CD4 Transcription Research Articles

Features of immunoregulation in patients with pulmonary tuberculosis with blood eosinophilia

The aimof the investigation was to determine the characteristics of the immune response regulation for pulmonary tuberculosis (TB) and to analyze the role of regulatory T cells in the immunopathogenesis of TB with eosinophilia in the blood, depending on the clinical form of the disease and sensitivity of Micobacterium tuberculosis to anti-TB drugs.Materials and methods.157 patients who were initially diagnosed with infiltrative and disseminated TB were examined. The material of the study was venous blood and culture of mononuclear leukocytes isolated from venous blood. The content of interleukin (IL) 4, IL-10 and transforming factor beta (TGFβ) in culture suspensions of mononuclear leukocytes in vitro and IL-5 in the blood was determined by enzyme-linked immunosorbent assay (ELISA) test. The expression of surface molecules CD4, CD20, CD25 and intracellular transcription factor Foxp3 by lymphocytes of the blood was evaluated by flow cytometry. The obtained results were analyzed by statistical methods.Results.It is shown that excessive generation of regulatory T cells in patients with TB is associated with eosinophilia of the blood and imbalance of immune response regulation mechanisms. In TB with eosinophilia, an increase in the number of Foxp3-positive regulatory T cells in the blood is combined with in vitro hypersecretion of anti-inflammatory cytokines TGFβ, IL-10, IL-4 and an increase in the content of CD20+ B lymphocytes and IL-5 in the blood. These changes are most pronounced in the disseminated form of TB in combination with drug resistance.Conclusion.Characteristics of immunoregulation at TB with blood eosinophilia are associated with activation of immunosuppression mechanisms and polarization of immune response towards Th2-dependent pathway.

Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development

The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism.

Upregulation of microRNA-3129 suppresses epithelial ovarian cancer through CD44.

The purpose of this work is to evaluate whether human microRNA-3129 (hsa-miR-3129) may functionally regulate cancer development, possibly through downstream target CD44 in human epithelial ovarian cancer (EOC). Direct targeting of hsa-miR-3129 on human CD44 transcript was evaluated using a dual-luciferase reporter assay. Gene expression of hsa-miR-3129 in immortal EOC cell lines was evaluated by qRT-PCR. Lentivirus-mediated hsa-miR-3129 upregulation or downregulation was conducted in SK-OV-3 and CAOV-3 cells, in which endogenous hsa-miR-3129 and CD44 expressions were then measured. In hsa-miR-3129 upregulated or downregulated EOC cells, functional assays were applied to evaluate EOC proliferation, bufalin chemoresistance in vitro, or xenotransplantation in vivo. Moreover, CD44 was ectopically overexposed in hsa-miR-3129 upregulated EOC cells to functionally evaluate the correlation between hsa-miR-3129 and CD44 in EOC. Dual-luciferase reporter assay confirmed hsa-miR-3129 directly binds CD44. QRT-PCR revealed that hsa-miR-3129 was substantially downregulated in EOC cell lines. In SK-OV-3 and CAOV-3 cells, lentivirus-induced hsa-miR-3129 upregulation downregulated CD44 whereas hsa-miR-3129 downregulation did not affect CD44 expression. Hsa-miR-3129 upregulation had significant anti-cancer effects by inhibiting EOC proliferation, increasing bufalin chemoresistance, and suppressing xenotransplantation. On the other hand, overexpressing CD44 reversed the anti-cancer functions by hsa-miR-3129 upregulation in EOC cells. In conclusion, Has-miR-3129 is a functional regulator, possibly through reverse targeting on CD44, in EOC.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Foxp3 expression in induced T regulatory cells derived from human umbilical cord blood vs. adult peripheral blood.

Foxp3 is essential for T regulatory cell (Treg) function. Broad complex-Tramtrack-Bric-a-brac domain (BTB) and Cap'n'collar (CNC) homology 1, transcription factor 2 (BACH2) stabilizes Treg immune homeostasis in murine studies. However, little is known regarding what role, if any, BACH2 may have in Foxp3 regulation in human-induced Treg (iTreg). We examined Foxp3 expression and regulation comparing iTreg differentiated from umbilical cord blood (UCB) vs. adult blood (AB) naive CD4+ T-cells. Foxp3 expression was higher in UCB vs. AB-derived iTreg, and was sustained during 21-day expansion in vitro. The number of Foxp3+ iTreg generated from UCB vs. AB naive CD4+ T-cells was higher in iTreg differentiation conditions. In addition, UCB iTreg were more potent in suppressing T-cell proliferation compared to AB iTreg. Naive UCB CD4+ T-cells highly expressed BACH2 protein compared to AB. Putative transcriptional BACH2 binding sites were identified at the Foxp3 promoter, using BACH2 consensus sequence. Cross-linking chromatin immunoprecipitation (ChIP) showed that BACH2 binds to the Foxp3 proximal promoter in UCB iTreg, but not AB iTreg. BACH2 was transcriptionally active, as shRNA-mediated BACH2 knockdown resulted in reduction of Foxp3 gene transcription in UCB CD4+ T-cells. In summary, BACH2 serves to stabilize robust Foxp3 expression in UCB CD4+ T-cell-derived iTreg.

CD4+ and Perivascular Foxp3+ T Cells in Glioma Correlate with Angiogenesis and Tumor Progression.

Angiogenesis and immune cell infiltration are key features of gliomas and their manipulation of the microenvironment, but their prognostic significance remains indeterminate. We evaluate the interconnection between tumor-infiltrating lymphocyte (TIL) and tumor blood-vasculatures in the context of glioma progression. Paired tumor tissues of 44 patients from three tumor-recurrent groups: diffuse astrocytomas (DA) recurred as DA, DA recurred as glioblastomas (GBM), and GBM recurred as GBM were evaluated by genetic analysis, immunohistochemistry for tumor blood vessel density, TIL subsets, and clinical outcomes. These cells were geographically divided into perivascular and intratumoral TILs. Associations were examined between these TILs, CD34+ tumor blood vessels, and clinical outcomes. To determine key changes in TIL subsets, microarray data of 15-paired tumors from patients who failed antiangiogenic therapy- bevacizumab, and 16-paired tumors from chemo-naïve recurrent GBM were also evaluated and compared. Upon recurrence in primary gliomas, similar kinetic changes were found between tumor blood vessels and each TIL subset in all groups, but only CD4+ including Foxp3+ TILs, positively correlated with the density of tumor blood vessels. CD4 was the predominant T cell population based on the expression of gene-transcripts in primary GBMs, and increased activated CD4+ T cells were revealed in Bevacizumab-resistant recurrent tumors (not in chemo-naïve recurrent tumors). Among these TILs, 2/3 of them were found in the perivascular niche; Foxp3+ T cells in these niches not only correlated with the tumor vessels but were also an independent predictor of shortened recurrence-free survival (RFS) (HR = 4.199, 95% CI 1.522-11.584, p = 0.006). The minimal intratumoral T cell infiltration and low detection of CD8 transcripts expression in primary GBMs can potentially limit antitumor response. CD4+ and perivascular Foxp3+ TILs associate with tumor angiogenesis and tumor progression in glioma patients. Our results suggest that combining antiangiogenic agents with immunotherapeutic approaches may help improve the antitumor efficacy for patients with malignant gliomas.

Roles of M1 and M2 Macrophages in Herpes Simplex Virus 1 Infectivity.

Macrophages are the predominant infiltrate in the corneas of mice that have been ocularly infected with herpes simplex virus 1 (HSV-1). However, very little is known about the relative roles of M1 (classically activated or polarized) and M2 (alternatively activated or polarized) macrophages in ocular HSV-1 infection. To better understand these relationships, we assessed the impact of directed M1 or M2 activation of RAW264.7 macrophages and peritoneal macrophages (PM) on subsequent HSV-1 infection. In both the RAW264.7 macrophage and PM in vitro models, HSV-1 replication in M1 macrophages was markedly lower than in M2 macrophages and unstimulated controls. The M1 macrophages expressed significantly higher levels of 28 of the 32 tested cytokines and chemokines than M2 macrophages, with HSV-1 infection significantly increasing the levels of proinflammatory cytokines and chemokines in the M1 versus the M2 macrophages. To examine the effects of shifting the immune response toward either M1 or M2 macrophages in vivo, wild-type mice were injected with gamma interferon (IFN-γ) DNA or colony-stimulating factor 1 (CSF-1) DNA prior to ocular infection with HSV-1. Virus replication in the eye, latency in trigeminal ganglia (TG), and markers of T cell exhaustion in the TG were determined. We found that injection of mice with IFN-γ DNA, which enhances the development of M1 macrophages, increased virus replication in the eye; increased latency; and also increased CD4, CD8, IFN-γ, and PD-1 transcripts in the TG of latently infected mice. Conversely, injection of mice with CSF-1 DNA, which enhances the development of M2 macrophages, was associated with reduced virus replication in the eye and reduced latency and reduced the levels of CD4, CD8, IFN-γ,and PD-1 transcripts in the TG. Collectively, these results suggest that M2 macrophages directly reduce the levels of HSV-1 latency and, thus, T-cell exhaustion in the TG of ocularly infected mice.IMPORTANCE Our findings demonstrate a novel approach to further reducing HSV-1 replication in the eye and latency in the TG by modulating immune components, specifically, by altering the phenotype of macrophages. We suggest that inclusion of CSF-1 as part of any vaccination regimen against HSV infection to coax responses of macrophages toward an M2, rather than an M1, response may further improve vaccine efficacy against ocular HSV-1 replication and latency.

Rituximab treatment circumvents the prognostic impact of tumor-infiltrating T-cells in follicular lymphoma patients

Previous immunohistochemical (IHC) studies showed controversial data about the prognostic value of tumor-infiltrating lymphocytes (TILs) in follicular lymphoma (FL). To clarify this issue, a large series of FL samples from rituximab-treated patients enrolled in the randomized PRIMA trial was examined. IHC was quantified using automated image analysis in 417, 287, 418, 406, 379, and 369 patients for CD3, CD4, CD8, PD1, ICOS, and FOXP3, respectively. RNAseq analysis was used to quantify TIL-related mRNA transcripts from 148 patients. When each IHC marker was used as a continuous variable in the whole cohort, high CD3 counts were associated with better progression-free survival (PFS) (P = .025). When an optimal IHC cut point was applied to the whole patient population, high CD3 counts and high PD1 counts were associated with better PFS (P = .011 and P = .044, respectively), whereas none of the other TIL markers had any significant correlation with outcome. When a stringent analysis was performed by dividing the whole cohort into a training set and a validation set, none of the TIL markers showed a prognostic significance in both groups. RNAseq analysis showed a significant correlation between high levels of CD3 and CD8 transcripts and better PFS (P = .001 and P = .037, respectively). No prognostic correlation was found as to the level of other immune gene transcripts. These results suggest that the IHC prognostic value of TILs is circumvented by rituximab treatment, although there is a trend for high numbers of CD3+ TILs to correlate with better PFS.

MSK‐1 mediated Histone H3 phosphorylation is critical for ethanol‐induced inhibition of IL‐2 gene transcription in CD4+ T lymphocytes

Immune suppression due to decreased CD4+ T cell function is a significant pathogenic component of multiple diseases including alcoholism, HIV infection and aging; however, the underlying mechanisms are not yet fully understood. We have previously shown that immunosuppressive effects of alcohol involve a decrease in the expression of interleukin‐2 (IL‐2), which critically regulates T cell proliferation, clonal expansion and homeostasis. In the present study we investigated the role of ethanol‐induced promoter histone modifications in the context of dysregulated IL‐2 gene expression. Chromatin immunoprecipitation (ChIP) analysis revealed that ethanol significantly prevented TCR inducible transcriptionally permissive histone H3 modifications at IL‐2 promoter in CD4+ T lymphocytes. Specifically, ethanol treatment inhibited phosphorylation of H3 serine 10 (H3S10ph) which then coordinately down regulated histone H3 lysine 4 tri‐methylation (H3K4Me3) and H3 lysine 9 acetylation (H3K9Ac). Notably, data showed that ethanol mediated transcriptional repression at IL‐2 promoter involves inhibition of Mitogen and Stress activated protein kinase (MSK‐1). Treatment of CD4+ T cells with MSK‐1 inhibitor further confirm the role of MSK‐1 mediated phosphorylation in regulating IL‐2 gene expression. Importantly, ethanol inhibited MSK‐1 activation via PP1 activation and could be reversed by okadaic acid‐phosphatase inhibitor treatment of CD4+ T lymphocytes. Overall, these observations suggest that, ethanol‐induced MSK‐1 inhibition and resultant decrease in promoter histone phosphorylation constitute a significant pathogenic mechanism underpinning the loss of IL‐2 expression and subsequent CD4+ T cell function associated with alcohol‐induced immune suppression.Support or Funding InformationThis work was supported by NIH grants.

Loss of CD45 cell surface expression in canine T-zone lymphoma results from reduced gene expression

Canine T-zone lymphoma (TZL) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant CD45-negative phenotype, commonly recognized by flow cytometry (FC). Recent studies have described clinical presentation and behavior, but to date the mechanisms behind the loss of CD45 protein expression have never been investigated.The aims of this study were: 1) to confirm the absence of CD45 in canine TZL via the concomitant use of FC and immunohistochemistry with two different sources of antibody; and 2) to investigate the amount of CD45 transcript and the presence of CD45 gene in the neoplastic cells of dogs affected by TZL.57 lymph node aspirates were included in the present study: 40 (70.2%) TZLs, 7 (12.3%) high grade T-cell lymphomas and 10 (17.5%) reactive lymph nodes. Neoplastic cells and normal T-cells were isolated from TZL and reactive lymph nodes, respectively, via cell sorting. Immunohistochemistry was performed on 2 TZL, 2 reactive lymph nodes and 2 Peripheral T-cell Lymphomas. Total RNA and genomic DNA were extracted from lymph-nodes aspirates. Two different quantitative real-time PCR experiments were designed, to determine the amount of the CD45 transcript and of the corresponding gene fragment.All TZL cases were negative for CD45 at immunohistochemistry. CD45 transcript amount was significantly lower in TZL compared to controls (p<0.001). This difference was not significant (p=0.584) for CD45 DNA load, that was similar between TZL and controls. Moreover, CD45 transcript amount was inversely correlated with the percentage of neoplastic cells in each TZL sample (p=0.010).These results confirm that CD45 protein is lacking on cell surface irrespective of the technique and antibody source adopted. This phenotypic aberrancy is apparently due to the absence of gene transcription, as CD45 DNA was present, whereas CD45 transcript was virtually absent in the neoplastic cells. The data here reported support further studies investigating possible factors impairing CD45 gene transcription.

HTLV-1 Tax activates HIV-1 transcription in latency models

HIV-1 latency is a major obstacle to HIV-1 eradication. Coinfection with HTLV-1 has been associated with faster progression to AIDS. HTLV-1 encodes the transactivator Tax which can activate both HTLV-1 and HIV-1 transcription. Here, we demonstrate that Tax activates HIV transcription in latent CD4+ T cells. Tax promotes the activation of P-TEFb, releasing CDK9 and Cyclin T1 from inactive forms, promoting transcription elongation and reactivation of latent HIV-1. Tax mutants lacking interaction with the HIV-1-LTR promoter were not able to activate P-TEFb, with no subsequent activation of latent HIV. In HIV-infected primary resting CD4+ T cells, Tax-1 reactivated HIV-1 transcription up to five fold, confirming these findings in an ex vivo latency model. Finally, our results confirms that HTLV-1/Tax hijacks cellular partners, promoting HIV-1 transcription, and this interaction should be further investigated in HIV-1 latency studies in patients with HIV/HTLV-1 co-infection.

Abstract PR06: Overcoming intratumor T-cell exclusion by modulation of lactate metabolism to improve immune checkpoint therapies in aggressive breast cancer

Abstract Background: Breast cancer (BC) has historically been considered immunologically silent; however, several observations indicate that potentiation of immune functions can benefit BC patients. Intratumor T-cell infiltration has prognostic significance in patients with BC across different molecular and histological categories. In addition, PD-L1 can be overexpressed in BC, in particular in the highly aggressive triple negative BC (TNBC) subtype. This is associated with poor prognosis specifically in patients with luminal B and basal-like phenotypes, thus making these subtypes rational targets of PD-1/PD-L1 axis blockade treatments. However, initial results from early-phase clinical trials show a modest activity of immune checkpoint blockade monotherapy against BC, with 19% TNBC and 3-12% hormone receptor-positive patients achieving a clinical response to anti-PD-1/PD-L1 therapies. Lack of inflammation and T-cell infiltration at the tumor site are characteristic features of tumors that do not respond to checkpoint blockade. Since BC is typically poorly infiltrated by T cells, having strategies that reverse this immune exclusion as well as non-invasive modalities to predict this effect are crucial to improve the clinical efficacy of immune checkpoint blockade. Experimental Design: Our working model is that tumor glycolysis (lactate) and T-cell infiltration are mechanistically interdependent, since we reasoned that a highly glycolytic tumor microenvironment (due to lactate production) could hamper survival, expansion and effector functions of T cells in BC lesions, thus explaining T-cell exclusion. Therefore, we tested whether genetic and/or pharmacologic inhibition of lactate production/consumption could restore intratumor T-cell infiltration and immune function in BC models, thus favoring tumor responsiveness to checkpoint blockade. Results: By interrogating a compendium of 4 BC patient gene expression datasets, we found that patients harboring tumors with high expression of lactate dehydrogenase A (LDH-A) or the lactate transporters MCT-1/4 have a significantly higher risk to develop metastases (p&amp;lt;10−16), whereas those having tumors with increased expression of CD3 and CD8 transcripts experience a better prognosis (p&amp;lt;10−16). Gene expression data from MSKCC's cBio Portal showed an inverse correlation between glycolysis- and immune-related gene expression signatures in BC patients, which was in agreement with our hypothesis. To mechanistically demonstrate the impact of lactate metabolism on intratumor T-cell infiltration in BC, we generated an LDH-A-knocked-down variant (LDH-A KD) of the metastatic TNBC murine model 4T1, and studied its growth and immune infiltrate in vivo in comparison with the control 4T1 tumor. Animals implanted with LDH-A KD 4T1 showed a 5-fold increase in tumor-infiltrating CD3+ T cells and a 4-fold reduction in tumor-associated macrophages, and experienced a significantly prolonged survival. LDH-A KD tumors could be completely eradicated in immunocompetent but not in immunodeficient mice, further supporting the immunologic basis of the antitumor effects of LDH-A inhibition. In vitro analyses showed that proliferation, activation and pro-inflammatory cytokine release of activated CD8 T cells were significantly hampered when they were co-cultured with 4T1 cells. Of note, blocking lactate transport with two new MCT-1/4 small-molecule inhibitors promoted immune functions of CD8 T cells cultured with 4T1, without showing significant toxicity in cultures of T cells alone. Citation Format: Roberta Zappasodi, Arnab Ghosh, Inna Serganova, Ivan Cohen, Yasin Senbabaoglu, Masahiro Shindo, Mayuresh M. Mane, Avigdor Leftin, Ellen Ackerstaff, Jason A. Koutcher, Jedd D. Wolchok, Ronald G. Blasberg, Taha Merghoub. Overcoming intratumor T-cell exclusion by modulation of lactate metabolism to improve immune checkpoint therapies in aggressive breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR06.

β-1,3/1,6-Glucan-supplemented diets antagonize immune inhibitory effects of hypoxia and enhance the immune response to a model vaccine

The diets of farmed salmon are usually supplemented with immunostimulants to improve health status. Because β-glucan is one of the most common immunostimulants used in diets, here we examined the effect of two β-1,3/1,6-glucan-supplemented diets on the expression of immune response genes of Atlantic salmon. The relative abundances of IFN-α1, Mx, IFN-γ, IL-12, TGF-β1, IL-10, and CD4 transcripts were evaluated in head kidney by qRT-PCR. We assessed the effects of the diets under normoxia and acute hypoxia, as salmon are especially sensitive to changes in the concentration of dissolved oxygen, which can also affect immunity. These effects were also tested on vaccinated fish, as we expected that β-1,3/1,6-glucan-supplemented diets would enhance the adaptive immune response to the vaccine. We found that administration of the Bg diet (containing β-1,3/1,6-glucan) under normoxia had no effects on the expression of the analyzed genes in the kidney of the diet-fed fish, but under hypoxia/re-oxygenation (90 min of acute hypoxia), the βg diet affected the expression of the antiviral genes, IFN-α1 and Mx, preventing their decrease caused by hypoxia. The Bax diet, which in addition to β-1,3/1,6-glucan, contained astaxanthin, increased IL-12 and IFN-γ in kidneys. With fish exposed to hypoxia/reoxygenation, the diet prevented the decrease of IFN-α1 and Mx levels observed after hypoxia. When fish were vaccinated, only the levels of IL-12 and CD4 transcripts increased, but interestingly if fish were also fed the Bax diet, the vaccination induced a significant increase in all the analyzed transcripts. Finally, when vaccinated fish were exposed to hypoxia, the effect of the Bax diet was greatly reduced for all genes tested and moreover, inducible effects completely disappeared for IL-12, IFN-α, and Mx. Altogether, these results showed that the tested β-1,3/1,6-glucan diets increased the levels of transcripts of key genes involved in innate and adaptive immune response of salmon, potentiating the response to a model vaccine and also antagonizing the effects of hypoxia.

CD4+ T cells with an activated and exhausted phenotype distinguish immunodeficiency during aviremic HIV-2 infection.

Objective:HIV type 2 (HIV-2) represents an attenuated form of HIV, in which many infected individuals remain ‘aviremic’ without antiretroviral therapy. However, aviremic HIV-2 disease progression exists, and in the current study, we therefore aimed to examine if specific pathological characteristics of CD4+ T cells are linked to such outcome.Design:HIV-seronegative (n = 25), HIV type 1 (HIV-1) (n = 33), HIV-2 (n = 39, of whom 26 were aviremic), and HIV-1/2 dually (HIV-D) (n = 13)-infected study participants were enrolled from an occupational cohort in Guinea-Bissau.Methods:CD4+ T-cell differentiation, activation, exhaustion, senescence, and transcription factors were assessed by polychromatic flow cytometry. Multidimensional clustering bioinformatic tools were used to identify CD4+ T-cell subpopulations linked to infection type and disease stage.Results:HIV-2-infected individuals had early and late-differentiated CD4+ T-cell clusters with lower activation (CD38+HLA-DR+) and exhaustion programmed death-1 (PD-1) than HIV-1 and HIV-D-infected individuals. We also noted that aviremic HIV-2-infected individuals possessed fewer individuals. CD4+ T cells with pathological signs compared to other HIV-infected groups. Still, compared to HIV-seronegative individuals, aviremic HIV-2-infected individuals had T-bet+ CD4+ T cells that showed elevated immune activation/exhaustion, and particularly the frequencies of PD-1+ cells were associated with a suboptimal percentage of CD4+ T cells.Conclusion:Increased frequencies of CD4+ T cells with an activated/exhausted phenotype correlate with exacerbated immunodeficiency in aviremic HIV-2-infected individuals. Thus, these findings encourage studies on the introduction of antiretroviral therapy also to individuals with aviremic HIV-2 infection.

Alternation of circadian clock modulates forkhead box protein-3 gene transcription in CD4+ T cells in the intestine

Alternation of circadian clock modulates forkhead box protein-3 gene transcription in CD4+ T cells in the intestine

The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region. Pyrosequencing of the TIM-3 promoter up to -1440bp revealed 11 hypermethylated CpG sites and 4 hypomethylated CpG sites in human CD4(+) T cells as well as in CD11b(+) cells. Dimethylation of histone H3 lysine 4 (H3K4), a mark of transcriptional activation, was predominantly found in the proximal TIM-3 promoter -954 to -34bp region, whereas trimethylation of H3K9 and H3K27, which are markers of transcriptional suppression, were mostly observed in the distal promoter -1549 to -1048bp region in human CD4(+) T cells and CD11b(+) cells. However, no change in the methylation status of CpG sites and the histone H3 in the TIM-3 promoter was found during induction of TIM-3 transcription in T cells. Finally, AP-1 involvement in TIM-3 transcription was shown in relation with the TIM-3 minimal promoter -146 to +144bp region. The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells.

Toll-interacting protein inhibits HIV-1 infection and regulates viral latency

HIV-1 latency is mainly characterized by a reversible silencing of long-terminal repeat (LTR)-driven transcription of provirus. The existing of repressive factors has been described to contribute to transcription silencing of HIV-1. Toll-interacting protein (Tollip) has been identified as a repressor of Toll like receptors (TLR)-mediated signaling. Our previous study has found that Tollip inhibited NF-κB-dependent HIV-1 promoter LTR-driven transcription, indicating the potential role of Tollip in governing viral latency. In this study, by using HIV-1 latently infected Jurkat T-cell and central memory CD4+ T-cells, we demonstrate the role of Tollip in regulating HIV-1 latency, as the knock-down of Tollip promoted HIV-1 reactivation from both HIV-1 latently infected Jurkat CD4+ T cells and primary central memory T cells (TCM). Moreover, we found that the activities of LTRs derived from multiple HIV-1 subtypes could be repressed by Tollip; Knock-down of Tollip promoted HIV-1 transcription and infection in CD4+ T cells. Our data indicate a key role of Tollip in suppressing HIV-1 infection and regulating viral latency, which provides a potential host target for combating HIV-1 infection and latency.

A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+ T Cells.

Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a "shock-and-kill" approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56(dim)CD16(+)NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4(+)and CD8(+)T cells. Furthermore, CD4(+)T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4(+)T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4(+)T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel "dumbbell-shape" structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4(+)T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4(+)T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.

Selective VIP Receptor Agonists Facilitate Immune Transformation for Dopaminergic Neuroprotection in MPTP-Intoxicated Mice.

Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating microglial activation and by slowing degradation of neuronal cell bodies and termini in MPTP-intoxicated mice. The protective mechanism arises from altering a Th1/Th2 immune cytokine response into an anti-inflammatory and neuronal sparing profile. These results are directly applicable for the development of novel PD therapies.

Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.