Natural Killer Receptor Expression Research Articles

Abstract 2641: Natural Killer Cell Receptors Distribution In Patients With Carotid Atherosclerosis According To Clinical Expressivity

Background: Natural killer (NK) cells are lymphocytes involved in innate immune defence against viral infections whose function is controlled by a balance of inhibitory and activating NK receptors (NKR), shared by some T cell subsets. Healthy individuals infected with human cytomegalovirus (HCMV), a pathogen related to the development of atherosclerosis, display variable increased proportions of NK cells expressing the CD94/NKG2C activating receptor specific for HLA-E. On the other hand, some infections including HCMV may also increase the numbers of NK and T cells bearing LILRB1, an inhibitory receptor specific for HLA class I molecules expressed at late differentiation stages. LILRB1+ NK and T cells have been recently associated to acute myocardial infarction and carotid intima-media thickness, supporting the infectious burden hypothesis in the pathogenesis of atherosclerosis. Objective: To evaluate NKR expression (NKG2C, NKG2A, LILRB1 and KIRs) in patients with carotid atherosclerosis according to their clinical expressivity seeking for a potential marker of high-risk carotid plaques. Patients and methods: Peripheral blood was drawn from 30 patients with carotid stenosis higher than 50% classified as asymptomatic (n=11) and symptomatic (transient ischemic attack, n=10; acute ischemic stroke, n=9). HCMV serology and plasma levels of CRP were analysed in all patients. NKR expression by NK cells and CD8+ T lymphocytes were evaluated by immunofluorescence staining and flow cytometry analysis. Results: Twenty-nine patients with carotid atherosclerosis were HCMV+. NKG2C expression by NK cells was similar in asymptomatic and symptomatic patients (17.6% ± 18.0 vs. 20 ± 19.2, p 0.7); moreover, both groups displayed as well a comparable distribution of other NKR, including LILRB1, by NK cells and CD8+ T lymphocytes. Yet, a positive correlation between the proportions of NKG2C+ NK cells and CRP plasma levels (Spearman correlation coefficient 0.5, p<0.05) was observed in symptomatic patients. In addition, LILRB1 expression by CD8+ T lymphocytes was directly related with age in the group of symptomatic patients (r 0.7, p<0.001) but not in asymptomatic patients (r 0.1, p=0.8). Conclusion: Patients at increased risk of developing an acute stroke secondary to carotid atherosclerosis display an NKR expression pattern that might be linked to chronic inflammation; its putative relationship with the infectious burden hypothesis deserves further attention.

Dynamic Shift from CD85j/ILT-2 to NKG2D NK Receptor Expression Pattern on Human Decidual NK during the First Trimester of Pregnancy

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1+/KIR2DL2/L3/S2+ subset towards the double negative subset, coupled with a decrease of the CD85j+/NKG2D− subset in favour of the CD85j−/NKG2D+ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14+ and CD3+ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14+ and CD3+ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14+ cells whereas it was not detected on CD3+ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells

AbstractSoluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G–mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56bright and CD56dim NK cells. Such sHLA-G–mediated down-modulations were reverted by adding anti–HLA-G or anti–ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G–treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G–mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G–mediated inhibition of NK-cell functions.

Role of NKG2D in Cytokine-Induced Killer (CIK) Cells Against Multiple Myeloma Cells

Abstract 5119Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures:No relevant conflicts of interest to declare.

Differential Expression of NK Receptors CD94 and NKG2A by T Cells in Rheumatoid Arthritis Patients in Remission Compared to Active Disease

ObjectiveTNF inhibitors (TNFi) have revolutionised the treatment of rheumatoid arthritis (RA). Natural killer (NK) cells and Natural Killer Cell Receptor+ T (NKT) cells comprise important effector lymphocytes whose activity is tightly regulated through surface NK receptors (NKRs). Dysregulation of NKRs in patients with autoimmune diseases has been shown, however little is known regarding NKRs expression in patients with TNFi-induced remission and in those who maintain remission vs disease flare following TNFi withdrawal.MethodsPatients with RA were recruited for this study, (i) RA patients in clinical remission following a minimum of one year of TNFi therapy (n = −15); (2) Active RA patients, not currently or ever receiving TNFi (n = 18); and healthy control volunteers (n = 15). Patients in remission were divided into two groups: those who were maintained on TNFi and those who withdrew from TNFi and maintained on DMARDS. All patients underwent full clinical assessment. Peripheral blood mononuclear cells were isolated and NKR (CD94, NKG2A, CD161, CD69, CD57, CD158a, CD158b) expression on T-(CD3+CD56−), NK-(CD3−CD56+) and NKT-(CD3+CD56+) cells was determined by flow cytometry.ResultsFollowing TNFi withdrawal, percentages and numbers of circulating T cells, NK cells or NKT cell populations were unchanged in patients in remission versus active RA or HCs. Expression of the NKRs CD161, CD57, CD94 and NKG2A was significantly increased on CD3+CD56-T cells from patients in remission compared to active RA (p<0.05). CD3+CD56-T cell expression of CD94 and NKG2A was significantly increased in patients who remained in remission compared with patients whose disease flared (p<0.05), with no differences observed for CD161 and CD57. CD3+CD56− cell expression of NKG2A was inversely related to DAS28 (r = −0.612, p<0.005).ConclusionHigh CD94/NKG2A expression by T cells was demonstrated in remission patients following TNFi therapy compared to active RA, while low CD94/NKG2A were associated with disease flare following withdrawal of therapy.

Natural killer cell phenotype and clinical response to interferon-beta therapy in multiple sclerosis

CD56(bright) NK cells, which may play a role in immunoregulation, are expanded in multiple sclerosis (MS) patients treated with immunomodulatory therapies such as daclizumab and interferon-beta (IFNβ). Yet, whether this NK cell subset is directly involved in the therapeutic effect is unknown. As NK receptor (NKR) expression by subsets of NK cells and CD8+ T lymphocytes is related to MS clinical course, we addressed whether CD56(bright) NK cells and NKR in IFNβ-treated MS patients differ according to the clinical response. IFNβ was associated to lower LILRB1+ and KIR+NK cells, and higher NKG2A+NK cell proportions, an immunophenotypic pattern mainly found in responders. After IFNβ treatment, a CD56(bright) NK cell expansion was significantly related to a positive clinical response. Our results reveal that IFNβ may promote in responders changes in the NK cell immunophenotype, corresponding to the profile found at early maturation stages of this lymphocyte lineage.

Expression Profiles of Peripheral CD160+ Lymphocytes During the Course of Healthy Human Pregnancy

CD160 receptor is expressed by natural killer (NK) and T-cell subsets, and after activation, it could enhance cytotoxicity or pro-inflammatory cytokine production on NK cells. Here, we investigated the phenotype of peripheral CD160+ cells during healthy pregnancy. We analyzed the expression of CD69 activation marker, gamma/delta TCR, and NKG2A or NKG2D NK cell receptors on CD160+ lymphocytes of non-pregnant and healthy pregnant women at four different stages of pregnancy by flow cytometry. In our hands, CD160 receptor-positive lymphocytes were present during pregnancy; however, they had different characteristics depending on gestational age. During implantation, CD160+ cells showed low activation rate, decreased NK receptor expression while 40% of Vδ2 + T cells expressed CD160 receptor. In turn, all the above parameters increased as pregnancy proceeds. Our results indicate that CD160+ lymphocytes could be able to play a role in the maintenance of healthy pregnancy.

CD200 expression suppresses natural killer cell function and directly inhibits patient anti-tumor response in acute myeloid leukemia

Upregulation of the immunosuppressive cell surface glycoprotein, CD200, is a common feature of acute myeloid leukemia (AML) and is associated with poor patient outcome. We investigated whether CD200 overexpression on AML cells could specifically compromise patient natural killer (NK) cell anti-tumor responses. We found that CD200(hi) patients showed a 50% reduction in the frequency of activated NK cells (CD56(dim)CD16(+)) compared with CD200(lo) patients. Additionally, NK receptor expression (NKp44 and NKp46) on these cells was also significantly downregulated in CD200(hi) patients. To assess whether NK cell activity was directly influenced by CD200 expression, we examined the effect of ectopic expression of CD200. These assays revealed that both NK cell cytolytic activity and interferon-γ response were significantly reduced toward CD200(+) leukemic targets and that these targets showed increased survival compared with CD200(-) cells. Similarly, NK cells isolated from AML patients were less functionally active toward CD200(hi) autologous blasts from both cytolytic and immunoregulatory perspectives. Finally, blocking CD200 alone was sufficient to recover a significant proportion of NK cell cytolytic activity. Together, these findings provide the first evidence that CD200 has a direct and significant suppressive influence on NK cell activity in AML patients and may contribute to the increased relapse rate in CD200(+) patients.

Age-related changes in natural killer cell receptors from childhood through old age

Most studies on natural killer (NK) cells and aging have focused on overall cell numbers and global cytotoxic activity. NK cell functions are controlled by surface receptors belonging to three major families: killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), and C-type lectins. The expression of these receptors was investigated from childhood through old age in T, NKT- and NK cells and also in the CD56dim (cytotoxic) and CD56bright (responsible for cytokine production) NK cell subsets. A decrease in the expression of activating receptors (NKp30 and NKp46) was observed in NK cells in elderly individuals. KIR expression was increased only in the CD56bright subset. Children presented similar results regarding expression of NKp30 and KIR, but not NKp46. NKG2D expression was decreased in T cells of elderly subjects. Analysis of KIR genotype revealed that KIR2DL5 and KIR2DS3 were significantly associated with old age. Cytotoxic activity was preserved from childhood through old age, suggesting that the increase of the absolute number of CD56dim, observed in elderly, may represent a compensatory mechanism for the receptor expression alterations. This initial study provides the framework for more focused studies of this subject, which are necessary to determine whether the changing balance of NK receptor expression may influence susceptibility to infectious, inflammatory, and neoplastic diseases.

Alteration of inhibitory and activating NK cell receptor expression on NK cells in HIV-infected Chinese

Natural killer (NK) cell function, based on the expression of activating and inhibitory natural killer receptors (NKRs), may become abnormal during human immunodeficiency virus (HIV) infection. In this study, we investigated changes in receptor expression with individual and combinational analysis on NK cell subsets in HIV-infected Chinese. The results showed that natural killer group 2 member D (NKG2D) expression on total NK cells decreased significantly in HIV infection, while the expressions of natural killer group 2 member A (NKG2A) and killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 (KIR3DL1) on total NK cells were not significantly different between any of the groups including HIV-positive treatment-naïve group, AIDS treatment-naïve group, HAART-treatment AIDS group and HIV-negative control group. Individual analysis of NKG2A + and KIR3DL1 + cells revealed no significant differences in expression in any NK cell subsets between any of the groups, but the combinational analysis of NKG2D −NKG2A +, and NKG2D −KIR3DL1 + on the NK CD56 dim cell subset in the AIDS group were increased compared to the HIV-negative control group. On the contrary, NKG2D −NKG2A + expression on the CD56 bright subset decreased in the AIDS group compared to the control group. Highly active antiretroviral therapy (HAART) treatment almost completely restored the levels of these receptor expressions. The results indicate that the distinct alteration of activating and inhibitory NKR expression on NK cells and its subsets occurred during HIV progression. Moreover, the imbalanced change of activating and inhibitory NKRs on NK cells and its subsets may explain the impaired NK cell immunity in HIV infected individuals.

TOX regulates the differentiation of human natural killer cells from hematopoietic stem cells in vitro

Natural killer (NK) cells act important roles in innate immunity and adaptive immunity. However, the mechanisms governing NK cell development have not been clearly elucidated. Previous studies have shown that an HMG (high-mobility group) protein, TOX, is important for regulating the differentiation program of developing T cells in mice. In this study, we examined the role of TOX in differentiation of human NK cells. Knockdown of TOX in differentiating cells decreased the NK cell population identified by expression of NK surface markers and receptors. In addition, over-expression of TOX enhanced the differentiation of NK cells which give rise to a population showing effector functions of mature NK cells. Moreover, TOX influenced expression of T-bet (T-box expressed in T cells, also as known as Tbx21) during NK cell development. Overall, these results suggest that TOX is required for IL-15-mediated NK cell differentiation and affected expression of T-bet that plays critical roles in NK differentiation and maturation.

IL-15 induces CD8+ T cells to acquire functional NK receptors capable of modulating cytotoxicity and cytokine secretion

During the last years several authors have described a small population of CD8+ T cells expressing NK receptors (NKRs). Although their origin remains largely unknown, we have recently demonstrated that IL-15 is capable of inducing NKR expression in purified human CD8+CD56− T cells. In this study we show that IL-15-driven NKR induction in CD8+ T cells was linked with CD56 de novo acquisition, consistent with an effector-memory phenotype, increased anti-apoptotic levels, high granzyme B/perforin expression and with the ability of displaying in vitro NK-like cytotoxicity. Interestingly, dissection of NKR functional outcome in IL-15-cultured CD8+ T cells revealed: (i) that NKG2D cross-linking was able per se to upregulate degranulation levels and (ii) that KIR and NKG2A cross-linking upregulated secretion of cytokines such as IFN-γ, TNF-α, IL-1β and IL-10. These results suggest that IL-15 is capable of differentiating CD8+ T cells into NK-like T cells displaying a regulatory phenotype.

Interleukin‐10 Promotes NK Cell Killing of Autologous Macrophages by Stimulating Expression of NKG2D Ligands

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.

Altering the specificity of NK:target cell interactions by genetic manipulation of NK receptor expression on primary mouse NK cells

A balance of signals generated via stimulatory and inhibitory NK receptors determines both target cell specificity and the outcome of NK–target cell interactions. The feasibility of introducing naturally occurring or genetically engineered chimeric NK receptors at the effector cell level may prove useful in NK cell-based immunotherapies. Here, we utilized a previously established lentiviral transduction system to over-express a model NKR-P1B inhibitory receptor on primary mouse NK cells. These genetically engineered NK cells became more sensitive to inhibitory signals delivered by target cells expressing the cognate NKR-P1B ligand, Ocil/Clr-b. This study demonstrated the utility of lentiviral vectors as a means to stably manipulate the target cell specificity of primary NK cells.

Expression of natural killer receptors in T- and NK-cells: comparison of healthy individuals, patients with prior stem cell transplant, and patients undergoing chemotherapy

We studied the expression of natural killer receptors (NKRs) on peripheral blood cytotoxic T lymphocytes and NK cells in patients who underwent an allogeneic stem cell transplant (SCT), and compared these findings with results from healthy individuals (CTRL) and patients undergoing chemotherapy (CHEMO), respectively. Peripheral blood mononuclear cells were analyzed by flow cytometry with antibodies against the NKRs CD158a, CD158b, CD158e (known as killer immunoglobulin-like receptors, KIRs), and CD94. Expression of NKRs was evaluated separately in CD56(+), CD57(+), and CD56/CD57 (double +) subsets of T and NK cells. We found mainly differences in CD158a and CD94 expression between the three cohorts, with the SCT and CHEMO groups usually showing similar changes, when compared to the CTRL population. None of the patients with SCT or CHEMO demonstrated patterns of restricted NKR expression. Our results provide a comprehensive overview of KIR and CD94 expression in T and NK cells following SCT and chemotherapy.

A Novel Method Of Genetically Engineered K562 Cells To Significantly Expand Cord Blood (CB) NK Cells And Increase CB NK Receptor Expression And NK Cell Activation

A Novel Method Of Genetically Engineered K562 Cells To Significantly Expand Cord Blood (CB) NK Cells And Increase CB NK Receptor Expression And NK Cell Activation

A Phase I Study of the Anti-Natural Killer Inhibitory Receptor (KIR) Monoclonal Antibody (1-7F9, IPH2101) in Elderly Patients with Acute Myeloid Leukemia (AML): Clinical and Immunological Effects of a Single Dose Followed by Repeated Dosing.

Abstract 632Background: Non cytotoxic treatments such as immunotherapy represent promising approaches for the post-remission therapy of elderly patients with AML for which relapse free survival is short. The therapeutic potential of natural Killer (NK) cells has been revealed by the KIR mismatch allogeneic transplant model where the anti-leukemic effect of the graft is due to unleashed NK cells towards AML blasts, as suggested by enhanced in vitro NK lytic activity of KIR-HLA mismatched donor NK against recipient blasts (Miller et al. 2005; Ruggeri et al. 2002). To mimic this effect with a pharmaceutical agent, a fully human IgG4 anti-KIR mAb specific for KIR2DL1/2/3 (HLA-C specific KIRs), the 1-7F9/IPH2101, was generated (Romagne et al., Blood 2009). We present the results of the First in Human phase I trial of this agent in patients with AML in complete remission (CR). Methods: Patients aged 60-80 years with non promyelocytic AML in first CR following induction and 1-6 cycles of consolidation chemotherapy, normal renal and hepatic functions, KIR-expression on NK-cells and who signed informed consent were eligible for Protocol IPH2101-101. Dose escalation (IPH2101: 0.0003, 0.003, 0.015, 0.075, 0.3, 1, 3 mg/kg as iv infusion) was studied using a 3+3 scheme. Patients still in CR who had tolerated the single dose administration were eligible for an extension trial (Protocol IPH2101-102) investigating repeated doses of IPH2101 (1/ month x 6 months) using the dose the patient was allocated to in protocol IPH2101-101. Pharmacokinetic (PK) and circulating cytokines (IL-1b, IL-6, IFN-g, MIP-1β and TNF-a) were measured by ELISA. KIR occupancy and activation markers (CD69) were monitored by flow cytometry. Results: 23 patients, median age 71 years (range 61-79) in first CR of AML for a median of 20 weeks have been included in IPH2101-101. 3 patients (13%) had achieved CR after 2 induction courses, 5 patients (22%) had unfavorable cytogenetics, and 5 patients (22%) had high WBC at diagnosis. Planned dose escalation has been completed and no Dose Limiting Toxicity (DLT) has been recorded. 2 patients (in DL 4 and 5) have been replaced (one relapsed before 4 weeks of follow-up; the other had no detectable antibody after injection). Related Adverse Events seen in 14/23 patients (61%) were mild (grade ≤2) and transient. For doses above 0.075 mg/kg, ½ life ranged between 11-28 days. For doses above 0.3mg/kg, full saturation (>90% KIR occupancy) has been observed for 28 days to up to 6 weeks (at the 3 mg/kg DL). IPH2101 had no effect on the number and distribution of the peripheral lymphocyte subsets or the expression of NK receptors (KIRs, CD94/NKG2A, CD85j, NKp46, NKp30 or NKG2D). NK cell function tested by ex vivo cytotoxicity assay was not impaired after treatment. From dose 1 mg/kg (5/6 patients), IL-1b and IL-6 increased 6h post-dosing (2 to 8 fold increase and 2 to 15 fold increase compare to pre-dose, respectively) and decreased to pre-dose concentrations within 24h. Furthermore increase in TNF-a during the first hour was often correlated with CD69 up-regulation on NK cells. 8 patients received repeated doses of IPH2101 (0.0003 mg/kg: 2 patients, 0.003 mg/kg: 2 patients, 0.015 mg/kg: 1 patient, 1 mg/kg: 3 patients, 3 mg/kg pending) as part of the extension trial. No grade 3-4 related toxicities were observed. 2 patients completed the 6 courses, 5 patients were withdrawn for relapse and 1 patient is still ongoing at 1mg/kg. In accordance with the pre-clinical PK/PD model there is a clear relationship between exposure (Cmax) and KIR occupancy. Moreover the inter-patient variability is low (PK data) to moderate (KIR occupancy data). Median follow-up is 47 weeks. Among the 21 evaluable patients 11 patients are alive, 15 relapsed with 10 death, 6 are still in remission (1 patient in DL1, 2 patients in DL6 and 3 patients in DL7). Conclusions: Anti KIR treatment is safe and well tolerated and MTD has not been reached for doses producing full KIR saturation for 4 weeks, confirming the specificity of the immunological effects. Retreatment for prolonged period of time is feasible and safe. Signs of NK cell activation can be observed for doses above 0.3 mg/kg. IPH2101 deserves further investigation in patients with AML. Disclosures:Vey:INNATE PHARMA: Consultancy. d'Arnoux:INNATE PHARMA: Employment. Romagne:INNATE PHARMA: Employment. Andre:INNATE PHARMA: Employment. Tiollier:INNATE PHARMA: Employment. Squiban:INNATE PHARMA: Employment. Blaise:INNATE PHARMA: Consultancy. Olive:INNATE PHARMA: Research Funding.

Significant Ex-Vivo Expansion and Functional Activation of Cord Blood (CB) Natural Killer (NK) Cells with A Concomittant Significant Decrease in CB T Cells Following Stimulation with Genetically Engineered K562 Cells (K562-mb15-41BBL).

Abstract 499 Introduction:CD56+ NK subsets exhibit differential NK receptors (NKR) such as cytotoxicity profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition, which, in part, may play a role in adoptive cellular immunotherapy (ACI) for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by triggering receptors such as NCR (i.e. NKp46), and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opin in Immunol, 2004, Marcenaro et al, Eur J Immunol, 2003). The major limitations of the use of NK cells in ACI include lack of tumor recognition and/or limited numbers of viable and functionally active NK cells (Shereck/Cairo et al. PBC, 2007). To circumvent these limitations, methods to expand and activate PB NK cells by genetic reengineering have been developed (Imai/Campana et al. Blood, 2005). It has been demonstrated that PB NK cells expanded with modified K562 cells expressing membrane bound IL-15 and 4-1BBL (K562-mb15-41BBL; Imai et al Blood, 2005) are significantly increased in number and maintain heterogeneous KIR expression (Fusaki/Campana et al, BJH, 2009) .We have previously reported the ex-vivo expansion, activation and cytolytic activity of CB NK cells with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006; Ayello/Cairo, Exp Hem, 2009, In Press). Objective:In this study, we compared CB NK expansion and activation following stimulation with genetically engineered K562 cells (K562-mb15-41BBL, generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, TN) with wild-type (WT) K562 cells and NK cell characterization expressing inhibiting and activating KIRs, c-lectin, NCRs and NK cytolytic activation. Methods: Following irradiation with 100Gy, K562-mb15-41BBL or WTK562 were incubated at a 1:1 ratio with fresh CB MNCs at 37C, 5% CO2 for 7 days in RPMI-1640+10IU IL-2. NKR expression (KIR2DS4, NKG2D, NKG2A, CD94, KIR3DL1, KIR2DL2, Nkp46) and LAMP-1 (CD107a) receptor expression and NK cell phenotype (CD56 dim and bright subsets) were determined by flow cytometry. Results: On Day 0, NK cells population was 3.9±1.3%. After 7 days in culture, CB NK cells were significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). Concomitantly, there was a significant decrease in CB T cells vs WTK562 or media alone (15±2 vs 36±2 vs 51±7%, p<0.001),respectively. There was a significant increase in CD56bright vs CD56dim populations (67 vs 33%, p<0.01) following stimulation with K562-mb15-41BBL. Also, there was a 10-fold increase in CB NK cells expressing KIR3DL1 following stimulation with K562-mb15-41BBL vs WTK562 (p<0.01) and a 5-fold increase in NK KIR2DS4 expression (p<0.05), respectively. There was a significant increase in the expression of NK activation marker, CD107a, compared to WTK562 (51±0.7 vs 32±1.1,p<0.05). There was no change in CB NK cell expression of the c-lectin receptor, CD94/NKG2A and CD94/NKG2D after stimulation with K562-mb15-41BBL. A standard cryopreserved CB unit (25 ml) contains approximately 750×106 MNC. By using the smaller 5-ml aliquot (20%) of a two-aliquot bag (150×106 MNCs × 3.9%=5.8×106 NK cells), this expansion method would hypothetically yield 200×106 CB NK cells after 7 days stimulation with K562-mb15-41BBL. Conclusion:These results suggest that CB MNC can be ex-vivo expanded with K562-mb15-41BBL resulting in specific expansion of CB NK cells with increased NK KIR expression (KIR2DS4 and KIR3DL1) and NK activation (CD107a), along with a significant decrease in CB T cells. This expansion provides a means to enhance specific CB NK cell expansion for possible use for adoptive cellular immunotherapy in the post UCBT setting Disclosures:No relevant conflicts of interest to declare.

NK Cytotoxicity Is Decreased During the Early Posttransplant Period Following Pediatric Allogeneic Transplantation From Unrelated Donors Using CD3/CD19-Depleted Graft.

Abstract 2434Poster Board II-411 Introduction and objectives:Unrelated donors, match unrelated (MUD) and haploidentical donors (HSCT), have been described as a therapeutic option for high-risk childhood acute leukemia. CD3/CD19 depleted graft has been used in order to decrease the incidence of graft versus host disease (GvHD) and post-transplant lymphoproliferative disease, in the unrelated transplantation setting. Donor-derived NK cell alloreactivity has been reported to mediate early graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation. NK cells are components of the innate immunity playing an important role in the surveillance of human tumors. NK cell recognition of malignant cells depends on a dynamic balance between activating and inhibitory receptors. NK cell alloreactivity can be predicted by donor Killer Immunoglobulin like Receptors (KIRs), Natural Killer Receptors (NCRs), C-type Lectin receptors (NKG2D), Toll Like Receptors (TLRs) and recipient human leukocyte antigen (HLA) class I alleles as ligands. Reduced risk of relapsed has been described in malignant cancer after haploidentical stem cell transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient (KIR–HLA receptor–ligand mismatch). We prospectively investigated NK function and NK reconstitution in 18 CD3/CD19 depleted graft unrelated hematopoietic stem cell transplantation (7 MUD and 11 HSCT) using fludarabine-based reduced intensity conditioning regimen. Results:NK cells peaked around day 30 after transplantation. The median number of NK cells on day +30 was 403±88/μL . On day 100 after transplantation the median number of NK cells/μL was 221±58. While the CD56bright NK cell subset was above normal during the first 100 days post-transplant, the “effector” NK cell subset, CD56dim CD16bright, was significantly reduced early after transplantation. The median percentage of CD56bright cells among NK cells in peripheral blood was 25.8±4.6% at day +30, and it was 24.5±5.7 at day +100. The decreased in CD56dim CD16bright NK cell subset was correlated with the decreased of the inhibitory KIR receptors (KIR2DL1, KIR2DL2, KIR3DL1) expression. We also observed a lower expression than donors of the activating receptors NKG2D, TLR4 at day +30, NKp46, TLR 9 at day 60 and NKp46, NKp30 at day +100. Although absolute NK-cell counts rapidly increased after transplant, their cytotoxicity against K562 was much lower compared to that of their donors. At day 100 after transplantation, patients NK cytotoxicity was lower than donor values. These results suggest that the low NK cell cytotoxicity could be related to an “immature” NK phenotype during the early period after HSCT. As other authors have published, activating receptors can be significantly upregulated in cytokine-stimulated NK cells. In our experience, overnight incubation with IL-15 overcomes this limitation, enhancing three times NK cytotoxicity, in vitro. Conclusion:The phenotype of NK cells and NK cytotoxicity ability are significantly altered early after allogeneic transplantation from unrelated donors using CD3/CD19-depleted graft. NK repertoire observed in patients was associated with the imbalance between CD56bright and CD56dim NK subsets and the expression of KIRs and NCRs. These data suggest a pattern consistent with an ongoing NK maturation after MUD and HSCT transplantation. In our experience, the phenotype and functional pattern of NK cells observed is suggestive of a cytokine-driven process. IL-15 stimulated NK cells could be helpful to optimize adoptive antitumor NK immunotherapy to enhance GvL effect as early as possible after transplantation. Disclosures:No relevant conflicts of interest to declare.

Analysis of NK Cells Receptors and NK Cells Ligands in Acute Myeloid Leukemia: Comparisons Between the Blood and the Bone Marrow.

Abstract 1643Poster Board I-669 BackgroundThe significant role of NK cells in the control of acute myeloid leukemia (AML) has been demonstrated in the setting of allogeneic stem cell transplantation. However, the implication of NK cells against autologous leukemic cells needs to be clarified.We have previously described deficient expression of NK activatory receptors in AML at diagnosis, in particular the natural cytotoxicity receptors (NCR) namely NKp30 and NKp46. So, defective cytotoxicity of AML cells can be explained by abnormalities of activating NK-receptor expression allowing immune escape from NK cells. However, immune escape can be also due to defective activating NK receptor-ligand interactions due to abnormal expression of their ligands on blasts cells. These defects have been observed in particular on NK cells or blasts cells isolated from the blood. Few studies have analysed the bone marrow component although blasts cells concentrate here. We postulated that abnormalities of NK cells receptors or ligands expression are more severe in bone marrow, in near contact with the blasts, compared with blood. We sought to identify disparities between deficient expression of NK or ligands in the bone marrow in comparison with the blood. MethodsWe realized a phenotypic analysis of NK cells and blasts cells at the diagnosis of AML. The level of activatory NK receptors (NKRa) knew to mediate NK cell recognition and lysis of AML blasts cells (NCR (NKp30 and NKp46) and DNAM-1) was investigated by flow cytometry. The expression of NKG2D ligands (MICA/B and ULBP1-3) and DNAM-1 ligands (Nectin-2 and PVR) receptors were also analysed. These analyses were realised with coupled specimens obtained in the same patient at diagnosis of AML (n=19), peripheral blood and bone marrow samples in order to detect discrepancies between these two sites. A control group (age-matched; n=15) for blood samples was included for this study. All biological samples were obtained from patients and healthy volunteers after informed consent. ResultsA total of 19 patients were included in this study. We included 6 cases of AML 5, 4 cases of AML 4, 4 cases of AML 2, 4 cases of AML 1 and one case of AML 0. Flow cytometry data for NKRa were only available for 11 patients. We confirmed the deficient expression of NKp30 and NKp46 receptors (as determined by MFI) on NK cells from blood of AML patients. In AML patient, the ratio MFI (MFI receptor/MFI control isotype) of NKp30 (4.27 +/- 2.97; p<0.0001) and of NKp46 (5.96 +/- 5.67; p<0.0001) significantly differed from healthy volunteers (NKp30 26.65 +/- 6.12; NKp46 39.73 +/- 9.66). Moreover, the deficient expression of these receptors was also observed on NK cells from the bone marrow (NKp30 3.66 +/- 2.22, p<0.0001; NKp46 6.71+/- 6.42, p<0.0001). However, we can not demonstrated significant differences between the NKRa expression on NK cells from blood versus from bone marrow (NKp30 p= 0.8438; NKp46 p= 0.9476 and DNAM-1 p= 0.3579).The expression of the ligands for NKRa was analysed to compare the expression on blasts cells isolated from the blood compared to blasts cells isolated from the bone marrow. Flow cytometry data for ligands were only available for 17 patients. We observed a strong expression of HLA class I molecules on blasts cells that was equivalent in the blood and in bone marrow. DNAM-1 ligands (PVR, Nectin 2) were expressed on blasts cells (see figure). NKG2D ligands were also expressed but to a lesser extent with predominant ULBP1 expression. However, we can not observed significant differences in the expression of ligands between the blood and the bone marrow. ConclusionsThe deficiency of activating NK cells receptors expression at AML diagnosis is significant, present in a majority of patients, and consistent across the 2 components, ie blood and bone marrow. These defects are one component of the immune escape from NK cells. We have speculated that these abnormalities were more pronounced in bone marrow, near blasts cells, because these abnormalities are in part induced by blasts cells. However, we can not demonstrated significant differences in the expression of activating receptors or ligands between blood and bone marrow. We are accumulating more data in order to detect differences between sub-groups of AML. DisclosuresNo relevant conflicts of interest to declare.