Natural Killer Susceptibility Research Articles

Inhibition of MICA and MICB Shedding Enhances Cytokine-Induced Memory-like NK Cell-Mediated Activity Against Multiple Myeloma

Introduction Natural Killer (NK) cells are important mediators of cytotoxic immune responses against MM, and the efficacy of existing MM therapies relies substantially on NK cell function. However, MM cells develop mechanisms to escape NK cell recognition by loss of ligands for activating receptors and by inducing functional defects. To overcome reduced NK cell functionality, adoptive transfer of cytokine-induced memory-like (CIML) NK cells are of particular interest, as these cells acquire memory-like features resulting in enhanced activity upon restimulation, greater IFN-γ response, and greater anti-tumor cytotoxicity. The success of adoptive transfer of CIML NK cells in MM is dependent on the ability of NK cells to recognize malignant plasma cells (PCs). NKG2D, one of the major activating receptors on NK cells, is upregulated on CIML NK cells. NKG2D plays an important role in the immune recognition of malignant PCs through interaction with its ligands MICA and MICB. However, MM cells evade this recognition by proteolytic shedding of MICA/B from the cell surface, leading to NKG2D internalization. Rationally designed antibodies like the 7C6 monoclonal antibody (mAb) targeting the proteolytic MICA α3 domain stabilize cell surface MICA/B expression and can enhance NK cell recognition and susceptibility to NK cell-mediated cytotoxicity. Furthermore, it has been shown that Bortezomib, an FDA-approved proteasome inhibitor with potent anti-MM activity, augments MICA expression in MM. We hypothesized that use of the 7C6 mAb would allow simultaneous blockade of MICA/B shedding and ADCC by CIML NK cells resulting in enhanced targeting of MM cells, which would further synergize with Bortezomib treatment. Results Flowcytometric analysis of surface MICA/B levels on 10 human myeloma cell lines (HMCLs) demonstrated heterogeneous protein expression. MICA/B transcript levels for corresponding HMCLs were queried from the CCLE database. There was no correlation between transcript and protein expression, suggesting substantial post-translational modifications, such as proteolytic cleavage, determining surface expression levels of MICA/B in MM. To determine if proteolytic cleavage occurs in MM, we treated HMCL with the 7C6 mAb, targeting the proteolytic cleavage site. Flowcytometric analysis of MM.1S revealed that 7C6 was able to upregulate basal expression of MICA/B in a dose-dependent manner as well as inhibit MICA/B shedding. These results suggest proteolytic cleavage of MICA/B occurs in MM and that this process can be inhibited by a MICA/B stabilizing antibody. Previously, it has been shown that MICA/B levels can be upregulated in MM by Bortezomib. As heterogeneity in MICA/B expression levels was seen among HMCLs, we hypothesized that the use of 7C6 would synergize with Bortezomib. Indeed, the addition of Bortezomib to 7C6 substantially increased MICA/B surface expression. To assess the functional consequences of MICA/B stabilization, we examined the effects on NK cell activation in co-culture assays of NK cells with MM.1S pretreated with 7C6 or IC mAb. We observed enhanced NK cell function as assessed by increased CD107a degranulation, IFN-γ and TNF-α production in co-culture assays of conventional NK cells with MM.1S pre-treated with 7C6 vs IC. Next, we measured the activity of CIML NK cells to myeloma targets. CIML NK cells exhibited enhanced CD107a in response to MM targets when compared to control NK cells from the same donors (Figure 1A). Furthermore, CIML NK cells demonstrated significantly higher cytotoxicity against 7C6 pretreated MM.1S, which was dependent on increased MICA/B and NKG2D levels. CIML NK cells in NSG mice treated with 7C6-hIgG1 mAb were significantly more effective in controlling early MM tumor cell outgrowth compared to CIML NK cells in mice treated with isotype control antibodies (Figure 1B). Conclusion Here we demonstrate that inhibition of MICA/B shedding with a novel antibody 7C6 enhances CIML NK cell-mediated function against MM in vitro and in a xenograft mouse model. Preliminary data demonstrate that this treatment strategy could synergize with current treatment regimens including Bortezomib. Our data support the hypothesis that combination strategies to enhance NK cell activation can be effective to enhance NK cell-mediated immune surveillance in MM. This combined approach highlights their potential use as novel immunotherapy in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance

SummaryThe heterotypic CIC structures formed of cancer and immune cells have been observed in tumor tissues. We aimed to assess the feasibility of using heterotypic CICs as a functional biomarker to predict NK susceptibility and drug resistance. The heterotypic CIC-forming cancer cells showed a lower response to NK cytotoxicity and higher proliferative ability than non-CIC cancer cells. After treatment with anticancer drugs, cancer cells that formed heterotypic CICs showed a higher resistance to anticancer drugs than non-CIC cancer cells. We also observed the formation of more CIC structures in cancer cells treated with anticancer drugs than in the non-treated group. Our results confirm the association between heterotypic CIC structures and anticancer drug resistance in CICs formed from NK and cancer cells. These results suggest a mechanism underlying immune evasion in heterotypic CIC cancer cells and provide insights into the anticancer drug resistance of cancer cells.

CEBPA mutants down-regulate AML cell susceptibility to NK-mediated lysis by disruption of the expression of NKG2D ligands, which can be restored by LSD1 inhibition

ABSTRACT NK group 2, member D (NKG2D) is one of the most critical activating receptors expressed by natural killer (NK) cells. There is growing evidence that acute myeloid leukemia (AML) cells may evade NK cell-mediated cell lysis by expressing low or no ligands for NKG2D (NKG2D-Ls). We hypothesized that CCAAT/enhancer-binding protein α (C/EBPα), one of the most studied lineage-specific transcription factors in hematopoiesis, might influence the expression of NKG2D-Ls. To test this hypothesis, we first examined the endogenous expression of wild-type C/EBPα (C/EBPα-p42) in human AML cell lines and demonstrated that its expression level was highly relevant to the sensitivity of AML cells to NK cell cytotoxicity. Induction of C/EBPα-p42 in the low endogenous CEBPA-expressing AML cell line increased the sensitivity to NK-induced lysis. Moreover, decreased expression of C/EBPα-p42 by RNA interference in AML cells abrogated NK-mediated cytotoxicity. We further showed that the increase in NK susceptibility caused by C/EBPα-p42 occurred through up-regulation of the NKG2D-Ls ULBP2/5/6 in AML cells. More importantly, chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing captured C/EBPα motif signatures at the enhancer regions of the ULBP 2/5/6 genes. Whilst, the AML-associated C/EBPα C-terminal mutant and N-terminal truncated mutant (C/EBPα-p30) diminished ULBP2/5/6 transcription. Finally, we identified that histone demethylase lysine-specific demethylase 1 (LSD1) inhibition can restore the expression of ULBPs via induction of CEBPA expression in AML cells, which may represent a novel therapeutic strategy for CEBPA-mutated AML. Abbreviations: C/EBPα: CCAAT/enhancer-binding protein α; TF: Transcription factor; AML: Acute myeloid leukemia; TAD: Transactivation domain; FS: Frameshift; NK: Natural killer; NKG2D: NK group 2, member D; NKG2D-Ls: Ligands for NKG2D; MHC: Major histocompatibility complex; MICA: MHC class I-related chain A; ULBP: UL16-binding protein; STAT3: Signal transducer and activator of transcription 3; LSD1: Lysine-specific demethylase 1; Ab: Antibody; PBMC: Peripheral blood mononuclear cell; PBS: Phosphate-buffered saline; CFSE: Carboxyfluorescein diacetate succinimidyl ester; PI: Propidium iodide; shRNA: Short hairpin RNA; ChIP: Chromatin immunoprecipitation; BM: Binding motif; HCNE: Highly conserved noncoding element; TSS: Transcription start site; HMA: Hypomethylating agent; AZA: Azacitidine/5-azacytidine; DAC: Decitabine/5-aza-29-deoxycytidine; 2-PCPA: Tranylcypromine; RBP: RNA-binding protein; MSI2: MUSASHI-2; HDACi: Inhibitor of histone deacetylases; VPA: Valproate; DNMTi: DNA methyl transferase inhibitor; SCLC: Small cell lung cancer

Killer cell immunoglobulin-like receptors (KIR) and human leukocyte antigen-C (HLA-C) allorecognition patterns in women with endometriosis

Endometriosis shares similarities with several autoimmune diseases. The human leukocyte antigen (HLA)-C genotype is associated with several human autoimmune diseases. HLA-C is a ligand of killer cell immunoglobulin receptors (KIRs) and is an essential regulator of natural killer cell activity, which is associated with endometriosis progression. Polymorphisms in HLA-C and KIR affect the activity of NK cells and susceptibility to several diseases. Therefore, we attempted to investigate an association between HLA-C genotype and KIR polymorphism and the occurrence of endometriosis. We tested the association of certain KIR and HLA-C combinations and the development of endometriosis by characterizing both KIR and HLA-C genes in 147 women with endometriosis and 117 controls. The HLA-C genotypes and KIR polymorphisms were analyzed via DNA-based method for higher-resolution genotyping. We found that the occurrence of HLA-C*03:03*01 was increased in endometriosis than in control groups. Analysis of various KIR haplotypes revealed differences between the endometriosis and control cohorts. The number of KIR centromeric A/A haplotypes was increased in the endometriosis group than controls. Moreover, the endometriosis cohort was characterized by reduced number of KIR2DS2-positive individuals in the Han Chinese population. Our current findings suggest that the KIR and HLA-C genotypes are associated with the pathogenesis of endometriosis.

Restoring Innate and Adaptive Immune Repertoire in Multiple Myeloma for Therapeutic Application

Despite advances and improvements in survival, majority of multiple myeloma (MM) patients ultimately relapses. Extensive analysis on the properties of tumor cells has provided interesting insights into the disease biology allowing for the identification of novel targets and development of related therapeutics. However, the key microenvironmental influences, especially immune microenvironment, that drive the disease and impact outcome remain to be fully characterized. We have now performed both single cell RNA-sequencing and high-dimensional CyTOF analysis of both peripheral blood (PBMC) and bone marrow mononuclear cells (BMMC) from MM patients and healthy donors (HD) and evaluated both phenotypic and functional state of the MM immune repertoire. In addition to humoral immunodeficiency impacting aberrations in B-cell subsets (B1a, B1b, B2 and Breg cells) and the disfunction in the adaptive immune system including an increase in the immunosuppressive cells (Tregs or myeloid-derived suppressor cells), a significant impairment of the innate immunity was identified in MM patients with a progressive decline in functional state of natural killer (NK) cells. Additionally, a lower expression of the activating receptors NKG2D and NKp46 as opposed to a higher expression of certain inhibitory KIR receptors was observed in NK cells from MM patients compared to HD. This immunosuppressive microenvironment allows tumor immune escape and ultimately myeloma cell growth. To overcome some of the immune dysfunction observed in MM, we have evaluated the role of IL15, a key player in both innate and adaptive immunity, with antitumor activity via enhancing both NK and memory T cell functions. Using extensive flow cytometry-based analysis, we evaluated impact of recombinant IL15 on PBMC from HD and MM patients at different stages of disease. Treatment with recombinant IL15 rescued the immune effector cell decline observed in MM patients. Specifically, a 2.9-fold-increase in CD8+ CD45RO+ CCR7- effector memory T cells and a 1.5-fold-increase in NK populations in both HD and MM PBMC was observed. For a subset of MM patients, we have also confirmed these positive effects on BMMC. Importantly, cytotoxicity tests revealed an improvement in NK cell effector functions leading to increased tumor cell recognition and killing, while we did not observe any direct effect of IL15 on growth and viability of MM cells. Integration of IL15 in the immunotherapeutic arsenal is limited by the unfavorable pharmacokinetic properties. Therefore, for clinical application and to restore both innate and adaptive immunity, we evaluated impact of NKTR-255, a polymer-conjugated IL15 receptor agonist designed to engage the IL-15 pathway to stimulate and expand natural killer (NK) cells and promote the survival and expansion of central memory CD8+ T cells without inducing suppressive regulatory T cells. Treatment with NKTR-255 enhanced the number and function of both NK and CD8+ effector memory T cell populations in PBMC from HD and MM patients in a dose dependent manner. Interestingly, the natural killer T (NKT) cells, heterogeneous group of T cells that share properties of both T cells and NK cells with important role in MM, were also increased in number by NKTR-255. To evaluate if NKTR-255 increases NK recognition of MM cells, cytotoxicity tests were performed using several MM cell lines and primary MM cells as targets, and both NK cells purified from PBMC of HD and MM patients as effectors. NKTR-255 was able to revert the inhibitory status of NK cells from MM patients (via induction of NKG2D expression), and significantly increase NK susceptibility of the MM cells in a dose dependent manner. Taken together, our data suggest a significant impact of NKTR-255 on the activation of effector cell function to efficiently target MM cells. This study has important translational implications and highlights the importance of restoring the balance between innate and adaptive immunity in MM. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Munshi:Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy.

TGF-β regulated leukemia cell susceptibility against NK targeting through the down-regulation of the CD48 expression

Transforming growth factor-β (TGF-β) is known to function as a dual role regulatory cytokine for being either a suppresser or promoter during tumor initiation and progression. In solid tumors, TGF-β secreted from tumor microenvironment acts as a suppresser against host immunity, like natural killer (NK) cells, to favor tumor evasion. However, besides solid tumors, the underlying mechanism of how TGF-β regulates leukemogenesis, tumor progression, immunoediting, and NK function is still not clear in detail. In this study, we found that TGF-β induced leukemia MEG-01 and U937 cells to become less sensitive to NK-92MI targeting by down-regulating CD48, a ligand for NK activating receptor 2B4, but not down-regulating other tumor-associated carbohydrate antigens (TACAs). In CD48-knockdown cells, cells responding to NK-92MI targeting displayed a phenotype of less NK susceptibility and cell conjugation. On the other hand, when NK cells were treated with TGF-β, TGF-β suppressed NK recognition, degranulation, and killing activity in time-dependent manner by regulating ICAM-1 binding capacity instead of affecting expressions of activating and inhibitory receptors. Taken together, both leukemia cells and immune NK cells could be regulated by TGF-β through suppressing leukemia cell surface CD48 to escape from host surveillance and down-regulating NK cell surface ICAM-1 binding activity to impair NK functions, respectively. Our results suggested that TGF-β had effect in leukemia similar to that observed in solid tumors but through different regulatory mechanism.

Mutated Von Hippel-Lindau-renal cell carcinoma (RCC) promotes patients specific natural killer (NK) cytotoxicity

BackgroundPrevious evidence demonstrated that restoration of wild type VHL in human renal cancer cells decreased in vitro NK susceptibility. To investigate on the role of tumoral VHL status versus NK capability in renal cancer patients, 51 RCC patients were characterized for VHL mutational status and NK function.MethodsVHL mutational status was determined by direct DNA sequencing on tumor tissue. NK cytotoxicity was measured against specific target cells K562, VHL-wild type (CAKI-1) and VHL-mutated (A498) human renal cancer cells through externalization of CD107a and IFN-γ production. Activating NK receptors, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NCAM-1 and FcγRIIIa were evaluated through quantitative RT-PCR. RCC tumoral Tregs were characterized as CD4+CD25+CD127lowFoxp3+ and Treg function was evaluated as inhibition of T-effector proliferation.ResultsVHL mutations were detected in 26/55 (47%) RCC patients. IL-2 activated whole-blood samples (28 VHL-WT-RCC and 23 VHL-MUT-RCC) were evaluated for NK cytotoxicity toward human renal cancer cells A498, VHL-MUT and CAKI-1, VHL-WT. Efficient NK degranulation and increase in IFN-γ production was detected when IL-2 activated whole-blood from VHL-MUT-RCC patients were tested toward A498 as compared to CAKI-1 cells (CD107a+NK: 7 ± 2% vs 1 ± 0.41%, p = 0.015; IFN-γ+NK: 6.26 ± 3.4% vs 1.78 ± 0.9% respectively). In addition, IL-2 activated NKs induced higher CD107a exposure in the presence of RCC autologous tumor cells or A498 as compared to SN12C (average CD107a+NK: 4.7 and 2.7% vs 0.3% respectively at 10E:1 T ratio).VHL-MUT-RCC tumors were NKp46+ cells infiltrated and expressed high NKp30 and NKp46 receptors as compared to VHL-WT-RCC tumors. A significant lower number of Tregs was detected in the tumor microenvironment of 13 VHL-MUT-RCC as compared to 13 VHL-WT-RCC tumors (1.84 ± 0.36% vs 3.79 ± 0.74% respectively, p = 0.04). Tregs isolated from VHL-MUT-RCC patients were less suppressive of patients T effector proliferation compared to Tregs from VHL-WT-RCC patients (Teff proliferation: 6.7 ± 3.9% vs 2.8 ± 1.1%).ConclusionsVHL tumoral mutations improve NKs effectiveness in RCC patients and need to be considered in the evaluation of immune response. Moreover therapeutic strategies designed to target NK cells could be beneficial in VHL-mutated-RCCs alone or in association with immune checkpoints inhibitors.

Grainyhead-like-2 confers NK-sensitivity through interactions with epigenetic modifiers

Natural Killer (NK) cells suppress tumor initiation and metastasis. Most carcinomas are heterogeneous mixtures of epithelial, mesenchymal and hybrid tumor cells, but the relationships of these phenotypes to NK susceptibility are understood incompletely. Grainyhead-like-2 (GRHL2) is a master programmer of the epithelial phenotype, that is obligatorily down-regulated during experimentally induced Epithelial-Mesenchymal Transition (EMT). Here, we utilize GRHL2 re-expression to discover unifying molecular mechanisms that link the epithelial phenotype with NK-sensitivity. GRHL2 enhanced the expression of ICAM-1, augmenting NK-target cell synaptogenesis and NK killing of target cells. The expression of multiple interferon response genes, including ICAM1, anti-correlated with EMT. We identified two novel GRHL2-interacting proteins, the histone methyltransferases KMT2C and KMT2D. Mesenchymal-epithelial transition, NK-sensitization and ICAM-1 expression were promoted by GRHL2-KMT2C/D interactions and by GRHL2 inhibition of p300, revealing novel and potentially targetable epigenetic mechanisms connecting the epithelial phenotype with target cell susceptibility to NK killing.

Transcription Factor IRF8 Orchestrates the Adaptive Natural Killer Cell Response

Natural killer (NK) cells are innate lymphocytes that display features of adaptive immunity during viral infection. Biallelic mutations in IRF8 have been reported to cause familial NK cell deficiency and susceptibility to severe viral infection in humans; however, the precise role of this transcription factor in regulating NK cell function remains unknown. Here, we show that cell-intrinsic IRF8 was required for NK-cell-mediated protection against mouse cytomegalovirus infection. During viral exposure, NK cells upregulated IRF8 through interleukin-12 (IL-12) signaling and the transcription factor STAT4, which promoted epigenetic remodeling of the Irf8 locus. Moreover, IRF8 facilitated the proliferative burst of virus-specific NK cells by promoting expression of cell-cycle genes and directly controlling Zbtb32, a master regulator of virus-driven NK cell proliferation. These findings identify the function and cell-type-specific regulation of IRF8 in NK-cell-mediated antiviral immunity and provide a mechanistic understanding of viral susceptibility in patients with IRF8 mutations.

Influence of Histone Deacetylase Inhibitors and DNA-Methyltransferase Inhibitors on the NK Cell-Mediated Lysis of Pediatric B-Lineage Leukemia

Epigenetic drugs like histone deacetylase inhibitors (HDACi) and DNA-methyltransferase inhibitors (DNMTi) have been shown to be effective against a variety of tumor entities. Among different molecular anticancer activities of epigenetic active substances, up-regulation of natural killer (NK) cell ligands was described to contribute to an enhanced NK cell-mediated killing of tumor cell lines. So far, no data is available on this effect in childhood acute lymphoblastic leukemia. We investigated the effect of two HDACi [vorinostat, valproic acid (VPA)] and two DNMTi (azacytidine, decitabine) on the viability, expression of NK ligands, and NK susceptibility of the pre-B-cell-ALL cell line MHH-CALL-4. Whereas vorinostat, azacytidine, and decitabine directly reduced viability of the cell line, VPA had no direct cytotoxic effect. NKG2D-ligands were expressed only at very low levels and not affected by epigenetic treatment. Higher expression was found for the DNAM-1 ligands with significant up regulation of CD112 after treatment with VPA (p = 0.02). No significant increase in lysis mediated by resting NK cells could be observed, whereas incubation of target cells with decitabine resulted in a significant increase in lysis mediated by IL-2 activated NK cells (p = 0.0051, p = 0.06 for azacytidine). Vorinostat and VPA could increase the lysis by expanded NK cells which was statistically not significant due to high inter-individual variability. Furthermore, HDACi but not DNMTi reduced the NK-mediated lysis of MHH-CALL-4 after incubation of effector cells. In conclusion, there is a synergistic effect between epigenetic drugs and NK cells against MHH-CALL-4 which is not as strong as in other tumor entities. In situations where NK-mediated control of leukemia is assumed or wanted, a sophisticated combination of single epigenetic drugs and ex vivo expanded NK cells is needed to maximize the synergistic effect of both treatment strategies and DNMTIs may be preferred based on the direct inhibitory effect of HDACi on NK cell cytotoxicity.

1,25‐Dihydroxyvitamin D3 enhances NK susceptibility of human melanoma cells via Hsp60‐mediated FAS expression

The active metabolite of vitamin D(3), 1α,25(OH)(2)D(3) , displays anticancer effects by regulating cell cycle and apoptosis in many cancer cells. However, it has not been determined whether 1α,25(OH)(2)D(3) increases the susceptibility of cancer cells to NK cells. Here, we investigated the anticancer effect of 1α,25(OH)(2)D(3) in human melanoma cell lines by investigating enhancement of NK susceptibility and elucidating the mediator of NK cytotoxicity. 1α,25(OH)(2)D(3)-resistant melanoma cells (G-361 and SK-MEL-5) treated with 1α,25(OH)(2)D(3) showed higher susceptibility to NK cells with up-regulation of Fas expression. Furthermore, G-361 cells treated with 1α,25(OH)(2)D(3) showed significantly increased caspase activity. In addition to Fas up-regulation, expression of heat shock protein 60 (Hsp60) was elevated by 1α,25(OH)(2) D(3) . Increased expression of Hsp60 by 1α,25(OH)(2)D(3) was related to not only up-regulation of Fas expression but also to NK susceptibility of G-361 cells. Taken together, our data suggest that 1α,25(OH)(2)D(3) acts as an anticancer agent by increasing expression of Fas on the surface of melanoma cells through Hsp60 induction and strengthens caspase sensitivity to Fas-mediated apoptotic pathway by NK cells. 1α,25(OH)(2)D(3) treatment may therefore have a preventive role in melanoma occurrence or potentiate the anticancer effects of NK-cell immune therapy.

Natural killer cells take two tolls to destruct bile ducts

Pit cells were first described by Wisse et al.1 in 1976. Later, Kaneda et al.2 referred to this cell type as “natural killer (NK) cells” due to their resemblance to large granular lymphocytes that are known to possess NK cell activity. In 1984, Kaneda et al.3 also observed that in autoimmune hepatitis, these pit cells (now known as liver-specific NK cells) were frequently seen in close contact with hepatocytes that eventually showed degenerative or immature features, providing the first evidence that NK cells may contribute to hepatocellular damage in autoimmune hepatitis. Although more than 2 decades have since passed and the accumulating evidence indicates that NK cells are involved in the pathogenesis of many types of human autoimmune diseases,4 little is known about the role of NK cells in autoimmune liver disease specifically. To date, there are several studies reporting that NK cell functions are dysregulated in autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis (PBC). These data show that in patients with PBC, there was increased cell-killing activity and decreased cytokine production in NK cells5 and that there appeared to be a genetic link between NK cell receptor ligand expression and susceptibility to primary sclerosing cholangitis.6 However, the mechanism by which NK cells are activated and contribute to the pathogenesis of autoimmune liver disease was largely unknown until Shimoda et al.7 published their recent data in this issue of Hepatology.

Overexpression of IL-32α Increases Natural Killer Cell-mediated Killing through Up-regulation of Fas and UL16-binding protein 2 (ULBP2) Expression in Human Chronic Myeloid Leukemia Cells

IL-32 was recently identified as a proinflammatory cytokine that is induced by IL-18 in natural killer (NK) cells and is highly correlated with inflammatory disorders. However, the relationship between IL-32 and tumor progression is still unknown. In this study, we investigated whether overexpression of IL-32 affects susceptibility of chronic myeloid leukemia (CML) cells to NK cells. Interestingly, IL-32α-overexpressing CML cell lines, K562, Kcl22, and BV173, showed higher NK cell-mediated killing. Flow cytometry analysis revealed that overexpression of IL-32α induced increased expression of Fas and UL16-binding protein 2 (ULBP2) in CML cells. The direct relationship between overexpression of surface molecules by IL-32α and increased NK cell-mediated killing was confirmed by Fas or ULBP2 siRNA transfection. IL-32α-induced Fas and ULBP2 expression are regulated p38 MAPK. In addition, the transcription factor Ets1 plays a key role in ULBP2 specific expression by IL-32α overexpression in ULBP family members. Taken together, these data show that IL-32α stimulates Fas and ULBP2 expression via activation of p38 MAPK, which increases NK susceptibility of CML cells. Enhanced NK cell susceptibility of CML cells by IL-32α overexpression may improve the efficiency of NK cell-based immunotherapy.

Transfer of MHC Class I by Coating Platelets Enables Tumor Cells to Evade NK Cell Immune Surveillance by Conferring a “Pseudo Self” Phenotype

AbstractAbstract 583NK cells are a central component of the cytotoxic lymphocyte compartment capable of lysing tumor cells without prior immune sensitization of the host. The mechanisms leading to activation of NK reactivity are described by the principles of ‘missing-self’ and ‘induced-self’, which imply that cells with a low or absent expression of MHC class I ('missing-self') and/or a stress-induced expression of ligands of activating NK receptors like e.g. NKG2D ('induced-self') are preferentially recognized and eliminated by NK cells. Thus, a balance of various activating and inhibitory signals determines whether NK cell responses are initiated or not. Tumor cells often downregulate expression of MHC class I to evade T cell-mediated immune surveillance, which results in enhanced NK susceptibility. Besides the direct interaction with their target cells, NK activity is further influenced by the reciprocal interplay with various other hematopoietic cells. We and others demonstrated previously that thrombocytopenia inhibits metastasis in murine models, which is reversed by additional depletion of NK cells (e.g., Jin et al., Nature Med. 2006, Palumbo et al., Blood 2005). However, the mechanisms by which platelets impair NK-tumor interaction are largely unclear, especially in humans. Recently we reported that platelets release TGF-β upon interaction with tumor cells causing downregulation of NKG2D on NK cells, which impairs anti-tumor immunity by disturbing the principle of “induced self” (Kopp et al., Cancer Res. 2009). Here we demonstrate that platelets further enable tumor cells to evade NK cell immune surveillance by preventing detection of “missing self”: We found that tumor cells rapidly get coated in the presence of platelets, the latter expressing large amounts of MHC class I on their surface. In case of MHC class I-negative or -low cancer cells, this process results in MHC class I “pseudoexpression” on the tumor cell surface as revealed by flow cytometry, immunofluorescent staining, and electron microscopy. Platelet-derived MHC class I was found to inhibit the reactivity of autologous NK, both upon activation with cytokines and, most importantly, in cultures with platelet-coated tumor cells. Using constitutively MHC class I-negative/low tumor cells we found that blocking MHC class I restored NK cytotoxicity and IFN-γ production against platelet-coated tumor cells, but did not alter NK reactivity against the tumor cells in the absence of coating platelets. Taken together, our data indicate that platelets enable a molecular mimicry of tumor cells, allowing the latter to downregulate MHC class I in order to escape T cell immunity without inducing sufficient NK tumor immune surveillance due to conferred platelet-mediated “pseudo self”. Disclosures:No relevant conflicts of interest to declare.

Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

Frequencies of killer immunoglobulin-like receptor genotypes influence susceptibility to spontaneous abortion

Natural killer (NK) cells are the most abundant lymphocyte population in the decidua. These cells express killer immunoglobulin-like receptors (KIRs), which upon recognition of HLA class I molecules on trophoblasts may either stimulate NK cells (activating KIRs) or inhibit them (inhibitory KIRs) to produce soluble factors necessary for the maintenance of pregnancy. KIR genes exhibit extensive haplotype polymorphism; individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes. This polymorphism affects NK cell reactivity and susceptibility to diseases, including gynecological disorders. Therefore we KIR-genotyped 149 spontaneously aborting women and 117 control multiparae (at least 2 healthy-born children). Several genotypes (i.e. combinations of various KIR genes) were differently distributed among the patients and control subjects. Differences were observed in the numbers and the ratios of activating to inhibitory KIRs between patients and healthy women: (i) genotypes containing 6 activating KIR genes were less frequent and those containing 6 inhibitory KIR genes were more frequent in patients than in control subjects, and (ii) an excess of inhibitory KIRs (activating-to-inhibitory KIR gene ratios of 0.33 to 0.83) was associated with miscarriage, whereas ratios close to equilibrium (0.86-1.25) seemed to be protective. In addition, the results suggest for the first time that sporadic and recurrent spontaneous abortions as well as miscarriage in the presence or absence of autoantibodies may have different KIR genotypic backgrounds.

NOVEL Pathways Modulate TUMOR CELL Susceptibility to NK CELLS.

Abstract 277NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures:No relevant conflicts of interest to declare.

Early Hematopoietic Zinc Finger Protein Prevents Tumor Cell Recognition by Natural Killer Cells

Early hematopoietic zinc finger/zinc finger protein 521 (EHZF/ZNF521) is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is down-regulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed up-regulation of HLA class I cell surface expression. This phenotypic change was associated with an increased level of HLA class I H chain, in absence of detectable changes in the expression of other Ag-processing machinery components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I Ag-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I Ag up-regulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells.

Early Hematopoietic Zinc Finger (EHZF/ZNF521) prevents tumor cell recognition by Natural Killer cells. (41.43)

Abstract EHZF/ZNF521 is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is downregulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed upregulation of HLA class I cell surface expression. This phenotypic change was associated with increased level of HLA class I heavy chain, in absence of detectable changes in the expression of other antigen processing machinery (APM) components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B-lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I antigen-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I antigen upregulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells.

NK Cell‐mediated Destruction of Influenza A Virus‐infected Peripheral but not Central Neurones

Peripheral neurones have the potential to transmit infectious agents to the central nervous system (CNS). This raises the possibility of existing host defence mechanisms that may prevent such spread. Natural killer (NK) cells can target infected cells, and by this ability serve to limit spread of infection prior to the development of adaptive immune responses. To address directly if NK cells can target infected peripheral neurones, we examined the expression of NK cell-activating ligands and susceptibility to NK cell-mediated cytolytic effects in ex vivo cultures of mouse peripheral dorsal root ganglia (DRG) neurones prior to and after infection with a neurotropic strain of influenza A virus, WSN/33. In infected DRG cultures, retinoic acid early inducible gene-1 (RAE-1) transcripts were induced and exposure to interleukin (IL)-2-activated NK cells resulted in a total destruction of neurites. Studies on cultures from interferon (IFN)-alpha/betaR-deficient mice suggest that the infection engages an IFN-alpha/beta-dependent signalling pathway to induce RAE-1 transcripts. In contrast, induction of RAE-1 transcripts or NK cell-mediated neurite destructions was not observed in central hippocampal neurones. This reveals distinct properties between peripheral DRG and central hippocampal neurones with respect to the ability to signal for immune destruction following infection.