Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation, which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo, antibody blockade of TNFSF15 (TL1A), which is the ligand for TNFR25, inhibits lung inflammation and production of Th2 cytokines such as IL-13, even when administered days after airway antigen exposure. Similarly, blockade of TNFR25 by a dominant-negative (DN) transgene, DN TNFR25, confers resistance to lung inflammation in mice. Allergic lung inflammation–resistant, NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells, but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung, and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics

Plants with genetically encoded autoluminescence

Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants

TNFR25 Expression on CD4+CD25+ T Cells: Down Modulation of Regulatory Activity

Abstract TNFR25 (“DR3”) is a member of the TNF receptor family that is expressed by activated CD4+ and CD8+ T cells. To determine if activated CD4+CD25+ T cells also expressed this TNFR family molecule, B6 CD4+CD25+ T cells were stimulated with anti-CD3/CD28 coated beads (kind gift of Dr. B. Blazar, U. Minn.) and expanded for 3–4 days. TNFR25 expression was readily detected on CD4+CD25+ FoxP3+ T cells. Since other members of the TNF receptor family (GITR, OX40, 4–1BB) are known to influence T regulatory cell function, we investigated whether TNFR25 signaling can regulate CD4+CD25+ T cell activity. TNFR25 triggering in B6-wt T regulatory CD4+CD25+ cells with the recombinant TNFR25 ligand TL1A or agonistic anti-TNFR25 antibody (4C12) resulted in reduction of their ability to suppress anti-CD3 induced ex-vivo proliferation of CD4+CD25− cells. 4C12 mediated TNFR25 signaling also reduced B6-wt Treg mediated inhibition of peptide induced proliferation of OVA-specific B6 CD8+ (OT-I) cells. To further investigate a role for TNFR25 in Treg cell regulation, TNFR25 (full length) transgenic mice were generated and bred onto the BL/6 background. CD4+CD25+ cells from these TNFR25 tg mice were found to possess diminished T regulatory activity in vitro as determined by their diminished inability to regulate proliferation by B6-wt CD4+ and OT-I CD8+ T cells. To assess their in vivo regulatory activity, B6-wt and B6 TNFR25 tg Treg cells were examined for their ability to inhibit graft vs. host disease (GVHD) following allogeneic MHC class I/II mismatched BMT. In contrast to B6-wt Treg cells, TNFR25 tg Treg cells exhibited significantly diminished ability to regulate the onset of GVHD in vivo as assessed by weight loss and clinical symptoms. Using agonistic antibody, stimulation of TNFR25 on transgenic Treg cells was also found to effectively remove the ex-vivo regulatory activity expressed by this population. To exclude any possible direct co-stimulatory effects of 4C12 antibody on the responding proliferating cells, CD4+CD25−T cells from TNFR25 dominant negative transgenic mice were employed. 4C12 mab again abolished Treg cell inhibitory activity. The effect of TNFR25 agonists on T reg cell activity in vivo is being further investigated in both mouse models of GVHD and IBD diseases. Initial observations administering 4C12 post-allogeneic BMT together with B6-wt Treg cells indicate a reduction in their ability to regulate GVHD. In total, these studies identify TNFR25 as a new potential target for augmenting CD4+ and CD8+ responses by concomitant direct co-stimulation of effecter cells and inhibition of T regulatory cell function

Molecular and Cellular Requirements for Enhanced Antigen Cross-Presentation to CD8 Cytotoxic T Lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents

Tumor-Induced Suppression of CTL Expansion and Subjugation by gp96-Ig Vaccination

Established tumors suppress anti-tumor immune responses and induce tolerance by incompletely characterized mechanisms, and this phenomenon is an important barrier to tumor immunotherapy. Single vaccination with tumor cells expressing gp96-Ig stimulates robust expansion of tumor-specific CTLs in tumor naïve mice and this expansion is inhibited by established tumors. Interestingly, frequent vaccinations restore anti-tumor immune responses in the presence of established tumors. Syngeneic EG7 tumor bearing mice have heterogeneous responses to frequent vaccination with EG7-gp96-Ig, with 32% complete responders and 68% partial responders. Comparison of responders to non-responders revealed an inverse correlation between tumor-specific CTL expansion in the peripheral blood and tumor size. To identify immune cells and molecules associated with effective anti-tumor immune responses, RT-PCR arrays were performed using cells isolated from the vaccination site. ELISAs, cellular phenotyping and tumor immunohistochemistry were also performed comparing vaccine responders to non-responders. These data demonstrate that upregulation of T-bet, RORγt, IFNγ, CCL8, CXCL9 and CXCL10 at the vaccination site are associated with vaccine-induced anti-tumor immunity. These data correlate with increased CTL expansion in the peripheral blood of responders, increased infiltration of responder tumors by CD8+ cells and IL-17+ cells and decreased infiltration of responder tumors by CD11b+Gr-1+ cells and FoxP3+ cells. Furthermore, serum ELISAs revealed a significant elevation of TGF-β in non-responders as compared to responders. Interestingly, CD8+ T cells isolated from responders and non-responders have equivalent cytotoxic activity in vitro. Taken together, our data suggest that established tumors may escape immunosurveillance by preventing clonal expansion of tumor specific CTL without inducing anergy

Tumor-induced suppression of CTL expansion and subjugation by gp96-Ig vaccination.

Abstract Established tumors suppress antitumor immune responses and induce tolerance by incompletely characterized mechanisms, and this phenomenon is an important barrier to tumor immunotherapy. Single vaccination with tumor cells expressing gp96-Ig stimulates robust expansion of tumor-specific CTLs in tumor-naïve mice and this expansion is inhibited by established tumors. Interestingly, frequent vaccinations restore antitumor immune responses in the presence of established tumors. Syngeneic EG7 tumor-bearing mice have heterogeneous responses to frequent vaccination with EG7-gp96-Ig, with 32% complete responders and 68% partial responders. Comparison of responders to nonresponders revealed an inverse correlation between tumor-specific CTL expansion in the peripheral blood and tumor size. To identify immune cells and molecules associated with effective antitumor immune responses, reverse transcription-PCR arrays were performed using cells isolated from the vaccination site. ELISAs, cellular phenotyping, and tumor immunohistochemistry were also performed comparing vaccine responders to nonresponders. These data show that up-regulation of T-bet, ROR;t, IFN;, CCL8, CXCL9, and CXCL10 at the vaccination site are associated with vaccine-induced antitumor immunity. These data correlate with increased CTL expansion in the peripheral blood of responders, increased infiltration of responder tumors by CD8+ cells and interleukin-17+ cells, and decreased infiltration of responder tumors by CD11b+Gr-1+ cells and FoxP3+ cells. Furthermore, serum ELISAs revealed a significant elevation of transforming growth factor-B in nonresponders as compared with responders. Interestingly, CD8+ T cells isolated from responders and nonresponders have equivalent cytotoxic activity in vitro. Taken together, our data suggest that established tumors may escape immunosurveillance by preventing clonal expansion of tumor-specific CTL without inducing anergy

Mice were immunized and subjected to ovalbumin aerosol according to our standard protocol

Administration of 50 μg blocking (L4G6) or nonblocking (L3A10) anti-Tl1A, or control hamster IgG i.p. was started at the time indicated relative to aerosol exposure and continued daily until analysis, which was at 76 h after aerosol. In the 72-h time points, anti-TL1A was administered 4 h before analysis. A, B, and C are separate experiments testing different schedules and controls. Note that nonblocking TL1A (L3A10) does not affect eosinophil exudation, similar to hamster IgG. (A and B) Data from three experiments with two mice. (C) Data from two experiments with five mice. (D and E) Histopathology of lung sections stained with HE and PAS. Five sections from each of three mice in each group were evaluated in a blinded fashion according to the scoring system described in Materials and methods. (F and G) Relative frequency of CD4 and CD8 cells in lung parenchyma after aerosol exposure and blockade of TL1A for different periods of time. Single-cell suspensions were analyzed by flow cytometry gating on the lymphocyte gate. (H–J) RNA was isolated from whole lungs and analyzed by real-time PCR as in . Error bars represent the mean ± the SEM.<p><b>Copyright information:</b></p><p>Taken from "Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1037-1048.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373837.</p><p></p

NKT-deficient Jα18 KO mice () were primed with ovalbumin and alum as in our standard protocol in Materials and methods

On day 11, the mice received 3.1 million purified NK/NKT cells containing 1 million WT NKT cells or DN TNFR25-tg NKT cells (DN NKT) or PBS by i.v. adoptive transfer, as indicated. The mice were exposed to ovalbumin aerosol on day 12 and analyzed on day 15. WT mice and Jα18 KO mice receiving WT NKT cells by adoptive transfer served as positive controls for induction of lung inflammation. Jα18 KO mice, immunized and ovalbumin aerosolized without adoptive cell transfer, served as negative controls. The data of three independent experiments and two mice in each group are shown. (A) Eosinophils in BALF. Error bars represent the mean ± the SEM. (B and C) Cytokine and TL1A mRNA expression in bronchial lymph nodes (LN; B) and lung parenchyma (C) determined by real time Taqman PCR. The fold increase or decrease of mRNA in Jα18 KO mice (Jα) adoptively transferred with WT NKT cells over mice adoptively transferred with DN TNFR25-tg NKT (DN NKT) cells is plotted.<p><b>Copyright information:</b></p><p>Taken from "Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1037-1048.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373837.</p><p></p

(A) TL1A is not expressed on resting lymphocytes and up-regulated on activated T cells (top 6 graphs)

Resting splenocyte cell suspensions were gated using the respective labeled antibody as a population marker and the TL1A histogram displayed. Red curve, anti-TL1A; black curve, isotype control (bottom 3 graphs). Splenocytes were activated for 24 h with plate-bound anti-CD3 or with LPS and stained with anti-TL1A and with the population marker, as indicated. After gating on the population marker, TL1A expression on activated cells is shown as blue/shaded histogram. Red curve, resting cells; black curve, isotype control. Representative of more than three experiments. (B) TNFR25 and TL1A expression on cDNA transfected P815 and EL4. Transfected (right curve in each histogram) and untransfected cells were stained with the appropriate antibody and isotype controls and analyzed by flow cytometry. (C) TNFR25 activates NF-κB when triggered by agonistic antibody 4C12, by soluble TL1A or by membrane-bound TL1A. NF-κB activation was measured in EL4 cells transfected with TNFR25 in response to TNFR25 triggering. Cells were treated with the agonistic anti-TNFR25 antibody 4C12 (5 μg/ml) for 50 min; soluble TL1A was given for 25, 50, or 70 min, as indicated in the form of 25% supernatants from TL1A-transfected EL4 cells; membrane-bound TL1A (MTL1A) was given for 50 min by adding TL1A-transfected EL4-cells directly to TNFR25-transfected EL4. Controls received EL4 (untransfected) supernatants for 50 min. Nuclear extracts were prepared and analyzed by EMSA; the arrow indicates activated NF-κB. (D) Anti-TL1A antibody L4G6 blocks TL1A induced cell death of TNFR25-transfected cells. Soluble TL1A harvested from supernatants of P815-TL1A–transfected cells were mixed with Cr-labeled P815-TNFR25 target cells. Different anti-murine TL1A monoclonal antibodies were added into the assay, and Cr release was analyzed 5 h later. L4G6 antibody completely blocked the ability of TL1A to induce apoptosis in TNFR25-transfected P815 cells.<p><b>Copyright information:</b></p><p>Taken from "Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1037-1048.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373837.</p><p></p

(A) Diminished cellular exudation in BALF in anti-TL1A (L4G6)–treated mice

Mice were primed i.p. on day 0 and 5 with 66 μg ovalbumin absorbed to alum. On day 12, mice were aerosol challenged with 0.5% ovalbumin in PBS for 1 h using an ultrasonic nebulizer. Mice received 4 daily doses of 50 μg purified L4G6-IgG i.p. (anti-TL1A), beginning 1 d before aerosol. Controls received the same amount and schedule of purified hamster IgG. All mice were analyzed 3 d after aerosol antigen exposure ( = 4; representative of >10 experiments). *, P < 0.05; **, P < 0.01. (B) TL1A-blocking antibody L4G6 suppresses mucus production and lung inflammation. Lung histology after PAS staining after treatment of mice with control IgG (top) or L4G6-IgG (anti-TL1A; bottom). Notice the lack of mucus production and cell infiltration in L4G6-treated animals (arrows point to mucus in mice treated with control IgG). Experiments were repeated three times. (C) Diminished IL-5 and -13 production by ovalbumin restimulated bronchial lymph node cells after TL1A blockade with L4G6. Bronchial lymph node cells were harvested 3 d after aerosol and restimulated in vitro with 100 μg/ml ovalbumin for 4 d. IL-4 was not detectable (not depicted), even in the absence of anti-TL1A. = 4; **, P < 0.01; ***, P < 0.001. Experiments were repeated more than three times. (D) Cytokine expression in lung parenchyma after ovalbumin aerosol exposure. Lungs were harvested 1, 2, or 3 d after ovalbumin aerosol treatment. RNA was extracted, and after reverse transcription it was analyzed by Taqman PCR. Values are normalized to GAPDH cDNA and expressed as the fold increase of ovalbumin aerosol–treated over untreated mice. (E) Blocking anti-TL1A antibody L4G6 suppresses ovalbumin-induced cytokine expression in lung parenchyma. Mice were immunized twice with ovalbumin/alum, as described. 1 d before ovalbumin aerosol and for the next 3 d, mice received 50 μg blocking TL1A antibody L4G6 or control IgG i.p. Lungs were analyzed for expression of cytokine mRNA on day 1–3 after aerosol administration by Taqman PCR, as above. Data are presented as anti-TL1A–induced suppression of cytokine mRNA over control IgG.<p><b>Copyright information:</b></p><p>Taken from "Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1037-1048.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373837.</p><p></p