CD16 pre-ligation by defucosylated tumor-targeting mAb sensitizes human NK cells to γc cytokine stimulation via PI3K/mTOR axis
Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.
KeywordsObinutuzumab CD16 Natural killer cells IFN-γ PI3K/mTOR miR-155
eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1
common γ chain
ethylene glycol tetraacetic acid
mammalian target of rapamycin
Natural Killer group 2 member D
real-time quantitative PCR
ribosomal protein S6 kinase
src homology 2 domain-containing leukocyte protein of 76 kDa
suppressor of cytokine signaling 1
NK cells doubly contribute to the therapeutic efficacy of tumor-targeting mAbs; besides the killing of tumor cells mediated by the engagement of the low affinity receptor for IgG, FcγRIIIA/CD16, activated NK cells secrete pro-inflammatory cytokines and chemokines which act in boosting the recruitment and activation of other immune effector cells and the development of long-lasting T cell immunity [2, 3]. In particular, NK-derived IFN-γ stands as a key immunoregulatory factor in the shaping of anti-tumor adaptive immune responses, by promoting the maturation of DC and the subsequent development of Th1 and CTL responses . Indeed, the current understanding of anti-tumor responses driven by tumor-targeting mAbs introduced a novel ground by which NK cells may contribute to the development of a vaccine-like effect required for the long-term protection of mAb-treated patients [5, 6, 7, 8, 9].
By virtue of an accessible Ifng locus, NK cells represent a prompt source of IFN-γ. Such cytokine is transcribed constitutively at low levels in NK cells; its increased production in response to cytokines or after the engagement of activating receptors is tightly regulated at transcriptional and post-transcriptional levels [10, 11, 12]. In this context, microRNA (miR)-155 functions as a positive regulator of IFN-γ production stimulated by CD16 and cytokines  by directly targeting the hematopoietic cell-specific inositol 5-phosphatase, SHIP-1, which negatively regulates the PI3K pathway . Downstream PI3K, the master metabolic regulator mammalian target of rapamycin (mTOR) promotes IFN-γ translation through the phosphorylation of the ribosomal protein S6 kinase (S6K) and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) [15, 16, 17, 18].
To reach an enhanced clinical efficacy, new mAbs with increased affinity for CD16 have been generated. Among them, obinutuzumab, recently approved for clinical use [19, 20, 21], is a type II glycoengineered anti-CD20 mAb with a defucosylated crystallizable fragment (Fc) domain that binds to a CD20 epitope in a different space orientation with respect to the reference molecule rituximab [22, 23].
Here, extending our previous observations , we demonstrate that following obinutuzumab pre-stimulation, NK cells undergo enhanced IFN-γ production in response to a subsequent re-stimulation with common γ chain (γc) cytokines IL-15 or IL-2, which correlates to the upregulation of miR-155 and to reduced SHIP-1 levels but not with the upregulation of IFN-γ mRNA levels; the increased IFN-γ competence depends on the PI3K/mTOR axis.
Such data add mechanistic insights into NK cell plasticity in therapeutic settings. Moreover, taking into account the current research efforts focused on the development of IL-2 and IL-15 cytokine variants with extended half-life and targeted action , our results suggest that obinutuzumab-based immunotherapy in combination with NK cell-activating cytokines may achieve a useful synergism for the development of long-lasting curative anti-tumor responses.
Materials and methods
The following anti-CD20 mAbs were used: the chimeric IgG1κ rituximab, the humanized IgG1κ obinutuzumab (GA101), and its non-glycoengineered parental molecule, GA101 wild type (WT), all kindly provided by Roche Innovation Center Zurich (Schlieren, Switzerland).
For functional assays, the following mAbs were used: anti-2B4 (clone:C1.7, #IM1607, Beckman Coulter Life Science), anti-NKp46 (clone: 9E2, #331902, Biolegend), anti-natural killer group 2 member D (NKG2D) (clone: 149810, #MAB139, R&D Systems), all mouse IgG1 isotype, and goat F(ab')2 fragment anti-mouse IgG (H + L) (#115-006-003, Jackson ImmunoResearch Laboratories). The following fluorochrome-conjugated mAbs were used for flow cytometric analysis: anti-CD25 APC (clone:M-A2511, #555434) and anti-CD215 PE (clone:JM7A4, #566589) were from BD Biosciences; the anti-pS6 ribosomal protein (S235/236) PE (clone: D57.2.2E, #5316S) was from Cell Signaling Technology. For immunoblot analysis, antibodies were obtained from the following sources: anti-SHIP-1 (clone:P1C1, #sc-8425) from Santa Cruz Biotechnology Inc); the anti-phospho-STAT4 (Tyr693) (clone:D2E4, #4134), anti-STAT4 (clone:C46B10, #2653), anti-Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) (#4958) and anti-Akt (#9272), all from Cell Signaling Technology.
Patients and healthy donors
PBMCs were obtained from anonymized healthy donors of Transfusion Center of Sapienza University (Rome, Italy) or CLL patients of Hematology Unit, S. Maria Goretti Hospital (Latina, Italy).
The diagnosis of CLL was based on criteria recommended by the International Workshop on Chronic Lymphocytic Leukemia (IWCLL); the stage of disease was assessed according to the Rai staging system . In all specimens, the percentage of CD5+CD19+ CLL cells was more than 70%. From each patient, part of PBMC was used to obtain primary cultured NK cells (see below) and the rest was cryopreserved and stored at − 160 °C. The day before the experiment, thawed samples were rested overnight in complete medium. Only samples with viability more than 90% were used.
Primary cultured human NK cells were obtained from healthy donors , or from CLL patients as previously described . Briefly, PBMC were co-cultured for 10 days with irradiated (3000 rad) Epstein–Barr virus positive RPMI 8866 lymphoblastoid cell line in 10% FCS and 1% l-glutamine containing RPMI 1640 (all from Euroclone). Experiments were performed on NK cell cultures which were more than 80% pure (CD3−CD56+).
Anti-CD20-experienced NK cell preparation and purification
The human CD20+ lymphoblastoid Raji cell line or primary B-CLL cells were loaded with 10 μγ/ml of EZ-Link Sulfo-NHS-SS-Biotin (#21331, Thermo Fisher Scientific) for 30 min at room temperature , washed twice with PBS (Euroclone) and then opsonized for 20 min at room temperature with saturating doses of rituximab, obinutuzumab or obinutuzumab WT. Not opsonized and anti-CD20-opsonized targets were mixed at 1:2 ratio with primary cultured NK cells for 18 h, and then washed twice with cold 5 mM EDTA containing PBS. NK cells were purified by negative selection on biotin-binder Dynabeads (#11047), followed by anti-CD3-coated Dynabeads (#11151D) (both from Invitrogen, Life Technologies), according to the manufacturer’s protocols. Experienced NK populations were checked by flow cytometry to assess purity.
Evaluation of IFN-γ release
Purified experienced NK cells (5 × 105/ml) were resuspended in complete medium and left untreated or stimulated for 18 h at 37 °C with 500 U/ml of IL-2 (#200-02), or 10 ng/ml of IL-12 (#200-12), or 100 ng/ml of IL-15 (#200-15) (all from PeproTech). Where required, 10 μM idelalisib (CAL-101, GS1101; #S2226, Selleck Chemicals) or 20 nM rapamycin (#R0395, Merck) was added 2 h before cytokine treatment, and maintained until the end of stimulation. An equivalent volume of DMSO (#D5879, Merck), as vehicle, was added to control samples.
Stimulation of activating receptors was performed by plastic-immobilized goat F(abʹ)2 anti-mouse IgG (H + L) (1 μg/106) followed by anti-NKp46 (0.5 μg/106), anti-NKG2D (1 μg/106) or anti-2B4 (0.24 μg/106) mAbs, used alone or in combination. IFN-γ was quantified in the supernatants by commercial ELISA kit (#EHIFNG2, Thermo Fisher Scientific), according to the manufacturer’s instructions.
Stimulation and analysis of S6 phosphorylation
To determine the phosphorylation status of ribosomal protein S6, purified experienced NK cells were resuspended in complete medium, rested for 2 h at 37 °C and stimulated with 500 U/ml of IL-2 or 100 ng/ml of IL-15 at 37 °C for different lengths of time. Following stimulation, samples were washed, fixed and permeabilized with commercial kit (# 00-5523-00, eBioscience, Thermo Fisher Scientific), according to the manufacturer’s instructions, and stained with PE-conjugated anti-pS6 (S235/236) rabbit mAb. Samples were acquired on a FACSCanto II (BD Bioscience) and analyzed with FlowJo v.9.3.2 (TreeStar) software.
miR and mRNA analyses
Total RNA, including miRs, was extracted from highly purified experienced NK cells before and after cytokine stimulation using Total RNA Purification Kit (#3755, Norgen Biotek Corp). The concentration and purity of extracted RNA were determined spectrophotometrically using NanoDrop 2000 (Thermo Fisher Scientific). cDNA was generated by means of High-Capacity cDNA Reverse Transcription kit (#4374966) or TaqMan MicroRNA Reverse Transcription kit (#4366596), using RT primers specific for miR-155, miR-29a, RNU-44 and RNU-48. mRNA, and miR levels were determined by real-time quantitative PCR (RT-qPCR) using TaqMan gene expression or miR assays, according to the manufacturer’s instructions. All reagents were obtained from Applied Biosystem, Life Technologies (Thermo Fisher Scientific) and the list of all primers employed is reported below. Reactions were performed in 96-well plate format by StepOnePlus real-time PCR system and data obtained were analyzed by StepOne Software v2.3 (Applied Biosystem). Water (no template) was loaded as a negative control. mRNA and miR contents were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or RNU-44 and RNU-48 endogenous controls, respectively, and relative quantification was evaluated by the comparative cycling threshold (ΔΔCT) method. The fold change was calculated according to the formula 2−ΔΔCT, setting the control population to 1. The following TaqMan gene expression assays (#4331182) were used: Hs00989291_m1 IFNG, Hs00183290_m1 INPP5D, Hs00705164_s1 suppressor of cytokine signalling 1 (SOCS-1), Hs03929097_g1 GAPDH. The following TaqMan miRNA assays (#4427975) were used: hsa-miR-155 (#002623), hsa-miR-29a-5p (#002447), RNU-44 (#001094), RNU-48 (#001006) all conjugated with fluorochrome carboxyfluorescein (FAM).
For assessing STAT4 phosphorylation, purified experienced NK populations were stimulated for 30 min at 37 °C with 500 U/ml of IL-2 or 100 ng/ml of IL-12, or left untreated, and lysed in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA, pH8, 1 mM ethylene glycol tetraacetic acid (EGTA), pH 8, 5 mM MgCl2, 5 mM NaF) supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 μg/ml each of aprotinin and leupeptin inhibitors. To analyze SHIP-1, SLP-76 and Akt levels, whole cell lysates of purified experienced NK cells were obtained by incubating with 1% Triton X-100 lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, pH 8, 1 mM MgCl2, 50 mM NaF) supplemented with protease and phosphatase inhibitors as above. Protein content was determined by Bradford Colorimetric Assay (#500-0006, Bio-Rad Laboratories) and equal amounts of proteins from each sample were separated by SDS-PAGE and transferred to nitrocellulose for immunoblot analysis. Quantification of specific bands was performed with ImageJ1.41o software (National Institutes of Health).
Wilcoxon matched-pairs signed rank or paired t tests were used to determine statistically significative differences (p value < 0.05) between two groups, as appropriate. Error bars represent the SEM. Analysis were performed using Prism v.6 (GraphPad Software).
Pre-stimulation of CD16 with obinutuzumab-opsonized targets leads to an amplified IFN-γ production in response to IL-2 or IL-15, independently of transcriptional regulation
To assess whether the increased IFN-γ production may be attributable to the modulation of specific cytokine receptor subunits, we evaluated IL-2 receptor (IL-2R) α-chain (CD25) and IL-15R α-chain (CD215) expression levels. While a robust upregulation of IL-2R α surface levels was observed in obinutuzumab- and, to a lesser extent, in rituximab-experienced cells, IL-15R α levels remained unaffected (Fig. 1c), thus indicating that the increased cytokine responsiveness does not completely rely on receptor affinity modulation.
Overall, these data demonstrate that in obinutuzumab-experienced cells the enhanced IFN-γ production in response to γc cytokine stimulation relies on post-transcriptional events.
miR-155 upregulation in obinutuzumab-experienced NK cells is associated with reduced SHIP-1 levels
We analyzed the levels of selected miRs previously involved in the regulation of IFN-γ production in NK cells [30, 31]. It is known that both miR-155 and miR-29a are poorly expressed in primary human NK cells and that their increased levels in activated cells act in the regulation of IFN-γ production [13, 32, 33, 34].
Remarkably, we found higher miR-155 levels in obinutuzumab- compared to rituximab-experienced NK cells also in primary NK cells derived from untreated CLL patients (Supplementary Table 1) stimulated for 18 h with anti-CD20-opsonized autologous leukemia (Fig. 3b). Similar results were obtained using RNU-48 as endogenous control (Supplementary Fig. 1).
Accordingly, we found a marked reduction of SHIP-1 at protein level of almost 65% in obinutuzumab-experienced cells, but not in rituximab- or GA101WT-stimulated samples, with respect to control population. A consensual downregulation of the signaling intermediate SLP-76, known to be a direct target of miR-155 too , was observed in obinutuzumab-experienced cells (Fig. 4b).
Collectively, these data demonstrate that the sustained CD16 ligation by means of obinutuzumab-opsonized targets induces the upregulation of miR-155 levels associated with the downregulation of both SHIP-1 and SLP-76.
Role of PI3K/mTOR pathway in the increased IFN-γ competence of obinutuzumab-experienced NK cells
Downstream of PI3K, mTOR acts in promoting mRNA translation . We assessed ribosomal protein S6 phosphorylation in response to cytokine stimulation. The basal phosphorylation levels are comparable between all the experimental groups (Supplementary Fig. 2). In control population, the stimulation with IL-2 or IL-15 induces a time-dependent increase of S6 phosphorylation up to 60 min (Supplementary Fig. 3a). In obinutuzumab-experienced cells, in response to γc cytokines, we observed higher levels of S6 phosphorylation, particularly at later time points of stimulation, with respect to rituximab- or not opsonized target-experienced cells, which behave comparably (Fig. 5b).
The functional role of the mTOR/S6K pathway was evaluated by assessing IFN-γ production in the presence of the mTOR inhibitor, rapamycin. Rapamycin treatment inhibits, in a dose-dependent manner, cytokine-induced S6 phosphorylation (Supplementary Fig. 3b); by comparing the effect of rapamycin on anti-CD20-experienced populations, we observed a more pronounced inhibition of IFN-γ production in obinutuzumab- with respect to rituximab-experienced cells (Δ between untreated and treated cells is 1158 for GA101-exp vs 647 for RTX-exp for IL-2 setting; 1051 for GA101-exp vs 530 for RTX-exp for IL-15 setting) (Fig. 5c). Indeed, rapamycin prevents the enhanced response of obinutuzumab-experienced cells.
All together, these data indicate that the enhanced IFN-γ production in response to IL-2 or IL-15 stimulation relies on a potentiated PI3K/mTOR/S6K axis.
Recently, Pahl et al. demonstrated that CD16 aggregation by a tetravalent bispecific therapeutic antibody AFM13 (CD30/CD16A) followed by long-term re-stimulation with IL-2 or IL-15 results in a global NK cell hyperresponsiveness, attributable to increased cytokine receptor expression levels . In our experimental conditions, we observed that the improved sensitivity of obinutuzumab-experienced cells to cytokine receptor stimulation coincided with increased CD25 expression levels, which, together with CD122 (IL-2Rβ) and CD132 (γc), assembles the trimeric high-affinity IL-2R , whereas IL-15R α chain, which by itself mediates the high-affinity binding [27, 39], remains unaffected upon CD16 stimulation. Such observations, along with the fully stimulatory doses of cytokines used, would exclude an increased receptor affinity as the unique mechanism responsible for the enhanced responsiveness.
The lack of correlation between IFN-γ protein and mRNA levels in γc cytokine-stimulated samples led us to hypothesize that post-transcriptional mechanisms may be responsible for the amplified response. In this context, our data confirm the existence of fundamental differences in the regulation of IFN-γ production induced by different cytokines [12, 40, 41]; in response to stimulation with IL-12, IFN-γ transcript increases almost 20-fold, whereas stimulation with IL-2 or IL-15 leads to a lower IFNG gene transcription, despite the efficient IFN-γ production. The stronger transcriptional effect of IL-12 is in line with a necessary role of STAT4 for maximal IL-12-mediated IFN-γ transcription [40, 41]. The increased IFN-γ competence is reminiscent of the recently described memory NK cell population which, in fact, exhibits enhanced IFN-γ production in response to CD16 stimulation ; however, memory NK cells display a defective responsiveness to IL-12 stimulation .
Several recent studies have demonstrated that IFN-γ production by NK cells is under the control of the metabolic sensor mTOR [16, 43, 44]. PI3K/Akt/mTOR pathway is an essential component of signaling via IL-15R and IL-2R [16, 45], with mTOR involved in translational activation through the phosphorylation of 4E-BP1 and S6K [15, 17, 18, 45].
In this context, our data evidence, in obinutuzumab-experienced cells, an amplified ribosomal protein S6 phosphorylation induced by γc cytokine stimulation along with a higher sensitivity to the mTOR inhibitor rapamycin, thus indicating that the increased IFN-γ competence depends on the mTOR pathway.
Based on these data, we reasoned that CD16 ligation in high-affinity conditions may lower the threshold for PI3K/mTOR pathway activation, thus leading to γc cytokine hyperresponsiveness.
SHIP-1 phosphatase acts in constraining PI3K/Akt-dependent signals  and negatively regulates IFN-γ production by monokines and CD16 stimulation in both human and mouse NK cells [35, 36]; its reduced expression levels in CD56bright with respect to CD56dim NK cells contribute to the increased IFN-γ producing potential of the former population . SHIP-1, along with SOCS-1, are directly targeted and repressed by miR-155, which is involved in the regulation of IFN-γ production stimulated by cytokines in NK cells [13, 14, 32, 33].
On this line, we evidence that the upregulation of miR-155 transcript, in obinutuzumab-experienced cells, associates with SHIP-1 but not SOCS-1 downregulation; the observation that both rituximab and obinutuzumab WT stimulation induce still significant but lower levels of miR-155 indicates that the strength of CD16 ligation dictates the capability to promote miR-155 expression. On this issue, a recent paper highlighted a correlation between the strength of TCR stimulation and miR-155 expression levels that impacts IFN-γ production by anti-tumor CD8+ T cells .
SLP-76, a direct target of miR-155 in NK cells , is known to orchestrate the formation of a molecular platform critical in signal integration downstream activating receptors . The reduction of SLP-76 levels, along with the degradation of FcεRIγ and CD3ξ adaptor chains and the Spleen-associated tyrosine kinase, Syk , may explain the profound impairment of IFN-γ production induced by the engagement of NKG2D and 2B4 activating receptors in anti-CD20-experienced cells. It should be noted that in a different experimental setting, based on shorter CD16 pre-ligation (90 min), we previously reported that the capability to produce IFN-γ in response to activating receptor stimulation was preserved . In such conditions, no miR-155 upregulation and consequently no SLP-76 downmodulation have occurred (C. Capuano, unpublished observation).
The role of PI3K in the priming of obinutuzumab-experienced cells is strengthened by the observation that the inhibition of PI3K greatly counteracts γc cytokine hyperresponsiveness, while, as previously described, it has a more marginal effect on NK cell viability and cytotoxicity .
NK cell plasticity is being exploited by new NK cell-based intervention strategies against cancer [49, 50, 51]; by recapitulating the signaling pathway responsible for CD16-dependent priming, this study evidences new aspects of NK cell adaptation in a therapeutic setting. Taking into account that NK-derived IFN-γ is a key immunoregulatory factor in the shaping of anti-tumor adaptive immune responses, obinutuzumab-based therapy may be envisaged as a driver of a mAb-mediated vaccinal effect [5, 6, 7, 8, 9] that could significantly impact on long-term therapeutic efficacy. Suitable clinical trials focused on the prospective evaluation of the emergence of adaptive anti-tumor responses in obinutuzumab-treated patients are needed to highlight the actual translational impact.
We are grateful to Prof. G. Cimino, Department of Translational and Precision Medicine, Cellular Biotechnology and Hematology, Hematology Unit (2U), S. Maria Goretti Hospital/ICOT, AUSL Latina, Italy, for CLL patients’ staging and recruitment. We also thank Dr. F.D. Batista (Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA) for providing Raji cell line and P. Birarelli and B. Milana for their technical support.
CC and CP designed the study, collected, analyzed and interpreted data and assisted to write the manuscript. RM and SB contributed to the experiments and to organize data. SM, GP, AS and CK contributed to data analysis and interpretation, and revised the manuscript. RG conceived and designed the study, analyzed and interpreted data and wrote the manuscript. All authors read and approved the final manuscript.
Cristina Capuano is supported by the Italian Ministry of University and Research (MIUR) (Grant number RBSI14022M). Simone Battella is a recipient of an Italian Association for Cancer Research (AIRC) fellowship. This work was supported by Roche Glycart AG, Schlieren, Switzerland.
Compliance with ethical standards
Conflict of interest
Christian Klein declares employment, patents and stock ownership with Roche Glycart AG, Schlieren, Switzerland. All other authors declare that they have no conflict of interests.
Ethical approval and ethical standards
All procedures involving human participants were in accordance with the ethical standards of the institutional research committee and with 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The study was approved by the Ethics Committee of Sapienza University of Rome (approval number 639/16 RIF/CE 4179).
All experiments were performed after obtaining written informed consent from patients and healthy donors. The informed consent was taken at the beginning of the study to use data and materials for research and publication of this study.
Cell line authentication
RPMI 8866 cell line was purchased by American Type Culture Collection, and Raji cell line was provided by Dr. F.D. Batista. Cells were authenticated (last testing May 2019) by morphology, growth and immunophenotypic characteristics, biologic behavior according to the provider recommendations, and routinely tested for mycoplasma contamination by EZ-PCR Mycoplasma test kit (#20–700-20, Biological Industries). All cell lines were kept in culture for less than two consecutive months.
- 1.Pighi C, Capuano C, Maggio R et al (2019) CD16 aggregation in high affinity conditions by tumor-targeting mAb obinutuzumab promotes a PI3K/mTOR-dependent priming of Natural Killer cells for IFN-gamma production, associated to miR-155 upregulation. II Joint Meeting of the German Society for Immunology (DGfl) and the Italian Society of Immunology, Clinical Immunology and Allergology (SIICA). Eur J Immunol 49(1):271–272. https://doi.org/10.1002/eji.201970300 (Munich, September 10–13, 2019 [meeting abstract number P366])
- 2.Long EO, Kim HS, Liu D, Peterson ME, Rajagopalan S (2013) Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol 31:227–258. https://doi.org/10.1146/annurev-immunol-020711-075005 CrossRefPubMedGoogle Scholar
- 5.Srivastava RM, Lee SC, Andrade Filho PA et al (2013) Cetuximab-activated natural killer and dendritic cells collaborate to trigger tumor antigen-specific T-cell immunity in head and neck cancer patients. Clin Cancer Res 19:1858–1872. https://doi.org/10.1158/1078-0432.CCR-12-2426 CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Sehn LH, Chua N, Mayer J et al (2016) Obinutuzumab plus bendamustine versus bendamustine monotherapy in patients with rituximab-refractory indolent non-Hodgkin lymphoma (GADOLIN): a randomised, controlled, open-label, multicentre, phase 3 trial. Lancet Oncol 17:1081–1093. https://doi.org/10.1016/S1470-2045(16)30097-3 CrossRefPubMedGoogle Scholar
- 26.Capuano C, Battella S, Pighi C et al (2018) Tumor-targeting anti-CD20 antibodies mediate in vitro expansion of memory natural killer cells: impact of CD16 affinity ligation conditions and in vivo priming. Front Immunol 9:1031. https://doi.org/10.3389/fimmu.2018.01031 CrossRefPubMedPubMedCentralGoogle Scholar
- 35.Parihar R, Trotta R, Roda JM et al (2005) Src homology 2-containing inositol 5'-phosphatase 1 negatively regulates IFN-gamma production by natural killer cells stimulated with antibody-coated tumor cells and interleukin-12. Cancer Res 65:9099–9107. https://doi.org/10.1158/0008-5472.CAN-04-4424 CrossRefPubMedGoogle Scholar
- 48.Herman SE, Gordon AL, Wagner AJ et al (2010) Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood 116:2078–2088. https://doi.org/10.1182/blood-2010-02-271171 CrossRefPubMedPubMedCentralGoogle Scholar
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