Creb coactivators direct anabolic responses and enhance performance of skeletal muscle.

During the stress response to intense exercise, the sympathetic nervous system (SNS) induces rapid catabolism of energy reserves through the release of catecholamines and subsequent activation of protein kinase A (PKA). Paradoxically, chronic administration of sympathomimetic drugs (ß-agonists) leads to anabolic adaptations in skeletal muscle, suggesting that sympathetic outflow also regulates myofiber remodeling. Here, we show that ß-agonists or catecholamines released during intense exercise induce Creb-mediated transcriptional programs through activation of its obligate coactivators Crtc2 and Crtc3. In contrast to the catabolic activity normally associated with SNS function, activation of the Crtc/Creb transcriptional complex by conditional overexpression of Crtc2 in the skeletal muscle of transgenic mice fostered an anabolic state of energy and protein balance. Crtc2-overexpressing mice have increased myofiber cross-sectional area, greater intramuscular triglycerides and glycogen content. Moreover, maximal exercise capacity was enhanced after induction of Crtc2 expression in transgenic mice. Collectively these findings demonstrate that the SNS-adrenergic signaling cascade coordinates a transient catabolic stress response during high-intensity exercise, which is followed by transcriptional reprogramming that directs anabolic changes for recovery and that augments subsequent exercise performance.

CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART.

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

PD-1 coinhibitory signals: the link between pathogenesis and protection.

In the majority of HIV-1 infected individuals, the adaptive immune response drives virus escape resulting in persistent viremia and a lack of immune-mediated control. The expression of negative regulatory molecules such as PD-1 during chronic HIV infection provides a useful marker to differentiate functional memory T cell subsets and the frequency of T cells with an exhausted phenotype. In addition, cell-based measurements of virus persistence equate with activation markers and the frequency of CD4 T cells expressing PD-1. High-level expression of PD-1 and its ligands PD-L1 and PD-L2 are found on hematopoietic and non-hematopoietic cells, and are upregulated by chronic antigen stimulation, Type 1 and Type II interferons (IFNs), and homeostatic cytokines. In HIV infected subjects, PD-1 levels on CD4 and CD8 T cells continue to remain high following combination anti-retroviral therapy (cART). System biology approaches have begun to elucidate signal transduction pathways regulated by PD-1 expression in CD4 and CD8 T cell subsets that become dysfunctional through chronic TCR activation and PD-1 signaling. In this review, we summarize our current understanding of transcriptional signatures and signal transduction pathways associated with immune exhaustion with a focus on recent work in our laboratory characterizing the role of PD-1 in T cell dysfunction and HIV pathogenesis. We also highlight the therapeutic potential of blocking PD-1-PD-L1 and other immune checkpoints for activating potent cellular immune responses against chronic viral infections and cancer.

CRTC1/MAML2 gain-of-function interactions with MYC create a gene signature predictive of cancers with CREB-MYC involvement.

Chimeric oncoproteins created by chromosomal translocations are among the most common genetic mutations associated with tumorigenesis. Malignant mucoepidermoid salivary gland tumors, as well as a growing number of solid epithelial-derived tumors, can arise from a recurrent t (11, 19)(q21;p13.1) translocation that generates an unusual chimeric cAMP response element binding protein (CREB)-regulated transcriptional coactivator 1 (CRTC1)/mastermind-like 2 (MAML2) (C1/M2) oncoprotein comprised of two transcriptional coactivators, the CRTC1 and the NOTCH/RBPJ coactivator MAML2. Accordingly, the C1/M2 oncoprotein induces aberrant expression of CREB and NOTCH target genes. Surprisingly, here we report a gain-of-function activity of the C1/M2 oncoprotein that directs its interactions with myelocytomatosis oncogene (MYC) proteins and the activation of MYC transcription targets, including those involved in cell growth and metabolism, survival, and tumorigenesis. These results were validated in human mucoepidermoid tumor cells that harbor the t (11, 19)(q21;p13.1) translocation and express the C1/M2 oncoprotein. Notably, the C1/M2-MYC interaction is necessary for C1/M2-driven cell transformation, and the C1/M2 transcriptional signature predicts other human malignancies having combined involvement of MYC and CREB. These findings suggest that such gain-of-function properties may also be manifest in other oncoprotein fusions found in human cancer and that agents targeting the C1/M2-MYC interface represent an attractive strategy for the development of effective and safe anticancer therapeutics in tumors harboring the t (11, 19) translocation.

Therapeutic Vaccination With Dendritic Cells Loaded With Autologous HIV Type 1-Infected Apoptotic Cells.

BACKGROUND: We report the results of a phase I/II, open-label, single-arm clinical trial to evaluate the safety and anti-human immunodeficiency virus type 1 (HIV-1) efficacy of an autologous dendritic cell (DC)-based HIV-1 vaccine loaded with autologous HIV-1-infected apoptotic cells. METHODS: Antiretroviral therapy (ART)-naive individuals were enrolled, and viremia was suppressed by ART prior to delivery of 4 doses of DC-based vaccine. Participants underwent treatment interruption 6 weeks after the third vaccine dose. The plasma HIV-1 RNA level 12 weeks after treatment interruption was compared to the pre-ART (ie, baseline) level. RESULTS: The vaccine was safe and well tolerated but did not prevent viral rebound during treatment interruption. Vaccination resulted in a modest but significant decrease in plasma viremia from the baseline level (from 4.53 log10 copies/mL to 4.27 log10 copies/mL;P= .05). Four of 10 participants had a >0.70 log10 increase in the HIV-1 RNA load in plasma following vaccination, despite continuous ART. Single-molecule sequencing of HIV-1 RNA in plasma before and after vaccination revealed increases in G>A hypermutants in gag and pol after vaccination, which suggests cytolysis of infected cells. CONCLUSIONS: A therapeutic HIV-1 vaccine based on DCs loaded with apoptotic bodies was safe and induced T-cell activation and cytolysis, including HIV-1-infected cells, in a subset of study participants. CLINICAL TRIALS REGISTRATION: NCT00510497.

Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies.

UNLABELLED: A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4(+) T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n = 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P = 0.0003 and P = 0.0004, respectively), while the frequency of CD4(+) T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals. IMPORTANCE: Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

Interleukin-7 promotes HIV persistence during antiretroviral therapy.

HIV persists in latently infected memory CD4(+) T cells during antiretroviral therapy (ART). When administered to HIV-infected subjects receiving suppressive ART, interleukin-7 (IL-7) increases the number of CD4(+) T cells by promoting their survival and proliferation. However, little is known about the impact of IL-7 on HIV persistence during ART. By isolating large numbers of CD4(+) T cells from HIV-infected subjects, we demonstrate that IL-7 enhances viral production in productively infected cells but does not disrupt viral latency in latently infected cells. When administered to virally suppressed subjects, IL-7 led to the rapid proliferation of memory CD4(+) T cells, which resulted in a 70% increase in the absolute number of circulating CD4(+) T cells harboring integrated HIV DNA 4 weeks after therapy. The genetic diversity of the viral reservoir increased transiently in the majority of the subjects studied before returning to baseline values. Altogether, our results indicate that IL-7 promotes the mechanisms of HIV persistence during ART by enhancing residual levels of viral production and inducing proliferation of latently infected cells, and suggest that IL-7 does not represent a suitable candidate therapeutic strategy for HIV eradication. This trial was registered at www.clinicaltrials.gov as #NCT00099671 (AIDS Clinical Trials Group protocol 5214).

Association of Monocyte Migration Marker CD11b With Pulmonary Function in People Living With HIV.

BACKGROUND: Maladaptive immune responses contribute to the pathogenesis of many chronic lung diseases. Here, we tested hypotheses that CD4 and CD8 T-cell and monocyte phenotypes are associated with lung function in people living with HIV and those without HIV. METHODS: Markers of T cell differentiation, activation, exhaustion and senescence, and markers of monocyte recruitment and migration were quantified in 142 HIV-positive and 73 HIV-negative participants of the Pittsburgh HIV Lung Cohort. All participants underwent lung function testing. RESULTS: CD4 or CD8 T-cell phenotypes were not associated with measures of lung function in HIV-positive or HIV-negative participants after adjustment for multiple comparisons. In HIV-positive participants, however, the percentage of classical monocytes that were CD11b+ had positive associations at the Bonferroni-adjusted significance threshold of P = 0.05/63 with prebronchodilator and postbronchodilator forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ratio (ß = 0.36; P = 0.00003 and ß = 0.31; P = 0.0003, respectively). In stratified analyses of n = 87 participants with CD4 ≥ 500 cells/µL, associations of percentage of classical monocytes that were CD11b+ with prebronchodilator and postbronchodilator FEV1/FVC ratio were stronger (ß = 0.48 and ß = 0.41, for pre- and post-, respectively) than in the entire HIV-positive study population. Significant associations of monocyte phenotypes were not observed in HIV-negative participants after adjustment for multiple comparisons. CONCLUSIONS: CD11b+ expression on classical monocytes is positively associated with FEV1/FVC ratio in people living with HIV including in those with CD4 T-cell recovery. Given the normal surveillance activity of monocytes, such association suggests this monocyte subset may play a role in preservation of pulmonary function in PLWH.