.v104.11.2449.2449 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2449-2449

Author(s):

Frits van Rhee ◽

Susann M. Szmania ◽

JuMei Shi ◽

Michele Cottler-Fox ◽

Justin W. Dyniewski ◽

...

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Nk Cells ◽

Natural Killer ◽

Cell Lines ◽

Nk Cell ◽

Allogeneic Transplantation ◽

Myeloma Cell ◽

Myeloma Cells

Abstract Recent observations in haplo-identical, T-cell-depleted allogeneic transplantation have focused attention on the rapid and remarkable anti-tumor effect mediated by Killer Inhibitory Receptor (KIR) ligand mismatched natural killer (NK) cells. Most data are confined to the role of KIR-ligand mismatched NK cells for acute leukemia after allogeneic transplantation. In this study, we report both the ability of KIR mismatched NK cells to kill primary myeloma cells, and the effects of interleukin (IL)2 and IL15 on NK cells in vitro. NK cells were prepared from healthy donor PBMCs by depletion of T-lymphocytes using immunomagnetic beads conjugated to anti-CD3 followed by depletion of monocytes using a simple adherence step. After overnight incubation in cytokines (IL2/IL15 as indicated) standard chromium release assays determined the cytotoxicity of NK cells against target cells and 3H-thymidine incorporation assessed NK cell proliferation. KIR-ligand mismatched NK cells incubated overnight in 300 IU/ml IL2 killed primary MM cells from four individuals (76, 64,60,48% lysis) as well as MM lines U266, NCI H929, and the NK sensitive line K562 (95, 86, 76% lysis, respectively) at an E:T ratio of 20:1. NK cells were not cytotoxic towards autologous phytohemagglutinin (PHA) or allogeneic PHA-induced T-cell blasts. Increasing the IL2 concentration during overnight NK cell incubation from 300 to 1000 IU/ml did not significantly enhance the cytotoxicity against U266 myeloma cells or control K562 cells. IL15 was more potent than IL2 in inducing NK cell proliferation. The optimal IL15 concentration was 300 IU/ml (range tested: 0–3000 IU/ml). The use of both cytokines in concert was less effective than the use of IL15 alone, probably due to homology of the IL2 and IL15 b and g receptors. We conclude that T cell and monocyte depletion of PBMC results in a preparation significantly enriched for NK cells (±50%) that effectively kills KIR-ligand mismatched primary myeloma cells and myeloma cell lines. IL15 is the superior cytokine for enhancing NK cell proliferation. The exciting anti-leukemic effects mediated by KIR-ligand mismatched NK cells have thus far only been reported in the haplo-identical transplant setting. In view of our positive in vitro data in MM, we will evaluate in a phase II trial wheter the repeated transfusion of KIR-ligand-mismatched, T-cell depleted NK cells from haplo-identical donors after immunosuppressive treatment with fludarabine and dexamethasone (to avoid rejection of donor NK cells) and tumor reduction with melphalan followed by a delayed auto-transplant can improve outcome in patients who have high risk MM or who have relapsed after a previous auto-transplant. This will be the first clinical application of KIR-ligand-mismatched NK cells in autologous transplantation and the first such trial in myeloma. Figure Figure

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In Vivo Antagonistic Effects of Dexamethasone On Lenalidomide-Induced NK Cell Activation.

Blood ◽

10.1182/blood.v114.22.1639.1639 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1639-1639 ◽

Cited By ~ 1

Author(s):

Hang Quach ◽

Hsu Andy ◽

David Ritchie ◽

Paul Neeson ◽

Kevin Lynch ◽

...

Keyword(s):

Cell Proliferation ◽

Nk Cells ◽

K562 Cell ◽

Cell Function ◽

Nk Cell ◽

Cell Lysis ◽

Cell Numbers ◽

And Function

Abstract Abstract 1639 Poster Board I-665 Dexamethasone (dex) and lenalidomide (len) is a potent treatment for multiple myeloma (MM). In vitro, len directly inhibits MM tumor cell proliferation via cell cyle arrest, and can also costimulate T cells and augment natural killer (NK) cell activity, leading to enhanced anti-tumour immunity. Conversely, dex also directly inhibits MM cell proliferation but is profoundly immuno-suppressive and may therefore subvert the full capacity of len to act via immune mechanisms against MM. We previously reported that MM patients responding to len-dex combination show an increase in Treg numbers, and little evidence in recovery of their B and T cell numbers (Quach et al. Blood 2008; 112: abstract 1696). We have since undertaken a prospective and systematic analysis of NK cell number and function in MM patients treated with len-dex, and evaluated the mechanisms by which dex downregulates len-induced NK activation in in vitro assays using patients' and normal donors' blood samples. 25 relapsed MM patients (aged 58-77 years) were treated with low dose len (15mg Days 1-21 of each 28-day cycle) and dex (20mg/day, Days 1-4,9-12,17-20). After a median of 9 (2-19) cycles, 19 patients responded (24% CR/VGPR, 52% PR). At baseline, NK cell numbers and function [assessed by % lysis of 51Cr labelled K562 target cells at 50 (effector):1 (target) ratio] in MM patients were similar to age matched controls (0.2 vs. 0.3× 105/ml in controls, p=0.09 and 49% K562 cell lysis vs. 58% in controls, p=0.44 respectively) (fig.1A). Whilst NK cell numbers slightly increased in vivo after len-dex treatment [2.0 (baseline) vs. 3.9×105/l (cycle 6), p=0.04, paired t test] (fig.1A), mean NK cell function progressively decreased compared to baseline after 6 and 9 len-dex cycles [mean 49% K562 cell lysis at baseline vs. 28% after 6 cycles (p=0.007) and 31% after 9 cycles (p=0.02)] (fig.1B). Following 72 hours of in vitro treatment with len (10mM), there was increased NK function in healthy donor peripheral blood mononuclear cells (PBMC) [mean 54% K562 cell lysis from len-treated PBMC vs. 38% lysis in untreated PBMC, p=0.04] (fig. 2). In PBMCs from MM patients at baseline, ex vivo treatment with len (10mM) did not significantly increase NK cell function [mean 47% K562 cell lysis (untreated) vs. 52% (len treated), p=0.17], nor did it increase NK cell function after 6 len-dex treatment cycles [mean 32% K562 cell lysis (untreated) vs. 30% (treated), p=0.4].Conversely, dex (0.1mM) decreased NK cell function in healthy donors' PBMC [mean 7.6% K562 cell lysis (dex treated) vs. 38% (untreated) p=0.01], even in the presence of len [mean 7% K562 cell lysis (len+dex) vs. 38% (untreated), p=0.002] (fig. 2). Dex-induced in vitro NK inhibition was dose dependent and could be rescued by the addition of IL-2 to normal donors [mean 7.6 % K562 cell lysis (dex) vs. 28% lysis (Dex +IL2),p=0.03] as well as PBMC from MM patients at baseline [mean lysis 16% (dex) vs. 59% (Dex+IL2) p=0.0002]. However, IL-2 was less able to rescue dex-induced NK dysfunction in PBMC from patients post 6 treatment cycles compared to patients at baseline [mean 59% K562 cell lysis (baseline) vs. 28% (C6), p=0.03]. Dex induced NK dysfunction was reversible as NK cell function recovered after a 3 days dex washout. In summary, NK function in MM patients, whilst similar to healthy controls at baseline, progressively decreases after prolonged len-dex treatment despite a clinical response. The observed decrease in NK function in vivo and in vitro is directly due to the effects of dex, which could not be reversed by the NK activating effects of len. Our results suggest that the efficacy of len and dex co-therapy is not due to augmentation of NK cytolytic activity, due to the immunosuppressive effects of dex against NK cells. This suggests that alternative dosing schedules of dex, after initial induction with len and dex co-therapy, may optimise len-induced immunostimulation of NK cells and subsequent sustained disease control via anti-MM immunity. Disclosures Lynch: Celgene Corporation: Employment. Prince:Celgene Corporation: Research Funding.

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