Renal graft function in transplanted patients correlates with CD45RC T cell phenotypic signature

International audienceBiomarkers that could predict the evolution of the graft in transplanted patients and that could allow to adapt the care of the patients would be an invaluable tool. Additionally, certain biomarkers can be target of treatments and help to stratify patients. Potential effective biomarkers have been identified but still need to be confirmed. CD45RC, one of the splicing variants of the CD45 molecule, a tyrosine phosphatase that is critical in negatively or positively regulating the TCR and the BCR signaling, is one marker already described. The frequency of CD8 + T cells expressing high levels of CD45RC before transplantation is increased in patients with an increased risk of acute rejection. However, single biomarkers have limited predictive reliability and the correlation of the expression levels of CD45RC with other cell markers was not reported. In this study, we performed a fluorescent-based high dimensional immunophenotyping of T cells on a cohort of 69 kidney transplant patients either with stable graft function or having experienced acute transplant rejection during the first year after transplantation or at the time of rejection. We identified combinations of markers and cell subsets associated with activation/inflammation or Tregs/tolerance (HLA-DR, PD-1, IFNγ, CD28) as significant biomarkers associated to transplant outcome, and showed the importance of cell segregation based on the CD45RC marker to identify the signature of a stable graft function. Our study highlights potential reliable biomarkers in transplantation to predict and/or monitor easily graft-directed immune responses and adapt immunosuppression treatments to mitigate adverse effects

Optineurin Regulates the Interferon Response in a Cell Cycle-Dependent Manner

International audienceViral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of inter-feron regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phos-phorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphoryla-tion of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production , suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis. The innate immune system has evolved to detect and neutralize viral invasion. Triggering of this defense mechanism relies on the production and secretion of soluble factors that stimulate an intracellular antiviral defense mechanism. The protein Optineurin was shown to negatively regulate this process. Importantly, we discovered the mechanism by which Optineurin inhibits antiviral activity and showed that this regulation is prevented during a critical step of cell division leading to enhancement of the cellular defense mechanism. This paper shows that the antiviral immune system is controlled during the cell cycle and that Optineurin-mediated induction of this system might serve to protect cells from infection during cell division

Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells

International audienceRat and human CD4(+) and CD8(+) Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4(+) and CD8(+) Foxp3(+) Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC(high) T cells through intrinsic cell signaling but preserved and potentiated CD4(+) and CD8(+) CD45RC(low/-) Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4(+) and CD8(+) CD45RC(low/-) Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human

Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/− Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice

Both CD4+ and CD8+ Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8+ Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8+CD45RClow/− Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8+CD45RClow/− Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8+ Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8+CD45RClow/− Tregs are equivalent to canonical CD4+CD25highCD127low/− Tregs for suppression of allogeneic immune responses in vitro. Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD

NKT cell-plasmacytoid dendritic cell cooperation via OX40 controls viral infection in a tissue-specific manner.

International audienceInvariant natural killer T (iNKT) cells promote immune responses to various pathogens, but exactly how iNKT cells control antiviral responses is unclear. Here, we showed that iNKT cells induced tissue-specific antiviral effects in mice infected by lymphocytic choriomeningitis virus (LCMV). Indeed, iNKT cells inhibited viral replication in the pancreas and liver but not in the spleen. In the pancreas, iNKT cells expressed the OX40 molecule and promoted type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) through OX40-OX40 ligand interaction. Subsequently, this iNKT cell-pDC cooperation attenuated the antiviral adaptive immune response in the pancreas but not in the spleen. The dampening of pancreatic anti-LCMV CD8(+) T cell response prevented tissue damage in transgenic mice expressing LCMV protein in islet beta cells. Thus, this study identifies pDCs as an essential partner of iNKT cells for mounting an efficient, nondeleterious antiviral response in peripheral tissue

Expanded human CD8+CD45RClowFoxp3+ Tregs secreting IFNγ, IL-10, IL-34 and TGFβinhibit human transplant rejection

International audienceTregs play a critical role in the control of immune responses and tolerance induction; however our understanding of CD8+ Tregs is limited while they are particularly promising for therapeutic application. We previously reported that CD8+CD45RC[low] Tregs use IFNγ and IL-34 to suppress donor responses in a rat model of allotransplantation (Guillonneau et al, JCI, 2007 ; Bezieetal, JCI, 2015). Here, we aim to characterize human CD8+ Tregs to assess their potential for cell therapy in transplantation in comparison to CD4+CD25[high]CD127- Tregs

Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/\textminus Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice

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Optn negative effect on the virus-induced interferon pathway requires its phosphorylation and ubiquitin-binding activity.

<p>(A) Phosphorylation status of Optn (on S177) and IRF3 (on S396) were determined by Western blot in uninfected HeLa cells and at different time after infection by Sendai virus. (B) Total cell lysates from HEK293T infected for 6h with SeV in the presence or absence of the TBK1-specific inhibitor BX795 were analyzed by Western blot using anti-pS177 Optn, anti-Optn, anti-pS396 IRF3, anti-IRF3 and anti-tubulin antibodies. (C) Total cell lysates extracted from HEK293T cells expressing either VSV-Optn (Optn, left panels) or VSV-Optn S162-170-171-173-174-177A (Optn-S₆A, right panels) were transfected with empty vector (-) or with TBK1-expressing plasmid (TBK1) and subjected to two-dimensional gel electrophoresis. Optn isoforms were analyzed by immunoblotting with an anti-VSV antiserum. As control, lysates were pre-treated with lambda phosphatase (λ-Phos.) at 30°C. (D) HEK293T cells were transiently transfected with Flag-TBK1. TBK1 kinase assays were carried out using Flag immunoprecipitates as enzyme and GST-Optn wt, GST-Optn S177A, GST-Optn S171-173A or GST-Optn S171-173-177A as substrates. (E) IFN-B mRNA levels were determined by RT-QPCR in control HeLa cells, Optn-deficient cells and deficient cells reconstituted with wt, D474N- or S177A-mutated forms of Optn following infection by SeV or stimulation with dsRNA (pIC) or dsDNA (poly(dA):(dT)) and presented as mRNA transcripts levels related to the mRNA of 18S (set at 100). Mean ± SD values of expression levels are shown.</p