IL-2 Activated NK Cells Research Articles

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.

CDX2 enhances natural killer cell-mediated immunotherapy against head and neck squamous cell carcinoma through up-regulating CXCL14.

(NK) cells are at the first line of defence against tumours, but their anti‐tumour mechanisms are not fully understood. We aimed to investigate the mechanism by which NK cells can mediate immunotherapy against head and neck squamous cell carcinoma (HNSCC). We collected fifty‐two pairs of HNSCC tissues and corresponding adjacent normal tissues; analysis by RT‐qPCR showed underexpression of CXCL14 in HNSCC tissues. Primary NK cells were then isolated from the peripheral blood of HNSCC patients and healthy donors. CXCL14 was found to be consistently under‐expressed in the primary NK cells from the HNSCC patients. However, CXCL14 expression was increased in IL2‐activated primary NK cells and NK‐92 cells. We next evaluated NK cell migration, IFN‐γ and TNF‐α expression, cytotoxicity and infiltration in response to CXCL14 overexpression or knockdown using gain‐ and loss‐of‐function approach. The results exhibited that CXCL14 overexpression promoted NK cell migration, cytotoxicity and infiltration. Subsequent in vivo experiments revealed that CXCL14 suppressed the growth of HNSCC cells via activation of NK cells. ChIP was applied to study the enrichment of H3K27ac, p300, H3K4me1 and CDX2 in the enhancer region of CXCL14, which showed that CDX2/p300 activated the enhancer of CXCL14 to up‐regulate its expression. Rescue experiments demonstrated that CDX2 stimulated NK cell migration, cytotoxicity and infiltration through up‐regulating CXCL14. In vivo data further revealed that CDX2 suppressed tumorigenicity of HNSCC cells through enhancement of CXCL14. To conclude, CDX2 promotes CXCL14 expression by activating its enhancer, which promotes NK cell–mediated immunotherapy against HNSCC.

Phase 1 Trial of Prophylactic Donor-Derived IL-2 Activated NK Cell Infusion after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Sibling Donor

Background: Donor lymphocyte infusion (DLI) is commonly used to enhance T-cell mediated graft-versus-tumor (GVT) effect after allogeneic stem cell transplantation (allo-HSCT). It was showed that prophylactic DLI can produce promising outcome but is also associated with a high rate of graft-versus-host disease (GVHD), limiting its use as maintenance treatment. By contrast with T cells, there are clinical and biological evidences that NK cells can provide strong GVT effect without inducing GVHD, making post allo-HSCT NK cell-based immunotherapy appealing. Thus, we designed a phase 1 clinical trial evaluating the safety of donor-derived ex vivo IL-2 activated NK cells (DLI-NK) as maintenance immunotherapy after allo-HSCT (DLI-NK: NCT01853358). Methods: We included patients who were in morphologic remission of hematological cancers 2 months after allo-HSCT from a matched sibling donor after reduced intensity conditioning regimen, without history of acute GVHD. NK cells were purified from the donor (CD3 depletion / CD56 positive selection) and cultured ex vivo during 7 days with IL-2 before prior to infusion in patients between day+60 and day+90 after allo-HSCT. The study included a first phase of dose escalation (3+3 method) to determine the maximal tolerated dose (MTD) using 3 dose levels: 1.0 (dose 1), 5.0 (dose 2), 5.0-50 (dose 3) million cells/kg. A second phase allowed the treatment of subsequent patients to reach a total amount of 10 patients treated at the MTD. As ancillary studies, we performed: 1) preclinical evaluation of donor-derived IL-2 stimulated NK cell therapy in mice; 2) phenotypical and functional characterization of the NK cell therapy product before and after 7-day IL-2 stimulation, and 3) longitudinal immunomonitoring of NK cell phenotype in patients. Results: We treated 16 patients without observing any dose-limiting toxicity (grade 3-4 adverse events related to DLI-NK within 30 days after infusion). During the follow up, 4 patients developed chronic GVHD (1 mild, 2 moderate and 1 severe forms). All of them were in complete remission of GVHD without immunosuppressive treatment at last contact. We observed one non-relapse death due to idiopathic pneumonia. From the four patients who relapsed (3 AML and 1 CLL), 2 died from their disease and 2 were in complete response after salvage therapy. At last follow up, 11 out of 16 treated patients were alive in CR without GVHD features and immunosuppressive treatment. Ancillary studies showed that IL-2 stimulated allogeneic NK cells did not induce GVHD and displayed strong antitumor effect in mice against YAC-1 tumor cells, supporting the efficacy of this approach. In human, we observed that IL-2-activated NK cells expressed high levels of activating receptors (DNAM-1, NKG2A, NKp46, NKp30) and of the inhibitory receptor NKG2A, and exhibited increased degranulation and cytokine production when challenged by K562 tumor cells, as compared to freshly isolated NK cells. Immunomonitoring of patient NK cells after DLI-NK do not reveal significant phenotypic or functional dysfunction, with a pattern of NK cell recovery that is similar to what was previously observed after allo-HSCT. Conclusion: We showed that prophylactic DLI-NK after matched sibling donor allo-HSCT was feasible and safe. We observed low incidence of GVHD as compared to previous reports of conventional prophylactic DLI, reinforcing the rationale of NK cell role in GVHD prevention. In addition, promising disease-free survival supports the design of future clinical trials evaluating the efficacy of DLI-NK as disease relapse prevention. Disclosures Harbi: Sanofi: Honoraria. Olive:Imcheck Therapeutics: Other: Co-Founder & shareholder; Emergence Therapeutics: Other: Co-Founder & shareholder; Alderaan Biotechnology: Other: Co-Founder & shareholder. Blaise:Jazz Pharmaceuticals: Honoraria.

Abstract 4552: Preclinical characterization of BMS-986218, a novel nonfucosylated anti–CTLA-4 antibody designed to enhance antitumor activity

Abstract Background: Blockade of the CTLA-4 pathway with ipilimumab (IPI) as monotherapy or in combination with nivolumab (anti–PD-1) is an effective treatment for a variety of cancers. Antibody-dependent cellular cytotoxicity (ADCC) is a cellular process through which effector immune cells that express an Fc receptor (FcR) recognize and eliminate antibody-bound targets. Anti–CTLA-4 has been shown to mediate the ADCC depletion of tumor-infiltrating regulatory T cells (Tregs), which play an important role in suppressing antitumor immune response in the tumor microenvironment. A version of IPI that is nonfucosylated (NF) in the Fc region (BMS-986218; anti–CTLA-4 NF) was created to increase binding affinity to activating Fcγ receptors (FcγR, CD16) and improve ADCC, thus increasing intratumoral Treg depletion. Methods: Antibody binding to CD16 was studied by surface plasmon resonance. IL-2 release from staphylococcal enterotoxin B (SEB)–stimulated normal human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells was measured; ADCC function was evaluated by IL-2–activated NK cell–induced lysis of CD4+ T cells or activated CD4+Foxp3+ Tregs. Tumor growth was measured in transgenic (human CD16) mice implanted with MC38 tumors. Peripheral pharmacodynamics (PD) was evaluated in cynomolgus (CYNO) macaques after administration of customized adenovirus-5 vector vaccines. Results: Binding affinity to human CD16 was enhanced by BMS-986218 compared with IPI. BMS-986218 induced greater NK cell–mediated lysis of CD4+ T cells (37%) and Tregs (100%) compared with that induced by IPI (7% and 58%, respectively). Anti–CTLA-4 NF was also more effective than IPI in enhancing IL-2 release from SEB-stimulated PBMCs. Addition of an anti-CD16 antibody blocked the IL-2 response, supporting the concept that CD16 was necessary for anti–CTLA-4 NF activity. In mice, anti–CTLA-4 NF produced greater dose-dependent tumor growth inhibition (99%) than anti–CTLA-4 (61%). Tumors from anti–CTLA-4 NF–treated mice compared with anti–CTLA-4–treated mice had a greater decrease in Treg levels (70% vs 30%, respectively) and a larger increase in CD8+ T-cell levels (82% vs 62%, respectively) and no changes in Treg or CD8+ cell levels in the spleen. A more pronounced vaccine-induced T-cell response in anti–CTLA-4 NF–treated vs anti–CTLA-4–treated CYNO macaques was observed over a 3-week period. Conclusions: Nonfucosylation of an anti–CTLA-4 antibody increased binding affinity to CD16, induced depletion of Tregs while increasing T-effector cells in mouse tumors, and enhanced tumor growth inhibition in a dose-dependent manner, thus demonstrating improved ADCC compared with IPI. An ongoing phase 1/2 study is evaluating the safety and antitumor activity of anti–CTLA-4 NF alone and in combination with nivolumab (NCT03110107) in patients with advanced solid cancers. Citation Format: John Engelhardt, Rahima Akter, John Loffredo, Paula So, Natalie Bezman, Karen Price. Preclinical characterization of BMS-986218, a novel nonfucosylated anti–CTLA-4 antibody designed to enhance antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4552.

Loss of NKG2D in murine NK cells leads to increased perforin production upon long-term stimulation with IL-2.

NK cells are innate lymphocytes responsible for lysis of pathogen‐infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46+ cells (NKG2DΔNK). NKG2DΔNK NK cells develop normally, have an unaltered IFN‐γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long‐term stimulation with IL‐2, NKG2D‐deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL‐2 activated NK cells.

Interleukin-15 Enhances Anti-GD2 Antibody-Mediated Cytotoxicity in an Orthotopic PDX Model of Neuroblastoma.

Immunotherapy with IL2, GM-CSF, and an anti-disialoganglioside (GD2) antibody significantly increases event-free survival in children with high-risk neuroblastoma. However, therapy failure in one third of these patients and IL2-related toxicities pose a major challenge. We compared the immunoadjuvant effects of IL15 with those of IL2 for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) in neuroblastoma. We tested ADCC against neuroblastoma patient-derived xenografts (PDX) in vitro and in vivo and examined the functional and migratory properties of NK cells activated with IL2 and IL15. In cell culture, IL15-activated NK cells induced higher ADCC against two GD+ neuroblastoma PDXs than did IL2-activated NK cells (P < 0.001). This effect was dose-dependent (P < 0.001) and was maintained across several effector-to-tumor ratios. As compared with IL2, IL15 also improved chemotaxis of NK cells, leading to higher numbers of tumorsphere-infiltrating NK cells in vitro (P = 0.002). In an orthotopic PDX model, animals receiving chemoimmunotherapy with an anti-GD2 antibody, GM-CSF, and a soluble IL15/IL15Rα complex had greater tumor regression than did those receiving chemotherapy alone (P = 0.012) or combined with anti-GD2 antibody and GM-CSF with (P = 0.016) or without IL2 (P = 0.035). This was most likely due to lower numbers of immature tumor-infiltrating NK cells (DX5+CD27+) after IL15/IL15Rα administration (P = 0.029) and transcriptional upregulation of Gzmd. The substitution of IL15 for IL2 leads to significant tumor regression in vitro and in vivo and supports clinical testing of IL15 for immunotherapy in pediatric neuroblastoma.

Targeting CD38high Acute Myeloid Leukaemia with “Affinity Optimized” Chimeric Antigen Receptor and Membrane Bound TRAIL Expressing Natural Killer Cells

Targeting CD38high Acute Myeloid Leukaemia with “Affinity Optimized” Chimeric Antigen Receptor and Membrane Bound TRAIL Expressing Natural Killer Cells

Applications of microfluidic devices in advancing NK-cell migration studies.

Understanding how NK cells interact with tumor cells under specific microenvironment will be informative in development of NK-cell based immunotherapy. Applications of microfluidic devices in in vitro studies of NK-cell migrations offer unique opportunities to examine NK-cell migrations at single-cell under controlled cellular microenvironments. Novel devices can be created and engineered to present precise configuration that mimics cellular microenvironments for cell migration studies. We established previously the first application of a simple Y-shaped device for imaging and analysis of the abilities of the immature and mature DC to regulate murine IL-2 activated NK cell migrations. Here we reported the application of our recent technical development of a novel microfluidic device, which is also called the triple docking device (i.e., D3-Chip), for the studies of NK-cell migrations in NK-4T1 breast cancer cell interactions in vitro. Key features of this microfluidic device are its pump-free gradient generation, and the three-parallel units design that supports easy setup and parallel comparison of multiple experimental conditions. The cell docking structure enables the prealignment of all NK cells at the same "start" position before their exposures to the test conditions. As a result, quantification of cell displacement toward a chemical gradient can be quantified by enumeration of the number of cells migrated out of the docking structure and their displacements. Such microfluidic devices can be further modified in future to mimic the complex in vivo microenvironments to support more advanced investigations of NK-cell migratory responses in vitro.

MiR-544 promotes immune escape through downregulation of NCR1/NKp46 via targeting RUNX3 in liver cancer

ObjectiveTo study the potential role of miR-544 in the immune escape mechanism of hepatoma cells.MethodsNatural killer (NK) cells were collected from healthy volunteers and patients with liver cancer. Interleukin (IL)-2 activated-NK-92 cells were transfected with miR-544 inhibitor/mimic or NC/pre-NC in HepG2 co-culture system. NK-92 cells were treated with control, IL-2, IL-2 + pre-NC, IL-2 + miR-544 mimic, IL-2 + miR-544 mimic + pcDNA and IL-2 + miR-544 mimic + pcDNA-runt-related transcription factor 3 (RUNX3) groups. Mice models of liver cancer were well established. Expression of miR-544, natural cytotoxicity receptor 1 (NCR1) and RUNX3 were evaluated by quantitative real-time PCR and western blotting. Flow cytometry and ELISA were used to determine NK cell cytotoxicity and the levels of INF-γ, respectively.ResultsMiR-544 was upregulated while NCR1 and RUNX3 was downregulated in NK cells of patients with liver cancer. The levels of IFN-γ and miR-544 expression were increased and decreased in IL-2 activated-NK cells, respectively. Inversely, miR-544 overexpression inhibited NK cell cytotoxicity by downregulating IFN-γ. However, miR-544 directly targeted RUNX3 and negatively regulated NCR1. Furthermore, miR-544 promoted immune escape of hepatoma cells in vivo and in vitro.ConclusionmiR-544 promoted the immune escape of liver cancer cells by downregulating NCR1 via targeting RUNX3.

Uptake of CCR7 by KIR2DS4⁺ NK cells is induced upon recognition of certain HLA-C alleles.

The KIR2DS4 receptor is the oldest KIR2DS expressed by human NK lymphocytes. The specificity of recognition of this receptor for various HLA class I alleles has been demonstrated; however it remains poorly understood whether these interactions may result in the activation of some specific functions in NK cells. Here, we examined the functional outcome of the KIR2DS4/HLA class I interaction by the use of an alternative functional system based on the ability of KIR2DS4 to regulate the mechanism of trogocytosis by NK cells. We demonstrate that KIR2DS4 can induce the uptake of CCR7 by KIR2DS4+ NKG2A+ NK cell clones after interacting with CCR7+ target cells expressing HLA-Cw4 and HLA-Cw6 alleles. However this interaction is not always sufficient to override the inhibition generated by NKG2A expressed on the same NK cells. The recognition of HLA-Cw4 was confirmed by experiments of cytotoxicity against HLA-C-transfected cells. We also show that, different from resting NK cells, the acquisition of CCR7 in response to IL-18 cannot occur in IL2-activated NK cells because of a marked downregulation in their IL-18Rα expression. As a consequence trogocytosis represents the major mechanism by which KIR2DS4+ activated NK cells acquire the expression of this chemokine receptor.

Nicotinamide (NAM) Increases Surface Expression of CD62L On in Vitro Expanded NK Cells

AbstractAbstract 4355Natural killer (NK) cells comprise a critical component of the innate immune system that controls infections and tumor transformation. Although proof of principle supporting the antitumor effects of adoptively infused autologous and allogeneic NK cells has been established, overall response rates appear low, possibly related to their inability to traffic to lymphoid tissues and the bone marrow where hematological malignancies such as AML, lymphomas, and multiple myeloma reside. CD62L receptors play a critical role in the recruitment of NK cells from the circulation to the bone marrow and lymph nodes. Recently, investigators have developed a number of approaches to expand NK cells ex vivo for adoptive infusion in patients with cancer. Although NK cells cultured with irradiated EBV transformed lymphoblastoid cell lines (EBV-LCL) can be expanded by more than 1000 fold and are more cytotoxic to tumor cells compared to resting or IL-2 activated NK cells, these expanded cells undergo a substantial reduction in CD62L surface expression which could hinder their ability to be recruited into the tumor microenvironment. NAM, a specific inhibitor of NAD (+) dependent enzymes, was shown to up-regulate surface expression of CD62L on freshly isolated NK cells cultured in feeder cell-free cytokine-containing media. Based on this observation, we hypothesized that CD62L expression would be increased on NK cells expanded ex vivo using EBV-LCL feeders by adding NAM to the culture media. Methods:Human NK cells were isolated from PBMCs of 5 healthy volunteers by depleting CD3+ T cells and subsequently selecting CD56+ cells using immuno-magnetic beads. NK cells were expanded ex vivo in flasks over 21 days by co-culturing with an irradiated EBV-LCL feeder cell line at 20:1 LCL to NK cell ratio in media containing 500 IU/mL of IL-2. NAM was added to the media on culture day 7 at a 5 mM or 7.5 mM concentration and expanded NK cells were evaluated every 2–3 days by FACS to assess their phenotype and viability and by cytotoxicity assays to assess their cytotoxicity against K562 and renal cell carcinoma targets. The effects of NAM on the proliferative capacity of ex vivo expanded NK cultures were likewise assessed. Results:NK cell expansion cultures containing NAM had substantially higher CD62L surface expression compared to cultures without NAM, with this effect being most pronounced in NK cell cultures containing the higher concentration (7.5 mM) of NAM (see figure). On NK cell culture days 12, 14, 16, and 21, the surface MFI of CD62L was a median 1.62, 2.44, 2.03 and 3.65 fold higher on NK cells cultured in 7.5 mM of NAM compared to cultures without NAM (p =0.017, 0.012, 0.037 and 0.046 respectively; paired t-test). No significant difference in surface expression of NK cell TRAIL, NKG2D, NKG2A, NKp30, NKp46, KIR2DL1, KIR3DL1, KIR2DL2/DL3, CD44, CD95, and CD200R was observed between NAM containing cultures and controls although NKp44 expression was slightly higher in cultures that did not contain NAM. NAM-treated and untreated NK cells had similar cytotoxic function against K562 and renal cell carcinoma (RCC) tumor cell lines. The viability of NK cells, assessed by Trypan Blue and Annexin V staining, was unaffected by NAM. The addition of NAM to cell culture media did result in a dose-dependent reduction in ex vivo NK cell expansion: on days 14 and 16, NK cells expanded a median 926 and 2963 fold in control media compared to a median 443 and 1151 fold in media containing 5 mM NAM (p=0.0029 and 0.0074 respectively) and a median 359 and 732 fold in media containing 7.5 mM NAM (p=0.0019 and 0.0003 respectively). Although the proliferation of NK cells was reduced, ex vivo expansion of NK cells still occurred in NAM containing media with NK cells expanding a median 2785 and 1360 fold by day 18 in cultures containing 5 and 7.5 mM of NAM, respectively. Conclusion:The addition of NAM to NK cell expansion cultures substantially increased surface expression of CD62L on NK cells without any deleterious effect on their cytotoxic function. Although NAM reduced ex vivo NK cell proliferation, NK cells still expanded more than 1000 fold in NAM containing media. These data suggest NAM-induced increases in CD62L surface expression could be used as a novel method to improve the homing capacity of ex vivo expanded NK cells to the bone marrow and the lymphoid organs, potentially enhancing their antitumor effects when adoptively infused in patients with hematological malignancies. [Display omitted] Disclosures:Peled:Gamida Cell Ltd. Cell Therapy Technologies: Employment. Frei:Gamida Cell Ltd. Cell Therapy Technologies: Employment.

Mice Lacking Ly49E Show Normal NK Cell Development and Provide Evidence for Probabilistic Expression of Ly49E in NK Cells and T Cells

Ly49E is an unusual member of the Ly49 family that is expressed on fetal NK cells, epithelial T cells, and NKT cells, but not on resting adult NK cells. Ly49E(bgeo/bgeo) mice in which the Ly49E gene was disrupted by inserting a β-geo transgene were healthy, fertile, and had normal numbers of NK and T cells in all organs examined. Their NK cells displayed normal expression of Ly49 and other NK cell receptors, killed tumor and MHC class I-deficient cells efficiently, and produced normal levels of IFN-γ. In heterozygous Ly49E(+/bgeo) mice, the proportion of epidermal T cells, NKT cells, and IL-2-activated NK cells that expressed Ly49E was about half that found in wild-type mice. Surprisingly, although splenic T cells rarely expressed Ly49E, IL-2-activated splenic T cells from Ly49E(bgeo/bgeo) mice were as resistant to growth in G418 as NK cells and expressed similar levels of β-geo transcripts, suggesting that disruption of the Ly49E locus had increased its expression in these cells to the same level as that in NK cells. Importantly, however, the proportion of G418-resistant heterozygous Ly49E(+/bgeo) cells that expressed Ly49E from the wild-type allele was similar to that observed in control cells. Collectively, these findings demonstrate that Ly49E is not required for the development or homeostasis of NK and T cell populations or for the acquisition of functional competence in NK cells and provide compelling evidence that Ly49E is expressed in a probabilistic manner in adult NK cells and T cells.

Induction of Cardiac Angiogenesis Requires Killer Cell Lectin-Like Receptor 1 and α4β7 Integrin Expression by NK Cells

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study, we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.

In Vitro Expanded NK Cells Have Increased Natural Cytotoxity Receptors, TRAIL and NKG2D Expression, and Superior Tumor Cytotoxicity Compared to Short-Term IL-2 –Activated NK Cells.

Abstract 463IL-2 activates NK cell enhancing their capacity to lyse tumor cells. To date, most clinical studies of adoptive NK cell transfer have utilized short-term (12-16 hours) IL-2-activated NK cells. Because IL-2 alone is ineffective in expanding NK cells in vitro, the relatively low numbers of NK cells obtained for infusion following short term IL-2 activation may limit the full therapeutic impact of this approach. To obtain larger numbers of NK cells, novel ex vivo expansion protocols that utilize irradiated EBV-LCL or K562 feeder cells have recently been developed. However, concerns exist that extensive ex vivo expansion might significantly reduce the in vivo proliferative potential and long-term viability of adoptively transferred NK cells. Here we investigated for differences in phenotype, tumor cytotoxicity and in vivo persistence between short-term IL-2 activated and long-term expanded NK cells. CD56+/CD3- NK cells were isolated from normal donors by immuno-magnetic bead selection and were either activated with IL-2 (500U/ml) for 12-16 hours or were expanded in vitro over 14 days using irradiated EBV-LCL feeder cells in IL-2 containing media (500U/ml). Short-term IL-2 activated NK cells did not expand in number in contrast to EBV-LCL stimulated NK cells which expanded 400-1000 fold by culture day 14. Flow cytometry analysis revealed no differences in expression of DNAM-1, 2B4, LFA-1 or granzyme B between short-term activated and expanded NK cells. However, expanded NK cells had significantly higher expression of TRAIL, NKG2D, and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 and a slight increase in KIR3DL1 and KIR2DL2/3. A 4-hour 51Cr-release assay showed expanded NK cells were significantly more cytotoxic against K562 cell compared to short-term IL-2 activated NK cells; at a 1:1 effector to target ratio, 67±6%, 26±1%, and 9±1% of K562 cells were killed by expanded, short term IL-2 activated and fresh NK cells respectively (p<0.05). Increased TRAIL expression on expanded NK cells was also associated with increased lysis of TRAIL-sensitive tumor cells (RCC tumors treated with bortezomib); at a 1:1 E:T ratio, 55±3% and 5±2% of bortezomib-treated RCC tumors were killed by expanded and short-term IL-2 activated NK cells respectively (p<0.05). We next assessed for differences in the in vivo longevity of these NK cell populations when transferred into immuno-deficient mice. Two million NK cells were labeled with a near infrared-dye (DiR; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) and injected intra-peritoneal (i.p.) into CB.17 SCID-beige mice. Mice were administered IL-2 (100,000U/ml bid i.p.) for five days then underwent bioluminescent imaging using the IVIS100 system. Although FACS analysis of NK cells performed immediately prior to injection showed increased DiR fluorescent intensity in short-term IL-2 activated vs. expanded NK cells, fluorescence signal in vivo was slightly higher in the first 24-96 hours in mice that received expanded NK cells; fluorescence intensity was 5-41% (p=0.003) stronger in recipients of expanded NK cells compared to mice receiving short-term IL-2 activated NK cells. We next evaluated the in vivo anti-tumor effects of activated vs. expanded NK cells. CB.17 SCID-beige mice were injected i.p. with luciferase transduced 526 human melanoma cells three days prior to receiving an i.p. injection of short term IL-2 activated vs. expanded NK cells (+ bid i.p. IL-2). Bioluminescent imaging measuring tumor flux to calculate tumor burden and tumor doubling time showed no difference in tumor progression between both NK cell cohorts.In conclusion, these results demonstrate that ex vivo expanded NK cells are phenotypically and functionally different than short-term IL-2 activated NK cells. Expanded NK cells have increased expression of natural cytotoxicity receptors, NKG2D and TRAIL and have greater TRAIL-mediated tumor cytotoxicity compared to IL-2 activated NK cells. Importantly, despite extensive ex vivo proliferation, expanded NK cells appear maintain similar longevity in vivo as non-expanded short term IL-2 activated NK cells. Disclosures:No relevant conflicts of interest to declare.

Romedepsin (RM), a HDACi, Significantly Increases the Expression of NKG2D Ligands, MIC A/B, in Leukemia/Lymphoma Cells (LL), in Part through the Glycogen Synthase Kinase-3 (GSK-3) Pathway, Resulting in Enhanced NK Cytotoxicity.

Abstract 3027Poster Board II-1003 IntroductionNatural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D-ligands, including MIC A/B, which are known to be expressed on epithelial tumors, resulting in tumor cytotoxicity (Ayello/Cairo et al, BBMT, 2006). The expression of MIC A/B on these tumors can be induced by in vitro exposure of these cells to HDACi, specifically Romedepsin (RM) (Skov et al, Cancer Res, 2005). Glycogen synthase kinase-3 (GSK-3), a constitutively active serine-threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, has also been shown to be an important regulatory enzyme in the expression of MIC A/B in response to RM (Doble et al, J cell Sci 2003; Frame et al, Biochem J 2001; Skov et al, Cancer Res, 2005). ObjectiveWe sought to determine of the expression of MIC A/B in response to RM in various leukemia and lymphoma cells (LL), its influence on NK cell mediated cytotoxicity and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. MethodsLL cells (106/ml, RS 4:11 [MLL-ALL], REH [pre-B cell ALL], Jurkat [T-cell ALL], Toledo [DLBCL], Ramos [Burkitt's Lymphoma]) were exposed to RM (10 ng/mL) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B antibody to determine surface expression of MIC A/B. Peripheral blood NK cells (CD3-/56+) were isolated via magnetic separation followed by IL-2 activation (3000IU/ml, 18 hrs). LL cells exposed to RM (generously supplied by Gloucester Pharmaceuticals) were subjected to NK cell mediated cytotoxicity assays (using an europium assay) at effector:target (E:T) ratio of 10:1, as we had previously described (Ayello/Cairo et al, BBMT 2006). LL cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity (Davies et al, Biochem J, 2000), to determine the role of this regulatory enzyme in the RM mediated expression of MIC A/B in these LL cells. Finally, blocking studies were also performed with anti-NKG2D receptor blockers to determine the specific role of NKG2D signal transduction pathway in NK cell mediated cytotoxicity. ResultsMIC A/B expression significantly increased in LL cells in response to RM ([RS4:11 0.2% vs 19.2%, p< 0.0001], [REH 0.2% vs 46%, p= 0.0003], [Jurkat 1.12% vs 44.7%, p< 0.0001], [Toledo 0.5% vs 15.8%, p=0.0001], [Ramos 0.57% vs 33.6%, p=0.0003]). In addition, the expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl (Jurkat [RM vs RM+LiCl] 85% vs 18%, p<0.0001; RS 4:11 [RM vs RM+LiCl] 82% vs 5%, p<0.0001; Ramos [RM vs RM+LiCl] 67% vs 35%, p<0.0001). Cytotoxicity assays revealed significant increases in-vitro cytotoxicty in RS 4:11, Ramos and REH cells at E:T ratio of 10:1. Mean specific release (MSR)(±SEM) was measured for four conditions in the assay: NK cells (A) vs NK cells with 10 ng/ml RM (C) vs IL-2 activated NK cells (B) IL-2 activated NK cells +10 ng/ml of RM (D). MSR for RS 4:11 10%±3.6% vs 108%±47% and 12%±4% vs 145%±45% p<0.05(A vs C, B vs D) for RS 4;11. MSR for REH was 13.2%±14.4% vs 50%±17.6% and 22%±18% vs 132%±29% p<0.01(A vs C, B vs D). MSR for Ramos was 7.6%±7.6% vs 79%±15.6% and 35%±13% vs 90%±22% p<0.05(A vs C, B vs D). NKG2D receptor-blocking experiments resulted in significant decrease in NK cell mediated cytotoxicity in REH (p<0.03) and Ramos cells (p<0.001). ConclusionOur data suggests that the surface expression of MICA/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D-mediated cytotoxicity by NK cells. Furthermore, up-regulation of MICA/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway. DisclosuresNo relevant conflicts of interest to declare.

Anti-tumor activity of patient-derived NK cells after cell-based immunotherapy – a case report

BackgroundMembrane-bound heat shock protein 70 (Hsp70) serves as a tumor-specific recognition structure for Hsp70-peptide (TKD) plus IL-2 activated NK cells. A phase I clinical trial has shown that repeated re-infusions of ex vivo TKD/IL-2-activated, autologous leukapheresis product is safe. This study investigated the maintenance of the cytolytic activity of NK cells against K562 cells and autologous tumor after 6 plus 3 infusions of TKD/IL-2-activated effector cells.MethodsA stable tumor cell line was generated from the resected anastomotic relapse of a patient with colon carcinoma (pT3, N2, M0, G2). Two months after surgery, the patient received the first monthly i.v. infusion of his ex vivo TKD/IL-2-activated peripheral blood mononuclear cells (PBMNC). After 6 infusions and a pause of 3 months, the patient received another 3 cell infusions. The phenotypic characteristics and activation status of tumor and effector cells were determined immediately before and at times after each infusion.ResultsThe NK cell ligands Hsp70, MICA/B, and ULBP-1,2,3 were expressed on the patient's anastomotic relapse. An increased density of activatory NK cell receptors following ex vivo stimulation correlated with an enhanced anti-tumoricidal activity. After 4 re-infusion cycles, the intrinsic cytolytic activity of non-stimulated PBMNC was significantly elevated and this heightened responsiveness persisted for up to 3 months after the last infusion. Another 2 re-stimulations with TKD/IL-2 restored the cytolytic activity after the therapeutic pause.ConclusionIn a patient with colon carcinoma, repeated infusions of ex vivo TKD/IL-2-activated PBMNC initiate an intrinsic NK cell-mediated cytolytic activity against autologous tumor cells.

Differential Effects of the BCR-ABL-Inhibitors Imatinib, Nilotinib and Dasatinib on NK Cell Reactivity against Chronic Myeloid Leukemia (CML)

CML is characterized by the BCR-ABL fusion protein, which mediates the oncogenic signaling. This led to the development of BCR-ABL inhibitors revolutionizing therapy of CML. However, as recently reported for Dasatinib (Schade et al., Blood 111:1366 (2008); Blake et al., Blood 111:4415 (2008)), these agents may impair the activity of immune effector cells like NK cells and T cells. After initiating oncogenic events, development and progression of clinically apparent malignancy is dependent on the evasion of the tumor cells from immunosurveillance. In light of the important role of NK cell reactivity against leukemia we compared the influence of Imatinib, Nilotinib and Dasatinib on the reactivity of both resting and IL-2 activated NK cells against CML cells to identify the compound with the least immuno-compromising side effects. First, the effects of the compounds on NK cell reactivity in concentrations corresponding to plasma peak levels were studied. Dasatinib (200nM) completely abolished NK cell granule mobilization, cytotoxicity and IFN-γ production, while no substantial inhibition was observed with Imatinib (5μM) and Nilotinib (3.6μM) mediated a minor but significant inhibition (p<0.05, Student's T-test). Presence of the compounds in concentrations corresponding to IC50 levels (Imatinib 600nM, Nilotinib 30nM, Dasatinib 10nM) revealed no influence of Imatinib and Nilotinib, while Dasatinib still significantly reduced NK cell cytotoxicity and IFN-γ production up to 60%. Since Dasatinib, in addition to BCR-ABL, potently inhibits SRC kinases, which are involved in the activation of MAPK pathways and thus crucial for NK cell cytotoxicity, we determined the influence of the compounds on ERK phosphorylation. While no inhibitory effect was observed using Imatinib and Nilotinib, Dasatinib markedly reduced ERK phosphorylation in NK cells. Our data demonstrate that NK cell anti-tumor reactivity is not inhibited by clinically relevant concentrations of Imatinib. While Nilotinib may mediate a minor effect, Dasatinib substantially impairs NK cell reactivity by inhibition of signaling pathways crucial for NK cell effector functions. For a given patient, the choice and dosing of the most suitable BCR-ABL inhibitor may thus require careful consideration of its influence on the immune system, especially in view of the important role of NK cells in the immunesurveillance of residual leukemia.

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.

Potential contribution of naïve immune effectors to oral tumor resistance: role in synergistic induction of VEGF, IL-6, and IL-8 secretion.

The aim of this study is to identify the phenotype of resistant oral tumors, and to delineate the contribution of immune effectors to resistance of oral tumors. UCLA-1 oral tumors which were resistant to NK cell mediated cytotoxicity secreted increased amounts of IL-6, IL-1beta, GM-CSF, and IL-8 when cultured with or without immune effectors. In addition, the levels of vascular endothelial growth factor (VEGF) secretion in the co-cultures of naïve immune effectors with UCLA-1 rose significantly when compared to tumor cells alone. IL-2 activated NK cells decreased VEGF secretion in all tumor cells. However, NK cells which were induced to undergo cell death with anti-CD16 antibody were not only unable to decrease VEGF secretion, but they also contributed further to the increase in VEGF secretion by oral tumors. Overall, we show in this paper that naïve as well as non-viable immune effectors may contribute to the growth and resistance of oral tumors by triggering the secretion of key tumor cell growth factors.