Mature Natural Killer Cells Research Articles

FT538: Preclinical Development of an Off-the-Shelf Adoptive NK Cell Immunotherapy with Targeted Disruption of CD38 to Prevent Anti-CD38 Antibody-Mediated Fratricide and Enhance ADCC in Multiple Myeloma When Combined with Daratumumab

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, though there remains meaningful opportunity to improve mAb efficacy by optimizing the interaction with natural killer (NK) cells to enhance antibody-dependent cellular cytotoxicity (ADCC). NK cells are an ideal effector cell for combined use with tumor-targeting mAbs, as NK cells effect both innate tumoricidal capacity and ADCC. CD38-targeting mAbs, such as daratumumab, are effective in treating multiple myeloma (MM) and achieve their efficacy through multiple mechanisms, including ADCC. However, because activated NK cells express high levels of CD38, daratumumab induces NK cell depletion through fratricide, potentially reducing treatment effectiveness. Adoptive NK cell immunotherapy therefore has the potential to augment daratumumab's ADCC activity if fratricide can be reduced or prevented. FT538 is an off-the-shelf adoptive NK cell immunotherapy product candidate designed for enhanced cellular persistence and ADCC while avoiding anti-CD38 mAb induced fratricide. It is derived from induced pluripotent stem cells (iPSC) engineered to lack CD38 expression, which we have previously shown to eliminate daratumumab-induced fratricide among iPSC-derived NK cells, resulting in enhanced long-term daratumumab-mediated ADCC. FT538 is engineered to express an IL-15 receptor alpha fusion protein (IL-15RF; IL-15 tethered to IL-15 receptor α) to enhance persistence and a high-affinity non-cleavable CD16 (hnCD16, FcRγIII) to increase ADCC. To support the clinical translation of FT538, and to enable the repeatable and scalable cell production to support off-the-shelf availability of a uniform NK cell product, a clinical-grade master pluripotent stem cell line was developed. The FT538 master pluripotent stem cell line was created by reprogramming donor fibroblasts into iPSCs using our non-integrating cellular reprogramming platform, and cells were further genetically edited by targeting IL-15RF and hnCD16 to the CD38 locus. Clonal iPSC lines were generated and screened for precise knock-in and knock-out edits at the CD38 locus and a lack of off-target genome integration (15% total success rate for CD38-/-IL-15RF+CD16+). Selected engineered iPSC clones were confirmed to be free of reprogramming transgenes and to maintain genomic stability. Engineered iPSC clones were additionally tested for their NK cell differentiation potential and function, and a single clone was selected to serve as the renewable starting material for cGMP manufacturing and clinical development. Upon differentiation and expansion FT538 demonstrated a mature NK cell phenotype with expression of NK cell receptors including NKp30, NKp46, NKG2D, KIR, NKG2A, and DNAM-1. The functional impact of CD38 knockout on FT538 NK cells was confirmed in an in vitro fratricide assay, where peripheral blood (PB)-NK cells exhibited fratricide at a frequency of 33% after 3 hr culture with increasing daratumumab concentrations. In contrast, FT538 cells were entirely resistant (<1% specific cytotoxicity) to daratumumab-induced fratricide. In vitro cytotoxic re-stimulation assays showed that repeat exposure of PB-NK cells to daratumumab plus MM target cells resulted in a loss of cytotoxic capacity (from 74% to 58% upon re-stimulation), and a similar effect was seen for non-engineered iPSC-derived NK cells. In contrast, FT538 NK cells maintained robust ADCC in during primary and secondary exposure to MM target cells and daratumumab. FT538 with daratumumab resulted in 86% cytotoxicity against MM target cells upon first exposure and 92% cytotoxicity upon re-stimulation, with a 20-fold increase in viable NK cells at the conclusion of the assay compared to non-engineered iPSC-derived NK cells. Additionally, the combined survival benefit of IL-15RF expression and fratricide resistance mediated by the CD38 knockout as well as the enhanced hnCD16-mediated ADCC allowed for greater cytotoxicity of FT538 against MM tumor spheroids. Together, these preclinical data support the clinical translation of FT538, an off-the-shelf adoptive NK cell immunotherapy product engineered for uniform hnCD16 and IL-15RF expression with CD38 elimination for enhanced ADCC in combination with daratumumab and other anti-CD38 mAbs for the treatment of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Fong:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:CytoSen: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Signatures of Immune Dysfunction and Identification of Prognostic Immune-Related Risk Factors in Patients with Acute Myeloid Leukemia

There is growing appreciation that immune system plays an important role in anti-leukemia effect in patients with acute myeloid leukemia (AML). However, comprehensive illustration of phenotypic and functional immune signatures of AML at diagnosis is still lacking. Here, we conducted a prospective study to perform a comprehensive peripheral blood immune profiling through multiparameter flow cytometry and investigated prognostic immune-related risk factors in AML patients. In cohorts of 50 newly-diagnosed patients and 24 healthy controls, we performed phenotypic and functional analysis of circulating lymphocytes including CD4+ T, CD8+ T, natural killer (NK), natural killer-liked T (NKT), gamma delta T (γδ T) and B cells to elucidate the nature of immune defects in AML development. Our findings suggest that immune defects in AML patients are operative in T and NK cells that are important component of antitumor immunity while B cell function remains unaffected. Both CD8+ T and CD4+ T cells from AML patients exhibit features of senescence and exhaustion at diagnosis. These terminally senescent and exhausted T cells are phenotypically characterized by increased expression of CD57 and PD-1 and decreased expression of CD28. In addition, lessened frequency of NK, NKT and γδ T cells indicate slack innate immunity in AML. One important parameter involved in NK cell function is the maturation stage, and we found similarly increased NK cell maturation in AML. NK cell dysfunction was further supported by downregulation of activating receptor NKG2D and NKP30, which may weaken the recognition and interaction between NK cells and AML blasts. Furthermore, diseased γδ T cells demonstrate a highly-activated or even exhausted state through a profound PD-1 upregulation and NKG2D downregulation. We next performed immunologic studies on paired pre- and post-chemotherapy samples to analyze whether immune dysfunction was recovered during the course of induction chemotherapy. Immune reconstitution occurs only in patients with therapeutic response. T and NK cell function restoration is principally reflected in recovered proportions, decreased extent of terminal differentiation and partly improved T cell senescence and exhaustion. Moreover, we extended studies to analyze the difference in immune signatures between CR group (achieved stable complete remission after chemotherapy, n = 24) and RR group (not achieved complete remission after chemotherapy or suffered a relapse, n = 20). Patients in RR group exhibit selectively more senescent and exhausted CD8+ T (CD28lo/CD57hi/PD-1hi) and CD4+ T (PD-1hi) cells, lower percentages of γδ T cells and excessive NK maturation state. More importantly, we demonstrated that specific immune phenotypes apparently correlate with overall survival (OS) and event-free survival (EFS, the "event" refers to progression, relapse and death) in AML patients. Median values of the complete cohort were used for grouping the patients. Poor OS correlated with select pre-chemotherapy circulating PD-1+CD8+ and CD28-CD57+CD8+ T subsets (Figure 1). Elevated day +30 post induction chemotherapy PD-1+CD8+ T frequency and increased NK cell maturation correlated with unfavorable OS and EFS after induction chemotherapy. Moreover, decreased day +30 post induction chemotherapy CD28-CD57+CD8+ T and increased γδ T cell proportion were associated with improved EFS, but not with OS (Figure 2 and Figure 3). This is the first study to provide insights into longitudinal immunological landscape at diagnosis and after chemotherapy in patients with AML. Select immune phenotypes correlate with EFS and OS, implying that non-invasive immune testing of blood samples can be applied to identify high risk for relapse and unfavorable prognosis. Personized immunotherapies, aimed at reactivating senescent T cells, reverting exhausted T cells or improving innate NK and γδ T immunity, may contribute to better therapeutic effects and reduced early relapse. Disclosures No relevant conflicts of interest to declare.

Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Aggressive Natural Killer Cell Leukemia: An Advantage of Cord Blood Transplantation

Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Aggressive Natural Killer Cell Leukemia: An Advantage of Cord Blood Transplantation

JAK/STAT Cytokine Signaling at the Crossroad of NK Cell Development and Maturation.

Natural Killer (NK) cells are cytotoxic lymphocytes of the innate immune system and play a critical role in anti-viral and anti-tumor responses. NK cells develop in the bone marrow from hematopoietic stem cells (HSCs) that differentiate through common lymphoid progenitors (CLPs) to NK lineage-restricted progenitors (NKPs). The orchestrated action of multiple cytokines is crucial for NK cell development and maturation. Many of these cytokines such as IL-2, IL-7, IL-12, IL-15, IL-21, IL-27, and interferons (IFNs) signal via the Janus Kinase / Signal Transducer and Activator of Transcription (JAK/STAT) pathway. We here review the current knowledge about these cytokines and the downstream signaling involved in the development and maturation of conventional NK cells and their close relatives, innate lymphoid cells type 1 (ILC1). We further discuss the role of suppressor of cytokine signaling (SOCS) proteins in NK cells and highlight their potential for therapeutic application.

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.

Impact of CMV Infection on Natural Killer Cell Clonal Repertoire in CMV-Naïve Rhesus Macaques.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56−CD16+ NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56+16− NK cells from HSPC. These CD56−CD16+ NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16+ NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies.

Human signal transducer and activator of transcription 5b (STAT5b) mutation causes dysregulated human natural killer cell maturation and impaired lytic function

Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent infections, and combined immune deficiency. In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b-/- mice. We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b-/- mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function. This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b-/- mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation. Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.

2366 Acute Liver Failure Caused by Hemophagocytic Lymphohistiocytosis Secondary to Aggressive Natural Killer Cell Leukemia and Epstein-Barr Virus Reactivation

INTRODUCTION: Acute liver injury is a common presentation of many systemic illnesses, including hematologic malignancies. The present case was an unusual presentation of acute liver failure (ALF) caused by hemophagocytic lymphohistiocytosis (HLH) secondary to aggressive natural killer cell leukemia(ANKL) associated with Epstein-Barr virus(EBV) infection reactivation. CASE DESCRIPTION/METHODS: A 24-year-old female presented with fever and altered mental status. Her physical examination revealed massive hepatosplenomegaly. Complete blood count showed pancytopenia with WBC 460/µL, microcytic anemia Hgb 7.8g/dL with MCV 79, thrombocytes 45,000/µL, AST 170IU/L, ALT 162IU/L, INR 1.5, albumin 1.3g/dl and total bilirubin 11.08mg/dL, triglycerides 1164mg/dL, ferritin 1792ng/ml. CT abdomen showed hepatosplenomegaly with hepatic steatosis. Her liver biopsy showed hepatic necrosis with moderate steatohepatitis and perisinusoidal fibrosis with normal liver copper level. She was initially treated with steroids and N-acetylcysteine. Flow cytometry and bone marrow biopsy revealed CD56+ and CD3+ positive cells with high serum soluble IL-2 receptor consistent with ANKL and HLH. EBV markers were found positive for EBV IgG antibody and EBV nuclear antigen with high EBV PCR and negative for EBV IgM antibody, which is consistent with EBV reactivation. A modified SMILE chemotherapy regimen with rituximab was initiated. Unfortunately, her clinical status continued to decline progressively with worsening acute encephalopathy, acute hypoxic respiratory failure and Pseudomonas septic shock which required vasopressors and IV antibiotics. The patient died on day-30 of hospitalization due to her hematologic malignancy, septic shock, and ALF. DISCUSSION: HLH is a consequence of abnormal immune activation which could be secondary to infections, malignancies, or autoimmune diseases; leading to life-threatening multi-organ inflammatory response, especially in the liver. Natural killer (NK) cells are types of lymphocytes that play an important role as part of innate immune system in fighting tumors and viral cells; NK cells are abundant in the liver. It was shown that NK cells might be involved in the intense immune response causing severe liver damage. ANKL is a rare malignant lymphoproliferative disorder of mature NK cells closely associated with EBV. EBV reactivation can occur in this disease setting. Careful evaluation of patients with ALF and abnormal hematologic profile is essential for early detection and therapy of the primary culprit.

CTLA4Ig-based reduced intensity conditioning and donor lymphocyte infusions for haploidentical transplantation in refractory aggressive B-cell lymphoma relapsing after an autograft: Early results from a pilot study

CTLA4Ig-primed donor lymphocyte infusions (DLIs) have been found to promote natural killer (NK) cell-mediated anti-leukemia effect following haploidentical hematopoietic cell transplantation (HCT). Incorporation of CTLA4Ig in conditioning aided long-term remission in myeloma probably by blocking the CD28-CD86 pro-survival pathway when combined with CTLA4Ig-primed DLI. We explored a similar approach in 12 patients (8-65 years) who had refractory aggressive B-cell lymphoma (R-ABCL) following autologous HCT. They received CTLA4Ig-based reduced-intensity conditioning and sequential CTLA4Ig-primed DLIs on days +7, +21, and +35. None developed acute graft-versus-host disease (GVHD). Two patients developed chronic GVHD. Only 3 patients had disease-progression at 100 days posttransplant with a progression-free and GVHD-free survival at 2 years of 75%. A higher expression of CD80 in tumor cells and a greater proliferation of CD56dim CD16+ NK cells were observed at days +30 and +60 in patients with progression-free survival. We hypothesize that CTLA4Ig, with a greater avidity for CD80, probably interferes with the anti-apoptotic effect mediated through this pathway, and together with early proliferation of mature NK cell when used in conjunction with DLI, this approach might provide a curative option for patients with R-ABCL.

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

IL-17 constrains natural killer cell activity by restraining IL-15–driven cell maturation via SOCS3

Increasing evidence demonstrates that IL-17A promotes tumorigenesis, metastasis, and viral infection. Natural killer (NK) cells are critical for defending against tumors and infections. However, the roles and mechanisms of IL-17A in regulating NK cell activity remain elusive. Herein, our study demonstrated that IL-17A constrained NK cell antitumor and antiviral activity by restraining NK cell maturation. It was observed that the development and metastasis of tumors were suppressed in IL-17A-deficient mice in the NK cell-dependent manner. In addition, the antiviral activity of NK cells was also improved in IL-17A-deficient mice. Mechanistically, ablation of IL-17A signaling promoted generation of terminally mature CD27-CD11b+ NK cells, whereas constitutive IL-17A signaling reduced terminally mature NK cells. Parabiosis or mixed bone marrow chimeras from Il17a-/- and wild-type (WT) mice could inhibit excessive generation of terminally mature NK cells induced by IL-17A deficiency. Furthermore, IL-17A desensitized NK cell responses to IL-15 and suppressed IL-15-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) via up-regulation of SOCS3, leading to down-regulation of Blimp-1. Therefore, IL-17A acts as the checkpoint during NK cell terminal maturation, which highlights potential interventions to defend against tumors and viral infections.

On the Way to Become a Natural Killer Cell.

Natural Killer (NK) cells are innate lymphocytes playing pivotal roles in host defense and immune-surveillance. The homeostatic modulation of germ-line encoded/non-rearranged activating and inhibitory NK cell receptors (NKRs) determines the capability of these innate lymphocytes to either spare “self” cells or to kill viral-infected, tumor-transformed and heterologous cell targets. However, despite being discovered more than 40 years ago, several aspects of NK cell biology remain unknown or are still being debated. In particular, our knowledge of human NK cell ontogenesis and differentiation is still in its infancy as the majority of our experimental evidence on this topic mainly comes from findings obtained in vitro or with animal models in vivo. Although both the generation and the maintenance of human NK cells are sustained by hematopoietic stem cells (HSCs), the precise site(s) of NK cell development are still poorly defined. Indeed, HSCs and hematopoietic precursors are localized in different anatomical compartments that also change their ontogenic commitments before and after birth as well as in aging. Currently, the main site of NK cell generation and maturation in adulthood is considered the bone marrow, where their interactions with stromal cells, cytokines, growth factors, and other soluble molecules support and drive maturation. Different sequential stages of NK cell development have been identified on the basis of the differential expression of specific markers and NKRs as well as on the acquisition of specific effector-functions. All these phenotypic and functional features are key in inducing and regulating homing, activation and tissue-residency of NK cells in different human anatomic sites, where different homeostatic mechanisms ensure a perfect balance between immune tolerance and immune-surveillance. The present review summarizes our current knowledge on human NK cell ontogenesis and on the related pathways orchestrating a proper maturation, functions, and distributions.

The deubiquitinase Otub1 controls the activation of CD8+ T cells and NK cells by regulating IL-15-mediated priming

CD8 T cells and natural killer (NK) cells, central cellular components of immune responses against pathogens and cancer, rely on IL-15 for homeostasis. Here we show that IL-15 also mediates homeostatic priming of CD8 T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a pivotal kinase for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8 T cells to IL-15, rendering naive CD8 T cells hyper-sensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Consistently, Otub1 deletion profoundly enhances anticancer immunity through unleashing the activity of CD8 T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8 T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming.

Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy.

Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their potential for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like receptor (KIR) mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK cells are platforms for genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting off-target activation. Current clinical studies have demonstrated the safety and efficacy of allogeneic NK cell adoptive transfer therapies as a means for treatment of hematologic malignancies and, to a lesser extent, solid tumors. However, challenges associated with sourcing allogeneic NK cells have given rise to controversy over the contribution of NK cells to graft-versus-host disease (GvHD). Specifically, blood-derived NK cell infusions contain contaminating T cells, whose activation with NK-stimulating cytokines has been known to lead to heightened release of proinflammatory cytokines and trigger the onset of GvHD in vivo. NK cells sourced from cell lines and stem cells lack contaminating T cells, but can also lack many phenotypic characteristics of mature NK cells. Here, we discuss the available published evidence for the varying roles of NK cells in GvHD and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.

The Food Additive tBHQ Impairs NK Cell Cytotoxicity Against Influenza Infection (FS12-02-19)

ObjectivesWe examined the effects of the food preservative, tert-Butylhydroquinone (tBHQ), on natural killer (NK) cells when infected with influenza in C57Bl/6 mice. Previously, we found tBHQ to negatively impact splenic NK cell effector function in vitro. Therefore, we hypothesized that the consumption of tBHQ would impair the NK cell response against a primary influenza virus infection. MethodsFemale, C57Bl/6 mice were fed either a diet containing 0.0014% tBHQ or a control diet two weeks prior to infection. Mice were instilled intranasally with 21.78 HAU of influenza A/PR/8/34 (H1N1) and were monitored for two days. Body weight was recorded as a measurement for infection. After day two of infection, lungs were collected and processed for analysis by flow cytometry, enzyme-linked immunosorbent assay (ELISA), and total antioxidant capacity (TAC). ResultsThe NK cell activation markers, CD25 and CD69, were not affected by tBHQ consumption. Additionally, tBHQ did not have an effect on maturation of NK cells or interferon (IFN)γ production. Notably, NK cell expression of granzyme B was significantly decreased with tBHQ in infected mice. Furthermore, mice on the tBHQ diet showed decreased TAC after two days of infection compared to mice on the control diet, suggesting increased oxidative stress in the tBHQ group. ConclusionstBHQ had no effect on NK cell activation, maturation, and IFNγ production after two days of influenza infection. However, decreased expression of granzyme B by NK cells suggests tBHQ may impair NK cell cytoxicity during the infection. Moreover, the decrease in TAC suggests increased oxidative stress in the tBHQ-treated mice. Funding SourcesThis study was funded by National Institute of Environmental Health Sciences (ES024966).

PF636 NATURAL KILLER CELL ALLOREACTIVE HAPLOIDENTICAL STEM CELL TRANSPLANTATION FOR MULTIPLE MYELOMA PATIENTS

Background:Notwithstanding the progress made over the past years in survival of Multiple Myeloma (MM) patients, MM remains incurable. Haploidentical bone marrow transplantation (Haplo‐BMT) is a treatment option with curative potential. Haplo‐BMT already has shown clinical results however, only in a minority of MM patients. We hypothesize that this observation might be due to differences in natural killer (NK) cell alloreactivity and in this study we set out to prospectively investigate if NK cell mismatch is relevant for the outcome of haplo‐BMT.Aims:The aim of this prospective phase 2 study is to evaluate if KIR‐ligand mismatched haplo‐BMT with post‐transplant cyclophosphamide will improve progression free survival in poor risk MM patients.Methods:Poor risk MM patients, aged < 66 years were enrolled if they were responsive to their last line of therapy. Poor risk was defined as high‐risk cytogenetics, relapse within a year after autologous SCT, or treatment with threeor more previous lines of therapy. A prerequisite of enrolment was the possibility of an NK cell mismatch and availability of a mismatched family donor. Patients were excluded if donor‐specific HLA‐antibodies were present. Patients receive a haplo‐BMT with a non‐myeloablative conditioning regimen and post‐transplant cyclophosphamide. Primary endpoint is PFS at 1,5 years. Secondary endpoints are engraftment, bone marrow reconstitution, NK cell reconstitution, graft versus host disease (GvHD), infections and non‐relapse mortality (NRM).Results:In total 12 poor risk patients were included in the study of which 10 could be evaluated for the primary end point. Graft failure (1/12) and disease progression before transplant (1/12) rendered the remaining two patients not evaluable. At this interim analysis, 5 patients have reached 1,5 years follow up. Off all included patients mortality was 4/12 (33%), only 2 patients were in remission at death (NRM 17%). Of the 8 ‐ for the primary endpoint evaluable‐ patients 7 patients relapsed within 1,5 years. The last included patient is still in a stringent very good partial response at day 120. Average time of relapse is 141 days (30–360 days). Though relapsed, only 4/7 patients had to start anti‐myeloma treatment (time to next treatment 188 days). Of the 11 engrafted patients, average time to neutrophil recovery was 18 days (12–30 days) and average time to platelet recovery was 31 days (23–49 days). At day 30, all of the 9 analyses patients showed NK cell recovery, though with an immature phenotype (NKG2A+, KIR−). At day 60 in both the peripheral blood as well as bone marrow, mature NK cells (KIR+) could be identified. Of the engrafted patients 7 developed aGvHD, 55% grade I‐II and 9% grade II‐IV. Four patients developed cGvHD, all 36% grade I‐II. Treatment related mortality was 25%, which was in all cases due to infections. Infections were common post‐transplant and 45% of patients suffered from severe infections.Summary/Conclusion:The majority of patients showed early disease progression after mismatched haplo‐BMT. We predefined that with a PFS of 25% at 1,5 years we would qualify this treatment option successful. With only one patient still in remission this goal will not be achieved and we hypothesize that the late reconstitution of functional mature NK cells is responsible for the lack of response. Though, not yet successful, we show that mismatched haplo‐BMT in MM patients is feasible, in terms of engraftment and late NK cell reconstitution, save and forms a possible platform for future immunotherapeutic strategies.

Survival disparities of diffuse large B-cell lymphoma in a community-based cancer center.

7541 Background: Non-Hodgkin’s Lymphoma (NHL) is a heterogeneous group of hematologic malignancy from immature or mature lymphocytes or natural killer cells. It is the 9th leading cause of death for both sexes. Diffuse Large B Cell Lymphoma (DLBCL), a subset of NHL, comprises 30 – 40% of all NHL. The etiology for the racial disparities in survival among patients with DLBCL is still unknown. The Revised International Prognostic Index (R-IPI), a tool which predicts the outcome of DLBCL patients, has not yet been fully validated in African Americans (AA). Methods: We conducted a single cohort study of patients diagnosed with DLBCL from January 1, 2007 to December 31, 2017 from our tumor registry in a single community-based cancer center. We abstracted demographic, clinical, histopathologic, treatment, and R-IPI variables. Our primary endpoint was overall survival (OS). Our secondary endpoints were factors that determine mortality through Cox multivariate analysis. Results: Among 381 patients with NHL, 181 (47.5%) patients had biopsy-proven DLBCL. Median age was 65 years old, 47% were males, 41% were AA, 44% were Caucasians, 46% were stage IV by Ann-Arbor staging. African-Americans (AA) had a median OS of 15.7 months (95% CI, 10.31 to 23.90) compared to non-AA 93.6 months (95% CI, 61.48 to 142.57). Even after adjusting for race, R-IPI was correlated with mortality on all patients. Lactate dehydrogenase levels were significantly higher in AA (P<0.001) but there was no difference in the interval from diagnosis to initiation of treatment in all groups. The presence of B symptoms HR 1.80 (95% CI, 1.17 to 2.77), African-American race HR 2.19 (95% CI, 1.38 to 3.52), positive HIV HR 2.16 (95% CI, 1.15 to 4.05), chronic liver disease HR 1.87 (95% CI, 1.09 to 3.23), and malnutrition HR 2.46 (95% CI, 1.30 to 4.65) were all independently associated with increased mortality. Histopathologic features such as the cell of origin, BCL6, and BCL2 rearrangements were not independently associated with increased mortality. Conclusions: There is an overwhelming disparity in survival among AA with DLBCL. The R-IPI is a reliable tool for predicting mortality in AA with DLBCL.

Hematopoietic-Specific Deletion of Foxo1 Promotes NK Cell Specification and Proliferation.

We previously reported that deletion of Foxo1, via Ncr1-iCre mice from the expression of NKp46 onward, led to enhanced natural killer (NK) cell maturation and effector function. In this model, however, the role of Foxo1 in regulating NK cell specification and early development remains exclusive. Herein, we utilized a murine model of hematopoietic-specific deletion of Foxo1 before lymphoid specification, by crossing mice carrying floxed Foxo1 alleles (Foxo1fl/fl) with Vav1-iCre mice, to revisit the role of Foxo1 on NK cell specification and early development. The data showed that hematopoietic-specific deletion of Foxo1 resulted in increased proportion and numbers of common lymphoid progenitors (CLP) (Lin−CD127+c-Kit+Sca-1+), pre-pro NK b cells (Lin−Sca-1+c-Kit−CD135−CD127+), as well as committed Lin−CD122+ cells and CD3−CD19−NKp46+ NK cells in bone marrow. Hematopoietic-specific deletion of Foxo1 also promoted NK cells proliferation in a cell-intrinsic manner, indicated by increased Ki-67 expression and more expansion of NK cell after ex vivo stimulation with IL-15. The reason for Foxo1 suppressing NK cell proliferation might be its direct transcription of the cell-cycle inhibitory genes, such as p21cip1, p27kip1, p130, Gadd45a, and Ccng2 (cyclin G2) in NK cells, supported by the evidence of decreased mRNA expression of p21cip1, p27kip1, p130, Gadd45a, and Ccng2 in Foxo1-deficient NK cells and direct binding of Foxo1 on their promoter region. Furthermore, hematopoietic-specific deletion of Foxo1 resulted in increased ratio of mature NK subsets, such as CD11b+CD27− and CD43+KLRG1+ NK cells, but decreased ratio of immature NK subsets, such as CD27+CD11b− and CD27+CD11b+ NK cells, consistent with the findings in the murine model of Ncr1-iCre mediated Foxo1 deletion. Conclusively, Foxo1 not only acts as a negative checkpoint on NK cell maturation, but also represses NK cell specification and proliferation. The relative higher expression of Foxo1 in CLP and early NK precursors may also contribute to the later NK cell proliferation and responsiveness, which warranties another separate study in the future.

The transcription factor TCF1 is a critical target of ID2 that limits natural killer cell maturation

Abstract ID2 is an inhibitor of the E protein transcription factors that is expressed in all innate lymphoid cells. In natural killer (NK) cells, ID2 prevents immature cells from acquiring a naïve T cell – like gene program that restricts NK cell maturation and function. Here we show that Tcf7 (encoding the transcription factor TCF1) is the critical E protein target gene in immature NK cells that maintains NK cell progenitors. TCF1 binds to a subset of key “naïve T cell” genes that are normally expressed at low, but detectable, levels in immature NK cells and whose expression is deregulated in the absence of ID2. Importantly, combined deficiency of TCF1 and ID2 restores expression of these naïve T cell genes to near wild-type levels. Moreover, in the absence of TCF1, ID2-deficient NK cells mature and acquire functional capacity but fail to expand to wild-type numbers. Our data indicate that ID2 is essential for NK cell maturation because it controls TCF1, whose heightened expression arrests NK cell maturation and activates a naïve – like gene program.

Kctd9 Deficiency Impairs Natural Killer Cell Development and Effector Function.

We previously showed that potassium channel tetramerization domain containing 9 (KCTD9) is aberrantly expressed in natural killer (NK) cells in patients with hepatitis B virus-associated acute-on-chronic liver failure and mice with experimental fulminant hepatitis. However, the mechanism underlying the regulation of NK cell function and fulminant hepatitis progression by KCTD9 is unknown. Here, we investigated the role of Kctd9 in regulation of early development, maturation, and function of NK cells using Kctd9-knockout mice. Compared to wild-type mice, Kctd9-deficient mice exhibited impaired NK cell lineage commitment, as evidenced by selective reduction in the refined NK progenitors, and incomplete NK cell maturation, as manifested by a higher proportion of CD11b− NK cells and a lower percentage of CD11b+ NK cells with high proliferative potential. Moreover, Kctd9-depleted NK cells displayed insufficient IFN-γ production, degranulation, and granzyme B production in response to cytokine stimulation, and attenuated cytotoxicity to tumor cells in vitro. The defect in NK cells was further supported by ameliorated liver damage and improved survival in Kctd9-deficient mice following murine hepatitis virus strain-3 (MHV-3) infection, which otherwise leads to immune-mediated fulminant hepatitis, a phenotype homologous to that caused by NK cell depletion in wild-type mice. Further investigation to identify the underlying mechanism revealed that Kctd9 deficiency hindered the expression of transcription factors, including Ets1, Nfil3, Eomes, and Id2 in NK cells. Collectively, our data reveal that Kctd9 acts as a novel regulator for NK cell commitment, maturation, and effector function.