Unstimulated NK Cells Research Articles

Moesin: A novel receptor on NK lymphocytes binds to TOMM40 on K562 leukemia cells initiating cytolysis

Natural killer lymphocytes (NK cells) are the first line of defense (innate immunity) against viral infections and leukemia since they do not require activation to deliver a lethal hit to infected/aberrant cells. In contrast, T lymphocytes require stimulation by a foreign/neo – antigen, which may take days before they are active against the pathogen (adaptive immunity). A number of receptors on activated NK cells that kill the prototypical leukemia target cell line, K562, have been identified. To date, the receptor(s) by which freshly isolated unstimulated NK cells (naïve, nNK) kill K562 has not been fully elucidated. We provide peptide sequence and immune-blot data from ligand pull down experiments that moesin, a protein that typically links the inner leaf of the plasma membrane to the cytoskeleton, additionally, in NK cells, localizes to the cell surface where it may bind to its ligand, TOMM40 (aka Haymaker, HYMKR), on leukemia cells thereby initiating their destruction. Flow cytometry experiments with a mouse monoclonal antibody (Mab) to a moesin peptide (554 to 565) were performed. Moesin was detected on the surface of CD3–, CD16+nNK cells but was not detected on the surface of freshly isolated unstimulated CD3+, CD16– T cells or CD19+, CD16– B cells from healthy subjects. Moesin, is therefore another marker that distinguishes unstimulated CD3–, CD16+ NK cells from other non-activated lymphocytes. The anti –moesin peptide Mab was highly effective (>95% inhibition) in blocking target cell cytolysis by CD16+ lymphocytes demonstrating that moesin–HYMKR interaction appears to be necessary for most of the observed cell death of K562 caused by unstimulated NK cells.

Phase I Trial of Prophylactic Donor-Derived IL-2-Activated NK Cell Infusion after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Sibling Donor.

Simple SummaryAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for high-risk hematologic malignancies. However, disease recurrence after allo-HSCT remains a critical issue, underlining the need to develop maintenance therapy. In this context, NK cell-based immunotherapies could enhance graft-versus-tumor effect without triggering graft-versus-host disease. In this prospective phase I clinical trial, we demonstrated the safety of donor-derived NK cell infusion as a prophylactic treatment after allo-HSCT for patients with hematological malignancies. This opens perspectives for future developments of NK cell based therapeutic strategies after allo-HSCT with low incidence of GVHD, representing an advantage over post-transplant T cell modulations that are commonly used in clinical routine.Background: NK cell-based immunotherapy to prevent relapse after allogeneic transplantation is an appealing strategy because NK cells can provide strong antitumor effect without inducing graft-versus-host disease (GVHD). Thus, we designed a phase-I clinical trial evaluating the safety of a prophylactic donor-derived ex vivo IL-2 activated NK cell (IL-2 NK) infusion after allo-HSCT for patients with hematologic malignancies. Methods: Donor NK cells were purified and cultured ex vivo with IL-2 before infusion, at three dose levels. To identify the maximum tolerated dose was the main objective. In addition, we performed phenotypical and functional characterization of the NK cell therapy product, and longitudinal immune monitoring of NK cell phenotype in patients. Results: Compared to unstimulated NK cells, IL-2 NK cells expressed higher levels of activating receptors and exhibited increased degranulation and cytokine production in vitro. We treated 16 patients without observing any dose-limiting toxicity. At the last follow up, 11 out of 16 treated patients were alive in complete remission of hematologic malignancies without GVHD features and immunosuppressive treatment. Conclusions: Prophylactic donor-derived IL-2 NK cells after allo-HSCT is safe with low incidence of GVHD. Promising survivals and IL-2 NK cell activated phenotype may support a potential clinical efficacy of this strategy.

NK-92 serial killers - characterization of potential limitations to cytotoxic capacity.

Abstract The NK cell line NK-92 and transgenic variants are receiving attention as adoptive transfer immunotherapies to treat a range of malignancies. However, since NK-92s are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. In the present study, we investigated NK-92 serial killing against various tumor cells as well as the effects of gamma-irradiation and ligation of the death receptor Fas (CD95) on NK-92 cells and the susceptibility of NK-92s to attack by primary NK cells. To evaluate serial killing, we used a 51Cr-release assay with very low effector to target (E:T) Raji, Daudi or K562 tumor cell ratios at 2, 4, 6, and 8 hour time points. NK-92s that were maintained in high concentrations of recombinant IL-2 were able to kill as many as 14 Raji cells per NK-92 cell in 8 hours at an E:T of 1:32. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but lost >50% activity 24 hours after just 7 Gy irradiation. Despite strong expression of CD95, NK-92s maintained viability after 24 hours of Fas-ligation with an anti-CD95 IgM as compared to cells treated with an isotype control antibody. While NK-92s survived 16 hours of exposure to unstimulated primary NK cells, they were killed when exposed to IL-2 activated primary LAK cells. In conclusion, we recommend that adoptive transfer of irradiated NK-92 cells into patients be made as quickly as possible post-irradiation of the NK-92s. Genetic modification of NK-92 cell lines to secrete IL-2 as an autocrine growth factor may need reconsideration because in vivo activation of endogenous LAK cells by IL-2 could reduce the number of NK-92 cells within tumors and thus decrease therapeutic efficacy.

Functional capacity of natural killer cells in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients

BackgroundNatural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection.MethodsThis cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30.ResultsThe frequency of NKp30+ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30–61] compared to controls (73%, IQR 54–79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a+ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals.ConclusionsNK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.

IL-18/IL-15/IL-12 synergy induces elevated and prolonged IFN-γ production by ex vivo expanded NK cells which is not due to enhanced STAT4 activation

The synergistic effect of IL-18/IL-15/IL-12 stimulation potently activates NK cells, inducing high levels of IFN-γ production. As a result of this potent stimulatory effect, NK cell pre-activation with IL-18/IL-15/IL-12 is being developed as a cancer immunotherapy. Ex vivo expansion of NK cells enables the efficient generation of large numbers of NK cells for wide-scale and repeated therapeutic use, and is thus an important source of NK cells for clinical application. However, the effects of IL-18/IL-15/IL-12 stimulation on ex vivo expanded NK cells have not yet been assessed. Thus, the present study assessed the effects of IL-18/IL-15/IL-12 stimulation on NK cells expanded ex vivo using K562-based artificial antigen presenting cells expressing membrane-bound IL-21. We report that ex vivo expanded NK cells stimulated with IL-18/IL-15/IL-12 produce high levels of IFN-γ and TNFα, have potent cytotoxicity, and maintain prolonged IFN-γ production following removal of stimulation. IL-18/IL-15/IL-12 stimulation induces a phenotypically unique IFN-γ-producing population with reduced CD16 expression and greater CD25 expression as compared to stimulated IFN-γ- NK cells and unstimulated NK cells. We elucidate that the mechanism of synergy for induction and maintenance of IFN-γ production is not due to a further enhancement of STAT4 activation compared to stimulation with IL-12 alone. Furthermore, we demonstrate that the synergistic increase in IFN-γ is not solely under translational regulation, as elevated levels of IFN-γ mRNA contribute to the synergistic increase in IFN-γ. Overall, this study characterizes the response of ex vivo expanded NK cells to IL-18/IL-15/IL-12 stimulation and supports the use of ex vivo expanded NK cells as a feasible and efficient source of IL-18/IL-15/IL-12 pre-activated NK cells for adoptive transfer in cancer immunotherapies.

IL-22 producing NK cells aid in the repair of damaged tissues (INC5P.326)

Abstract Natural killer (NK) cells are lymphocytes of the innate immune system that are associated with killing of virally infected and neoplastic cells. They have been shown to produce large amounts of pro-inflammatory cytokines, such as IFN-γ, and cytotoxic molecules, such as perforin and granzyme. Recent studies have identified unique subsets of natural killer cells, with distinct effector functions not limited to their classic cytotoxic function. These functions include producing a new array of cytokines, including IL-22, which has been shown to aid in tissue regeneration. Here, we provide evidence for a NK cell subset with the capacity to aid in the repair of tissue damage following viral infection as well as radiation damage. Using an in vitro co-culture system where we culture unstimulated NK cells with virally infected fibroblasts or irradiated epithelial cells, we were able to identify triggers that induce the expression of IL-22. In order to test the biological significance of IL-22 production by NK cells, we next used an in vivo murine cytomegalovirus (MCMV) model where we are able to deplete or adoptively transfer the IL-22 producing subset. With this model we are able show differences in liver pathology following MCMV infection, dependent on an IL-22 producing NK cell subset. A better understanding of IL-22 production by natural killer cells, and delineating a specific IL-22 producing subset could offer therapeutic benefits to patients who receive radiation therapy.

Membrane-Type 6 Matrix Metalloproteinase Regulates the Activation-Induced Downmodulation of CD16 in Human Primary NK Cells

CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2-induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector-target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.

Impaired CD4+ T cell stimulation of NK cell anti-fibrotic activity may contribute to accelerated liver fibrosis progression in HIV/HCV patients

HIV/HCV co-infection is characterized by a faster progression to liver fibrosis compared to HCV mono-infection. Epidemiologic studies found an association between low CD4(+) T cell counts and advanced stages of liver fibrosis. However, the mechanisms underlying this association remain unclear. CD4(+) T cells critically modulate NK cell activity. Of note, NK cells have been shown to display anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Thus, we speculated that CD4(+) T cells might modulate fibrosis progression by interacting with NK cells. NK cells from HCV(+) (n=35), HIV(+)/HCV(+) (n=28), HIV(+) (n=8) patients, and healthy controls (n=30) were used in this study. NK cells were cultured in the presence or absence of supernatants from CD3/CD28-stimulated CD4(+) cells. Then, NK cells were co-incubated with activated HSC and studied for degranulation, IFN-γ secretion, and induction of HSC apoptosis. Following incubation with CD4(+) T cell supernatants, NK cells displayed a significantly increased activity against primary HSC as compared to unstimulated NK cells. This effect was, at least in part, mediated via an IL-2 dependent upregulation of NKG2D expression. HCV/HIV co-infection was associated with an impaired IL-2 secretion of CD4(+) T cells resulting in an ineffective stimulation of anti-fibrotic NK cell function. Here, we show that CD4(+) T cells are able to stimulate anti-fibrotic NK cell activity via IL-2 mediated upregulation of NKG2D. HIV-induced loss of CD4(+) T cells together with an impaired activity of CD4(+) T cells may contribute to accelerate progression of liver fibrosis observed in co-infection.

Reduced NK cell activity and abnormal expression of CCR7 and CXCR1 by NK cells analysis in patients with DOCK8 deficiency

DOCK8-deficiency is an autosomal recessive primary immunodeficiency that is characterized by multiple abnormalities of the immune system, including a defect of NK cell cytotoxicity which could not be restored after IL2 stimulation. Nevertheless, unanswered questions remain regarding how the absence of DOCK8 leads to predisposition for malignancy, viral, fungal, and bacterial infections. To address these questions we have analyzed NK cell phenotype and functions in patients with DOCK8 deficiency. We observed that NK cells derived from five DOCK8-deficient patients displayed dramatically reduced cytotoxicity which was partially restored after IL-2 stimulation. Analysis of activating and inhibitory NK receptors, including KIRs molecules, chemokine receptors and activation markers on gated CD56+cells by cytofluorimetric analysis showed a substantial defect of CCR7 expression by CD56bright NK cells. Noteworthy, we have also detected the expression of NKG2C and of the chemokine receptor CXCR1 on CD56dull NK cells in DOCK8-deficient cells. Because CCR7 expression by NK cells can be induced after cell culture with IL18 we stimulated NK cells from DOCK-8 deficient patients with IL18. Despite unstimulated CD56bright NK cells from DOCK8 patients showed reduced CCR7 expression, we could not detect any increase of CCR7 on CD56 dull and bright NK cells of DOCK8-deficient patients, whereas CCR7 expression on NK cells derived from healthy donors significantly increased from 5% to 17%. Taken together our results suggest that NK cells of DOCK-8 deficient patients show reduced cytotoxicity and abnormal expression of the chemokine receptors CXCR1 and CCR7 suggesting an abnormal recruitment of these cells to secondary lymphoid organs.

Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies Overcome Resistance of Ovarian Cancer Cells to Targeted Therapy and Natural Cytotoxicity

The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.

Modulation of NK Cell Autocrine-Induced Eosinophil Chemotaxis by Interleukin-15 and Vitamin D3: A Possible NK-Eosinophil Crosstalk via IL-8 in the Pathophysiology of Allergic Rhinitis

Natural killer cells (NK) secrete eosinophilotactic cytokines, however, whether they contribute to eosinophil chemotaxis by secreting IL-8 is not known. We investigated the ability of CD56+CD3-ve (NK cells) to induce chemotaxis of peripheral blood eosinophils from allergic rhinitis (AR) patients, through IL-8 secretion, and the effects of IL-15, the NK cell proactivating cytokine, and calcitriol: 1α, 25-dihydroxy Vitamin D3 (vitamin D3), the immunomodulator agent, in this scenario. Herein, it is shown that supernatants from unstimulated NK cells exhibited chemotactic activity against eosinophil. This effect was significantly augmented by IL-15 (1 ng/mL) treatment, resulting in an increase in the chemotactic index of approximately 3 folds and was abrogated by neutralizing antibody (Ab) to IL-8 in a dose-dependent fashion. The amount of IL-8 secreted by NK cells was increased by IL-15 treatment from levels of 88.64 ± 21.5 to 178.9 ± 23.6 Pg/mL and was significantly reduced by 10−6 M vitamin D3 to levels of 59.2 ± 16.3 Pg/mL. Our results indicate a novel inflammatory crosstalk between NK cells and eosinophils via IL-15/IL-8 axis that can be modulated by vitamin D3.

Interferon-γ (IFN-γ) Mediates a Direct Fungicidal Effect of Human Natural Killer (NK) Cells Against Aspergillus Fumigatus

AbstractAbstract 2779NK cells are innate immune effector cells, which are well recognized for their role against cells transformed by malignant processes or by an intracellular infection. Although, there is increasing evidence of an interaction of NK cells also with extracellular pathogens, less is known about their interplay with moulds and especially with A. fumigatus. The interest for this interaction is mainly driven by the high mortality characterizing invasive aspergillosis (IA), an infection of immunosuppressed patients caused mainly by this fungus. In our study, we investigated the in vitro interaction of human NK cells with A. fumigatus. Methods. NK cells were magnetically depleted from the peripheral blood of healthy volunteers followed by pulsing for 24h with 500 U/ml recombinant interleukin–2 (rhIL-2). The induction of IFN-γ and Tumor Necrosis Factor-α (TNF-α) by the fungus was measured by real time RT-PCR and ELISA assays after 0, 3, 6 and 12h of co-culture. The lethal impact of NK cells and culture supernatants on A. fumigatus was evaluated by plate killing and 2,3-bis[2-methoxy-4-nitro-5-sulpho-phenyl]2H-tetrazolium-5-carboxanilide (XTT) assays. Transwell permeable membranes with pores of 0.4 μm, prohibiting the direct contact of cells placed on opposite sides but allowing the free circulation of molecules were used to evaluate the relevance of a direct cell – fungal contact on cytokine induction and fungal damage. The degranulation markers CD107a and CD107b were measured to assess the involvement of cytotoxic proteins on the anti-Aspergillus effect. The supernatant was magnetically depleted of IFN-γ. Recombinant human IFN-γ (rhIFN-γ) was used to mimic the effect of IFN-γ released by NK cells. Results.Co-cultivating human NK cells with resting (conidia) and germinated (germlings) fungal morphologies, only germlings induce a Th1 like cytokine response of NK cells, with the major representative IFN-γ (p<.05). Consistently, NK cells demonstrate a strong fungicidal effect only towards germlings (p<.05). Using transwell membranes to investigate the mechanism of this interaction, we found that physical contact was a prerequisite for the induction of cytokines (p<.05), but also for the release of a soluble factor, exhibiting fungicidal properties. Once the soluble factor was released, germlings were killed without the presence of NK cells (p<.05). To confirm this finding, germlings were incubated with supernatant obtained from the culture of NK cells with A. fumigatus. After 3 h of culture, 40% killing (p<.001) was demonstrated. To determine the soluble factor(s) causing the fungicidal effect, we explored the degranulation of cytotoxic proteins like perforin, granzymes and granulysin. FITC-labeled antibodies against the degranulation markers CD 107a, b were co-incubated with unstimulated NK cells, NK cells challenging germlings and NK cells challenging the cell line K562. In the presence of A. fumigatus, there was no expression of CD 107a, b, while the expression of these markers was significant in the culture with K562 cells. This result suggests that the granule proteins are not the means of the NK cell cytotoxicity against A. fumigatus. Moreover, the protective effect of IFN- γ for the host defense against IA is well known. So far, this effect was attributed to the influence of IFN- γ on innate immune cells, increasing their antifungal activity. In our study, we hypothesized that IFN- γ might demonstrate direct antifungal properties, mediating the fungicidal activity of NK cells. Thus, we compared the fungal killing caused by IFN- γ partially depleted supernatant and un-depleted supernatant and we found that the depletion of IFN- γ diminished the fungicidal effect (p<.05). Moreover, we exposed germlings to culture medium enriched with rhIFN- γ at concentrations equivalent to those detected in the supernatants and we observed a similar killing rates. To summarize, our study demonstrates that human NK cells interact with A. fumigatus germlings, which induce the release of Th1 like cytokines and cause significant fungal killing. Our data show for the first time that IFN-γ, released by NK cells, is a soluble molecule mediating direct fungicidal activity. These results attribute new characteristics to human NK cells, suggesting them as a potential alternative therapeutic tool against IA. Disclosures:No relevant conflicts of interest to declare.

In vitro preclinical study results for a phase I adoptive immunotherapy trial using cetuximab with allogeneic NK cells in KRAS oncogene-mutated metastatic colorectal cancer patients.

e14057 Background: Prognosis for metastatic colorectal-cancer (mCRC) patients is unfavorable. Futhermore mCRC patients with tumors harboring a mutation in the KRAS gene do not derive benefit from the administration of cetuximab. We hypothesized that KRAS mutated CRC cells may be susceptible to cetuximab-induced ADCC mediated by healthy donor NK cells. Methods: Purified allogeneic NK cells or preincubated with IL-2 or IL-15 were used as effectors. Seven different CRC cell lines (DLD1, HCT-116, HT-29, LOVO, SW620, SW48, SW480) labeled with 51Cr were used as target cells alone or preincubated with cetuximab for ADCC assay. Results: All but one (SW620) cell lines express EGFR and MHC molecules; 4/7 harbor KRAS mutation (DLD 1, HCT-116, SW620, SW480). All cell lines were susceptible to unstimulated NK cells lysis (mean 20% ± 4.57, at ratio 25:1; mean 15% ± 3.65, at ratio 12:1) and lytic activity is strongly enhanced by IL-2 or IL-15 (mean 48% ± 5.8, at ratio 25:1; mean 38% ± 4.3, at ratio 12:1) (p < 0.05). No difference in lytic activity was observed between IL-2 vs. IL-15 and thawed vs. fresh NK cells. The SW48, a KRAS wilde-type cell line was killed by NK cells from 38% of lysis without cetuximab to 50% with cetuximab (E/T ratio of 50:1, p < 0.05). Similarly the HCT-116, a KRAS mutated cell line was killed by NK cells from 20% without cetuximab to 36% with cetuximab (E/T ratio of 50:1, p < 0.05). When IL-2 or IL-15 activated NK cells were used as effectors, both targets lysis increased from 50% without cetuximab to 71% with cetuximab (E/T ratio of 100:1, p < 0.05). When SW620 (EGFR expression 0%) was used as target cell line, pre-coating with cetuximab did not enhanced unstimulated or pre-activated NK lysis capacity. Conclusions: allogeneic NK cells are cytotoxic versus all tested cell lines. Moreover lytic activity of thawed and fresh NK cells is similar. In ADCC assay NK cell lytic activity is significantly enhanced by cetuximab; surprisingly cetuximab mediated ADCC activity is independent from CRC cell lines KRAS oncogene mutational status and require EGFR membrane expression. Moreover lytic activity of IL-2 and IL-15 activated NK cells is increased by cetuximab. No significant financial relationships to disclose.

In Vitro Preclinical Study Results for a Phase I Adoptive Immunotherapy Trial Using Cetuximab in Association with Allogeneic NK Cell Infusion in KRAS Oncogene Mutated Metastatic Colorectal Cancer Patients.

Abstract 5119 PurposePrognosis for metastatic colorectal-cancer (mCRC) patients is unfavorable, median survival from diagnosis varying between sixteen and twenty-four months. Numerous clinical trials have demonstrated a clinical advantage for patients treated with a combination of chemotherapy and cetuximab. However recent retrospective evidence from several randomized studies, has established that mCRC patients with tumors harboring a mutation in the KRAS gene do not derive benefit from the administration of cetuximab. We hypothesized that KRAS mutated CRC cells may be susceptible to cetuximab-induced ADCC mediated by healthy donor NK cells. Experimental Design7 different CRC cell lines (DLD 1, HCT-116, HT-29, LOVO, SW620, SW48, SW480) were analyzed for EGFR membrane expression levels, tested for KRAS mutational status and MHC class I-II expression. CD56+CD3- NK cells were purified from volunteer healthy donors peripheral blood mononuclear cells, by a two-steps CD3+ cells negative selection followed by CD56+ cells positive selection by MACS cell separation columns (Miltenyi biotec). Purified NK cells were incubated overnight with medium alone or stimulated with IL-2 (100U/ml) or IL-15 (20U/ml). CRC cell lines, were labeled with 51Cr overnight, and used as target cells for NK assay or incubated at 4°C for 30 minutes with Cetuximab 100 ug/ml for ADCC assay. Cytotoxic activity was evaluated in a 4-hour cytotoxicity assay, at an effector/target (E/T) ratio ranging between 100:1 and 2:1. Resultsall but one (SW620) CRC cell lines express EGFR (range 40-90%) and MHC class I-II molecules; 4/7 harbor KRAS mutation (DLD 1, HCT-116, SW620, SW480). All CRC cell lines were susceptible to unstimulated allogeneic NK cells lysis (mean 20% ± 4,57, at ratio 25:1; mean 15% ± 3,65, at ratio 12:1) and lytic activity is significantly enhanced (p<0.05) by either IL-2 or IL-15 pre-activation (mean 48% ± 5,8, at ratio 25:1; mean 38% ± 4,3, at ratio 12:1, respectively). No difference in lytic activity was observed between IL-2 vs IL-15 pre-activation and between thawed vs fresh NK cells. SW48, KRAS wilde-type (EGFR expression 74%) and HCT-116 KRAS mutated (EGFR expression 61%) cell lines were chosen as targets for ADCC assay. When unstimulated NK cells were used as effectors, SW48 lysis increased from 38% without cetuximab pre-coating to 50% with cetuximab pre-coating (E/T ratio of 50:1, p<0.05). Similarly, HCT-116 lysis increased from 20% without cetuximab pre-coating to 36% with cetuximab pre-coating (E/T ratio of 50:1, p<0.05). When IL-2 or IL-15 pre-activated NK cells were used as effectors, both targets lysis increased from 50% without cetuximab pre-coating to 71% with cetuximab pre-coating (E/T ratio of 100:1, p<0.05). Pre-coating with cetuximab did not enhanced unstimulated or pre-activated NK lysis against SW620 (EGFR expression 0%). Conclusionsallogeneic NK cells from different healthy donors are able to lyse all tested CRC lines (4 KRAS mutated). IL-2 and IL-15 activation significantly enhances NK cytotoxicity; moreover lytic activity of thawed and fresh NK cells is similar allowing the use of both populations in clinical trials. In ADCC assay NK cell lytic activity is significantly enhanced by cetuximab; surprisingly cetuximab mediated ADCC activity is independent from CRC cell lines KRAS oncogene mutational status and require EGFR membrane expression. Pre-coating with cetuximab also increase lytic activity of IL-2 and IL-15 activated NK cells. Further experiments are in progress to evaluate the susceptibility to the lysis of primary tumor cells derived from KRAS mutated and wild type CRC patients, in NK and ADCC assays in order to confirm data obtained against established CRC cell lines and to correlate cetuximab-mediated ADCC with EGFR membrane expression. These findings support a phase I study with allogeneic NK cells infusions in association with cetuximab and IL-2 in mCRC patients harboring a mutation in the KRAS gene. DisclosuresNo relevant conflicts of interest to declare.

Patients Receiving IL-2 Activated Donor NK Cells Show Lower Incidence of Severe GvHD after Haploidentical SCT.

To improve the antileukemic effectiveness of haploidentical stem cell transplantation (HSCT) and strengthen the cell mediated immune response, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation. In addition, we activated the transferred NK-cell population ex vivo in short term cultures with high doses of IL-2 in order to enhance the antileukemic activity and inhibit the occurrence of severe GvHD.Method: In a phase-II study, 17 patients (10 AML, 1 MDS, 1 HD, 2 CML, 3 ALL, median age 37 yrs, range 17–48 yrs) were transplanted in late phases of their disease (6 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Seven patients received activated NK cells and 10 patients received unstimulated NK cells. Cells were activated by 16h incubation with IL-2 (500U/1x107 cells).Results: After selection and subsequent overnight activation of the NK cells with IL-2 (7 out of 17 patients), a mean number of 8.3 x 106/kg CD56+CD3− NK cells was transferred at day 2 after transplantation. The purity was 76%, due to contaminating CD56+ monocytes. The mean number of contaminating CD3+ cells in the transfused NK product was 2.1 x 104/kg. Activation of donor NK cells with IL-2 resulted in an increase of cytotoxic activity, when cells were tested against target cell lines. No differences in yield or number of contaminating T cells were observed between IL-2 activated and not activated NK cells. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Comparing the rate of high-grade GvHD revealed an interesting result. Whereas only one patient developed GvHD ≥ grade II after treatment with IL-2 activated NK cells, seven out of ten patients showed GvHD ≥ grade II after transfer of non-activated NK cells (p<0.05). When the correlation between GvHD and the presence of a KIR mismatch was analyzed no significant difference was observed. Moreover immunocytometric analysis of lymphocyte subpopulations at different time points after transplant revealed a long-lasting cellular immunodeficiency in all patients, with slow recovery of CD4+ lymphocytes. As for the incidence of GvHD, there was also a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells. Patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant, whereas no differences in the number of granulocytes were present between the two groups. Based on these findings we can assume that the use of IL-2 for NK cell activation could play a role in reducing the incidence of severe GvHD after haploidentical HSCT.

NK-Cell Recovery and Immune Reconstitution after Haploidentical Hematopoietic Cell Transplantation Using Either CD34 Selected Grafts and Adoptive NK-Cell Transfer or CD3/CD19 Depleted Grafts: Comparison of Two Strategies for NK Cell Based Immunotherapy.

NK-cells have been shown to play a pivotal role in haploidentical hematopoietic cell transplantation (HHCT) for engraftment, GvL effects and to combat infectious complications. Different strategies have been employed to hasten NK-cell recovery after HHCT. Here we compare the immune recovery of 17 patients after CD34 selected HHCT receiving additional adoptive CD3-depleted CD56-enriched NK cells 2 days after HHCT (adoptive NK-cells), with 18 patients receiving CD3/CD19 depleted grafts (CD3/CD19) for HHCT. Transplantations were performed at two different institutions with a median follow-up of >1 year. Conditioning consisted of 12 Gy TBI, thiotepa (10mg/kg), fludarabine (150 mg/m2) and OKT3 (day −4 to +2) in the group receiving CD34 selected grafts and adoptive NK-cell transfusions. All patients in the CD3/CD19 group received conditioning with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (day −5 to +14). No postgrafting immunosuppression was used in both groups. Seven out of the 17 patients in the adoptive NK-cell group received IL-2 activated NK cells. Median age was 37 years in the adoptive NK-cell group compared to 40 years in the CD3/CD19 group. Diagnoses in the adoptive NK-cell group included AML (n=10), ALL (n=3), CML (n=2), and Hodgkin's disease (n=1) and MDS (n=1). Diagnoses in the CD3/CD19 group were AML (n=10), ALL (n=5), NHL (n=1), CML (n=1) and multiple myeloma (n=1). The grafts contained a median of 12.5x10E6 CD34+ cells/kg and 1.1×10E4 CD3+ cells/kg in the CD34 selected group versus 9.2×10E6 CD34+cells/kg and 2.3×10E4 CD3+cells/kg in the CD3/CD19 group. The number of transferred CD56+ cells was 8.3×10E6/kg in the adoptive NK-cell group and 7.2×10E7/kg cells in the CD3/CD19 group. Hematopoietic recovery was similar in both groups. Among the patients receiving adoptive NK-cells we observed a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells: patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant (p=<0.05). In addition, we compared the immune recovery of the patients in the CD3/CD19 group and the 10 patients in the adoptive NK cells group receiving unstimulated NK-cells. There was a significant faster recovery of CD4+ T cells in the adoptive NK-cell group with a median day 40 count of 179 versus 2 cells/μl (p<0.05). However, patients in the CD3/CD19 group showed a significant faster and more sustained recovery of NK-cells with a median day 40 CD56+ count of 1464 versus 254 cells/μL (p<0.05). After day 50 no significant difference between the two groups was observed. The incidence of GvHD≥II was similar with 47% in the adoptive NK-cell group versus 50% in the CD3/CD19 group. In conclusion, patients receiving CD3/19 depleted grafts show a faster and more sustained NK-cell reconstitution but a slower T cell recovery in the early phase after transplant. The clinical impact of these differences warrants evaluation in further prospective studies.

Molecular Analysis of NTB-A Signaling: A Role for EAT-2 in NTB-A-Mediated Activation of Human NK Cells

Engagement of NTB-A on human NK cells by homophilic interaction with NTB-A-expressing target cells can trigger NK cell cytotoxicity, cytokine production, and proliferation. To better understand how NTB-A can activate NK cells, we analyzed the molecular mechanisms of NTB-A signaling. We show that NTB-A is tyrosine phosphorylated in unstimulated human NK cells and associates with SLAM-associated protein (SAP) and EAT-2. This phosphorylation of NTB-A is mediated by Src family kinases and is most likely a result of the homophilic interaction of NTB-A among neighboring NK cells. Stimulation of NK cells by NTB-A-positive targets results in increased NTB-A phosphorylation. The cytoplasmic tail of NTB-A contains three tyrosines, two of which are embedded within an immunoreceptor tyrosine-based switch motif. We generated a NTB-A-negative NK cell line, in which we expressed different mutants of NTB-A. Functional studies showed that the second tyrosine is sufficient and essential for NTB-A-mediated cytotoxicity. EAT-2, but not SAP, is recruited to this second tyrosine, indicating that SAP may be dispensable for this NTB-A function. To further investigate this, we silenced SAP expression in NK cell lines. Functional analysis of these cells showed that NTB-A can mediate NK cell cytotoxicity in the absence of SAP, probably via EAT-2. In contrast, NTB-A-mediated IFN-gamma production was greatly reduced in the absence of SAP, demonstrating that cytokine production and cytotoxicity are differentially dependent on SAP and possibly EAT-2.

Activated human NK cells can stimulate resting B cells to secrete immunoglobulin.

In addition to their cytotoxic function, NK cells can either suppress or enhance Ab production. Upon activation, NK cells generally amplify Ig secretion by activated B cells. We now report that activated NK cells prepared from human peripheral blood can induce resting B cells to produce IgM and IgG. This was demonstrated by adding IL-2 or pokeweed mitogen to purified NK cells and B cells. The addition of IL-2-activated NK cells to B cells also had this effect. The responding B cells did not require activation. Moreover, following fractionation of B cells on Percoll gradients, responsive B cells were found in the high density as well as the low density fractions. The NK cell stimulatory effect was a two-step process. The first step induced B cells to respond to soluble mediators and was mediated by unstimulated NK cells, but not CD4 nor CD8 T cells. Physical contact between NK cells and B cells was required, and mAbs to CD11a and CD54 blocked this interaction. The second step was the production of as yet unidentified cytokine(s) by activated NK cells. These direct T cell-independent stimulatory effects of NK cells on B cells may be important in autoimmune and other chronic inflammatory diseases in which the suppressive effects of NK cells may be blocked.

NK cell recognition of MHC class I. NK cells are sensitive to peptide-binding groove and surface alpha-helical mutations that affect T cells.

NK cells directly or indirectly recognize MHC class I molecules, but the exact structures recognized remain poorly defined. We address the hypothesis that NK cells, like T cells, directly contact peptide/MHC class I complexes. This hypothesis predicts that NK-mediated killing is inhibited by amino acid substitutions in the MHC class I peptide-binding groove and in solvent-accessible alpha-helical residues proposed to contact the TCR. In our model system, target cell HLA-B*0702 inhibited killing by unstimulated peripheral blood NK cells. NK-mediated killing was increased significantly by 6 of 11 peptide-binding groove mutations and 6 of 12 TCR contact site mutations, but only 1 of 6 mutations outside these sites. Many of the mutations that inhibited NK-mediated killing prohibited killing by 12 alloreactive CTL clones. These data suggest that NK receptors directly contact HLA-B*0702, focusing on the peptide-binding groove and surrounding alpha-helices. NK cell lines exhibited multiple HLA recognition patterns, which is consistent with nonuniform expression of MHC receptors by NK cells. We propose that NK cells, like alpha beta T cells and some anti-MHC Abs, directly or indirectly recognize MHC-bound peptides.

Inhibition of interferon stimulation of natural killer cell activity in mice anesthetized with halothane or isoflurane.

Basal cytotoxic activity of NK cells, a subtype of lymphocytes involved in the nonspecific immune response to viruses, tumors, and some bacteria, is altered in the postoperative period. The current study examines the effects of halothane and isoflurane on interferon-induced stimulation of NK cell cytotoxicity in vivo and in vitro. Mice were exposed to either anesthetic on days 10, 5, 1, or -1 relative to interferon treatment on day 0. NK cytotoxicity was assessed 24 h later. Similarly, splenic mononuclear cells containing NK cells were treated with interferon, before or after in vitro exposure with either halothane or isoflurane, and cytotoxicity was determined. In vivo, isoflurane or halothane inhibited subsequent interferon-induced NK cell stimulation (> 90% and 67%, respectively). No inhibition occurred if interferon was given before anesthetic exposure. Significant inhibition of interferon-induced NK cell stimulation could be observed 11 days after anesthesia. In vitro, both anesthetics inhibited the subsequent stimulation of NK cytotoxicity by interferon, however, cytotoxicity of NK cells treated with interferon before anesthetic exposure was comparable to untreated interferon-stimulated NK cells. Halothane and isoflurane inhibit interferon stimulation of NK cytotoxicity in naive (unstimulated) NK cells of the splenic mononuclear cell pool without affecting the cytotoxicity of previously stimulated (interferon) NK cells. This could occur directly by preventing the NK cell from responding or indirectly by altering other cells in the splenic mononuclear cell pool (T cells, macrophages), which then inhibit NK cell induction.