Specific N-cadherin-dependent pathways drive human breast cancer dormancy in bone marrow.

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-ß-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Exosomes from differentially activated macrophages influence dormancy or resurgence of breast cancer cells within bone marrow stroma.

Breast cancer (BC) cells (BCCs) can retain cellular quiescence for decades, a phenomenon referred to as dormancy. BCCs show preference for the bone marrow (BM) where they can remain dormant for decades. Targeting BCCs within the BM is a challenge since the dormant BCCs reside within BM stroma, also residence for hematopoietic stem cells (HSCs). Dormant BCCs could behave as cancer stem cells (CSCs). The CSCs and HSCs are similar by function and also, by commonly expressed genes. The method by which dormant BCCs transition into clinically metastatic cells remains unclear. This study tested the hypothesis that macrophages (MΦs) within BM stroma, facilitates dormancy or reverse this state into metastatic cells. MΦs exhibiting an M2 phenotype constitute ~10% of cultured BM stroma. The M2 MΦs form gap junctional intercellular communication (GJIC) with CSCs, resulting in cycling quiescence, reduced proliferation and carboplatin resistance. In contrast, MΦs expressing the M1 phenotype reversed BC dormancy. Activation of M2a MΦs via the toll-like receptor 4 (TLR4) switched to M1 phenotype. The switch can occur by direct activation of M2a MΦs, or indirectly through activation of mesenchymal stem cells. M1 MΦ-derived exosomes activated NFкB to reverse quiescent BCCs to cycling cells. Using an in vivo model of BC dormancy, injected Mi MOs sensitized BCCs to carboplatin and increased host survival. In summary, we have shown how BM stromal MΦs, through exosomes, regulate the behavior of BCCs, by either inducing or reversing dormancy.

Learning Science Communication Skills Using Improvisation, Video Recordings, and Practice, Practice, Practice.

Doctoral students in science disciplines spend countless hours learning how to conduct cutting-edge research but very little time learning to communicate the nature and significance of their science to people outside their field. To narrow this disparity, we created an unusual course titled Communicating Science for doctoral science trainees at Rutgers University. Our goal was to help students develop an advanced ability to communicate their research clearly and accurately and to emphasize its value and significance to diverse audiences. Course design included classroom instruction supplemented with improvisation, video recordings, and ample opportunity for students to practice and receive immediate, constructive feedback in a supportive environment. A multidisciplinary faculty with expertise in science, education, communication, and theater arts taught this course. PhD students came from diverse scientific disciplines, ranging from biology and chemistry to civil engineering. Students also completed a capstone project in which they worked with a professional in the academic or private sector to explore a possible career aspiration. Assessment was in the form of feedback on students' oral and poster presentations, and written abstracts about their research. Student evaluations and comments about course format and content were mostly positive and also provided input for ways to improve the course. We discovered that the diversity of scientific backgrounds among our students enhanced their ability to learn how to communicate their science to others outside their disciplines. We are leveraging the success of our initial course offering to reach other student and faculty groups at Rutgers.

Teaching Medical Students to Communicate With Empathy and Clarity Using Improvisation.

PROBLEM: Medical educators widely accept that health care providers need strong communication skills. The authors sought to develop a course incorporating improvisation to teach health professions students communication skills and build empathy. APPROACH: Teaching health care professionals to communicate more effectively with patients, the public, and each other is a goal of the Alan Alda Center for Communicating Science at Stony Brook University. The authors designed an interprofessional elective for medical, nursing, and dental students that differed in several respects from traditional communication training. The Communicating Science elective, which was offered by the Alda Center from 2012 to 2016, used verbal and nonverbal exercises, role-playing, and storytelling, including improvisation exercises, to teach students to communicate with empathy and clarity. OUTCOMES: In course evaluations completed by 76 students in 2012 and 2013, 100% said they would recommend the course to fellow students, saw the relevance of the course content to their careers, and desired more of the course content in their school's curriculum. As a result of this positive feedback, from 2014 to 2016, 10 hours of instruction pairing empathy and communication training was embedded in the preclinical curriculum at the Stony Brook University School of Medicine. NEXT STEPS: This course could be an effective model, and one that other institutions could employ, for improving communication skills and empathy in the next generation of health care professionals. Next steps include advocating for communication skills training to be embedded throughout the curriculum of a four-year medical school program.

Steroid-Mediated Decrease in Blood Mesenchymal Stem Cells in Liver Transplant could Impact Long-Term Recovery.

Orthotopic liver transplant (OLT) remains the standard of care for end stage liver disease. To circumvent allo-rejection, OLT subjects receive gluococorticoids (GC). We investigated the effects of GC on endogenous mesenchymal stem (stromal) cells (MSCs) in OLT. This question is relevant because MSCs have regenerative potential and immune suppressor function. Phenotypic analyses of blood samples from 12 OLT recipients, at pre-anhepatic, anhepatic and post-transplant (2 h, Days 1 and 5) indicated a significant decrease in MSCs after GC injection. The MSCs showed better recovery in the blood from subjects who started with relatively low MSCs as compared to those with high levels at the prehepatic phase. This drop in MSCs appeared to be linked to GC since similar change was not observed in liver resection subjects. In order to understand the effects of GC on decrease MSC migration, in vitro studies were performed in transwell cultures. Untreated MSCs could not migrate towards the GC-exposed liver tissue, despite CXCR4 expression and the production of inflammatory cytokines from the liver cells. GC-treated MSCs were inefficient with respect to migration towards CXCL12, and this correlated with retracted cytoskeleton and motility. These dysfunctions were partly explained by decreases in the CXCL12/receptor axis. GC-associated decrease in MSCs in OLT recipients recovered post-transplant, despite poor migratory ability towards GC-exposed liver. In total, the study indicated that GC usage in transplant needs to be examined to determine if this could be reduced or avoided with adjuvant cell therapy.

Mesenchymal Stem Cell-Derived Exosomes Stimulate Cycling Quiescence and Early Breast Cancer Dormancy in Bone Marrow.

Dormant breast cancers resurge as metastatic disease after a long dormancy period in the bone marrow, where cancer cells interact with mesenchymal stem cells (MSC). However, the nature of early interactions between breast cancer cells and MSCs in the bone marrow microenvironment that facilitate adaptation to a quiescent state remains poorly understood. Here, we report that breast cancer cells prime MSC to release exosomes containing distinct miRNA contents, such as miR-222/223, which in turn promotes quiescence in a subset of cancer cells and confers drug resistance. Building on these results, we developed a novel, nontoxic therapeutic strategy to target dormant breast cancer cells based on systemic administration of MSC loaded with antagomiR-222/223. In an immunodeficient mouse model of dormant breast cancer, this therapy sensitized breast cancer cells to carboplatin-based therapy and increased host survival. Overall, our findings illuminate the nature of the regulatory interactions between breast cancer cells and MSCs in the evolution of tumor dormancy and resurgence in the micrometastatic microenvironment of the bone marrow. Cancer Res; 76(19); 5832-44. ©2016 AACR.

Maternal inflammation promotes fetal microglial activation and increased cholinergic expression in the fetal basal forebrain: role of interleukin-6.

BACKGROUND: Perinatal exposure to infectious agents with associated maternal immune activation (MIA) leads to neuroanatomical and behavioral dysregulation reminiscent of autism spectrum disorders. Persistent microglial activation as well as increased choline acetyltransferase (ChAT) activity in the basal forebrain (BF) are characteristic of autistic subjects. Previous studies have shown that medium from activated microglia promotes cholinergic differentiation of precursors in the BF. We sought to determine whether MIA in vivo would lead to a similar effect on developing BF neurons. METHODS: Pregnant mice were treated with the viral mimic polyinosinic-polycytidylic acid (poly(I:C)) or saline. RESULTS: Poly(I:C) treatment resulted in increased production of cytokines and chemokines in fetal microglia and increased ChAT activity and cholinergic cell number in the perinatal BF. Whether microglial activation causes these changes is unclear. Examination of fetal brains from mice lacking interleukin-6 (IL-6 KOs) revealed an elevation in non-microglial-derived cytokines and chemokines over wild-type controls. Moreover, IL-6 KO offspring showed an elevation of ChAT activity even in the absence of poly(I:C) administration. CONCLUSION: These data suggest that elevations in cytokines and/or chemokines caused either by maternal poly(I:C) administration or by the absence of IL-6 are associated with alterations in cholinergic development in the BF.

Maternal immune stimulation during pregnancy shapes the immunological phenotype of offspring.

Epidemiological studies have associated infection during pregnancy with increased risk of neurodevelopmental disorders in children, which is modeled in rodents by stimulating the immune system of pregnant dams with microorganisms or their mimics, such as poly(I:C) or LPS. In two prenatal mouse models, we show that in utero exposure of the fetus to cytokines/inflammatory mediators elicited by maternal immune stimulation with poly(I:C) yields offspring that exhibit a proinflammatory phenotype due to alterations in developmental programming of their immune system. Changes in the innate and adaptive immune elements of these pro-inflammatory offspring result in more robust responses following exposure to immune stimuli than those observed in control offspring from PBS-injected pregnant dams. In the first model, offspring from poly(I:C)-injected immunologically naïve dams showed heightened cellular and cytokine responses 4 h after injection of zymosan, a TLR2 agonist. In the second model, using dams with immunological memory, poly(I:C) injection during pregnancy produced offspring that showed preferential differentiation toward Th17 cell development, earlier onset of clinical symptoms of EAE, and more severe neurological deficits following immunization with MOG35-55. Such "fetal programming" in offspring from poly(I:C)-injected dams not only persists into neonatal and adult life, but also can have profound consequences on health and disease.

Pharmacokinetics and modeling of immune cell trafficking: quantifying differential influences of target tissues versus lymphocytes in SJL and lipopolysaccharide-treated mice.

BACKGROUND: Immune cell trafficking into the CNS and other tissues plays important roles in health and disease. Rapid quantitative methods are not available that could be used to study many of the dynamic aspects of immune cell-tissue interactions. METHODS: We used pharmacokinetics and modeling to quantify and characterize the trafficking of radioactively labeled lymphocytes into brain and peripheral tissues. We used variance from two-way ANOVAs with 2 × 2 experimental designs to model the relative influences of lymphocytes and target tissues in trafficking. RESULTS: We found that in male CD-1 mice, about 1 in 5,000 intravenously injected lymphocytes entered each gram of brain. Uptake by brain was 2 to 3 times higher in naïve SJL females, but uptake by spleen and clearance from blood was lower, demonstrating a dichotomy in immune cell distribution. Treatment of CD-1 mice with lipopolysaccharide (LPS) increased immune cell uptake into brain but decreased uptake by spleen and axillary nodes. CONCLUSIONS: Differences in brain uptake and in uptake by spleen between SJL and CD-1 mice were primarily determined by lymphocytes, whereas differences in uptake with LPS were primarily determined by lymphocytes for the brain but by the tissues for the spleen and the axillary lymph node. These results show that immune cells normally enter the CNS and that tissues and immune cells interact in ways that can be quantified by pharmacokinetic models.

Development and characterization of a preclinical ovarian carcinoma model to investigate the mechanism of acquired resistance to trastuzumab.

Trastuzumab (Herceptin®) is a humanized monoclonal antibody designed to bind and inhibit the function of the human epidermal growth factor receptor 2 (HER2)/erbB2 receptor. Trastuzumab has demonstrated clinical activity in several types of HER2-overexpressing epithelial tumors, such as breast and metastatic gastric or gastroesophageal junction cancer. Relapse and therapeutic resistance, however, still occur in a subset of patients treated with regimens including trastuzumab, despite significant improvements in response rates, survival and quality of life. To investigate the potential mechanisms of acquired therapeutic resistance to trastuzumab, we developed a preclinical model of human ovarian cancer cells, SKOV-3 Herceptin-resistant (HR), and examined the corresponding changes in gene expression profiles. SKOV-3 HR cells were developed by in vivo serial passaging of parental trastuzumab-sensitive SKOV-3 cells. Following four rounds of serial transplantation of 'break-through' xenograft tumors under trastuzumab treatment, significant and reproducible differences in the effects of trastuzumab treatment between SKOV-3 HR and SKOV-3 cells in vivo and in vitro were revealed. SKOV-3 HR cells retained HER2 protein expression but were unaffected by the antiproliferative effects of trastuzumab. The trastuzumab binding affinity for SKOV-3 HR cells was diminished, despite these cells having more binding sites for trastuzumab. Microarray expression profiling (MEP) was performed to determine the genes involved in the resistance mechanism. Functional analysis revealed the differential expression of genes potentially involved in angiogenesis, metastasis, differentiation and proliferation, such as mucin1 (MUC1). Immunohistochemical staining of SKOV-3 HR cells demonstrated a marked overexpression of MUC1. Based on these data, we hypothesize that the overexpression of MUC1 may hinder trastuzumab binding to HER2 receptors, abrogating the antitumor effects of trastuzumab and thus could contribute to resistance to therapy. Moreover, the resultant MEP preclinical gene signature in this preclinical model system may provide the basis for further investigation of potential clinical mechanisms of resistance to trastuzumab.

Maternal immune stimulation during pregnancy affects adaptive immunity in offspring to promote development of TH17 cells.

Behavioral abnormalities in offspring of murine dams that receive immune stimulation with (poly)I:C during pregnancy are well-documented. In this prenatal model, (poly)I:C-induced maternal cytokines, particularly IL-6, appear involved in the etiology of the behavioral abnormalities. While much has been published on the abnormal behaviors of offspring in this model, much less is known about how maternal immune stimulation affects the adaptive immune system of the offspring, and its possible role in the observed pathophysiology. In the present study, pregnant dams were stimulated with (poly)I:C at E12, and 24h later cytokine levels were measured in maternal sera and amniotic fluids. Lymphocytes from offspring were also analyzed for T Helper (TH) cell subsets. The results demonstrate that lymphocytes from offspring of pregnant dams stimulated with (poly)I:C develop into TH17 cells upon in vitro activation. This preferential TH17 cell differentiation occurs in offspring of pregnant dams with an immunological "memory" phenotype, but not in offspring of immunologically "naive" dams. Comparable levels of IL-6 were found in the sera of immune and naïve pregnant dams, however, there was a disparity between levels of IL-6 in maternal sera and amniotic fluids of (poly)I:C-injected dams. In matings between IL-6 KO dams (IL-6-/-) and wild-type males (IL-6+/+) there was no IL-6 in sera from (poly)I:C-injected dams, but there were high levels of IL-6 in their amniotic fluids. Analysis of supernatants of cultured placental cell preparations from these IL-6 KO dams confirmed that the IL-6 was produced from the fetal (IL-6+/-) component, and heterozygous IL-6+/- offspring could also produce IL-6.

Epigenetic regulation of beta2-adrenergic receptor expression in T(H)1 and T(H)2 cells.

We showed previously that murine naive CD4(+) T cells and T(H)1 cell clones express the beta2-adrenergic receptor (ß(2)AR), while T(H)2 cell clones do not. We report here that naive CD4(+) T cells that differentiated for 1-5 days under T(H)1 driving conditions increased ß(2)AR gene expression, while cells cultured under T(H)2 driving conditions decrease ß(2)AR gene expression. Chromatin immunoprecipitation revealed that the increase in ß(2)AR gene expression in T(H)1 cells is mediated by an increase in histone 3 (H3) and H4 acetylation, as well as an increase in histone 3 lysine 4 (H3K4) methylation. Conversely, the decrease in ß(2)AR gene expression in T(H)2 cells is mediated by a decrease in H3 and H4 acetylation and a decrease in H3K4 methylation, as well as an increase H3K9 and H3K27 methylation. The histone changes could be detected as early as 3 days of differentiating conditions. Genomic bisulfite sequencing showed that the level of methylated CpG dinucleotides within the promoter of the ß(2)AR gene was increased in T(H)2 cells as compared to naive and T(H)1 cells. Collectively, these results suggest that epigenetic mechanisms mediate maintenance and repression, respectively, of the ß(2)AR gene expression in T(H)1- and T(H)2-driven cells, providing a potential mechanism by which the level of ß(2)AR expression might be modulated pharmacologically within immune cells and other cell types in which the expression profile may change during a disease process.

Preferential development of Th17 cells in offspring of immunostimulated pregnant mice.

Pregnant mice were stimulated at day 12 of gestation with the nucleotide poly(I:C). At 24h after stimulation, serum levels of maternal cytokines were measured, and at postnatal ages 2 and 3 weeks, offspring were analyzed for T helper (Th) cell subsets. Lymphocytes from offspring of poly(I:C)-injected (vs. control PBS-injected) pregnant dams preferentially developed into T helper 17 (Th17) cells upon in vitro activation. This occurred in offspring of pregnant dams who exhibited an immunological "memory" phenotype, but not in offspring of immunologically "naïve" dams. Preferential development of Th17 cells in these offspring may be facilitated by the higher levels of pro-inflammatory cytokines such as IL-6, found in immune vs. naïve pregnant dams. Murine immune stimulation during pregnancy is frequently used to model human neurological disorders, such as autism and schizophrenia. However, immune stimulation of women during pregnancy occurs in the context of an immunological "memory" phenotype, resulting from previous immunizations and/or natural exposure to micro-organisms and other antigens. Therefore, use of previously immunized female mice with a similar immunological memory phenotype to study maternal immune stimulation during pregnancy presents a more biologically relevant experimental strategy to investigate developmental, behavioral, and immunological sequelae of offspring in such rodent models.

Immunological response to peptide nucleic acid and its peptide conjugate targeted to transactivation response (TAR) region of HIV-1 RNA genome.

Anti-human immunodeficiency virus-1 (HIV-1) polyamide (peptide) nucleic acids (PNAs) conjugated with cell-penetrating peptides (CPPs) targeted to the viral genome are potent virucidal and antiviral agents. Earlier, we have shown that the anti-HIV-1 PNA(TAR)-penetratin conjugate is rapidly taken up by cells and is nontoxic to mice when administered at repeat doses of as high as 100 mg/kg body weight. In the present studies we demonstrate that naked PNA(TAR) is immunologically inert as judged by the proliferation responses of splenocytes and lymph node cells from PNA(TAR)-immunized mice challenged with the immunizing antigen. In contrast, PNA(TAR)-penetratin conjugate is moderately immunogenic mainly due to its penetratin peptide component. Cytokine secretion profiles of the lymph node cells from the conjugate-immunized mice showed marginally elevated levels of proinflammatory cytokines, which are known to promote proliferation of T lymphocytes. Since the candidate compound, PNA(TAR)-penetratin conjugate displays potent virucidal and antiviral activities against HIV-1, the favorable immunological response together with negligible toxicity suggest a strong therapeutic potential for this class of compounds.

Immunostimulatory effects of mesenchymal stem cell-derived neurons: implications for stem cell therapy in allogeneic transplantations.

Mesenchymal stem cells (MSCs) differentiate along various lineages to specialized mesodermal cells and also transdifferentiate into cells such as ectodermal neurons. MSCs are among the leading adult stem cells for application in regenerative medicine. Advantages include their immune-suppressive properties and reduced ethical concerns. MSCs also show immune-enhancing functions. Major histocompatibility complex II (MHC-II) is expected to be downregulated in MSCs during neurogenesis. Ideally, "off the shelf" MSCs would be suited for rapid delivery into patients. The question is whether these MSC-derived neurons can reexpress MHC-II in a milieu of inflammation. Western analyses demonstrated gradual decrease in MHC-II during neurogenesis, which correlated with the expression of nuclear CIITA, the master regulator of MHC-II expression. MHC-II expression was reversed by exogenous IFNY. One-way mixed lymphocyte reaction with partly differentiated neurons showed a stimulatory effect, which was partly explained by the release of the proinflammatory neurotransmitter substance P (SP), cytokines, and decreases in miR-130a and miR-206. The anti-inflammatory neurotransmitters VIP and CGRP were decreased at the peak time of immune stimulation. In summary, MSC-derived neurons show decreased MHC-II expression, which could be reexpressed by IFNY. The release of neurotransmitters could be involved in initiating inflammation, underscoring the relevance of immune responses as consideration for stem cell therapies.

Cytokine levels during pregnancy influence immunological profiles and neurobehavioral patterns of the offspring.

The underlying causes of autism spectrum disorders (ASD) are unknown, but clinical and experimental studies indicate immune mechanisms, in general, and cytokine dysregulation, in particular, as contributing factors in their etiology. We developed a prenatal mouse model of autism to demonstrate that circulating levels of defined cytokines in pregnant dams could influence fetal development and behavioral characteristics in their offspring. We administered daily injections of murine IL-2 (0.4 mug in phosphate-buffered saline [PBS]) to pregnant mice during mid-gestation, and analyzed their offspring (IL-2 pups) in comparison to offspring of pregnant mice injected with vehicle only (PBS pups). Significant levels of IL-2 were present in amniotic fluid and tissues from embryos of dams given radiolabeled IL-2, indicating that the injected IL-2 crossed the placenta and entered the fetuses. Lymphocytes from IL-2 pups demonstrated accelerated T cell development, with a skewing toward TH1 cell differentiation. IL-2 pups also showed in vitro proliferative and cytotoxicity responses that were significantly higher than control PBS pups when stimulated with syngeneic B lymphoma cells or allogeneic spleen cells. In addition to their previously shown increases in open-field activity, grooming and rearing behavior, offspring of IL-2-injected (vs. PBS-injected) dams also displayed abnormal new motor learning as assessed through acquisition of the classically conditioned eyeblink response. These results suggest that increases in maternal levels of IL-2 during pregnancy induce in their offspring long-lasting increased vulnerability to neurobehavioral abnormalities associated with autism, and provide a valid animal model to determine the underlying immunological mechanisms.

CpG oligodeoxynucleotide-induced immunity prevents growth of germinal center-derived B lymphoma cells.

Therapeutic efficacy of CpG oligodeoxynucleotide (ODN) ISS 1018 was tested in a murine B cell lymphoma model. Previous studies showed that the B lymphoma cells of SJL mice stimulate vigorous proliferation of host CD4(+) TH cells that is unaccompanied by development of tumor-specific CTL. In the presence of ISS 1018, however, tumor cells stimulated high levels of CTL activity in vitro, and this cytotoxic activity was inhibited when anti-IL-12 mAb was added to the cultures. Tumor cells pre-incubated with ISS 1018 were also able to generate CTL without addition of exogenous ODN, and FACS analysis revealed that following incubation with ISS 1018 for 24 h, tumor cells exhibited upregulation of MHC I, MHC II, and co-stimulatory molecule CD80. Finally, tumor-injected mice treated with ISS 1018 showed significantly less growth of tumor cells in lymph nodes and spleen, and exhibited prolonged survival compared to mice treated with a control ODN. The documented effects of CpG ODNs to stimulate cytokines, such as IL-12, from antigen presenting cells, and to upregulate expression of MHC Class I and Class II, as well as co-stimulatory molecules on tumor cells, are also the likely mechanisms by which CTL are generated by ISS 1018 in the SJL B cell lymphoma model.

Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma.

Mesenchymal stem cells (MSCs) are mostly found around the vasculature system of the adult bone marrow (BM). They function as immune suppressors, express MHC-II, are phagocytic, and support T-cell cytotoxicity. We hypothesize that these contradictory properties of MSCs are important for BM homeostasis and occur partly through antigen presentation (antigen-presenting cells [APCs]) within a narrow window. Indeed, we have verified APC functions of MSCs to recall antigens, Candida albicans and Tetanus toxoid. The target cells have been identified to be CD4(+) T cells. APC assays with IFNgamma-knockdown MSCs and with anti-IFNgamma receptor confirmed that MHC-II expression requires autocrine stimulation by IFNgamma. During APC functions, as IFNgamma levels become elevated, there was a concomitant decrease in MHC-II on MSCs. This observation was correlated with flow cytometry studies showing a gradual decrease in MHC-II expression as IFNgamma levels were increased. The reduced levels of MHC-II correlated with losses in their allogeneic potential, as indicated in mixed lymphocyte reaction. In summary, endogenous and low levels of IFNgamma are required for MHC-II expression on MSCs, and for APC functions. APC functions occur during a narrow window before IFNgamma levels are increased. The study has implications for BM protection against infection and exacerbated inflammatory responses.

A paradoxical role for IFN-gamma in the immune properties of mesenchymal stem cells during viral challenge.

OBJECTIVE: The functional "plasticity" and immune-suppressive effects of human bone marrow (BM)-derived mesenchymal stem cells (MSC) provide them with the potential to be used across allogeneic barriers. The immunosuppressive properties of MSC may be detrimental in a clinical setting in which viral exposure is common. The study hypothesizes that MSC-derived IFN-gamma could offset the immune-suppressive functions of MSC and mediate partial CTL responses during viral infection. METHODS: CTL responses were studied in bioassays with (51)Cr-P815 targets and PBMC (uninfected or infected) as effectors. Immunofluorescence studied the relative expression of CD8(+) cells. Cytokine analyses were performed with microarrays. Roles for IFN-gamma in CTL responses were studied with IFNgammaRI mAb or with MSC knockdown for IFN-gamma by siRNA (pPMSKH1-IFNgamma). RESULTS: MSC showed no significant effect on circulating CTL of healthy subjects. For virus-induced CTL, MSC demonstrated approximately 50% suppression. CD8(+) cell expansion could not explain the suppressive effects of MSC. Soluble factors produced by MSC were responsible for the retention of 50% CTL responses. Cytokine microarray analyses, noncontact cultures, and functional assays identified a role for IFN-gamma. MSC were identified as the relevant source of IFN-gamma. CONCLUSION: The results show a facilitating role of IFN-gamma on CTL responses, although paradoxical in light of the veto properties of MSC. This report shows that in cases where MSC are used in transplantation for repair of damaged tissue, they can exert an additional role by protecting the host to viral challenges and thereby protect from its immunosuppressive properties.