CD1d-Expressing Breast Cancer Cells Modulate NKT Cell-Mediated Antitumor Immunity in a Murine Model of Breast Cancer Metastasis

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer

A new extract of the plant calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation

BACKGROUND: Phytopharmacological studies of different Calendula extracts have shown anti-inflamatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS: An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS: The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION: These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells

Prevention of Diabetes in NOD Mice by Repeated Exposures to a Contact Allergen Inducing a Sub-Clinical Dermatitis

BACKGROUND: Type 1 diabetes is an autoimmune disease, while allergic contact dermatitis although immune mediated, is considered an exposure driven disease that develops due to epicutaneous contact with reactive low-molecular chemicals. The objective of the present study was to experimentally study the effect of contact allergens on the development of diabetes in NOD mice. As the link between contact allergy and diabetes is yet unexplained we also examined the effect of provocation with allergens on Natural Killer T (NKT) cells, since involvement of NKT cells could suggest an innate connection between the two diseases. METHOD: NOD mice 4 weeks of age were exposed, on the ears, to two allergens, p-phenylenediamine and 2,4-dinitrochlorobenzene respectively, to investigate the diabetes development. The mice were followed for a maximum of 32 weeks, and they were either repeatedly exposed to the allergens or only sensitized a week after arrival. The stimulation of NKT cells by the two allergens were additionally studied in C57BL/6 mice. The mice were sensitized and two weeks later provocated with the allergens. The mice were subsequently euthanized at different time points after the provocation. RESULTS: It was found that repeated application of p-phenylenediamine reduced the incidence of diabetes compared to application with water (47% vs. 93%, P = 0.004). Moreover it was shown that in C57BL/6 mice both allergens resulted in a slight increment in the quantity of NKT cells in the liver. Application of the allergens at the same time resulted in an increased number of NKT cells in the draining auricular lymph node, and the increase appeared to be somewhat allergen specific as the accumulation was stronger for p-phenylenediamine. CONCLUSION: The study showed that repeated topical application on the ears with a contact allergen could prevent the development of diabetes in NOD mice. The contact allergens gave a non-visible, sub-clinical dermatitis on the application site. The preventive effect on diabetes may be due to stimulation of peripheral NKT cells, as shown for provocation with p-phenylenediamine in the C57BL/6 mouse. This epicutaneous procedure may lead to new strategies in prevention of type 1 diabetes in humans

Ameliorated ConA-Induced Hepatitis in the Absence of PKC-theta

Severe liver injury that occurs when immune cells mistakenly attack an individual's own liver cells leads to autoimmune hepatitis. In mice, acute hepatitis can be induced by concanavalin A (ConA) treatment, which causes rapid activation of CD1d-positive natural killer (NK) T cells. These activated NKT cells produce large amounts of cytokines, which induce strong inflammation that damages liver tissues. Here we show that PKC-θ−/− mice were resistant to ConA-induced hepatitis due to essential function of PKC-θ in NKT cell development and activation. A dosage of ConA (25 mg/kg) that was lethal to wild-type (WT) mice failed to induce death resulting from liver injury in PKC-θ−/− mice. Correspondingly, ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in PKC-θ−/− mice. Peripheral NKT cells had developmental defects at early stages in the thymus in PKC-θ−/− mice, and as a result their frequency and number were greatly reduced. Furthermore, PKC-θ−/− bone marrow adoptively transferred to WT mice displayed similar defects in NKT cell development, suggesting an intrinsic requirement for PKC-θ in NKT cell development. In addition, upon stimulation with NKT cell-specific lipid ligand, peripheral PKC-θ−/− NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells also requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cell to induce hepatitis, and thus, is a potential drug target for prevention of autoimmune hepatitis

Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells

Invariant natural killer T cells (iNKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled gene expression in iNKT cells during ontogeny and in peripheral subsets as part of the Immunological Genome Project. High-resolution comparative transcriptional analyses defined developmental and subset-specific programs of gene expression by iNKT cells. In addition, we found that iNKT cells shared an extensive transcriptional program with NK cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Notably, the program shared by NK cells and iNKT cells also operated constitutively in γδ T cells and in adaptive T cells after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes

The functional cancer map: A systems-level synopsis of genetic deregulation in cancer

<p>Abstract</p> <p>Background</p> <p>Cancer cells are characterized by massive dysegulation of physiological cell functions with considerable disruption of transcriptional regulation. Genome-wide transcriptome profiling can be utilized for early detection and molecular classification of cancers. Accurate discrimination of functionally different tumor types may help to guide selection of targeted therapy in translational research. Concise grouping of tumor types in cancer maps according to their molecular profile may further be helpful for the development of new therapeutic modalities or open new avenues for already established therapies.</p> <p>Methods</p> <p>Complete available human tumor data of the Stanford Microarray Database was downloaded and filtered for relevance, adequacy and reliability. A total of 649 tumor samples from more than 1400 experiments and 58 different tissues were analyzed. Next, a method to score deregulation of KEGG pathway maps in different tumor entities was established, which was then used to convert hundreds of gene expression profiles into corresponding tumor-specific pathway activity profiles. Based on the latter, we defined a measure for functional similarity between tumor entities, which yielded to phylogeny of tumors.</p> <p>Results</p> <p>We provide a comprehensive, easy-to-interpret functional cancer map that characterizes tumor types with respect to their biological and functional behavior. Consistently, multiple pathways commonly associated with tumor progression were revealed as common features in the majority of the tumors. However, several pathways previously not linked to carcinogenesis were identified in multiple cancers suggesting an essential role of these pathways in cancer biology. Among these pathways were 'ECM-receptor interaction', 'Complement and Coagulation cascades', and 'PPAR signaling pathway'.</p> <p>Conclusion</p> <p>The functional cancer map provides a systematic view on molecular similarities across different cancers by comparing tumors on the level of pathway activity. This work resulted in identification of novel superimposed functional pathways potentially linked to cancer biology. Therefore, our work may serve as a starting point for rationalizing combination of tumor therapeutics as well as for expanding the application of well-established targeted tumor therapies.</p

The ROS Scavenger, NAC, Regulates Hepatic Vα14iNKT Cells Signaling during Fas mAb-Dependent Fulminant Liver Failure

Uncontrolled systemic activation of the immune system is an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In this study, we demonstrate that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished Fas mAb-induced FLF through suppression of Vα14iNKT cell activation, IFN-γ signaling, apoptosis and nitrotyrosine formation in liver. In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18−/− mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions. In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF. Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses

NK Cell–Like Behavior of Vα14i NK T Cells during MCMV Infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Vα14 invariant natural killer T cell response to MCMV has not been examined. We found that Vα14i NK T cells become activated and produce significant levels of IFN-γ, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Vα14i NK T cells into MCMV-infected CD1d−/− mice demonstrate that CD1d is dispensable for Vα14i NK T cell activation. In contrast, both IFN-α/β and IL-12 are required for optimal activation. Vα14i NK T cell–derived IFN-γ is partially dependent on IFN-α/β but highly dependent on IL-12. Vα14i NK T cells contribute to the immune response to MCMV and amplify NK cell–derived IFN-γ. Importantly, mortality is increased in CD1d−/− mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Vα14i NK T cells that act as effector T cells during bacterial infection, but have NK cell–like behavior during the innate immune response to MCMV infection

Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8α+ and CD4-CD8α- Invariant Natural Killer T Cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4+, CD8α+ and CD4−CD8α− double-negative (DN) subsets. CD4+ iNKT cells expanded more readily than CD8α+ and DN iNKT cells upon mitogen stimulation. CD8α+ and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d+ cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4+ and CD8α+ fractions, respectively. Only CD4+ iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α+, DN or CD4+ iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease

Human Natural Killer T Cells Are Heterogeneous in Their Capacity to Reprogram Their Effector Functions

BACKGROUND: Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery. RESULTS: We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4− NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed. CONCLUSIONS AND SIGNIFICANCE: Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4− NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines