Investigating the Role of the Mammalian RAS-Association Domain Family Member 7 (RASSF7)

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Hyperglycaemia does not affect antigen-specific activation and cytolytic killing by CD8+ T cells in vivo

Metabolism is of central importance for T cell survival and differentiation. It is well known that T cells cannot function in the absence of glucose, but it is less clear how they respond to excessive levels of glucose. In the present study, we investigated how increasing levels of glucose affect T-cell-mediated immune responses. We examined the effects of increased levels of glucose on CD8+ T-cell behaviour in vitro by assessing activation and cytokine production, as well as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and intracellular signalling. In addition, we assessed in vivo proliferation, cytokine production and cytolytic activity of cells in chemically induced diabetic C57BL/6 mice. Elevated levels of glucose in in vitro cultures had modest effects on proliferation and cytokine production, while in vivo hyperglycaemia had no effect on CD8+ T-cell proliferation, interferon γ (IFNγ) production or cytolytic killing

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells

Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production.

Type 1 diabetes is caused by the destruction of insulin producing beta cells by the immune system. The p110δ isoform of PI3K is expressed primarily in cells of haematopoietic origin and the catalytic activity of p110δ is important for the activation of these cells. Targeting of this pathway offers an opportunity to reduce immune cell activity without unwanted side effects. We have explored the effects of a specific p110δ isoform inhibitor, IC87114, on diabetogenic T cells both in vitro and in vivo, and find that although pharmacological inhibition of p110δ has a considerable impact on the production of pro-inflammatory cytokines, it does not delay the onset of diabetes after adoptive transfer of diabetogenic cells. Further, we demonstrate that combination treatment with CTLA4-Ig does not improve the efficacy of treatment, but instead attenuates the protective effects seen with CTLA4-Ig treatment alone. Our results suggest that decreased IL-10 production by Foxp3+ CD4+ T cells in the presence of IC87114 negates individual anti-inflammatory effects of IC8114 and CTLA4-Ig.This work was supported by Grant number 09/0003840, Diabetes UK https://www.diabetes.org.uk/ (MW); Grant number 5-2006-229, Juvenile Diabetes Research Foundation https://www.jdrf.org.uk/ (KO); Grant number BBS/E/B/0000C236 Biotechnology and Biological Sciences Research Council (KO); Grant number health-f5-2009-241883 European Research Council 7th Frame Programme http://ec.europa.eu/research/fp7/index_en.cfm (AC); and Grant number 02BX12ACYD, the Britain Israel Research and Academic Exchange Partnership (BIRAX) http://www.britishcouncil.org.il/en/programmes/science/birax.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.014651

Promoter optimisation of lentiviral vectors for efficient insulin gene expression in canine mesenchymal stromal cells: potential surrogate beta cells.

BACKGROUND: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re-implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy. In the present study, we optimised insulin production using lentiviral transduced canine MSCs (cMSCs), aiming to evaluate their ability for use as surrogate beta cells. METHODS: Canine MSCs were derived from bone marrow and validated by measuring the expression of MSC lineage specific markers. Lentivirus vectors encoding the proinsulin gene (with or without a Kozak sequence) under the control of spleen focus forming virus, cytomegalovirus, elongation factor 1α and simian virus 40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. The insulin-producing capacity of transduced primary cMSCs was assessed by measuring the concentration of C-peptide produced. RESULTS: Primary cMSC could be readily expanded in culture and efficiently transduced using lentiviral vectors encoding proinsulin. Increasing the multiplicity of infection from 3 to 20 led to an increase in C-peptide secretion (from 1700 to 4000 pmol/l). The spleen focus forming virus promoter conferred the strongest transcriptional ability. CONCLUSIONS: The results of the present study suggest that optimised lentiviral transduction of the insulin gene into primary cMSCs renders these cells capable of secreting insulin over both the short- and long-term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.The study was funded by the Lollipop Trust. Work in the laboratory is supported by the Biomedical Research Centre.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/jgm.290

Research data supporting publication - title to be confirmed.

Microarray analysis of sorted, previously transferred GFP+ Th1 cells from NOD mice treated with either control antibody (NT) or aglycosyl anti-CD3 (T).This work was supported by the ERC and NC3R [grant numbers NC/M001083/1 and health-f5-2009-241883]

Administration of IC87114 does not prevent diabetes after adoptive transfer of naïve diabetogenic cells.

<p>CD4⁺ T cells were isolated by cell sorting from spleen and lymph nodes of BDC2.5 TCR transgenic mice. 1x10⁶ CD4⁺ T cells were transferred i.p. route to NOD-<i>scid</i> mice. Mice received IC87114 treatment (30mg/Kg) by oral gavage (2x/day) from day 0 to 10 (A). Positive controls are mice that received CD4⁺ T cells from BDC2.5 mice without any other treatment. After this time, blood glucose levels were checked every day, and mice were considered irrevocably diabetic and sacrificed when they reached two consecutive blood glucose levels >13mM (B).</p