Neuroprotective Effects of Ex Vivo Expanded Regulatory T Cells on Trimethyltin Induced Neurodegeneration in Mice

Abstract BackgroundTrimethyltin (TMT) is a potent neurotoxicant that leads to hippocampal neurodegeneration. Regulatory T cells (Tregs) play an important role in maintaining the immune balance in the central nervous system (CNS), but their activities are impaired in neurodegenerative diseases. In this study, we aimed to determine whether adoptive transfer of Tregs, as a living drug, ameliorates hippocampal neurodegeneration in TMT-intoxicated mice.MethodsCD4+CD25+ Tregs were expanded in vitro and adoptively transferred to TMT-treated mice. First, we explored the effects of Tregs on behavioral deficits using the Morris water maze and elevated plus maze tests. Biomarkers related to memory formation, such as cAMP response element-binding protein (CREB), protein kinase C (PKC), neuronal nuclear protein (NeuN), nerve growth factor (NGF), and ionized calcium binding adaptor molecule 1 (Iba1) in the hippocampus were examined by immunohistochemistry after mouse sacrifice. To investigate the neuroinflammatory responses, the polarization status of microglia was examined in vivo and in vitro using real-time reverse transcription polymerase chain reaction (rtPCR) and Enzyme-linked immunosorbent assy (ELISA). Additionally, the inhibitory effects of Tregs on TMT-induced microglial activation were examined using time-lapse live imaging in vitro with an activation-specific fluorescence probe, CDr20.ResultsAdoptive transfer of Tregs improved spatial learning and memory functions and reduced anxiety in TMT-intoxicated mice. Additionally, adoptive transfer of Tregs reduced neuronal loss and recovered the expression of neurogenesis enhancing molecules in the hippocampi of TMT-intoxicated mice. In particular, Tregs inhibited microglial activation and pro-inflammatory cytokine release in the hippocampi of TMT-intoxicated mice. The inhibitory effects of TMT were also confirmed via in vitro live time lapse imaging in a Treg/microglia co-culture system.ConclusionsThese data suggest that adoptive transfer of Tregs ameliorates disease progression in TMT-induced neurodegeneration by promoting neurogenesis and modulating microglial activation and polarization.

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IL-9 Production by Regulatory T Cells Recruits Mast Cells That Are Essential for Regulatory T Cell-Induced Immune-Suppression

Blood ◽

10.1182/blood.v116.21.2782.2782 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2782-2782

Author(s):

Anna Maria Wolf ◽

Dominik Wolf ◽

Andrew McKenzie ◽

Marcus Maurer ◽

Alexander R Rosenkranz ◽

...

Keyword(s):

T Cells ◽

Mast Cells ◽

Regulatory T Cells ◽

Adoptive Transfer ◽

Conflicts Of Interest ◽

Cell Populations ◽

Perfect Agreement ◽

Immunosuppressive Cell

Abstract Abstract 2782 Tipping the balance between effector and regulatory cell populations is of critical importance in the pathogenesis of various autoimmune disorders. Both, mast cells (MC) and regulatory T cells (Treg) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MC and Treg have been shown to play a protective role. We recently provided evidence that adoptive transfer of wild-type (wt) Treg into wt recipients almost completely prevents development of NTS. We here show that Treg transfer induces a profound increase of MC in the kidney draining lymph nodes (LN). In contrast, transfer of wt Treg into animals deficient in MC, which are characterized by an exaggerated susceptibility to NTS, do not prevent acute renal inflammation. Blocking the pleiotropic cytokine IL-9, which is known to be critically involved in MC recruitment and proliferation, by means of an antagonizing monoclonal antibody in animals receiving wt Treg abrogated protection from NTS. Moreover, we provide clear evidence that Treg-derived IL-9 is critical for MC recruitment as mediators of their full immune-suppressive potential, as adoptive transfer of IL-9 deficient Treg failed to protect from NTS. In line with our hypothesis, absence of Treg-derived IL-9 does not induce MC accumulation into kidney-draining LN, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Treg, as shown by in vitro testing of their functional capacities. Finally, we observed a significantly decreased expression of the MC chemoattractant Cxcl-1 in the LN of mice receiving IL-9 deficient Treg as compared to mice receiving wt Treg or control CD4+CD25− T cells, which might at least in part explain the deficient MC recruitment under these conditions. In summary, our data provide the first evidence that the immunosuppressive effects of adoptively transferred Treg depend on IL-9-mediated recruitment of MC to the kidney draining LN in NTS. This data is in perfect agreement with our previous report showing that CCR7-mediated LN occupancy of Treg is a prerequisite for their immune-suppressive potential and furthers adds a piece of information to the functional understanding of the in vivo anti-inflammatory effects of Treg. Disclosures: No relevant conflicts of interest to declare.

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Generation of Functional Regulatory T Cells By FOXP3 Gene Transfer into CD4 T Cells from Scurfy Mice and IPEX Patients

Blood ◽

10.1182/blood.v128.22.2526.2526 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2526-2526 ◽

Cited By ~ 2

Author(s):

Marianne Delville ◽

Emmanuelle Six ◽

Florence Bellier ◽

Nelly Sigrist ◽

David Zemmour ◽

...

Keyword(s):

T Cells ◽

Gene Transfer ◽

Regulatory T Cells ◽

Immune Tolerance ◽

Adoptive Transfer ◽

Cd4 T Cells ◽

Hematopoietic Stem ◽

Foxp3 Gene ◽

Human Pathology

Abstract IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that a gene therapy approach designed to selectively induce a Treg program in T cells by expressing FOXP3 could be a promising potential cure for IPEX. However, several issues might compromise the success of this strategy: (i) will the introduction of FOXP3 alone be sufficient to induce a stable Treg program or will it require additional transcription factors to lock the Treg function and sustain the stability of transduced cells? (ii) Targeting effector CD4+ T cells might be an issue in terms of T-cell receptor repertoire, since the TCR repertoire of nTregs is different from the one of effector CD4+ T cells, (iii) will FOXP3-transduced T cells be able to migrate to appropriate tissues to control auto-immune reactions?, (iv) infusion of nTreg prevents the appearance of some autoimmune manifestations in murine models, however the infusion was done in prophylaxis before the appearance of the symptoms. In order to address these questions, we have developed a mouse scurfy model to evaluate the functional and stability of the correction in vivo in parallel to the characterization of gene corrected human CD4 T cells from IPEX patients. Scurfy mice develop a disease very close to human pathology due to a spontaneous mutation of Foxp3 gene. We improved Scurfy mice model to improve animal production and increase the timeline of treatement. We demonstrated that FOXP3 gene transfer into murine CD4+ T cells enable the generation of potent regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. The ability of CD4-FOXP3 to prevent Scurfy disease by adoptive transfer in the first days of life is currently under evaluation. Similarly in humans, we demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent regulatory T cells, as shown through the functional in vitro suppressive properties of the generated CD4IPEX-FOXP3. Moreover comparison of the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells to natural Treg by RNA-seq analysis demonstrated a good repression of cytokine transcripts (IL4/5/13/CSF2, CD40L), a strong repression of IL7R, a strong induction of IL1R2, and a moderate activation of typical Treg genes (IL2RA, IKZF2, CTLA4). Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression. Disclosures No relevant conflicts of interest to declare.

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Activated regulatory T-cells attenuate myocardial ischaemia/reperfusion injury through a CD39-dependent mechanism

Clinical Science ◽

10.1042/cs20140672 ◽

2015 ◽

Vol 128 (10) ◽

pp. 679-693 ◽

Cited By ~ 22

Author(s):

Ni Xia ◽

Jiao Jiao ◽

Ting-Ting Tang ◽

Bing-Jie Lv ◽

Yu-Zhi Lu ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Reperfusion Injury ◽

Myocardial Ischaemia ◽

Adoptive Transfer ◽

Ischaemia Reperfusion Injury ◽

Ischaemia Reperfusion ◽

Treg Population ◽

Dependent Mechanism

Regulatory T-cells (Tregs) are generally regarded as key immunomodulators that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. However, its role in myocardial ischaemia/reperfusion injury (MIRI) remains unknown. The purpose of the present study was to determine whether Tregs exert a beneficial effect on mouse MIRI. We examined the role of Tregs in murine MIRI by depletion using ‘depletion of regulatory T-cell’ (DEREG) mice and adoptive transfer using Forkhead box P3 (Foxp3)–GFP knockin mice and the mechanisms of cardio protection were further studied in vivo and in vitro. Tregs rapidly accumulated in murine hearts following MIRI. Selective depletion of Tregs in the DEREG mouse model resulted in aggravated MIRI. In contrast, the adoptive transfer of in vitro-activated Tregs suppressed MIRI, whereas freshly isolated Tregs had no effect. Mechanistically, activated Treg-mediated protection against MIRI was not abrogated by interleukin (IL)-10 or transforming growth factor (TGF)-β1 inhibition but was impaired by the genetic deletion of cluster of differentiation 39 (CD39). Moreover, adoptive transfer of in vitro-activated Tregs attenuated cardiomyocyte apoptosis, activated a pro-survival pathway involving Akt and extracellular-signal-regulated kinase (ERK) and inhibited neutrophil infiltration, which was compromised by CD39 deficiency. Finally, the peripheral blood mononuclear cells of acute myocardial infarction (AMI) patients after primary percutaneous coronary intervention (PCI) revealed a decrease in CD4+CD25+CD127low Tregs and a relative increase in CD39+ cells within the Treg population. In conclusion, our data validated a protective role for Tregs in MIRI. Moreover, in vitro-activated Tregs ameliorated MIRI via a CD39-dependent mechanism, representing a putative therapeutic strategy.

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