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Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15).Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice.Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a “nonexhausted” effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor.Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817–30. ©2016 AACR.

Supplementary Figure 1. IL-15 availability promotes proliferation and maintenance of transferred CD8+ T in the spleen of recipient mice; Supplementary Figure 2. Gating strategy for the identification of adoptively transferred Pmel-1 cells and endogenous CD8+ T cells infiltrating the tumor; Supplementary Figure 3. Absolute counts of splenic Pmel-1 and CD8+ T cells are profoundly affected by hetIL-15 treatment; Supplementary Figure 4. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating Pmel-1 cells; Supplementary Figure 5. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating CD8+ T cells; Supplementary Table 1. Hematological parameters during ACT treatments.

The COVID-19 pandemic has a unprecedented impact on the way individuals make sense of the interconnected nature of themselves in relation to the world. This study investigated the mediating role of transcendental awareness and compassion in the association of interconnectedness with a sense of civic duty and collective action participation during COVID-19 using a longitudinal design. A total of 336 young adult participants were recruited at baseline and were asked to complete measures of interconnectedness, transcendental awareness, compassion, civic duty, and collective action participation at three time points over a 6-month period. Path analysis was used to test the hypothesized mediation model. The results showed that compassion fully mediated the positive association between interconnectedness and collective action participation and partially mediated the positive association between interconnectedness and civic duty. Transcendental awareness also partially mediated the positive association between interconnectedness and civic duty but not collective action participation. This study highlighted the potential of interconnectedness in promoting civic duty and engagement in collective action through transcendental awareness and compassion during the COVID-19 pandemic.

BACKGROUND Internet-based mental health interventions have been demonstrated to be effective in alleviating psychological distress and promoting mental well-being. However, real-world uptake and engagement of such interventions have been low. Rather than being stand-alone interventions, situating internet-based interventions under a stratified stepped care system can support users to continue with mental health practice and monitor their mental health status for timely services that are commensurate with their needs. A user-centered approach should be used in the development of such web-based platforms to understand the facilitators and barriers in user engagement to enhance platform uptake, usability, and adherence so it can support the users’ continued adoption and practice of self-care for their mental health. OBJECTIVE The aim of this study was to describe the design process taken to develop a web-based stratified stepped care mental health platform, TourHeart+, using a user-centered approach that gathers target users’ perceptions on mental self-care and feedback on the platform design and incorporates them into the design. METHODS The process involved a design workshop with the interdisciplinary development team, user interviews, and 2 usability testing sessions on the flow of registration and mental health assessment and the web-based self-help interventions of the platform. The data collected were summarized as descriptive statistics if appropriate and insights are extracted inductively. Qualitative data were extracted using a thematic coding approach. RESULTS In the design workshop, the team generated empathy maps and point-of-view statements related to the possible mental health needs of target users. Four user personas and related processes in the mental health self-care journey were developed based on user interviews. Design considerations were derived based on the insights drawn from the personas and mental health self-care journey. Survey results from 104 users during usability testing showed that the overall experience during registration and mental health assessment was friendly, and they felt cared for, although no statistically significant differences on preference ratings were found between using a web-based questionnaire tool and through an interactive chatbot, except that chatbot format was deemed more interesting. Facilitators of and barriers to registering the platform and completing the mental health assessment were identified through user feedback during simulation with mock-ups. In the usability testing for guided self-help interventions, users expressed pain points in course adherence, and corresponding amendments were made in the flow and design of the web-based courses. CONCLUSIONS The design process and findings presented in the study are important in developing a user-centric platform to optimize users’ acceptance and usability of a web-based stratified stepped care platform with guided self-help interventions for mental well-being. Accounting for users’ perceptions and needs toward mental health self-care and their experiences in the design process can enhance the usability of an evidence-based mental health platform on the web. CLINICALTRIAL

Introduction: Perceived loneliness and depression are risk factors threatening mental well-being of older people. Yet, hardiness, a modifiable internal resource that supports functional resilience,...

OBJECTIVE: Hormonal contributions to the sex-dependent development of both obsessive-compulsive disorder (OCD) and obesity have been described, but the underlying mechanisms are incompletely understood. A-kinase anchoring protein 13 (AKAP13) significantly augments ligand-dependent activation of estrogen receptors alpha and beta. The hypothalamus and pituitary gland are implicated in the development and exacerbation of OCD and obesity and have strong AKAP13 expression. The AKAP13 localization pattern observed in these key brain regions together with its effects on sex steroid action suggest a potential role for AKAP13 in compulsive-like behaviors. Here we tested the role of AKAP13 in compulsive-like behavior and body weight using an Akap13 haploinsufficient murine model. MATERIALS AND METHODS: Targeted deletion of the Akap13 gene generated haploinsufficient (Akap13+/−) mice in a C57BL6/J genetic background. Established behavioral assays were conducted, video recorded, and scored blindly to assess compulsive-like behavior based on genotype and gender. Tests included: marble-burying, grooming, open-field and elevated plus-maze. Brain and body weights were also obtained. Mean levels of test outcomes were compared using multi-way ANOVA to test for genotype, sex, genotype*sex, and genotype*sex*age interaction effects with Bonferroni adjustment for multiple comparisons, to further explain any significant interactions. RESULTS: The marble-burying and grooming assays revealed significant sex-dependent increases in perseverative, compulsive-like behaviors in female Akap13 haploinsufficient mice compared to female wild type (WT) mice by demonstrating increased marble-burying activity (p=0.0025) and a trend towards increased grooming behavior (p=0.06). Male Akap13 haploinsufficient mice exhibited no behavioral changes (p>0.05). Elevated plus-maze and open-field test results showed no overt anxiety-like behavior in Akap13 haploinsufficient mice irrespective of sex (p>0.05, both). No differences in brain weight were found in Akap13 haploinsufficient mice compared to WT mice (p>0.05). However, female Akap13 haploinsufficient mice weighed more than female WT mice in the 4 to 0.05) at any age range examined. CONCLUSION: Akap13 haploinsufficiency led to sex-dependent, compulsive-like behavioral changes in a murine model. Interestingly, Akap13 haploinsufficiency also led to a sex-dependent increase in body weight. These results revealed a requirement for AKAP13 in murine behavior, particularly in female mice, and is the first report of AKAP13 involvement in murine behavior. Future studies may show AKAP13 involvement in the pathophysiology of OCD in female humans and may contribute to a better understanding of the role of AKAP13 and sex hormones in the development and exacerbation of OCD.

Progesterone is essential for pregnancy maintenance and menstrual cycle regulation. Hormone action has been primarily ascribed to the well-characterized classical signaling pathway involving ligand binding, activation of nuclear progesterone receptors (PRs), and subsequent activation of genes containing progesterone response elements (PREs). Recent studies have revealed progesterone actions via non-classical signaling pathways, often mediated by non-genomic signaling. Progesterone signaling, in conjunction with growth factor signaling, impacts on the function of growth factors and regulates important physiological actions such as cell growth and remodeling, as well as apoptosis. This review focuses on non-classical progesterone signaling pathways, both including and excluding PR, and highlights how research in this area will provide a better understanding of progesterone actions and may inform novel therapeutic strategies.

We examined the incidence and predictors of threat perceptions toward people who oppose government action (i.e., protestors) following the Umbrella Movement in Hong Kong (September 28th to December 15th, 2014). A population-representative sample of 1,208 citizens (mean age = 46.89 years; 52.4% female) was recruited two months after the conclusion of the Movement using random digit dialing. Upon giving their informed consent, respondents reported sociodemographics, perceived threats of protestors to the prospects of democracy, ways of life, and the economy, anxiety symptoms (STAI), and depressive symptoms (PHQ-9). More than half disagreed that protestors threatened the prospects of democracy (54.7%, 95% CI = .52, .57), ways of life (52.2%, 95% CI = .49, .55), and the economy (51.4%, 95% CI = .49, .54). Regression analyses revealed that male sex was associated with lower odds of perceiving threats to the prospects of democracy and ways of life. Being unmarried was associated with lower odds of perceiving threats to the economy. Secondary education level and depressive symptoms were associated with higher odds of perceiving threats to ways of life and the economy, respectively. This is one of the first population-based studies that measured socioeconomic and mental health correlates of political attitudes immediately following pro-democracy movements.

Context Uterine leiomyomata (fibroids) are prevalent sex hormone‒dependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood. Objective Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids. Methods and Results Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferase‒binding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus. Conclusion These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.

IL-15 is an important cytokine for the regulation of lymphocyte homeostasis. However, the role of IL-15 in the generation, maintenance and cytotoxic potential of antigen specific T cells is not fully understood. Because the route of antigenic delivery and the vaccine modality could influence the IL-15 requirement for mounting and preserving cytotoxic T cell responses, we have investigated the immunogenicity of DNA-based vaccines in IL-15 KO mice. DNA vaccination with SIV Gag induced antigen-specific CD4(+) and CD8(+) T cells in the absence of IL-15. However, the absolute number of antigen-specific CD8(+) T cells was decreased in IL-15 KO mice compared to WT animals, suggesting that IL-15 is important for the generation of maximal number of antigen-specific CD8(+) T cells. Interestingly, antigen-specific memory CD8 cells could be efficiently boosted 8 months after the final vaccination in both WT and KO strains of mice, suggesting that the maintenance of antigen-specific long-term memory T cells induced by DNA vaccination is comparable in the absence and presence of IL-15. Importantly, boosting by DNA 8-months after vaccination revealed severely reduced granzyme B content in CD8(+) T cells of IL-15 KO mice compared to WT mice. This suggests that the cytotoxic potential of the long-term memory CD8(+) T cells is impaired. These results suggest that IL-15 is not essential for the generation and maintenance of adaptive cellular responses upon DNA vaccination, but it is critical for the preservation of maximal numbers and for the activity of cytotoxic CD8(+) T cells.

More empirical investigation is needed to understand the mechanisms through which psychological resilience impacts psychiatric symptoms. This study aimed to examine the mediating role of emotion-focused positive rumination and relationship satisfaction in the inverse associations between trait resilience and anxiety and depressive symptoms. A sample of 284 Chinese college students were recruited and asked to self-administer questionnaires measuring trait resilience, emotion-focused positive rumination, relationship satisfaction, and anxiety and depressive symptoms. Structural equation modeling revealed that emotion-focused positive rumination and relationship satisfaction independently and jointly mediated the inverse association between trait resilience and anxiety symptoms (data-model fit: CFI = 1.000; TLI = 1.022; IFI = 1.004; RMSEA = .000; SRMR = .014). Relationship satisfaction, alone, mediated the inverse association between trait resilience and depressive symptoms (data-model fit: CFI = 1.000; TLI = 1.021; IFI = 1.003; RMSEA = .000; SRMR = .014). The results suggested possible underlying mechanisms of the psychological benefit of trait resilience. Feasible directions for fostering resilience among college populations were discussed.

Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15). Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice. Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a “nonexhausted” effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor. Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817–30. ©2016 AACR.

Glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, whereas AMP-activated protein kinase (AMPK) is the master regulator of energy homeostasis, sensing energy depletion and stimulating pathways of increasing fuel uptake and saving on peripheral supplies. We showed here that AMPK regulates glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. Transcriptomic analysis in the liver suggested marked overlaps between the AMPK and glucocorticoid signaling pathways directed mostly from AMPK to glucocorticoid actions. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive glucocorticoid-inducible leucine zipper protein, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Because increased action of glucocorticoids is associated with the development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacological interventions to these pathologies.

This study investigated the associations of savoring with cancer-specific physical symptoms, psychological distress, and psychological well-being and the moderating effect of savoring in the associations between physical symptoms and psychological outcomes among cancer patients.A total of 263 Chinese adults recently diagnosed with cancer (mean time since diagnosis = 43.72 days, SD = 38.20) were recruited and administered a questionnaire assessing cancer-specific physical symptoms, perceived capability of savoring the moment, psychological distress, and psychological well-being within six months following diagnosis.Structural equation modeling revealed significant associations of savoring with physical symptoms and psychological distress and well-being (β = -0.41-0.54, p 0.0001). Savoring significantly moderated the association between physical symptoms and depressive symptoms. Simple slope tests revealed that the association was not significant at higher levels of savoring (estimate = 0.15, z = 0.49, p = 0.62) whereas it was stronger at lower (estimate = 1.11, z = 4.81, p 0.001) and medium (estimate = 0.63, z = 3.04, p 0.01) levels of savoring. The effects of demographic and medical covariates were controlled for in all models.The findings suggest that savoring is positively associated with physical and psychological functioning among people with cancer. The link between physical symptoms and depressive symptoms could be exacerbated at lower levels of savoring. Fostering savoring beliefs and practices could be a significant psychological component of symptom management among cancer patients. Copyright © 2016 John WileySons, Ltd.

Recovery experience including psychological detachment from caregiving and savoring positive moments in life could be complementary coping processes for cancer caregivers. This study aims to examine the nature of their associations with caregiving burden and anxiety and depressive symptoms among Chinese cancer caregivers in Hong Kong.A total of 155 Chinese caregivers of recently diagnosed cancer patients (mean time since diagnosis = 42.57 days, SD = 39.25) were recruited from two major government-funded hospitals and administered a questionnaire assessing psychological detachment, savoring, caregiving burden, anxiety and depressive symptoms, and demographics.Controlling for demographic and medical covariates, structural equation modeling revealed significant associations of detachment, savoring, and their interaction term with caregiving burden and anxiety and depressive symptoms. Detachment and savoring were inversely associated with caregiving burden only when the other was at lower/medium levels. Detachment was inversely associated with anxiety and depressive symptoms at lower/medium levels of savoring, but savoring was inversely associated with anxiety and depressive symptoms across all levels of detachment.Detachment and savoring could overshadow the positive impact of the other on caregiving burden if either one is at higher levels, while they could demonstrate concurrent positive impact on burden when both are at lower/medium levels. Savoring could have a prioritized role in ameliorating caregivers' anxiety and depressive symptoms, supplemented by detachment. Copyright © 2015 John WileySons, Ltd. Copyright © 2015 John WileySons, Ltd.

Background This study examined the prevalence and critical predictors of anxiety and depressive symptoms and self-rated health, following the Umbrella Movement in Hong Kong. Methods: Random digit dialing recruited a population-representative sample of 1208 Chinese Hong Kong citizens (mean age=46.89 years; 63% female) in the first two weeks of February 2015. Respondents gave their informed consent and reported personal, social, and economic resource loss since the Umbrella Movement (Conservation of Resources-Evaluation), current anxiety symptoms (State-Trait Anxiety Inventory) and depressive symptoms (Patient Health Questionnaire-9), and self-rated health (1= very good , 4= very bad ). Results A total of 47.35% (95% CI=44.55, 50.17) respondents reported moderate/severe anxiety symptoms and 14.4% (95% CI=12.54, 16.50) reported moderate/severe depressive symptoms; 9.11% (95% CI=7.61, 10.86) reported “poor” or “very poor” health. Multivariable regressions revealed that personal and social resource loss was associated with higher anxiety and depressive symptoms and greater odds of “very poor” health (adjusted odds ratios/incidence rate ratios=5–102%), independent of lower education level and income and being unmarried. Limitations This study was cross-sectional in nature and thus could not determine causality from the associations between resource loss and outcome variables. Second, the telephone survey relied on self-reports; response bias and social desirability could influence respondents' answers and discount data validity. Third, potential confounders such as preexisting mental and physical health issues and concurrent predictors like exposure to the Umbrella Movement were not assessed. Conclusions This is one of the first studies following any recent political movement (e.g., The Arab Spring) to quantify distress and the associated correlates of distress among affected citizens. Perceived psychosocial resource losses were critical predictors of poor outcomes.

This study examined whether and how changes in positive affect and mindfulness predicted changes in cortisol secretion and psychological distress in adaptation to examination stress.A sample of 105 college students completed a questionnaire set and provided salivary samples before (T1), during (T2) and after (T3) an examination period.Latent change score modelling revealed that T1-T2 and T2-T3 increases in mindfulness were associated with larger T2-T3 decrease in area-under-the-curve ground of cortisol awakening response (CARg), whereas T2-T3 increases in both positive affect and mindfulness were associated with larger T2-T3 decrease in anxiety symptoms (comparative fit index = .96; Tucker-Lewis index = .93-.95; root-mean-square error of approximation = .04-.08; standardised root-mean-square residual = .08-.10). T1-T2 and T2-T3 increases in positive affect were not associated T2-T3 decrease in CARg, whereas T1-T2 increases in positive affect and mindfulness were not associated with T2-T3 decrease in anxiety symptoms.The levels of post-stress recovery from anxiety symptoms could depend on concurrent increases in positive affect and mindfulness, whereas the levels of post-stress decline in cortisol secretion could depend on increases in mindfulness both during and after stress. Directions for translating the present findings into stress management programmes in college settings are discussed.

We have shown that the physiologically relevant Interleukin-15 molecule in vivo is a heterodimeric molecule consisting of the IL-15 polypeptide chain and the so-called IL-15 Receptor alpha chain (IL-15Ra), a membrane protein. There are two forms of IL-15 in the body: membrane bound heterodimer of IL-15 and IL-15Ra and soluble heterodimeric IL-15 produced by a specific proteolytic cleavage of the heterodimer at the extracellular domain of IL-15Ra. Our results indicate that IL-15Ra polypeptide is not a receptor but the other half of the heterodimeric IL-15 cytokine. We have characterized the soluble heterodimeric IL-15 (hetIL-15) and developed methods for its efficient production and purification. Injection of hetIL-15 resulted in a significant delay in tumor growth, in preclinical models of tumor-bearing mice. Intratumoral delivery of hetIL-15 induced regression of established tumors. hetIL-15 has the unique property to increase intratumoral CD8 cells and to also increase dramatically the ratio of CD8 to Treg. We dissect the mechanisms leading to tumor control in hetIL-15-treated mice, revealing a novel interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated subcutaneous administration of purified hetIL-15 in macaques resulted in sustained plasma IL-15 levels and in dose-dependent expansion of NK and T cells in blood and tissues, demonstrating pharmacokinetics and in vivo bioactivity superior to monomeric IL-15. On the basis of these data, we have produced and characterized a cGMP preparation of hetIL-15 and we have initiated a Phase 1 dose escalation clinical trial in metastatic cancer to assess safety and activity of this cytokine.

The hypothalamic-pituitary-adrenal axis plays a central role in the adaptive response to stress including infection of pathogens through glucocorticoids. Physical and/or mental stress alter susceptibility to viral infection possibly by affecting this regulatory system, thus we explored potential cellular targets and mechanisms that underlie this phenomenon in key immune components dendritic cells (DCs). Dexamethasone (DEX) treatment and subsequent Newcastle disease virus (NDV) infection most significantly and cooperatively stimulated mRNA expression of the interleukin (IL)-10 in murine bone marrow-derived DCs among 89 genes involved in the Toll-like receptor signaling pathways. NDV increased DEX-induced IL-10 mRNA and protein expression by 7- and 3-fold, respectively, which was observed from 3 hours after infection. Conventional DCs (cDCs), but not plasmacytoid DCs (pDCs) were major sources of IL-10 in bone marrow-derived DCs treated with DEX and/or infected with NDV. Murine cytomegalovirus and DEX increased serum IL-10 cooperatively in female mice. Pre-treatment of DCs with the extracellular signal-regulated kinase (ERK) inhibitor U0126 abolished cooperative induction of IL-10 by DEX and NDV. Further, ERK overexpression increased IL-10 promoter activity stimulated by wild-type human GR but not by its mutant defective in serine 203, whereas ERK knockdown abolished NDV/DEX cooperation on IL-10 mRNA and phosphorylation of the mouse GR at serine 213. NDV also increased DEX-induced mRNA expression of three known glucocorticoid-responsive genes unrelated to the Toll-like receptor signaling pathways in DCs. These results indicate that virus and glucocorticoids cooperatively increase production of anti-inflammatory cytokine IL-10 by potentiating the transcriptional activity of GR in DCs, through which virus appears to facilitate its own propagation in infected hosts. The results may further underlie in part known exacerbation of IL-10/T helper-2-related allergic disorders by stress and viral infection.

Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Lymphodepletion of the host is associated with favorable ACT outcomes but it may cause detrimental effects in humans. Lymphodepletion results in high level of homeostatic cytokines including Interleukin-15 (IL-15) that support survival and proliferation of the transferred lymphocytes. We tested the hypothesis that exogenous IL-15 enhances ACT in the absence of lymphodepletion. We have previously shown that bioactive IL-15 in vivo comprises a stable complex of the IL-15 chain with the IL-15 Receptor alpha chain, termed heterodimeric IL-15 (hetIL-15). We therefore evaluated the effects of the combination regimen ACT+hetIL-15 in absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice. hetIL-15 promoted infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells in the tumor. Following lymphodepletion by irradiation, Pmel-1 cells also localized to tumor sites efficiently, but their persistence was severely reduced in comparison to mice treated with hetIL-15. Importantly, we found that hetIL-15 preferentially enriched Pmel-1 cells in the tumor sites. hetIL-15 treatment also supported proliferation and cytotoxic function of tumor-infiltrating Pmel-1 cells, while reducing their expression level of PD-1, and resulted in improved tumor control and survival benefit. Thus, hetIL-15 administration improves the outcome of ACT in non-lymphodepleted hosts, and can be a valuable addition to strategies for cancer immunotherapy. Citation Format: Sinnie Ng, Cristina Bergamaschi, Bethany Nagy, Candido Alicea, Shawn M. Jensen, Bernard A. Fox, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 enhances tumor infiltration, persistence and effector functions of adoptively transferred tumor-specific T cells in the absence of lymphodepletion. [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 LB-088.

Interleukin-15 (IL-15) is a common γ-chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). We investigated the anti-tumor efficacy of hetIL-15 in several mouse cancer models. Repeated injections of hetIL-15 were effective in delaying tumor growth in the MC38 colon carcinoma and the B16 melanoma models. The beneficial effects of hetIL-15 therapy included a significantly reduced onset of lung metastasis in 4T1 breast cancer-bearing mice. We study the mechanisms of antitumor effects of hetIL-15 in several tumor models. hetIL-15 administration promoted a dramatic increase of tumor infiltration and persistence of CD8+ T cells, including tumor specific T cells, and resulted in an increased CD8+/Treg ratio. Tumor-resident CD8+ T cells showed features of effectors cells and were characterized by increased proliferation (Ki67+) and high cytotoxic potential (Granzyme B+). In the absence of hetIL-15, the smaller population of tumor-infiltrating T cells exhibited high levels of the exhaustion marker PD-1, potentially limiting their anti-cancer effectiveness. Provision of hetIL-15 resulted in a significant decrease in lymphocyte expression of PD-1, alleviating one potential mechanism for the exhaustion phenotype. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells.

GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism.

Context and Objective: Circulating cortisol fluctuates diurnally under the control of the ‘‘master’’ circadian CLOCK, while the peripheral ‘‘slave’’ counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. Design and Participants: We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoidresponsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. Results: GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. Conclusions: Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, genespecific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.

Context and Objective: Circulating cortisol fluctuates diurnally under the control of the “master” circadian CLOCK, while the peripheral “slave” counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. Design and Participants: We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. Results: GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock-and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. Conclusions: Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.

Interleukin-15 (IL-15) is a common γ -chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). HetIL-15 produced from engineered human cell lines showed favorable pharmacokinetics and bioactivity compared to monomeric IL-15 in both mice and macaques. We investigated the anti-tumor efficacy of hetIL-15, using the MC38 colon carcinoma mouse model. hetIL-15 resulted in a significant delay in tumor growth, when administered intraperitoneally every 2 days for 2 weeks. In addition, intratumoral delivery of hetIL-15 induced regression of established tumors and cured 50% of mice. Tumor-free mice were resistant to tumor challenge, demonstrating the development of specific anti-tumor immune responses. We further dissected the mechanism leading to tumor control in hetIL-15-treated mice, discovering a new interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated injections of hetIL-15 resulted in an increased CD8 + /Treg cells ratio in spleen, lymph nodes and tumor microenvironment. Interestingly, hetIL-15 induced a transient accumulation of CD8 + T cells expressing the high affinity IL-2 receptor CD25, that compete with CD25 + Treg for the available IL-2. In addition, IL-15 decreased the frequency of IL-2-producing CD4 + T cells, while increasing their ability to secrete IFN γ . These data suggest that hetIL-15 treatment limits Treg fitness through a concerted action of both CD8 + and CD4 + T cells, that results in an IL-2 deprived environment. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells.

Allergic diseases such as asthma and allergic dermatitis are associated with the degranulation of mast cells. Chymase, a mast-cell-specific protease, is the major component in mast cell granules that can induce eosinophil infiltration into inflammatory sites. We examined the immunopathological mechanisms for the activation of eosinophils by chymase in allergic inflammation. Cytokines were measured by cytometric bead array Flex Sets multiplex assay using flow cytometry and enzyme-linked immunosorbent assay. Adhesion molecules, migration and intracellular signalling pathways were assessed by flow cytometry, Boyden chamber assay and Western blot, respectively. Chymase suppressed the apoptosis of eosinophils and induce the release of the cytokine interleukin-6 (IL-6) and chemokines CXCL8, CCL2 and CXCL1 by eosinophils dose-dependently. It also up-regulated the surface expression of adhesion molecule CD18 and stimulated the chemokinetic migration of eosinophils. The expressions of adhesion molecules, cytokines and chemokines, and chemokinetic migration were differentially regulated by the activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Akt, Janus-activated kinase and nuclear factor-kappaB pathways. Chymase therefore plays a pivotal immunological role in the interaction between mast cells and eosinophils in allergic diseases such as allergic dermatitis by inducing adhesion molecule-mediated chemokinetic migration and inflammatory cytokines and chemokines of eosinophils, through multiple intracellular signalling molecules and transcription factor. Our results therefore provide a further biochemical basis for the pathogenesis of allergic inflammation consequent on the interaction between mast cells and eosinophils, and give insight for the development of new therapies.

Interleukin-15 (IL-15) is a common γ-chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). HetIL-15 produced from engineered human cell lines showed favorable pharmacokinetics and bioactivity compared to monomeric IL-15 in both mice and macaques. We investigated the anti-tumor efficacy of hetIL-15, using the MC38 colon carcinoma mouse model. hetIL-15 resulted in a significant delay in tumor growth, when administered intraperitoneally every 2 days for 2 weeks. In addition, intratumoral delivery of hetIL-15 induced regression of established tumors and cured 50% of mice. Repeated injections of hetIL-15 resulted in an increased CD8+/Treg cells ratio in spleen, lymph nodes and tumor microenvironment. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells.

DCs, versatile regulators of host immune response against viral infection, capture antigens and present them to T-cells and subsequently activate both innate and acquired immunity. NRs, on the other hand, are intracellular receptors which function as hormone-dependent transcription factors. Some of the NRs, such as the glucocorticoid receptor and the peroxisome proliferators-activated receptors (PPARs), suppress inflammatory reactions. Thus, we examined with real-time PCR using our custom NR PCR arrays for the mRNA expression of all mice NRs and their coregulators in bone marrow derived DCs (BMDCs) infected with viruses. At the baseline, BMDCs expressed 21 NRs. PPARγ, thyroid hormone receptor-α, retinoid X receptor-α, NGF-induced factor B and Nur related factor 1 were significantly down-regulated by virus infection (15-, 6-, 6-, 50- and 15-fold reduction, respectively), while liver X receptor-α and neuron-derived orphan receptor-1 were strongly upregulated (6- and 20-fold induction, respectively). All coregulators were expressed in BMDCs at the baseline, whereas histone deacetylase 4, 6 and 10, nuclear receptor corepressor-1 and Set-TAF-Iβ were significantly down-regulated by viruses (10-, 4-, 12-, 4- and 2-fold reduction, respectively). These results indicate that viral infection alters mRNA, and possibly, protein expression of some NRs and their coregulators. Our results suggest that NR signaling pathways are integral components of the global response of DCs to viral infection.

We investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in mouse bone marrow-derived dendritic cells (DCs) upon infection with Newcastle Disease virus (NDV) or murine cytomegalovirus (MCMV). These viruses regulated mRNA expression of some NRs among which NOR1 and LXRα were highly induced at mRNA and protein levels. Exogenous expression of the latter NRs repressed IRF3- or IRF7-induced transactivation of the interferon β promoter and NDV infection further potentiated their repressive effect. The viral infection also significantly regulated mRNA expression of some coregulators, including HDAC1. Toll-like receptor ligands regulated NR and coregulator mRNA expression similar to the viruses. Thus, NRs and coregulators are integral components of DC-organizing anti-viral response wherein NOR1 and LXRα participate in regulating interferon production.