High Numbers Of Natural Killer Cells Research Articles

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Identification and Validation of Molecular Markers of Tumor Cell Resistance to Natural Killer Cells through CRISPR-Based Screens and Large-Scale Phenotypic Screens of Pooled Tumor Cell Lines

Natural killer (NK) cellsexhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors. The in vivo anti-tumor effects of NK cells are not confounded by graft vs. host disease, providing a key advantage over strategies exploiting T cell reactivity. Indeed, infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. Because of these considerations, NK cells had been viewed as a promising therapeutic approach for a disease like multiple myeloma (MM), which remains incurable, even after initial responses to novel immunotherapies, including anti-CD38 antibodies; combinations of thalidomide derivatives with immune checkpoint blockade; and even CAR-T cells. However, our prior studies and more recent unpublished work indicate that tumor cells can exhibit decreased response to NK due to the protective effect of nonmalignant mesenchymal stromal cells and depending on the genetic background of the tumor cells. To our knowledge, there have been limited, if any, reports of systematic screens to quantify NK cell cytotoxicity against large panels of molecularly characterized tumor cell lines. Moreover, while there have been earlier RNAi-based studies, to the best of our knowledge there have not been any genome-wide CRISPR-based screens for candidate NK cell resistance genes. To address this void, we sought to identify and validate mechanisms regulating tumor cell response to NK cells, by performing orthogonal screens in 2 systems: (i) Using similar approaches as in other screens conducted by our lab in other models, we performed a genome-wide CRISPR gene editing screen (GECKO library) in the NK cell-sensitive colorectal Ca line HCT15 and identified candidate genes for which knockout confers resistance to the NK cell line KHYG1. (ii) We studied the cytotoxicity of NK cells (primary NK cells or KHYG1; at different time points and effector-to-tumor [E2T] ratios) against a pool of DNA-barcoded 568 lineage diverse solid tumor lines from the Cancer Cell Line Encyclopedia (CCLE), using the PRISM multiplexed cell viability screening technology. This PRISM screen expanded on a previous screen across 100 cancer cell lines and yielded consistent results supporting the reproducibility of the method. We correlated NK cell cytotoxicity with the CCLE genomic feature data set (mRNA expression, copy number variation, and gene mutational statues) for each of the 568 cell lines. The screens provided candidate “hits” which were concordant in both studies and included recognizable regulators of NK cell biology (e.g. loss-of-function of death receptors such as FAS or gain-of-function of HLA-E correlating with NK cell resistance); as well as other plausible, but previously under-appreciated candidates, which will be considered for further studies in this project. We did not observe significant tumor type-specific differences in NK cell responses in this screen, suggesting that candidate markers identified in these studies may be relevant across lineages, including MM and blood cancers (even though these lines were not included so far in our PRISM or CRISPR screens). Additional ongoing “multi-omic” analyses seek to further examine the links between the phenotypic responses to NK cells with the molecular features of these cell lines (e.g. integrating mutations, DNA methylation, proteomic [RPPA] etc.). Our study highlights the feasibility of combining CRISPR/Cas9-based functional genomic screens (on genome-wide and sub-genome scale) on individual cell lines with large scale phenotypic screens on pooled barcoded cell lines to identify candidate biomarkers of tumor cell sensitivity vs. resistance to NK cells. Notably our PRISM screen represents, to our knowledge, the largest effort to-date to correlate the molecular annotation of tumor cells with their response to uniform treatment, pre-clinical or clinical, to a cell-based immunotherapy. DisclosuresMitsiades:Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; Ono: Research Funding; TEVA: Research Funding; Abbvie: Research Funding; Takeda: Other: Employment of family member.

Expression of the Fractalkine Receptor (CX3CR1) Is Significantly Increased in Immune Cells of Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma patients with Active Disease

Fractalkine (CX3CL1) is a chemokine constitutively expressed in many hematopoietic and non-hematopoietic tissues (Bazan J.F. et al., Nature, 1997). It mediates different biological activities by binding to its receptor CX3CR1, expressed on different cell types such as natural killer (NK) cells, monocytes, cytotoxic effector T cells and B cells. The CX3CL1/CX3CR1 axis is involved in the pathogenesis of several cancers including various B-cell malignancies (Ferretti E. et al.,Mediators Inflamm, 2014). CX3CR1 was found both at the mRNA and protein levels in several subtypes of lymphoma (Andreasson, U. et al.,Cancer Lett, 2008). In this study, we analysed the expression of the fractalkine receptor (CX3CR1) in peripheral blood mononuclear cells (PBMC) of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) patients (pts) with active disease (n=22), pts with newly diagnosed diffuse large B-cell lymphoma (DLBCL) (n=21) as well as classical Hodgkin lymphoma (cHL) (n=19) and compared with healthy donors (n=14). PBMC populations were assessed by flow-cytometry including T cells, NK cells and monocytes. Statistical comparisons between groups were done using the non-parametric Mann-Whitney U test. CLL/SLL pts had a median age of 75 years with equal sex distribution. Thirteen pts had high-risk and 9 intermediate-risk disease according to the modified Rai staging system; 1/21 had 17p deletion. Median lymphocyte count was 103 x 109/L (range 1.5-312). Of the 19 cHL pts, 12 had limited (stage 1-2A) and 7 had advanced (stage 2B-4) disease. Of the 21 DLBCL pts, 10 had stage 1, 4 had stage 2, 2 had stage 3 and 5 had stage 4 disease. CLL/SLL pts had significantly higher absolute number of CD4+and CD8+ T cells compared to controls, DLBCL and cHL patients (p=0.02, p=0.0001 and p<0.0001 for CD4+ T cells and p=0.0007, p<0.0001 and p=0.0002 for CD8+ T cells, respectively). DLBCL and cHL pts had lower CD4+ numbers compared to controls (p<0.005), while no difference was observed for CD8+ cells (Fig. 1A). The absolute numbers of CD4+ and CD8+ cells expressing the CX3CR1 were also significantly higher in CLL pts compared to controls and the other patient groups (p≤0.0001 for all comparisons). No significant difference was observed between either DLBCL or cHL pts and controls (Fig. 1B). Both the CD56bright and the CD56dim NK cell populations were also studied. CLL pts were found to have a significantly higher numbers of NK cells (p=0.001) and CD56dim NK cells (p=0.0027) compared to controls, while no difference was found for the other patient groups. No difference was observed between CLL pts and controls with regard to the CD56bright NK cells, which, however, were lower in both DLBCL and cHL pts than in controls (p<0.05) (Fig. 2A). The absolute numbers of NK cells expressing CX3CR1 was also significantly higher in CLL compared to controls (p=0.002). This was observed in both the CD56dim (p=0.004) and the CD56bright (p=0.04) subpopulations. In the other patient subgroups, no significant difference was observed with the exception of CX3CR1+CD56bright NK cells, which were lower in both DLBCL and cHL pts compared to controls (Fig. 2B). The median monocyte count was not significantly different among the patient groups or controls. Monocyte sub-populations were then defined by flow-cytometry: classical (CD14highCD16low), intermediate (CD14highCD16high) and non-classical (CD14lowCD16high). The median percentage of monocytes expressing CX3CR1 was >95% in all the patient groups and did not significantly differ from controls. No significant difference was observed for the monocyte subpopulations either. In conclusion, CLL/SLL pts with active disease have significantly higher numbers of T and NK cells expressing the fractalkine receptor (CX3CR1) compared to both controls and pts with DLBCL and cHL. No difference was observed for the monocytes. CX3CR1 might be a therapeutic target in CLL/SLL. Small molecule inhibitors of this axis are in clinical development. Disclosures Palma: Takeda: Research Funding. Olin:Kancera AB: Employment, Equity Ownership. Schultz:Kancera AB: Employment, Equity Ownership. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Österborg:Kancera AB: Research Funding; Abbvie: Research Funding; Janssen: Research Funding; BeiGene: Research Funding; Gilead: Research Funding.

NK Cell-Based Immunotherapy for Hematological Malignancies.

Natural killer (NK) lymphocytes are an integral component of the innate immune system and represent important effector cells in cancer immunotherapy, particularly in the control of hematological malignancies. Refined knowledge of NK cellular and molecular biology has fueled the interest in NK cell-based antitumor therapies, and recent efforts have been made to exploit the high potential of these cells in clinical practice. Infusion of high numbers of mature NK cells through the novel graft manipulation based on the selective depletion of T cells and CD19+ B cells has resulted into an improved outcome in children with acute leukemia given human leucocyte antigen (HLA)-haploidentical hematopoietic transplantation. Likewise, adoptive transfer of purified third-party NK cells showed promising results in patients with myeloid malignancies. Strategies based on the use of cytokines or monoclonal antibodies able to induce and optimize NK cell activation, persistence, and expansion also represent a novel field of investigation with remarkable perspectives of favorably impacting on outcome of patients with hematological neoplasia. In addition, preliminary results suggest that engineering of mature NK cells through chimeric antigen receptor (CAR) constructs deserve further investigation, with the goal of obtaining an “off-the-shelf” NK cell bank that may serve many different recipients for granting an efficient antileukemia activity.

Automated closed-system large-scale expansion of clinical-grade natural killer cells

Background & Aim The field of cellular immunotherapy is growing rapidly. Natural killer (NK) cells are part of the innate immune system and have wide potential for therapeutic application. Although CAR-T cells are now commercially available, NK cells are still in pre-commercial development and testing. It is likely that much higher numbers of NK cells will be required to achieve therapeutic results compared to that of antigen-specific T cells. With the development of feeder cells expressing membrane-bound IL-21, it has become possible to achieve large-scale expansion of non-senescent NK cells. To date, most studies have expanded NK cells at clinical scale by sequential application of open systems (G-rex) or small-volume closed systems. Achieving large-scale expansion of GMP-grade NK cells requires multiple systems operating in parallel, which increase labor costs, material costs and potential for contamination. To develop a large-scale process for NK-cell expansion in compliance with regulatory requirements, we combined the CliniMACS Prodigy (Miltenyi Biotec) and Xuri W25 (GE Healthcare) into one closed-system process. Methods, Results & Conclusion In collaboration with Miltenyi, we optimized a custom program for the Prodigy to perform Ficoll density-gradient selection of mononuclear cells, CD3-based magnetic depletion of T cells, periodic addition of and forced interaction with feeder cells, and on-demand addition of media for the first phase of NK-cell culture, culminating in transfer of the cells to a secondary culture bag. We then applied a semi-continuous perfusion program on the Xuri for the second phase. We identified optimal glucose and pH levels to support log-phase expansion, and refined the Prodigy program to optimize CD3-depletion and allow efficient volume reduction steps for media exchanges. On the Xuri, pH and dissolved oxygen (DO) monitoring was utilized to identify optimal rocking rates, rocking angles and perfusion parameters to allow sufficient NK cell-feeder cell contact and achieve high cell densities. Finally, NK cells produced by this approach demonstrated very high cytotoxicity (mean 65.2% at 1.25:1 E:T ratio). With these optimized protocols, we achieved high-purity (≥99%, n=3) and high-density (∼107 cells/mL) conditions to yield 5 × 1010 high-activity NK cells in a 5L final culture volume from one donor buffy coat. We anticipate realizing both reduced cost and increased regulatory compliance of NK cell production by using the automated closed systems of Prodigy and Xuri in sequence.

Identification of Molecular Markers of Tumor Cell Sensitivity and Resistance to Natural Killer Cells through Genome-Wide CRISPR Activation and CRISPR Editing Screens in Multiple Myeloma Cell Lines: Implications for Anti-Myeloma Immunotherapy

Natural killer (NK) cells represent a promising immunotherapeutic approach as they can potently kill tumor cells without triggering graft-versus-host reactions. Indeed, infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. However, prior studies and early clinical trials indicate that tumor cells can exhibit decreased response to NK due to the protective effect of nonmalignant mesenchymal stromal cells; and depending on the genetic background of the tumor cells. To our knowledge, since earlier subgenome-scale RNAi-based studies, there have been no genome-wide CRISPR-based screens to identify candidate markers conferring tumor cell resistance or sensitivity to NK cells in multiple myeloma (MM). To address this void, and building on a recent loss-of-function (LOF) study by our group on solid tumors, we sought to identify genes regulating the response of MM cells to the cytotoxic activity of NK cells by conducting a genome-wide CRISPR/Cas9-based gene editing (Brunello library of sgRNA) and gene activation (Calabrese library of sgRNA) screens in MM.1S cells co-cultured with primary NK (pNK) cells (effector-to-target [E:T] ratio of 3.75:1) derived from healthy donor peripheral blood mononuclear cells (PBMCs) cultured in vitro in GMP SCGM medium with IL-2. Briefly, MM.1S cells engineered to stably express the nuclease SpCas9 (Brunello) or a catalytically inactive programmable RNA-dependent DNA-binding protein (dCas9)-VP64 (Calabrese) were also transduced with lentiviral particles for a pool of ~70,000 (Brunello library) or ~120,000 (Calabrese) sgRNAs, targeting exons of ~20,000 genes (plus non-targeting control sgRNAs), under conditions of transduction which allow for an average of no more than 1 sgRNA to be incorporated in a given cell. This allowed us to convert the initial population of MM.1S cells into heterogeneous pools in which each gene is subject to individual LOF or gain-of-function (GOF), due to Cas9-mediated editing, by only 1 sgRNA. Flow cytometry was performed to verify pNK viability, purity (CD56 and CD3), and expression of p46 receptor, surrogate marker of NK cell activity. These screens identified genes whose knock-out (Brunello sgRNA library) or activation (Calabrese sgRNA library) led to NK cells resistance or potential sensitivity. The hits observed in the current MM-oriented study exhibited, compared to our similar studies in solid tumor model, substantial gene level differences, but notable overlap at the pathway level (including death receptor pathways, NK activating pathways), which suggests that mechanisms determining tumor cell response vs. resistance to NK cells operate through modules consistent across tumors, but manifested through potentially different members of the respective pathways in different neoplasms. For instance, in this MM-oriented study, we identified that NK cell sensitivity of tumor cells is modulating by activation of several metabolic and homeostatic genes, receptor kinases, and interestingly membrane-bound proteins of the mucin family, e.g. MUC1, and MUC4, which have been reported to play a role in NK-mediated tumor killing in other types of cancer. MUC1 in particular has a clinical relevance as a small molecule inhibitor with prior preclinical studies in MM is available. Interestingly, our GOF screen identified as potential NK cell sensitizers TNFRSF10B, a death receptor related to TNFRSF10A (a hit identified in our studies in solid tumors), the putative death receptor adaptor TRADD, and the NK ligands PVR and ULBP1. Interestingly, genes such as PTEN and TP53, commonly associated with high-risk MM, didn't affect the response to NK cell, suggesting that NK cell-based therapies may potentially have a role in treatment of MM patients with high-risk clinical or biological features. In conclusion, this is the first study applying both LOF and GOF genome-wide screens to NK cell response in MM. The combination of such screens performed in parallel provide complementary and orthogonal information that allows us to identify genes that might not have been appreciated if only either LOF or GOF alone screens had been performed. We envision that the methodology and results presented herein will provide a framework towards validation of molecular markers which can help to optimize and individualize the use of NK cell-based therapy in MM. DisclosuresMitsiades:Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/ Johnson & Johnson: Research Funding; Takeda: Other: employment of a relative.

Umbilical Cord Blood-Derived Natural Killer Cells Combined with Bevacizumab for Colorectal Cancer Treatment.

Colorectal cancer (CRC) is among the cancers with the highest incidence globally, and it currently ranks as the fourth leading cause of cancer-related deaths worldwide. Novel strategies for the treatment of advanced CRC are urgently needed, and adoptive transfer of allogeneic natural killer (NK) cells represents an attractive option. In this study, we successfully expanded NK cells from umbilical cord blood (UCB) with membrane-bound interleukin (IL)-21, termed eUCB-NK cells. eUCB-NK cells efficiently lysed CRC cell lines in vitro and secreted significantly higher levels of interferon-γ, tumor necrosis factor-α, granulocyte-macrophage colony stimulating factor, and chemokine ligand 3 compared with IL-2-stimulated NK cells. Adoptive transfer of these NK cells significantly inhibited the growth of HT29 xenografts, whereas LoVo tumors were not effectively controlled with eUCB-NK cells. Higher numbers of NK cells inside HT29 tumors, not seen in LoVo tumors, might contribute to the differences in response to eUCB-NK cells. Bevacizumab increased extravasation of adoptively transferred NK cells into LoVo tumors and improved the therapeutic activity of eUCB-NK cells. These results justify clinical translation of UCB-derived NK cell-based therapeutics, used alone or in combination with bevacizumab, as a novel treatment option for patients with CRC.

PO-414 Stage IV melanoma patients with tumoural MHC class i loss only respond to anti-PD-1 therapy in the presence of high NK cell density

IntroductionPrior to 2011 there were no effective systemic therapies for advanced stage melanoma patients and the overall survival was 7.5 months. Recently, immune checkpoint inhibitors, anti-PD-1 and anti-CTLA-4 inhibitors, have produced response rates as high as 50% and doubled overall survival, but only 30% of patients have a durable response. One important resistance mechanism to immunotherapies is the downregulation of MHC Class I, resulting in the expansion of melanoma cells resistant to CD8 +T cell killing. Natural Killer (NK) cells monitor MHC Class I expression and eliminate cells that fail to express it; thus, NK cells are likely critical in preventing resistance to anti-PD-1 therapy. This study sought to investigate whether tumour-infiltrating NK cells in the presence of MHC class I loss improved survival outcome of patients treated with anti-PD-1.Material and methodsTwenty-five stage IV metastatic melanoma patients treated with anti-PD-1 therapy were categorised into responders (CR/PR/SD >6 mo, n=13) and non-responders (SD <6 mo/PD, n=12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on formalin-fixed-paraffin embedded pre-treatment tumour samples. Flow cytometry was used to confirm novel NK phenotypes in melanoma lymph node metastases of treatment naïve stage IV melanoma patients (n=5).Results and discussionsDifferential expression analysis identified nine up-regulated NK cell specific genes in responders when compared to non-responders (adjusted p<0.05). Immunofluorescent staining of biopsies confirmed a significantly higher density of intratumoural and peritumoural CD16+ (intratumoural p=0.0015 and peritumoural p=0.0039) and granzyme B+ (intratumoural p=0.019 and peritumoural p=0.011) NK cells in responding patients. Flow cytometry demonstrated 46%±8% of the NK cells were identified as expressing PD-1. When stage IV melanoma patients were further stratified by MHC class I loss, responding patients with MHC class I loss, had a higher NK cell density and better survival outcome, compared to non-responders (p=0.012).ConclusionThis study showed that the presence of higher numbers of NK cells are associated with improved responses to anti-PD-1 therapy. Most importantly, responding patients with MHC class I loss had higher NK cell densities, suggesting that NK cells play an important role in mediating response to anti-PD-1 in patients whose tumour down regulates MHC class I expression.

NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: dynamics and clinical implications

AbstractThe use of posttransplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (HSCT), allowing safe infusion of unmanipulated T cell–replete grafts. PT-Cy selectively eliminates proliferating alloreactive T cells, but whether and how it affects natural killer (NK) cells and their alloreactivity is largely unknown. Here we characterized NK cell dynamics in 17 patients who received unmanipulated haploidentical grafts, containing high numbers of mature NK cells, according to PT-Cy–based protocols in 2 independent centers. In both series, we documented robust proliferation of donor-derived NK cells immediately after HSCT. After infusion of Cy, a marked reduction of proliferating NK cells was evident, suggesting selective purging of dividing cells. Supporting this hypothesis, proliferating NK cells did not express aldehyde dehydrogenase and were killed by Cy in vitro. After ablation of mature NK cells, starting from day 15 after HSCT and favored by the high levels of interleukin-15 present in patients' sera, immature NK cells (CD62L+NKG2A+KIR−) became highly prevalent, possibly directly stemming from infused hematopoietic stem cells. Importantly, also putatively alloreactive single KIR+ NK cells were eliminated by PT-Cy and were thus decreased in numbers and antileukemic potential at day 30 after HSCT. As a consequence, in an extended series of 99 haplo-HSCT with PT-Cy, we found no significant difference in progression-free survival between patients with or without predicted NK alloreactivity (42% vs 52% at 1 year, P = NS). Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are lost upon PT-Cy administration, blunting NK cell alloreactivity in this transplantation setting.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3-) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO™10, CellGro®, TexMACS™, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Abstract 621: Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models

Abstract TUSC2, a pro-apoptotic tumor suppressor gene whose expression is lost or decreased in most lung cancers, activates the innate immune system through initiation of broad spectrum cytokine secretion and natural killer (NK) cell activation. TUSC2 delivered systemically by nanovesicles has mediated tumor regression in metastatic non-small cell lung cancer clinical trials. We studied the effect of TUSC2 on immune cell populations and the anti-tumor activity of TUSC2 in combination with anti-PD1 checkpoint blockade in two syngeneic mouse models: C57BL/6 mice subcutaneously injected with murine lung adenocarcinoma cell line CMT/167-luc cells (KrasG12V mutation) and 344SQ (KrasG12D allele and a knock-in Trp53R172HΔG allele) adenocarcinomas which metastasize to the lung in 129S2 mice. Tumor growth was monitored by scoring ex-vivo luminescence using the IVIS Imaging System 200. Multi-color flow cytometry was used for immune profiling of circulating immune cells after nanovesicle mediated TUSC2 intravenous injection. Cytokine gene expression in response to TUSC2 in sorted immune subpopulations was determined by real-time PCR. Tumor growth was significantly reduced with TUSC2 treatment compared with no treatment in both subcutaneous and metastatic mouse models. Synergistic anti-tumor activity was observed when TUSC2 was combined with anti-PD1 verified in five independent experiments. In the lung metastasis model, mice treated with TUSC2 + anti-PD1 lived significantly longer than with single agent treatment. Circulating NK cells increased three fold following TUSC2 nanovesicle intravenous injection both in tumor free and tumor bearing mice which correlated with tumor regression and survival. Cytotoxic T lymphocyte responses were increased whereas Tregs and MDSCs decreased with TUSC2 alone and TUSC2+anti-PD1 treatment. The levels of T cell checkpoint markers PD1, CTLA-4, LAG-3, and TIM-3 evaluated by flow cytometry were decreased after TUSC2 treatment. TUSC2 anti-tumor response was abolished when NK cells were depleted indicating NK cells are important mediators of the TUSC2 treatment effect. Single cell suspension analysis by flow cytometry showed high numbers of NK cells infiltrating lung tumor metastases after TUSC2 treatment. The number of tumor nodules in the lung was significantly less following treatment with TUSC2 nanovesicles compared with control. IL-15 gene expression which mediates NK cell proliferation, was increased by TUSC2. In conclusion, systemic TUSC2 nanovesicle immunogene therapy combined with checkpoint blockade showed synergistic anti-tumor efficacy and activated the immune system through upregulation of NK cells and CTL and downregulation of regulatory cells. Citation Format: Ismail M. Meraz, Mourad Majidi, RuPing Shao, Meng Feng, Xiaobo Cao, David Rice, Boris Sepesi, Lin Ji, Jack Roth. Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 621. doi:10.1158/1538-7445.AM2017-621

OP9 Feeder Cells Are Superior to M2-10B4 Cells for the Generation of Mature and Functional Natural Killer Cells from Umbilical Cord Hematopoietic Progenitors.

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), mainly because UCB is one of the most accessible HSC sources. In our study, we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4–5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin, and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point toward the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.

Development and Scale-up of a Novel GMP Method for Enrichment and Expansion of Terminally Differentiated Adaptive Natural Killer Cells (FATE-NK100) with Enhanced Anti-Tumor Function

Natural killer (NK) cells are innate lymphoid cells that mediate immune responses against pathogens and cancer. Human NK cells are distinguished by the surface phenotype CD3-CD56+ and differential expression of the CD56 surface antigen defines subsets. CD56bright NK cells are presumed to be precursors of CD56dim NK cells, and terminal maturation of CD56dim NK cells is associated with acquisition of CD57. Rather than being an immunosenescence marker CD57 acquisition represents a shift toward greater effector function, including increased CD16 signaling (Fc receptor responsible for triggering antibody-dependent cellular cytotoxicity), enhanced cytotoxicity and decreased responsiveness to interleukin (IL)-12 and IL-18 stimulation. Cytomegalovirus (CMV) infection is uniquely associated with expansion of CD57+ NK cells expressing the activating receptor NKG2C.We have reported that in vivo expanded of CD57+NKG2C+ NK cells (referred to as adaptive NK cells) persist for over one year and are directly associated with reduced leukemia relapse after reduced intensity hematopoietic cell transplantation. Ex vivo expansion to enrich the subset of cells with the adaptive NK cell phenotype represents a new strategy to obtain high numbers of NK cells with enhanced effector function for use in adoptive transfer to treat cancer patients. The main challenge in enriching for CD57+ NK cells using current ex vivo expansion protocols is that IL-15, the cytokine that drives NK cell proliferation and is critical for NK cell survival preferentially expands less mature NK subsets that fail to terminally differentiate in culture. Our group has developed a novel NK cell expansion method that overcomes this barrier. Peripheral blood mononuclear cells from CMV seropositive donors are depleted of CD3+ T cells and CD19+ B cells and cultured for 7-9 days with IL-15 and a small molecule inhibitor of glycogen synthase kinase 3-beta (GSK3β), a multifunctional kinase downstream of the PI(3)K pathway. Compared to vehicle control, addition of the GSK3β inhibitor led to a substantial increase (2.2-fold ± 0.19, n=23, p<0.0001) in the CD57+ NK cell population. NK cells were highly enriched (90.9% ± 2.2) relative to the pre-cultured population post CD3/CD19 depletion (23.3% ± 2.5) (p<0.0001) Sorting experiments where purified NK cell subsets were cultured with monocytes obtained from the same donor and either vehicle or GSK3β inhibitor clearly demonstrated that the GSK3β inhibitor enhanced NK cell maturation. Importantly, NK cells expanded in the presence of the GSK3β inhibitor exhibited enhanced interferon (IFN)-γ production relative to the vehicle control in response to leukemia cells in vitro (2.15-fold ± 0.60, n=7, p=0.0002)and were superior in their ability to control tumor growth out to at least one month in a xenogeneic adoptive transfer model. To determine whether inhibition of GSK3β acted directly or indirectly on NK cells to drive terminal differentiation, purified CD56+ NK cells were cultured with or without monocytes in the presence or absence of the GSK3β inhibitor.The presence of differentiated monocytes was required for effective terminal NK cell differentiation, suggesting a monocyte-dependent, indirect effect of GSK3β (Figure 1A). Monocytes cultured with the GSK3β inhibitor exhibited markedly higher surface expression of an array of markers associated with monocyte maturation including HLA-DR, IL-15Rα, CD80, CD83 and CCR7 (Figure 1B). Thus, our data demonstrate that efficient NK cell differentiation is dependent upon the maturation state of the co-cultured monocytes.We have scaled our process to manufacture a GMP product (referred to as FATE-NK100) for clinical use. Using a representative apheresis product from a CMV seropositive donor containing 19.8 x 108 CD57+ NK cells and 1.7 x 108 CD57+NKG2C+ adaptive NK cells, we achieved 6.4-fold NK cell expansion resulting in a final GMP-grade product containing 142.2 x 108 CD57+ NK cells and 15.8 x 108 CD57+NKG2C+ adaptive NK cells.The cytotoxicity of activity of these ex vivo expanded adaptive NK cells in response to tumor targets is superior that of CD3/CD19-depleted haploidentical NK cells activated overnight with either IL-2 or IL-15 in the NK products in current clinical trials.These data have been presented to the FDA in preparation for a clinical trial of FATE-NK100 in lymphodepleted patients with advanced AML anticipated for Q1 2017. [Display omitted] DisclosuresCichocki:Fate Therapeutics, Inc: Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cooley:Fate Therapeutics: Research Funding. Bjordahl:Fate Therapeutics, Inc: Employment. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Rogers:Fate Therapeutics, Inc: Employment. Green:Fate Therapeutics, Inc: Employment. McKenna:Fate Therapeutics, Inc: Research Funding. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Infusion of Umbilical Cord Blood-Derived Natural Killer Cells in Immune-Deficient Mice for the Treatment of Resistant Acute Myeloid Leukaemia

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against acute myeloid leukaemia (AML). Success of this NK cell immunotherapy is dependent on obtaining high numbers of functional NK cells that have the potential to survive in vivo. The use of umbilical cord blood (UCB) CD34+ cells as a source of allogenic NK cells is an interesting method that can generate a readily available, non-invasive, off the shelf cellular product.We developed a cytokine-based culture method for the generation of NK cell products derived from CD34+hematopoietic progenitor cells (HPC) isolated from fresh UCB units. Immuno-phenotyping of ex vivo expanded NK cells showed typical inhibitory and activating NK receptors except for CD16 and the KIR receptors.UCB-derived NK cells displayed good cytolytic activity against NK-sensitive K562 cells with a percentage of specific lysis varying from 30 to 50%. Cytolysis was directly correlated to CD94 expression since CD94-sorted NK cells were responsible for all the in vitro cytolytic function of differentiated NKs against K562 cells. There was an inconstant susceptibility of patient-derived primary AML cells to UCB-derived NK lysis in vitro with a specific lysis ranging from 0 to 25%.We further characterized UCB-derived NK cells by investigating their toxicity, biodistribution, and anti-leukemic potential in vivo. As adoptive transfer of NK cells is an attractive approach for treating refractory leukemia, immune deficient mice were engrafted with a patient derived AML strain resistant to NK-mediated lysis and doxorubicin.After successful engraftment and randomization, leukemic mice were injected with either UCB- derived NK cells or NK cells from healthy donors (NKhds) or doxorubicin, with one control group that didn't receive any treatment. Mice were sacrificed after 2 weeks of treatment and leukemia load along with NK distribution were evaluated by flow cytometry in the blood, bone marrow (BM) and spleen.There was no evidence of toxicity of UCB-derived or healthy donors NK cells in mice. Both types of cells were preferentially found in the blood and in the spleen, even though NKhds reached much higher levels than UCB-derived NKs.As for treatment efficacy, none of our treatment showed anti-leukemic potential based on the absence of decrease of leukemic cells in BM, blood, and spleen. In vivo microenvironment didn't overcome resistance of the patient derived AML cell to NK lysis or to doxorubicin.Remarkably, all of the UCB derived NK cells founded in vivo expressed the CD94 whereas not more than 20% of the injected cells were positive for this marker. Whether it was by in vivo selection or by in vivo differentiation must be investigated.Interestingly, a small cell population with CD56 and CD34 double staining was distinguished in UCB-derived NK and NK healthy donor treated leukemic mice suggesting in vivo interaction between leukemic and NK cells. Further characterization of this population may help to understand the molecular mechanism of leukemic recognition by NK cells and resistance of leukemic cells to cytolysis.In conclusion, UCB-derived NK generation is feasible. Investigation of the role of CD94 in these cells is needed, as cell sorting by CD94 selection in addition to the CD56 could be an interesting approach in the future to select highly functional expanded NK cells before therapeutic use. Furthermore, infusion of UCB-derived NK cells into immune-deficient mice is achievable and non-toxic. However, in vivo environment didn't overcome primary in vitro resistance of AML cells despite an established interaction. Additional elucidation of AML resistance mechanisms to NK lysis is mandatory before therapeutic application. DisclosuresNo relevant conflicts of interest to declare.

Natural Killer Cell Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide: Elimination of Donor-Derived Mature Alloreactive NK Cells, but Favorable Conditions for Adoptive Immunotherapy

INTRODUCTIONThe use of high-dose cyclophosphamide as post-transplant Graft versus Host Disease (GvHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (haplo-HSCT), allowing the safe infusion of T cell replete grafts. The efficacy of post-transplant cyclophosphamide (PT-Cy) has its basis in its capacity to selectively eliminate proliferating cells, including alloreactive T cells. It is however to date unknown whether PT-Cy affects the reconstitution of Natural Killer (NK) cells, whose alloreactivity is known to play a major role in T cell-depleted haplo-HSCT.PATIENTS AND METHODSWe analyzed the grafts and serial peripheral blood (PB) and bone marrow (BM) samples from 14 patients who received T cell replete haplo-HSCT followed by PT-Cy at the San Raffaele Scientific Institute, Milan (n=10, OSR) or the Johns Hopkins University, Baltimore (n=4, JHU). OSR patient received a myeloablative conditioning, PB stem cell grafts, and sirolimus and mycophenolate as pharmacological GvHD prophylaxis. JHU patients received the "classical" Baltimore nonmyeloablative conditioning, unmanipulated BM grafts, and tacrolimus and mycophenolate as pharmacological GvHD prophylaxis.To characterize NK cells reconstitution, we monitored absolute counts and employed a 27-marker flow cytometry panel with high dimensional single-cell analysis using the bh-SNE algorithm.We used intracellular staining to determine the frequency of Ki67+ proliferating cells and expression of Aldehyde Dehydrogenase (ALDH), known to confer resistance to PT-Cy. Interleukin-15 (IL-15) serum concentration was quantified using the Bio-Plex Pro Human Cytokine 4-plex assay.To directly assess the effect of PT-Cy on proliferating NK cells we exposed graft NK cells to IL-15 and mafosfamide, a cyclophosphamide analogue active in vitro. Functional assays against leukemic cell lines and primary AML blasts were performed measuring CD107A degranulation on NK cells and Annexin V on targets.RESULTSAll patients received high numbers of mature donor NK cells as part of the graft (OSR median 17x106/kg, JHU median 7.25x106/kg ), and donor-derived NK cells were detectable as early as day 3 after HSCT and throughout the entire follow-up.At day 3 after HSCT, all subsets of NK cells, including single KIR+ alloreactive cells, were actively proliferating (mean 61.23% of Ki-67+ cells for OSR patients, and 58% for JHU patients), possibly driven by the high levels of IL-15 detected in patient serum after conditioning (Fig.1A).After PT-Cy, a marked reduction in the frequency and counts of proliferating NK cells was evident irrespectively of the transplantation platform, suggesting selective killing of dividing cells by PT-Cy.In line with this hypothesis, NK cells from the graft and from patient PB at day 3 after HSCT showed no detectable ALDH expression, and NK cells prompted to proliferate in vitro were killed in a dose-dependent manner by mafosfamide (Fig.1B).The phenotype of NK cells also changed upon PT-Cy: whereas before the infusion they resembled their mature counterparts from the graft, after PT-Cy an immature phenotype, CD62L+NKG2A+KIR-, became prevalent, suggesting derivation from donor HSCs rather than from infused NK cells (Fig.1C). Accordingly, bhSNE maps demonstrated differential clustering of NK cells from the graft and analyzed 30 days after HSCT (Fig.1D).In line with these features, we detected very low numbers of putatively alloreactive single KIR+ NK cells both in the PB and in the BM of patients at day 30 after HSCT, and these NK cells displayed impaired cytotoxic potential against leukemic targets.Finally, consistent with these observations, when we analyzed the impact of predicted NK alloreactivity in an extended series of 99 patients who received myeloablative haplo-HSCT with PT-Cy, we detected no significant difference in progression-free survival (Fig.1E).CONCLUSIONOur data suggest that the majority of mature NK cells infused with unmanipulated grafts are eliminated upon PT-Cy administration and that in this transplantation platform NK cell alloreactivity might be blunted by the elimination of donor single KIR+ NK cells and by the competition between reconstituting NK and T cells. Still, the high levels of IL-15 detected in patients' sera at early time-points might provide a biological rationale for the infusion of mature donor NK cells early after PT-Cy administration. [Display omitted] DisclosuresBonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.

The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells.

Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy

Background aimsEx vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell–based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. MethodsEnriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus–transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. ResultsAutomated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (±0.9) × 109 activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-γ and tumor necrosis factor-α and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell–activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. ConclusionsWe established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.

The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1 Stimulates Expression of NK Cell Related Transcription Factors, Thereby It Facilitates Generation of Highly Functional NK Cells in Vitro

IntroductionAllogeneic stem cell transplantation (SCT) can be a curative treatment for hematological malignancies. The therapeutic effectiveness is attributed to the graft-versus-tumor (GVT) effect, mediated by alloreactive T cells and natural killer (NK) cells. Although T cells can induce a potent GVT effect, they can also induce graft-versus-host disease (GVHD), causing high morbidity and mortality. Interestingly, after non-myeloablative allogeneic SCT, early NK cell repopulation has been associated with decreased relapse rates, without increasing GVHD incidence, illustrating a possible role for donor NK cell adoptive transfer after allogeneic SCT. However, isolation of sufficient numbers of activated NK cells from donor origin is challenging. Recently, it has been described that antagonizing the aryl hydrocarbon receptor (AhR) using the antagonist StemReginin1 (SR1) promotes expansion of human hematopoietic stem cells. Furthermore, AhR turned out to regulate differentiation of multiple immune cells like dendritic cells, regulatory T cells, γδ T cells, and also NK cells. Therefore, we investigated if SR1 could enhance NK cell generation in a cytokine-based culture system.MethodsCD34+ hematopoietic progenitor cells (HPCs) were isolated using immunomagnetic beads from G-CSF mobilized aphaeresis material. These HPCs were expanded during 14 days in medium containing SCF, Flt3L, TPO, IL-7 and IL-15 and subsequently differentiated into NK cells using IL-15 and IL-2. HPC-NK cell expansions were performed with or without SR1. RNA was collected from the cultures weekly and expression of NK cell related genes was analyzed using qPCR. After 35 days, HPC-NK cells were assessed for their cytolytic functions against acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines and primary AML blasts. In addition, expression levels of typical NK-activating receptors and differentiation markers were analyzed by flow cytometry.ResultsInterestingly, SR1 induced expression of TOX, ID2, EOMES, GATA3 and SH2D1B, which are important factors involved in early and late NK cell differentiation. In addition, SR1 improved the expansion, differentiation, and functionality of the NK cells generated. In the presence of SR1, we were able to expand PB-derived HPCs up to 1000-fold in 5 weeks. The SR1-generated HPC-NK cell products contained 80 – 92% NK cells, which expressed high levels of activating NKG2D and natural cytotoxicity receptors. Furthermore, functional analysis showed marked degranulation and cytokine release upon co-culture with AML and MM cell lines and efficient lysis of primary AML blasts at low NK-target ratios.ConclusionAddition of the AHR antagonist SR1 in our culture system induces expression of transcription factors involved in NK cell differentiation and thereby facilitates the generation of high numbers of functional NK cells from G-CSF mobilized CD34+ progenitor cells. These NK cells hold great promise for future donor NK cell-mediated therapy after allogeneic SCT. DisclosuresNo relevant conflicts of interest to declare.

P57 The IL28B gene SNP rs12979860 CC haplotype, predictor of response to treatment in chronic HCV infection, is associated to high numbers of CD56bright NK cells, low numbers of CD3-CD56-CD16+ NK cells and low HCV-specific IL-10 production

IntroductionSingle nucleotide polymorphisms (SNPs) rs12979860 and rs8099917 near the IL28B gene predict response to treatment in chronic hepatitis C (CH-C). Control of HCV infection is linked to strong immune responses....

Lymphocyte subset recovery and outcome after T-cell replete allogeneic hematopoietic SCT

Rapid recovery of lymphocytes after T-cell depleted hematopoietic SCT (HSCT) protects from relapse of myeloid malignancies. Whether lymphocyte reconstitution has a similar role after non-manipulated transplantation is controversial. We assessed numbers of CD4 and CD8 T-cells, natural killer (NK) cells and B-cells, before and 1, 3, 6, 12 and 24 months after T-cell replete transplantation in 345 patients. Lymphocyte subset counts up to 6 months post transplant had no effect on relapse. Elevated number of NK cells 12 months post transplant protected from relapse. As a novel finding, early recovery of NK cells was associated with significant protection from TRM already at the 3 and 6 months time points (P=0.03, P=0.02). In Cox multivariable models, patients with NK cells above 150/μL were significantly protected from TRM (hazard ratio (HR) 0.45, 95% confidence interval (95% CI) 0.21-0.95, P=0.03), an effect comparable in magnitude with that of carrying >200 CD4 T-cells/μL (HR 0.37, 95% CI 0.19-0.74, P=0.005). CD8 T-cell and B-cell recovery did not affect the rates of relapse or TRM. Early reconstitution of NK cells and CD4 T-cells in patients undergoing T-cell replete HSCT independently protected from TRM. Only a weak protection from disease relapse was noted for patients with high numbers of NK cells, and this occurred only late after transplantation.