Activating Natural Killer Receptors 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.

Yet another role for natural killer cells: Cytotoxicity in immune regulation and viral persistence

At early times during viral infections, natural killer (NK) cells can contribute to direct antiviral defense (1, 2). The effect is most dramatic when they are stimulated to release the contents of their cytolytic granules for lysis of virus-infected target cells. Type 1 interferons (IFNs), produced during innate responses to infections, induce elevated NK cell cytotoxicity. Killing, however, is ultimately dependent on signaling through NK activating receptors by ligands expressed on target cell surfaces. A complex repertoire of germ-line genes encodes NK receptors (2). Some of these are conserved, whereas others are polygenic and polymorphic. At later times, adaptive immunity, such as that delivered by cytotoxic CD8 T cells, mediates protection. The kinetics and magnitudes of viral replication compared with developing T-cell responses can shift the balance between the virus and an infected host through three states: viral clearance with long-lived immunity, immune-mediated pathology with life-threatening conditions, and functional exhaustion of T cells with resulting chronic infection. The report by Lang et al. in PNAS (3) provides evidence of a role for NK cell cytotoxicity in limiting CD8 T-cell responses, to result in persistent viral infection as well as elevated infection-induced disease. This immunoregulatory function suggests that NK cells direct the balance between the virus and the host, but the results must be considered in the context of the targets of NK cell-mediated lysis, the race in establishing states of infection, and the complexities associated with the experimental systems used to identify the effects.

Defective Triggering of NK Cells Results in Primary CLL Cells Resistance to Cytotoxicity,

Abstract 3876Chronic lymphocytic leukemia (CLL) remains an incurable disease except after allogenic transplantation. Natural killer (NK) cells are one of the main effectors of immune surveillance involved in tumor control. Alterations of NK cells functions have been well characterized in myeloid malignancies. However the role of NK cells in immune escape of CLL in less known and controversial. Here we describe extensive phenotypic and functional characterization of NK cells and primary CLL cells and their interactions in vitro and in vivo.Twenty eight untreated CLL patients, twenty four age-matched healthy donors and ten AML patients were enrolled in the study.We have previously shown that expression and function of NK cell-triggering receptors is defective in AML. We then assessed the phenotypic and functional properties of NK cells from CLL patients. Unlike the results found in AML, no significant differences were observed in term of activating receptors, NKp46, DNAM-1, NKG2D, 2B4 and CD16. Only the natural cytotoxicity receptor (NCR) NKp30 was weakly decreased compared to healthy donors (p=0.0107). There wasn't any difference in the expression of inhibitory receptors CD158a, b, e, ILT2 and NKG2A. Looking at the spontaneous NK-mediated cytotoxicity, CLL NK cells displayed a cytolytic activity similar to that of healthy donors against K562 cell line. To further evaluate the functional consequences of the decreased expression of NKp30, mAb redirected killing assays was performed against P815 cell lines. The NK cells killing was slightly lower in CLL patients compared to healthy donors when anti-NKp30 was used although no difference could be observed with anti-NKp46 and anti-CD16. All these results supported that NK cells cytotoxicity should be effective in CLL.We then studied the susceptibility of CLL B cells to allogenic NK killing both in vitro and in vivo. Unlike AML cells and K562 cells, CLL cells were resistant to NK cytotoxicity mediated by resting cells. Exogenous stimulation of allogenic NK cells with IL2 and IL15 restored partially CLL killing, which was nevertheless still lower than AML blasts and K562 cells killing (p=0.0288 and <0.0001 respectively). Murine xenotransplantation model using NOD/SCID g null (NSG) mice allowed us to study the anti-leukemic capacity of purified NK cells after activation with IL2. We didn’t observe any clearance of CLL cells after allogenic NK cell injection while CLL and NK cells were checked to be present in blood, bone marrow, spleen and liver. These experiments confirmed the CLL resistance to NK-mediated killing.To investigate the potential mechanisms of this resistance, we analyzed the surface expression of ligands for activating and inhibitory NK receptors on CLL cells. CLL cells displayed poor expression of ligands for activating NK receptors MICA/B, ULBP1-3, PVR, nectin-2 and CD54. Interestingly, this profile of surface expression was similar to that of normal B cells except a slight increase of ULBP3 expression on CLL cells. Regarding ligands for inhibitory NK receptors, HLA-class I molecules were significantly down-regulated while HLA-E tended to be up-regulated on CLL cells compared to normal B cells.Finally, we tested ADCC in order to overcome the resistance of CLL cells to NK killing: the presence of rituximab increased significantly CLL lysis. Of note, priming of NK cells with IL2+IL15 still increased CLL cytotoxicity (p<0.0001).Our findings demonstrate that primary CLL cells are resistant to NK mediated killing. This defect is mainly due to the lack of ligands for NK receptors on CLL cells surface leading to deficient triggering of NK cells. However NK cells of CLL patients are fully competent. Attempts to optimize NK cell therapy for treatment of CLL will require overcoming the low immunogenicity of B-CLL cells. Our xenograft model provides the tools for such preclinical development. Disclosures:No relevant conflicts of interest to declare.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

Reduced NKG2D ligand expression in hepatocellular carcinoma correlates with early recurrence

The activating receptor natural killer group 2, member D (NKG2D) and its ligands play a crucial role in immune response to tumors. NKG2D ligand expression in tumors has been shown to be associated with tumor eradication and superior patient survival, but the involvement of NKG2D ligands in the immune response against hepatocellular carcinoma (HCC) still remains to be elucidated. We investigated the expression of NKG2D ligands in HCC tissues collected from 54 patients and HCC cell lines. We also examined the proteasome expression and the effect of inhibition of proteasome activity on NKG2D ligand expression in HCC tissues and cell lines. In dysplastic nodules (DN), well-differentiated (well-HCC), and moderately-differentiated HCCs (mod-HCC), UL16-binding protein (ULBP) 1 was expressed predominantly in tumor cells, but not in poorly-differentiated HCCs (poor-HCC). Remarkably, recurrence-free survival of patients with ULBP1-negative HCC was significantly shorter than that of patients with ULBP1-positive HCC (p=0.006). Cox regression analysis revealed that loss of ULBP1 expression was an independent predictor of early recurrence (p=0.008). We confirmed that ULBP1 was expressed in the well- and mod-HCC cell lines, but not in the poor-HCC cell line KYN-2. However, inhibition of proteasome activity resulted in significant up-regulation of ULBP1 expression in KYN-2. Moreover, we found that 20S proteasome expression was more abundant in KYN-2 than that in the well- and mod-HCC cell lines. ULBP1 is prevalently expressed in DN to mod-HCC, but loss of its expression correlates with tumor progression and early recurrence.

Clinical-Grade Generation of Active NK Cells from Cord Blood Hematopoietic Progenitor Cells for Immunotherapy Using a Closed-System Culture Process

Natural killer (NK) cell-based adoptive immunotherapy is a promising treatment approach for many cancers. However, development of protocols that provide large numbers of functional NK cells produced under GMP conditions are required to facilitate clinical studies. In this study, we translated our cytokine-based culture protocol for ex vivo expansion of NK cells from umbilical cord blood (UCB) hematopoietic stem cells into a fully closed, large-scale, cell culture bioprocess. We optimized enrichment of CD34+ cells from cryopreserved UCB units using the CliniMACS system followed by efficient expansion for 14 days in gas-permeable cell culture bags. Thereafter, expanded CD34+ UCB cells could be reproducibly amplified and differentiated into CD56+CD3− NK cell products using bioreactors with a mean expansion of more than 2,000 fold and a purity of >90%. Moreover, expansion in the bioreactor yielded a clinically relevant dose of NK cells (mean: 2×109 NK cells), which display high expression of activating NK receptors and cytolytic activity against K562. Finally, we established a versatile closed washing procedure resulting in optimal reduction of medium, serum and cytokines used in the cell culture process without changes in phenotype and cytotoxic activity. These results demonstrate that large numbers of UCB stem cell-derived NK cell products for adoptive immunotherapy can be produced in closed, large-scale bioreactors for the use in clinical trials.

Induction ofIn VitroandIn VivoNK Cell Cytotoxicity Using High-Avidity Immunoligands Targeting Prostate-Specific Membrane Antigen in Prostate Carcinoma

Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cells by targeting the activatory receptor NKG2D. Prostate-specific membrane antigen (PSMA) is an integral non-shed type 2 membrane protein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Here, we compare the impact of various anti-PSMA immunoligand formats on the therapeutic efficacy against prostate carcinoma cells by activating NK cells via NKG2D. Shortening of the linker separating the heavy and light chain antibody domain leads to the formation of dimers, trimers, and higher molecular mass oligomers. NK cells are most efficiently activated by multimeric immunoligands, thus showing an altered cytokine release pattern. The high avidity format is also superior in in vitro NK-mediated tumor cell targeting as shown in cytotoxicity assays. Finally, the efficacy of a multimeric immunoligand is shown in a prostate carcinoma mouse xenograft model showing a strong activity against advanced established tumors.

DNAM-1 ligand expression on Ag-stimulated T lymphocytes is mediated by ROS-dependent activation of DNA-damage response: relevance for NK–T cell interaction

AbstractAn important role for natural killer (NK) cells in the regulation of T-cell responses is emerging, although the receptor pairs regulating the NK–T-cell interaction have still not been identified. We found that superantigen-stimulated T cells express Nectin-2 (CD112) and poliovirus receptor (PVR; CD155), the ligands of the activating NK receptor DNAX accessory molecule-1 (DNAM-1; CD226). Interestingly, only PVR was present at the T cell surface, particularly on cells in the S and G2/M phases of the cell cycle. The up-regulation of PVR expression involves DNA-damage response (DDR)–dependent pathways, because we found that pharmacologic inhibition of ATM and ATR kinases reduced PVR expression and that PVR was almost exclusively induced on cells expressing the DDR marker γH2AX. Oxidative stress contributed to DDR activation, and our results showed impaired PVR levels in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), being monocytes the main ROS source needed for optimal PVR expression on activated T cells. Interestingly, in accordance with ligand expression, NK cells lysed allogeneic proliferating more efficiently than nonproliferating T lymphocytes, with a mechanism requiring the cooperation between DNAM-1 and NKG2D. These results could contribute to unraveling the role of NK cells in the down-regulation of T-cell responses in physiologic and pathologic processes such as autoimmunity or GVHD.

Abstract 1785: IL-15 and IL-2 improve Cetuximab mediated cytotoxicity EGFR-expressing triple negative breast cancer cell lines

Abstract Background and Objective: Approximately 10 – 15% of Breast Cancers (BC) are known to be ‘triple-negative (TN) receptor’, not expressing Estrogen receptor, Progesterone receptor and not exhibiting overexpression and/or gene amplification of HER2-neu. TNBC is associated with a more aggressive clinical course and worse clinical outcome than non-TNBC. As patients with TNBC are not likely to benefit from anti-estrogen or anti-HER2 therapy, first-line treatment usually consists of conventional cytotoxic chemotherapy. Taking into account the limited treatment possibilities in TNBC, in the present work we studied a potential therapy based on Cetuximab-mediated immune activity by Natural Killer (NK) cells. Results: We performed in vitro studies on a human cell line, IIB-BR-G, previously established in our laboratory and its metastatic in vivo variant, IIB-BR-G MT. The immunohistochemical analysis of both cell lines showed a TNBC phenotype with high expression of EGFR, the target for Cetuximab antibody. We also determined by DNA sequencing that both cell lines have a mutated K-RAS status (38 G&amp;gt;A at codon 12) which is the main Cetuximab predictive lack-of-response marker. Consequent to that, Cetuximab did not inhibit cellular proliferation or induced apoptosis, suggesting that it does not interfere with the EGFR signaling pathway, even at a concentration of 200 μg/ml (p&amp;gt;0.05). To investigate the immune mechanisms triggered by Cetuximab, we performed in vitro cytotoxicity assays. K-RAS mutated cell lines treated with 1 μg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity activity (ADCC), reaching a lytic activity of 40 ± 2.5% using as effectors peripheral blood mononuclear cells, and 50 ± 10% using enriched NK cells (p&amp;lt;0.001). Furthermore, IL-2 or IL-15 pretreatment of PBMC increased the effect of Cetuximab-mediated ADCC, reaching a cellular lysis of 75 ± 3 % in IIB-BR-G and 80 ± 3.5 % in IIB-BR-G MT (p&amp;lt;0.001), consistent with the upregulation of NK activating receptors CD16 and NKG2D and the enhancement of IFN-γ production in cocultures (p&amp;lt;0.001). Conclusions: EGFR-expressing TNBC cell lines were not affected by Cetuximab inhibition of its tyrosine kinase signaling, in concordance with their mutated K-RAS status. However, they could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could replace IL-2 in most of its immunologic activities, stimulating the cytotoxicity of NK cells and its ability to produce IFN-γ, paralleling the upregulation of activating receptors. This in vitro study shows the involvement of an antibody-mediated immune response rather than the blockade of intracellular signaling in the antitumor activity of Cetuximab in K-RAS mutated TNBC. The combination of Cetuximab with IL-2 or IL-15 and NK cells may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1785. doi:10.1158/1538-7445.AM2011-1785

Identification of a genomic region within MCMV critical for the export of m4/Kb complexes (105.17)

Abstract The murine cytomegalovirus (MCMV) protein m4/gp34 binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), and this stable m4/gp34-MHC class I complex is then exported to the cell surface. m4/gp34 can impact antigen presentation to CD8 T cells either positively or negatively. However, its main role may be to interact with natural killer (NK) cells. Recent evidence indicates that m4/gp34 in combination with the MHC class I molecule H-2Dk stimulates the activating NK receptor Ly49P. m4/gp34 is retained in the ER unless it binds to MHC class I. The goal of this study was to understand the molecular basis for export of the m4/gp34-MHC class I complex. We found that when m4/gp34 was expressed in a gutless adenovirus vector, it readily formed complexes with MHC class I. However, the complexes were retained in the ER. Superinfection with MCMV lacking m4 restored the export of m4/gp34-Kb complexes. Screening a panel of virus mutants revealed that a region of the MCMV genome containing open reading frames (ORFs) m166-m170 was needed for efficient export of m4/gp-34-Kb complexes. We will present data describing the impact of individual ORFs within this region on the export of m4/gp34-Kb complexes and on the biological function of m4/gp34.

Human and Mouse Mast Cells Express and Secrete the GPI-Anchored Isoform of CD160

CD160 is expressed by human and mouse natural killer (NK) cells and other cytotoxic lymphocyte subpopulations. CD160 is mostly expressed as a trimeric 83 kDa glycosylphosphatidylinositol (GPI)-anchored activating NK receptor, cleaved upon IL-15 stimulation in a secreted trimeric soluble form (sCD160) that binds to major histocompatibility complex (MHC) class I molecules, while a transmembrane isoform appears. sCD160 exhibits immunoregulatory function as it inhibits CD8(+) T-lymphocyte cytotoxic activity. We show that human mast cells (MCs) express CD160. In human and mouse skin, resident MCs expressed CD160, whereas in C57BL/6-Kit(W-sh/W-sh) mice, CD160(+) cells were only identified at the site of reconstitution with syngeneic cultured MCs. In the human mast cell line, HMC-1, we only identified the transcripts of the GPI-anchored CD160 isoform. Furthermore, CD160 was identified in HMC-1 and mouse MC supernatants, suggesting that MCs release sCD160. Supporting this hypothesis, HMC-1 express the GPI-specific phospholipase D variant 2 involved in the NK lymphocyte membrane cleavage of CD160, and morphological studies highlighted a relative loss of CD160 expression in inflammatory skin sites, where MC degranulation is expected to occur. We also demonstrated an inhibition of T-cell cytotoxicity by HMC-1 supernatant that was partially reversed by anti-CD160 mAb. In conclusion, sCD160, produced by MCs, may have a role in T-cell-MC interactions in vivo.

IL-15 and IL-2 increase Cetuximab-mediated cellular cytotoxicity against triple negative breast cancer cell lines expressing EGFR

Triple negative breast cancer (TNBC) patients are not likely to benefit from anti-estrogen or anti-HER2 therapy and this phenotype is associated with a more aggressive clinical course and worse clinical outcome. Taking into account the limited treatment possibilities in TNBC, the aim of the present work was to study a potential therapy based on Cetuximab-mediated immune activity by natural killer (NK) cells. We performed in vitro studies on human breast cancer (BC) cell lines, IIB-BR-G, and the in vivo metastatic variant IIB-BR-G MT. The immunohistochemical analysis showed a TNBC phenotype with high but different levels of EGFR expression on each cell line, measured by flow cytometry. DNA sequencing showed that both cell lines have a mutated K-RAS status, 38 G > A at codon 13. Consequently, Cetuximab did not inhibit cellular proliferation or induce apoptosis. We investigated if Cetuximab could trigger immune mechanisms, and we determined that both cell lines treated with 1 μg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity (ADCC), mediated by peripheral blood mononuclear cells (PBMC). At 50:1 effector:target ratio, lytic activity was 34 ± 2% against IIB-BR-G and 27 ± 6% against IIB-BR-G MT cells. PBMC pretreatment with IL-2 allowed reaching 65 ± 3% of Cetuximab-mediated ADCC against IIB-BR-G and 63 ± 6.5% against IIB-BR-G MT. Furthermore, IL-15 pretreatment increased the ADCC up to 71 ± 3% in IIB-BR-G and 79 ± 3.5% in IIB-BR-G MT. We suggest that NK cells are the effectors present in PBMC since they were able to induce ADCC at lower effector:target ratios. Besides, IL-2- and mainly IL-15-induced upregulation of NK activating receptors CD16 and NKG2D and enhanced IFN-γ production. EGFR-expressing TNBC could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could advantageously replace IL-2 in most of its immunologic activities, stimulating the ability to produce IFN-γ, and paralleling the up-regulation of activating receptors.

Mutations of the von Hippel–Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma

The tumor suppressor gene von Hippel-Lindau (VHL) is involved in the development of sporadic clear-cell renal cell carcinoma (RCC). VHL interferes with angiogenesis and also controls cell adhesion and invasion. Therapies that target VHL-controlled genes are currently being evaluated in RCC patients. RCC is a immunogenic tumor and treatment with interleukin-2 (IL2) or interferon (IFN)-α results in regression in some patients. We used two renal tumor cell lines (RCC6 and RCC4) carrying VHL loss-of-function mutations to investigate the role of mutant VHL in susceptibility to natural killer (NK) cell-mediated lysis. The RCC6 and RCC4 cell lines were transfected with the wild-type gene to restore the function of VHL. The presence of the gene in RCC cells downregulated hypoxia-inducible factor (HIF)-1α and subsequently decreased vascular endothelial growth factor (VEGF) production. Relative to control transfectants and parental cells, pVHL-transfected cell lines activated resting and IL2-activated NK cells less strongly, as assessed by IFNγ secretion, NK degranulation and cell lysis. NKG2A, a human leukocyte antigen (HLA)-I-specific inhibitory NK receptor, controls the lysis of tumor targets. We show that HLA-I expression in RCC-pVHL cells is stronger than that in parental and controls cells, although the expression of activating receptor NK ligands remains unchanged. Blocking NKG2A/HLA-I interactions substantially increased lysis of RCC-pVHL, but had little effect on the lysis of VHL-mutated RCC cell lines. In addition, in response to IFNα, the exponential growth of RCC-pVHL was inhibited more than that of RCC-pE cells, indicating that VHL mutations may be involved in IFNα resistance. These results indicate that a decreased expression of HLA-I molecules in mutated VHL renal tumor cells sensitizes them to NK-mediated lysis. These results suggest that combined immunotherapy with anti-angiogenic drugs may be beneficial for patients with mutated VHL.

Natural killer cells and tumor control

After hematopoietic cell transplantation (HCT) donor-derived natural killer (NK) cells kill tumor cells to prevent relapse and mediate other beneficial clinical effects including control of infections without inducing graft-vs.-host disease (GVHD). Understanding the determinants of NK cell alloreactivity and function will support improvements in the design of HCT and adoptive cellular therapies. Refinements to the model of NK cell education or licensing have been made which will inform strategies to develop functional alloreactive NK cells for therapeutic use. Differences in NK cell function have been shown to be dependent on the nature of the stimuli. Recent advances have been made in our understanding of the role of activating NK receptors on education and outcome after HCT. The use of adoptively transferred NK cells to treat hematopoietic malignancies has been expanding. New approaches to modulate target sensitivity to NK cell-mediated killing are under development. NK cells play an important role in the therapeutic efficacy of HCT, with effects on control of infections, GVHD, engraftment and relapse prevention. Recent advances in our understanding of NK cell biology will support improvements in our ability to exploit NK cells to treat cancer.

Cytotoxicity of Activated Natural Killer Cells against Pediatric Solid Tumors

To develop new therapies for children with solid tumors, we tested the cytotoxicity of natural killer (NK) cells expanded by coculture with K562-mb15-41BBL cells. We sought to identify the most sensitive tumor subtypes, clarify the molecular interactions regulating cytotoxicity, and determine NK antitumor potential in vivo. We tested in vitro cytotoxicity of expanded NK cells against cell lines representative of Ewing sarcoma (EWS; n = 5), rhabdomyosarcoma (n = 4), neuroblastoma (n = 3), and osteosarcoma (n = 3), and correlated the results with expression of inhibitory and activating NK receptor ligands. We also compared expanded and primary NK cells, determined the effects of activating receptor ligation and of chemotherapeutic drugs, and assessed the therapeutic effect of NK cell infusions in xenografts. In 45 experiments, EWS and rhabdomyosarcoma cell lines were remarkably sensitive to expanded NK cells, with median cytotoxicities at 1:1 effector/target ratio of 87.2% and 79.1%, respectively. Cytotoxicity was not related to levels of expression of NK receptor ligands, nor was it affected by pretreatment of target cells with daunorubicin or vincristine, but was markedly inhibited by preincubation of NK cells with a combination of antibodies against the NK-activating receptors NKGD2 and DNAM-1. Expanded NK cells were considerably more cytotoxic than unstimulated NK cells, and eradicated EWS cells engrafted in nonobese diabetic/severe combined immunodeficient Il2rgnull mice. Among pediatric solid tumors, EWS and rhabdomyosarcoma are exquisitely sensitive to expanded NK cells. The NK expansion method described here has been adapted to large-scale conditions and supports a phase I clinical study including patients with these malignancies.

Natural killer cells: alloreactive effects in hematopoietic stem cell transplantation

Background and Objectives Natural Killer (NK) cells are capable of recognizing target cells altered by pathologic states such as viral infection and malignant transformation. The NK cell’s capacity to recognize and kill abnormal cells while sparing normal cells relies on the integration of activating signals and tolerance to cells expressing self‐MHC molecules. Our understanding of how NK receptors mediate these signals and control NK cell function has advanced to the point where models of NK biology can be tested and revised in the clinical setting of hematopoietic stem cell transplantation.Materials and Methods This review article highlights several of the seminal contributions to the field of NK biology and their relevance to allogeneic and autologous hematopoietic cell transplantation.Results and Conclusions Inhibitory NK receptors are critical for achieving tolerance to cells expressing self‐MHC molecules and recognizing target cells lacking self‐MHC class I molecules. Activating NK receptors can circumvent NK tolerance and directly signal activation. Both inhibitory and activating receptors play an important role in hematopoietic stem cell transplantation outcome for hematologic and solid tumors. Incorporation of NK receptor genotyping in donor selection algorithms may benefit recipients of both HLA‐matched and ‐mismatched allografts.

Reconstitution of repertoire of natural killer cell receptors after transplantation: just a question of time?

Natural killer (NK) cells, key members of the natural immune system which each single individual possesses, are known to have a crucial role in innate defence against viruses, as well as against tumour cells.1, 2 In a self-environment, NK lymphocytes sense and kill target cells lacking MHC class I molecules and release various cytokines upon activation.3 The discovery of HLA class I-specific inhibitory receptors (namely, killer Ig-like receptors, KIRs), as well as of various activating receptors and their ligands,1, 2, 3, 4, 5 provided the basis for understanding the molecular mechanism of NK cell activation and function, thus permitting exploitation of the GVL effect mediated by these cells of innate immunity in the successful therapy of patients with high-risk leukaemias given allogeneic haematopoietic SCT (HSCT).6, 7, 8, 9 The most important human inhibitory KIRs are (i) KIR2DL1, which recognizes HLA-C alleles characterized by Lys at position 80 (HLA-CLys80 also named C2 group); (ii) KIR2DL2/3 recognizing HLA-C alleles characterized by Asn at position 80 (HLA-CAsn80 C1 group); and (iii) KIR3DL1, which recognizes HLA-B alleles sharing the Bw4 supertypic specificity (HLA-BBw4 Bw4 group).10 In an allogeneic setting, this type of inhibitory KIR repertoire can lead to alloreactivity in HLA-disparate HSCT through the mechanism of ‘missing self’-recognition.11 In detail, donor-origin NK cells can kill recipient-derived leukaemia cells when they express inhibitory KIRs, which do not recognize any of the HLA-class I alleles (that is, KIR ligands) present on recipient cells, this leading to a KIR/KIR-ligand mismatch in graft-vs-host direction. Alloreactive NK cells have been found to positively affect the outcome of HSCT from an HLA-haploidentical relative in adults with AMLs, as well as in children with ALL.7, 8, 9, 10 The peculiar GVL effect mediated by NK cells is completely separated by the occurrence of GVHD, this indicating that NK cells are able to kill leukaemia targets (as well as cells of haematopoietic origin as suggested by data obtained in the animal model,7) while sparing normal tissues. The molecular basis of the resistance of recipient normal tissues, other than haematopoietic cells, to the NK cell-mediated attack is the lack of ligands for the activating NK receptors.12 Indeed, there is large experimental evidence that killing of allogeneic cells also depends on the surface density of activating receptors on NK cells and on the expression of their ligands, these latter being expressed on target cells of different histotypes only upon cell stress infection or tumour transformation.13

Biology and clinical effects of natural killer cells in allogeneic transplantation

Following allogeneic hematopoietic cell transplantation, donor-derived natural killer (NK) cells target recipient hematopoietic cells, resulting in an antileukemia effect and a lower incidence of graft rejection. NK cells do not mediate and may diminish graft versus host disease. Here we review the determinants of NK cell alloreactivity and their implications for adoptive NK cell therapy. NK cell alloreactivity has been defined by the absence of recipient MHC class I ligands for donor inhibitory killer immunoglobulin-like receptor (KIR) receptors, as predicted by a number of algorithms. Recently, the role of activating NK receptors and their cognate ligands has received more attention. The beneficial clinical effect of NK-cell alloreactivity has not been uniformly demonstrated, likely reflecting differences in conditioning regimens, graft components and posttransplant immune suppression. Investigations of NK cell phenotype and function after transplantation have helped demonstrate which NK cell subsets mediate the graft versus leukemia effect. These advances have proceeded in parallel with increasing facility in GMP-grade bulk purification and administration of NK cell preparations. NK cells are a heterogeneous population of lymphocytes with diverse patterns of target-cell recognition and effector function. Further clinical and functional correlations will help maximize their potential for clinical benefit.

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells

Tumor resistance to lysis by resting natural killer (NK) cells may be overcome by priming of NK cells with cytokines or by binding of NK activating receptors to ligands expressed on target cells. In this study, major histocompatibility complex class I (MHC-I)-negative LNCaP and MHC-I-positive DU145 cells were infected with genetically modified influenza A virus lacking the non-structural gene 1 (NS1 IAV). The cells were used to investigate the influence of NS1 IAV infection on NK cell lysis of tumor cells as well as to prime NK cells for lysis of LNCaP and DU145 cells. While LNCaP cells infected with DeltaNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% +/- 1.47 vs. 52% +/- 0.74), both mock-infected and DeltaNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with DeltaNS1 IAV-infected LNCaP/DU145 cells effectively lysed resistant DU145 and sensitive LNCaP cells to a greater extent than NK cells primed with mock-infected LNCaP/DU145 or non-primed NK cells. Also, NK cell priming with DeltaNS1 IAV-infected tumor cells enhanced extracellular signal-regulated kinase phosphorylation and increased granule release in NK cells. The increased granule release was specifically mediated by NKp46, which eventually potentiated NK cells primed with DeltaNS1 IAV-infected tumor cells to overcome the inhibitory effects posed by MHC-I expression on DU145 cells. These findings show that in addition to direct lytic activity of NK cells, DeltaNS1 IAV may influence anti-tumoral responses by priming NK cells.