OX40 (CD134) Controls Memory T Helper 2 Cells that Drive Lung Inflammation

Asthma is caused by memory Th2 cells that often arise early in life and persist after repeated encounters with allergen. Although much is known regarding how Th2 cells develop, there is little information about the molecules that regulate memory Th2 cells after they have formed. Here we show that the costimulatory molecule OX40 is expressed on memory CD4 cells. In already sensitized animals, blocking OX40–OX40L interactions at the time of inhalation of aerosolized antigen suppressed memory effector accumulation in lung draining lymph nodes and lung, and prevented eosinophilia, airway hyperreactivity, mucus secretion, and Th2 cyto-kine production. Demonstrating that OX40 signals directly regulate memory T cells, antigen-experienced OX40-deficient T cells were found to divide initially but could not survive and accumulate in large numbers after antigen rechallenge. Thus, OX40–OX40L interactions are pivotal to the efficiency of recall responses regulated by memory Th2 cells

Critical Role of the Programmed Death-1 (PD-1) Pathway in Regulation of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon γ–producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE

Induction of Tumor-specific T Cell Immunity by Anti-DR5 Antibody Therapy

Because tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells and plays a critical role in tumor surveillance, its receptor is an attractive target for antibody-mediated tumor therapy. Here we report that a monoclonal antibody (mAb) against the mouse TRAIL receptor, DR5, exhibited potent antitumor effects against TRAIL-sensitive tumor cells in vivo by recruiting Fc receptor–expressing innate immune cells, with no apparent systemic toxicity. Administration of the agonistic anti-DR5 mAb also significantly inhibited experimental and spontaneous tumor metastases. Notably, the anti-DR5 mAb-mediated tumor rejection by innate immune cells efficiently evoked tumor-specific T cell immunity that could also eradicate TRAIL-resistant variants. These results suggested that the antibody-based therapy targeting DR5 is an efficient strategy not only to eliminate TRAIL-sensitive tumor cells, but also to induce tumor-specific T cell memory that affords a long-term protection from tumor recurrence

OR.107. TIM-1 Plays a Crucial Role in the Expansion of Autopathogneic T-Cells and Regulation of Autoimmunity [abstract only]

T-cell immunoglobulin and mucin (TIM) family Members are differentially expressed on Th1 and Th2 cells. Polymorphisms of TIM-1 have been associated with susceptibility to asthma; however, its role in regulating autoimmunity has not been studied. Here, we have used an agonistic antiTIM-1 antibody (Ab, Clone 3B3) which has previously been shown to costimulate T-cell activation and expansion, to analyze the role of TIM-1 in the development and regulation of experimental autoimmune encephalomyelitis (EAE). Treatment with 3B3 dramatically enhances the severity of EAE as well as the frequency of encephalitogenic CD4+ T-cells and the production of IFN-g and IL-17 by these cells. Furthermore, administration of 3B3 breaks self-tolerance and induces EAE in the disease resistant B10.S strain. We have utilized another anti-TIM-1 Ab (RMT1-10) that does not costimulate T-cell activation in vitro. In contrast to 3B3, treatment with RMT1-10 inhibits the development of EAE and reduces the frequency of encephalitogenic CD4+ T-cells with a commensurate decrease in the production of IFN-g and IL-17. Treatment with RMT1-10 causes CD4+ T-cells to produce more IL-4 and IL-10. We provide evidence that both 3B3 and RMT1-10 bind to the same epitope in the Ig domain of TIM-1, but the binding affinity of 3B3 is much higher than that of RMT1-10. These data suggest that TIM-1 engagement with the agonistic Ab, along with TcR ligation, costimulates T-cell expansion with pro-inflammatory IFN-g and IL-17 production resulting in the breakdown of self-tolerance and development of autoimmunity, whereas blocking anti-TIM-1 Ab causes a decrease in the autopathogenic Th1/ThIL-17 responses. This study demonstrates that TIM-1 is a key cell surface molecule that regulates effector T-cell response and depending on hopw the molecule is engaged, autoimmune responses can be either enhanced or inhibited in vivo

Expression and Function of Inducible Costimulator on Peripheral Blood CD4 ؉ T Cells in Behçet's Patients with Uveitis: A New Activity Marker?

PURPOSE. Inducible costimulator (ICOS) is an important costimulatory molecule involved in T-cell activation. In this study, the role of ICOS in the pathogenesis of uveitis in Behçet's disease (BD) was investigated. METHODS. Peripheral blood mononuclear cells (PBMCs) were obtained from BD patients with uveitis in the active or remission phase and in healthy subjects. Total RNA was isolated from PMBCs, and mRNA expression was analyzed on an oligonucleotide microarray. ICOS expression on CD4 ϩ T cells was determined by flow cytometry, and the functional costimulatory effect of ICOS/B7RP-1 interaction was assessed on stimulation with concanavalin A (conA) or IRBP in the presence or absence of anti-ICOS mAb. RESULTS. As the result of microarray analysis, ICOS in PBMCs showed the greatest difference in expression in BD patients with uveitis compared with healthy control subjects. ICOS expression on CD4 ϩ T cells in BD patients with uveitis was significantly higher than that in healthy individuals, both before and after conA stimulation. Among the BD patients, ICOS expression on CD4 ϩ T cells was significantly higher in those with active uveitis than in those with remitted uveitis. Blockade of ICOS/B7-related protein-1 (B7RP-1) interaction by anti-ICOS mAb significantly decreased IFN-␥, IL-17, and TNF-␣ production by PBMCs when stimulated with conA or IRBP in BD with active uveitis. CONCLUSIONS. High ICOS expression in BD patients with uveitis contributed to the upregulation of IFN-␥, IL-17, and TNF-␣ production, suggesting that abnormal ICOS costimulation may play an immunopathologic role in the pathogenesis of uveitis in BD. (Invest Ophthalmol Vis Sci. 2010;51:5099 -5104) DOI: 10.1167/iovs.10-5286 E ndogenous uveitis such as Behçet's disease (BD), VogtKoyanagi-Harada syndrome, sympathetic ophthalmia, birdshot retinochoroidopathy, and sarcoidosis is a potentially blinding disease in humans and is responsible for 10% to 15% of the acquired blindness in Japan. 1 Although endogenous uveitis covers a spectrum of clinical entities, all forms are believed to share immunohistologic similarities characterized by the infiltration of mainly T cells. Behçet's disease (BD) is a systemic inflammatory disease characterized by oral and genital ulcers as well as ocular, cutaneous, arthritic, vascular, and neurologic lesions. 3-5 An increasing number of reports indicate that aberrant cellular immunity, such as pathogenic CD4 ϩ T-cell[b]-mediated autoimmunity via the Th1/Th17 pathway, plays a key role in the pathophysiological process in BD with uveitis, 6 -10 although the mechanisms of ocular inflammation in BD remain largely unknown. During recent years, the understanding of immunologic mechanisms involved in uveitis has advanced greatly through investigations of experimental autoimmune uveoretinitis (EAU), an animal model of human uveitis that can be induced by immunization of susceptible animals with interphotoreceptor retinoid-binding protein (IRBP) or with an eye-specific retinal antigen or by adoptive transfer of CD4 ϩ T cells specific for retinal antigens. 11 EAU resembles certain human uveitic conditions in various aspects 14,15 Although CD28 regulation has substantial effects on immunity, its function appears to reside predominantly in the control of primary, but not secondary, immune responses in various autoimmune diseases. 16 -18 The CD28 homolog inducible costimulator (ICOS) has recently been identified as a novel member of the CD28 costimulator family and is expressed by activated T cells in both humans and mice. To identify new genes that may cause or contribute to the disease process of ocular BD, we used cDNA microarrays that provide the expression profile of more than 54,000 genes, some of which are immune-related, whereas others are involved in the cell cycle, cell growth, intracellular signaling, cellular adhesion, and transport, and we compared the expression profiles of healthy individuals and patients. One of the genes found to be upregulated in patients with ocular BD is ICOS, an activation marker expressed on activated T cells that binds B7RP-1-expressing monocytes. The engagement of ICOS with B7RP-1 along with an appropriate antigen provide a positive signal that promotes T-cell differentiation, cytokine secretion, and effector function in the absence of CD28. ϩ T cells and B7RP-1 expressed on infiltrating APCs are upregulated directly after disease onset. 30 Therefore, we speculated that in active BD, pathogenic CD4 ϩ T cells that infiltrate the eye express ICOS in the inflamed eye and would be an appropriate target for the treatment of human ocular BD. Moreover, we have demonstrated that blockade of the ICOS/ B7RP-1 costimulatory pathway inhibits ocular inflammation in the effector phase of murine EAU. 30 Therefore, to investigate the role of ICOS in the pathogenesis of ocular BD, we assessed its expression on peripheral blood CD4 ϩ T cells and functional roles in patients with ocular BD. The results suggest that upregulation of ICOS is associated with the pathogenesis of uveitis in BD. MATERIALS AND METHODS PBMC Samples Thirty-five patients with BD (27 men and 8 women, mean age; 37.8 Ϯ 11.1 years) were enrolled in the study Heparinized blood samples were obtained from patients and healthy individuals, and PBMCs were isolated within 2 hours by density gradient centrifugation. The PBMCs were washed twice and resuspended at 1 ϫ 10 6 cells/mL in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 1 mM L-glutamine, 100 U/mL penicillin, and 100 g /mL streptomycin). Cell suspension was dispensed in 24-well plates (Falcon; BD Biosciences, Mountain View, CA) at 1 mL/well and incubated with or without concanavalin A (ConA; 10 g/mL) for 12 hours. Analysis of Gene Expression with cDNA Array Gene expression profile of human PBMCs was analyzed by microarray, as described previously. 37 Briefly, total RNA was isolated from pooled PBMCs obtained from seven patients with BD and active uveitis by an extraction method (Isogen Nippon Gene, Tokyo, Japan), and portions (2 g) of the preparation were subjected to amplification of mRNA with T7 RNA polymerase. Biotin-labeled cRNA (10 g) synthesized from the amplified RNA was subjected to hybridization with the Human Whole Genome Bioarray chip (Amersham Biosciences, Piscataway, NJ) that contains oligonucleotides corresponding to a total of approximately 55,585 human genes. Detection and digitization of hybridization signals were performed (G2565; Agilent Technologies, San Diego, CA) and analyzed (CodeLink Expression Analysis software version 4.0; Amersham Biosciences). The microarray data of various costimulatory molecules are summarized in Reagents FITC-conjugated anti-human CD4 (L3T4) was purchased from eBioscience (San Diego, CA), and anti-human ICOS (DX29) from BD PharMingen (San Diego, CA). ConA was obtained from Vector Laboratories (Burlingame, CA). Preparation of IRBP Fresh swine IRBP was purified according to the method described by Fukai et al. Immunofluorescence and Flow Cytometry The results of ICOS gene expression obtained from microarray were confirmed by flow cytometry. PMBCs were isolated by density gradient centrifugation of heparinized blood samples obtained from patients and healthy individuals. Each PBMC sample was divided into two aliquots: one for direct flow cytometry analysis of ICOS expression and the other for flow cytometry analysis after activation with conA at 10 g/mL for 12 hours. ICOS analysis was performed by using the following procedures: A cocktail of FITC-conjugated anti-CD4 and PE-conjugated anti-ICOS was added to the PBMCs. After the cells were washed with PBS, the stained cells (live-gated based on forward-and sidescatter profiles and propidium iodide exclusion) were passed through a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) and the results analyzed (CellQuest program; BD Biosciences). ICOS expression on human CD4 ϩ T cells was calculated by gating for the CD4 ϩ T cell population, and 10,000 cells were analyzed in each experiment. Cytokine Assay Purified PBMCs (5 ϫ 10 5 /well) from BD patients with active uveitis were cultured in 96-well microtiter plates, with or without stimulation with conA (3 g/mL) or IRBP (10 g/mL) plus anti-ICOS mAb (10 g/mL) or control IgG at 37°C for 48 hours. The unstimulated PBMCs served as negative control samples. After stimulation, cell-free supernatants were collected at 48 hours and assayed for IFN-␥, TNF-␣, IL-2, and IL-6 by cytometric bead array (CBA) kits (BD PharMingen) and IL-17 by ELISA (Human IL-17 Quantikine ELISA kit; R&D Systems, Minneapolis, MN), according to the manufacturers' instructions. The minimum levels detected by CBA kits in either the culture supernatant or serum/plasma were 2.6 pg/mL for IL-2, 3.0 pg/mL for IL-6, 7.1 pg/mL for IFN-␥, and 2.8 pg/mL for TNF-␣; the minimum level in the IL-17 ELISA was 7.8 pg/mL. In a preliminary study, serial concentrations of conA (1, 3, 5, and 10 g/mL) or IRBP (1, 5, and 10 g/mL) were used in the assays. The results showed that optimal concentrations of cytokines were obtained at a concentration of 3 g/mL for conA and 10 g/mL for IRBP, as previously described. Statistical Analysis Data were analyzed (JMP 5; SAS Institute, Inc., Cary, NC) and the results expressed as the mean Ϯ SD. Statistical analysis was performed by using a paired or unpaired t-test. P Ͻ 0.05 was considered significant. RESULTS Analysis of Microarray Results in BD Patients with Active Uveitis To identify the genes involved in the pathogenesis of ocular BD, we compared phosphorescent images of the arrays hybridized with cDNA probes generated from RNA preparations from BD patients with active uveitis and healthy individuals. The array membrane that we used contained 360 positive bacterial controls and 384 negative controls. All positive controls were detected on the microarray. No signals were observed for the negative spots, indicating that the hybridization was highly specific. The DNA screening can evaluate more than 54,000 genes for a single sample. Among these genes, we focused on the immune-related ones, especially those of costimulatory molecules. Among the seven genes of costimulatory molecules, ICOS showed the greatest difference in expression in BD patients with active uveitis compared with that in healthy control subjects ICOS Expression on CD4 ؉ T Cells of BD Patients with Uveitis Flow cytometry was performed to confirm the results of gene array analysis. ICOS expression (median fluorescence intensity [MFI] of ICOS on CD4 ϩ T cells; Comparison of ICOS Expression on CD4 ؉ T Cells in BD Patients with Active or Inactive Uveitis Next, we compared ICOS expression on CD4 ϩ T cells in BD patients with active or inactive uveitis. ICOS expression (MFI of ICOS on CD4 ϩ T cells, Cytokine Production Stimulated with ConA and Uveitogenic Antigen To determine whether ICOS has functional costimulatory activity, we first measured the amounts of pathogenic Th1 and Th17 cytokines (IFN-␥, IL-17, TNF-␣, IL-6, and IL-2) produced by nonspecific stimulation (conA) of PBMCs obtained from BD patients with active uveitis. When ICOS costimulation was blocked with anti-ICOS mAb, the amounts of IFN-␥, IL-17, and TNF-␣ (Figs. 3A-C) produced by PBMCs were both significantly reduced. Since PBMCs from BD patients are known to be sensitized to the two most uveitogenic retina-specific antigens, S-antigen and IRBP, 1 we then examined the amounts of pathogenic Th1 and Th17 cytokines produced by stimulation with swine IRBP protein as the specific antigen. DISCUSSION The precise pathogenesis of uveitis associated with BD is unknown. Accumulated data obtained from animal models such as EAU suggest that Th1 and Th17 cells have major roles IOVS, October 2010, Vol. 51, No. 10 New Activity Marker for Ocular Behçet's Disease 5101 Downloaded from iovs.arvojournals.org on 06/30/2019 in its pathogenesis. We have previously shown that blockade of the ICOS ϩ costimulatory pathway has an ameliorating effect during the effector phase of EAU, by suppressing the expansion and effector function of pathogenic Th1 cells. In conclusion, our data provide additional evidence of the potential utility of ICOS expression on CD4 ϩ T cells as a marker of disease activity and as a promising therapeutic target for ocular BD via its inhibition of Th1 and Th17 cytokines. As the population studied was small and heterogeneous, further studies are needed to confirm the findings

Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti–Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-γ and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti–Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-γ and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti–Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged