Natural killer cells and cytotoxic T lymphocytes are required to clear solid tumor in a patient-derived xenograft.

Existing patient-derived xenograft (PDX) mouse models of solid tumors lack a fully tumor donor-matched, syngeneic, and functional immune system. We developed a model that overcomes these limitations by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in 83% to 89% of TIL-PDX mice, and these were seen to harbor exhausted immuno-effector as well as functional immunoregulatory cells persisting for at least 6 months postengraftment. Combined treatment with interleukin-15 stimulation and immune checkpoint inhibition resulted in complete or partial tumor response in this model. Further, depletion of cytotoxic T lymphocytes and/or natural killer cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.

IL-10 normalizes aberrant amygdala GABA transmission and reverses anxiety-like behavior and dependence-induced escalation of alcohol intake.

Alcohol elicits a neuroimmune response in the brain contributing to the development and maintenance of alcohol use disorder (AUD). While pro-inflammatory mediators initiate and drive the neuroimmune response, anti-inflammatory mediators provide an important homeostatic mechanism to limit inflammation and prevent pathological damage. However, our understanding of the role of anti-inflammatory signaling on neuronal physiology in critical addiction-related brain regions and pathological alcohol-dependence induced behaviors is limited, precluding our ability to identify promising therapeutic targets. Here, we hypothesized that chronic alcohol exposure compromises anti-inflammatory signaling in the central amygdala, a brain region implicated in anxiety and addiction, consequently perpetuating a pro-inflammatory state driving aberrant neuronal activity underlying pathological behaviors. We found that alcohol dependence alters the global brain immune landscape increasing IL-10 producing microglia and T-regulatory cells but decreasing local amygdala IL-10 levels. Amygdala IL-10 overexpression decreases anxiety-like behaviors, suggesting its local role in regulating amygdala-mediated behaviors. Mechanistically, amygdala IL-10 signaling through PI3K and p38 MAPK modulates GABA transmission directly at presynaptic terminals and indirectly through alterations in spontaneous firing. Alcohol dependence-induces neuroadaptations in IL-10 signaling leading to an overall IL-10-induced decrease in GABA transmission, which normalizes dependence-induced elevated amygdala GABA transmission. Notably, amygdala IL-10 overexpression abolishes escalation of alcohol intake, a diagnostic criterion of AUD, in dependent mice. This highlights the importance of amygdala IL-10 signaling in modulating neuronal activity and underlying anxiety-like behavior and aberrant alcohol intake, providing a new framework for therapeutic intervention.

Alcohol dependence promotes systemic IFN-γ and IL-17 responses in mice.

Alcohol use disorder (AUD) is a chronic relapsing disorder characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences. AUD is associated with a variety of physiological changes and is a substantial risk factor for numerous diseases. We aimed to characterize systemic alterations in immune responses using a well-established mouse model of chronic intermittent alcohol exposure to induce alcohol dependence. We exposed mice to chronic intermittent ethanol vapor for 4 weeks and analyzed the expression of cytokines IFN-γ, IL-4, IL-10, IL-12 and IL-17 by different immune cells in the blood, spleen and liver of alcohol dependent and non-dependent control mice through multiparametric flow cytometry. We found increases in IFN-γ and IL-17 expression in a cell type- and organ-specific manner. Often, B cells and neutrophils were primary contributors to increased IFN-γ and IL-17 levels while other cell types played a secondary role. We conclude that chronic alcohol exposure promotes systemic pro-inflammatory IFN-γ and IL-17 responses in mice. These responses are likely important in the development of alcohol-related diseases, but further characterization is necessary to understand the initiation and effects of systemic inflammatory responses to chronic alcohol exposure.

Human natural killer cells mediate adaptive immunity to viral antigens.

Adaptive immune responses are defined as antigen sensitization-dependent and antigen-specific responses leading to establishment of long-lived immunological memory. Although natural killer (NK) cells have traditionally been considered cells of the innate immune system, mounting evidence in mice and nonhuman primates warrants reconsideration of the existing paradigm that B and T cells are the sole mediators of adaptive immunity. However, it is currently unknown whether human NK cells can exhibit adaptive immune responses. We therefore tested whether human NK cells mediate adaptive immunity to virally encoded antigens using humanized mice and human volunteers. We found that human NK cells displayed vaccination-dependent, antigen-specific recall responses in vitro, when isolated from livers of humanized mice previously vaccinated with HIV-encoded envelope protein. Furthermore, we discovered that large numbers of cytotoxic NK cells with a tissue-resident phenotype were recruited to sites of varicella-zoster virus (VZV) skin test antigen challenge in VZV-experienced human volunteers. These NK-mediated recall responses in humans occurred decades after initial VZV exposure, demonstrating that NK memory in humans is long-lived. Our data demonstrate that human NK cells exhibit adaptive immune responses upon vaccination or infection. The existence of human memory NK cells may allow for the development of vaccination-based approaches capable of establishing potent NK-mediated memory functions contributing to host protection.

CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities.

Tissue-resident Natural Killer (NK) cells vary in phenotype according to tissue origin, but are typically CD56bright, CXCR6+, and CD69+. NK cells appear very early in fetal development, but little is known about when markers of tissue residency appear during gestation and whether the expression of these markers, most notably the chemokine receptor CXCR6, are associated with differences in functional capability. Using multi-parametric flow cytometry, we interrogated fetal liver and spleen NK cells for the expression of a multitude of extracellular markers associated with NK cell maturation, differentiation, and migration. We analyzed total NK cells from fetal liver and spleen and compared them to their adult liver and spleen counterparts, and peripheral blood (PB) NK. We found that fetal NK cells resemble each other and their adult counterparts more than PB NK. Maturity markers including CD16, CD57, and KIR are lower in fetal NK cells than PB, and markers associated with an immature phenotype are higher in fetal liver and spleen NK cells (NKG2A, CD94, and CD27). However, T-bet/EOMES transcription factor profiles are similar amongst fetal and adult liver and spleen NK cells (T-bet-/EOMES+) but differ from PB NK cells (T-bet+EOMES-). Further, donor-matched fetal liver and spleen NK cells share similar patterns of expression for most markers as a function of gestational age. We also performed functional studies including degranulation, cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) assays. Fetal liver and spleen NK cells displayed limited cytotoxic effector function in chromium release assays but produced copious amounts of TNFα and IFNγ, and degranulated efficiently in response to stimulation with PMA/ionomycin. Further, CXCR6+ NK cells in fetal liver and spleen produce more cytokines and degranulate more robustly than their CXCR6- counterparts, even though CXCR6+ NK cells in fetal liver and spleen possess an immature phenotype. Major differences between CXCR6- and + NK cell subsets appear to occur later in development, as a distinct CXCR6+ NK cell phenotype is much more clearly defined in PB. In conclusion, fetal liver and spleen NK cells share similar phenotypes, resemble their adult counterparts, and already possess a distinct CXCR6+ NK cell population with discrete functional capabilities.

HIV Prevalence and Sexual Behaviors Among Transgender Women in Tehran, Iran.

To date, no study has looked at the prevalence of HIV and the high-risk behaviors among transgender women in Iran. Between May 2013 and February 2014, 104 transgender women were recruited for participation in this study. Inclusion criteria consisted of having an official letter from the Tehran Psychiatric Institute, or a well-known psychiatrist, that showed a diagnosis of gender dysphoria and/or completed Gender-Affirming Surgery at least 6 months prior to this study. Of the 104 participants, 2 were diagnosed with HIV, which translates to a HIV prevalence of 1.9%. Condom use with a non-paying partner, casual partner, and paying partner was respectively 39.7%, 34.6%, and 53.3%. A high percentage of transgender women in Tehran engage in high-risk sexual behaviors including condomless receptive anal sex, which is of particular concern given the low rates of HIV testing. Targeted public intervention programs and research are desperately needed for this high-risk group.

p53 Nongenotoxic Activation and mTORC1 Inhibition Lead to Effective Combination for Neuroblastoma Therapy.

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling.Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53.Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination.Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629-39. ©2017 AACR.

HIV, HCV, HBV, HSV, and syphilis prevalence among female sex workers in Tehran, Iran, by using respondent-driven sampling.

To find out the prevalence of HIV, HCV, HBV, HSV, and syphilis infections among female sex workers (FSWs) in Tehran, a cross-sectional study by using respondent-driven sampling (RDS) method was conducted. From December 2012 to April 2013 FSWs in Tehran were recruited. Inclusion criteria consisted of trading sex during the 12 months prior to this study and selling sex for at least 6 months in participants' lifetime. Among 161 consenting participants, 5% were infected with HIV. Moreover, 8.1% of FSWs were HCV positive, 37.9% were of HSV type1/type2, 1.2% of participants were infected with HBV, and none of the participants were infected with syphilis. HIV-positive participants were significantly more likely to be co-infected with HSV type1/type2, be younger, have more sexual partners and especially more clients during seven days prior to this study and report more history of having at least one of sexually transmitted infections symptoms in 12 months prior the study. In the multiple logistic regression analysis, being infected with HSV and also being under 25 years of age were found to be independently associated with HIV infection. Compared with the prevalence of HIV among general population of Tehran, relatively high prevalence of HIV and other viral infections among FSWs should be considered. All in all, it is critical to commence effective counter-measures for this high-risk group if the aim is to prevent spreading of these viruses to general population.

S-nitrosylation-mediated redox transcriptional switch modulates neurogenesis and neuronal cell death.

Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade.

Isobaric tagging-based quantification by mass spectrometry of differentially regulated proteins in synaptosomes of HIV/gp120 transgenic mice: implications for HIV-associated neurodegeneration.

HIV/gp120 transgenic mice manifest neuropathological features similar to HIV-associated neurocognitive disorders (HAND) in humans, including astrogliosis, microglia activation, and decreased neuronal synapses. Here, proteomic screening of synaptosomes from HIV/gp120 transgenic mice was conducted to determine potential neuronal markers and drug targets associated with HAND. Synaptosomes from 13 month-old wild-type (wt) and HIV/gp120 transgenic mouse cortex were subjected to tandem mass tag (TMT) labeling and subsequent analysis using an LTQ-Orbitrap mass spectrometer in pulsed-Q dissociation (PQD) mode for tandem mass spectrometry (MS/MS). A total of 1301 proteins were identified in both wt and HIV/gp120 transgenic mice. Three of the most differentially-regulated proteins were validated by immunoblotting. To elucidate putative pathways associated with the proteomic profile, 107 proteins manifesting a ≥1.5 fold change in expression were analyzed using a bioinformatics pathway analysis tool. This analysis revealed direct or indirect involvement of the phosphotidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, a well-known neuronal survival pathway. Immunoblots confirmed a lower phospho (p)Akt/Akt ratio in synaptosomes from HIV/gp120 transgenic animals compared to wt, suggesting that this neuroprotective pathway was inactivated in the HIV/gp120 transgenic brain. Based on this information, we then compared immunoblots of pAkt/Akt in the forebrains of these mice as well as in human postmortem brain. We observed a significant decrease in the pAkt/Akt ratio in synaptosomes and forebrain of HIV/gp120 transgenic compared to wt mice, and a similar decrease in human forebrain from HAND patients compared to neurologically unimpaired HIV+ and HIV- controls. Moreover, mechanistic insight into an additional pathway for decreased Akt activity in HIV/gp120 mouse brains and human HAND brains was shown to occur via S-nitrosylation of Akt protein, a posttranslational modification known to inhibit Akt activity and contribute to neuronal cell injury and death. Thus, MS proteomic profiling in the HIV/gp120 transgenic mouse predicted dysregulation of the PI3K/Akt pathway observed in human brains with HAND, providing evidence that this mouse is a useful disease model and that the Akt pathway may provide multiple drug targets for the treatment of HIV-related dementias.