Cell Immune Surveillance Research Articles

2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells

ABSTRACTMutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein–Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8+ T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a−/− CD8+ T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a−/− CD8+ T cells responded equivalently to wild-type CD8+ T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8+ T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8+ T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8+ T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

Regulation of NKG2D+CD8+ T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

ABSTRACTThe natural killer (NK) group 2D (NKG2D) receptor, which displays on mouse and human NK cells, activates CD8+ T cells and small subsets of other T cells. NKG2D+CD8+ T cells play critical roles in both innate and adaptive immunity upon engagement with NKG2D ligands to eliminate tumor and infected cells. Despite the important role of NKG2D+CD8+ T cells in immune surveillance, the mechanisms of how NKG2D expression on CD8+ T cells is regulated remain poorly defined. We treated mouse and human CD8+ T cells with CD80 recombinant protein, plus a pharmacologic model with small molecular inhibitors to determine which signaling pathway leads to NKG2D regulation on CD8+T cells. This study revealed that CD28 activation gives rise to sustained NKG2D expression on both mouse and human CD8+ T cells in a signal transducer and activator of transcription 3 (STAT3) phosphorylation-dependent manner. Further, we found that CD28 activation stimulated sustained activation of the tyrosine kinase Lck, which recruits and triggers Janus kinase/STAT3 signaling to phosphorylate STAT3, and in turn increases NKG2D expression. Moreover, NKG2D induction on CD8+ T cells exerts cytolytic activity against target tumor cells in vitro, as well as significantly improves the antitumor therapeutic effects in vivo in an NKG2D-dependent manner. Taken together, these results elucidated a novel mechanism of NKG2D regulation by phosphorylated STAT3 (pSTAT3) on CD8+ T cells upon CD28 activation. This mechanism may shed light on the effectiveness of CD80-based, NKG2D-dependent antitumor immunotherapy.

NK cell activating receptor ligand expression in lymphangioleiomyomatosis is associated with lung function decline.

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women that leads to progressive cyst formation and accelerated loss of pulmonary function. Neoplastic smooth muscle cells from an unknown source metastasize to the lung and drive destructive remodeling. Given the role of NK cells in immune surveillance, we postulated that NK cell activating receptors and their cognate ligands are involved in LAM pathogenesis. We found that ligands for the NKG2D activating receptor UL-16 binding protein 2 (ULBP2) and ULBP3 are localized in cystic LAM lesions and pulmonary nodules. We found elevated soluble serum ULBP2 (mean = 575 pg/ml ± 142) in 50 of 100 subjects and ULBP3 in 30 of 100 (mean = 8,300 pg/ml ± 1,515) subjects. LAM patients had fewer circulating NKG2D+ NK cells and decreased NKG2D surface expression. Lung function decline was associated with soluble NKG2D ligand (sNKG2DL) detection. The greatest rate of decline forced expiratory volume in 1 second (FEV1, -124 ± 30 ml/year) in the 48 months after enrollment (NHLBI LAM Registry) occurred in patients expressing both ULBP2 and ULBP3, whereas patients with undetectable sNKG2DL levels had the lowest rate of FEV1 decline (-32.7 ± 10 ml/year). These data suggest a role for NK cells, sNKG2DL, and the innate immune system in LAM pathogenesis.

Tumor Cell Recovery from Senescence Induced by Radiation with PARP Inhibition.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are clinically used as single-agent therapy for tumors with BRCA1 or BRCA2 mutations. One approach to expanding the use of PARP inhibitors to a wider range of tumors is to combine them with cytotoxic chemotherapy or radiotherapy. Preclinical studies in experimental animals and tumor cells in culture indicate that PARP inhibition modestly sensitizes most tumor cells to ionizing radiation. Studies of cell behavior after these combined treatments show that radiosensitization is manifested predominantly in an increase in prolonged growth arrest and senescence, with little or no contribution from apoptosis. The secretory phenotype associated with senescence can target these tumor cells for immune surveillance, and therefore increased senescence can effectively contribute to tumor control. However, the possible recovery of senescent cells and re-entry into cell cycle after prolonged arrest also needs to be considered. Such recovery could lead to tumor recurrence, yet may not be reflected in short-term assays commonly used to assess radiosensitization.

Abstract # 1776 Upper-respiratory viral infection triggers EAE onset in autoimmune prone T-cell receptor transgenic mice

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system. Most MS patients experience periods of symptom exacerbation (relapses) followed by periods of partial recovery (remission). Interestingly, upper-respiratory viral infections increase the risk for relapse. Here, we utilized an autoimmune-prone T-cell receptor transgenic mouse (2D2) and a mouse-adapted human influenza virus to test the hypothesis that upper-respiratory viral infection can cause glial activation, promote immune cell trafficking to the CNS and trigger disease. Specifically, we inoculated C57BL/6 mice with the H1N1 strain of influenza A virus (Puerto Rico/8/34; PR8) then measured: Transcriptomic alterations occurring in the cerebellum and spinal cord, immune cell surveillance of the CNS by flow cytometry and chemokine production from primary glial cultures by ELISA. Infected 2D2 mice were monitored for symptoms of inflammatory demyelination. Infection caused temporal alterations in the transcriptome of both the cerebellum and spinal cord that were consistent with glial activation. Infection of C57BL/6 mice increased CD3+CD4+ T-cell trafficking to the brain at day 8 p.i. Cytokine stimulation of primary glial cultures implicated astrocytes as a major source of chemokine production. Finally, clinical and histological EAE was observed in ∼ 27% of infected 2D2 mice. These data identify a new model with which to study how viral infection progresses disease in MS patients and suggests that peripheral infection induced glial activation precedes relapse.

Cutaneous T-Cell Lymphoma. A hypothesis on disease pathophysiology involving deficiency in DNA repair.

Cutaneous T-cell lymphoma (CTCL) is a rare disease occurring in Europe among two persons per million per year. It affects men more often than women (2:1). It is primarily a skin disease. In about 20% of patients, it becomes fatal with tumours in the skin and spreading to lymph glands. Approximately 3% of patients show a leukemic form called Sezary's syndrome, where malignant cells are present in blood with accompanying erythrodermia. CTCL is a T-lymphocyte disease occurring late in life as the average age of patients is around 66 years in Europe, Japan and the US. This article focuses on cell lines and immune surveillance in CTCL, and especially the pronounced chromosomal instability. It leads to the hypothesis that chromosomal changes is the key event linked to DNA repair deficiencies, which in a subpopulation of T cells leads to CTCL development over years.

Abstract 5143: MHC class I polypeptide-related sequence A (MICA) as a factor of aggressive prostate cancer

Abstract Prostate cancer (PCa) remains the second most common cancer in American men, with higher incidence and death rates in African American men (AAM) relative to Caucasian American men (CAM). MHC class I polypeptide-related sequence A (MICA) is a transmembrane protein responsible for activation of immunocytes, by binding the NKG2D receptor. The expression of MICA under normal conditions is low. However under stress conditions such as those found in neoplasms, MICA expression is increased. The soluble form of MICA (sMICA) is released by tumor cells and promotes immune evasion by binding and downregulating the NKG2D receptor in immune surveillance cells. Increased levels of sMICA have been found in patient sera of several types of cancer, including PCa. We hypothesized that low oxygen levels, a condition of the microenvironment present in aggressive tumors, would modify the levels of sMICA in PCa cell lines. Likewise we speculated that when compared to CAM, MICA would be differentially expressed in tumors from AAM, known to suffer a more aggressive disease. To address those questions, LNCaP cells were cultured under 20% O2 (normoxia) and 2% O2 (hypoxia) at various time points, supernatants were collected and the presence of sMICA in culture supernatant and patient sera was detected by ELISA. After 48 hours, the cells grown in hypoxia expressed about 5-fold more sMICA than cells grown in normoxia. When assessed in patient plasma, PCa patients had 2-fold more sMICA compared to healthy controls. In additional experiments cells growing under different oxygen levels were lysed and used in Western blot analysis to detect cellular MICA. Normal oxygen levels increased MICA protein by 3.5-fold. Hypoxia reduced MICA expression by 11%. A tissue microarray (TMA) was stained with MICA polyclonal antibody. The TMA was composed of 34 tumor samples and 30 controls. The samples came from 32 CAM and 32 AAM. The staining intensity was scored as 0, 1, 2 or 3 and scores 2 and 3 were defined as high intensity. Association of the staining result to presence of tumor was evaluated using the Fisher's exact test. A higher proportion of high intensity in tumor site (50%) was found when compared to non-tumor site (20%, p = 0.019). Interestingly, stronger association was expressed in CAM (p = 0.036) than in AAM (p = 0.265). The results found in the cell line suggest that different oxygen levels affect the expression of sMICA. Higher expression of MICA in prostate tumor tissue and sMICA in patient sera are consistent with data previously reported in other cancers. Lower intensity in MICA staining in AAM patients may be related to better capacity of the tumor to evade the immune system. Overall our findings suggest the involvement of MICA on aggressive PCa. More studies are needed to decipher the biological mechanisms of MICA's immune evasion in prostate tumors and to establish its contributing role to aggressive disease in minorities. Funding sources: PCRP W81XWH-14-1-0151, UMMC Office of Research Citation Format: Marcelo J. Sakiyama, Angel Garcia, Ingrid Espinoza, Jack Lewin, Xu Zhang, Elizabeth Tarsi, Tara Craft, Logan Fair, Jesus Monico, Lisa Sullivan, Charles R. Pound, Srinivasan Vijayakumar, Christian R. Gomez. MHC class I polypeptide-related sequence A (MICA) as a factor of aggressive prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5143.

Abstract 3235: IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands

Abstract Background: Diffuse gliomas are fatal primary brain tumors that are poorly immunogenic. The basis for insufficient anti-tumor immunity in diffuse gliomas is not understood. Gain of function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. Methods: We analyzed the TCGA database for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligands expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and in genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza- 2’deoxycytodine (Decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. Results: All IDH mutant glioma stem-like cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes were resistant to NK cell- mediated lysis. The hypomethylating agent Decitabine increased NKG2D ligand expression, and restored NK-mediated lysis of IDH mutant cells in an NKG2D- dependent manner. Conclusions: IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULPB3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell- mediated immune surveillance in patients with IDH mutated diffuse gliomas. Citation Format: Aparna Rao, Xiaoran Zhang, Christopher Deibert, Paola Sette, Tim Chan, Paola Grandi, Nduka Amankulor. IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3235.

Mathematical model of tumor–immune surveillance

We present a novel mathematical model involving various immune cell populations and tumor cell populations. The model describes how tumor cells evolve and survive the brief encounter with the immune system mediated by natural killer (NK) cells and the activated CD8+ cytotoxic T lymphocytes (CTLs). The model is composed of ordinary differential equations describing the interactions between these important immune lymphocytes and various tumor cell populations. Based on up-to-date knowledge of immune evasion and rational considerations, the model is designed to illustrate how tumors evade both arms of host immunity (i.e. innate and adaptive immunity). The model predicts that (a) an influx of an external source of NK cells might play a crucial role in enhancing NK-cell immune surveillance; (b) the host immune system alone is not fully effective against progression of tumor cells; (c) the development of immunoresistance by tumor cells is inevitable in tumor immune surveillance. Our model also supports the importance of infiltrating NK cells in tumor immune surveillance, which can be enhanced by NK cell-based immunotherapeutic approaches.

749. Escape from Tumor Dormancy Following Gene- or Viro-Therapies Is Mediated by Acquisition of a Phenotype in Which Innate Immune Surveillance Actively Drives Tumor Cell Recurrence

When established (~0.3-0.5cm) primary murine B16 tumors are treated with either cytotoxic gene therapy (HSVtk/GCV), oncolytic virotherapy (intra-tumoral reovirus) or adoptive T cell therapy, a proportion of mice will become apparently tumor free. However, if the animals are observed for prolonged periods of time (>60-100 days), the majority of mice will develop aggressively growing recurrent tumors. Excision of the site of tumor injection during the period of tumor dormancy (no palpable tumor) indicated that residual tumor cells are readily detectable in most mice by histology. Unexpectedly, it was extremely difficult to re-grow the tumor cells from these specks of minimal residual disease in vitro. However, by screening multiple cytokines for their ability to support re-growth of recurrent B16 cells, we identified TNF-α as a major growth factor for recurrent cells. Conversely, treatment of primary B16 populations with TNF-α led to highly significant reductions in tumor cell viability. Similarly, depletion of NK cells, or antibody blockade of TNF-α, from C57Bl/6 mice led to significantly increased tumorigencity of primary B16 tumors. In contrast, depletion of NK cells allowed for decreased tumorigenicity of TNF-α stimulated recurrent B16 cells recovered from mice in a state of minimal residual disease. These data suggest that tumor recurrence may be mediated by a distinct phenotypic switch in vivo. Thus, initially, primary tumor cells are highly sensitive to innate immune cell surveillance through NK cells and TNF-α. However, escape from dormancy in vivo is associated with acquisition of a phenotype in which such TNF-α-mediated immune surveillance actively drives tumor growth. TNF-α growth promoted recurrent cells also expressed high levels of PD-L1 compared to primary tumor cells, suggesting that this may be a further mechanism by which recurrences could emerge in vivo, even in the presence of anti tumor T cell responses. Consistent with this hypothesis, recurrence was significantly inhibited in vivo following gene, viro- or adoptive T cell therapies, by treatment with TNF-α blockade or systemic checkpoint inhibitor therapy.

SLAM family receptors 2B4 and CD48 are key drivers of CD8 T cell priming by B cells

Abstract Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we utilized SAP-deficient (Sh2d1a−/−) mice to investigate the hypothesis that SAP is critical for CD8 T cell immune surveillance of antigen-expressing B cells or B lymphoma cells. Sh2d1a−/− CD8 T cells exhibited greatly diminished proliferation relative to wild type when antigen-presenting-B cells or -B lymphoma cells served as the primary APC. By contrast, Sh2d1a−/− CD8 T cells responded equivalently to wild type CD8 T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed antigen presentation. Through application of SLAM family receptor blocking antibodies or SLAM family receptor-deficient CD8 T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the antigen-driven CD8 T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naïve CD8 T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligand expression.

Diffuse gliomas are poorly immunogenic, fatal brain tumors. The basis for insufficient antitumor immunity in diffuse gliomas is unknown. Gain-of-function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. We analyzed the database of The Cancer Genome Atlas for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligand expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza-2'deoxycytodine (decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. IDH mutant glioma stemlike cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes are resistant to NK cell-mediated lysis. Decitabine increases NKG2D ligand expression and restores NK-mediated lysis of IDH mutant cells in an NKG2D-dependent manner. IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULBP3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.

Targeting NK Cells for Anticancer Immunotherapy: Clinical and Preclinical Approaches.

The recent success of checkpoint blockade has highlighted the potential of immunotherapy approaches for cancer treatment. Although the majority of approved immunotherapy drugs target T cell subsets, it is appreciated that other components of the immune system have important roles in tumor immune surveillance as well and thus represent promising additional targets for immunotherapy. Natural killer (NK) cells are the body’s first line of defense against infected or transformed cells, as they kill target cells in an antigen-independent manner. Although several studies have clearly demonstrated the active role of NK cells in cancer immune surveillance, only few clinically approved therapies currently exist that harness their potential. Our increased understanding of NK cell biology over the past few years has renewed the interest in NK cell-based anticancer therapies, which has lead to a steady increase of NK cell-based clinical and preclinical trials. Here, the role of NK cells in cancer immune surveillance is summarized, and several novel approaches to enhance NK cell cytotoxicity against cancer are discussed.

The role of glycans in the development and progression of prostate cancer.

Prostate cancer is a unique and heterogeneous disease. Currently, a major unmet clinical need exists to develop biomarkers that enable indolent disease to be distinguished from aggressive disease. The prostate is an abundant secretor of glycoproteins of all types, and alterations in glycans are, therefore, attractive as potential biomarkers and therapeutic targets. Despite progress over the past decade in profiling the genome and proteome, the prostate cancer glycoproteome remains relatively understudied. A wide range of alterations in the glycoproteins on prostate cancer cells can occur, including increased sialylation and fucosylation, increased O-β-N-acetylglucosamine (GlcNAc) conjugation, the emergence of cryptic and high-mannose N-glycans and alterations to proteoglycans. Glycosylation can alter protein function and has a key role in many important biological processes in cancer including cell adhesion, migration, interactions with the cell matrix, immune surveillance, cell signalling and cellular metabolism; altered glycosylation in prostate cancer might modify some, or all of these processes. In the past three years, powerful tools such as glycosylation-specific antibodies and glycosylation gene signatures have been developed, which enable detailed analyses of changes in glycosylation. Thus, emerging data on these often overlooked modifications have the potential to improve risk stratification and therapeutic strategies in patients with prostate cancer.

Natural killer cells require monocytic Gr-1+/CD11b+ myeloid cells to eradicate orthotopically engrafted glioma cells

ABSTRACTMalignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1−/− and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1+ myeloid cells in RAG1−/− mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1+/CD11b+ cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1+/CD11b+ myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1+/CD11b+ myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.

Large molecular systems landscape uncovers T cell trapping in human skin cancer.

Immune surveillance of tumour cells is an important function of CD8 T lymphocytes, which has failed in cancer for reasons still unknown in many respect but mainly related to cellular processes in the tumour microenvironment. Applying imaging cycler microscopy to analyse the immune contexture in a human skin cancer we could identify and map 7,000 distinct cell surface-associated multi-protein assemblies. The resulting combinatorial geometry-based high-functional resolution led to discovery of a mechanism of T cell trapping in the epidermis, which involves SPIKE, a network of suprabasal keratinocyte projections piercing and interconnecting CD8 T cells. It appears initiated by clusters of infrabasal T and dendritic cells connected via cell projections across a fractured basal lamina to suprabasal keratinocytes and T lymphocytes.

NK cells and cancer: you can teach innate cells new tricks.

Natural killer (NK) cells are the prototype innate lymphoid cells endowed with potent cytolytic function that provide host defence against microbial infection and tumours. Here, we review evidence for the role of NK cells in immune surveillance against cancer and highlight new therapeutic approaches for targeting NK cells in the treatment of cancer.

Normal hematopoiesis and lack of β-catenin activation in osteoblasts of patients and mice harboring Lrp5 gain-of-function mutations

Osteoblasts are emerging regulators of myeloid malignancies since genetic alterations in them, such as constitutive activation of β-catenin, instigate their appearance. The LDL receptor-related protein 5 (LRP5), initially proposed to be a co-receptor for Wnt proteins, in fact favors bone formation by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5A214V) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5 mutation, we show here that Lrp5 GOF mutations in both humans and mice do not activate β-catenin signaling in osteoblasts. Consistent with a lack of β-catenin activation in their osteoblasts, Lrp5A214V mice have normal trilinear hematopoiesis. In contrast to leukemic mice with constitutive activation of β-catenin in osteoblasts (Ctnnb1CAosb), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5A214V mice. Likewise, peripheral blood count analysis in HBM patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

Abstract C107: Preclinical response of murine syngeneic tumor models to immune checkpoint inhibitor antibodies

Abstract Tumor cell viability and proliferation is regulated by the balance between tumor self-survival and host elimination mechanisms. There are multiple mechanisms whereby tumors successfully evade host immune cell surveillance, permitting continued growth and proliferation. Immunotherapies are aimed at bolstering the host immune system. Delivery of these agents has been shown to actively eliminate disease in a number of patients to date. We evaluated several syngeneic mouse tumor lines for anti-tumor activity with anti-CTLA-4 and/or anti-PD-L1 antibodies, as well as the immunological changes occurring following treatment with these checkpoint inhibitors. The effectiveness of these inhibitors was assessed in the B16.F10 (melanoma), RIF-1 (sarcoma), 4T1-luc (mammary) and CT26 (colon) murine tumor models. Cells or tumor fragments (RIF-1) were implanted SC in the axilla. The anti-CTLA-4 9D9 antibody clone from Bio X Cell (West Lebanon, NH) was employed for responsiveness in each model. All treatments were initiated 3 days post implantation, prior to the appearance of established tumor, at inoculums known to have 100% take rates. The CT26 model was highly responsive with all animals showing tumor regressions and 70% tumor-free survivors at study end. The RIF-1 model was moderately responsive with several mice showing long term progression-free survival and a 56% increase in lifespan vs. the isotype control group. Both the B16.F10 and the 4T1-luc models were non-responsive. Tumor responsiveness to the anti-PD-L1 10F.9G2 antibody clone (Bio X Cell) was also compared in the RIF-1 and CT26 models. In contrast to the anti-CTLA-4 antibody studies, RIF-1 was found to be non-responsive and CT26 was moderately responsive to the anti-PD-L1 antibody. Evaluating immunological endpoints by flow cytometry is critical when assessing the effectiveness of immune checkpoint inhibitors. To this end, the immune profiles of both responsive (CT26) and non-responsive (4T1 and B16.F10) models were examined in the absence or presence of antibody treatments. Initial studies were focused on analysis of T cells (CD4 and CD8) as well as T regulatory cells (Tregs; CD4/CD25/FoxP3) and myeloid derived suppressive cells (MDSC; CD11b/Gr1). The 4T1 model was found to be highly MDSC driven and would be suitable to evaluate agents that target myeloid-derived cell populations. While this model was not very responsive to anti-CTLA-4 treatment, a trend toward decreased MDSCs following treatment was observed with anti-CTLA-4 and not the isotype control antibody. In contrast, the CT26 model was more highly mixed and contained populations of both Tregs and MDSCs. Treatment with anti-CTLA-4 increased the CD8+ T cell population and decreased the Treg cell population with minimal effect on the MSDC population. However, in this same model, anti-PD-L1 treatment increased CD8+ T cells and decreased both Tregs and MDSCs. A more complete mechanistic understanding of how the immune system responds to checkpoint inhibitors permits identification of appropriate models for monotherapy, as well as the rationale for designing drug combination studies. Data enhancing understanding of the above, along with tumor profiling on additional syngeneic models and more flow cytometry multiplexing, is expected from further ongoing research. Citation Format: Maryland Franklin, Matt Thayer, Chris Elders, Dan Saims, Scott Wise. Preclinical response of murine syngeneic tumor models to immune checkpoint inhibitor antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C107.

Psychological stress and cancer

Psychological or psychosocial stress has been emerging as one of the key factors associated with cancer initiation, growth, and metastasis. Accumulating data, however, has mainly focused on cancer progression due to the inconsistent results of cancer etiology caused by emotional stress. Depression is closely linked to stress and induces hypothalamic-pituitary-adrenal (HPA) axis activation as well as sympathetic nervous system (SNS) down to immune cell surveillance. This review briefly describes the activation of neuroendocrine system by psychological stress/depression and its effect on cancer occurrence and metastasis, which may be useful for the design of new cancer treatments.