Multiparametric Flow Cytometry Evaluation of CD200L/CD200R- LSC/NK Synapse Including Leukemia Stem Cell (LSC) Fraction As a Potential Therapeutic Target and Marker of NK Cell Exhaustion in Pediatric AML-Conect-AML French Collaborative Network

Abstract BACKGROUND: NK cells play a crucial role in the immune surveillance of malignant hemopathies. They undergo fine regulation by the microenvironment and by integrating activating and inhibiting signals trough several receptor/ligand couple interactions, hereafter referred to as "NK synapse". The ligands are expressed by a variety of cell types in the hematopoietic niche, including most immature leukemic stem cells CD34+CD38-. High expression of inhibiting ligands on AML (acute myeloid leukemia) blasts was associated with adverse clinical outcome . This observation highlights the relevance of identifying new ligand/receptor (L/R) pairs that could be targeted to prevent inhibiting interactions at the NK synapse. Relevant interactions to be blocked would display both ligand and receptor expressions on the leukemic cells and NK cells respectively. PATIENTS AND METHODS: 23 pediatric AML patients from the pediatric MyeChild01 protocol including in CONECT-AML French national collaborative network project diagnosed between 2018 and 2019 were included in this study. Reference bone marrows used were regenerative (4) or healthy bone marrows (5) . Multicolour flowcytometry protocole used fresh EDTA bone marrow at AML diagnosis and immunostaining with fluorochrome-coupled antibodies using 14 colour panel of L/R couples (Figure 1). Data was acquired on the FORTESSA Becton Dickinson with the Diva software and analysis using script R-PCA and FlowJo . RESULTS: We studied 5 inhibiting NK synapses (iinhibitory ligand/receptor pairs) . Four out of five inhibiting synapses (TIGIT/CD155; PD1-1/PD-L1; CD94/HLA-E and KIR2DL/HLA-A-B-C), showed not significant expression of ligand associated with the corresponding receptor expression. The CD200/CD200R synapse was the only one in which high ligand expression in blasts was significantly associated with high receptor expression on NK cells (Figure 2). This synapse could thus be of interest to develop targeting therapies for CD200-positive pediatric AML, with the strong advantage that patient eligibility could be easily identified at diagnosis. We then realized a principal component analysis, using the R software (PCA), integrating the MFIs of the 5 inhibiting NK synapses and 6 activating NK synapses (Figure 1) for the pediatric AML cohort (ID #1 to #23 ) together with reference bone marrows (healthy donors (n=5; ID #24 to #28) and regenerative bone marrows (n=4; ID #29 to #32)) . The CD200/CD200R synapse was identified as the main variable, explaining the distribution of patients and healthy donors as both CD200 and CD200R expressions happened to be among the most contributive to PCA axes. Interestingly, healthy donors clustered together, close to regenerative bone marrows. Pediatric AML patients distributed heterogeneously (Figure 3). In parallel, we evaluated whether CD200 expression on bulk leukemia blasts including most immature CD34+CD38- LSC was associated with exhaustion markers on NK cells. We found that patients with high and intermediate expression of CD200 on blasts (MFI > 3 rd quartile and comprised between 2 nd and 3 rd quartile, respectively) displayed strong PD-1 and TIGIT expressions on NK cells. Reciprocally, patients with low CD200 expression (MFI< 2 nd quartile) displayed a moderate PD-1 expression on NK cells, and TIGIT expression was more heterogeneous among individuals (Figure 4). CONCLUSIONS: Here, we identified CD200 expression in AML blasts including LSC as a marker that could be associated with NK cell exhaustion. at diagnosis. A PCA strategy allowed to observe that this marker differentiated pediatric AML patients NK synapse profiles from healthy donors and regenerative bone marrows sugesting a potential deregulation of bone marrow niche including NK-LSC escape. This suggests that CD200 expression assessment on blasts at diagnosis could be a tool to evaluate NK cell antitumor potential. Indeed, direct NK cell assessment by flow cytometry can be challenging because of blast invasion in the bone marrow. Nevertheless, it remains to be elucidated whether this clustering and exhaustion markers on NK cells correlated with patient clinical outcomes and MRD kinetics including CD34+CD38- LSC flow frequency evaluation that should be useful in most clinical trials to overcome chemoresistance of LSC. These results should be confirmed in a prospectively larger cohort of patients in future clinical trials. Figure 1 Figure 1. Disclosures Renard: Jazz Pharmaceuticals: Research Funding.

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T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3781.3781 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3781-3781

Author(s):

Eolia Brissot ◽

Sawa Ito ◽

Kit Lu ◽

Carly Cantilena ◽

B. Douglas Smith ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Research Funding ◽

Immune Escape ◽

Nk Cell ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an immune editing effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

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Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽

10.1182/blood.v104.11.3244.3244 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3244-3244

Author(s):

Gabriele Multhoff ◽

Catharina Gross ◽

Anne Dickinson ◽

Ernst Holler

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Nk Cell ◽

Granzyme B ◽

Hla Class I ◽

K562 Cells ◽

Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

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The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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Cytokine Activation Induces CD25 Expression and a Signaling-Competent High-Affinity IL-2 Receptor On CD56dim Human NK Cells.

Blood ◽

10.1182/blood.v120.21.2159.2159 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2159-2159

Author(s):

Julie M Chase ◽

Jeffrey W Leong ◽

Rizwan Romee ◽

Todd A. Fehniger

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Low Dose ◽

Nk Cell ◽

Surface Expression ◽

Receptor Expression ◽

Published Data ◽

High Affinity ◽

Cd25 Expression ◽

Cytokine Activation

Abstract Abstract 2159 Introduction. NK cells are innate immune lymphocytes important for host defense against infection, and also mediate anti-tumor responses. NK cell effector functions are triggered by ligand-mediated engagement of activating receptors and by cytokine stimulation. Human NK cells may be divided into developmental and functional CD56bright and CD56dim subsets with distinct biology. Both NK subsets have constitutive expression of CD122 (IL-2/15Rβ) and CD132 (γc), allowing for responsiveness to IL-15 and high doses (1 nM) of IL-2. The high affinity heterotrimeric IL-2 receptor is formed by CD122, CD132, and CD25 (IL-2Rα), allowing for responses to very low (10 pM) concentrations of IL-2. Previous studies demonstrated that resting CD56bright, but not CD56dim, NK cells constitutively express a functional high-affinity IL-2Rαβγ, as picomolar concentrations of IL-2 result in proliferation and co-stimulate IFN-γ production. We hypothesized that cytokine activation, which occurs at the site of an inflammatory response or interaction with a priming dendritic cell, may induce the expression of CD25 on CD56dimNK cells, allowing for enhanced responsiveness to IL-2. Methods. Purified normal donor NK cells (>95% CD56+CD3-) were cultured for 16h in media containing various cytokines, including IL-15 (100ng/mL) + IL-18 (50ng/mL), IL-15 (100ng/mL) + IL-12 (10ng/mL) or low dose IL-15 (1ng/mL) as a control (to support survival). Following stimulation, cells were washed and analyzed for surface expression of CD25 by antibody staining and flow cytometry. To assess the functional capacity of an induced IL-2Rα chain, NK cells were purified and treated with cytokines as above, washed extensively and replated in media devoid of all cytokines. At 3d after initial stimulation, cultures were briefly (15min) stimulated with IL-2 (10pM, 100pM and 1nM) or IL-15 (100ng/mL) to induce phosphorylation of STAT5. Cells were immediately fixed, permeablized, and assessed for intracellular phosphoSTAT5 by flow cytometry. Results. Pretreatment with IL-15 + IL-18 or IL-12 + IL-18, but no single cytokine, induced marked upregulation of CD25 on both CD56bright as well as CD56dim NK cells, with 93.6 ±1.9% of CD56bright and 97.7 ±1.2% (n=4) of CD56dim NK cells positive for CD25 expression immediately following the initial 16h stimulation with IL-15+18, compared to 20.7 ±6.8 and 3.6 ±1.4% for control treated (low dose IL-15) CD56bright and CD56dim NK. In preliminary experiments, CD25 appears to be markedly upregulated at the mRNA level, following treatment with IL-15+18. While kinetic analyses revealed that the absolute surface receptor expression was maximal at time points early after stimulation, 75.8 ±4.1% of CD56bright and 84.9 ±5.7% (n=4) of CD56dimNK cells pretreated with IL-15+18 retained CD25 surface expression at 7d post-stimulation. Importantly, CD25 induced on both CD56dim and CD56bright NK cells resulted in a signaling-competent high affinity IL-2 receptor, as stimulation with low dose IL-2 at 3d following initial cytokine pre-activation revealed increased production of phosphoSTAT5, versus control treated NK cells. The greatest enhancement was noted in CD56dim NK cells, showing an 8-fold increase in responsiveness to low dose (10pM) IL-2 stimulation (48.2±7.9% pSTAT5+ in IL-15+18 pretreated cells vs. 5.8±2.4% in control treated cells, p<0.02). Cytokine-pretreated CD56bright NK cells demonstrated no enhancement in phosphoSTAT5 following stimulation with low dose IL-2 (10pM) (27.6 ±8.3% in IL-15+18 pretreated vs. 26.0 ±6.4% in control) at this time-point. However, these results are in agreement with published data, which describe expression of CD25 on resting CD56bright but not CD56dimNK cells. Conclusions. Here, we report the induction of CD25, and a signal-competent high-affinity component of the IL-2 receptor, on CD56dim human NK cells, following cytokine pre-activation. These results have implications for the function of both CD56bright and CD56dim NK cells in the context of inflammation and potential for cross-talk with IL-2-producing T cells during an adaptive immune response. In addition, since rhIL-2 is clinically available and used following NK cell adoptive transfer in leukemia patients, these data provides a rationale for low dose IL-2 following allogeneic NK cell pre-activation with combinations of IL-15, IL-12, and IL-18. Disclosures: No relevant conflicts of interest to declare.

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Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia

Frontiers in Immunology ◽

10.3389/fimmu.2021.665541 ◽

2021 ◽

Vol 12 ◽

Author(s):

Vlad Andrei Cianga ◽

Lydia Campos Catafal ◽

Petru Cianga ◽

Mariana Pavel Tanasa ◽

Mohamad Cherry ◽

...

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Expression Patterns ◽

Receptor Expression ◽

Cell Maturation ◽

Target Cells ◽

Cell Subpopulations

Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.

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Individuals Expressing FcγRIIIA-158 V/V and V/F Show Increased NK Cell Surface Expression of FcgRIIIA (CD16), Rituximab Binding, and Demonstrate Higher Levels of ADCC Activity in Response to Rituximab.

Blood ◽

10.1182/blood.v106.11.776.776 ◽

2005 ◽

Vol 106 (11) ◽

pp. 776-776 ◽

Cited By ~ 2

Author(s):

Evdoxia Hatjiharissi ◽

Daniel Ditzel Santos ◽

Lian Xu ◽

Sigitas Verselis ◽

Michael Modica ◽

...

Keyword(s):

Monoclonal Antibody ◽

Nk Cells ◽

Nk Cell ◽

Basic Mechanism ◽

Surface Expression ◽

Receptor Expression ◽

Cell Surface Expression ◽

Protein Levels ◽

Healthy Donors ◽

Anti Cd20

Abstract The homozygous expression of phenylalanine (F/F) at codon 158 of FcγRIIIa (CD16) is associated with inferior clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with indolent non-Hodgkin’s lymphoma, in contrast to higher response rates observed for Waldenstrom’s macroglobulinemia patients expressing at least one valine (V/F, V/V) and follicular NHL patients who are homozygous for valine (V/V). We attempted elucidate the potential basic mechanism(s) behind these clinical observations by analyzing all the potential implications of this polymorphism among the 3 polymorphic subgroups at FcγRIIIA-158 (F/F, V/F, and V/V). We therefore used peripheral blood isolated natural killer (NK) cells from a pool of 52 unrelated healthy donors who were genotyped for FcγRIIIA-158. We evaluated allele-specific differences for FcγRIIIa gene expression by quantitative RT-PCR and demonstrated higher transcript levels among V/V (23.2 ng/mL) versus V/F (6.7 ng/mL) and F/F (6.2 ng/mL) (p<0.0001). We then determined protein levels for CD16 in the same subgroup of donors using quantitative flow cytometry. The number of CD16 receptors per NK cell was 105,947, 94,863, and 69,130 for the V/V, V/F and F/F donors, respectively and was significantly higher among donors who expressed at least one valine (V/V and V/F) versus F/F (p=0.033). We next determined rituximab binding affinity to NK cells from V/V, V/F and F/F donors following incubation at concentrations of 10–200 ug/ml, and use of an indirect competitive assay with the anti-CD16 monoclonal antibody 3G8 (Cancer Research 64:4664). Rituximab binding to NK cells was higher among donors expressing at least one valine at all concentrations evaluated, with mean rituximab binding (defined as % of inhibition of mean fluorescence intensity for 3G8) as follow: V/V (72%); V/F (53%); and FF (37%); (p=0.017). Lastly, we assessed rituximab dependent NK cell mediated cytotoxicity (ADCC) using CD20 expressing ARH-77 B-cells and observed higher levels of ADCC killing among V/V (82%) and V/F (80%) versus F/F (23%) donors at an effector: target cell ratio of 20:1. Taken together, these studies suggest that individuals who express at least one valine at FcγRIIIA-158 might have better clinical outcomes to rituximab therapy on the basis of increased FcγRIIIA-158 receptor expression, rituximab binding and ADCC mediated killing of tumor cells.

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Interleukin-15 Upregulates the Expression of NK-Cell Activating Receptors and Concomitantly Increases the NK Cytotoxicity in Patients with Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.2298.2298 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2298-2298

Author(s):

Miroslaw J. Szczepanski ◽

Malgorzata Czystowska ◽

Marta Szajnik ◽

Ann Welsh ◽

Kenneth A. Foon ◽

...

Keyword(s):

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Receptor Expression ◽

Activating Receptors ◽

Healthy Donors ◽

Interleukin 15 ◽

Nk Cell Cytotoxicity ◽

Nk Cytotoxicity ◽

Acute Myeloid

Abstract Natural killer (NK) cells lyse malignant cells without prior antigen-specific priming and play a critical role in the innate immune response. A balance of signals from activating and inhibiting receptors expressed on each NK cell controls its activity. The growth, differentiation and survival of NK cells have been found to be dependent on interleukin-15 (IL-15). Using multicolor flow cytometry we investigated the receptor repertoire and also measured the NK cell activity in twenty three patients with newly diagnosed acute myeloid leukemia (AML) prior to any treatment. Further, we investigated the ex-vivo effect of IL-15 on the NK cell repertoire and NK cell cytotoxicity. The percentage of circulating NK cells was lower (p<0.0001) in the AML patients (6%± 0.7, range 1–17%) compared to the NK cells of healthy donors (12%± 1, range 9–17%). The expression of the activating natural cytotoxicity (NCR) receptors NKp30 and NKp46 and the C-type lectin receptors NKG2D and NKG2C was significantly decreased in the AML patients compared to the NK cells of healthy donors: NKp30 24 vs 51% p<0.0001, NKp46 32 vs 73% p<0.0001, NKG2D 43 vs 83% p<0.0001, NKG2C 17 vs 28% p<0.03. In addition, the receptor expression (mean fluorescence intensity, MFI) was also significantly lower in AML patients compared to healthy donors. No significant differences in the expression of the NCR NKp44 and the NK- cell inhibitory receptors were observed. Furthermore, the NK cytotoxicity in the AML patients at diagnosis was significantly lower (p<0.0003) compared to the NK cytotoxicity of healthy donors (4 vs 75 LU). When NK cells obtained from AML patients were cultured with IL-15, significant increases in the expression of the NK- cell activating receptors (Table 1) were observed. The upregulation of the activating receptors was associated with a concomitant significant increase (p<0.001) of the NK cell cytotoxicity (4 vs 70 LU). The data suggest that IL-15, a homeostatic NK cell cytokine, can upregulate the expression of activating receptors and concomitantly increase the NK lytic activity. The use of IL-15 as a platform for NK- based therapies for AML patients should be considered in the future. Table 1. The effect of IL-15 on the receptor expression in AML patients NK cell Activating Receptors NK cells in AML pts at diagnosis NK cells in AML pts after IL-15 stimulation p value % positive, MFI % positive, MFI NKp30 24, 2.6 84, 6.0 0.001 NKp44 4, 2.4 71, 6.7 0.001 NKp46 32, 2.6 83, 7 0.002 NKG2C 17, 2.2 58, 6.9 0.005 NKG2D 43, 2.5 87, 7.8 0.01

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Forced Fucosylation With ASC-101 Enhances The Binding Of Ex Vivo Expanded Human NK Cells To E-Selectin: A Novel Method To Improve The Homing Of Adoptively Transferred NK Cells To The Bone Marrow In Patients With Hematological Malignancies

Blood ◽

10.1182/blood.v122.21.4499.4499 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4499-4499 ◽

Cited By ~ 2

Author(s):

Toshihiro Onishi ◽

Maria Berg ◽

Robert Reger ◽

Leonard Miller ◽

Steve Wolpe ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Nk Cells ◽

Hematological Malignancies ◽

Nk Cell ◽

Ex Vivo ◽

Binding Capacity ◽

Surface Expression ◽

Selectin Ligands

Introduction The ability of adoptively infused NK cells to home and traffic to the microenvironment where the tumor resides may be a critical determinant of their ability to mediate clinically meaningful anti-tumor effects. The initial step in leukocyte emigration from post-capillary venules, referred to as “tethering”, is a low-affinity interaction between leukocyte ligands with selectins expressed on endothelial cells. Since E-selectin is constitutively expressed on endothelium of skin and bone marrow in humans, leukocyte recruitment to bone marrow is thought to be largely dependent on E-selectin-binding. Among E-selectin ligands, only ligands bearing sialyl Lewis X with a terminal fucose (fucosylated) are functional forms that actively bind to E-selectin. One of the pitfalls of ex vivo NK cell expansion for adoptive infusion in humans is that expanded NK cells express predominantly non-fucosylated E-selectin ligands. We hypothesized that ex vivo fucosylation could enhance the binding capacity of E-selectin ligands on NK cells improving their homing into bone marrow where hematological malignancies reside. Methods CD56+/CD3- NK cells were isolated from normal human subjects by immuno-magnetic bead selection and were expanded ex vivo over 7-21 days by co-culturing with irradiated EBV-LCL feeder cells in IL-2 containing medium. Expanded NK cells were incubated for 30 minutes at room temperature with GDP-fucose and alpha1,3 fucosyltransferase-VI (ASC-101). The levels of fucosylation were determined by CLA surface expression measured by flow cytometry using the antibody HECA-452. After fucosylation, NK cells were analyzed by flow cytometry to assess for phenotype changes, viability and stability of fucosylation. Chromium release assays were performed to assess NK cell cytotoxicity against tumor cells. To determine whether fucosylated NK cells had enhanced binding to E-selectin, the binding capacity of NK cells to human recombinant E-selectin/Fc Chimera protein was evaluated by flow cytometry. Results Expanded human NK cells had low levels of baseline fucosylation, ranging from only 10-25%. Expanded NK cells were successfully fucosylated with ASC-101 in a dose-dependent manner (figure); the MFI of CLA on NK cells peaked at 25 ug/ml of ASC-101, with nearly 100% of NK cells being fucosylated (CLA positive). Fucosylation did not affect NK cell viability nor was it associated with changes in NK cell phenotype including surface expression of CD16, CD56, KIR2DL1, KIR2DL2/3, KIR3DL1, NKG2A, NKG2D, TRAIL, perforin, or granzymes A/B. Ex vivo cell culture showed fucosylation was sustained at nearly 100% for 48 hours, but then rapidly declined returning to baseline levels by 96 hours. NK cell cytotoxicity against tumor targets including K562 cells and myeloma cells was preserved and unaffected by fucosylation. Fucosylation significantly enhanced the binding capacity of NK cells to human E-selectin. Further, NK cell binding to recombinant human E-selectin/Fc chimera protein directly correlated with the degree of NK cell fucosylation (figure), which was dose-dependent on the ASC-101 concentration. The effects of forced fucosylation on the ability of human NK cells to home to the bone marrow following adoptive transfer into immuno-deficient mice is currently being explored. Conclusion Expanded NK cells primarily express non-glycosylated ligands for E-selectin, potentially limiting their ability to home to the bone marrow following adoptive transfer in humans with hematological malignancies. Ligands for E-selectin on the surface of expanded NK cells can be glycosylated ex vivo rapidly and to high degrees using ASC-101, significantly enhancing their ability to bind E-selectin. These data suggest forced fucosylation of NK cells could be used as a novel approach to improve the antitumor effects of adoptive NK cell infusions in patients with hematological malignancies. Disclosures: Miller: America Stem Cell Inc: Employment. Wolpe:American Stem Cell, Inc: Employment. Koh:America Stem Cell Inc: Employment.

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A Rare Case of Chronic Lymphoproliferative Disorder of NK Cells with an Unusual Expression of CD57

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa161.221 ◽

2020 ◽

Vol 154 (Supplement_1) ◽

pp. S101-S101

Author(s):

H Laharwani ◽

K Perrizo ◽

S Jain ◽

J Lam

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Nk Cells ◽

Peripheral Blood ◽

Lymphoproliferative Disorder ◽

Nk Cell ◽

Clinical Course ◽

Deficiency Anemia ◽

Variable Expression

Abstract Introduction/Objective Natural Killer cells (NK-cells) malignancies are extremely rare and the two NK-cell disorders involving the peripheral blood and bone marrow include chronic lymphoproliferative disorder of NK-cells (CLPDNK) and aggressive NK-cell leukemia (ANKL). ANKL is EBV associated with a prevalence in Asian adults and a fulminant clinical course leading to death within 2 months. Methods A 76-year-old female with a history of iron deficiency anemia presented for evaluation of lymphocytosis (78 TH/mm) and negative EBV with extreme fatigue, weight loss, and worsening weakness from the past 8 months. Results The peripheral and bone marrow aspirate showed an increase in granular lymphocytes with bland nuclei and abundant pale-staining cytoplasm containing fine to coarse azurophilic granules with no evidence of nuclear atypia. Flow cytometry demonstrated an atypical lymphocytic cell population comprising >50% of total marrow and peripheral blood lymphocytes demonstrating loss of CD56 and diminished CD7 expression. The analysis showed atypical lymphocytes with the following immunophenotype: CD2, CD8 (the majority, at least 75%),CD7 (dim), CD38, CD11c (subset) with no expression of CD3, CD5, CD4, CD56, or CD11c. Flow cytometry is important as it helps in the phenotypic detection of aberrancy. A broad panel of immunohistochemical stains revealed scattered positivity for CD3, CD4, CD7, CD8, TIA-1, granzyme, perforin, and tryptase and negative for CD56 and CD57. Our top differential with a similar clinical course included T-cell large granular lymphocytic leukemia which is characterized by a clonal proliferation of mature cytotoxic T cells and also has an indolent course. They show CD3 positivity by flow cytometry along with co-expression of NK cell-associated markers (CD16 and CD57), and variable expression of other pan T cell markers such as CD2, CD5, CD7. It is found incidentally in patients having cytopenias with a persistent increase in circulating mature NK-cells. Of note, the PET scan revealed splenomegaly with no other significant findings.The patient was started on steroids and is currently doing well. Conclusion Thus, overall morphologic, immunophenotypic, and molecular results with negative expression of surface or cytoplasmic CD3 as well as the absence of TCR expression were most consistent with CLPD-NK with an unusual expression of CD57 expression detected in flow cytometry.

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690 TIGIT blockade improves anti-tumor activity of ex vivo expanded NK cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.690 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A718-A718

Author(s):

Md Faqrul Hasan ◽

Alicja Copik

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Statistical Significance ◽

Adoptive Cell Therapy ◽

Cell Activity ◽

K562 Cells ◽

Inhibitory Receptors ◽

Cell Exhaustion

BackgroundNatural killer (NK) cells are innate immune cells that directly kill and coordinate responses against cancer prompting interest in using ex vivo expanded NK cells as an adoptive cell therapy for treatment of cancer. NK cells express a set of activating and inhibitory receptors that regulate their activity. Inhibitory receptor TIGIT (T cell Immunoreceptor with Ig and ITIM domain) is upregulated on intratumoral NK cells in some cancers, inhibits NK cell activity and promotes NK cell exhaustion. In this study, the effect of TIGIT blockade on the anti-tumor activities of ex vivo expanded NK cells was evaluated.MethodsNK cells were activated overnight with cytokines or ex vivo expanded with PM21-particles. Their TIGIT expression was determined with qRT-PCR and flow cytometry. Cytotoxicity was assessed by kinetic, imaging-based assay (Incucyte S3) against A549 and NCI-H1299 cells cultured in 3D. Cytotoxicity was calculated based on untreated controls at different time-points. Results from multiple donors were normalized to cytotoxicity of NK cells with isotype for individual donors and was compared to the cytotoxicity of NK cells with anti-TIGIT. Unpaired t test was used to determine statistical significance. K562 cells stably expressing Polio Virus Receptor (PVR), were used to restimulate A549 spheroid-exposed NK cells to measure IFNγ, TNFα and degranulation. Furthermore, phenotypic changes of NK cells upon TIGIT blockade were examined by analyzing a set of activating and inhibitory receptors by flow cytometry.ResultsThe effect of NK cell expansion/activation on TIGIT expression was assessed. TIGIT was upregulated on expanded and cytokine-activated NK cells both on mRNA and protein level. The effect of TIGIT blockade on NK cell cytotoxicity was examined by co-culturing PM21-NK cells with cancer cells in the presence of anti-TIGIT antibodies or respective isotypes. TIGIT blockade significantly increased cytotoxicity of PM21-NK cells against A549 (1.3 fold, P < 0.0001) and NCI-H1299 (1.3 fold, P = 0.0003) spheroids after 48 h. To access exhaustion, NK cells exposed to A549 spheroids for 7 days were restimulated with PVR+ K562 cells. TIGIT blockade prevented NK cell exhaustion resulting in increased expression of IFNγ, TNFα and surface CD107a on restimulated NK cells. TIGIT blockade did not result in changes to the surface phenotype of NK cells.ConclusionsTIGIT was highly expressed on expanded and cytokine-activated NK cells. TIGIT blockade improved anti-tumor activities of PM21-NK cells. Thus, PM21-NK cells and TIGIT antibodies have translational potential as a combination therapy to improve anti-tumor response.

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