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Expression of T Cell Protein Tyrosine Phosphatase (PTPN2) is Enhanced in Mucosal T Cells in IBD Patients
AGA Abstracts
colitis (UC) patients and 10 healthy controls. Phenotypic analysis was performed by eightcolor flow cytometry and by Reverse-Transcription Polymerase Chain Reaction (Q-RT-PCR). Lymphocytes were stimulated with PMA and ionomycin before intracellular staining (IL17, IFNγ and TNFα). To study the effect of NKG2D stimulation, fresh PBLs or LPLs from CD patients were stimulated with P815 cell line transfected or not with NKG2D ligands (MICA/ B, ULPB 1/2/3), coated with an agonist anti-CD3 antibody (TCR stimulation) or control isotype followed by cytokine staining as above. To assess the Th1 and Th17 cytokine polarization of CD4+NKG2D+ and CD4+NKG2D- T cells, LPMCs were cultured with antiCD3/anti-CD28 activation beads with different cocktails of cytokines (IL12 for Th1 and IL1β and IL23 Th17 polarization). Results: LP CD4+NKG2D+ T cells were highly positive for IL17 intracellular staining (34.5±25.3%) and expressed significantly higher levels of IL17 than their LP CD4+NKG2D- counterparts (8.8±6%, p=0.001). NKG2D expression on PB CD4+ T cells correlated significantly with IL17 production (p=0.01). In Q-RT-PCR, messenger RNAs of IL-17 and RORC were expressed preferentially in LP CD4+NKG2D+ T cells compared to the CD4+NKG2D- subset (p=0.02). CCR6 and IL23R were significantly more expressed on LP and PB CD4+NKG2D+ T cells as compared to CD4+NKG2D- T cells (p<0.05). A high proportion of IL17 producing T cells co-expressed both NKG2D and CD161, as compared to cells not producing IL17 (52.5±23.7% vs. 4.5±3.2%, p=0.003). Targeting NKG2D by its ligands expressed on P815 cell lines in co-stimulation with the TCR significantly increased the production of IL17 by CD4+ T cells (4.5%±2.1) compared to the stimulation through the TCR alone (1.3%±1.1, p<0.05). LP CD4+NKG2D+ population secreted two times more IL17 in the presence of IL1β and/or IL23 as compared to the IL15 condition, whereas the CD4+NKG2D- showed almost no stimulation under these conditions. Conclusion: The subset of CD4+ T cells expressing NKG2D represents a major source of IL17 in CD, and has typical features of Th17 cells. Interactions between NKG2D and its ligands may strongly influence IL17 production in CD. NKG2D pathway could represent a promising therapeutic target in CD.
in mesenteric lymph nodes from anti-PD-L1 treated mice expressed significantly higher levels of α4β7 and CCR9 than isotype treated controls. This suggests that PD-L1 negatively regulates the expression of gut homing molecule by CD8 T cells upon antigen encounter in gut associated lymphoid tissue. Currently we are carrying out studies to define the specific cellular and molecular mechanisms by which PD-L1 regulates of intestinal imprinting of T cells by CD8+ DCs. The PD-L1 pathway is well known to have a significant role in regulating T cell activation, but its effect on T cell expression of α4β7 and CCR9 has not previously been evaluated. Further investigation into the mechanisms of this interaction may provide additional targets for therapeutic manipulations in the context of autoimmunity and inflammation in the intestine. Su1751 GM-CSF is a Physiologic Regulator of CD11c+ and Possesses AntiInflammatory Properties Satheesh K. Sainathan, Kumar S. Bishnupuri, Mark A. Schroeder, Miriam Steinberg, Rodney D. Newberry, John F. Dipersio, Brian K. Dieckgraefe Crohn's disease (CD) is a chronic inflammatory disorder of the gastro-intestinal tract (GI). A clinical study has shown that human recombinant Granulocytes Macrophage Colony Stimulating Factor (GM-CSF) was effective in the treatment of patients with moderate to severe Crohn's disease (NEJM 2005; 352:2193-2201). Similar study demonstrated that recombinant pegylated murine GM-CSF (peg-mGM) ameliorated experimental DSS-induced colitis (IBD 2008; 14: 88-99). We hypothesized that GM-CSF is a physiologic regulator of dendritic cells (CD11c+) subsets with potent anti-inflammatory properties in-vitro. We further investigated the role of non-pathogenic luminal bacteria as regulators of mucosal GM-CSF expression. Methods: Expression of murine GM-CSF was examined in mice reared under germ-free conditions, and following conventionalization with flora from the normal mouse cecum. GM-CSF mRNA was determined by RT-PCR regionally throughout the GI tract. Balb/c mice also were treated with peg-GM (5mcg IP 4d) or anti-GM-CSF (500μg, IP d1, 3, 5, 7). Spleen samples were analyzed for myeloid, lymphoid and plasmacytoid dendritic cell (440c+; SiglecH+ IPCs) populations by flow cytometry. CD11c+ isolated from spleen cells of GM-CSF treated mice were stimulated with TLR agonists and IL10 ELISPOT assay was performed. We have identified a novel cellular phenotype mobilized by peg-mGM in peripheral blood (SiglecH+, B220-, Ly6c+, CD11b+, CD11c+, GR-1 intermediate, and F4/ 80+/-), which demonstrated suppressor activity in MLR equivalent to the addition of T regulatory cells (ASH 2010). Cells were sorted for SiglecH+B220- population or B220+SiglecH- (control cells) and stimulated with CpG DNA. Supernatant and cells were collected for IL 10 ELISA and real time RT-PCR analysis respectively. Results: Endogenous GM-CSF is regulated by bacteria in GI tract, proportionate to regional bacterial concentrations. Following conventionalization with flora from the normal mouse cecal flora, we observed a 3-4-fold induction of GM-CSF mRNA expression. Peg-mGM treatment significantly expands total CD11c+ cell population (16% vs. untreated 4.1%) including this unique SiglecH+ population. Moreover, antagonism of endogenous GM-CSF with a neutralizing mAb, leads to near disappearance of the total SiglecH+ cell population (1.2% vs. untreated 14.4%, or peg-mGM 17.8%). CD11c+ cells isolated from peg-mGM treated mice significantly increased IL10 production in response to defined stimulants like IFN-γ and S. aureus. Isolated SiglecH+ B220- cells stimulated with CpG DNA demonstrated a significant increase in IL10 production. Conclusion. GM-CSF is regulated at the mucosal-bacterial interface by luminal bacteria. GM-CSF regulates the function and expression of CD11c+ and unique SiglecH+ suppressor populations. These unique cell populations may contribute to the therapeutic response to GM-CSF.
Su1749 Regulation of MicroRNAs by NOD2 Oliver Brain, Philip Allan, Tica Pichulik, Elham Khatamzas, Peter Simpson, Derek P. Jewell, Alison Simmons Background: Crohn's Disease (CD) is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th1/ Th17 immune response to commensal gut flora. CARD15 polymorphisms confer susceptibility to terminal ileal CD. Nod2 responds to muramyl dipeptide (MDP), leading to NF-κB activation and release of pro-inflammatory cytokines. CD-associated CARD15 mutations appear to be predominantly loss-of-function, which raises the question as to how they predispose to a pro-inflammatory disease. Published data have revealed Nod2 cross-talks with toll-like receptors (TLR). This study investigates the role of microRNAs in regulating Nod2 signalling and Nod2/TLR cross-talk. MicroRNAs (miRNAs) are short non-coding RNAs that prevent the translation of mRNA into protein, and can negatively regulate the innate immune response. Loss of these negative regulators might lower the threshold to the development of a pro-inflammatory state in mucosal tissue. Aims: 1) Identify miRNAs differentially expressed in human dendritic cells (DCs) following Nod2, and combined Nod2/ TLR, stimulation. 2) Determine the targets of these miRNAs. 3) Determine the functional consequence of miRNA expression, and loss-of-expression. Methods: Monocyte-derived DCs, expressing either WT or 1007fsinsC CARD15, were stimulated with MDP, Pam3CSK4 (TLR2 ligand), or Flagellin (TLR5 ligand), before lysis and extraction of RNA. MiRNA microarray analysis was conducted using Illumina miRNA microarrays. MiRNA expression was validated with quantitative PCR. Potential miRNA targets were identified using available algorithms, and from published data. MiRNA mimic and antimir transfection were used to confirm targets by western blot, elisa and 3'UTR lucferase assay. Further targets and functional effects were determined by dual-colour Affymetrix cDNA array following miRNA mimic transfection into DCs. Functional consequence of loss of miRNA expression determined by intracellular bacterial killing assay, confocal microscopy, FACS analysis, and T-cell proliferation assay. Results: Nod2 stimulation is required for expression of two miRNA clusters, in combination with either TLR2 or TLR5 triggering. MiRNA up-regulation is dependent on Ripk2 expression. DCs from patients homozygous for 1007fsinsC Nod2 fail to up-regulate these miRNA clusters. MiRNA cluster over-expression down-regulates the IL12-p40 subunit in human DCs at both mRNA and protein level. This subunit is a component of both IL-12 and IL-23 cytokines. IL-12p40 down-regulation can also be induced in FS1007insC DCs by miRNA mimic expression. Conclusion: DCs from patients with CD-associated mutations fail to up-regulate two miRNA clusters. These clusters regulate a critical component of the Th1/ Th17 immune response. These findings may help link CARD15 polymorphisms and the pathogenesis of CD.
Su1752 Expression of T Cell Protein Tyrosine Phosphatase (PTPN2) is Enhanced in Mucosal T Cells in IBD Patients Anja Schirbel, Stephanie Bussières-Marmen, Jean-Paul Achkar, Florian Rieder, Gail West, Manijeh H. Phillips, Michel L. Tremblay, Claudio Fiocchi Background TCPTP (T-cell protein tyrosine phosphatase, PTPN2) is a key negative regulator of T cell activation. Genome wide association studies have revealed a link between PTPN2 variants and Crohn's disease (CD), but how such variants may impact on T cell function is unknown. We investigated the TCPTP expression and function in IBD and control mucosa, lamina propria (LPT) and peripheral blood T cells (PBT). Methods TCPTP levels were evaluated by immunohistochemistry, RT-PCR, immunoblotting and confocal microscopy of fresh, anti-CD3/28- or anti-CD2/CD28-activated LPT and PBT from CD, ulcerative colitis (UC) and control subjects. To evaluate TCPTP biological activity, TCPTP was blocked by sodium orthovanadate (SOV), a phosphatase inhibitor or siRNA for TCPTP. The modulation of STAT3 phosphorylation was assessed by immunoblotting. Additionally, substrate trapping experiments were performed with LPT from inflamed and non-inflamed tissue. Results TCPTP was abundantly expressed in the intestinal mucosa, with higher levels in CD and UC compared to controls. Immunoblotting showed strong TCPTP expression in freshly isolated LPT, with higher levels in mucosal T cells isolated from CD and UC compared to controls. When LPT and PBT were rested in culture, PTPN2 expression almost disappeared, but was restored by T cell activation with anti-CD3/28 and even more with anti-CD2/28 stimulation. These results were confirmed by confocal microscopy. Interestingly, PBT express TCPTP stronger than LPT, regardless whether cells were freshly isolated or activated. Activation of LPT increased TCPTP mRNA expression of both the p45 and p48 isoform. Spontaneous tyrosine phosphorylation was stronger in PBT than LPT, and increased proportionally with T cell activation. Blockade of phosphatase activity with SOV markedly upregulated tyrosine phosphorylation in both LPT and PBT. Knockdown of TCPTP using siRNA increased the phosphorylation of STAT3 in PBT. Substrate trapping experiments revealed different substrates of TCPTP in LPT isolated from inflamed and non-inflamed tissue from the same patient, with common substrates at 250 and 55kDa but differences at 100 and 125kDa. Conclusion These results provide the first evidence that the expression of TCPTP is increased in IBD. The spontaneous expression of TCPTP by mucosal T cells directly isolated from normal mucosa and its loss upon resting in culture suggest that the gut microenvironment induces this specific TC-PTP. In addition, different substrates of TCPTP are processed in
Su1750 Novel Role of PD-L1 in Regulating T Cell Homing to the Small Intestine Mikelle Bassett, Gordon Freeman, Arlene Sharpe, Shannon Turley Immunological homeostasis typically exists in the intestinal microenvironment, preventing penetration of pathogenic and non-pathogenic bacteria while avoiding unnecessary and potentially harmful immune responses to innocuous antigens such as commensal bacteria. To play their integral role in achieving this balance, activated T cells must home to the intestinal tract where the antigen was encountered. If these regulatory mechanisms are altered, pathogen invasion or induction of chronic intestinal inflammation in response to nonpathogenic bacteria can occur. T cells homing to either peripheral or mucosal sites have been activated by dendritic cells (DCs), then express certain ligands which allow them to preferentially migrate to the site where the DC initially encountered the antigen. For intestinal homing specifically, expression of the integrin α4β7 and the chemokine receptor CCR9 allow the activated T cell to interact with and transmigrate across the intestinal epithelium. We previously reported that blockade of programmed death ligand 1 (PD-L1) leads to lethal autoimmune enteritis following CD8 T cell mediated destruction of the epithelial lining of the terminal ileum. More recently, we have shown that CD8 T cells upregulate the gut homing molecules α4β7 and CCR9 upon stimulation by lymph-node resident CD8α+ DCs presenting antigen derived from the intestinal epithelium. We have also found CD8+ T cells
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beads immunoprecipitation for TNFα, TNFα inhibitors, Jagged-1, or BSA with consequent western. ELISAs detected cytokine secretion. Results Mucosal Notch-1 was detectable in normal intestine. In inflamed IBD tissue, Notch-1 expression was significantly lower (P ≤ .01) than in uninflamed mucosal controls. In autologous tissues from UC patients, Notch1 expression was again significantly lower in the inflamed than in non-inflamed tissue (P ≤ .05). Linking Notch and TNFα, we showed that 1) ADA and IFX abrogated activationinduced Notch-1 cell surface expression with a concurrent increase of intracellular Notch1. 2) Notch-1 silencing averted the anti-TNFα-induced T cell cycle arrest and de-activation. 3) TNFα, but not TNFα inhibitors, binds directly to Notch-1. 4) Blockade of Notch signaling distinctively regulates Pro-inflammatory cytokine secretion. Conclusion Our results reveal for the first time decreased mucosal Notch-1 in IBD. Moreover, we provide novel evidence that Notch-1 mediates the inhibitory effects of ADA and IFX on T cell cycling and activation, possibly via direct contact inhibition. In sum, we provide not only a new mode of action, but also an underlying signalling pathway by which biologicals act in IBD.
Su1753 NK Cells Regulate CD62L- CD44- T Cell Subset in the Development of Pathogenic T Cells in a Murine Model of Colitis Osamu Yamaji, Teruji Totsuka, Takashi Nagaishi, Michio Onizawa, Masahiro Suzuki, Naoto Tsuge, Takanori Kanai, Mamoru Watanabe [Background and Aim] Natural killer (NK) cells are associated with regulation of acquired immune responses related to inflammatory diseases such as experimental autoimmune encephalomyelitis and colitis. However, the detail mechanism of NK cell-regulation of pathogenic T cell in inflammation remains unclear. We therefore examine the role of NK cells in a murine model of chronic colitis. [Methods and Results] Anti-asialo GM1 (ASGM1) Ab was injected into RAG deficient (RAG-/-) mice in order to deplete NK cells, followed by adoptive transfer of CD62L+ CD44- (naïve) T cells. The NK cell depletion interestingly resulted in an increase of CD62L- CD44- T cells in the spleen and the mesenteric lymph nodes 5 days after T cell reconstitution despite slight exacerbation of colitis compared to mice without NK cell depletion. Flow cytometric analysis showed the CD62L- and CD44- T cell subset to be Qa-1- DR5Lo IL-7R+, while the CD62L- CD44+ effecter/memory T cells (TEM) expressed Qa-1+ DR5Hi IL-7R+, suggesting that CD62L- CD44- T cells are regulated by NK cells in a different mechanism from that of TEM by NK cells via apoptosis. Neither the cytotoxic activity nor the surface markers of NK cells, such as NKG2A/C/E, NKG2D, Ly49, CD94, KLRG1, CD11b and CD27, were affected in the presence or absence of IL-7. Our interest in IL-7 stems from our previous report that IL-7 deficiency completely abrogates colitis in RAG-/- mice receiving naïve T cells. It had been suggested that such abrogation of colitis may be associated with not only the lack of IL-7 but also another mechanism by which the development of colitis is suppressed at the early stage. Therefore, anti-ASGM1 Ab was injected into RAG-/- IL-7-/- (DKO) mice receiving naïve T cells. The NK cell depletion at the early stage during the induction of colitis resulted in severe colitis in the DKO mice with associated increase in the production of proinflammatory cytokines such as IFN-γ. [Conclusions] Our study suggests that the development of TEM, which induces chronic inflammation, through CD62L- CD44- T cell subset may be affected by both the presence of NK cells and IL-7, and regulating these mechanisms could be a potential therapeutic target for IBD.
Su1756 Expansion of Vδ2+T-Cells Occurs in Crohn's Disease Patients and Their Siblings: Impact of Therapy and Implications for Pathogenesis Charlotte R. Hedin, Neil E. McCarthy, Shaumick Bhattacharjee, Georgina James, Kevin Whelan, James O. Lindsay, Andrew J. Stagg INTRODUCTION: Crohn's disease (CD) is driven by inappropriate inflammatory responses to gut microbiota. Circulating, microbe-responsive Vγ9Vδ2+(δ2)T-cells express 'gut-homing‘ integrin β7 and may contribute to intestinal inflammation. HYPOTHESIS: Increased intestinal permeability and microbe exposure in CD leads to activation and expansion of δ2T-cells. AIM: To compare δ2T-cells in CD patients, unaffected siblings and healthy controls (HC). METHODS: Flow cytometry was used to gate T-cell subsets in 36 CD patients, 13 of their healthy siblings, and 13 HC. δ2T-cell activation and cytokine production in culture of HC and CD peripheral blood mononuclear cells (PBMCs) was assayed upon stimulation with synthetic microbial phosphoantigen (HDMAPP) In Vitro. RESULTS: When HC and CD PBMCs were activated by HDMAPP, δ2T-cells proliferated, produced high levels of IFNγ and TNFα, and maintained high integrin β7 levels In Vitro. In CD patients, the variation in numbers of circulating δ2T-cells was significantly (p=0.02) greater than in HC (0.1-138.4 vs. 6.2-37.8 cells per μl blood; 0.1-13.0% vs. 0.5-2.9% of total T-cells). Of the 19 CD patients not treated with thiopurines (TP), 9 had expanded δ2T-cells (number or proportion above upper limit of HC) and of these, 8 (89%) had inactive disease (HBI<5, p<0.05). There was no difference in age, age at diagnosis, CRP, disease location, behavior or duration between expanded and non-expanded non-TP treated patients. Strikingly, four of 13 siblings also had expanded δ2T-cells (up to 6-10-fold higher than HC values). In long-term TPtreated CD (n=17), mean δ2T-cell numbers were significantly lower than both HC (3.5 vs. 19.7μl-1, p<0.001; 0.5% vs. 1.3%, p<0.01), and TP-naive patients (20.6μl-1 p=0.02; 2.61%, p=0.01) and this effect was selective for δ2 over αβT-cells (mean absolute counts 17.4% vs. 34.3% of the mean in HC, p=0.02). This effect was not evident in 12 patients with TP therapy of <3 months. In Vitro, therapeutic azathioprine (AZA) levels (5μM) equally blocked proliferation of αβ and δ2T-cells, although effects on δ2T-cells were achieved at lower [AZA] than for αβT-cells (0.005μM vs. 0.05μM). CONCLUSION: Circulating δ2T-cells are disturbed in CD due both to expansion in some individuals as well as depletion in TPtreated patients. Selective depletion of δ2T-cells was not observed during TP induction despite enhanced AZA-sensitivity of stimulated δ2T-cells In Vitro. We speculate that repeated microbial stimulation under the cover of immunosuppression may be required to selectively deplete δ2T-cells. δ2T-cell expansion in patients and siblings may imply a role for δ2Tcells in CD pathogenesis and could be a marker of CD risk.
Su1754 Both Innate Immune and Epithelial HSP70 Are Essential for Regulation of Intestinal Homeostasis and Development of Experimental Colitis Yuji Shimada, Xiaorong Zhu, Mark W. Musch, David L. Boone, Bana Jabri, Sumio Watanabe, Eugene B. Chang Background and aims: Heat shock protein 70 (Hsp70) is an inducible, multi-functional, highly conserved molecule that is expressed by intestinal epithelial and laminar propria immune cells. Its expression in the colon is maintained by signals provided by the enteric microbiota. Colonocyte Hsp70 is believed to be cytoprotective, whereas Hsp70 expressed in innate immune cells may be anti-inflammatory. In experimental and human IBD, the expression of Hsp70 is selectively down-regulated, rendering the mucosa susceptible to inflammation-associated stress and injury. This study therefore sought to define the relative roles of intestinal epithelial versus innate immune Hsp70 in mitigating injury from dextran sodium sulfate (DSS)-induced colitis. Methods: Three groups of bone marrow chimeric mice were created: (1) WT > WT (wild-type mice receiving wild-type bone marrow), (2) KO > WT (wild-type mice receiving Hsp70-/- bone marrow) and (3) WT > KO (Hsp70-/mice receiving wild-type bone marrow). All mice were on C57BL/6 background, female, and 6-8 weeks of age at the time of bone marrow transplantation (BMT). After six weeks from BMT, experimental colitis was induced by 2% DSS in drinking water for five days. To assess the disease activity, mice were monitored daily for body weight, rectal bleeding, and stool consistency. Results: Following administration of DSS, the survival rate for KO > WT mice was only 25.0%. In WT > KO mice, 57.1% survived, while all mice survived in WT > WT mice (100%). At day 7, body weight decreased to 77.6% of their initial body weight in KO > WT mice, 81.0% in WT > KO and 89.4% in WT > WT mice. Conclusion: Intestinal epithelial as well as myeloid-derived immune cell Hsp70 appear to be important for maintaining intestinal homeostasis and in determining the course of DSS-induced colitis. The down-regulation of Hsp70 in human IBD may be an important factor contributing to the extent, severity and chronicity of mucosal injury.
Su1757 WASp Alters Regulatory T Cell Induction via Reduced Strength of T Cell Receptor Signaling Elisa K. Boden, Paul M. Arnaboldi, Christopher J. Moran, Deanna D. Nguyen, David Dunkin, Lloyd Mayer, Scott B. Snapper Background: The Wiskott-Aldrich Syndrome is a primary immunodeficiency that results from mutations in the Wiskott-Aldrich Syndrome protein (WASp). WASp is expressed in hematopoietic cells and links cell surface receptor signals to the actin cytoskeleton via ARP2/ 3, a protein complex linked to ulcerative colitis in genome-wide association studies. WASPdeficient(WKO) mice and up to 10% of patients develop a spontaneous colitis that is associated with quantitative and qualitative defects in CD4+CD25+Foxp3+ regulatory T cells (Tregs). We have previously shown defects in de novo induction of Tregs in WKO T cells. In this study, we explore the mechanism by which altered Treg induction results from WASp-deficiency. Methods: We assessed whether increased antigen dose could overcome defects in Treg induction in WKO T cells by culturing naive (CD4+CD25-) CFSE-labeled T cells from ovalbumin-specific TCR transgenic wild type(WT) or WKO mice with WT irradiated T-depleted splenocytes and ova peptide (0-10 mcg/mL) in the presence of TGFβ. Cells were analyzed by flow cytometry at 72 hours for expression of FoxP3 and dilution of CFSE. Supernatants were analyzed by ELISA for IL-2 and IFNγ. To investigate Treg induction In Vivo, naïve WT or WKO CFSE-labeled transgenic T cells were adoptively transferred into WT mice. Recipient mice were injected intravenously with ova peptide (0-50 mcg) and 6 days later, T cells within the lymph nodes were evaluated for CFSE dilution and FoxP3 expression. Results: Antigen-specific WKO T cells demonstrated significantly reduced proliferation, IL-2 and IFNγ production compared to WT at antigen concentrations between 0.1 and 10 mcg/mL ova peptide in the presence of TGFβ. WKO T cells required 10-fold increased concentration of ova peptide to achieve the same level of proliferation as WT. This correlated with an altered threshold for the maximal induction of Tregs in WKO T cells. While peak induction of Tregs occurred in WT at an antigen dose of 0.1 mcg/mL (33%+/-4), WKO T cells demonstrated reduced induction of Tregs at this antigen concentration (19%+/-4, p <.05). However, Treg induction could be rescued by increasing antigen dose, as an ova concentration of 1 mcg/mL induced peak Treg numbers in WKO T cells equivalent to WT(39% +/- 3). WASp-deficiency was also found to alter Treg differentiation In Vivo. WASpdeficient T cells demonstrated reduced proliferative response to antigen (Figure 1A) and
Su1755 Notch-1 is a Novel Player in the Mode of Action TNFα Inhibitors in Inflammatory Bowel Diseases Lael Werner, Uta Berndt, Andreas Sturm Introduction Notch proteins are key regulators of immature leukocyte development and differentiation. Recently, Notch has emerged as a central regulator of mature leukocyte function. In inflammatory bowel diseases (IBD), the Notch target gene HES1, is upregulated in epithelial cells, yet the functional role of Notch is unknown. Several reports describe reciprocal modulation of the TNFα and Notch pathways. However, the plausible link of Notch to TNFα inhibitors has not been studied yet. Thus, the aim of our study was to elucidate the role of Notch in the mode of action of TNFα inhibitors, identifying Notch-1 as a key mediator. Methods Immunohistochemistry was performed on paraffin embedded tissues. PBMC from normal or IBD patients were isolated, activated by anti-CD3 Ab and cultured with Infliximab (IFX), Adalimumab (ADA), TACE inhibitors (TAPI), Notch inhibitors and/or Notch ligands for 48 h. Small interfering (si) RNA for Notch1 was employed to block protein synthesis. Flow cytometry determined cell surface Notch1, and immunoblotting determined Notch1 intracellular expression. Notch-1 binding was performed using Dynal
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inflamed and non-inflamed tissue. This indicates a crucial regulatory role of TCPTP in normal mucosal T cell function, and its upregulation during inflammation. If PTPN2 genetic variants induce a loss of function, TCPTP capacity to suppress T cell phosphorylation may be limited, causing sustained T cell activation and thus contributing to IBD pathogenesis.
colitis (UC) patients and 10 healthy controls. Phenotypic analysis was performed by eightcolor flow cytometry and by Reverse-Transcription Polymerase Chain Reaction (Q-RT-PCR). Lymphocytes were stimulated with PMA and ionomycin before intracellular staining (IL17, IFNγ and TNFα). To study the effect of NKG2D stimulation, fresh PBLs or LPLs from CD patients were stimulated with P815 cell line transfected or not with NKG2D ligands (MICA/ B, ULPB 1/2/3), coated with an agonist anti-CD3 antibody (TCR stimulation) or control isotype followed by cytokine staining as above. To assess the Th1 and Th17 cytokine polarization of CD4+NKG2D+ and CD4+NKG2D- T cells, LPMCs were cultured with antiCD3/anti-CD28 activation beads with different cocktails of cytokines (IL12 for Th1 and IL1β and IL23 Th17 polarization). Results: LP CD4+NKG2D+ T cells were highly positive for IL17 intracellular staining (34.5±25.3%) and expressed significantly higher levels of IL17 than their LP CD4+NKG2D- counterparts (8.8±6%, p=0.001). NKG2D expression on PB CD4+ T cells correlated significantly with IL17 production (p=0.01). In Q-RT-PCR, messenger RNAs of IL-17 and RORC were expressed preferentially in LP CD4+NKG2D+ T cells compared to the CD4+NKG2D- subset (p=0.02). CCR6 and IL23R were significantly more expressed on LP and PB CD4+NKG2D+ T cells as compared to CD4+NKG2D- T cells (p<0.05). A high proportion of IL17 producing T cells co-expressed both NKG2D and CD161, as compared to cells not producing IL17 (52.5±23.7% vs. 4.5±3.2%, p=0.003). Targeting NKG2D by its ligands expressed on P815 cell lines in co-stimulation with the TCR significantly increased the production of IL17 by CD4+ T cells (4.5%±2.1) compared to the stimulation through the TCR alone (1.3%±1.1, p<0.05). LP CD4+NKG2D+ population secreted two times more IL17 in the presence of IL1β and/or IL23 as compared to the IL15 condition, whereas the CD4+NKG2D- showed almost no stimulation under these conditions. Conclusion: The subset of CD4+ T cells expressing NKG2D represents a major source of IL17 in CD, and has typical features of Th17 cells. Interactions between NKG2D and its ligands may strongly influence IL17 production in CD. NKG2D pathway could represent a promising therapeutic target in CD.
in mesenteric lymph nodes from anti-PD-L1 treated mice expressed significantly higher levels of α4β7 and CCR9 than isotype treated controls. This suggests that PD-L1 negatively regulates the expression of gut homing molecule by CD8 T cells upon antigen encounter in gut associated lymphoid tissue. Currently we are carrying out studies to define the specific cellular and molecular mechanisms by which PD-L1 regulates of intestinal imprinting of T cells by CD8+ DCs. The PD-L1 pathway is well known to have a significant role in regulating T cell activation, but its effect on T cell expression of α4β7 and CCR9 has not previously been evaluated. Further investigation into the mechanisms of this interaction may provide additional targets for therapeutic manipulations in the context of autoimmunity and inflammation in the intestine. Su1751 GM-CSF is a Physiologic Regulator of CD11c+ and Possesses AntiInflammatory Properties Satheesh K. Sainathan, Kumar S. Bishnupuri, Mark A. Schroeder, Miriam Steinberg, Rodney D. Newberry, John F. Dipersio, Brian K. Dieckgraefe Crohn's disease (CD) is a chronic inflammatory disorder of the gastro-intestinal tract (GI). A clinical study has shown that human recombinant Granulocytes Macrophage Colony Stimulating Factor (GM-CSF) was effective in the treatment of patients with moderate to severe Crohn's disease (NEJM 2005; 352:2193-2201). Similar study demonstrated that recombinant pegylated murine GM-CSF (peg-mGM) ameliorated experimental DSS-induced colitis (IBD 2008; 14: 88-99). We hypothesized that GM-CSF is a physiologic regulator of dendritic cells (CD11c+) subsets with potent anti-inflammatory properties in-vitro. We further investigated the role of non-pathogenic luminal bacteria as regulators of mucosal GM-CSF expression. Methods: Expression of murine GM-CSF was examined in mice reared under germ-free conditions, and following conventionalization with flora from the normal mouse cecum. GM-CSF mRNA was determined by RT-PCR regionally throughout the GI tract. Balb/c mice also were treated with peg-GM (5mcg IP 4d) or anti-GM-CSF (500μg, IP d1, 3, 5, 7). Spleen samples were analyzed for myeloid, lymphoid and plasmacytoid dendritic cell (440c+; SiglecH+ IPCs) populations by flow cytometry. CD11c+ isolated from spleen cells of GM-CSF treated mice were stimulated with TLR agonists and IL10 ELISPOT assay was performed. We have identified a novel cellular phenotype mobilized by peg-mGM in peripheral blood (SiglecH+, B220-, Ly6c+, CD11b+, CD11c+, GR-1 intermediate, and F4/ 80+/-), which demonstrated suppressor activity in MLR equivalent to the addition of T regulatory cells (ASH 2010). Cells were sorted for SiglecH+B220- population or B220+SiglecH- (control cells) and stimulated with CpG DNA. Supernatant and cells were collected for IL 10 ELISA and real time RT-PCR analysis respectively. Results: Endogenous GM-CSF is regulated by bacteria in GI tract, proportionate to regional bacterial concentrations. Following conventionalization with flora from the normal mouse cecal flora, we observed a 3-4-fold induction of GM-CSF mRNA expression. Peg-mGM treatment significantly expands total CD11c+ cell population (16% vs. untreated 4.1%) including this unique SiglecH+ population. Moreover, antagonism of endogenous GM-CSF with a neutralizing mAb, leads to near disappearance of the total SiglecH+ cell population (1.2% vs. untreated 14.4%, or peg-mGM 17.8%). CD11c+ cells isolated from peg-mGM treated mice significantly increased IL10 production in response to defined stimulants like IFN-γ and S. aureus. Isolated SiglecH+ B220- cells stimulated with CpG DNA demonstrated a significant increase in IL10 production. Conclusion. GM-CSF is regulated at the mucosal-bacterial interface by luminal bacteria. GM-CSF regulates the function and expression of CD11c+ and unique SiglecH+ suppressor populations. These unique cell populations may contribute to the therapeutic response to GM-CSF.
Su1749 Regulation of MicroRNAs by NOD2 Oliver Brain, Philip Allan, Tica Pichulik, Elham Khatamzas, Peter Simpson, Derek P. Jewell, Alison Simmons Background: Crohn's Disease (CD) is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th1/ Th17 immune response to commensal gut flora. CARD15 polymorphisms confer susceptibility to terminal ileal CD. Nod2 responds to muramyl dipeptide (MDP), leading to NF-κB activation and release of pro-inflammatory cytokines. CD-associated CARD15 mutations appear to be predominantly loss-of-function, which raises the question as to how they predispose to a pro-inflammatory disease. Published data have revealed Nod2 cross-talks with toll-like receptors (TLR). This study investigates the role of microRNAs in regulating Nod2 signalling and Nod2/TLR cross-talk. MicroRNAs (miRNAs) are short non-coding RNAs that prevent the translation of mRNA into protein, and can negatively regulate the innate immune response. Loss of these negative regulators might lower the threshold to the development of a pro-inflammatory state in mucosal tissue. Aims: 1) Identify miRNAs differentially expressed in human dendritic cells (DCs) following Nod2, and combined Nod2/ TLR, stimulation. 2) Determine the targets of these miRNAs. 3) Determine the functional consequence of miRNA expression, and loss-of-expression. Methods: Monocyte-derived DCs, expressing either WT or 1007fsinsC CARD15, were stimulated with MDP, Pam3CSK4 (TLR2 ligand), or Flagellin (TLR5 ligand), before lysis and extraction of RNA. MiRNA microarray analysis was conducted using Illumina miRNA microarrays. MiRNA expression was validated with quantitative PCR. Potential miRNA targets were identified using available algorithms, and from published data. MiRNA mimic and antimir transfection were used to confirm targets by western blot, elisa and 3'UTR lucferase assay. Further targets and functional effects were determined by dual-colour Affymetrix cDNA array following miRNA mimic transfection into DCs. Functional consequence of loss of miRNA expression determined by intracellular bacterial killing assay, confocal microscopy, FACS analysis, and T-cell proliferation assay. Results: Nod2 stimulation is required for expression of two miRNA clusters, in combination with either TLR2 or TLR5 triggering. MiRNA up-regulation is dependent on Ripk2 expression. DCs from patients homozygous for 1007fsinsC Nod2 fail to up-regulate these miRNA clusters. MiRNA cluster over-expression down-regulates the IL12-p40 subunit in human DCs at both mRNA and protein level. This subunit is a component of both IL-12 and IL-23 cytokines. IL-12p40 down-regulation can also be induced in FS1007insC DCs by miRNA mimic expression. Conclusion: DCs from patients with CD-associated mutations fail to up-regulate two miRNA clusters. These clusters regulate a critical component of the Th1/ Th17 immune response. These findings may help link CARD15 polymorphisms and the pathogenesis of CD.
Su1752 Expression of T Cell Protein Tyrosine Phosphatase (PTPN2) is Enhanced in Mucosal T Cells in IBD Patients Anja Schirbel, Stephanie Bussières-Marmen, Jean-Paul Achkar, Florian Rieder, Gail West, Manijeh H. Phillips, Michel L. Tremblay, Claudio Fiocchi Background TCPTP (T-cell protein tyrosine phosphatase, PTPN2) is a key negative regulator of T cell activation. Genome wide association studies have revealed a link between PTPN2 variants and Crohn's disease (CD), but how such variants may impact on T cell function is unknown. We investigated the TCPTP expression and function in IBD and control mucosa, lamina propria (LPT) and peripheral blood T cells (PBT). Methods TCPTP levels were evaluated by immunohistochemistry, RT-PCR, immunoblotting and confocal microscopy of fresh, anti-CD3/28- or anti-CD2/CD28-activated LPT and PBT from CD, ulcerative colitis (UC) and control subjects. To evaluate TCPTP biological activity, TCPTP was blocked by sodium orthovanadate (SOV), a phosphatase inhibitor or siRNA for TCPTP. The modulation of STAT3 phosphorylation was assessed by immunoblotting. Additionally, substrate trapping experiments were performed with LPT from inflamed and non-inflamed tissue. Results TCPTP was abundantly expressed in the intestinal mucosa, with higher levels in CD and UC compared to controls. Immunoblotting showed strong TCPTP expression in freshly isolated LPT, with higher levels in mucosal T cells isolated from CD and UC compared to controls. When LPT and PBT were rested in culture, PTPN2 expression almost disappeared, but was restored by T cell activation with anti-CD3/28 and even more with anti-CD2/28 stimulation. These results were confirmed by confocal microscopy. Interestingly, PBT express TCPTP stronger than LPT, regardless whether cells were freshly isolated or activated. Activation of LPT increased TCPTP mRNA expression of both the p45 and p48 isoform. Spontaneous tyrosine phosphorylation was stronger in PBT than LPT, and increased proportionally with T cell activation. Blockade of phosphatase activity with SOV markedly upregulated tyrosine phosphorylation in both LPT and PBT. Knockdown of TCPTP using siRNA increased the phosphorylation of STAT3 in PBT. Substrate trapping experiments revealed different substrates of TCPTP in LPT isolated from inflamed and non-inflamed tissue from the same patient, with common substrates at 250 and 55kDa but differences at 100 and 125kDa. Conclusion These results provide the first evidence that the expression of TCPTP is increased in IBD. The spontaneous expression of TCPTP by mucosal T cells directly isolated from normal mucosa and its loss upon resting in culture suggest that the gut microenvironment induces this specific TC-PTP. In addition, different substrates of TCPTP are processed in
Su1750 Novel Role of PD-L1 in Regulating T Cell Homing to the Small Intestine Mikelle Bassett, Gordon Freeman, Arlene Sharpe, Shannon Turley Immunological homeostasis typically exists in the intestinal microenvironment, preventing penetration of pathogenic and non-pathogenic bacteria while avoiding unnecessary and potentially harmful immune responses to innocuous antigens such as commensal bacteria. To play their integral role in achieving this balance, activated T cells must home to the intestinal tract where the antigen was encountered. If these regulatory mechanisms are altered, pathogen invasion or induction of chronic intestinal inflammation in response to nonpathogenic bacteria can occur. T cells homing to either peripheral or mucosal sites have been activated by dendritic cells (DCs), then express certain ligands which allow them to preferentially migrate to the site where the DC initially encountered the antigen. For intestinal homing specifically, expression of the integrin α4β7 and the chemokine receptor CCR9 allow the activated T cell to interact with and transmigrate across the intestinal epithelium. We previously reported that blockade of programmed death ligand 1 (PD-L1) leads to lethal autoimmune enteritis following CD8 T cell mediated destruction of the epithelial lining of the terminal ileum. More recently, we have shown that CD8 T cells upregulate the gut homing molecules α4β7 and CCR9 upon stimulation by lymph-node resident CD8α+ DCs presenting antigen derived from the intestinal epithelium. We have also found CD8+ T cells
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beads immunoprecipitation for TNFα, TNFα inhibitors, Jagged-1, or BSA with consequent western. ELISAs detected cytokine secretion. Results Mucosal Notch-1 was detectable in normal intestine. In inflamed IBD tissue, Notch-1 expression was significantly lower (P ≤ .01) than in uninflamed mucosal controls. In autologous tissues from UC patients, Notch1 expression was again significantly lower in the inflamed than in non-inflamed tissue (P ≤ .05). Linking Notch and TNFα, we showed that 1) ADA and IFX abrogated activationinduced Notch-1 cell surface expression with a concurrent increase of intracellular Notch1. 2) Notch-1 silencing averted the anti-TNFα-induced T cell cycle arrest and de-activation. 3) TNFα, but not TNFα inhibitors, binds directly to Notch-1. 4) Blockade of Notch signaling distinctively regulates Pro-inflammatory cytokine secretion. Conclusion Our results reveal for the first time decreased mucosal Notch-1 in IBD. Moreover, we provide novel evidence that Notch-1 mediates the inhibitory effects of ADA and IFX on T cell cycling and activation, possibly via direct contact inhibition. In sum, we provide not only a new mode of action, but also an underlying signalling pathway by which biologicals act in IBD.
Su1753 NK Cells Regulate CD62L- CD44- T Cell Subset in the Development of Pathogenic T Cells in a Murine Model of Colitis Osamu Yamaji, Teruji Totsuka, Takashi Nagaishi, Michio Onizawa, Masahiro Suzuki, Naoto Tsuge, Takanori Kanai, Mamoru Watanabe [Background and Aim] Natural killer (NK) cells are associated with regulation of acquired immune responses related to inflammatory diseases such as experimental autoimmune encephalomyelitis and colitis. However, the detail mechanism of NK cell-regulation of pathogenic T cell in inflammation remains unclear. We therefore examine the role of NK cells in a murine model of chronic colitis. [Methods and Results] Anti-asialo GM1 (ASGM1) Ab was injected into RAG deficient (RAG-/-) mice in order to deplete NK cells, followed by adoptive transfer of CD62L+ CD44- (naïve) T cells. The NK cell depletion interestingly resulted in an increase of CD62L- CD44- T cells in the spleen and the mesenteric lymph nodes 5 days after T cell reconstitution despite slight exacerbation of colitis compared to mice without NK cell depletion. Flow cytometric analysis showed the CD62L- and CD44- T cell subset to be Qa-1- DR5Lo IL-7R+, while the CD62L- CD44+ effecter/memory T cells (TEM) expressed Qa-1+ DR5Hi IL-7R+, suggesting that CD62L- CD44- T cells are regulated by NK cells in a different mechanism from that of TEM by NK cells via apoptosis. Neither the cytotoxic activity nor the surface markers of NK cells, such as NKG2A/C/E, NKG2D, Ly49, CD94, KLRG1, CD11b and CD27, were affected in the presence or absence of IL-7. Our interest in IL-7 stems from our previous report that IL-7 deficiency completely abrogates colitis in RAG-/- mice receiving naïve T cells. It had been suggested that such abrogation of colitis may be associated with not only the lack of IL-7 but also another mechanism by which the development of colitis is suppressed at the early stage. Therefore, anti-ASGM1 Ab was injected into RAG-/- IL-7-/- (DKO) mice receiving naïve T cells. The NK cell depletion at the early stage during the induction of colitis resulted in severe colitis in the DKO mice with associated increase in the production of proinflammatory cytokines such as IFN-γ. [Conclusions] Our study suggests that the development of TEM, which induces chronic inflammation, through CD62L- CD44- T cell subset may be affected by both the presence of NK cells and IL-7, and regulating these mechanisms could be a potential therapeutic target for IBD.
Su1756 Expansion of Vδ2+T-Cells Occurs in Crohn's Disease Patients and Their Siblings: Impact of Therapy and Implications for Pathogenesis Charlotte R. Hedin, Neil E. McCarthy, Shaumick Bhattacharjee, Georgina James, Kevin Whelan, James O. Lindsay, Andrew J. Stagg INTRODUCTION: Crohn's disease (CD) is driven by inappropriate inflammatory responses to gut microbiota. Circulating, microbe-responsive Vγ9Vδ2+(δ2)T-cells express 'gut-homing‘ integrin β7 and may contribute to intestinal inflammation. HYPOTHESIS: Increased intestinal permeability and microbe exposure in CD leads to activation and expansion of δ2T-cells. AIM: To compare δ2T-cells in CD patients, unaffected siblings and healthy controls (HC). METHODS: Flow cytometry was used to gate T-cell subsets in 36 CD patients, 13 of their healthy siblings, and 13 HC. δ2T-cell activation and cytokine production in culture of HC and CD peripheral blood mononuclear cells (PBMCs) was assayed upon stimulation with synthetic microbial phosphoantigen (HDMAPP) In Vitro. RESULTS: When HC and CD PBMCs were activated by HDMAPP, δ2T-cells proliferated, produced high levels of IFNγ and TNFα, and maintained high integrin β7 levels In Vitro. In CD patients, the variation in numbers of circulating δ2T-cells was significantly (p=0.02) greater than in HC (0.1-138.4 vs. 6.2-37.8 cells per μl blood; 0.1-13.0% vs. 0.5-2.9% of total T-cells). Of the 19 CD patients not treated with thiopurines (TP), 9 had expanded δ2T-cells (number or proportion above upper limit of HC) and of these, 8 (89%) had inactive disease (HBI<5, p<0.05). There was no difference in age, age at diagnosis, CRP, disease location, behavior or duration between expanded and non-expanded non-TP treated patients. Strikingly, four of 13 siblings also had expanded δ2T-cells (up to 6-10-fold higher than HC values). In long-term TPtreated CD (n=17), mean δ2T-cell numbers were significantly lower than both HC (3.5 vs. 19.7μl-1, p<0.001; 0.5% vs. 1.3%, p<0.01), and TP-naive patients (20.6μl-1 p=0.02; 2.61%, p=0.01) and this effect was selective for δ2 over αβT-cells (mean absolute counts 17.4% vs. 34.3% of the mean in HC, p=0.02). This effect was not evident in 12 patients with TP therapy of <3 months. In Vitro, therapeutic azathioprine (AZA) levels (5μM) equally blocked proliferation of αβ and δ2T-cells, although effects on δ2T-cells were achieved at lower [AZA] than for αβT-cells (0.005μM vs. 0.05μM). CONCLUSION: Circulating δ2T-cells are disturbed in CD due both to expansion in some individuals as well as depletion in TPtreated patients. Selective depletion of δ2T-cells was not observed during TP induction despite enhanced AZA-sensitivity of stimulated δ2T-cells In Vitro. We speculate that repeated microbial stimulation under the cover of immunosuppression may be required to selectively deplete δ2T-cells. δ2T-cell expansion in patients and siblings may imply a role for δ2Tcells in CD pathogenesis and could be a marker of CD risk.
Su1754 Both Innate Immune and Epithelial HSP70 Are Essential for Regulation of Intestinal Homeostasis and Development of Experimental Colitis Yuji Shimada, Xiaorong Zhu, Mark W. Musch, David L. Boone, Bana Jabri, Sumio Watanabe, Eugene B. Chang Background and aims: Heat shock protein 70 (Hsp70) is an inducible, multi-functional, highly conserved molecule that is expressed by intestinal epithelial and laminar propria immune cells. Its expression in the colon is maintained by signals provided by the enteric microbiota. Colonocyte Hsp70 is believed to be cytoprotective, whereas Hsp70 expressed in innate immune cells may be anti-inflammatory. In experimental and human IBD, the expression of Hsp70 is selectively down-regulated, rendering the mucosa susceptible to inflammation-associated stress and injury. This study therefore sought to define the relative roles of intestinal epithelial versus innate immune Hsp70 in mitigating injury from dextran sodium sulfate (DSS)-induced colitis. Methods: Three groups of bone marrow chimeric mice were created: (1) WT > WT (wild-type mice receiving wild-type bone marrow), (2) KO > WT (wild-type mice receiving Hsp70-/- bone marrow) and (3) WT > KO (Hsp70-/mice receiving wild-type bone marrow). All mice were on C57BL/6 background, female, and 6-8 weeks of age at the time of bone marrow transplantation (BMT). After six weeks from BMT, experimental colitis was induced by 2% DSS in drinking water for five days. To assess the disease activity, mice were monitored daily for body weight, rectal bleeding, and stool consistency. Results: Following administration of DSS, the survival rate for KO > WT mice was only 25.0%. In WT > KO mice, 57.1% survived, while all mice survived in WT > WT mice (100%). At day 7, body weight decreased to 77.6% of their initial body weight in KO > WT mice, 81.0% in WT > KO and 89.4% in WT > WT mice. Conclusion: Intestinal epithelial as well as myeloid-derived immune cell Hsp70 appear to be important for maintaining intestinal homeostasis and in determining the course of DSS-induced colitis. The down-regulation of Hsp70 in human IBD may be an important factor contributing to the extent, severity and chronicity of mucosal injury.
Su1757 WASp Alters Regulatory T Cell Induction via Reduced Strength of T Cell Receptor Signaling Elisa K. Boden, Paul M. Arnaboldi, Christopher J. Moran, Deanna D. Nguyen, David Dunkin, Lloyd Mayer, Scott B. Snapper Background: The Wiskott-Aldrich Syndrome is a primary immunodeficiency that results from mutations in the Wiskott-Aldrich Syndrome protein (WASp). WASp is expressed in hematopoietic cells and links cell surface receptor signals to the actin cytoskeleton via ARP2/ 3, a protein complex linked to ulcerative colitis in genome-wide association studies. WASPdeficient(WKO) mice and up to 10% of patients develop a spontaneous colitis that is associated with quantitative and qualitative defects in CD4+CD25+Foxp3+ regulatory T cells (Tregs). We have previously shown defects in de novo induction of Tregs in WKO T cells. In this study, we explore the mechanism by which altered Treg induction results from WASp-deficiency. Methods: We assessed whether increased antigen dose could overcome defects in Treg induction in WKO T cells by culturing naive (CD4+CD25-) CFSE-labeled T cells from ovalbumin-specific TCR transgenic wild type(WT) or WKO mice with WT irradiated T-depleted splenocytes and ova peptide (0-10 mcg/mL) in the presence of TGFβ. Cells were analyzed by flow cytometry at 72 hours for expression of FoxP3 and dilution of CFSE. Supernatants were analyzed by ELISA for IL-2 and IFNγ. To investigate Treg induction In Vivo, naïve WT or WKO CFSE-labeled transgenic T cells were adoptively transferred into WT mice. Recipient mice were injected intravenously with ova peptide (0-50 mcg) and 6 days later, T cells within the lymph nodes were evaluated for CFSE dilution and FoxP3 expression. Results: Antigen-specific WKO T cells demonstrated significantly reduced proliferation, IL-2 and IFNγ production compared to WT at antigen concentrations between 0.1 and 10 mcg/mL ova peptide in the presence of TGFβ. WKO T cells required 10-fold increased concentration of ova peptide to achieve the same level of proliferation as WT. This correlated with an altered threshold for the maximal induction of Tregs in WKO T cells. While peak induction of Tregs occurred in WT at an antigen dose of 0.1 mcg/mL (33%+/-4), WKO T cells demonstrated reduced induction of Tregs at this antigen concentration (19%+/-4, p <.05). However, Treg induction could be rescued by increasing antigen dose, as an ova concentration of 1 mcg/mL induced peak Treg numbers in WKO T cells equivalent to WT(39% +/- 3). WASp-deficiency was also found to alter Treg differentiation In Vivo. WASpdeficient T cells demonstrated reduced proliferative response to antigen (Figure 1A) and
Su1755 Notch-1 is a Novel Player in the Mode of Action TNFα Inhibitors in Inflammatory Bowel Diseases Lael Werner, Uta Berndt, Andreas Sturm Introduction Notch proteins are key regulators of immature leukocyte development and differentiation. Recently, Notch has emerged as a central regulator of mature leukocyte function. In inflammatory bowel diseases (IBD), the Notch target gene HES1, is upregulated in epithelial cells, yet the functional role of Notch is unknown. Several reports describe reciprocal modulation of the TNFα and Notch pathways. However, the plausible link of Notch to TNFα inhibitors has not been studied yet. Thus, the aim of our study was to elucidate the role of Notch in the mode of action of TNFα inhibitors, identifying Notch-1 as a key mediator. Methods Immunohistochemistry was performed on paraffin embedded tissues. PBMC from normal or IBD patients were isolated, activated by anti-CD3 Ab and cultured with Infliximab (IFX), Adalimumab (ADA), TACE inhibitors (TAPI), Notch inhibitors and/or Notch ligands for 48 h. Small interfering (si) RNA for Notch1 was employed to block protein synthesis. Flow cytometry determined cell surface Notch1, and immunoblotting determined Notch1 intracellular expression. Notch-1 binding was performed using Dynal
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inflamed and non-inflamed tissue. This indicates a crucial regulatory role of TCPTP in normal mucosal T cell function, and its upregulation during inflammation. If PTPN2 genetic variants induce a loss of function, TCPTP capacity to suppress T cell phosphorylation may be limited, causing sustained T cell activation and thus contributing to IBD pathogenesis.