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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-07-2157. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-07-2157v1 101/2/621    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kuwana, M. Articles by Ikeda, Y. Search for Related Content PubMed PubMed Citation Articles by Kuwana, M. Articles by Ikeda, Y. Related Collections Hemostasis, Thrombosis, and Vascular Biology Immunobiology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 January 2003, Vol. 101, No. 2, pp. 621-623 IMMUNOBIOLOGY Brief report Suppression of autoreactive T-cell response to glycoprotein IIb/IIIa by blockade of CD40/CD154 interaction: implications for treatment of immune thrombocytopenic purpura Masataka Kuwana, Yutaka Kawakami, and Yasuo Ikeda From the Institute for Advanced Medical Research and Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.     Abstract Top Abstract Introduction Study design Results and discussion References The potential immunosuppressive effect of an anti-CD154 monoclonal antibody (mAb) on the pathogenic autoreactive T-cell response was evaluated using an in vitro culture system with glycoprotein IIb/IIIa (GPIIb/IIIa)-reactive T cells from patients with immune thrombocytopenic purpura (ITP). The anti-CD154 mAb did not inhibit T-cell proliferation, but suppressed anti-GPIIb/IIIa antibody production, in bulk peripheral blood mononuclear cell cultures stimulated with GPIIb/IIIa. Repeated antigenic stimulation of GPIIb/IIIa-reactive CD4+ T-cell lines in the presence of anti-CD154 mAb resulted in the loss of proliferative capacity and helper function for promoting anti-GPIIb/IIIa antibody production. These anergic T-cell lines showed a cytokine profile of low interferon  and high interleukin 10 and suppressed anti-GPIIb/IIIa antibody production. Our results indicate that blockade of the CD40/CD154 interaction induces generation of autoantigen-specific anergic CD4+ T cells with regulatory function and could be a therapeutic option for suppressing pathogenic autoimmune responses in patients with ITP. (Blood. 2003;101:621-623) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet destruction caused by antiplatelet autoantibodies, which mainly target glycoprotein IIb/IIIa (GPIIb/IIIa).1,2 We have recently found that GPIIb/IIIa-reactive CD4+ T cells from patients with ITP have helper activity that promotes production of anti-GPIIb/IIIa antibodies capable of binding to normal platelets, indicating that these autoreactive T cells are involved in the pathogenic process of ITP.3-5 Therefore, the GPIIb/IIIa-reactive T cell is a reasonable target for a therapeutic strategy that selectively suppresses the pathogenic autoimmune response in patients with ITP. One candidate strategy is the disruption of a costimulatory signal by blocking the interaction between CD40 on antigen-presenting cells (APCs) and CD154 (also known as CD40 ligand) on activated CD4+ T cells; this interaction is essential for the T cell-dependent humoral immune response.6,7 The efficacy of blocking this interaction therapeutically with anti-CD154 monoclonal antibody (mAb) has been shown in animal models for various autoimmune diseases.8-10 Thus, blockade of the CD40/CD154 interaction has been proposed as a strategy for treating autoimmune diseases11 and is currently being used in preclinical and clinical studies.12,13 In animal models, the CD40/CD154 blockade inhibits the expansion and effector functions of pathogenic autoreactive T cells9,10 and even induces long-term antigen-specific tolerance,14 although the details of the immunoregulatory action remain to be elucidated. In this study, to examine the potential of applying CD40/CD154-targeted intervention to the treatment of ITP, we investigated the ability of anti-CD154 mAb to suppress the T-cell response to GPIIb/IIIa in patients with ITP using in vitro culture systems.     Study design Top Abstract Introduction Study design Results and discussion References Patients We studied cells from 5 patients with ITP (ITP1, 10, 11, 14, and 19), all of whom had been enrolled in our previous study.4 All samples were obtained after the patients gave their written informed consent, approved by the Institutional Review Board of Keio University School of Medicine, Tokyo. Effects of anti-CD154 mAb on GPIIb/IIIa-induced T-cell responses in bulk T-cell cultures Peripheral blood mononuclear cells (PBMCs) were used to analyze the direct effects of anti-CD154 mAb on T-cell proliferation and anti-GPIIb/IIIa antibody production induced by trypsin-digested human GPIIb/IIIa.3 IgG anti-GPIIb/IIIa antibodies in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) using purified human GPIIb/IIIa as an antigen.3 Serial dilutions of anti-CD154 mAb (0.25-2 µg/mL; Ancell, Bayport, MN), or anti-HLA-DR, anti-HLA-DQ, or isotype-matched control mAb (1 µg/mL; Leinco Technologies, Ballwin, MO) were added at the initiation of the cultures.15 Repeated treatment of GPIIb/IIIa-reactive T-cell lines with anti-CD154 mAb We used 2 GPIIb/IIIa-reactive CD4+ T-cell lines, SiM4 and WY9, generated from ITP14 and ITP10, respectively. These lines had a Th0 cytokine profile with the capacity to induce production of anti-GPIIb/IIIa antibodies that bound to intact platelets.4 SiM4 and WY9 recognized recombinant glutathione S-transferase fusion proteins encompassing amino acids 18-259 of GPIIb (IIb18-259) and 22-262 of GPIIIa (IIIa22-262), respectively, in an HLA-DR-restricted manner.4 Two tetanus toxoid (TT)-reactive CD4+ T-cell lines generated from a healthy donor were used as controls.16 After being rested for 10 days, T-cell lines (2 × 105) were stimulated with IIb11-259, IIIa22-262, or TT (5 µg/mL), interleukin 2 (IL-2; 50 U/mL), and irradiated autologous lymphoblastoid B-cell line (106) as APCs for 7 days in the presence of anti-CD154 or isotype-control mAb (2 µg/mL). This treatment was repeated for up to 5 rounds. The viable T cells were recovered and subsequently examined for their ability to proliferate and promote anti-GPIIb/IIIa antibody production from autologous B cells in response to antigenic stimulation.4 In some experiments, anti-CD154 mAb-treated GPIIb/IIIa-reactive T-cell line WY9 was washed twice, serially diluted, and added to cultures of untreated WY9 and autologous B cells to evaluate its effect on anti-GPIIb/IIIa antibody production in the presence or absence of anti-IL-10 mAb (10 µg/mL; R & D Systems, Minneapolis, MN). To determine cytokine profiles of GPIIb/IIIa-reactive T-cell lines treated with anti-CD154 or isotype-control mAb, T cells were stimulated with phorbol myristate acetate and ionomycin for 48 hours, and the amounts of interferon  (IFN-), IL-2, IL-4, IL-6, and IL-10 in culture supernatants were measured using commercial ELISA kits (Biosource International, Camarillo, CA).16 Statistical analysis All comparisons were tested for statistical significance using the Mann-Whitney U test.     Results and discussion Top Abstract Introduction Study design Results and discussion References The effects of anti-CD154 mAb on T-cell responses induced by trypsin-digested GPIIb/IIIa were examined in PBMC cultures from 5 patients with ITP, and similar results were obtained from all samples. As shown in Figure 1A-B, anti-CD154 mAb had no effect on the proliferative response of T cells with GPIIb/IIIa, but inhibited anti-GPIIb/IIIa antibody production in a dose-dependent manner. A similar inhibitory effect of anti-CD154 mAb on in vitro T cell-dependent antibody production was reported for an anti-TT antibody response in human splenocyte cultures17 as well as autoantibody responses in patients with autoimmune diseases.15,18,19 View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of anti-CD154 mAb on GPIIb/IIIa-induced T-cell responses in bulk PBMC cultures and GPIIb/IIIa-reactive CD4+ T-cell lines. (A) PBMCs from patient ITP14 were stimulated with trypsin-digested GPIIb/IIIa (5 µg/mL) for 7 days in the presence or absence of anti-HLA-DR (1 µg/mL), anti-HLA-DQ (1 µg/mL), anti-CD154 (0.5, 1, and 2 µg/mL), or isotype-matched control mAb (2 µg/mL), and [3H]thymidine incorporation was measured by liquid scintillation counting. The results shown are the means ± SDs of quadruplicate values. Asterisk indicates significant inhibition of T-cell proliferation by the mAb treatment in comparison with the culture with isotype-control mAb. (B) PBMCs from ITP14 were cultured with trypsin-digested GPIIb/IIIa (5 µg/mL) and pokeweed mitogen (1 µg/mL) for 10 days in the presence or absence of anti-HLA-DR (1 µg/mL), anti-HLA-DQ (1 µg/mL), anti-CD154 (0.25, 0.5, and 1 µg/mL), or isotype-matched control mAb (1 µg/mL), and IgG anti-GPIIb/IIIa antibody levels were measured by ELISA. The results shown are the means ± SDs of duplicate values. Asterisks indicate significant inhibition of IgG anti-GPIIb/IIIa antibody production by the mAb treatment compared with the culture with isotype-matched control mAb. (C) GPIIb/IIIa-reactive CD4+ T-cell lines SiM4 and WY9 (2 × 105) were repeatedly treated with irradiated autologous lymphoblastoid B-cell line (106), recombinant fragment (IIb18-252 for SiM4 or IIIa22-262 for WY9; 5 µg/mL), and IL-2 (50 U/mL) in the presence of anti-CD154 or isotype-control mAb (2 µg/mL), and examined for their capacity to proliferate on stimulation with the antigenic recombinant fragment or glutathione S-transferase in quadruplicate cultures. Results are expressed as a stimulation index, which was calculated as the mean counts per minute incorporated into cultures with the antigenic recombinant fragment divided by the mean counts per minute incorporated into the cultures with glutathione S-transferase. A result representative of 3 independent experiments is shown. (D) Lines SiM4 and WY9, which were repeatedly treated with anti-CD154 or isotype-matched control mAb, were examined for their capacity to stimulate autologous B cells to produce IgG anti-GPIIb/IIIa antibodies on stimulation with IIb18-252 (SiM4) or IIIa22-262 (WY9). Results shown are the means ± SDs of duplicate values. Asterisks indicate significant inhibition of anti-GPIIb/IIIa antibody production by treatment with anti-CD154 mAb compared with the treatment with isotype-control mAb. The IgG anti-GPIIb/IIIa antibody levels in cultures of B cells alone were 0.020 ± 0.005 (SiM4) and 0.085 ± 0.011 (WY9). The results were similar in 3 independent experiments. It has been proposed that failure to deliver the essential costimulatory signal during the T cell-APC interaction would result in a state of T-cell anergy, a cellular state in which T cells fail to proliferate when optimally restimulated by an antigen.20 To test whether blockade of the CD40/CD154 interaction induces an anergic state in autoreactive T cells, GPIIb/IIIa-reactive T-cell lines were repeatedly stimulated by a GPIIb/IIIa fragment, APCs, and anti-CD154 mAb. The number of viable cells recovered apparently decreased after 2 rounds of the anti-CD154 mAb treatment, and too few T cells to analyze were recovered after 4 rounds of treatment. As shown in Figure 1C, antigen-specific proliferation of GPIIb/IIIa-reactive T cells gradually decreased after repeated anti-CD154 mAb treatment, but not after treatment with isotype-matched control mAb. The anti-CD154 mAb-treated T cells lost their helper activity for promoting anti-GPIIb/IIIa antibody production (Figure 1D). The treatment of GPIIb/IIIa-reactive T cells with TT or without antigen and the treatment of TT-reactive T-cell lines with IIb18-259 failed to induce an anergic state, indicating that anti-CD154 mAb-induced T-cell anergy is antigen dependent and antigen specific. In GPIIb/IIIa-reactive T-cell lines treated twice with anti-CD154 mAb, production of IFN-, IL-2, and IL-6 was greatly reduced, but IL-10 production was preserved (Figure 2A), resulting in a cytokine profile with low IFN- and high IL-10, similar to the profile of CD4+ regulatory T cells.21 The anergic GPIIb/IIIa-reactive T-cell line WY9 inhibited anti-GPIIb/IIIa antibody production mediated by untreated WY9 (Figure 2B). Neutralization of IL-10 by anti-IL-10 mAb abolished the suppressive effect of the anti-CD154 mAb-treated anergic T cells, indicating that the effector function of GPIIb/IIIa-reactive CD4+ T cells could be suppressed by bystander GPIIb/IIIa-reactive anergic T cells, partly through the relatively augmented IL-10 production. View larger version (27K): [in this window] [in a new window]   Figure 2. Cytokine profiles and regulatory function of GPIIb/IIIa-reactive CD4+ T-cell lines treated twice with anti-CD154 mAb. GPIIb/IIIa-reactive CD4+ T-cell lines SiM4 and WY9 were cultured twice with autologous lymphoblastoid B-cell line, antigenic recombinant fragment, and IL-2 in the presence of anti-CD154 or isotype-matched control mAb. (A) The T-cell lines treated with anti-CD154 or isotype-matched control mAb (2 × 105) were stimulated with phorbol myristate acetate (1 µg/mL) and ionomycin (25 ng/mL) for 48 hours, and the levels of IFN-, IL-2, IL-4, IL-6, and IL-10 in the culture supernatants were measured by ELISA. The results shown are the means ± SDs of triplicate values. Asterisks indicate significant differences in cytokine levels between the anti-CD154 mAb-treated and isotype-matched control mAb-treated T-cell lines. The results of 1 of 2 experiments with similar results are shown. (B) Line WY9 (3 × 105) was cultured with autologous B cells (3 × 105), IIIa22-262 (5 µg/mL), and pokeweed mitogen (1 µg/mL) for 10 days in the presence or absence of serially diluted WY9-treated twice with anti-CD154 mAb (0.5 to 2 × 105) or treated with isotype-matched control mAb (2 × 105). Anti-IL-10 mAb (10 µg/mL) was added at the start of some cultures. IgG anti-GPIIb/IIIa antibodies in culture supernatants were measured by ELISA. The results shown are the means ± SDs of duplicate values. Asterisks indicate significant difference between anti-GPIIb/IIIa antibody levels in cultures with and without the anti-CD154 mAb-treated WY9 cells. The results of 1 of 2 experiments with similar results are shown. Several mechanisms for the in vivo suppressive effects of anti-CD154 mAb on the specific T-cell response have been proposed, including suppression of Th1-type immune response,9,10 up-regulation of cytotoxic T lymphocyte-associated antigen 4 on T cells,10 and induction of regulatory cells sharing properties of natural killer and dendritic cells.22 Here, using a human in vitro culture system, we found a novel mechanism, that is, suppression of T-cell effector function through the induction of antigen-specific anergic T cells with potential regulatory function. The in vitro suppressive effect of anti-CD154 mAb on the pathogenic GPIIb/IIIa-reactive T-cell responses strongly suggests potential utility of CD40/CD154-targeted immune interventions in therapy for ITP and other autoimmune diseases. However, anti-CD154 mAb has been reported to induce thromboembolic events in primates and humans.23 Therefore, when anti-CD154 mAb is used therapeutically, we have to pay special attention to its potential effects on formation and stabilization of thrombi, which are mediated by CD154 expressed on activated platelets.24,25     Footnotes Submitted July 19, 2002; accepted August 9, 2002. Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-07-2157. Supported by the Keio University Medical Science Fund, by a grant from the Japanese Ministry of Health and Welfare, and by the Terumo Life Science Foundation. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Masataka Kuwana, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; e-mail: kuwanam{at}sc.itc.keio.ac.jp.     References Top Abstract Introduction Study design Results and discussion References 1. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med. 2002;346:995-1008[Free Full Text]. 2. Karpatkin S. Autoimmune (idiopathic) thrombocytopenic purpura. Lancet. 1997;349:1531-1536[CrossRef][Medline] [Order article via Infotrieve]. 3. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb/IIIa in immune thrombocytopenic purpura: role in production of anti-platelet autoantibody. J Clin Invest. 1998;102:1393-1402[Abstract/Free Full Text]. 4. Kuwana M, Kaburaki J, Kitasato H, et al. Immunodominant epitopes on glycoprotein IIb/IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura. Blood. 2001;98:130-139[Abstract/Free Full Text]. 5. Kuwana M, Okazaki Y, Kaburaki J, Kawakami Y, Ikeda Y. Spleen is a primary site for activation of platelet-reactive T and B cells in patients with immune thrombocytopenic purpura. J Immunol. 2002;168:3675-3682[Abstract/Free Full Text]. 6. Laman JD, Claassen E, Noelle RJ. Functions of CD40 and its ligand, gp39 (CD40L). Crit Rev Immunol. 1996;16:59-108[Medline] [Order article via Infotrieve]. 7. van Kooten C, Banchereau J. CD40-CD40 ligand. J Leukoc Biol. 2000;67:2-17[Abstract]. 8. Mohan C, Shi Y, Laman JD, Datta SK. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J Immunol. 1995;154:1470-1480[Abstract/Free Full Text]. 9. Howard LM, Miga AJ, Vanderlugt CL, et al. Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis. J Clin Invest. 1999;103:281-290[Abstract/Free Full Text]. 10. Im SH, Barchan D, Maiti PK, Fuchs S, Souroujon MC. Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4. J Immunol. 2001;166:6893-6898[Abstract/Free Full Text]. 11. Datta SK, Kalled SL. CD40-CD40 ligand interaction in autoimmune disease. Arthritis Rheum. 1997;40:1735-1745[Medline] [Order article via Infotrieve]. 12. Kirk AD, Burkly LC, Batty DS, et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat Med. 1999;5:686-693[CrossRef][Medline] [Order article via Infotrieve]. 13. Davis JC Jr, Totoritis MC, Rosenberg J, Sklenar TA, Wofsy D. Phase I clinical trial of a monoclonal antibody against CD40-ligand (IDEC-131) in patients with systemic lupus erythematosus. J Rheumatol. 2001;28:95-101[Medline] [Order article via Infotrieve]. 14. Kenyon NS, Chtzipetrou M, Masetti M, et al. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys with humanized anti-CD154. Proc Natl Acad Sci U S A. 1999;96:8132-8137[Abstract/Free Full Text]. 15. Kuwana M, Medsger TA Jr, Wright TM. T-B cell collaboration is essential for the autoantibody response to DNA topoisomerase I in systemic sclerosis. J Immunol. 1995;155:2703-2714[Abstract]. 16. Kuwana M, Kaburaki J, Wright TM, Kawakami Y, Ikeda Y. Induction of antigen-specific human CD4+ T cell anergy by peripheral blood DC2 precursors. Eur J Immunol. 2001;31:2547-2557[CrossRef][Medline] [Order article via Infotrieve]. 17. Brams P, Black A, Padlan EA, et al. A humanized anti-human CD154 monoclonal antibody blocks CD154-CD40 mediated human B cell activation. Int Immunopharmacol. 2001;1:277-294[CrossRef][Medline] [Order article via Infotrieve]. 18. Kuwana M, Medsger TA Jr, Wright TM. Analysis of soluble and cell surface factors regulating anti-DNA topoisomerase I autoantibody production demonstrates synergy between Th1 and Th2 autoreactive cells. J Immunol. 2000;164:6138-6146[Abstract/Free Full Text]. 19. Arai T, Yoshida K, Kaburaki J, et al. Autoreactive CD4+ T cell clones to 2-glycoprotein I in patients with antiphospholipid syndrome: preferential recognition of the major phospholipid-binding site. Blood. 2001;98:1889-1896[Abstract/Free Full Text]. 20. Schwartz RH. Models of T cell anergy: is there a common molecular mechanism? J Exp Med. 1996;184:1-8[Medline] [Order article via Infotrieve]. 21. Groux H, O'Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997;389:737-742[CrossRef][Medline] [Order article via Infotrieve]. 22. Homann D, Jahreis A, Wilfe T, et al. CD40L blockade prevents autoimmune diabetes by induction of bitypic NK/DC regulatory cells. Immunity. 2002;16:403-415[Medline] [Order article via Infotrieve]. 23. Kawai T, Andrews D, Colvin RB, Sachs DH, Cosimi AB. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand [letter]. Nat Med. 2000;6:114[CrossRef]. 24. Henn V, Steinbach S, Büchner K, Presek P, Kroczek RA. The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40. Blood. 2001;98:1047-1054[Abstract/Free Full Text]. 25. André P, Srinivasa KS, Denis CV, et al. CD40L stabilizes arterial thrombi by a 3 integrin-dependent mechanism. Nat Med. 2002;8:247-252[CrossRef][Medline] [Order article via Infotrieve]. © 2003 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: A. Solanilla, J.-M. Pasquet, J.-F. Viallard, C. Contin, C. Grosset, J. Dechanet-Merville, M. Dupouy, M. Landry, F. Belloc, P. Nurden, et al. Platelet-associated CD154 in immune thrombocytopenic purpura Blood, January 1, 2005; 105(1): 215 - 218. [Abstract] [Full Text] [PDF] R. Stasi and D. Provan Management of Immune Thrombocytopenic Purpura in Adults Mayo Clin. Proc., April 1, 2004; 79(4): 504 - 522. [Abstract] [PDF] M. Kuwana, S. Nomura, K. Fujimura, T. Nagasawa, Y. Muto, Y. Kurata, S. Tanaka, and Y. Ikeda Effect of a single injection of humanized anti-CD154 monoclonal antibody on the platelet-specific autoimmune response in patients with immune thrombocytopenic purpura Blood, February 15, 2004; 103(4): 1229 - 1236. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-07-2157v1 101/2/621    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kuwana, M. Articles by Ikeda, Y. Search for Related Content PubMed PubMed Citation Articles by Kuwana, M. Articles by Ikeda, Y. Related Collections Hemostasis, Thrombosis, and Vascular Biology Immunobiology Brief Reports Social Bookmarking                   What's this?

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