Upregulation Of NKG2D Ligands Research Articles

SETDB1 Suppresses Interferon Responses and NK Cell-Mediated Immunosurveillance Specifically in Monocytic AML

Acute myeloid leukemia (AML) with monocytic differentiation (monocytic AML) responds poorly to current treatments, including venetoclax-based therapy. To identify novel therapeutic targets in monocytic AML, we conducted in vivo and in vitro CRISPR/Cas9 library screens using a pooled sgRNA library targeting epigenetic regulators with a mouse monocytic AML model driven by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations). We ranked all the epigenetic genes according to the in vivo specific essentiality, and selected several candidates which were more depleted in mice rather than in cell cultures. The top-ranked gene was Setdb1, which encodes a histone H3 lysine 9 (H3K9) methyltransferase. In addition, several H3K9 trimethylation (H3K9me3) regulators, ATF7IP and TRIM33, were also more important in vivo than in vitro in our screens. ATF7IP is a cofactor of SETDB1 to enhance its enzymatic activity. TRIM33 is also involved in H3K9me3 modification. These data suggest the critical role of the SETDB1-ATF7IP-TRIM33 H3K9 methyltransferase complex in the development of AML in vivo. To validate the results of our screenings and to investigate the role of immune cells in our transplantation assay, we next transplanted control and Setdb1-knockout (KO) cSAM cells into three types of mice with different immune systems: the immunocompetent C57BL/6 mice, the immunodeficient NSG mice lacking T, B and natural killer (NK) cells, and the C57BL/6J- Rag2-/- mice lacking mature T and B cells but possessing NK cells. Although Setdb1 depletion did not inhibit the growth of cSAM cells in NSG mice, it showed the strong growth-inhibitory effect in C57BL/6 mice and Rag2-/- mice (Fig 1A). Since both C57BL/6 mice and Rag2-/- mice have NK cells, these data suggest that SETDB1 promotes leukemogenesis by suppressing NK cell-mediated cytotoxicity in vivo. Mechanistically, loss of Setdb1 induced upregulation of NKG2D ligands, interferon-stimulated genes (ISGs), and endogenous retrovirus (ERV) sequences through H3K9me3 reduction, thereby rendering cSAM cells more susceptible to NK cell-mediated cytotoxicity. Setdb1 depletion also induced myeloid maturation of cSAM cells towards macrophages and downregulation of major histocompatibility complex class I (MHC-I), which contributed to the increased susceptibility of Setdb1-depleted cSAM cells to NK cell-mediated cytotoxicity. Next, we investigated whether SETDB1 inactivation also sensitizes human AML cells to NK cells using the co-culture assay with an IL-2 dependent NK cell line NK-92. Loss of SETDB1 rendered monocytic AML cell lines, NOMO-1, THP-1, OCI-AML3, U937, and NB4, more susceptible to NK-92 cell-mediated cytotoxicity. SETDB1 depletion also induced overactivation of interferon signaling and intrinsic apoptosis in these monocytic AML cells. In contrast, non-monocytic AML cells, such as K562, TF-1, and Kasumi-1, were not sensitive to SETDB1 depletion. SETDB1 depletion neither increased the susceptibility to NK cell-mediated cytotoxicity nor induced apoptosis in non-monocytic AML cells. Subsequent analyses revealed that SETDB1 depletion reduced H3K9me3 at the monocyte-specific enhancer regions with ERV sequences to upregulate NK cell-activating ligands and ISGs. Because the “ERV enhancers” are active only in monocyte lineage, SETDB1 is essential specifically in monocytic AML. Finally, we identified MNDA and its mouse counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. MNDA is one of the ISGs and is involved in myeloid cell differentiation. Human monocytic AML cells that are dependent on SETDB1 expressed higher MNDA at both RNA and protein levels compared to other subtypes of AML. We also found that Ifi203-high mouse AML cells transformed by MLL-AF9 were sensitive to Setdb1 depletion, whereas Setdb1 was dispensable in Ifi203-low mouse AML cells transformed by RUNX1-RUNX1T1. In summary, our findings reveal the critical and selective role of SETDB1 in monocytic AML. SETDB1 mediates H3K9me3 at the monocyte-specific ERV enhancers to repress the expression of NK cell-activating ligands and ISGs, thereby suppressing immunogenicity and apoptosis in monocytic AML (Fig 1B). SETDB1 as well as its binding partners could be promising therapeutic targets to treat monocytic AMLs that are relatively resistant to the current standard therapies.

Stem cell-assisted enzyme/prodrug therapy makes drug-resistant ovarian cancer cells vulnerable to natural killer cells through upregulation of NKG2D ligands.

Cancer stem-like cells (CSCs) are believed to be responsible for cancer recurrence and metastasis. Therefore, a therapeutic approach is needed to eliminate both rapidly proliferating differentiated cancer cells and slow-growing drug-resistant CSCs. Using established ovarian cancer cells lines as well as ovarian cancer cells isolated from a patient with high-grade drug-resistant ovarian carcinoma, we demonstrate that ovarian CSCs consistently express lower levels of NKG2D ligands (MICA/B and ULBPs) on their surfaces, a mechanism by which they evade natural killer (NK) cells' surveillance. Here, we discovered that exposure of ovarian cancer (OC) cells to SN-38 followed by 5-FU not only acts synergistically to kill the OC cells, but also makes the CSCs vulnerable to NK92 cells through upregulation of NKG2D ligands. Since systemic administration of these two drugs is marred by intolerance and instability, we engineered and isolated an adipose-derived stem cell (ASC) clone, which stably expresses carboxylesterase-2 and yeast cytosine deaminase enzymes to convert irinotecan and 5-FC prodrugs into SN-38 and 5-FU cytotoxic drugs, respectively. Co-incubation of ASCs and prodrugs with drug-resistant OC cells not only led to the death of the drug-resistant OC cells but also made them significantly vulnerable to NK92 cells. This study provides proof of principle for a combined ASC-directed targeted chemotherapy with NK92-assisted immunotherapy to eradicate drug-resistant OC cells.

Immunomodulatory effect of NEDD8-activating enzyme inhibition in Multiple Myeloma: upregulation of NKG2D ligands and sensitization to Natural Killer cell recognition

Multiple Myeloma (MM) is an incurable hematologic malignancy of terminally differentiated plasma cells (PCs), where immune interactions play a key role in the control of cancer cell growth and survival. In particular, MM is characterized by a highly immunosuppressive bone marrow microenvironment where the anticancer/cytotoxic activity of Natural Killer (NK) cells is impaired. This study is focused on understanding whether modulation of neddylation can regulate NK cell-activating ligands expression and sensitize MM to NK cell killing. Neddylation is a post-translational modification that adds a ubiquitin-like protein, NEDD8, to selected substrate proteins, affecting their stability, conformation, subcellular localization, and function. We found that pharmacologic inhibition of neddylation using a small-molecule inhibitor, MLN4924/Pevonedistat, increases the expression of the NK cell-activating receptor NKG2D ligands MICA and MICB on the plasma membrane of different MM cell lines and patient-derived PCs, leading to enhanced NK cell degranulation. Mechanistically, MICA expression is upregulated at mRNA level, and this is the result of an increased promoter activity after the inhibition of IRF4 and IKZF3, two transcriptional repressors of this gene. Differently, MLN4924/Pevonedistat induced accumulation of MICB on the plasma membrane with no change of its mRNA levels, indicating a post-translational regulatory mechanism. Moreover, inhibition of neddylation can cooperate with immunomodulatory drugs (IMiDs) in upregulating MICA surface levels in MM cells due to increased expression of CRBN, the cellular target of these drugs. In summary, MLN4924/Pevonedistat sensitizes MM to NK cell recognition, adding novel information on the anticancer activity of neddylation inhibition.

BCMA-Specific ADC MEDI2228 and Daratumumab Induce Synergistic Myeloma Cytotoxicity via IFN-Driven Immune Responses and Enhanced CD38 Expression.

Efforts are required to improve the potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma. We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate (ADC) MEDI2228 which can augment efficacy of these immunotherapies. MEDI2228-induced transcriptional and protein changes were investigated to define significantly impacted genes and signaling cascades in multiple myeloma cells. Mechanisms whereby MEDI2228 combination therapies can enhance cytotoxicity or overcome drug resistance in multiple myeloma cell lines and patient multiple myeloma cells were defined using in vitro models of tumor in the bone marrow (BM) microenvironment, as well as in human natural killer (NK)-reconstituted NOD/SCID gamma (NSG) mice bearing MM1S tumors. MEDI2228 enriched IFN I signaling and enhanced expression of IFN-stimulated genes in multiple myeloma cell lines following the induction of DNA damage-ATM/ATR-CHK1/2 pathways. It activated cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades and increased CD38 expression in multiple myeloma cells but did not increase CD38 expression in BCMA-negative NK effector cells. It overcame CD38 downregulation on multiple myeloma cells triggered by IL6 and patient BM stromal cell-culture supernatant via activation of STAT1-IRF1, even in immunomodulatory drug (IMiD)- and bortezomib-resistant multiple myeloma cells. In vitro and in vivo upregulation of NKG2D ligands and CD38 in MEDI2228-treated multiple myeloma cells was further associated with synergistic daratumumab (Dara) CD38 MoAb-triggered NK-mediated cytotoxicity of both cell lines and autologous drug-resistant patient multiple myeloma cells. These results provide the basis for clinical evaluation of combination MEDI2228 with Dara to further improve patient outcome in multiple myeloma.

An inhibitor of programmed death ligand 1 enhances natural killer cell-mediated immunity against malignant melanoma cells.

Since ionizing radiation has showed the dramatic effect to kill the cancer cells through direct DNA damage as well as triggering anti-cancer immune responses including induction of NKG2D ligands, it has used for long time to treat many cancer patients. However, it has been known that radiotherapy might promote the remnant cancer cells to escape immune system and metastasis. One of the suggested ways of immune evasion is induction of a ligand for programmed death-1 (PD-L1) in head and neck cancer, bladder cancer and lung cancer cells which engages the receptor, programmed death-1 (PD-1) in immune cells. PD-1/PD-L1 axis transduces the inhibitory signal and suppresses the adaptive immunity. However, their role in innate immunity remains poorly understood. Therefore, we investigated whether ionizing radiation could change the expression of PD-L1 in malignant melanoma cells and the receptor, programmed death-1 (PD-1), in NK-92 cells. Surface PD-L1 levels on melanoma cells were increased by ionizing radiation in a dose-independent manner but the level of PD-L1 was not changed significantly in NK-92 cells. Radiation-induced PD-L1 suppressed the activity of the NK-92 cells against melanoma cells despite of upregulation of NKG2D ligands. Furthermore, activated NK cells had high level of PD-1 and could not kill PD-L1+ melanoma cells effectively. When we used PD-L1 inhibitor or silenced PD-L1 gene, inhibited PD-1/PD-L1 axis reversed the activity of the suppressed NK cells. Through these results, we supposed that PD-1/PD-L1 blockade could enhance the immune responses of NK cells against melanoma cells after radiotherapy and might overcome the PD-L1 mediated radioresistance of cancer cells.

Induction of NKG2D ligand expression on tumor cells by CD8+ T-cell engagement-mediated activation of nuclear factor-kappa B and p300/CBP-associated factor.

The ligands for the natural killer group 2 (NKG2D) protein render tumor cells susceptible to NKG2D-dependent immune cell attack. However, cancer cells escape from immune surveillance by downregulating NKG2D ligands. We previously discovered that engagement of activated CD8+ T cells and tumor cells induces NKG2D ligands on tumor cells, but the underlying mechanism remains to be defined. Both in vivo mouse tumor models and in vitro cell assays were performed to study the downstream signaling. Our results supported the notion that, upon engagement with the cognate receptors, CD137 ligand and CD40 initiates activation of nuclear factor-kappa B (NF-κB) signaling in tumor cells even in the absence of CD8+ T cells. Like tumor and CD8+ T cell contact-dependent NKG2D ligand induction, this CD137L/CD40-mediated signaling activation was associated with elevated levels of acetyltransferase P300/CBP-associated factor (PCAF), whereas inhibition of phosphorylated NF-κB abrogated PCAF induction. Although stimulation of CD137L/CD40-mediated signaling is vital, inflammatory cytokines, including interferon gamma (IFNγ) and TNFα, also facilitate NKG2D ligand–induced immune surveillance via both facilitating T cell chemotaxis and CD137L/CD40 induced NF-κB/PCAF activation. Collectively, our results unveil a novel mechanism of NKG2D ligand upregulation involving reverse signaling of CD40 and CD137L on tumor cells which, along with inflammatory cytokines IFNγ and TNFα, stimulate downstream NF-κB and PCAF activation. Understanding this mechanism may help in development of induced NKG2D ligand–dependent T cell therapy against cancers.

Abstract 1196: Induction of NKG2D ligand expression by CD8+ T cells through a nuclear factor-kappa B and P300/CBP-associated factor-dependent mechanism

Abstract The ligands for the natural-killer group 2 (NKG2D) protein are frequently expressed on tumor cell surfaces, rendering tumor cells susceptible to NKG2D-dependent immune cell attack. However, cancer cells escape from immune surveillance by downregulating NKG2D ligands. We previously discovered that the engagement of activated CD8+ T cells and tumor cells induces NKG2D ligands on tumor cells, but the underlying mechanism remains to be defined. TCGA database analyses suggested that the expression of CD40 and CD137L (their cognate receptors are CD40L and CD137, respectively, expressed on CD8+T cells) is associated with pan-NKG2D ligand expression in sarcoma and colorectal adenocarcinoma patients (Pearson’s correlation coefficient r=0.2225 and 0.1947, respectively; and P=.0003 and .0002, respectively). In agreement with these human data analysis results, both in vivo mouse tumor models and in vitro cell assays validated that upon the engagement with the cognate receptors, the tumor necrosis factor receptor superfamily (TNFRSF), particularly CD137 ligand and CD40, initiates the activation of nuclear factor-kappa B (NF-κB) signaling in tumor cells even in the absence of CD8+ T cells. The same as tumor and CD8+ T contact-dependent NKG2D ligand induction, this CD137L/CD40-mediated signaling activation was associated with elevated levels of acetyltransferase P300/CBP-associated factor (PCAF), whereas inhibition of phosphorylated NF-κB abrogated PCAF induction. Although the stimulation of CD137L/CD40-mediated signaling by cognate receptors is vital for NKG2D ligand upregulation, the inflammatory cytokines, including interferon γ (IFNγ) and tumor necrosis factor α (TNFα) released from CD8+ T cells also facilitate NKG2D ligand-induced immune surveillance. These inflammatory cytokines not only trigger NF-κB signaling and PCAF activation, and they also boost T cell chemotaxis, recruit large numbers of CD8+T cells to tumor sites, and result in high yield expression of NKG2D ligands. Collectively, our results unveil a novel mechanism of NKG2D ligand upregulation involving reverse signaling of CD40 and CD137L on in tumor cells, which along with inflammatory cytokines IFNγ and TNFα, stimulate downstream NF-κB and PCAF activation. Understanding this mechanism may help to develop induced NKG2D ligand-dependent T-cell therapy against cancers. Citation Format: Jiemiao Hu, Xueqing Xia, Shulin Li. Induction of NKG2D ligand expression by CD8+ T cells through a nuclear factor-kappa B and P300/CBP-associated factor-dependent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1196.

Abstract 5574: Retinoic acid as a therapeutic adjuvant in low-grade gliomas

Abstract Introduction: Malignant gliomas are incurable brain tumors and the most common primary brain tumors in adults. Mutations in isocitrate dehydrogenase (IDH) are enriched in a subset of malignant low-grade gliomas (MLGGs) that arise as lower grade neoplasms and steadily progress to more aggressive and universally fatal glioblastomas (GBMs) usually within 5-10 years after initial diagnosis. Gliomas with IDH1/2 mutations are characterized by hypermethylation of multiple genes, including immune genes, thereby rendering them highly immune evasive. Retinol binding protein 1 (RBP1) gene, that encodes a protein involved in retinoic acid (RA) generation, is significantly hyper-methylated in IDH mutant cells. Since RA is instrumental in the induction of multiple immune genes including immune-activating ligands such as NKG2D ligands (NKG2D-L), we hypothesized that treatment of IDH mutant cells with RA will increase the expression of NKG2D-L on the tumor cells, leading to increased recognition and lysis by immune cells. In addition, we hypothesized that RBP1 methylation was crucial to development of the IDH mutant immune-evasive phenotype. Methods: Primary glioma cells (IDH WT and mutant) were treated with 1μM RA for 48 hours. The cells were assessed for changes in expression of NKG2D-L and other type-1 immune response genes. Effect of RA treatment on NK killing and immune cell chemotaxis were evaluated. We also assessed the impact of RBP1 knock-down in IDH WT cells. Finally, we evaluated the efficacy of RA as a therapeutic modality for low-grade gliomas in a murine model. Results: RA-treated IDH mutant gliomas had a higher expression of NKG2D-L and a higher sensitivity to killing by NK cells, when compared with untreated IDH mutant cells. RA treatment of IDH mutant cells also induced increased chemotaxis of NKs and CD8+ T cells in a CCL2-dependant manner. In addition, RA demonstrated superior anti-tumor efficacy in vivo, compared with controls. Finally, we demonstrated that RBP1 knock-down in IDH WT cells was sufficient to replicate the immune evasive phenotype observed in IDH mutant cells. Conclusions: RA treatment reverses the immune evasion in IDH mutant cells primarily by inducing the upregulation of NKG2D-L and by mediating increased NK and CD8+ T cell chemotaxis via CCL2 induction. Additionally, we established that RBP1 gene silencing is crucial for the development of the immune evasive IDH mutant phenotype. Note: This abstract was not presented at the meeting. Citation Format: Aparna Rao, Xiaoran Zhang, Jason Kim, Alexander Pomerantz, Youngmi Kim, Eric Holland, Nduka Amankulor. Retinoic acid as a therapeutic adjuvant in low-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5574. doi:10.1158/1538-7445.AM2017-5574

IL-18-dependent NKG2D ligand upregulation on accessory cells is mediated by the PI3K/GSK-3 pathway.

NK cells are critical for the control of viral infections. Studies have shown that efficient NK cell activation in response to infection with VV in vivo requires multiple pathways, including the NKG2D pathway. We have recently shown that IL-18 is necessary for the activation of NK cells through upregulation of the NKG2D ligand Rae-1 on DCs upon VV infection. However, how IL-18R signaling on the accessory cells contributes to Rae-1 up-regulation remains to be defined. In this study, we found IL-18-mediated Rae-1 up-regulation in accessory cells, including macrophages and DCs, to be dependent on the MyD88-PI3K pathway. We further found that IL-18 signaling through PI3K led to inhibition of GSK-3, which we found to be a negative regulator of Rae-1. Finally, we demonstrated that in vivo inhibition of GSK-3 could restore Rae-1 up-regulation on IL18R-/- DCs and partially rescue NK-cell activation against VV, leading to improved viral control in IL-18R-/- mice. Our results showed that IL18-dependent Rae-1 up-regulation on accessory cells is mediated by the MyD88-PI3K-GSK3 pathway. These observations may provide important insights into the design of effective NK cell-based immunotherapies.

Abstract IA20: Targeting molecules mediating the dialogue between epithelial cells and local T cells

Abstract Some extraordinary clinical successes of immunotherapy have had game-changing effects on the way we view cancer biology and the interaction of the immune system with our tissues. The conventional context for considering cancer immunology is one in which dendritic cells and/or monocytes present cancer antigens, particularly neo-antigens generated by somatic mutation, to systemic T cells within cancer-draining lymph nodes, as a result of which an expansive adaptive, cancer-specific T lymphocyte-driven immune response develops. Furthermore, cancer-specific T cells may become tissue-associated, maintaining a so-called Tissue-Resident Memory (TRM) pool. However, although this perspective readily explains many basic biological and clinical data, it leaves several issues unanswered. For example, does the immune system recognize tumor cells that do not carry high mutational loads, many of which cancers have shown disappointing responses to immunotherapy? Related to this, what is the nature of cancer immunogenicity that is required to first activate the immune system, thereby promoting the presentation of cancer antigens by DC and/ or monocyte? We have addressed these issues by considering a separate T cell compartment that is constitutively tissue-resident, rather than being primed in the lymphoid circulation. Prominent within this T cell compartment are intraepithelial gamma delta T cells that have been particularly well characterized in mice, wherein they have been shown to limit sensitivity to carcinogens, and to promote tissue integrity1,2. Moreover, a recent bio-informatic analysis showed a tumor-associated gamma delta T cell signature to be the strongest gene expression correlate with overall survival in over 18,000 patients across 39 tumor types3. Furthermore, another very strong correlate was the expression of NKG2D, a cell surface receptor expressed on tissue-resident gamma delta T cells and whose ligands are upregulated on tumor cells by dysregulation of the EGF-receptor pathway4. Given these observations, we have asked several questions: [1] what is the nature of tissue-resident gamma delta T cell compartments in human skin, gut, and breast tissue? [2] do such cells become activated solely by the upregulation of NKG2D ligands, thereby offering an explanation for tumor immunogenicity? [3] what are the effector functions and potentials of such cells? [4] what are the molecular checkpoints by which the cells are regulated? [5] can the cells be explanted, grown, and cultured in a manner that preserves their function(s) and that can provide a source of such cells to be used for adoptive immunotherapy? [6] might such cells be applied “off-the-shelf” rather than requiring autologous transfusions? Emerging answers to these questions will be provided. Thus, by examining over 100 donors, we have found that human skin, gut, and breast reproducibly harbor large and complex T cell subsets that include substantive gamma delta T cell compartments clearly distinct from most blood-borne gd T cells. These tissue-resident gamma delta T cells show strong, innate-like responses to NKG2D-ligands and to cytokines that were not shown by CD8+ TRM cells harvested in parallel from the same sites. Additionally, however, tissue-resident gamma delta T cells are uniquely and powerfully regulated at steady-state by novel, CD80/B7/PDL1-like molecules known as butyrophilin-like (BTNL) proteins that are expressed by epithelial cells in a tissue-specific fashion, and that de facto constitute local checkpoints. These emerging insights create a new context in which to consider the interaction of immune cells with developing cancers, and possibly to explain the patient benefit of a strong gamma delta T cell signature. At the same time, we have developed novel methods for maintaining and expanding tissue-derived gamma delta T cells that offer a practical off-the-shelf approach to tissue-targeted cellular immunotherapy, and that may be particularly suited to adenocarcinomas of low mutation load or with high rates of tumor evolution and antigenic drift. Citation Format: Oliver Nussbaumer, Yin Wu, Fernanda Kyle, Rick Woolf, Robin Dart, Deborah Enting, Pierre Vantourout, Adrian Hayday. Targeting molecules mediating the dialogue between epithelial cells and local T cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA20.

STAT3 contributes to NK cell recognition by modulating expression of NKG2D ligands in adriamycin-resistant K562/AO2 cells.

Leukemic cells can survive after chemotherapy by acquisition of multidrug resistance genes, but other phenotypes related to escape from immune recognition remain elusive. Adriamycin-resistant K562/AO2 cells are less susceptible to elimination by NK cells compared with wild type K562 cells due to lower expression of NKG2D ligands. Treatment of K562/AO2 cells with STAT3 inhibitor VII resulted in reduced expression of multidrug resistance gene P-glycoprotein, and up-regulation of NKG2D ligands on K562/AO2 cells. Meanwhile, K562/AO2 cells treated with STAT3 inhibitor proliferated less and were more susceptible to killing by NK cells than untreated K562/AO2 cells. The enhanced cytotoxicity of NK cells against K562/AO2 cells was partly blocked by treatment of NK cells with anti-NKG2D antibodies. These data suggest that STAT3 contributes to NK cell recognition by modulating NKG2D ligands in K562/AO2 cells, which may a mechanism by which cells survive and cause relapse of leukemia.

Drug-induced hyperploidy stimulates an antitumor NK cell response mediated by NKG2D and DNAM-1 receptors

ABSTRACTFormation of polyploid or aneuploid cells is a pathological hallmark of malignant tumors. Cell cycle checkpoint mechanisms play a crucial role in ensuring genomic integrity during mitosis, avoiding the generation of aneuploid cells. Additionally, cancer cell DNA ploidy is subjected to extrinsic controls operated by activation of adaptive immune responses mediated by T cells. NK cells exert a central role in the innate anticancer immunity; however, the mechanisms involved in the recognition of tumor cells by NK cells have not been fully elucidated. Herein, we report that drug-induced polyploidy in cancer cells activates antitumor responses mediated by NK cells. Thus, hyperploidy-inducing chemotherapeutic agents strongly upregulate the tumor expression of ligands for the NK cell activating receptors NKG2D and DNAM-1. Drug-induced hyperploidy modulated the repertoire of activating receptors and the cytokine profile of NK cells, rendering tumor cells more susceptible to NK cell-mediated lysis through the activation of NKG2D and DNAM-1 receptors. In addition, hyperploidization stimulated the production of IL-2 by CD4 T cells, which induced NK cell proliferation and activity. The stimulation of MICA, a key NKG2D ligand, in hyperploid cells was mainly mediated by ATM protein kinase. Likewise, pharmacological inhibition of key regulators of endoplasmic reticulum stress in certain cell models supports a role for this pathway in NKG2D ligand upregulation. Overall, our findings indicate that, besides the cytotoxic effect on tumor cells, the therapeutic activity of anti-mitotic drugs may be mediated by the induction of a coordinated antitumor immune response involving NK and T cells.

Combination doxorubicin and interferon-α therapy stimulates immunogenicity of murine pancreatic cancer Panc02 cells via up-regulation of NKG2D ligands and MHC class I.

Pancreatic adenocarcinoma is a malignant gastrointestinal cancer with significant morbidity and mortality. Despite severe side effects of chemotherapy, the use of immunotherapy combined with chemotherapy has emerged as a common clinical treatment. In this study, we investigated the efficacy of the combined doxorubicin and interferon-α (IFN-α) therapy on murine pancreatic cancer Panc02 cells in vitro and in vivo and underlying mechanisms. A Panc02-bearing mouse model was established to determine whether doxorubicin and interferon-α (IFN-α) could effectively inhibit tumor growth in vivo. Cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) was evaluated using a standard LDH release assay. To evaluate the relevance of NK cells and CD8 T cells to the combination therapy-mediated anti-tumor effects, they were depleted in tumor-bearing mice by injecting anti-asialo-GM-1 antibodies or anti-CD8 antibodies, respectively. Finally, the influence of doxorubicin+interferon-α (IFN-α) on the ligands of NK and T cells was assessed by flow cytometry. The combination therapy group demonstrated a significant inhibition of growth of Panc02 in vivo, resulting from activated cytotoxicity of NK cells and CTLs. Depleting CD8 T cells or NK cells reduced the anticancer effects mediated by immunochemotherapy. Furthermore, the doxorubicin+IFN-a treatment increased the expression of major histocompatibility complex class I (MHC I) and NKG2D ligands on Panc02 cells, suggesting that the combined therapy may be a potential strategy for enhancing immunogenicity of tumors. All these data indicate that the combination therapy using doxorubicin and interferon-α (IFN-α) may be a potential strategy for treating pancreatic adenocarcinoma.

NK cell-extrinsic IL-18 signaling is required for efficient NK-cell activation by vaccinia virus.

NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR-dependent and -independent pathways, as well as the NKG2D activating receptor that recognizes host stress-induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study using C57BL/6 mice, we first showed that IL-18 is critical for NK-cell activation and viral clearance. We then demonstrated that IL-18 signaling on both NK cells and DCs is required for efficient NK-cell activation upon VV infection in vitro. We further showed in vivo that efficient NK-cell activation in response to VV is dependent on DCs and IL-18 signaling in non-NK cells, suggesting an essential role for NK cell-extrinsic IL-18 signaling in NK-cell activation. Mechanistically, IL-18 signaling in DCs promotes expression of Rae-1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell-extrinsic IL-18 signaling in NK-cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK-cell-based therapies for viral infections and cancer.

Upregulation of NKG2D ligands in acute lymphoblastic leukemia and non-Hodgkin lymphoma cells by romidepsin and enhanced in vitro and in vivo natural killer cell cytotoxicity

Background aimsThere is a critical need to prevent and/or treat hematological relapse after allogeneic hematopoietic stem cell transplantation. The activating NKG2D receptor expressed on natural killer (NK) cells, when engaged by its corresponding ligands (MIC A/B), activates NK cells to become cytotoxic against malignant cells. MethodsWe incubated acute lymphoblastic leukemia and non-Hodgkin lymphoma cells for 24 h with 10 ng/mL of romidepsin. Flow cytometry was performed to demonstrate changes in surface expression of NKG2D ligands MIC A/B. In vitro and in vivo cytotoxicity was measured by means of modified Europium assay, and non-obese diabetic/severe combined immunodeficiency mice were xenografted with RS 4:11 cells. ResultsWe demonstrated an approximately 50, 200, 1300 and 180-fold increase in the number of cells positive for the surface expression of MIC A/B in RS 4:11 (P < 0.001), REH (P < 0.001), Ramos (P < 0.001) and Jurkat cells (P < 0.001), respectively. We further demonstrated a significant increase in NK cell–mediated in vitro cytotoxicity against RS 4:11 (P < 0.004), Ramos (P < 0.05), Jurkat (P < 0.001) and REH cells (P < 0.01), respectively. Romidepsin-mediated NK cytotoxicity was blocked by pre-incubating NK cells with anti-NKG2D-Fc in RS 4:11 (P < 0.03) and Ramos cells (P < 0.01), respectively. Finally, non-obese diabetic/severe combined immunodeficiency mice xenografted with RS 4:11 cells had a significant increase in survival (P < 0.02) in mice treated with romidepsin and interleukin-2–activated NK cells compared with each of these other treatment groups. ConclusionsRomidepsin significantly enhanced in vitro and in vivo NK cell cytotoxicity mediated in part by increased MIC A/B expression on malignant cells. This translational approach of the use of romidepsin and interleukin-2–activated NK cells should be considered in patients with relapsed/refractory leukemia or lymphoma.

5-Fluorouracil and interleukin-2 immunochemotherapy enhances immunogenicity of non-small cell lung cancer A549 cells through upregulation of NKG2D ligands.

The aim of this study was to investigate the anti-cancer effects and mechanisms of immunochemotherapy of 5-fluorouracil (5-FU) and interleukin-2 (IL-2) on non-small cell lung cancer (NSCLC) A549 cells. In order to detect whether 5-FU+IL-2 could effectively inhibit tumor growth in vivo, we established an A549-bearing nude mouse model. The cytotoxicity of natural killer (NK) cells was evaluated using a standard chromium release assay. To evaluate the relevance of NK cells in 5-FU+IL-2- mediated tumor inhibitory effects, we depleted NK cells in A549-bearing mice by injecting anti-asialo-GM-1 antibodies. Effects of 5-FU+IL-2 on the expression and promoter activity of NKG2D ligands (MICA/MICB) in A549 cells in vitro were also assessed. In A549-bearing nude mice, combination therapy significantly inhibited tumor growth in comparison with monotherapy with 5-FU or IL-2 and enhanced the recognition and lysis of tumor cells by NK cells. Further study of mechanisms showed that NK cells played a vital role in the anticancer immune response of 5-FU+IL-2 immunochemotherapy. In addition, the combination therapy synergistically stimulated the expression and promoter activity of MICA/MICB. 5-FU and IL-2 immunochemotherapy significantly inhibited tumor growth and activated NK cytotoxicity in vivo, and these effects were partly impaired after depleting NK cells in tumor-bearing mice. Combination treatment of 5-FU and IL-2 upregulated the expression and the promoter activity of MICA/MICB in A549 cells, which enhanced the recognition of A549 cells by NK cells. All of the data indicated that immunochemotherapy of 5-FU and IL-2 may provide a new treatment option for patients with lung cancer.

Role of NKG2D in the induction of mouse natural killer cell exhaustion (IRC8P.473)

Abstract Natural Killer (NK) cells have been used as a cancer therapy against multiple cancers. Despite initial promises, NK-based immunotherapy has resulted in limited success and therefore, new approaches are needed. Our group described the induction of NK cell exhaustion (NCE) upon tumor exposure that resulted in reduced cytotoxic function associated with lower expression of several molecules such as NKG2D and the transcription factors Eomesodermin and T-bet. Here we show that NCE induced by tumor cells is MHC-I independent and is not mediated by the expression of NKG2D ligands (NKG2DL) on tumor cells. However, when NKG2D-deficient NK cells were exposed to tumor cells limited proliferation and higher IFNg production was observed suggesting a role for NKG2D in NCE. Moreover, NCE patters were observed at later stages of in vitro IL2-activation that were reduced in NKG2D-deficient NK cells. Interestingly, concomitant downregulation of NKG2D and upregulation of NKG2DL (MULT1, Rae-1) was also detected. Upregulation of NKGDL expression was particularly seen after the cells were acid-stripped which was shown to disrupt MHC-I-Ly49s cis-interaction complexes. NCE was also observed after prolonged in vivo IL2 stimulation that impaired NK cell maturation. In conclusion, we demonstrated a role for NKG2D in the induction of NCE that could represent a self-regulatory mechanism important in controlling NK cell activation utilized by tumor cells to prevent NK cell anti-tumor responses.

Identification and Characterization Of Human NK Lineage Restricted Progenitor (NKP)

Natural Killer (NK) cells, the third lymphoid lineage after T and B cells, are large granular lymphocytes belonging to innate immune system, classically defined as CD3-CD56+CD16+ in human. NK cells can kill target cells without prior activation, based on the missing self-hypothesis, either due to the lack of MHC-class I molecule or up-regulation of NKG2D ligands, two properties commonly associated with transformed cells (D.H. Raulet, N. Guerra, 2009). Moreover, by secreting cytokines (such as TNF-α and IFN-γ), NK cells can also activate the response of the adaptive immune system. Experiments in mice have shown that NK cells are primarily responsible for the in vivo elimination of transplanted tumor cells (J. Wu, L. L. Lanier, 2003) and, in human, a direct correlation between low NK cytotoxicity and elevated cancer incidence was reported in a large cohort of Japanese aged over 40 (K. Imai et al., 2000). The important anti-tumor role for alloreactive NK cells has been shown in patients with acute myeloid leukemia where transplanted donor NK cells, expressing mismatch inhibitory receptors, provided graft versus leukemia effect in the absence of graft versus host disease (L. Ruggeri et al., 2006). These and many other findings are the basis of a large number of ongoing studies and clinical trials for NK cell immunotherapy against cancer (M. Terme et al., 2008). However, in contrast to B and T cell development where both cellular and regulatory pathways are well studied, the development of NK cells from hematopoietic stem cells (HSCs) is not well understood and the identity of restricted NK cell progenitor (NKP) is unknown (S. Doulatov et al., 2012).To identify human NKP, we applied the expression of known early lymphoid markers (CD45RA, CD10, CD7) and cytokine receptors (IL-7R: CD127) important for lymphoid development. Using multicolor flow cytometry, Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+/-CD127+/- populations were sorted from human bone marrow (hBM) and umbilical cord blood (hUCB). Purified candidate NKPs were cultured on OP9, OP9DL1 stroma and in Terasaki cultures in the presence of specific cytokines and generation of CD3-CD56+CD16+NKp46+ NK cells, CD19+ B, CD3+ CD4+ CD8+ T and CD33+CD14+ myeloid cells was investigated. To study the ability of NKP candidate to generate NK cells after transplantation, purified NKPs were injected into new-born NOD/SCID γcnull (NSG) mice and at 11 weeks after transplantation the phenotype of generated progeny was evaluated. In addition, NK cells generated in stroma cultures and after transplantation were tested for their cytotoxic activity against K562 leukemic cell line in degranulation assay, where the surface expression of membrane glycoprotein LAMP-1 (CD107a) is measured after activation of specific killing receptors.The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate population sorted from hUCB has a robust NK cell potential in vitro at the single cell level and lacked the ability to generate T, B and myeloid cells. Furthermore, NK cells generated from this candidate NKP were functionally mature: show cytotoxic activity against K562 cells and produce cytokines: IFNγ and TNFα after activation in vitro. Further phenotypic characterization showed that the candidate NKP is highly positive for lymphoid markers CD244 and CD62L, lacks the expression of mature NK cell markers NKp46 and NKG2D, and 50% of NKPs express c-kit and Flt3 receptors. At 11 weeks after transplantation, 9 out of 17 NSG mice injected with 600 Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKPs had a significant human cells engraftment and only CD3-CD56+NKp46+CD16+/- NK cells were found in the peripheral blood, bone marrow and spleen; whereas 15 out 17 NSG mice injected with CD34+ cells were positive for the presence of human B, T, NK and myeloid cells in these tissues. Human NK cells generated after transplantation into NSG mice were functionally active and able to kill K562 leukemic cells. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate was also found in adult hBM and showed NK-lineage restriction.Our results indicate that the Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- cells, found in human BM and UCB, represent NK-lineage restricted progenitors that generate fully mature functional NK cells. Disclosures:No relevant conflicts of interest to declare.

Transient NKG2D Blockade Attenuates Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Effects

In allogeneic hematopoietic stem cell transplantation (HSCT), identification of mechanisms to control GVHD yet maintain GVL responses is of critical importance. One key effector cell that mediates both GVHD and GVL is the CD8+ T cell, which expands in response to T cell receptor (TCR) stimulation by allogeneic MHC class I molecules during allogeneic HSCT. In addition, co-stimulatory molecules facilitate the TCR-mediated activation process and the effector function of CD8+ T cells. Recent data suggest that NKG2D may play a co-stimulatory role in activation and in augmenting anti-tumor cytotoxic responses of CD8+ T cells. NKG2D is an NK cell-associated receptor that is also expressed on all human CD8+ T cells and on activated/memory mouse CD8+ T cells. NKG2D recognizes a diverse array of MHC-related ligands that are expressed by many tumors and induced on cells under stress such as myeloablative conditioning during HSCT. As the role of NKG2D in allogeneic HSCT is unknown, we hereby investigated the role of NKG2D on CD8+ T cells in a mouse model of GVHD and GVL.Our results show that a large fraction (40-50%) of mouse CD8+ T cells inducibly express NKG2D upon activation by allogeneic MHC in vitro and in vivo. To test the role of NKG2D in GVHD pathogenesis, we employed a major MHC-mismatched mouse model of GVHD involving the transplantation of C57BL/6-derived CD8+ T cells and bone marrow (BM) into lethally irradiated Balb/c mice (B6→Balb/c). Using 3 different approaches to block NKG2D on CD8+ T cells (shRNA-mediated silencing, germline NKG2D deficiency, and antibody blockade), we found that weight loss, clinical score, and survival were significantly improved in transplanted mice with NKG2D blockade. The attenuation in GVHD correlated with a significant reduction in TNFα and IFNγ production, cytotoxicity, and proliferation (BrdU incorporation) by CD8+ T cells. Although CD4+ T cells did not express NKG2D, a protective effect of NKG2D blockade was still observed in GVHD induced by a mixture of CD8+ and CD4+ T cells, albeit to a lesser extent.We next tested the effects of NKG2D on CD8+ T cell-mediated GVL. To this end, irradiated Balb/c mice were transplanted with C57BL/6-derived CD8+ T cells and BM, challenged intravenously with luciferase-positive A20 leukemia cells, and followed by total body imaging of luciferase-expressing cells. Given that NKG2D ligands are constitutively expressed on many tumor cells and plays an important role in their eradication, we predicted that continuous NKG2D blockade would inhibit GVL effects. However, as NKGD ligands are upregulated only transiently on stressed normal tissue, we reasoned that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8+ T cells to regain their GVL function. To test this hypothesis, we compared the effect of anti-NKG2D antibody as continuous treatment or as 5-day transient treatment to mice receiving isotype control antibody. As expected, mice that received isotype control antibody cleared the A20 cells but developed severe GVHD. Continuous anti-NKG2D antibody-mediated blockade improved GVHD but also blunted the GVL response leading to increased A20 growth. In contrast, a large proportion of mice transiently treated with anti-NKG2D antibody cleared the A20 cells, while maintaining the attenuated GVHD state. Together, these data support a positive role of NKG2D on CD8+ T cells in mediating GVHD and GVL. Given the transient nature of NKG2D ligand upregulation on stressed tissues, a window of opportunity may exist where transient NKG2D blockade could provide a novel therapeutic strategy for treatment of acute GVHD while preserving the GVL function of CD8+ T cells after allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.

A Role for Host Activation-Induced Cytidine Deaminase in Innate Immune Defense against KSHV

Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens.