NK Cell-dependent Manner Research Articles

Abstract IA018: Determinants of B cell fate and function in cancer

Abstract Effector B cell responses in solid malignancies are associated with favorable response to immunotherapy. B cells can amplify anti-tumor immune responses via antibody production, antigen presentation, and pro-inflammatory cytokine release; yet B cells in cancer patients and tumor-bearing mice often fail to support these functions. We have evaluated the contribution of either systemic inflammatory cues or antigenic quality to B cell differentiation and function in cancer. First, we identify dysregulated transcriptional programs activated in pancreatic cancer-associated B cells that promote differentiation of naïve into immunosuppressive B cells and inhibit differentiation of anti-tumor plasma cells. Second, we find that systemic exposure to STING agonists potentiates expansion of immunosuppressive regulatory human and mouse IL35+ B lymphocytes. Finally, using a system where a B cell neoantigen is either soluble or tethered to the surface of cancer cells, we demonstrate that membrane-tethered antigen is sufficient to elicit activation of anti-tumor immunity and reduce tumor growth in a NK cell-dependent manner. Thus, inflammation and quality of antigen impact basic mechanisms governing B cell differentiation and the resulting immune response to solid malignancy with implications for vaccine engineering. Citation Format: Yuliya Pylayeva-Gupta. Determinants of B cell fate and function in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr IA018.

Abstract B047: Antigenic properties determine the outcome of B cell driven anti-tumor immunity

Abstract B cells have the capacity to elicit both anti- and pro-tumorigenic effects; yet the determinants of productive B cell responses in cancer remain elusive. Using a system where a B cell neoantigen is either soluble or tethered to the surface of pancreatic cancer cells, we demonstrate that membrane-tethered antigen is sufficient to elicit activation of anti-tumor immunity and reduce tumor growth in an NK cell-dependent manner. We also show that decreased antigen affinity to the BCR promotes regulatory B cell function which can be overcome by increased avidity of membrane-tethered antigen. Furthermore, soluble and membrane-tethered antigens drive qualitatively distinct germinal center, plasma cell, memory B cell, and Treg responses. Finally, we describe two classes of membrane-associated antigens that correlate with GC B cell signature and improved survival in pancreatic cancer patients. Thus, antigen localization impacts basic mechanisms governing B cell differentiation and the resulting immune response to solid malignancy with implications for vaccine engineering. Citation Format: Colleen Sturdevant, Nancy Kren, Harrison Silva, Ismael Gomez, Austin Hepperla, Yuliya Pylayeva-Gupta. Antigenic properties determine the outcome of B cell driven anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B047.

THEMIS2 Impairs Antitumor Activity of NK Cells by Suppressing Activating NK Receptor Signaling.

NK cells are cytotoxic innate lymphocytes that play a critical role in antitumor immunity. NK cells recognize target cells by using a repertoire of activating NK receptors and exert the effector functions. Although the magnitude of activation signals through activating NK receptors controls NK cell function, it has not been fully understood how these activating signals are modulated in NK cells. In this study, we found that a scaffold protein, THEMIS2, inhibits activating NK receptor signaling. Overexpression of THEMIS2 attenuated the effector function of human NK cells, whereas knockdown of THEMIS2 enhanced it. Mechanistically, THEMIS2 binds to GRB2 and phosphorylated SHP-1 and SHP-2 at the proximity of activating NK receptors DNAM-1 and NKG2D. Knockdown of THEMIS2 in primary human NK cells promoted the effector functions. Furthermore, Themis2-deficient mice showed low metastatic burden in an NK cell-dependent manner. These findings demonstrate that THEMIS2 has an inhibitory role in the antitumor activity of NK cells, suggesting that THEMIS2 might be a potential therapeutic target for NK cell-mediated cancer immunotherapy.

Pharmacologic tumor PDL1 depletion as a novel approach to overcome treatment resistance

Abstract Tumor surface PDL1 canonically engages immune cell PD1 to inhibit anti-tumor immunity. However, PDL1 also mediates tumor-intrinsic pathologic signals, including promoting the DNA damage response (DDR) and suppressing immunogenic STING. Genetic tumor PDL1 depletion (CRISPR/Cas9, shRNA) induces novel treatment vulnerabilities that are not replicated by αPDL1 antibodies, but genetic tumor PDL1 depletion is not clinically feasible. We hypothesized that pharmacologic tumor PDL1 depletion could replicate genetic PDL1 depletion effects as a highly translational strategy to create novel treatment vulnerabilities. We conducted a high-throughput drug screen that identified 15 tumor PDL1 depleting drugs (PDDs) that potently reduce tumor PDL1 and are FDA-approved. Cefepime (β-lactam antibiotic) is a PDD that induced PDL1-dependent Chk2 DDR protein depletion (immunoblots), Chk1 inhibitor sensitivity in vitro (MTT, p<0.0001) and in vivo in wild-type and NSG mice (p=0.03), and immunogenic STING signals (immunoblots, RT-qPCR). The PDD telmisartan (angiotensin II receptor blocker) induced PARP inhibitor sensitivity in vitro (p<0.0001), impaired homologous recombination DDR (DR-GFP reporter, immunoblots), and induced STING signals and immunogenic cell death (flow cytometry). The PDD chlorambucil (alkylating agent) selectively depleted tumor PDL1 in vivo and sensitized αPDL1-resistant tumors to αPDL1 in a tumor PDL1 and NK cell-dependent manner. Thus, PDDs deplete tumor PDL1 to inhibit its pathologic cell-intrinsic signals, induce novel treatment vulnerabilities, improve tumor immunogenicity, and are rapidly translatable. Supported by grants from NIH (Graduate Research in Immunology Program training grant T32 AI138944, TL1 TR002647, T32GM113896, CA204965, CA054515) and the Clayton Foundation (no grant number).

Abstract 4166: Effects of cyproheptadine in eliciting natural killer cell-mediated immune response in bladder cancer

Abstract Bladder cancer is one of the most common occurring cancers in urothelial carcinoma. The previous study suggested that cancer cells undergo immunoevasion due to epigenetic silencing of NKG2DL, in which NKG2D-NKG2DL interaction induced natural killer (NK) cell-mediated cytotoxicity. We hypothesized that NK-mediated cytotoxicity can be reactivated by restoring the expression of NKG2DL on tumor cells. Our previous studies demonstrated that cyproheptadine (CPH) is a novel HDAC inhibitor exhibiting antitumor activity by cell cycle arrest and apoptosis. To understand the mechanism behind, RNA-seq showed an upregulation of ULBP2, a NKG2DL, in bladder cancer cells treated with CPH. Interestingly, upregulation of ULBP2, was accompanied with the enrichment of H3K27Ac and H3K4me3 at ULBP2 promoter, was observed in cells treated with CPH. Increased NK-specific lysis was observed in CPH-treated cells co-cultured with NK-92 cells. More importantly, treatment of CPH enhanced the efficacy of immune checkpoint inhibitor in a syngeneic mice bladder cancer model. In conclusion, our results suggested that CPH may be a novel HDAC inhibitor which may elicit immune response in NK-cell dependent manner. Citation Format: Himani Kumari, Guan-Ling Lin, Chih-Chieh Yeh, Ciao-Ni Chen, Yu-Ming Chuang, Wan-Hong Huang, Wen-Long Huang, Steven Lin, Michael W. Chan. Effects of cyproheptadine in eliciting natural killer cell-mediated immune response in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4166.

A phase 1/2 multicenter investigator-initiated trial of DKN-01 as monotherapy or in combination with docetaxel for the treatment of metastatic castration-resistant prostate cancer (mCRPC).

5048 Background: Dickkopf-1 (DKK1) is a secreted Wnt signaling modulator that is upregulated in prostate cancers with low androgen receptor (AR) expression and co-occurring mutations in Wnt signaling family genes. DKN-01, a potent humanized monoclonal antibody (IgG4) with neutralizing activity against DKK1, delays prostate cancer growth in pre-clinical DKK1-expressing models in an NK cell dependent manner. These data provided the rationale for a prospective clinical trial testing DKN-01 in patients with mCRPC and elevated DKK1. Here, we report the safety and efficacy results of the phase 1 dose escalation cohorts. Methods: This is an investigator-initiated parallel-arm non-randomized phase 1/2 clinical trial testing DKN-01 alone or in combination with docetaxel 75 mg/m2 for men with mCRPC who progressed on ≥1 AR signaling inhibitor. Eligible patients who had progressed on or were intolerant of docetaxel were assigned to the monotherapy cohort whereas taxane-naïve patients were assigned to the DKN-01 plus docetaxel combination cohort. DKK1 status was determined by RNA in-situ expression. The primary endpoint of the phase 1 dose escalation cohorts was safety, characterized by dose-limiting toxicity (DLT). A secondary endpoint of the study was to correlate anti-tumor activity, DKK1 expression (cutoff H-score ≥ 1), and clinical evidence of aggressive variant prostate cancer (AVPC). Results: 13 pts were enrolled in the completed phase 1 portion of this study – 7 patients in the monotherapy cohort and 6 patients in the combination cohort. No DLTs were observed at DKN-01 300mg or 600mg dose levels as monotherapy or in combination with docetaxel. No treatment-related serious adverse events occurred in either cohort. A best overall response of stable disease occurred in 2 out of 7 patients in the monotherapy cohort. In the combination cohort, all 5 evaluable patients had a partial response (PR) – 3 confirmed and 2 unconfirmed. All evaluable combination patients had ≥ 50% reduction in either PSA or CEA. Confirmed PRs in the combination cohort were observed in both DKK1 low (DKK1 H-score < 1) and high expressing tumors (H-score ≥1), including in 2 out of 3 patients with AVPC. Conclusions: DKN-01 600mg was well tolerated and selected as the recommended phase 2 dose as monotherapy and in combination with docetaxel. DKN-01 in combination with docetaxel showed promising clinical activity in prostate cancers regardless of DKK1 expression and was particularly promising in patients with AVPC. Further accrual into the phase 2 portion of this study is ongoing alongside preclinical and correlative studies aiming to investigate the mechanism of action of this combination therapeutic strategy. Clinical trial information: NCT03837353.

Tim-3 Hampers Tumor Surveillance of Liver-Resident and Conventional NK Cells by Disrupting PI3K Signaling.

Natural killer (NK) cells are enriched within the liver. Apart from conventional NK (cNK) cells, recent studies identified a liver-resident NK (LrNK) subset, which constitutes about half of hepatic NK cells and exhibits distinct developmental, phenotypic, and functional features. However, it remains unclear whether and how LrNK cells, as well as cNK cells, participate in the development of hepatocellular carcinoma (HCC) individually. Here, we report that both LrNK and cNK cells are significantly decreased in HCC. The T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was significantly upregulated in both tumor-infiltrating LrNK and cNK cells and suppressed their cytokine secretion and cytotoxic activity. Mechanistically, phosphatidylserine (PtdSer) engagement promoted phosphorylation of Tim-3, which then competed with PI3K p110 to bind p85, inhibiting downstream Akt/mTORC1 signaling and resulting in malfunctioning of both NK-cell subsets. Tim-3 blockade retarded HCC growth in a NK-cell-dependent manner. These studies for the first time report the presence and dysfunction of LrNK cells in HCC and show that Tim-3-mediated PI3K/mTORC1 interference is responsible for the dysfunction of both tumor-infiltrating cNK and LrNK cells, providing a new strategy for immune checkpoint-based targeting. SIGNIFICANCE: Tim-3 enhances hepatocellular carcinoma growth by blocking natural killer cell function.

Induction of oligoclonal CD8 T cell responses against pulmonary metastatic cancer by a phospholipid-conjugated TLR7 agonist

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8+ cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.

Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines.

The development of a vaccine against human cytomegalovirus (CMV) has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV) expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1) (MULT-1MCMV) inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.

Efficient Attenuation of NK Cell–Mediated Liver Injury through Genetically Manipulating Multiple Immunogenes by Using a Liver-Directed Vector

Adenovirus or adenoviral vectors were reported to induce serious liver inflammation in an NK cell-dependent manner, which limits its clinical applicability for liver gene therapy. We tried to develop an efficient liver-directed therapeutic approach to control hepatic NK cell function via simultaneously manipulating multiple immune genes. Based on our previous study, we found that CCL5 knockdown synergistically enhanced the attenuating effect of silencing CX3CL1 (fractalkine [FKN]) in adenovirus-induced acute liver injury. In addition, the combined treatment of human IL-10 expression with FKN knockdown would further strengthen the protective effect of silencing FKN. We used a hepatocyte-specific promoter to construct a hepatocyte-specific multiple function vector, which could simultaneously overexpress human IL-10 and knock down CCL5 and FKN expression. This vector could attenuate adenovirus-induced acute hepatitis highly efficiently by reducing liver NK cell recruitment and serum IFN-γ and TNF-α. The multiple function vectors could be delivered by nonviral (hydrodynamic injection) and viral (adenovirus) approaches, and maintained long-term function (more than 1 month in mice). Our results suggest a possible strategy to ameliorate the acute liver injury induced by adenovirus by modulating multiple immune genes. The novel multifunction vector has an extensive and practical use for polygenic and complex liver diseases such as malignancies and hepatitis, which correlate with multiple gene disorders.

Abstract 493: Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model.

Abstract Natural Killer cells (NK cells) are lymphocytes able to recognize and kill tumors for which the expression of Major Histocompatibility Complex (MHC) class I molecules is altered. This “missing self” recognition is mediated in humans by the lack of engagement of MHC class I i.e. Human Leucocyte Antigens (HLA) molecules with NK cell inhibitory receptors that include Killer Immunoglobulin-like Receptors (KIR). Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. Consequently, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, BMS-986015/IPH2102, is a human IgG4 that is being developed for treating both hematologic malignancies and solid tumors. In rodents, the MHC class I inhibitory system regulating NK cell activation is based on lectin-like family Ly49 but the KIR molecules are not expressed. The objective of this study was to develop a preclinical model to assess the efficacy of the drug candidate used in clinical trials, BMS-986015/IPH2102. Mice expressing the human NK inhibitory KIR2DL3, on the surface of NK cells were generated on a RAG-1deficient background (KIRtgRAG mice). The human B cell lymphoma cell line, 721.221, transduced with HLA-Cw3 molecule, a ligand of KIR2DL3, was intra-venously engrafted in these mice. The expression of HLA-C by tumor cells was sufficient to allow them to escape control of NK cells, leading to mice death in around 30 days. IPH2102 treatment increased mice survival in a dose dependent manner when injected at the same time as the tumor challenge. This protective effect was NK cell mediated and directly correlated with the duration of KIR saturation. Interestingly, BMS-986015/IPH2102 treatment also improved survival in therapeutic conditions i.e. when the antibody was injected 5 days after the tumor, also in a NK cell dependent manner. In conclusion, this study showed efficacy of BMS-986015/IPH2102 as single agent in a HLA-Cw3-expressing tumor model and this xenogenic pre-clinical model will be an excellent tool to investigate the therapeutic benefits of combination treatments. Citation Format: Caroline Sola, Fabien Chanuc, Ariane Thielens, Nicolas Fuseri, Isabelle Palacios, Mathieu Blery, Pascale André, Eric Vivier, Sophie Ugolini, Robert Graziano, François Romagne, Cécile Bonnafous. Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2013-493

Plasma membrane proteomics identifies bone marrow stromal antigen 2 as a potential therapeutic target in endometrial cancer

This report utilizes a novel proteomic method for discovering potential therapeutic targets in endometrial cancer. We used a biotinylation-based approach for cell-surface protein enrichment combined with isobaric tags for relative and absolute quantitation (iTRAQ) technology using nano liquid chromatography-tandem mass spectrometry analysis to identify specifically overexpressed proteins in endometrial cancer cells compared with normal endometrial cells. We identified a total of 272 proteins, including 11 plasma membrane proteins, whose expression increased more than twofold in at least four of seven endometrial cancer cell lines compared with a normal endometrial cell line. Overexpression of bone marrow stromal antigen 2 (BST2) was detected and the observation was supported by immunohistochemical analysis using clinical samples. The expression of BST2 was more characteristic of 118 endometrial cancer tissues compared with 59 normal endometrial tissues (p < 0.0001). The therapeutic effect of an anti-BST2 antibody was studied both in vitro and in vivo. An anti-BST2 monoclonal antibody showed in vitro cytotoxicity in BST2-positive endometrial cancer cells via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In an in vivo xenograft model, anti-BST2 antibody treatment significantly inhibited tumor growth of BST2-positive endometrial cancer cells in an NK cell-dependent manner. The anti-BST2 antibody had a potent antitumor effect against endometrial cancer both in vitro and in vivo, indicating a strong potential for clinical use of anti-BST2 antibody for endometrial cancer treatment. The combination of biotinylation-based enrichment of cell-surface proteins and iTRAQ analysis should be a useful screening method for future discovery of potential therapeutic targets.

IL-17D mediated cancer rejection (162.26)

Abstract Immune system interaction with cancers can either enhance or prevent tumor formation. Cytokine secretion by immune infiltrates and tumor cells are important in regulating this process. IL-17A, a pro-inflammatory member of the IL-17 family, has been shown to have conflicting results in regards to cancer progression or regression. Other members of the IL-17 family, such as IL-17D, have no known roles in immune responses to cancer, or endogenous function. Our lab possesses matched methylcholanthrene (MCA)-induced tumor cell lines that will continuously grow (Progressors) or reject (Regressors) after transplantation into syngeneic WT mice. The regresssor cell lines can grow in immune-deficient RAG2-/- mice, indicating that their rejection in WT mice requires an intact immune system. Our lab has used these cell lines to identify differential cytokine expression profiles from microarray analysis. We have obtained data that shows IL-17D transcript and protein are significantly overexpressed in regressor cell lines when compared to progressor cell lines. Overexpression of IL-7D in progressor tumor cell lines leads to their rejection in WT mice in a NK cell dependent manner. We hypothesize that IL-17D expression in regressor cell lines leads to immune-mediated tumor rejection by recruiting NK cells that polarize M1 macrophages in the tumor microenvironment.

IFN-γ Treatment at Early Stages of Influenza Virus Infection Protects Mice from Death in a NK Cell-Dependent Manner

Influenza pandemics are imminent and represent a major world health concern. Since vaccinations are expected to be less efficient in the coming years due to newly emerging influenza virus strains, novel antiviral therapies are urgently needed. Here, we show that influenza-infected mice, capable of clearing the virus in the early stages of infection, failed to control inflammation and death. Sequential administration of Interferon-gamma (IFN-gamma) at early stage of the infection protected infected mice from death in a NK cell-dependent manner. IFN-gamma treatment stimulated NK cell proliferation and function and increased their number in the bone marrow, blood, spleen, and infected lungs, keeping viral clearance intact. In parallel, IFN-gamma treatment significantly reduced the number of T cells and NKT cells in the lungs at the inflammatory phase following infection. Thus, rapidly clearing the virus and reducing inflammation by shaping the cellular and cytokine profiles in the early stages of infection may favorably change the fate of influenza pathogenesis.

Vaccination with survivin as a self TAA and SA-4-1BBL is effective in eradicating established lung carcinomas in CD8+ T cell and NK cell dependent manner (131.29)

Abstract Costimulatory ligands of TNF family have great potential as adjuvant for therapeutic vaccines due to their critical role in the expansion of T cells, survival, and long-term memory. Particularly, 4-1BBL has the most potent T cell activity among the TNF costimulatory members. However, the use of 4-1BBL as the immunomodulatory component of conventional therapeutic vaccine has been limited due to its lack of activity in soluble form. We have recently generated a chimeric 4-1BBL with streptavidin (SA-4-1BBL) that exists as tetramers/oligomers with potent activity in soluble form. We herein formulated a subunit protein based vaccine containing SA-4-1BBL and survivin as an autologous tumor associated antigen. The therapeutic efficacy of this vaccine was tested in the transplantable survivin expressing 3LL metastatic lung tumor model. Vaccination with the survivin and SA-4-1BBL resulted in the eradication of established 3LL tumors in 75% of the animals. Importantly, animals with successful immunotherapy developed a robust NK cell cytotoxicity, antigen specific T cell proliferation, and IFN-γ and IL-2 responses. Depletion of various immune cell types demonstrated the indispensable role of CD8+ T cells and important role of NK cells for vaccine efficacy. Chimeric SA-4-1BBL hence prime CD8/NK cell axis for tumor eradication and has potential to be used as a specific immunological adjuvant to develop therapeutic vaccines against tumors with well characterized tumor associated antigens.

Requirements for control of B‐cell lymphoma by NK cells

The role of NK cells in the control of endogenously arising tumors is still unclear. We monitored activation and effector functions of NK cells in a c-myc-transgenic mouse model of spontaneously arising lymphoma. At early stages, tumors demonstrated reduced MHC class I expression and increased expression of natural killer group 2D ligands (NKG2D-L). NK cells in these tumors showed an activated phenotype that correlated with the loss of tumor MHC class I. With increasing tumor load however, NK-cell effector functions became progressively paralyzed or exhausted. In later stages of disease, tumors re-expressed MHC class I and lost NKG2D-L, suggesting a role of these two signals for NK cell-mediated tumor control. Testing a panel of lymphoma cell lines expressing various MHC class I and NKG2D-L levels suggested that NK cell-dependent tumor control required a priming and a triggering signal that were provided by MHC class I down-regulation and by NKG2D-L, respectively. Deleting either of the "two signals" resulted in tumor escape. At early disease stages, immune stimulation through TLR-ligands in vivo efficiently delayed lymphoma growth in a strictly NK cell-dependent manner. Thus, NK-receptor coengagement is crucial for NK-cell functions in vivo and especially for NK cell-mediated tumor surveillance.

Cyclophosphamide Chemotherapy Sensitizes Tumor Cells to TRAIL-Dependent CD8 T Cell-Mediated Immune Attack Resulting in Suppression of Tumor Growth

BackgroundAnti-cancer chemotherapy can be simultaneously lymphodepleting and immunostimulatory. Pre-clinical models clearly demonstrate that chemotherapy can synergize with immunotherapy, raising the question how the immune system can be mobilized to generate anti-tumor immune responses in the context of chemotherapy.Methods and FindingsWe used a mouse model of malignant mesothelioma, AB1-HA, to investigate T cell-dependent tumor resolution after chemotherapy. Established AB1-HA tumors were cured by a single dose of cyclophosphamide in a CD8 T cell- and NK cell-dependent manner. This treatment was associated with an IFN-α/β response and a profound negative impact on the anti-tumor and total CD8 T cell responses. Despite this negative effect, CD8 T cells were essential for curative responses. The important effector molecules used by the anti-tumor immune response included IFN-γ and TRAIL. The importance of TRAIL was supported by experiments in nude mice where the lack of functional T cells could be compensated by agonistic anti-TRAIL-receptor (DR5) antibodies.ConclusionThe data support a model in which chemotherapy sensitizes tumor cells for T cell-, and possibly NK cell-, mediated apoptosis. A key role of tumor cell sensitization to immune attack is supported by the role of TRAIL in tumor resolution and explains the paradox of successful CD8 T cell-dependent anti-tumor responses in the absence of CD8 T cell expansion.

CD1d Activation and Blockade: A New Antitumor Strategy

CD1d is expressed on APCs and presents glycolipids to CD1d-restricted NKT cells. For the first time, we demonstrate the ability of anti-CD1d mAbs to inhibit the growth of different CD1d-negative experimental carcinomas in mice. Anti-CD1d mAbs systemically activated CD1d(+) APC, as measured by production of IFN-gamma and IL-12. Tumor growth inhibition was found to be completely dependent on IFN-gamma and IL-12 and variably dependent on CD8(+) T cells and NK cells, depending upon the tumor model examined. Anti-CD1d mAb induced greater CD8(+) T cell-dependent tumor suppression where regulatory CD1d-restricted type II NKT cells have been implicated, and were less effective in a NK cell-dependent manner against tumors where T regulatory cells were immunosuppressive. The ability of anti-CD1d mAbs to coincidently activate CD1d(+) APCs to release IL-12 and inhibit CD1d-restricted type II NKT cells makes CD1d an exciting new target for immunotherapy of cancer based on tumor immunoregulation.

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects.

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

Triggering of Murine NK Cells by CD40 and CD86 (B7-2)

NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab')2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)-/- or CD28-/- mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L-/- and CD28-/- mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.