eNK Cells Research Articles

Feeder cell training shapes the phenotype and function of in vitro expanded natural killer cells.

Natural killer (NK) cells are candidates for adoptive cell therapy, and the protocols for their activation and expansion profoundly influence their function and fate. The complexity of NK cell origin and feeder cell cues impacts the heterogeneity of expanded NK (eNK) cells. To explore this, we compared the phenotype and function of peripheral blood-derived NK (PB-NK) and umbilical cord blood-derived NK (UCB-NK) cells activated by common feeder cell lines, including K562, PLH, and 221.AEH. After first encounter, most PB-NK cells showed degranulation independently of cytokines production. Meanwhile, most UCB-NK cells did both. We observed that each feeder cell line uniquely influenced the activation, expansion, and ultimate fate of PB eNK and UCB eNK cells, determining whether they became cytokine producers or killer cells. In addition, they also affected the functional performance of NK cell subsets after expansion, that is, expanded conventional NK (ecNK) and expanded FcRγ- NK (eg-NK)cells. Hence, the regulation of eNK cell function largely depends on the NK cell source and the chosen expansion system. These results underscore the significance of selecting feeder cells for NK cell expansion from various sources, notably for customized adoptive cell therapies to yield cytokine-producing or cytotoxic eNK cells.

Unraveling the early transcriptional signature of peripheral blood NK cell transition to uterine residency

Abstract Tissue-resident uterine NK cells are essential for successful pregnancy, yet the molecular mechanisms guiding their tissue residency programming early after infiltration of blood-based precursors remain unknown. We examined the transition of CD56+ peripheral blood NK cells to a uterine resident state by performing scRNA-seq on enriched CD56+ cells from a matched peripheral blood (pb) and endometrial (e) biopsy taken during the secretory phase. Our integrated analysis comparing eNK vs pbNK cells identified a tissue adaptation signature. Key components of this signature included NR4A2 (94% vs 0%, p=9.46e-22), FOS (90% vs 9%, p=9.05e-14), and JUNB (77% vs 16%, p=3.15e-07) respectively. This signature also mirrored genes previously identified in activated murine intestinal CD8+ TRM cells early after LCMV infection. This signature was found in all endometrial CD56+ cells, including CD56bright CD16- eNKs, NKTs, and conventional eNKs (CD56dimCD16+). Pathway analysis implicated IL-17 and IL-18 cytokines, along with NFAT and VEGF signals in tissue residency programming. In conclusion, these data expand our understanding of early transcriptional programs in lymphocyte tissue adaptation, revealing the involvement of cytokines beyond TGF-β. Our data further indicate that signals associated with early tissue adaptation are conserved across species and cell types independent of an antigen receptor.

Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Abstract 1336: Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma

Abstract Malignant pleural mesothelioma is a malignant tumor that is caused by exposure to asbestos. Because it is a rare cancer, there are few drugs that can be used to treat it. Healios is developing iPSC-derived NK cells (eNK cells) (HLCN061) engineered with NKG2D, DAP10, IL-15, CD16, CCL19, and CCR2B genes for the treatment of refractory solid tumors. This study evaluated the antitumor effect of eNK cells against human malignant pleural mesothelioma cell lines.(Method) The antitumor effect of eNK cells on human malignant pleural mesothelioma cell lines in vitro was evaluated using the Viability/Cytotoxicity Multiplex Assay Kit (WST-8/LDH).In vivo, immunodeficient mice were subcutaneously inoculated with human malignant pleural mesothelioma cell lines. After subcutaneous tumor formation, eNK cells were inoculated into or near the tumor, the size of the subcutaneous tumor was measured, and the tumor volume was compared with that of an untreated group. After the study was completed, the tumors were removed and the tumors in the untreated group and the eNK cell-administered group were analyzed using RNA-seq.(Results) The antitumor effect of eNK cells against human malignant pleural mesothelioma cell lines was confirmed in vitro. In a mouse subcutaneous tumor model, the eNK cell treatment group suppressed tumor growth compared to the untreated group (p≦0.05). RNA-seq showed increased expression of granulysin, IL2RB, cathepsin W, etc. in subcutaneous tumors treated with eNK cells. Citation Format: Seiji Matsumoto, Yoshiko Fujita, Kumiko Goto, Yuka Sato, Fusako Nishigaki, Yasuyuki Higashi, Hironobu Kimura, Kouichi Tamura. Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1336.

Dynamic Changes in miRNA Expression during the Generation of Expanded and Activated NK Cells.

Therapies based on allogenic Natural Killer (NK) cells are becoming increasingly relevant, and our laboratory has produced expanded and activated NK (eNK) cells that are highly cytotoxic against several hematological cancers when used alone or in combination with currently approved therapeutic monoclonal antibodies. In order to produce eNK cells, healthy human donor NK cells undergo a 20-day expansion protocol with IL-2, IL-15 and Epstein-Barr virus (EBV)-transformed lymphoblastoid feeder cells. In order to produce an even more potent eNK-based therapy, we must elucidate the changes our protocol produces within healthy NK cells. To understand the post-transcriptional changes responsible for the increased cytolytic abilities of eNK cells, we performed microRNA (miRNA) expression analysis on purified NK cells from day 0 and day 20 of the protocol using quantitative reverse transcription PCR (RT-qPCR). Of the 384 miRNAs profiled, we observed changes in the expression of 64 miRNAs, with especially significant changes in 7 of them. The up-regulated miRNAs of note were miRs-146a, -124, -34a, and -10a, which are key in the regulation of cell survival through the modulation of pro-apoptotic genes such as PUMA. The down-regulation of miRs-199a, -223, and -340 was also detected and is associated with the promotion of NK cell cytotoxicity. We validated our analysis using immunoblot and flow cytometry studies on specific downstream targets of both up- and down-regulated miRNAs such as PUMA and Granzyme B. These results corroborate the functional importance of the described miRNA expression patterns and show the wide variety of changes that occur in eNK cells at day 20.

Killer Timing: The Temporal Uterine Natural Killer Cell Differentiation Pathway and Implications for Female Reproductive Health.

Natural killer (NK) cells are the predominant maternal uterine immune cell component, and they densely populate uterine mucosa to promote key changes in the post-ovulatory endometrium and in early pregnancy. It is broadly accepted that (a) immature, inactive endometrial NK (eNK) cells in the pre-ovulatory endometrium become activated and transition into decidual NK (dNK) cells in the secretory stage, peri-implantation endometrium, and continue to mature into early pregnancy; and (b) that secretory-stage and early pregnancy dNK cells promote uterine vascular growth and mediate trophoblast invasion, but do not exert their killing function. However, this may be an overly simplistic view. Evidence of specific dNK functional killer roles, as well as opposing effects of dNK cells on the uterine vasculature before and after conception, indicates the presence of a transitory secretory-stage dNK cell (s-dNK) phenotype with a unique angiodevelopmental profile during the peri-implantation period, that is that is functionally distinct from the angiomodulatory dNK cells that promote vessel destabilisation and vascular cell apoptosis to facilitate uterine vascular changes in early pregnancy. It is possible that abnormal activation and differentiation into the proposed transitory s-dNK phenotype may have implications in uterine pathologies ranging from infertility to cancer, as well as downstream effects on dNK cell differentiation in early pregnancy. Further, dysregulated transition into the angiomodulatory dNK phenotype in early pregnancy will likely have potential repercussions for adverse pregnancy outcomes, since impaired dNK function is associated with several obstetric complications. A comprehensive understanding of the uterine NK cell temporal differentiation pathway may therefore have important translational potential due to likely NK phenotypic functional implications in a range of reproductive, obstetric, and gynaecological pathologies.

Abstract 2755: Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies

Abstract Natural Killer (NK) cells have shown great promise as a viable cell-based cancer immunotherapy. We have developed a protocol that produces expanded and highly activated NK (eNK) cells from healthy human donors that have been shown to be effective alone and in combination with monoclonal antibodies in a variety of hematological malignancies. The eNK cells showed a dramatic increase in cytolytic capacity after their expansion and activation using IL-2, IL-15, and 721.221 feeder cells. In order to elucidate the underlying mechanisms behind the changes, mRNAseq and miRNA arrays were performed on mRNA and miRNA samples, respectively, extracted from several NK expansions, taken at Day 0 (before activation) and Day 20 (after activation and expansion). Bioinformatic analysis was performed to identify differential gene expression changes and their downstream effects. Based on the genes with greatest differential expression, we quantified their corresponding proteins using flow cytometry, western blots, and ELISAs. Results reveal several discrete changes in the transcriptome that are reflected in changes in protein expression levels within the eNK cells. We conclude that the increased cytolytic capacity of eNK cells is due to the synergistic effect of variations in different genes responsible for target recognition, effector function, cell cycle progression and cell survival. These data can be used in the future to produce better eNK cells as treatment for hematological malignancies. Citation Format: Chantal Reina-Ortiz, Pilar Mozas Alonso, Alberto Cebollada, David Ovelleiro, Luis Alberto Anel Bernal. Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2755.

Uterine Natural Killer Cells: A Rising Star in Human Pregnancy Regulation.

Uterine natural killer (uNK) cells are an immune subset located in the uterus. uNK cells have distinct tissue-specific characteristics compared to their counterparts in peripheral blood and lymphoid organs. Based on their location and the pregnancy status of the host, uNK cells are classified as endometrial NK (eNK) cells or decidua NK (dNK) cells. uNK cells are important in protecting the host from pathogen invasion and contribute to a series of physiological processes that affect successful pregnancy, including uterine spiral artery remodeling, fetal development, and immunity tolerance. Abnormal alterations in uNK cell numbers and/or impaired function may cause pregnancy complications, such as recurrent miscarriage, preeclampsia, or even infertility. In this review, we introduce recent advances in human uNK cell research under normal physiological or pathological conditions, and summarize their unique influences on the process of pregnancy complications or uterine diseases. Finally, we propose the potential clinical use of uNK cells as a novel cellular immunotherapeutic approach for reproductive disorders.

Decidual Natural Killer Cells Are Essential for a Successful Pregnancy (Review)

Pregnancy is a complex physiological process involving several interconnected systems. Many researchers were concerned that the formation of a fetus with different genetic components may contradict the normal state of immunity, which attempts to reject and fight foreign bodies. This piqued the interest of biologists and immunologists, who set out to discover the immune system’s composition and mode of response in the uterus. According to several studies, natural killer (NK) cells are present in a significant percentage that differs from what is seen in peripheral blood. As a result, several scientific studies have been conducted on uterine NK cells, investigating their types, characteristics, receptors, secretions, and interactions with the surrounding environment. Research has also indicated the capacity of uterine NK cells to strike a balance between eradicating uterine infections and effectively contributing to different phases of pregnancy. Various studies have shown that NK cell activity is intimately related to the success or failure of pregnancy. In this review, we describe the uterine NK cell subtypes; decidual (dNK) cells and endometrial NK cells (eNK) cells and their important role during different phases of pregnancy.

Expanded NK cells from umbilical cord blood and adult peripheral blood combined with daratumumab are effective against tumor cells from multiple myeloma patients

ABSTRACT In this study we evaluated the potential of expanded NK cells (eNKs) from two sources combined with the mAbs daratumumab and pembrolizumab to target primary multiple myeloma (MM) cells ex vivo. In order to ascertain the best source of NK cells, we expanded and activated NK cells from peripheral blood (PB) of healthy adult donors and from umbilical cord blood (UCB). The resulting expanded NK (eNK) cells express CD16, necessary for carrying out antibody-dependent cellular cytotoxicity (ADCC). Cytotoxicity assays were performed on bone marrow aspirates of 18 MM patients and 4 patients with monoclonal gammopathy of undetermined significance (MGUS). Expression levels of PD-1 on eNKs and PD-L1 on MM and MGUS cells were also quantified. Results indicate that most eNKs obtained using our expansion protocol express a low percentage of PD-1+ cells. UCB eNKs were highly cytotoxic against MM cells and addition of daratumumab or pembrolizumab did not further increase their cytotoxicity. PB eNKs, while effective against MM cells, were significantly more cytotoxic when combined with daratumumab. In a minority of cases, eNK cells showed a detectable population of PD1+ cells. This correlated with low cytotoxic activity, particularly in UCB eNKs. Addition of pembrolizumab did not restore their activity. Results indicate that UCB eNKs are to be preferentially used against MM in the absence of daratumumab while PB eNKs have significant cytotoxic advantage when combined with this mAb.

Expanded and activated allogeneic NK cells are cytotoxic against B-chronic lymphocytic leukemia (B-CLL) cells with sporadic cases of resistance

Adoptive transfer of allogeneic natural killer (NK) cells is becoming a credible immunotherapy for hematological malignancies. In the present work, using an optimized expansion/activation protocol of human NK cells, we generate expanded NK cells (eNK) with increased expression of CD56 and NKp44, while maintaining that of CD16. These eNK cells exerted significant cytotoxicity against cells from 34 B-CLL patients, with only 1 sample exhibiting resistance. This sporadic resistance did not correlate with match between KIR ligands expressed by the eNK cells and the leukemic cells, while cells with match resulted sensitive to eNK cells. This suggests that KIR mismatch is not relevant when expanded NK cells are used as effectors. In addition, we found two examples of de novo resistance to eNK cell cytotoxicity during the clinical course of the disease. Resistance correlated with KIR-ligand match in one of the patients, but not in the other, and was associated with a significant increase in PD-L1 expression in the cells from both patients. Treatment of one of these patients with idelalisib correlated with the loss of PD-L1 expression and with re-sensitization to eNK cytotoxicity. We confirmed the idelalisib-induced decrease in PD-L1 expression in the B-CLL cell line Mec1 and in cultured cells from B-CLL patients. As a main conclusion, our results reinforce the feasibility of using expanded and activated allogeneic NK cells in the treatment of B-CLL.

Myeloma Patient-Derived Bone Marrow Serum Negatively Regulates Natural Killer Cell Activity

IntroductionHighly activated expanded NK cells (ENKs) can be generated in large numbers from the PBMCs of healthy donors or myeloma patients by coculture with K562 transfectants (cells expressing membrane-bound interleukin (IL)15 and 4-1BBL). These ENKs expand, persist, and retain their cytolytic ability further in vivo post adoptive transfer (Garg et al. 2012, Szmania et al. 2015). We have previously treated high-risk multiple myeloma (MM) patients in frank relapse with ENKs. These cells expanded in the patients after adoptive transfer, but rapidly lost their activation state. We hypothesized that the ENKs were adversely affected in the myeloma micro-environment, which contains a wide range of immunosuppressive soluble factors, such as shed NK ligands, cytokines and myeloma-derived exosomes. We therefore studied the effect of patient-derived bone marrow serum on activating ENK cell surface repertoire, immune synapse formation and cytolytic ability.MethodsENKs were generated from healthy donors (n=7) and incubated either with myeloma patients' pooled serum (PT-P), fetal bovine serum (FBS) or normal human AB serum (AB). Subsequently, the ENKs were evaluated for their viability, immune synapse (IS) formation and cytolytic ability. Immunophenotypic expression of these cells was assessed by flow cytometry to evaluate the receptors/markers for activation (NKp46, NKp30, NKp44, NKG2D), costimulation (2B4, NTB-A, NKp80, DNAM-1, CD26, CD69), adhesion (LFA-1, CD54) as well as molecules related to cytotoxicity (perforin, granzyme B, TRAIL). Global gene expression profile (GEP) of ENKs (n=7 pairs) was performed using U133 Plus 2.0 microarrays.ResultsENKs exposed to PT-P exhibited a significant drop in the viability compare to FBS or AB in a time- and dose-dependent manner in MTT assays (range 34%-53.8% reduction, p<0.001) and in trypan blue counts (range 11%-34.7% reduction, p<0.0001). This drop in viability was accompanied by induction of apoptosis in ENKs treated with PT-P (range 14.9%-31.7%, p<0.01). Flow cytometric analysis showed that activation markers (CD26, CD69, CD70), and receptors for activation (NKp46, NKp30, NKp44, NKG2D), costimulation (2B4, DNAM-1), adhesion (LFA-1, CD54) as well as molecules related to cytotoxicity (perforin, granzyme B, TRAIL) were down-regulated in the ENK cells incubated with PT-P. Imaging flow cytometry (ImageStream) revealed that PT-P treated ENKs formed significantly fewer immune synapses with OPM2, U266, JJN3 and K562 cells (range 12%-41% down, p<0.001). Blocking of LFA1, a receptor for the adhesion molecules ICAM-1, -2 and -3, on ENK cells in cytotoxicity assays showed reduced killing further confirming that immune synapse formation is critical to NK-mediated lysis. PT-P treated ENKs demonstrated a significant decrease in specific lysis of the MM cell lines OPM2, U266 and JJN3 in cytotoxicity assays compared to ENKs treated with FBS/AB at different effector to target ratios (14.2%-41.9%, p<0.05). Adding IL2 (300U/ml) in media augmented ENK-mediated specific lysis >1.6 to 3.6 fold compare to PT-P treated ENKs in myeloma cells (OPM2, JJN3 and U266).Microarray analysis revealed that ENK cells incubated with PT-P led to the expression of a unique set of 38 probes compare to AB+FBS (35 up and 3 down with FDR<0.15 cutoff). Genes encoding cell cycle, cell growth, survival, and motility (ANAPC16, AREG, DDIT4, TSC22D3, CXCR4), intracellular signaling molecules (FKBP5, ISG20, DEFB132, ZFP36L2), cell energy and metabolism (RAP1GDS1, GALM, PARP8) are up-regulated in PT-P treated ENKs as a response to PT-P serum. Over-expression of TSC22D3 is linked to anti-apoptosis and may be up-regulated as a protection from PT-P-induced apoptosis in these cells. Proteomics on patient serum to characterize soluble factors responsible for the down modulation of ENKs is in progress and the data will be presented.ConclusionMyeloma bone marrow serum derived soluble factors are immunosuppressive and induce apoptosis in ENKs. In surviving ENKs, there is adverse modulation of the cell surface receptor repertoire, and impaired immune synapse formation resulting in significantly reduced anti-myeloma cytolytic ability. Further characterization of patient serum by proteomics followed by functional analysis is in progress to pinpoint the individual immunosuppressive factors. DisclosuresDavies:Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR subpopulation expansions in eNK and pbNK cells, respectively. In contrast to the pbNK cell population, the expansions present in the eNK cell population were independent of CMV status and HLA-C genotype. Moreover, the typical NKG2C imprint induced by CMV infection on pbNK cells was not observed on eNK cells from the same female, suggesting a rapid local turnover of eNK cells and/or a distinct licensing process. Based on our previous work and the parameters studied here, menstrual blood-derived eNK cells closely resemble biopsy-derived eNK cells. However, sampling is not done at the exact same time during the menstrual cycle, and therefore we cannot exclude some, as yet undetected, differences. Our data reveals that NK cells in the pre-implantation endometrium appear to have a dedicated tissue-specific phenotype, different from NK cells in peripheral blood. This may indicate that eNK cells are finely tuned to receive an allogeneic fetus. Studying the endometrial NKR repertoire of women with pregnancy related problems could provide clues to understand the pathogenesis of pregnancy complications. No external funding was obtained for the present study. None of the authors has any conflict of interest to declare. NA.

Natural killer cells and telomerase in the endometrium of patients with endometriosis

BackgroundAlthough endometriosis is a common cause of pelvic pain and infertility, the pathogenesis of the disease remains poorly understood. Several theories have been proposed to explain the mechanisms involved in the development of this disease, including the role of the immunological system.ObjectivesTo evaluate the percentage of endometrial natural killer (eNK) cells and telomerase expression in the endometrium of patients with and without endometriosis.Materials and methodsEndometrium samples were obtained during the mid-secretory phase from two groups of women: 12 women with endometriosis diagnosed by laparoscopy and confirmed by histological evaluation of the lesion, and 13 fertile women (control group) who underwent tubal sterilization at the same institution and in whom no signs of endometriosis were detected at surgery. Specific antibodies were used for the immunohistochemical assessment of eNK and telomerase. Results: eNK cells were significantly higher in the group of women without endometrio...

HHV-6A Infection of Endometrial Epithelial Cells Induces Increased Endometrial NK Cell-Mediated Cytotoxicity.

Background: We have recently reported the presence of Human herpesvirus-6A (HHV-6A) DNA in the 43% of endometrial epithelial cells from primary idiopathic infertile women, with no positivity in fertile women. To investigate the possible effect of HHV-6A infection in endometrial (e)NK cells functions, we examined activating/inhibitory receptors expressed by eNK cells and the corresponding ligands on endometrial cells during HHV-6A infection.Methods: Endometrial biopsies and uterine flushing samples during the secretory phase were obtained from 20 idiopathic infertile women and twenty fertile women. HHV-6A infection of endometrial epithelial cells was analyzed by Real-Time PCR, immunofluorescence and flow cytometry. eNKs receptors and endometrial ligands expression were evaluated by immunofluorescence and flow cytometry.Results: We observed the presence of HHV-6A infection (DNA, protein) of endometrial epithelial cells in the 40% of idiopathic infertile women. The eNK from all the subgroups expressed high levels of NKG2D and NKG2A receptors. Functional studies showed that NKG2D activating receptor and FasL are involved in the acquired cytotoxic function of eNK cells during HHV-6A infection of endometrial epithelial cells. In the presence of HHV-6A infection, eNK cells increased expression of CCR2, CXCR3 and CX3CR1 chemokine receptors (p = 0.01) and endometrial epithelial cells up-modulated the corresponding ligands: MCP1 (Monocyte chemotactic protein 1, CCL2), IP-10 (Interferon gamma-induced protein 10, CXCL10) and Eotaxin-3 (CCL26).Conclusion: Our results, for the first time, showed the implication of eNK cells in controlling HHV-6A endometrial infection and clarify the mechanisms that might be implicated in female idiopathic infertility.

Adoptive natural killer cell therapy is effective in reducing pulmonary metastasis of Ewing sarcoma

ABSTRACTThe survival of patients with metastatic or relapsed Ewing sarcoma (ES) remains dismal despite intensification of combination chemotherapy and radiotherapy, precipitating the need for novel alternative therapies with minimal side effects. Natural killer (NK) cells are promising additions to the field of cellular immunotherapy. Adoptive NK cell therapy has shown encouraging results in hematological malignancies. Despite these initial promising successes, however, NK cell therapy for solid tumors remains to be investigated using in vivo tumor models. The purpose of this study is to evaluate the efficacy of ex vivo expanded human NK cells in controlling primary and metastatic ES tumor growth in vitro and in vivo. Using membrane-bound IL-21 containing K562 (K562-mbIL-21) expansion platform, we were able to obtain sufficient numbers of expanded NK (eNK) cells that display favorable activation phenotypes and inflammatory cytokine secretion, along with a strong in vitro cytotoxic effect against ES. Furthermore, eNK therapy significantly decreased lung metastasis without any significant therapeutic effect in limiting primary tumor growth in an in vivo xenograft model. Our data demonstrate that eNK may be effective against pulmonary metastatic ES, but challenges remain to direct proper trafficking and augmenting the cytotoxic function of eNK to target primary tumor sites.

Myeloma-Derived Exosomes and Soluble Factors Suppress Natural Killer Cell Function

IntroductionThe complex makeup of the tumor microenvironment (ME) exerts selective pressure on cancer cells leading to immune escape, and novel immunotherapeutic interventions have emerged from evolving knowledge of the immune system and tumor cells. Natural killer (NK) cells are innate immune cells that exert potent anti-tumor effects. Previously we have reported that ex vivo expansion of NK cells by co-culture PBMCs with K562mbIL15-41BBL can generate large numbers of highly active expanded NK cells (ENKs). These ENKs expand further upon adoptive transfer in vivo both in a murine model and in patients (Garg et al. 2012, Szmania et al. 2015), and have been shown to persist and retain their cytolytic ability. We are currently applying ENK therapy in a Phase II clinical trial at our institute in gene expression profiling-defined high-risk multiple myeloma (MM), a patient population which fares poorly despite the use of novel drugs and autologous stem cell transplantation.A potential obstacle to successful NK cell-based therapies is the suppression of NK cells in the MM bone marrow ME (BM-ME) by immunosuppressive cells, various soluble factors (SF), microRNAs, and exosomes. Exosomes are endosomal-derived, 30-130nm microvesicles present in almost all body fluids. Their number is significantly higher in cancer patients. Tumor-derived exosomes contain a wide range of bioactive molecules, such as microRNA, RNA, DNA and protein, and play a major role in immune escape, promoting tumor progression. Their size, structure, and presence in serum allow them to transport their cargo to distant targets. This study was designed to characterize the potential adverse effects of myeloma-derived exosomes (MEXs) and myeloma-derived SF (MSF) on NK cell function and determine if such inhibition can be overcome by cytokine support.MethodsMEXs were isolated from OPM2 myeloma cell line-derived conditioned media (MCM) using the Total Isolation Reagent (Life Technologies, Carlsbad CA). Transmission electron microscopy (TEM), flow cytometry, and western blot (WB) analysis were used for characterization of exosomes. Fresh NK cells (non-activated) and ENKs were incubated with MCM or MEXs and evaluated for their viability and cytolytic ability in standard 4-hour chromium release assays. Flow cytometry was used to evaluate the immunophenotype of these cells, including activation, costimulatory, inhibitory receptors, and adhesion molecules.ResultsTEM confirmed the presence of exosomes in MCM (size and morphology). Interestingly, OPM2-derived MEXs did not express the exosome-specific marker CD9, but did express CD63, and CD81. Flow cytometry showed that MEXs contain MICA/B, TGFβ, TRAIL-R1, TRAIL-R2, MHC class I, HLA-E, and ICAM3. NK cells exposed to MEXs demonstrated a dose-dependent, significant decrease in specific lysis of the MM cell lines JJN3, OPM2, and U266 in cytotoxicity assays compared to control NK cells (13%-51%, p<0.0005). In addition, a time-dependent decrease in NK cell-mediated lysis was observed in these MM cell lines at 24hours (14%-34%) versus 48hours (30%-48%; p<0.0005). A similar downward trend in the activity of ENKs incubated with MEXs was also noted but to a lesser extent. We hypothesize that highly-activated ENKs are able to partially overcome MEX-mediated inhibition compare to resting NK cells. We also noted a considerable decrease in the cytolytic ability of ENKs incubated with MCM which contains suppressive soluble factors in addition to MEXs (28%-58%, p<0.0005). Further, this suppression in ENK activity was partly rescued by fresh IL2 incubation (18-36%, p<0.01). Many of the activating receptors (NKp46, NKp30, NKp44, NKG2D), costimulatory receptors (2B4, NTB-A, NKp80, DNAM-1), activation markers (CD26, CD69), and adhesion molecules (LFA-1, CD54) were down regulated on the ENK cells incubated with MCM. However, differences were not as significant in these receptors on ENK cells incubated with MEX.ConclusionMEXs and other SF released from myeloma cells are capable of modulating the function and phenotype of NK cells and ENKs. MCM is more immunosuppressive as it contains both MEX and MSF. Cytolytic ability of ENKs could be partially restored by incubation in fresh IL2 medium. Further characterization of MEXs and MCM by proteomics is in progress. (Data will be presented). DisclosuresDavies:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Impact Of Elotuzumab Therapy On Circulating and Ex Vivo Activated/Expanded Autologous Natural Killer (Auto-ENK) Cell Activity

CS1 is an ideal target for multiple myeloma (MM) therapy as it is highly expressed on MM while having a very limited expression profile in normal tissues. Elotuzumab (elo), a humanized monoclonal antibody (mAb) targeting CS1, has an acceptable safety profile and clinical activity in relapsed/refractory MM when combined with the immune modulator lenalidomide (len) and low dose dexamethasone (dex). The primary mechanism of action for elo is NK cell-mediated antibody-dependent cellular cytotoxicity. Here we report on a patient who was given elo/len via a single patient IND 6 months after receiving therapy with ex vivo activated auto-ENK cells and low dose IL2 as previously described (Szmania et al, Blood ASH Annual Meeting Abstracts 2012;120:1912). The patient had relapsing GEP70 high-risk MM with cytogenetic abnormalities and had failed multiple lines of prior therapy including 3 auto-PBSC transplants and further salvage treatments including len, bortezomib, pomalidomide and carfilzomib. Although ENK cell therapy did not induce a response, subsequent disease progression was slow. IV elo was started 187 days after ENK cell infusion, and given every 14 days at the currently studied dose of 10mg/kg. Len at 15mg/day was given on days 1-21 of a 28-day cycle. Dex premedication (p.o. 38mg; IV, 10mg) was added after a grade 2 infusion reaction was observed to elo dose #1. While on the ENK cell protocol this patient had a dramatic increase in circulating NK cell counts peaking 9 days after infusion (6300 NK/µL, a 48-fold increase from baseline). Although still in the high range, NK cell levels at the time of elo treatment had normalized somewhat (539 NK/ml), and the cell surface expression of key activating receptors was consistent with a resting phenotype. NK cell count remained stable after the first dose of elo (530 NK/ml) but subsequently dipped to 179 NK/µL after elo dose #2. Since dex has been reported to affect NK cell counts, it is important to note that an additional dose was taken prior to elo dose #2 due to a travel delay (in total 66 mg of dex was taken on this occasion). Circulating NK cells (collected pre-elo and 11, 25, and 57 days after elo dose #1) had similar low activity against auto-MM collected prior to elo treatment (effector:target ratio 10:1, 0-5% specific lysis) and killing against MM collected after 5 elo doses was only modestly increased (3-12%). However, the same circulating NK cells exhibited significantly increased cytolytic ability when additional elo (10µg/mL) was added during the in vitro E:T co-incubation (3-11 fold increase in killing over isotype control, p=0.0008) suggesting that the MM targets were not saturated with mAb. Bound mAb may have been reduced in part during target cell isolation and freeze/thaw. Freshly prepared auto-ENK cells exhibited an activated immunophenotype and induced significantly higher killing of pre-elo MM (45%) compared to non-expanded NK. ENK killing was higher still against MM collected after 5 doses of elo (61%). When elo was present during the assay, ENK demonstrated the most effective killing of auto-MM, reaching levels equivalent to that of the NK sensitive target K562 (85% vs. 82% lysis). Successful mAb therapy for MM is now moving forward as target antigens with selective, high and homogeneous expression, such as CS1, are identified. However, the activity of responding effector cells is a critical issue to consider. Inadequate NK cell count and activity level has been reported in MM and steroids typically given to debulk and preempt mAb-induced infusion reactions may exacerbate this problem. Immunomodulatory agents given to enhance immune cell activity are not sufficient to reverse the negative effect of steroids. We have previously shown that large doses of highly activated auto-ENK cells can be safely infused and that these cells expand further after infusion. In this study we show that ENK cells have significant activity in vitro against auto-MM and that elo further enhances this activity. Combination therapy incorporating saturating doses of mAb followed by infusion of NK effector cells with optimized activity against auto-MM is an innovative approach that warrants investigation. Infusing highly activated effector cells after dex/elo may be one way to reap the benefits of combining these modalities while circumventing steroid-induced immune suppression. Disclosures:Barlogie:Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.

Multiple Myeloma Cells Modulate ICAM-3 To Evade Natural Killer Cell-Mediated Lysis

IntroductionEx vivo activated/expanded natural killer (ENK) cells can induce myeloma cell lysis both in vitro and in murine models and are currently being studied clinically in the setting of high-risk relapsing disease and asymptomatic disease at high risk of progression. This prompted us to study, in myeloma cell lines, whether intrinsic resistance to ENK cell lysis exists, whether repeated challenge with ENK leads to increased resistance, and what the underlying mechanisms of resistance are. Of 11 myeloma cell lines tested in standard 4h chromium release assays, 8 were avidly killed (78-89% lysis, E:T Ratio 10:1) whereas 3 lines were less sensitive (41-65% lysis). Repeated exposure to ENK challenge decreased sensitivity in 4 of 11 lines, that was at least in part due to down-regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors on the myeloma cell surface (Garg et al, Blood 2012, 120:4020). In this study we investigated the resistance issue further via metabolomics, gene expression profiling (GEP) and flow cytometry analysis of OPM2, which was intrinsically resistant and developed further resistance after challenge with ENK cells. MethodsMetabolomics was studied using a quantitative proteomic strategy entailing stable isotope labeling with amino acids in cell culture – mass spectrometry (SILAC-MS). Resistant and parental OPM2 cells were grown either in medium with heavy amino acids (13C6 L-Lysine and 13C6 L-Arginine) or with light amino acids (12C6 L-Lysine and 12C6L-Arginine). Reverse labeling with heavy or light amino acids was also done to confirm the results. Cell lysates from heavy and light amino acid labeled cells were pooled, simultaneously resolved on SDS-PAGE, protein bands were excised and analyzed on a mass spectrometer after trypsin digestion. GEP was performed using the Affymetrix U133 Plus 2.0 microarray platform (Santa Clara, CA). The fold change of signal intensity for genes and proteins in resistant vs. parental OPM2 was calculated. The most differentially expressed genes (top 150-fold up or down) and proteins (up or down by 1.3-fold) were compared for commonality. Cell surface protein expression was determined via flow cytometry. The ability of ENK to lyse myeloma cell targets in the presence of isotype control or ICAM-3 blocking antibody was tested in 4h chromium release assays. ResultsMetabolomics identified >3800 proteins and revealed that the abundance of 352 proteins was significantly altered in resistant myeloma cells. These altered proteins were mainly associated with cell cycle, morphology, organization, cellular compromise, immune response, and survival. Further, a comparison of these differentially expressed proteins with GEP data revealed 3 commonly up-regulated molecules: TBC1D8B, HSPA1A and IFI16; and 2 down-regulated molecules: intercellular adhesion molecule (ICAM-3) and BAI3. Of these, ICAM-3, a ligand for leukocyte function-associated antigen-1 (LFA-1) and a potent signaling molecule, was selected for further studies. Flow cytometry confirmed that ICAM-3 cell surface expression was > 8-fold lower on resistant versus parental OPM2 cells. Further, blocking of ICAM-3 in cytotoxicity assays resulted in decreased lysis (43% blocked, E:T ratio 5:1), suggesting that this molecule is functionally important and takes part in ENK cell-mediated killing. ConclusionIn conclusion, quantitative proteomic analysis demonstrated dynamic changes in the ENK-resistant OPM2 myeloma cells that correlated with GEP and differences in ICAM-3 expression may have functional implications. Studies evaluating the expression of ICAM-3 in myeloma patients at diagnosis and relapse are in progress. Myeloma cells may down-regulate ICAM-3 as a mechanism of escape from immune surveillance and therefore, ICAM-3 may be a useful biomarker to predict sensitivity to ENK cell-mediated killing and aid in the selection of patients most likely to benefit from ENK cell therapy. Disclosures:Barlogie:Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.

Cyclic Changes and Relationship between Peripheral and Endometrial NK Cells from Women with Repeated Failure after Artificial Insemination by Donor Sperm

Cyclic changes of peripheral natural killer (pNK) cells and/or endometrial NK (eNK) cells during menstrual cycle remain controversial, and their relationship remains uncertain. Peripheral blood and endometrial biopsies were simultaneously obtained from women (n = 23) undergoing artificial insemination with donor sperm (AID) for at least three cycles at both proliferative (days 9-11) and secretory phases (days 20-23) of menstrual cycle. The percentages of CD3(-) CD56(+), CD3(-) CD56(dim) CD16(+), CD3(-) CD56(bright) CD16(-) pNK, and eNK cell subsets within lymphocytes were determined by three-color flow cytometry. The correlation between the percentages of pNK and eNK cells was further analyzed by Spearman's test. The percentages of CD3(-) CD56(+), CD3(-) CD56(dim) CD16(+), and CD3(-) CD56(bright ) CD16(-) pNK cells were not statistically different between the proliferative and secretory phases (P > 0.05, respectively). However, the percentages of CD3(-) CD56(+) and CD3(-) CD56(bright ) CD16(-) eNK cells were significantly decreased at the secretory phase, compared with those in the proliferative phase (P < 0.05, respectively). No correlation between the percentages of all pNK cell parameters and those of CD3(-) CD56(bright ) CD16(-) eNK cells (the major subset of NK cells in uterus) was found in the same women throughout the menstrual cycle (P > 0.05). We found a menstrual-cycle-dependent change in the percentage of eNK cells in women undergoing AID treatment, but not pNK cells. Moreover, the percentage of pNK cells may not reflect that of eNK cells during menstrual cycle.