Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses.

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.

Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma.

Background: Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods: The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results: GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions: Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Sex Differences in Associations of Hostile and Non-hostile Criticism with Relationship Quality.

Three studies examine whether the type of criticism (hostile vs. non-hostile) or the sex of the person perceiving criticism affect the association of criticism with relationship satisfaction. Analyses of the samples of undergraduates (Studies 1 and 2) and community couples (Study 3) indicated that hostile criticism was negatively associated with relationship functioning, whereas non-hostile criticism was positively associated with relationship functioning. The former association was stronger for women than for men (Studies 2 and 3), while the latter association was stronger for men than for women (Study 1). The results also suggest that hostile criticism may be more strongly associated with negative relationship processes in women than men, whereas non-hostile criticism may be more strongly associated with positive relationship processes for men than women.

Coinhibitory Pathways in the B7-CD28 Ligand-Receptor Family.

Immune responses need to be controlled for optimal protective immunity and tolerance. Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis and defense and protect tissue integrity. These coinhibitory signals limit the strength and duration of immune responses, thereby curbing immune-mediated tissue damage, regulating resolution of inflammation, and maintaining tolerance to prevent autoimmunity. Tumors and microbes that cause chronic infections can exploit these coinhibitory pathways to establish an immunosuppressive microenvironment, hindering their eradication. Advances in understanding T cell coinhibitory pathways have stimulated a new era of immunotherapy with effective drugs to treat cancer, autoimmune and infectious diseases, and transplant rejection. In this review we discuss the current knowledge of the mechanisms underlying the coinhibitory functions of pathways in the B7-CD28 family, the diverse functional consequences of these inhibitory signals on immune responses, and the overlapping and unique functions of these key immunoregulatory pathways.

Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

Emotion Regulation and Perceptions of Hostile and Constructive Criticism in Romantic Relationships.

Perceptions of hostile criticism (PHC) from close others are associated with poor individual functioning and low relationship satisfaction, whereas perceptions of constructive criticism (PCC) are associated with better relationship satisfaction. There is little empirical knowledge, however, regarding individual factors that contribute to such perceptions. The present study examined associations of overall emotion regulation difficulties, as well as the specific use of expressive suppression and cognitive reappraisal, with PHC and PCC. Both partners of 63 community couples completed global self-report measures. Sixty-one couples also completed similar measures immediately following each of three discussions during a laboratory session. Multilevel modeling analyses of global data indicated that individuals' reports of PHC were higher when they used more suppression and when both they and their partners reported greater difficulty in emotion regulation. Results with discussion-specific data were similar: Participants reported higher PHC in discussions when both they and their partners reported using more suppression or when they had more difficulties in emotion regulation during the discussions. Individuals reported higher levels of PCC when their partners reported using less suppression, both globally and in discussions. Finally, participants also reported higher levels of PCC in discussions when they reported using more reappraisal.

Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model.

Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti-PD-1, anti-PD-L1, or anti-CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti-CTLA-4 plus anti-PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8(+) and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma.

Autophagy regulation in cancer development and therapy.

Autophagy is a cellular process to degrade long-lived or malfunctioning proteins and obsolete or damaged organelles. It maintains cellular homeostasis and helps cells survive stressful conditions. Tumor suppressors mostly positively regulate autophagy, whereas oncogene products usually inhibit autophagy. Alterations in key autophagy genes have also been shown to affect cancer development. However, the role of autophagy in cancer depends on the status of the cells and can either suppress or promote tumor growth. In the present review, we report on the current state of knowledge about the reciprocal regulation of autophagy and the potential role of autophagy played in cancer development and therapy.

The iron chelator Dp44mT inhibits the proliferation of cancer cells but fails to protect from doxorubicin-induced cardiotoxicity in spontaneously hypertensive rats.

PURPOSE: The iron chelator Dp44mT is a potent topoisomerase IIα inhibitor with novel anticancer activity. Doxorubicin (Dox), the current front-line therapy for breast cancer, induces a dose-limiting cardiotoxicity, in part through an iron-mediated pathway. We tested the hypothesis that Dp44mT can improve clinical outcomes of treatment with Dox by alleviating cardiotoxicity. METHODS: The general cardiac and renal toxicities induced by Dox were investigated in the presence and absence of Dp44mT. The iron chelating cardioprotectant Dexrazoxane (Drz), which is approved for this indication, was used as a positive control. In vitro studies were carried out with H9c2 rat cardiomyocytes and in vivo studies were performed using spontaneously hypertensive rats. RESULTS: Testing of the GI(50) profile of Dp44mT in the NCI-60 panel confirmed activity against breast cancer cells. An acute, toxic dose of Dox caused the predicted cellular and cardiac toxicities, such as cell death and DNA damage in vitro and elevated cardiac troponin T levels, tissue damage, and apoptosis in vivo. Dp44mT alone caused insignificant changes in hematological and biochemical indices in rats, indicating that Dp44mT is not significantly cardiotoxic as a single agent. In contrast to Drz, Dp44mT failed to mitigate Dox-induced cardiotoxicity in vivo. CONCLUSIONS: We conclude that although Dp44mT is a potent iron chelator, it is unlikely to be an appropriate cardioprotectant against Dox-induced toxicity. However, it should continue to be evaluated as a potential anticancer agent as it has a novel mechanism for inhibiting the growth of a broad range of malignant cell types while exhibiting very low intrinsic toxicity to healthy tissues.

Measuring social anxiety related interpersonal constraint with the flexible iterated prisoner's dilemma.

Data from self-report and observational studies make it clear that problematic social anxiety confers interpersonal constraint that may contribute to the interpersonal dysfunction reported by individuals with social anxiety disorder. Direct observation of interpersonal behavior in established relationships is rare and difficult to obtain for practical reasons. We tested a flexible iterated prisoner's dilemma computer task to examine whether it might capture interpersonal constraint related to social anxiety. Our first study suggested that, at least for men, the task directly captures social anxiety's constraint on responses to friendly giving. Our second study more strongly supported the assertion that the task directly assesses interpersonal constraint. The data support a model in which social anxiety directly confers trait-like tendencies toward interpersonal constraint and indirectly constrains behavior in specific interactions. These studies provide further evidence that constrained responses to positive social behaviors are characteristic of people with problematic social anxiety and may be particularly important to dysfunctions in the close relationships of these individuals.

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells.

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.

The antioxidant transcription factor Nrf2 negatively regulates autophagy and growth arrest induced by the anticancer redox agent mitoquinone.

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q(10.) Inhibition of cancer cell growth by MitoQ was associated with G(1)/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G(1) cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

Premenstrual mood symptoms: study of familiality and personality correlates in mood disorder pedigrees.

We sought to determine whether premenstrual mood symptoms exhibit familial aggregation in bipolar disorder or major depression pedigrees. Two thousand eight hundred seventy-six women were interviewed with the Diagnostic Interview for Genetic Studies as part of either the NIMH Genetics Initiative Bipolar Disorder Collaborative study or the Genetics of Early Onset Major Depression (GenRED) study and asked whether they had experienced severe mood symptoms premenstrually. In families with two or more female siblings with bipolar disorder (BP) or major depressive disorder (MDD), we examined the odds of having premenstrual mood symptoms given one or more siblings with these symptoms. For the GenRED MDD sample we also assessed the impact of personality as measured by the NEO-FFI. Premenstrual mood symptoms did not exhibit familial aggregation in families with BP or MDD. We unexpectedly found an association between high NEO openness scores and premenstrual mood symptoms, but neither this factor, nor NEO neuroticism influenced evidence for familial aggregation of symptoms. Limitations include the retrospective interview, the lack of data on premenstrual dysphoric disorder, and the inability to control for factors such as medication use.

The iron chelator Dp44mT causes DNA damage and selective inhibition of topoisomerase IIalpha in breast cancer cells.

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G(1) cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (gamma-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIalpha (top2alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2alpha knockout cells (top2alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2alpha(+/+) or top2beta(-/-) cells. Specificity for top2alpha was confirmed using top2alpha and top2beta small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2alpha. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2alpha activity.