Cord Blood NK Research Articles

Identification of a novel cord blood NK cell subpopulation expressing functional programmed death receptor-1.

Natural Killer cells (NKs) represent the innate counterpart of TCRαβ lymphocytes and are characterized by a high anti-tumor and an anti-viral cytotoxic activity. Recently, it has been demonstrated that NKs can express PD-1 as an additional inhibitory receptor. Specifically, PD-1 was identified on a subpopulation of terminally differentiated NKs from healthy adults with previous HCMV infection. So far it is unknown whether PD-1 appears during NK-cell development and whether this process is directly or indirectly related to HCMV infection. In this study, we analyzed the expression and function of PD-1 on Cord Blood derived NKs (CB-NKs) on a large cohort of newborns through multiparametric cytofluorimetric analysis. We identified PD-1 on CB-NKs in more than of half the newborns analyzed. PD-1 was present on CD56dim NKs, and particularly abundant on CD56neg NKs, but only rarely present on CD56bright NKs. Importantly, unlike in adult healthy donors, in CB-NKs PD-1 is co-expressed not only with KIR, but also with NKG2A. PD-1 expression was independent of HCMV mother seropositivity and occurs in the absence of HCMV infection/reactivation during pregnancy. Notably, PD-1 expressed on CB-NKs was functional and mediated negative signals when triggered. To our understanding, this study is the first to report PD-1 expression on CB derived NKs and its features in perinatal conditions. These data may prove important in selecting the most suitable CB derived NK cell population for the development of different immunotherapeutic treatments.

A phase 1 trial of umbilical cord blood–derived tumor-reactive PD-L1+ natural killer cells engineered to express soluble IL-15 (TRACK-NK) in patients with non–small-cell lung cancer (NSCLC) refractory to PD-1/PD-L1 inhibitors.

TPS2665 Background: The mechanism by which patients with PD-L1 – tumors can respond favorably to anti-PD-L1 monoclonal antibody therapy has been shown to involve the PD-L1+ NK cell in the tumor microenvironment (TME). PD-L1 is induced on the cell surface when NK cells recognize tumors, leading to enhanced NK-cell function without exhaustion. The PD-L1 + NK cell is capable of trafficking to the lung TME, recognizing and killing tumor cells as well as inducing activation of endogenous T cells via potent cytokine secretion, all without the expression of a chimeric antigen receptor (CAR). TRACK-NK cells are PD-L1+ NK cells derived from cord blood (CB) and engineered to express soluble IL-15 (sIL-15). The TRACK-NK cells express high levels of tumor reactive receptors that recognize "tumor stress" including DNAM-1, NKp30 and NKG2D. TRACK-NK cells induced significant reduction in tumor volume in mice injected with human A549 NSCLC cells compared to mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation. Survival was also prolonged in the H460 NSCLC model compared to vehicle alone. We hypothesized that TRACK-NK cells can be safely administered, and will provide meaningful clinical benefit in patients with NSCLC. Methods: NCT05334329 is a first-in-human, phase 1 dose finding study of allogeneic, off-the shelf frozen and thawed TRACK-NK cells in patients with NSCLC progressing on treatment with PD-1/PD-L1 inhibitors. TRACK-NK cells (CB NK cells engineered with retroviral transduction to express sIL-15 and ex vivo expanded and activated to express PD-L1) are administered in four weekly intravenous infusions per cycle following lymphocyte depletion (Fludarabine/Cyclophosphamide x 3 days) in the outpatient setting. Patients not experiencing disease progression (per RECIST v1.1) following cycle 1 may receive a second cycle. Primary endpoints include: 1. Toxicity/adverse events as per NCI-CTCA version 5.0, and ASTCT Consensus Grading for Cytokine Release Syndrome (CRS) and Neurotoxicity; and 2. Magnitude/duration of TRACK-NK cell persistence in peripheral blood via droplet digital PCR. We are employing a Utility Based-Bayesian Optimal Interval design to direct optimal dose determination. Dose limiting toxicity is defined as any ≥ grade 3 non-hematologic toxicity; any grade 3 CRS that does not resolve to ≤ grade 1 within 7 days; any ≥ grade 4 CRS; any ≥ grade 2 neurotoxicity that does not resolve to grade 1 within 72 hours; any ≥ grade 3 autoimmune, hypersensitivity, and acute graft-versus-host reactions. Correlatives also include cytokines levels, quantity and activation of CD4 and CD8 cells in peripheral blood, and NK and T cell trafficking and TME changes in paired tumor biopsies using quantitative immunofluorescence and gene expression analysis. Clinical trial information: NCT05334329 .

Umbilical cord blood as a promising source of NK cells for immunotherapy

Currently, a large number of studies on genetic modification of cord blood NK cells (UCB-NK) are carried out at both clinical and preclinical levels. Immunotherapy based on UCB-NK cells has great potential for antitumor therapy. However, despite having known several advantages over peripheral blood NK cells (PB- NK), including a high concentration in cord blood and low virulence rate, UCB-NK cells are predominantly characterized in the scientific literature as immature and low-functioning NK cells. In this work, we studied the phenotypic characteristics of UCB-NK cells and the possibility of stimulatory compensation of the decreased functional activity of UCB-NK cells. Our studies revealed UCB-NK cells can be characterized as poorly differentiated and weakly activated cells with high level of inhibitory receptor NKG2A and low level of activating receptor NKG2C and HLA-DR, accordingly with the literature data. Two types of stimuli were chosen to stimulate freshly isolated UCB-NK cells: 1) 100 units of IL-2; 2) combinations of 100 units IL-2 and K-562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). It was shown the degranulation (LAMP-1) and proliferative activity was higher than for parallel cultured ex vivo PB-NK cells under the same conditions for UCB-NK cells stimulated for 7 days with IL-2 + K562-mbIL21. Moreover, stimulation in the way of IL-2 + K562-mbIL21 seemed to be a more perspective way to obtain a large number of proliferatively active UCB-NK cells compared to stimulation with IL-2 only. Since genetic modification of NK cells is a promising way to improve the antitumor properties of NK cells, retroviral transduction procedure was performed to study of the stimulated UCB-NK cells. UCB-NK cells stimulated with IL-2 + K562-mbIL21 were transduced on day 8 of cultivation. In this study, we used targeted overexpression of the adaptor molecule DAP12, which is involved in the signaling of activating NK cell receptors. PB-NK cells and UCB-NK cells were transduced under the equal experimental conditions in same volume of viral particles. As a result, the transduction efficiency was found to be more than 4-fold higher for UCB-NK cells compared to PB-NK cells. Thus, UCB-NK cells appear to be a promising tool for further research in cancer immunotherapy.

Abstract CT003: Innate cell engager (ICE®) AFM13 combined with preactivated and expanded cord blood (CB)-derived NK cells for patients with refractory/relapsed CD30+ lymphoma

Abstract Pts with R/R CD30+ lymphoma have few effective therapies available. We previously developed a promising therapy using CB-derived NK that were first IL-12/IL-15/IL-18-preactivated followed by expansion (P+E) with K562 feeder cells ex vivo and subsequently complexed with ICE® AFM13 (a first-in-class CD30/CD16A bispecific antibody construct) prior to infusion. These cytokine-induced memory-like NK showed greater in vivo antitumor activity than either P+E NK or AFM13 alone. This single-center phase I-II trial (NCT04074746) evaluates AFM13-precomplexed CB-NK cells in pts ages 15-75 with R/R CD30+ lymphoma. Pts receive 2 cycles of fludarabine/cyclophosphamide (days −5 to −3) followed by AFM13-CB NK cells (day 0) and 3 weekly intravenous infusions of AFM13 (200 mg, days 7, 14 and 21). Pts were enrolled at 3 dose levels: DL1 (106 NK/Kg), DL2 (107 NK/Kg) and DL3 (108 NK/Kg). 19 pts have been treated to date at DL1 (N=3), DL2 (N=3) and DL3 (N=13); 18 have completed both planned cycles. All pts had active progressive disease at enrollment and no bridging therapy was given. Cords were selected for the 37 cycles without consideration for HLA match, which was 0/6 (N=16), 1/6 (N=17), 2/6 (N=2), 3/6 (N=1) and 4/6 (N=1). There were no cases of CRS, ICANS or GVHD; 6 infusion-related reactions (1 G3, 5 G2) in 110 infusions of AFM13 alone and no reactions to the AFM13-loaded NK cells. DL3 was established as the RP2D. There were 17/19 responses (8 CR, 9 PR, 2 PD). All 13 pts treated at the RP2D responded (6 CR, 7 PR). 2 pts subsequently received an autologous SCT. At median follow-up of 6 (2-16) months PFS/OS across all dose levels are 58%/79%. Expansion of CB NK cells occurred as early as 3 days post infusion and persisted for up to a month. In conclusion, the preliminary results of this first clinical trial of ICE®-precomplexed NK cells for R/R CD30+ lymphoma indicate excellent tolerability and high activity and warrant further investigation of this approach. Baseline patient characteristics (N=19) Age, median (range) 37 (20-68) Gender (male/female) 13/6 Diagnosis (Hodgkin/T-NHL) 17/2 No. prior lines therapy, median (range) 6 (1-14) Prior brentuximab vedotin 19 Prior anti-PD-1 18 Prior SCT (autologous/allogeneic) 13 (8/5) Prior cellular therapy (CAR-T) 2 No. prior relapses/progressive disease, median (range 5 (1-14) Citation Format: Yago Nieto, Pinaki Banerjee, Indreshpal Kaur, Roland Bassett, Lucila Kerbauy, Rafet Basar, Mecit Kaplan, Lori Griffin, Daniel Esqueda, Christina Ganesh, Melissa Barnett, Amin Alousi, Chitra Hosing, Jeremy Ramdial, Neeraj Saini, Samer Srour, Karenza Alexis, Andreas Harstrick, Elizabeth J. Shpall, Katayoun Rezvani. Innate cell engager (ICE®) AFM13 combined with preactivated and expanded cord blood (CB)-derived NK cells for patients with refractory/relapsed CD30+ lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT003.

Decidualization-derived cAMP promotes decidual NK cells to be angiogenic phenotype.

During early pregnancy, a large number of CD56bright natural killer cells (NKs) are accumulated in the decidua; unlike peripheral and cord blood NK cells (pNK and cNK), these decidual NK cells (dNK) display a great capacity to secrete a series of angiogenic/vascular factors, which are essential for placental development. However, the mechanism underlying the formation of dNK cells with an angiogenic phenotype remains unclear. First, we compared the difference between dNK and cNK/pNK cells in terms of the expression of CD56 and VEGF, and the regulation of the tube formation. The effect of cAMP on the differentiation of NK cells was evaluated by its analog and inhibitor stimulation. We further analyzed the differences in the phenotype of dNK cells and the expression of VEGF in dNK cells from normal pregnancies and miscarriages. Different from cNK and pNK, dNK cells displayed high expression of CD56 and VEGF. And dNK cells showed a higher capacity of inducing tube formation of HUVEC by trophoblast. Meanwhile, we observed thatcAMP-analogue increased the percentage of CD56bright NK population in cNK cells with up-regulated VEGF secretion and tube formation of HUVEC by trophoblast, which could be inhibited by pretreatment with VEGFR neutralizing antibody. Similar changes occurred when co-culturing cNK cells with DSCs but not ESCs. Interestingly, the inhibitor of cAMP signaling (Metadoxine, META) could significantly inhibit the upregulation of VEGF in cNK cells by DSCs. Furthermore, DSCs could secret much more cAMP than ESCs. Notably, decreased CD56bright NK population and VEGF secretion by dNK were related to pregnancy loss. These findings suggest that dNK cells display an angiogenic phenotype that can be induced by decidualized cAMP signaling. Our study indicates the significance of decidualization-derived cAMP in regulating angiogenesis of decidual NKs and reveals complex crosstalk between different cell types in a critical period during early pregnancy.

Generation of CAR-NK Cells Using a Notch-Mediated Cell Expansion and Differentiation Platform

An important challenge in the generation of therapeutic chimeric antigen receptor expressing natural killer (CAR-NK) cells is poor gene transfer efficiencies in mature NK cells requiring large quantities of viral vectors, significantly increasing the production cost. We have an established clinical stem cell expansion platform used to generate off-the-shelf hematopoietic stem/progenitor cell products that have been evaluated in 6 clinical trials to date. This proprietary Notch-mediated expansion platform has now been optimized to generate large numbers of functional NK cells by first ex vivo expansion and priming of CD34+ cells isolated from cord blood (CB), followed by directed differentiation to NK cells. The final NK cell product can be cryopreserved with post-thaw maintenance of viability (≥ 80%) and function. With respect to generation of CAR-NK cells, this manufacturing process is scalable and cost-effective and importantly, allows transduction using a viral vector early in the expansion phase of culture without compromising the robust expansion or phenotype of the developing cells. This significantly reduces both the number of cells to be transduced and the amount of viral vector needed to <5% of that required to transduce the final NK product. As proof of concept, we developed CB-derived CAR NK (CB-CAR-NK) cells with specificity against mesothelin (MSLN). Mesothelin is a cell surface adhesion molecule that is highly upregulated in 30-50% of AML cases in pediatric and adult cohorts and lacks expression in normal hematopoiesis. The AML-restricted expression of mesothelin makes it an ideal tumor-associated antigen for immunotherapeutic targeting in AML. CB derived CD34+ cells are transduced with a mesothelin-directed CAR vector during the expansion phase in serum-free expansion media supplemented with cytokines. Transduction efficiencies were assessed periodically in bulk, CD34+ and CD56+ cells throughout the culture duration by flow cytometry analysis of truncated CD19, which is linked to CAR expression by a cleavable T2A. We measured cytolytic activity of CB-CAR-NK and CB-NK cells against NOMO-1 AML cells, which express endogenous MSLN, or NOMO-1 MSLN knockout cells by flow cytometry analysis of cell viability in the target cells. Our manufacturing process generates over 2000 CD56+ NK cells per CD34+ cell initiated in culture. Following transduction and differentiation, we achieve a robust cell product that is >75% CD56+ NK cells and ~22% CD56+CAR+ using a multiplicity of infection of 10 virus particles per cell. The ex vivo generated CAR-NK cells demonstrate an immunophenotype similar to the unmodified NK cells, expressing the NK canonical surface markers NKG2A, NKG2D, NKp46, CD16, NKp44 and NKp30. Despite a modest transduction efficiency, the CB-CAR-NK cells demonstrate superior cytotoxicity against NOMO-1 cells over the unmodified CB NK cells (~4-fold increase). This enhanced killing is highly specific as CB-CAR-NK and CB-NK exhibited similar tumor-killing efficiency in NOMO-1 MSLN KO cells. Having demonstrated the functional potential of our CAR-NK cells in vitro, we are now working toward assessing their function in vivo in a mouse xenograft model. We will also explore other optimal leukemia-specific antigens as targets for our CAR-NK cells and optimize our CAR construct to further enhance transduction efficiency. The vast majority of anti-cancer cellular immunotherapies are patient specific, require apheresis, high volumes of viral vector and complex manufacturing processes that are expensive with a risk of delayed or no treatment if the production fails, all contributing to poor clinical adoption despite transformative outcomes. We have developed our platform to overcome these hurdles with the goal of delivering safe and cost-effective allogeneic off-the-shelf immunotherapies for improved clinical access. Disclosures Chandrasekaran: Deverra Therapeutics: Current Employment, Current equity holder in private company. Prieve:Deverra Therapeutics: Current Employment, Current equity holder in private company. Stoltzman:Deverra Therapeutics: Current Employment, Current equity holder in private company. Cook:Deverra Therapeutics: Current Employment, Current equity holder in private company. Blake:Deverra Therapeutics: Current Employment, Current equity holder in private company. Massey:Deverra Therapeutics: Current Employment, Current equity holder in private company. Delaney:Deverra Therapeutics: Current Employment, Current equity holder in private company.

Purification, Culture, and CD19-CAR Lentiviral Transduction of Adult and Umbilical Cord Blood NK Cells.

Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role that NK cells play is analogous to that of cytotoxic T cells in that they provide rapid responses to virus-infected cells and responses to tumor formation. Unmodified NK cells have long been used in various immunotherapies to treat different tumors, with only marginal success. However, in the last few years, NK cells modified to express chimeric antigen receptors (CAR-NK cells) have emerged as particularly ideal cellular platforms for antigen-specific antitumor agents. Unlike CAR-T cells, they do not elicit allogeneic responses or graft-versus-host disease and therefore can be administered to recipients with differing MHC expression. This article outlines protocols to obtain CD19-CAR-NK cells, focusing on the importance of obtaining and culturing a purified NK cell population and how to attain good transfection efficiency. © 2020 Wiley Periodicals LLC Basic Protocol 1: Purification and culture of adult peripheral blood and umbilical cord blood NK cells Basic Protocol 2: CD19-CAR lentiviral transduction of adult peripheral blood or umbilical cord blood NK cells Support Protocol: Production of lentiviral supernatant.

Abstract IA07: Engineering natural killer cells for the treatment of cancer

Abstract Natural killer (NK) cells play critical roles in host defense against cancer. Our group is exploring a number of avenues to enhance NK cell function against hematologic malignancies and solid tumors. These include novel strategies to expand off-the-shelf cord blood (CB)-derived NK cells based on their coculture with genetically modified feeder cells that express membrane-bound cytokines and costimulatory molecules. To redirect NK cell specificity and enhance their in vivo persistence, we have successfully transduced expanded CB NK cells with a retroviral vector incorporating the genes for CAR-CD19, IL-15, and inducible caspase-9-based suicide gene (iC9). Data from a first-in-human phase I/II clinical study to test the safety and efficacy of escalating doses of off-the-shelf iC9/CAR.19/IL-15 CB-NK cells in patients with relapsed or refractory B-lymphoid malignancies will be presented. Citation Format: Katayoun Rezvani. Engineering natural killer cells for the treatment of cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr IA07.

ISSUE HIGHLIGHTS ‐ March 2020

ISSUE HIGHLIGHTS ‐ March 2020

Adult peripheral blood and umbilical cord blood NK cells are good sources for effective CAR therapy against CD19 positive leukemic cells

Among hematological cancers, Acute Lymphoblastic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL) are the most common leukemia in children and elderly people respectively. Some patients do not respond to chemotherapy treatments and it is necessary to complement it with immunotherapy-based treatments such as chimeric antigen receptor (CAR) therapy, which is one of the newest and more effective treatments against these cancers and B-cell lymphoma. Although complete remission results are promising, CAR T cell therapy presents still some risks for the patients, including cytokine release syndrome (CRS) and neurotoxicity. We proposed a different immune cell source for CAR therapy that might prevent these side effects while efficiently targeting malignant cells. NK cells from different sources are a promising vehicle for CAR therapy, as they do not cause graft versus host disease (GvHD) in allogenic therapies and they are prompt to attack cancer cells without prior sensitization. We studied the efficacy of NK cells from adult peripheral blood (AB) and umbilical cord blood (CB) against different target cells in order to determine the best source for CAR therapy. AB CAR-NK cells are slightly better at killing CD19 presenting target cells and CB NK cells are easier to stimulate and they have more stable number from donor to donor. We conclude that CAR-NK cells from both sources have their advantages to be an alternative and safer candidate for CAR therapy.

Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56Dim NK cells.

Unrelated cord blood (CB) is an excellent alternative as an allogeneic donor source for stem cell transplantation. CB transplantation is associated with lower incidence of severe acute graft versus host disease (GVHD) and chronic GVHD but similar rates of malignant relapse compared with other unrelated donor cell transplants. NK cells are critical innate immune components and the comparison of CB vs. peripheral blood (PB) NK cells is relatively unknown. NK cell receptor expression, cell function, and maturation may play a role in the risk of relapse after CB transplant. We investigated CB vs. PB NK cell subset cytotoxicity, function, receptor expression, and genomic and proteomic signatures. The CB CD56dim compared with PB CD56dim demonstrated significantly increased expression of NKG2A and NKG2D, respectively. CB vs. PB CD56dim NK cells had significantly decreased in vitro cytotoxicity against a variety of non-Hodgkin lymphoma targets. Various proteins were significantly under- and over-expressed in CB vs. PB CD56dim NK cells. Microarray analyses and qRT-PCR in CB vs. PB CD56dim demonstrated significantly increased expression of genes in cell regulation and development of apoptosis, respectively. In summary, CB vs. PB CD56dim NK cells appear to be earlier in development, have decreased functional activity, and increased capacity for programmed cell death, suggesting that CB NK cells require functional and maturational stimulation to achieve similar functional levels as PB CD56dim NK cells.

Establishment of A Serum-Free Culture System Based on Heparin and Anti-CD16 Antibody for Expansion of Human Cord Blood NK Cells

To establish a novel method for ex vivo expansion of natural killer cells from human umbilical blood, so as to provide the basis for NK cell therapy. Mononucleated cells from human umbilical blood were harvested and suspended in a serum-free medium containing 5% autologous plasma, recombinant human IL-15 (50 ng/ml) and hydrocortisone sodium succinate (5×10-8 mol/L) at a concentration of 1.5×106/ml, then the cells were seeded into flasks pre-coated with heparin sodium (100 U/cm2) or/and anti-human CD16 antibody (1 µg/cm2). After culture for 2 weeks, the cells were harvested and counted. Ratios of CD3-/CD56+ of the cells were determined by flow cytometry. MTT test was performed to assess the cytotoxicity against K562 cells with graded ratios of effector/target cells. In contrast to the cells in flasks without pre-coating, the attached colonies appeared predominantly within 1 week of culture from heparin- and antibody-coated groups. The cell numbers from the pre-coated groups were significantly higher than that of uncoated one after culture for 2 weeks. Furthermore, the ratios of CD3-/CD56+ cells were much higher in pre-coated groups, and that of the cells from flasks pre-coated with heparin and antibody were the highest (all the P values <0.01). MTT test showed that the cytotoxic activity of the cells stimulated by precoating were much more potent than that of the cells without the stimulation. Advantageous expansion of NK cells can be achieved by precoating with heparin and anti-CD16 antibody, and also by supplement with IL-15 and hydrocortisone into the media, so the umbilical NK cells with high purity and potent cytotoxicity can be obtained.

Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity

Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity

JAK/STAT Signaling of Natural Killer (NK) Cells Following Cytokine Stimulation

Abstract Natural Killer (NK) cells are an important component of the innate immune system, capable of providing a fast and effective response against virally infected cells. NK cells are mainly characterized by their cytotoxic function and their ability to secrete cytokines. It has been shown that infant NK cells have decreased cytotoxicity and cytokine-secreting function, suggesting hyporesponsiveness of NK cells during the first year of life. Because NK cell activation is dependent on cytokine stimulation, we hypothesized that infant NK cells are hyporesponsive due to deficiencies in signaling following cytokine stimulation. We examined the activation of human infant and adult NK cells in response to IL2, IL12 or IFNα stimulation, cytokines important in NK survival and function. Using Phosflow analysis, we measured the phosphorylation of transcription factors, STATs, specific for the distinct cytokines. Cord blood NK cells showed significantly lower pSTAT activation when compared to adult NK cells when treated with IL2 or IFNα but not IL12. However, despite pSTAT4 activation in response to IL12, no nuclear translocation was observed by ImageStreamX Mark II analysis. NK cells in 6 and 12-month-old infants showed similar frequencies of pSTAT NK cells compared to adults when treated with IL2, IL12 or IFNα, suggesting that cytokine signaling in NK cells is age-dependent. Surprisingly, pSTAT activation was restored in cord blood NK cells when treated with a cocktail of IL2, IL12 and IL15. These results suggest that JAK/STAT signaling in infant NK cells is impaired at different steps in the pathway in response to specific cytokines.

Differential activation of cord blood and peripheral blood natural killer cells by cytokines

Background aimsNatural killer (NK) cells play important roles in the clearance of infection and transformed cells. Cord blood (CB) is currently used as a source of hematopoietic stem cells for transplantation and is a potential source of NK cells for immunotherapy. We previously showed that CB NK cells are immature and less cytotoxic as compared with peripheral blood (PB) NK cells. We aimed to identify which cytokines, among interleukin (IL)-2, IL-12, IL-15 and IL-18 and their combinations, could fully activate CB NK cells as compared with PB NK cells. MethodsWe performed a comprehensive analysis of phenotype and functionality of cytokine-activated NK cells. ResultsOur results show that the lower responsiveness of CB NK cells to IL-2 is associated with lower levels of expression of IL-2 receptors and decreased phosphorylation of STAT5 as compared with PB NK cells. Activation of CB NK cells with IL-15+18 led to the most robust proliferative response and higher interferon-γ and tumor necrosis factor-α secretion, whereas activation with IL-15+2 promoted enhanced cytotoxicity. PB NK cells responded significantly better to IL-2 than to CB NK cells but were also fully activated with other cytokine treatments including IL-15, IL-15+2 or IL-15+18. It was also possible to use cytokines to generate memory-like NK cells, with sustained ability to produce interferon-γ, from both CB and PB. ConclusionsCB NK cells are fully functional on activation with IL-15+2 or IL-15+18 rather than IL-2 alone as observed for PB NK cells. These cytokines should be considered in the future to activate CB NK cells for therapeutic purposes.

Cord blood CD4+CD25+ regulatory T cells fail to inhibit cord blood NK cell functions due to insufficient production and expression of TGF-beta1

Although CD4+CD25+ Treg (Treg) cells are known to modulate NK cell functions, the modulation mechanism of these cells in cord blood has not been fully clarified. The purpose of this study was to clarify the mechanism whereby cord blood Treg cells modulate cord NK cells. By performing various cultures of purified NK cells with or without autologous Treg cells, diminished inhibitory effects of cord Treg cells towards cord NK cell functions, including activation, cytokine production, and cytotoxicity, were observed. We also observed lower secretion of sTGF-beta1 and lower expression of mTGF-beta1 by cord Treg cells than by adult Treg cells. These data revealed the capability of adult Treg cells to suppress rhIL-2-stimulated NK cell function by TGF-beta1, both membrane-bound and soluble types. The reduced inhibitory capabilities of cord Treg cells compared with adult Treg cells is thought to be due to insufficient expression of TGF-beta1.

TLR3 impairment in human newborns

Newborns are highly susceptible to viral infections. We hypothesized that this susceptibility could be due to a dysregulated expression of innate virus-sensing receptors, i.e., TLR3, TLR7, TLR8, and TLR9 and the cytosolic receptors retinoic acid-inducible gene I, melanoma differentiation-associated protein 5, protein kinase R, and IFN-γ-inducible protein 16. Cord blood mononuclear cells (CBMCs) expressed mRNA for all these receptors except for TLR3. In peripheral blood mononuclear cells (PBMCs), TLR3 mRNA was preferentially expressed in cytotoxic cells, particularly CD56(dim) NK cells. Cord NK cells in contrast showed low TLR3 mRNA expression and lacked TLR3 protein expression. Cord NK cells did not produce IFN-γ in response to polyinosinic-polycytidylic acid [poly(I:C)], whereas strong IFN-γ production was observed in poly(I:C)-stimulated adult NK cells. Cord NK cells had poor cytotoxic function that was only marginally enhanced by exposure to the TLR3 ligand poly(I:C). Opposite to NK cells from adults, their cytotoxicity was not improved by herpes simplex virus (HSV) exposure and they were unable to kill HSV-infected cells. There were no differences in the TLR3 mRNA levels among men, women, and pregnant women, implying that TLR3 is not under sex hormone control. However, decidual NK cells expressed low levels of TLR3 mRNA, which was attributed to their CD56(bright) phenotype. Our data show that cord blood NK cells have deficient TLR3 expression associated with an inability to respond to poly(I:C) and HSV activation and to kill HSV-infected cells. This might explain why newborns are particularly sensitive to neonatal HSV infections.

Monocytes Play an IL-12-Dependent Crucial Role in Driving Cord Blood NK Cells to Produce IFN-g in Response to Trypanosoma cruzi

We previously reported that foetuses congenitally infected with Trypanosoma cruzi, the agent of Chagas disease, mount an adult-like parasite-specific CD8+ T-cell response, producing IFN-g, and present an altered NK cell phenotype, possibly reflecting a post-activation state supported by the ability of the parasite to trigger IFN-g synthesis by NK cells in vitro. We here extended our knowledge on NK cell activation by the parasite. We compared the ability of T. cruzi to activate cord blood and adult NK cells from healthy individuals. Twenty-four hours co-culture of cord blood mononuclear cells with T. cruzi trypomastigotes and IL-15 induced high accumulation of IFN-g transcripts and IFN-g release. TNF-a, but not IL-10, was also produced. This was associated with up-regulation of CD69 and CD54, and down-regulation of CD62L on NK cells. The CD56bright NK cell subset was the major IFN-g responding subset (up to 70% IFN-g-positive cells), while CD56dim NK cells produced IFN-g to a lesser extent. The response points to a synergy between parasites and IL-15. The neonatal response, observed in all newborns, remained however slightly inferior to that of adults. Activation of IL-15-sensitized cord blood NK cells by the parasite required contacts with live/intact parasites. In addition, it depended on the engagement of TLR-2 and 4 and involved IL-12 and cross-talk with monocytes but not with myeloid dendritic cells, as shown by the use of neutralizing antibodies and cell depletion. This work highlights the ability of T. cruzi to trigger a robust IFN-g response by IL-15-sensitized human neonatal NK cells and the important role of monocytes in it, which might perhaps partially compensate for the neonatal defects of DCs. It suggests that monocyte- and IL-12- dependent IFN-g release by NK cells is a potentially important innate immune response pathway allowing T. cruzi to favour a type 1 immune response in neonates.

Isolation, expansion and function of cord blood natural killer cells. (P2226)

Abstract Rate of immune reconstitution (IR) directly correlates with the number of hematopoietic stem cells (HSC) infused and is particularly delayed in patients undergoing cord blood (CB) transplantation (CBT). Methods to increase the number of CB progenitors have the potential to accelerate IR after CBT. NK cells are the first lymphocyte subset to recover after HSC transplant (HCT) and are crucial in preventing early relapse. Unfortunately, NK cells in CB are low in number, incomplete in maturation and require activation for effective function. Here, we report a clinically relevant ex vivo expansion method that optimizes maturation as well as activation of CB NK cells. CB NK cells cultured 14-21 days with CB mononuclear cells, IL-2 and IL-15 result in a multi-log increase in cell number. Potential killer cell Ig-like receptor (KIR) ligand and adhesion molecule(s) responsible for NK cell cytotoxicity were examined prior to and after culture. Phenotypic analysis showed that the percent of NK cells expressing activating receptors and adhesion molecules increased (CD336 +39.11%) while inhibitory receptor expression decreased (CD159a -25.09%). Furthermore, cytotoxic function of CB NK cells against NK cell sensitive (K562) and resistant (MV4-11) cancer cells lines were enhanced after culture. The data suggest that this ex vivo expansion method of matured and activated NK cells is a potential clinically relevant method to prevent early relapse after CBT.

Both the nature of KIR3DL1 alleles and the KIR3DL1/S1 allele combination affect the KIR3DL1 NK‐cell repertoire in the French population

NK-cell functions are regulated by many activating and inhibitory receptors including KIR3DL1. Extensive allelic polymorphism and variability in expression can directly alter NK-cell phenotype and functions. Here we investigated the KIR3DL1(+) NK-cell repertoire, taking into account the allelic KIR3DL1/S1 polymorphism, KIR3DL1 phenotype, and function. All 109 studied individuals possessed at least one KIR3DL1 allele, with weak KIR3DL1*054, or null alleles being frequently present. In KIR3DL1(high/null) individuals, we observed a bimodal distribution of KIR3DL1(+) NK cells identified by a different KIR3DL1 expression level and cell frequency regardless of a similar amount of both KIR3DL1 transcripts, HLA background, or KIR2D expression. However, this bimodal distribution can be explained by a functional selection following a hierarchy of KIR3DL1 receptors. The higher expression of KIR3DL1 observed on cord blood NK cells suggests the expression of the functional KIR3DL1*004 receptors. Thus, the low amplification of KIR3DL1(high) , KIR3DL1*004 NK-cell subsets during development may be due to extensive signaling via these two receptors. Albeit in a nonexclusive manner, individual immunological experience may contribute to shaping the KIR3DL1 NK-cell repertoire. Together, this study provides new insight into the mechanisms regulating the KIR3DL1 NK-cell repertoire.