Project description:Analysis perfomed on four paired samples with two treatments and paired untreated sample Overall design: We purified naïve CD45RA+CD4+ T cells from PBMCs collected from SLE patients. These cells were maintained for two days in culture medium supplementd with 20 IU of IL-2. Thereafter cells were harvetd and total RNA was purified using the kit from InVitroen. The sequencing was perfomed using Ion Torrent Kit. The RNA-seq was perfomed on Ion Torrent Sequncer.
Project description:RNA sequencing of the chromatin associated RNA and nucleoplasm associated RNA of Naive CD4+ T cells to identify novel chromatin associated RNAs containing TEs. RNA sequencing of Naive CD4+ T cells or Activated Naive CD4+ T cells treated with Scr or LINE1 antisense oligonucleotides (ASO).
Project description:Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus-cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high-potential-energy viral entry machines that are normally activated by receptor binding.
Project description:In addition to progressive CD4(+) T cell immune deficiency, HIV infection is characterized by generalized immune activation, thought to arise from increased microbial exposure resulting from diminishing immunity.Here we report that, in a virus-free mouse model, conditional ablation of activated CD4(+) T cells, the targets of immunodeficiency viruses, accelerates their turnover and produces CD4(+) T cell immune deficiency. More importantly, activated CD4(+) T cell killing also results in generalized immune activation, which is attributable to regulatory CD4(+) T cell insufficiency and preventable by regulatory CD4(+) T cell reconstitution. Immune activation in this model develops independently of microbial exposure. Furthermore, microbial translocation in mice with conditional disruption of intestinal epithelial integrity affects myeloid but not T cell homeostasis.Although neither ablation of activated CD4(+) T cells nor disruption of intestinal epithelial integrity in mice fully reproduces every aspect of HIV-associated immune dysfunction in humans, ablation of activated CD4(+) T cells, but not disruption of intestinal epithelial integrity, approximates the two key immune alterations in HIV infection: CD4(+) T cell immune deficiency and generalized immune activation. We therefore propose activated CD4(+) T cell killing as a common etiology for both immune deficiency and activation in HIV infection.
Project description:Previous studies in murine systems have demonstrated that CD8(+) Treg cells down-regulate immune responses in vivo through suppressing activated CD4(+) T cells. Here we describe novel regulatory CD8(+) T-cell clones isolated from healthy human peripheral blood following in vitro stimulation with autologous Epstein-Barr virus (EBV)-specific CD4(+) T cells. TCR activation of CD4(+) target T cells was required for CD8(+) Treg cells to exert suppressive activity, which was mediated through lysis of CD4(+) targets in a cell contact-dependent manner. Suppression was independent of Foxp3 expression in CD8(+) Treg cells, HLA compatibility between CD8(+) Treg cells and CD4(+) target cells and antigen-specificity of CD4(+) target T cells. CD8(+) Treg clones expressed CD3 and a variety of TCR V(?) chains as well as CD56, CD69, CD62L and CD95 but did not express CD16, CD161, CXCR4 and CCR7. When used together, antibodies specific for CD11a/CD18 and CD8 inhibited suppressive activity of CD8(+) Treg clones. The ability to establish clonal CD8(+) T cells that maintain regulatory function in vitro will facilitate further studies to define this population in vivo and to identify the mechanisms used for recognition and suppression of activated target cells.
Project description:Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1?. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1? production induced by activated memory T-cells concealed this effect. Priming with IFN? decreased pro-IL-1? production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFN? suppresses NLRP3 inflammasome activation through an indirect mechanism involving decreased P2X7R signaling. The inhibition of pro-IL-1? production and suppression of NLRP3 inflammasome activation by IFN?-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFN?. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFN? in MS.
Project description:Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4+ T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis.
Project description:We use next generation sequencing to investigate the different transcriptomes of closely related CD4+ T-cells from healthy human donors to elucidate the genetic programs that underlie their specialized immune functions. Six cell types were included: Regulatory T-cells (CD25hiCD127low/neg with >95% FOXP3+ purity), regulatory T-cells activated using PMA/ionomycin, CD25-CD45RA+ ('naive' helper T-cells), CD25-CD45RO+ ('memory' helper T-cells), activated Th17 cells (>98% IL17A+ purity) and activated IL17-CD4+ T-cells (called 'ThPI'). Poly-T capture beads were used to isolate mRNA from total RNA, and fragment sizes of ~200 were sequenced from both ends on Illumina's genome analyzer. We confirm many of the canonical signature genes of T-cell populations, but also discover new genes whose expression is limited to specific CD4 T-cell lineages, including long non-coding RNAs. Additionally, we find that genes encoded at loci linked to multiple human autoimmune diseases... (for more see dbGaP study page.)
Project description:Galectin-9 (Gal-9) is a tandem repeat-type member of the galectin family and is a ligand for T-cell immunoglobulin mucin domain 3 (Tim-3), a type-I glycoprotein that is persistently expressed on dysfunctional T cells during chronic infection. Studies in autoimmune diseases and chronic viral infections show that Tim-3 is a regulatory molecule that inhibits Th1 type immune responses. Here we show that soluble Gal-9 interacts with Tim-3 expressed on the surface of activated CD4(+) T cells and renders them less susceptible to HIV-1 infection and replication. The Gal-9/Tim-3 interaction on activated CD4(+) T cells, leads to down-regulation of HIV-1 coreceptors and up-regulation of the cyclin-dependent kinase inhibitor p21 (also known as cip-1 and waf-1). We suggest that higher expression of Tim-3 during chronic infection has evolved to limit persistent immune activation and associated tissue damage. These data demonstrate a novel mechanism for Gal-9/Tim-3 interactions to induce resistance of activated CD4(+) T cells to HIV-1 infection and suggest that Gal-9 may play a role in HIV-1 pathogenesis and could be used as a novel microbicide to prevent HIV-1 infection.