Adult NK Cells Research Articles

Abstract 3195: Preclinical development of SENTI-202, an off-the-shelf logic gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML

Abstract CD33 and FLT3 are validated targets for myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). These malignancies have poor prognosis and high clinical unmet need, and current targeted therapies present ample limitations. One challenge is that untargeted leukemic stem cells (LSCs) can contribute to eventual relapse; however, targeting the LSC markers FLT3 and CD33 can contribute to off-tumor toxicities against normal hematopoietic stem cells and progenitor cells (HSCs/HPCs), leading to bone marrow toxicities such as thrombocytopenia, neutropenia, and infectious complications. SENTI-202 is designed to address these challenges and provide a broader therapeutic window. SENTI-202 is a preclinical CAR-NK cell product candidate engineered with an OR and NOT logic gated gene circuit onto allogeneic healthy adult peripheral blood NK cells along with multi-arming via expression of calibrated release IL-15 (crIL-15) to enhance therapeutic activity and tolerability. The OR gate is implemented using a bivalent CAR (aCAR) that binds both CD33 and FLT3 to target both AML blasts and LSCs. Additionally, the NOT gate is implemented using an inhibitory CAR (iCAR) that recognizes the healthy cell protective antigen endomucin (EMCN) and targets endomucin which is designed to prevent CAR-mediated cytotoxicity against healthy cells, such as HSCs and early HPCs, potentially reducing on-target/off-tumor toxicities. Finally, crIL-15 provides NK cell activation and persistence via simultaneous autocrine, juxtracrine, and paracrine activities. Preclinical studies demonstrated the [antitumor] activity and tolerability of SENTI-202. All SENTI-202 components are stably expressed via a single g-retroviral vector. SENTI-202 preclinical pharmacokinetics and secondary pharmacodynamics demonstrated CAR target engagement, function, as well as mechanism of action for all components in different AML models. SENTI-202 CAR-NK cells demonstrated cytotoxic activity when co-cultured with AML target cells and primary patient-derived samples and had increased antitumor capacity compared to non-engineered NK cells. SENTI-202 induced the production of cytotoxic cytokines and increased expression of activation and cytotoxicity markers consistent with CAR-mediated activation of the NK cells. The presence of EMCN iCAR reduced cytotoxicity in an EMCN-dependent manner in target cells and primary HSCs. crIL-15 expression yielded functional pSTAT5 signaling and increased persistence of SENTI-202 in vitro and in vivo. These results demonstrate the antitumor activity and tolerability of SENTI-202 and warrant further clinical development as a novel therapeutic option for patients with CD33+ and/or FLT3+ tumors. Citation Format: Alba Gonzalez, Enping Hong, Gozde Yucel, Elizabeth Leitner, Pearley Chinta, Han Deng, Ian Li, Alice Lam, Abla Bakir, Brandon Lee, Papia Chakraborty, Carmina Blanco, Chen-Ting Lee, Niran Almudhfar, Mengxi Tian, Wenqi Song, Andrew Banicki, Otto Contreras, Martin Gieldin, Brian Garrison, Timothy K. Lu, Kanya Rajangam. Preclinical development of SENTI-202, an off-the-shelf logic gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3195.

Abstract 2905: SENTI-301A, an off-the-shelf multi-armed preclinical CAR-NK cell therapy, for the treatment of GPC3 expressing tumors

Abstract Purpose: SENTI-301A is a novel off-the-shelf CAR-NK product candidate that uses gene circuits to enhance NK cell persistence and targeted cytotoxicity to address unmet needs in GPC3 expressing solid tumors such as hepatocellular carcinomas (HCC). Experimental procedures: SENTI-301A is a Multi-Armed, targeted chimeric antigen receptor (CAR) NK cell preclinical product candidate with a GPC3 CAR and calibrated-release interleukin-15 (crIL-15) engineered onto allogeneic healthy adult peripheral blood NK cells. The activating CAR is designed to target GPC3, an antigen overexpressed in several cancers, such as HCC. crIL-15 is a unique technology attaching wild type IL-15 to the membrane via a linker that can be cleaved by a ubiquitous protease. This results in membrane-bound and secreted IL-15 to provide both autocrine support to the CAR NK cells, and paracrine stimulation to other surrounding immune cells in the tumor microenvironment for enhanced antitumor response. Results: SENTI-301A exhibited increased crIL-15-driven survival and expansion (>30 days) without exogenous cytokine support compared to unengineered NK cells (~6 days). SENTI-301A also showed significantly higher in vitro cytotoxic activity (~20%) against GPC3 expressing cell lines compared to isogenic cells lacking the antigen, indicating CAR-mediated activation and antigen specificity. SENTI-301A also showed significant tumor killing function, IFNg, and Granzyme-B production, when co-cultured with HCC and other GPC3 expressing tumor cell lines at different effector:target ratios. SENTI-301A maintained the antitumor function in the presence of full-length soluble GPC3, reducing the risk of competition from shedding GPC3 with surface GPC3 for CAR-NK cell binding. In HCC mouse xenograft models, SENTI-301A showed NK cells tumor infiltration, and displayed enhanced persistence, antitumor function and increased median survival (p< 0.01), in comparison to unengineered NK cells. As next steps, the potential positive impact of immune checkpoint inhibitors (CPIs) in combination with SENTI-301A to enhance anticancer function is being assessed. Conclusions: SENTI-301A is a novel off-the-shelf preclinical CAR-NK cell therapy candidate that targets GPC3-expressing tumors in an antigen-specific manner and exhibits increased persistence via crIL-15. We are exploring the potential synergy between SENTI-301A and CPIs to enhance the existing antitumor functions of SENTI-301A. SENTI-301A is planned for clinical development with an investigational new drug application (IND) expected in 2023. Citation Format: Marcela Guzman Ayala, Deepika Kaveri, Enping Hong, Elizabeth Leitner, Priscilla Wong, Ronni Ponek, Lawrence Naitmazi, Pearley Chinta, Wesley Gorman, Mengxi Tian, Niran Almudhfar, Kelly Lee, Nicholas Frankel, Alba Gonzalez Junca, Russell Gordley, Philip Lee, Timothy Lu, Kanya Rajangam. SENTI-301A, an off-the-shelf multi-armed preclinical CAR-NK cell therapy, for the treatment of GPC3 expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2905.

Zebrafish Model of Severe Combined Immunodeficiency (SCID) Due to JAK3 Mutation.

JAK3 is principally activated by members of the interleukin-2 receptor family and plays an essential role in lymphoid development, with inactivating JAK3 mutations causing autosomal-recessive severe combined immunodeficiency (SCID). This study aimed to generate an equivalent zebrafish model of SCID and to characterize the model across the life-course. Genome editing of zebrafish jak3 created mutants similar to those observed in human SCID. Homozygous jak3 mutants showed reduced embryonic T lymphopoiesis that continued through the larval stage and into adulthood, with B cell maturation and adult NK cells also reduced and neutrophils impacted. Mutant fish were susceptible to lymphoid leukemia. This model has many of the hallmarks of human SCID resulting from inactivating JAK3 mutations and will be useful for a variety of pre-clinical applications.

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.

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.

New insights on the natural killer cell repertoire from a thorough analysis of cord blood cells.

Although CB NK cells are characterized as immature lymphocytes, their impressive expansion and efficient graft-versus-leukemia response have been highlighted early after UCBT. To better evaluate their potential as source of effective NK cells, we revisited the study of NK cell repertoire from a large cohort of CB samples. Our study showed that the CB NK cell repertoire appears to be constructed early, depending on KIR gene content, but not on the autologous HLA environment. NKG2A was expressed on a large proportion of CB NK cells that inversely correlated with KIR(+) NK cell frequency. Self-HLA class I molecule-educated CB KIR(+) NK cells present a lower spontaneous lysis than do their adult counterparts, which is probably related to the low expression of activating NK receptors. We describe for the first time a proliferative and cytotoxic NKG2C(+) NK cell subset representing more than 10% of CB NK cells. NKG2A strongly inhibited CB NK cell degranulation, and its coexpression on NKG2C(+) NK cells may contribute to limiting their activation. Overall, the CB NK cell repertoire is constructed early and harbors numerous functional abilities shared by adult NK cells. In addition, their naïve viral status and fast expansion confer numerous advantages in immunotherapy on CB NK cells.

Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4β7(-) and α4β7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life.

HMGB1-Promoted and TLR2/4-Dependent NK Cell Maturation and Activation Take Part in Rotavirus-Induced Murine Biliary Atresia

Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.

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.

Cutting Edge: Ly49C/I− Neonatal NK Cells Predispose Newborns to Autoimmune Ovarian Disease Induced by Maternal Autoantibody

NK cells are critical in immune responses against pathogens. However, their role in autoimmunity is still controversial. In this study, we demonstrate that neonatal NK cells render newborns more susceptible to neonatal autoimmunity induced by maternal autoantibodies (neonatal autoimmune ovarian disease); thus, neonatal but not adult NK cells are pathogenic after transfer into NK cell-deficient pups. The inhibitory receptors Ly49C/I are expressed in ∼5% of neonatal and ∼50% of adult NK cells. In this study, we show that the presence of Ly49C/I⁺ adult NK cells inhibits neonatal autoimmune ovarian disease induction. Thus, the ontogenetic regulation of Ly49C/I expression determines the propensity to autoantibody-induced autoimmunity. In summary, this study provides definitive evidence of a pathogenic role of NK cells in neonatal autoimmunity and also elucidates a novel mechanism by which neonatal NK cells render newborns more susceptible to autoantibody-induced autoimmunity.

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.

The unique properties of neonatal NK cells predispose newborns to autoimmune ovarian disease (nAOD) induced by maternal autoantibody (171.37)

Abstract The transfer of maternal autoantibody (autoAb) to the progeny can cause neonatal autoimmune diseases. An example is the fatal congenital heart block elicited by maternal SSA/SSB antibodies in Sjogren’s patients. Disease mechanism is unknown but can now be investigated in a nAOD model developed in our laboratory. Ovarian inflammation and atrophy occurs in C57BL/6 Rag ko pups after the exposure to maternal autoAb against ovarian zona pellucida 3 within 1-5 days of life. Resistance to nAOD induction in both NK cell-depleted Rag ko pups, and Rag/common gamma chain (Rag/γc) double ko pups demonstrates the critical role of NK cells. Notably, neonatal but not adult NK cells restore nAOD in Rag/γc ko pups. Compared to adult NK cells, neonatal NK cells have lower expression of Ly49 receptors and higher frequency of the CD27+ subset. We now show that the expression of CD27, CXCR3 and IFNγ by neonatal NK cells are crucial for nAOD. Furthermore, Ly49C/I-depleted adult NK cells acquire the ability to induce nAOD. This suggests that the low expression of Ly49 reduces the activation threshold and enhances the pathogenic capacity of neonatal NK cells. Lastly, FcgRIII expression on both neonatal NK cells and ovarian resident macrophages and dendritic cells (APC) is obligatory for nAOD induction. However, ovarian APC requirement is not ontogenetically regulated. We conclude that the unique properties of neonatal NK cell predispose newborns to autoimmune disease by maternal autoAb.

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

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

A Highly Efficient Method to Expand CD3-CD56+ NK Cells from Cord Blood Segments

Background: The infusion of in-vitro expanded cord blood derived CD3−CD56+ NK cells could potentially be used to enhance graft-vs-tumor effects following cord blood transplantation. In order to infuse NK cells derived from the same cord unit used during allogeneic transplantation, we sought to develop a highly efficient culture method to expand large numbers of NK cells in vitro from < 1 ml of cord blood.Methods: Immunomagnetic beads were used to deplete CD3+ T-cells from thawed cord blood. CD3 depleted mononuclear cells (<0.1% CD56+) were co-cultured with either irradiated EBV-LCL feeder cells or 41BB transduced K562 cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 47 days.Results: Day 12 EBV-LCL expanded NK cell cultures contained up to 90% CD3−CD56+ NK cells with less than 0.5% CD3+CD56+ cells. Expanded cord blood derived CD56+NK cells had similar expression of CD16, NKG2D, LFA-1, perforin, and granzymes A and B and had similar cytotoxic function as NK cells expanded from adult PBMC. Surface expression of NK cell TRAIL increased dramatically with in vitro expansion. By day 12, TRAIL surface expression by FACS was at similar levels observed on expanded adult NK cells, although expression gradually declined with prolonged cell culture; on days 12, 20, and 34, 89%, 57% and 11% of cord derived NK cells expressed TRAIL respectively. NK cell cultures expanded with 41BB-transduced APCs had a similar phenotype and cytotoxic function against K562 cells and renal cell carcinoma (RCC) cells as EBV-LCL expanded cells. Furthermore, NK cell cytotoxicity against RCC tumor targets treated with 10 nM bortezomib for 18 hrs (bortezomib upregulates RCC surface expression of DR5) was higher than untreated RCC cells confirming the functional cytolytic activity of TRAIL expressed on cord blood derived NK cells. We next evaluated the feasibility of expanding CD56+ NK cells from thawed segments attached to the umbilical cord blood units. TNC numbers of each segment ranged from 3–9 × 106 cells. Unmanipulated thawed segments were co-cultured with irradiated EBV-LCL feeder cells as described above. On Day 12, 43% of viable cells were CD3+, 21.5% were CD3+CD56+ and 36% were CD3− CD56+. CD3−CD56+ NK cells from thawed segments increased 200 – 300 fold with in vitro expansion when maintained in culture for 2–3 weeks (Table). To enrich for pure NK cell populations, subsequent expansions were performed on CD3 depleted cells pooled from three thawed segments stimulated with EBV-LCL; cultures on day 12 contained a pure population of CD3−CD56+ NK cells with virtually an identical phenotypic and cytotoxicity profile as NK cells expanded from larger aliquots taken directly from the thawed cord blood unit. By day 33, CD3−CD56+ NK cells expanded by up to 30,000 fold; in one experiment, 391 ×106 CD3-CD56+ NK cells were expanded from only 13,000 NK cells obtained from a pool of three thawed cord segments.Conclusions: In vitro-expansion of a pure population of CD3−CD56+ cells derived from cord blood can be achieved using EBV-LCL or 41BB transduced feeder cells. Expanded cells have increased NKG2D and TRAIL expression and enhanced TRAIL-mediated tumor cytotoxicity. Even with very low starting numbers of TNCs, substantial numbers of CD3+, CD3+56+ and CD3−CD56+ cells can be expanded in vitro from thawed segments using EBV-LCL feeder cells in advance of thawing of the cord unit. These methods are being optimized to allow for clinical scale expansion of NK cells from the same cord unit used for hematopoietic cell transplantation.Cell numbers after expansion of individual segments with EBV – LCL feeder cellsDay 0 (×10e6)Day 16 (× 10e6)Fold expansionTNC3–678–11220–30CD3+0.42–0.9133–4750–70CD3+CD56-0.015–0.03216–24700–1000CD3-CD56+0.081–0.17528–40200–300Cell numbers after CD3+ depletion of pooled segments and expansion with EBV – LCL feeder cellsDay 0 (×10e6)Day 33 (× 10e6)Fold expansionTNC4.1640196CD3+00-CD3+CD56-00-CD3-CD56+0.013239129621

IFN‐α production by cord blood plasmacytoid dendritic cells is totally deficient.

Introduction: The interaction between natural killer cells (NK) and plasmacytoid dendritic cells (pDC) has been suggested to play a role during the early phase of the immune response to viral infections and tumors. NK cells can lyse tumor and virus‐infected cells and their cytolytic activity has long been known to be enhanced by interferon‐alpha (IFN‐α). Human pDC are a major source of IFN‐α following viral stimulation or upon exposure to TLR‐7 or TLR‐9 ligands. Objective: In order to study the cross‐talk between NK and pDC in human newborns, the effect of IFN‐α on NK cells was compared between cord and adult blood, and the response of pDC to CpG or HSV‐1 was studied. Results: We have recently reported that neonatal NK cells are mature and express a reduced cytolytic activity. In this study, we show that IFN‐α‐stimulated‐NK cells display an activated phenotype and a cytolytic activity similar to adult NK cells. Moreover, the production of IFN‐α in response to CpG or HSV‐1 is dramatically impaired in cord blood. Cord blood pDC present a decreased expression of TLR9 and IRF7 which may explain the deficient production of IFN‐α. This lower expression of IRF7 and IFN‐α production can be partially restored by pre‐treatment of IFN‐β. However, although all cord blood show a deficiency in IFN‐α production, most of cord blood samples (2/3) exhibit a normal maturation of pDC in response to CpG, as shown by the up‐regulation of CD80, CD83, CD40, HLA‐DR and CD54. Conclusions: The defect in IFN‐α production by cord blood plasmacytoid dendritic cells can be explained by the low level of IRF7 and TLR9 expression. This defect can be partially restored by increasing IRF7 expression by pre‐treatment of IFN‐β. These findings might explain the increased susceptibility of human newborns to infections.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

A Novel Triple Umbilical Cord Blood Transplant (UCBT) Strategy To Promote NK Cell Immunotherapy (Unit 1) with a Fully Ablative Preparative Regimen Followed by a Double UCBT in Patients with Refractory AML.

Treatment options for refractory AML are usually ineffective. Previously, we tested adoptive transfer of haploidentical peripheral blood (PB) derived NK cells without transplantation and demonstrated correlation between in vivo NK cell expansion and those who achieved a complete remission. This therapy is limited by:the inability to expand NK cells in most patients,prolonged neutropenia in some patients andinconsistent efficacy.UCB, in contrast to adult PB, is rich in NK precursors with CD34+/CD7−, CD34+/CD7+ and CD34−/CD7+ phenotypes. We hypothesized that UCB-derived NK cells may show better in vivo expansion than adult derived NK cells after cytoreduction. Therefore, we tested our triple UCBT strategy in patients with refractory relapsed AML who were <45 years old, without active infection and eligible for myeloablative conditioning. The UCB NK product (unit 1) was CD3 depleted (using a CliniMacs system) and activated with IL-2 (1000U/ml for 16–20 hours). The UCB-derived NK cells (matched at 3 HLA loci and KIR-ligand mismatched when possible) were infused on day -12 after conditioning with cyclophosphamide 120 mg/kg, fludarabine 125 mg/m2 and TBI 1320 cGy on days -19 to -13. Subcutaneous IL-2 (10 MU) was was given on days -12, -10, -8, -6, -4 and -2 to facilitate in vivo NK cell expansion. On day 0, two UCB units (≥4/6 match) were transplanted for hematopoietic rescue and followed by mycophenolate mofetil and cyclosporine for GVHD prophylaxis. Compared to pre-treatment levels, endogenous IL-15 was markedly increased after the preparative regimen at the time of the NK UCB unit. The NK UCB units contained both precursor and mature NK cells. Three product samples were cultured for 28 days in limiting dilution on a murine stromal feeder, demonstrating cloning frequencies of 1:5, 1:9 and 1:12 infused UCB cells giving rise to NK progeny. Two of the 3 patients had partial chimerism derived from the NK product on day -1. Unexpectedly, these same two patients demonstrated prompt neutrophil engraftment on days 3 and 7 after hematopoietic stem cell rescue. In both instances, chimerism was achieved from the NK product. Of the non-NK and non-T cells in the NK UCB units from these 2 patients there were 9.6% and 5.3% CD34+/CD7− cells. In the third patient, the NK UCB unit had only 2% CD34+/CD7− cells and they did not contribute to neutrophil engraftment which occurred on day 36 after UCBT. Of note, 3/3 tolerated the NK infusion without toxicity and were leukemia-free at the time of engraftment. Two remain alive (one died of TRM) with one relapse before day 100. These data suggest UCB NK cells may be administered safely and, despite CD3 depletion and IL-2 activation (ex vivo and in vivo), provide long term engraftment potential that may dominate over unmanipulated UCB infused subsequently. In summary, UCB is a rich source of NK precursors capable of in vivo expansion which are potentially better suited than adult NK cells for use in treatment of patients with refractory AML.

Expression of rearranged TCRγ genes in natural killer cells suggests a minor thymus-dependent pathway of lineage commitment

AbstractNatural killer (NK) cells are thought to develop from common lymphoid progenitors in the bone marrow. However, immature thymocytes also retain NK potential. Currently, the contribution of the thymus-dependent pathway in normal steady-state NK-cell development is unknown. Here, we show that TCRγ genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells, and similar levels are detected in NK cells from TCRβ,δ double-knockout mice, excluding the possibility of T-cell contamination. NK-cell TCRγ gene rearrangement is thymus dependent because this rearrangement is undetectable in nude mouse NK cells. These results change the current view of NK-cell development and show that a subset of NK cells develops from immature thymocytes that have rearranged TCRγ genes.

Neonatal Natural Killer Cells Produce Chemokines and Suppress HIV Replicationin Vitro

The innate immune system may be critical in the prevention of perinatal HIV infection. Since neonates have limited immunological experience, they may rely more on the ability of the innate immune system to defend against infection than their adult counterparts. To assess the potential of human neonatal natural killer (nNK) cells to suppress HIV infection in a noncytolytic manner, we evaluated their ability to secrete chemokines and inhibit HIV replication in vitro. nNK cells were cocultured with autologous, HIV-infected CD4(+) T cells and their suppressive activity against HIV was compared to nCD8(+) T cells and adult NK cells. We found that nNK cells could suppress HIV replication in autologous CD4(+) T cells infected with a CCR5-utilizing virus, but were unable to suppress replication by a CXCR4-utilizing virus. nNK cell-mediated suppression of HIV replication was comparable to that of nCD8(+) T cells and greater than that of NK cells from adults. Suppression was mediated by soluble factors, and was abrogated by the addition of an excess of anti-CC-chemokine Ab directed at CCR5 ligand chemokines. In contrast, inhibition of HIV replication by autologous nCD8(+) T cells was not fully abrogated with anti-CC-chemokine Abs indicating, as previously reported in HIV-infected adults, that other factors in addition to chemokines play a role in CD8(+) T cell-mediated suppression of HIV replication. Our results show that nNK cells can inhibit HIV replication via a chemokine-mediated mechanism, and support a potential role for the innate immune system in preventing perinatal transmission of HIV in a noncytolytic manner.

Acquisition of MHC-specific receptors on murine natural killer cells.

NK cells in adult mice express two families of MHC class I-specific receptors, namely, Ly49 and CD94/NKG2. Co-expression of these receptors in various combinations generates diverse receptor repertoires. The expression of individual receptors is mostly stochastic and independent of each other. NK cells acquire the receptors as they develop from progenitors in the bone marrow in adult mice. In vivo as well as in vitro studies have shown that the acquisition of the receptors is ordered and regulated by the host MHC class I. Developing NK cells first acquire CD94/NKG2 and subsequently various Ly49 receptors in an ordered manner. Unlike adult NK cells, most fetal and neonatal NK cells express CD94/NKG2 but not Ly49. During the first several weeks after birth, NK cells expressing various Ly49 receptors slowly accumulate, while CD94/NKG2+ NK cells decrease to approximately 50% of the population. The acquisition of NK receptors following hematopoietic stem cell transplantation is also a slow and apparently preprogrammed process, mimicking the ontogeny, regardless of whether stem cells from fetal liver or adult bone marrow are used as donors. The regulation of the transcription of individual receptor genes is rather complex, since two promoters have been identified for the genes encoding Ly49 and CD94.