scholarly journals c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells

2009 ◽  
Vol 206 (13) ◽  
pp. 3001-3014 ◽  
Author(s):  
Iwao Isomura ◽  
Stephanie Palmer ◽  
Raelene J. Grumont ◽  
Karen Bunting ◽  
Gerard Hoyne ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Intrinsic Defect ◽  
Thymocyte Development ◽  
Thymic Development ◽  
Homeostatic Expansion ◽  
Factor C

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-κB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel−/− mice, thymic T reg cell numbers are markedly reduced as a result of a T cell–intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-β conversion of peripheral CD4+CD25− T cells into CD4+Foxp3+ cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel−/− mice, the residual peripheral c-rel−/− T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

scholarly journals Identification of a Late Stage of Small Noncycling pTα−  Pre-T Cells as Immediate Precursors of T Cell Receptor α/β+  Thymocytes

1998 ◽  
Vol 188 (8) ◽  
pp. 1401-1412 ◽  
Author(s):  
César Trigueros ◽  
Almudena R. Ramiro ◽  
Yolanda R. Carrasco ◽  
Virginia G. de Yebenes ◽  
Juan P. Albar ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Developmental Model ◽  
Developmental Expression ◽  
Thymocyte Development ◽  
Cell Stage

During thymocyte development, progression from T cell receptor (TCR)β to TCRα rearrangement is mediated by a CD3-associated pre-TCR composed of the TCRβ chain paired with pre-TCRα (pTα). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here, we show that developmental expression of pTα is time- and stage-specific, and is confined in vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3. This restricted expression pattern allowed the identification of a novel subset of small CD3− thymocytes lacking surface pTα, but expressing cytoplasmic TCRβ, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation of T early α transcription are coincident events associated with cell cycle arrest, and immediately preceding TCRα gene expression. Importantly, thymocytes at this late pre-T cell stage are shown to be functional intermediates between large pTα+ pre-T cells and TCRα/β+ thymocytes. The results support a developmental model in which pre-TCR–expressing pre-T cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small resting pre-T cells, before the onset of TCRα gene expression.

scholarly journals CD4+ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance

2009 ◽  
Vol 206 (2) ◽  
pp. 421-434 ◽  
Author(s):  
Randall H. Friedline ◽  
David S. Brown ◽  
Hai Nguyen ◽  
Hardy Kornfeld ◽  
JinHee Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Cytotoxic T Lymphocyte ◽  
Critical Role ◽  
Lymphoproliferative Disease ◽  
In Trans ◽  
And Function

Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3+ regulatory T cells. CTLA-4–deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4–deficient and –sufficient bone marrow (BM)–derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4−/− T cells in trans by CTLA-4–sufficient T cells is a reversible process that requires the persistent presence of FOXP3+ regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3+ regulatory T cell function in vivo.

scholarly journals T-cell lineage commitment and cytokine responses of thymic progenitors

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1850-1860 ◽  
Author(s):  
TA Moore ◽  
A Zlotnik
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cultured Cells ◽  
Critical Role ◽  
Cell Lineage ◽  
Lineage Commitment ◽  
Organ Cultures ◽  
Cell Commitment

The earliest steps of intrathymic differentiation recently have been elucidated. It has been reported that both CD4lo (CD44+ CD25- c-kit+ CD3- CD4lo CD8-) and pro-T cells (CD44+ CD25+ c-kit+ CD3- CD4- CD8-, representing the next step in maturation) exhibit germline T-cell receptor beta and gamma loci, suggesting that neither population is exclusively committed to the T-cell lineage. Several groups have shown that CD4lo cells retain the capacity to generate multiple lymphoid lineages in vivo; however, the lineage commitment status of pro-T cells is unknown. To determine when T-cell lineage commitment occurs, we examined the ability of sorted CD4lo and pro-T cells to generate lymphoid lineage cells in vivo or in fetal thymic organ cultures (FTOCs). When intravenously injected into scid mice, CD4lo cells generated both T and B cells, whereas the progeny of pro-T cells contained T cells exclusively. Fetal thymic organ cultures repopulated with CD4lo cells contained both T and natural killer (NK) cells, whereas cultures repopulated with pro-T cells contained T cells almost exclusively. These observations strongly suggest that T-cell lineage commitment occurs during the transition of CD4lo to pro-T cells. Because it is likely that the thymic microenvironment plays a critical role in T-cell commitment, we compared the responses of CD4lo and pro-T cells to various cytokine combinations in vitro, as well as the ability of the cultured cells to repopulate organ cultures. Cytokine combinations that maintained T-cell repopulation potential for both CD4lo and pro-T cells were found. CD4lo cells proliferated best in response to the combination containing interleukin-1 (IL-1), IL-3, IL- 6, IL-7, and stem cell factor (SCF). Unlike CD4lo cells, pro-T cells were much more dependent upon IL-7 for proliferation and FTOC repopulation. However, combinations of cytokines lacking IL-7 were found that maintained the T-cell repopulating potential of pro-T cells, suggesting that, whereas this cytokine is clearly very important for normal pro-T cell function, it is not an absolute necessity during early T-cell expansion and differentiation.

scholarly journals TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

scholarly journals Conditional Knockout Mice Reveal Distinct Functions for the Global Transcriptional Coactivators CBP and p300 in T-Cell Development

2006 ◽  
Vol 26 (3) ◽  
pp. 789-809 ◽  
Author(s):  
Lawryn H. Kasper ◽  
Tomofusa Fukuyama ◽  
Michelle A. Biesen ◽  
Fayçal Boussouar ◽  
Caili Tong ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Conditional Knockout ◽  
Specific Cell ◽  
Creb Binding Protein ◽  
Thymocyte Development ◽  
Transcriptional Coactivators

ABSTRACT The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300 flox ) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300 flox and a CBP conditional knockout allele (CBP flox ) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4− CD8− double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.

scholarly journals NKAP is required for T cell maturation and acquisition of functional competency

2011 ◽  
Vol 208 (6) ◽  
pp. 1291-1304 ◽  
Author(s):  
Fan-Chi Hsu ◽  
Anthony G. Pajerowski ◽  
Molly Nelson-Holte ◽  
Rhianna Sundsbak ◽  
Virginia Smith Shapiro
Keyword(s):  
T Cells ◽  
T Cell ◽  
Conditional Knockout ◽  
Cell Maturation ◽  
Intrinsic Defect ◽  
Thymic Development ◽  
Cell Pool ◽  
Single Positive ◽  
Maturation Defect ◽  
T Cell Maturation

Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.

scholarly journals The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1134
Author(s):  
Won-Ju Kim ◽  
Gil-Ran Kim ◽  
Hyun-Jung Cho ◽  
Je-Min Choi
Keyword(s):  
Drug Delivery ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Autoimmune Encephalomyelitis ◽  
Optimal Sequence ◽  
T Cell Function

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 588-588
Author(s):  
Karrune Woan ◽  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Jennifer Rock-Klotz ◽  
Zi Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Egfp Expression ◽  
In Vivo Models

Abstract Abstract 588 We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100. 2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures: No relevant conflicts of interest to declare.

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 976-976 ◽  
Author(s):  
John C. Riches ◽  
Ajanthah Sangaralingam ◽  
Shahryar Kiaii ◽  
Tracy Chaplin ◽  
Demet Cekdemir ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Family Member ◽  
Cell Function ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Healthy Donors

Abstract Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Expanded CD4+Foxp3+ Regulatory T Cells through DR3 Signaling Have a Distinct Immunophenotype and Abrogate the Lethal Acute-Graft and Host Disease after Allogeneic Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1876-1876
Author(s):  
Hidekazu Nishikii ◽  
Byung-Su Kim ◽  
Yasuhisa Yokoyama ◽  
Jeanette Baker ◽  
Antonio Pierini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Proliferation ◽  
Agonistic Antibody ◽  
Host Disease ◽  
Acute Graft

Abstract Background : CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain self-tolerance and enhance immune tolerance after transplantation. Several groups have demonstrated that donor-derived Treg prevent the development of lethal acute graft and host disease (GVHD) in murine allogeneic transplant models. However, the low frequency of Treg limits clinical translation. To overcome the paucity of Treg, several strategies have been developed for Treg expansion. However, the activation of other immune cells and the instability of Foxp3 expression in ex vivo culture are problematic for widescale clinical usage. Recently, we showed that a single dose of agonistic antibody to DR3 (Death receptor 3, also called tumor necrosis factor super family 25; TNFSF25) into donor mice resulted in the expansion of donor derived Treg and prevented acute GVHD (Blood. 2015). Although the treatment with DR3 antibodies can preferentially expand Treg in vivo, the precise role of DR3 signaling in Treg has not been fully elucidated. In this study, we investigated the immune phenotype, gene expression profiles, and function of Treg after activation with DR3 signaling. Methods: To analyze the heterogeneous immunophenotype of Treg after DR3 signal activation, we comprehensively analyzed multicolor cytometry data using viSNE (visualization of stochastic neighbor embedding algorithm). For gene expression analysis using microarray (Affymetrix GeneChip 2.0 ST Array), CD4+Foxp3+ cells from Foxp3-GFP mice with or without DR3 activation were sorted by FACS. Normalized expression data was analyzed using TIGR Multi Experiment Viewer (MeV, version 4.9). To investigate the function of Treg after DR3 activation, CD4+CD25+Treg from wild type (WT) C57BL/6 mice (H2kb) with or without treatment of agonistic antibody to DR3 were isolated by FACS and then injected into lethally irradiated (8Gy in total) BALB/c mice (H2kd) together with 5x106 T cell depleted bone marrow (from WT C57BL/6 mice) and 1x106 T cells (C57BL/6-luciferase mice). The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking and survival. Results: The results of viSNE showed the heterogenic elevated expression level of Nrp1, Helios (natural occurring Treg marker/transcription factor), CD103, KLRG1, CD44, ICOS, PD-1, Lag3, TIGIT (effector or inhibitory molecules), and Ki67 (proliferation marker) in Treg after DR3 activation. On the other hand, the expression of CD25, the receptor for IL-2 was down regulated. In the microarray data, a significant elevated level (>2 fold relative expression levels in DR3 activated Treg) of chemokine/cytokine (ccr3, cxcl10) and effector molecules (CD74, Gzmb) were observed. These data suggest that the effect of DR3 signaling in Treg results in not only the expansion of Treg but also their activation. In transplantation experiments, the mice that received DR3 activated Treg (5X105/mouse) showed significantly lower donor T cell proliferation compared with the mice that received non-activated Tregs (n=5 in each group, P<0.01 on day 7 and 10 after transplant). Interestingly, even a smaller number (1x105/mouse) of DR3 treated Treg suppressed donor T cell proliferation in host mice (n=5 in each group, P<0.05 on day7 and day10), and the survival of the mice in the DR3 activated Treg group was also improved compared with control GVHD group (n=10 in each group, P<0.01 in Log-rank test). These data suggested that Treg isolated after DR3 activation were more functional for the prevention in GVHD. Conclusion: In conclusion, our data demonstrate that the activation of DR3 signaling can induce Treg populations with enhanced function in vivo. These observations support for future clinical testing using human DR3 signal modulation. Disclosures No relevant conflicts of interest to declare.

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