scholarly journals Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

2010 ◽  
Vol 207 (9) ◽  
pp. 1923-1938 ◽  
Author(s):  
Aradhana Awasthi ◽  
Asanga Samarakoon ◽  
Haiyan Chu ◽  
Rajasekaran Kamalakannan ◽  
Lawrence A. Quilliam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Synapse Formation ◽  
Nk Cell ◽  
Scaffolding Protein ◽  
Microtubule Organizing Center ◽  
Chemokine Production ◽  
Immune Synapse ◽  
Cell Functions ◽  
Organizing Center

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.

scholarly journals Cdc42-interacting protein–4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse

2007 ◽  
Vol 204 (10) ◽  
pp. 2305-2320 ◽  
Author(s):  
Pinaki P. Banerjee ◽  
Rahul Pandey ◽  
Rena Zheng ◽  
Megan M. Suhoski ◽  
Linda Monaco-Shawver ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Nk Cell ◽  
Filamentous Actin ◽  
Microtubule Organizing Center ◽  
Interacting Protein ◽  
Lytic Granules ◽  
Organizing Center ◽  
Microtubule Networks

An essential function of the immunological synapse (IS) is directed secretion. NK cells are especially adept at this activity, as they direct lytic granules to the synapse for secretion, which enables cytotoxicity and facilitates host defense. This initially requires rearrangement of the actin cytoskeleton and, subsequently, microtubule-dependent trafficking of the lytic granules. As these two steps are sequential, specific linkages between them are likely to serve as critical regulators of cytotoxicity. We studied Cdc42-interacting protein–4 (CIP4), which constitutively interacts with tubulin and microtubules but focuses to the microtubule organizing center (MTOC) after NK cell activation, when it is able to associate with Wiskott-Aldrich syndrome protein (WASp) and the actin filament–rich IS. WASp deficiency, overexpression of CIP4, or parts of CIP4 interfere with this union and block normal CIP4 localization, MTOC polarization to the IS, and cytotoxicity. Reduction of endogenous CIP4 expression using small interfering RNA similarly inhibits MTOC polarization and cytotoxic activity but does not impair actin filament accumulation at the IS, or Cdc42 activation. Thus, CIP4 is an important cytoskeletal adaptor that functions after filamentous actin accumulation and Cdc42 activation to enable MTOC polarization and NK cell cytotoxicity.

scholarly journals Dose of Retroviral Infection Determines Induction of Antiviral NK Cell Responses

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Elisabeth Littwitz-Salomon ◽  
Simone Schimmer ◽  
Ulf Dittmer
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Activity ◽  
High Dose ◽  
Nk Cell Activity ◽  
Retroviral Infection ◽  
Cell Functions ◽  
Virus Dose ◽  
Antiviral Activities

ABSTRACT Natural killer (NK) cells are part of the innate immune system and recognize virus-infected cells as well as tumor cells. Conflicting data about the beneficial or even detrimental role of NK cells in different infectious diseases have been described previously. While the type of pathogen strongly influences NK cell functionality, less is known about how the infection dose influences the quality of a NK cell response against retroviruses. In this study, we used the well-established Friend retrovirus (FV) mouse model to investigate the impact of virus dose on the induction of antiviral NK cell functions. High-dose virus inoculation increased initial virus replication compared to that with medium- or low-dose viral challenge and significantly improved NK cell activation. Antiviral NK cell activity, including in vivo cytotoxicity toward infected target cells, was also enhanced by high-dose virus infection. NK cell activation following high-dose viral challenge was likely mediated by activated dendritic cells (DCs) and macrophages and the NK cell-stimulating cytokines interleukin 15 (IL-15) and IL-18. Neutralization of these cytokines decreased NK cell functions and increased viral loads, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we demonstrate that virus dose positively correlates with antiviral NK cell activity and function, which are at least partly driven by IL-15 and IL-18. Our results suggest that NK cell activity may be therapeutically enhanced by administering IL-15 and IL-18 in virus infections that inadequately activate NK cells. IMPORTANCE In infections with retroviruses, like HIV and FV infection of mice, NK cells clearly mediate antiviral activities, but they are usually not sufficient to prevent severe pathology. Here we show that the initial infection dose impacts the induction of an antiviral NK cell response during an acute retroviral infection, which had not investigated before. High-dose infection resulted in a strong NK cell functionality, whereas no antiviral activities were detected after low- or medium-dose infection. Interestingly, DCs and macrophages were highly activated after high-dose FV challenge, which corresponded with increased levels of NK cell-stimulating cytokines IL-15 and IL-18. IL-15 and IL-18 neutralization decreased NK cell functions, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we show the importance of cytokines for NK cell activation in retroviral infections; our findings suggest that immunotherapy combining the well-tolerated cytokines IL-15 and IL-18 might be an interesting approach for antiretroviral treatment.

scholarly journals Regulation of human NK-cell cytokine and chemokine production by target cell recognition

Blood ◽  
2010 ◽  
Vol 115 (11) ◽  
pp. 2167-2176 ◽  
Author(s):  
Cyril Fauriat ◽  
Eric O. Long ◽  
Hans-Gustaf Ljunggren ◽  
Yenan T. Bryceson
Keyword(s):  
Nk Cells ◽  
Target Cell ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Recognition ◽  
Chemokine Production ◽  
Relative Contribution ◽  
Cytokines And Chemokines ◽  
Tnf Α ◽  
Ifn Γ

AbstractNatural killer (NK)–cell recognition of infected or neoplastic cells can induce cytotoxicity and cytokine secretion. So far, it has been difficult to assess the relative contribution of multiple NK-cell activation receptors to cytokine and chemokine production upon target cell recognition. Using Drosophila cells expressing ligands for the NK-cell receptors LFA-1, NKG2D, DNAM-1, 2B4, and CD16, we studied the minimal requirements for secretion by freshly isolated, human NK cells. Target cell stimulation induced secretion of predominately proinflammatory cytokines and chemokines. Release of chemokines MIP-1α, MIP-1β, and RANTES was induced within 1 hour of stimulation, whereas release of TNF-α and IFN-γ occurred later. Engagement of CD16, 2B4, or NKG2D sufficed for chemokine release, whereas induction of TNF-α and IFN-γ required engagement of additional receptors. Remarkably, our results revealed that, upon target cell recognition, CD56dim NK cells were more prominent cytokine and chemokine producers than CD56bright NK cells. The present data demonstrate how specific target cell ligands dictate qualitative and temporal aspects of NK-cell cytokine and chemokine responses. Conceptually, the results point to CD56dim NK cells as an important source of cytokines and chemokines upon recognition of aberrant cells, producing graded responses depending on the multiplicity of activating receptors engaged.

scholarly journals Interplay Between SNX27 and DAG Metabolism in the Control of Trafficking and Signaling at the IS

2020 ◽  
Vol 21 (12) ◽  
pp. 4254
Author(s):  
Natalia González-Mancha ◽  
Isabel Mérida
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Communication ◽  
Negative Regulator ◽  
Cell Junction ◽  
Microtubule Organizing Center ◽  
Immune Synapse ◽  
Organizing Center ◽  
Cell Cell

Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell–cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell–cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell–cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell–APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.

Lenalidomide Enhancement of NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Is Mediated by Granzyme B and FasL and Is Associated with Modification of SHIP-1, PLC-g2 and pERK and Enhanced Chemokine Production.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4885-4885 ◽  
Author(s):  
Lei Wu ◽  
Mary Adams ◽  
Peter Schafer ◽  
George Muller ◽  
David Stirling ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Activation ◽  
Nk Cell ◽  
Granzyme B ◽  
Lymphocytic Leukemia ◽  
Activation Markers ◽  
Chemokine Production ◽  
Early Results

Abstract Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo and we have previously shown that in an in vitro ADCC system lenalidomide is able to directly enhance human NK cell and monocyte function in response to rituximab (chimeric anti-CD20 mAb) and other IgG1 antibodies. We have now addressed the effect of lenalidomide on NK cell signaling pathways and have shown that compared to the levels seen with FC gR stimulation (with IgG) plus IL-12, there is an inhibitory effect of lenalidomide on SHIP-1 with concomitant enhancement of PLC g2 and pERK. This is associated with increased secretion of a variety of chemokines as well as GM-CSF and decreased IL-6 production. In functional ADCC assays, NHL cell lines were pre-coated with rituximab and exposed to either NK cells or monocytes pre-treated with lenalidomide. After 4 (NK cells) or 15 (monocytes) hours in culture the viability of the tumor cells was assessed. The consistent enhancement of ADCC in the presence of lenalidomide is associated with greatly increased expression of granzyme B and FasL, although perforin is maximally elevated without lenalidomide. We found that ADCC is totally blocked by an inhibitor of granzyme B and is partially blocked by anti-FasL. In conclusion, we have shown that the ability of lenalidomide to enhance ADCC-mediated killing of rituximab treated CD20+ve NHL cell lines is mediated by granzyme B and FasL and is associated with clear effects on NK cell signaling and chemokine production.

Lenalidomide Alone and in Combination with Rituximab Enhances NK Cell Immune Synapse Formation in Chronic Lymphocytic Leukemia (CLL) Cells in Vitro through Activation of Rho and Rac1 GTPases.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3441-3441 ◽  
Author(s):  
Svetlana Gaidarova ◽  
JianWu Li ◽  
Laura G Corral ◽  
Emilia Glezer ◽  
Peter H Schafer ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Synapse Formation ◽  
Nk Cell ◽  
Employment Equity ◽  
Immune Synapse ◽  
Immunological Synapses ◽  
Immune Synapses ◽  
Cd20 Expression ◽  
Equity Ownership

Abstract Abstract 3441 Poster Board III-329 Background CLL is characterized by the progressive accumulation of monoclonal B lymphocytes. One theory to explain how CLL cells avoid elimination through immune surveillance mechanisms is through a defect in the ability of T-cells to form immunological synapses with antigen-presenting tumor B-cells (Ramsay et al JCI 2008). Lenalidomide is an immunomodulatory agent with clinical activity in the treatment of B-cell malignancies. Recent laboratory studies showed that lenalidomide not only stimulates T- and natural killer (NK)-cell-mediated ADCC, it also restores the T-cell-mediated ability to form immunological synapses with CLL tumor cells. Since NK cells also exert cytotoxicity through immune synapse formation, here we explore how lenalidomide affects NK-cell-mediated cytotoxicity mechanisms and whether this activity is altered in the presence of rituximab since published studies showed that lenalidomide-pretreated B-cells have a down-regulated surface CD20 expression. Further, we investigated the molecular events associated with immune synapse formation and the effect of lenalidomide. Methods Immune synapse formation was assessed in NK cells (from healthy donors PBMCs) co-cultured with either B-CLL cells derived from pts or with K562 cells (positive control). Cells were fixed and the ability to form synapses was assessed via immunohistochemisty co-staining for either F-actin and CD2, or F-actin and perforin (a cytolytic protein found in NK cells). Synapse formation was visualized by microscopy and measured via relative mean fluorescent intensity. Activity of RhoA, Rac1, Cdc42 were measured using Rho GTPases assay kits. Inhibition of lenalidomide-mediated immune synapse activity was assayed using the cell permeable Rho inhibitor C3 (0.5 mM). Flow cytometry was used to measure changes in surface CD20 and CD54 (ICAM-1) expression in B-CLL samples from 3 pts after treatment with lenalidomide. Results Lenalidomide induced the formation of immunological synapses between NK cells and primary B-CLL cells (p<.01) or the K562 cell line. Lenalidomide activated NK cells regardless of the presence of target cells, as measured by F-actin and perforin staining. RhoA and Rac1 were activated at the immunological synapse in the presence of lenalidomide. Inhibition of RhoA by the C3 inhibitor blocked F-actin localization, as well as perforin accumulation induced by lenalidomide at cell-cell contact sites, indicating inhibition of immune synapses and the associated cytolytic activity. This was also observed with Rac1 inhibition, but to a lesser degree than with RhoA inhibition. Functionality of formed synapses was confirmed by co-localization of F-actin and perforin at the synapse sites. 3 CLL pt samples treated ex vivo with lenalidomide demonstrated variable changes in CD20 expression: a 20-30% decrease in CD20 expression was observed in 2 B-CLL pt samples, whereas CD20 levels remained unchanged in the third. In the presence of rituximab, lenalidomide-induced synapse formation between NK cells and B-cells from CLL patients was further enhanced. This was accompanied by upregulation of costimulatory and adhesion molecule CD54 on B-CLL cells suggesting increased antigen presentation, which might contribute to the increased synapse formation. Conclusion Lenalidomide can directly activate NK-cell-mediated anti-tumor activity through enhanced formation of immune synapses via the regulation of Rho and Rac1 GTPases and the cytoskeleton. Despite some down-modulation of CD20 expression in lenalidomide-pretreated B-CLL cells, the immune synapse activity increases when lenalidomide is combined with rituximab suggesting that combining lenalidomide and anti-CD20 antibodies warrants exploration in the CLL clinical setting. Disclosures Gaidarova: Celgene: Employment, Equity Ownership. Li:Celgene: Employment. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Xie:Celgene: Employment. Lopez-Girona:Celgene: Employment.

Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2663-2663
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Immune Suppression ◽  
Direct Contact ◽  
Myeloid Leukemia ◽  
Synapse Formation ◽  
Nk Cell ◽  
Immune Synapse ◽  
Acute Myeloid

Abstract Abstract 2663 Poster Board II-639 Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Natural killer cell immune synapse formation and cytotoxicity are controlled by tension of the target interface

2021 ◽  
pp. jcs.258570
Author(s):  
Daniel Friedman ◽  
Poppy Simmonds ◽  
Alexander Hale ◽  
Leoma Bere ◽  
Nigel W. Hodson ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Synapse Formation ◽  
Nk Cell ◽  
Killer Cell ◽  
Blind Spot ◽  
Immune Synapse ◽  
Lytic Granules ◽  
Planar Substrates ◽  
Soft Targets

Natural Killer (NK) cells can kill infected or transformed cells via a lytic immune synapse. Diseased cells may exhibit altered mechanical properties but how this impacts NK cell responsiveness is unknown. We report that human NK cells were stimulated more effectively to secrete granzymes A and B, FasL, granulysin and IFNγ, by stiff (142 kPa) compared to soft (1 kPa) planar substrates. To create surrogate spherical targets of defined stiffness, sodium alginate was used to synthesise soft (9 kPa), medium (34 kPa), or stiff (254 kPa) cell-sized beads, coated with antibodies against activating receptor NKp30 and the integrin LFA-1. Against stiff beads, NK cells showed increased degranulation. Polarisation of the microtubule-organising centre (MTOC) and lytic granules were impaired against soft targets, which instead resulted in the formation of unstable kinapses. Thus, by varying target stiffness to characterise the mechanosensitivity of immune synapses, we identify soft targets as a blind spot in NK cell recognition.

scholarly journals CD38 contributes to human natural killer cell responses through a role in immune synapse formation

2018 ◽  
Author(s):  
Mathieu Le Gars ◽  
Christof Seiler ◽  
Alexander W. Kay ◽  
Nicholas L. Bayless ◽  
Elsa Sola ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Synapse Formation ◽  
Cell Function ◽  
Nk Cell ◽  
Critical Role ◽  
Target Cells ◽  
Immune Synapse ◽  
Ifn Γ

AbstractNatural killer (NK) cells use a diverse array of activating and inhibitory surface receptors to detect threats and provide an early line of defense against viral infections and cancer. Here, we demonstrate that the cell surface protein CD38 is a key human NK cell functional receptor through a role in immune synapse formation. CD38 expression marks a mature subset of human NK cells with a high functional capacity. NK cells expressing high levels of CD38 display enhanced killing and IFN-γ secretion in response to influenza virus-infected and tumor cells. Inhibition of CD38 enzymatic activity does not influence NK cell function, but blockade of CD38 and its ligand CD31 abrogates killing and IFN-γ expression in response to influenza-infected cells. Blockade of CD38 on NK cells similarly inhibits killing of tumor cells. CD38 localizes and accumulates at the immune synapse between NK cells and their targets, and blocking CD38 severely abrogates the ability of NK cells to form conjugates and immune synapses with target cells. Thus, CD38 plays a critical role in NK cell immune synapse formation. These findings open new avenues in immunotherapeutic development for cancer and infection by revealing a critical role for CD38 in NK cell function.

Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3029-3029
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Nk Cell ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Cytolytic Activity ◽  
Lymphocyte Function ◽  
Immune Synapse

Abstract Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. Disclosures No relevant conflicts of interest to declare.

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