Eur. J. Immunol. 2005. 35: 2239–2249
Clinical immunology
Clinical immunology
A novel apoptosis-inducing anti-PSGL-1 antibody for T cell-mediated diseases Chiu-Chen Huang, Yi-Fen Lu, Shi-Ni Wen, Wen-Chuan Hsieh, Yu-Chin Lin, Meng-Ru Liu, Evelyn Chiang, Chung-Nan Chang and Rong-Hwa Lin AbGenomics Co., Neihu, Taipei, Taiwan, R.O.C. We previously discovered a hamster monoclonal antibody, TAB4, against mouse PSGL1/CD162 that can induce death of activated T cells. Here, we further investigated the potential of TAB4 in treating two murine models of T cell-mediated diseases. The results showed that administration of TAB4 suppressed incidence and severity of both GVHD and type I diabetes. Analyses of apoptotic T cells ex vivo shortly after antibody injection revealed a higher percentage of apoptosis among activated T cells in the TAB4-treated group than in the control group. Furthermore, restoration of functional donor T cells was observed in TAB4-treated mice. As TAB4 does not affect the binding of P-selectin to activated T cells, our data suggest that its long-lasting therapeutic effect on inhibiting disease progression is attained by specifically inducing apoptosis of activated T cells. These data hence extend our previous finding of the novel property of PSGL-1 and strongly indicate that the PSGL-1-specific apoptosis-inducing antibody is a new therapeutic agent possessing a great potential for controlling GVHD and other T cellmediated autoimmune diseases.
Introduction Abnormal activation of T cells is the prime cause of chronic inflammatory responses, which in turn lead to several diseases such as autoimmune diseases, allergic reaction, transplantation rejection and graft-versus-host disease (GVHD). Despite the fact that the activity of the potentially aggressive T cells can be controlled by immunosuppressants [1], repeated or even life-long treatment is required and often accompanied with profound systemic side effects. Reports have shown that depleting T cells in the donor marrow prior to transplantation efficiently ameliorated the incidence or severity of GVHD, yet it was also associated with The first two authors contributed equally to this study. Correspondence: Rong-Hwa Lin, AbGenomics Co., 2F No. 32 Lane 358, Juikuang Rd., Neihu, Taipei, Taiwan, R.O.C. 114 Fax: +886-2-26272708 e-mail:
[email protected] Abbreviations: AIF: Apoptosis-inducing factor PSGL-1: P-selectin glycoprotein ligand-1 SCID: Severe combined immunodeficiency f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received 15/11/05 Revised 29/4/05 Accepted 23/5/05 [DOI 10.1002/eji.200525849]
Key words: Graft-vs.-host disease Apoptosis Autoimmunity Antibodies T lymphocytes
higher rates of graft failure, leukemia relapse and opportunistic viral infections [2]. Similar difficulties have been observed in treating other T cell-mediated diseases. Therefore, strategies that could specifically target and remove the disease-causing T cells while sparing a functional T cell repertoire should be exploited. Apoptotic cell death has long been recognized as a primary feature in a variety of biological processes, including shaping the T cell repertoire during development, homeostasis and regulation of immune responses [3]. Appropriate use of ligands or antibodies recognizing the death receptors should succeed in removing abnormally activated T cells. However, attempts to use Fas or TNFR2 molecules in treating diseases have been hampered by the fact that these molecules are expressed not only on immune cells, but also on several other important organ systems such as liver [4, 5]. Recently, we described a novel property of P-selectin glycoprotein ligand-1 (PSGL-1) that can be triggered by antibody ligation and that can induce the activated T cells to undergo apoptosis [6]. Using the monoclonal antibody TAB4 generated in our laboratory, we demonwww.eji.de
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strated that cross-linking of PSGL-1 on activated T cells initiates a non-classical apoptosis pathway, which is caspase independent and involves mitochondrial release of apoptosis-inducing factors (AIF) and cytochrome c. Taking advantage of this important discovery, we further explored the potential of anti-PSGL-1 antibody in elimination of disease-causing T cells in vivo as a therapeutic strategy for GVHD and autoimmune diabetes. The results indicated that administration of TAB4 antibody efficiently caused death of activated T cells, resulting in not only amelioration of type I diabetes but also likely induction of antigen-specific tolerance in GVHD.
Results Exposure to TAB4 preferentially induces apoptosis of activated T cells in vitro In the previous report, we showed that TAB4, which recognizes mouse PSGL-1 (CD162), can induce apoptosis of Con A-activated or antigen-stimulated mouse T cells [6]. To clarify whether TAB4 has differential effects on activated and resting T cells when both populations are present, apoptosis was examined in a mixed culture of Con A-activated T cells and freshly
Eur. J. Immunol. 2005. 35: 2239–2249
isolated naive splenocytes, incubated with TAB4 or control antibody. In order to imitate the physiological conditions in which activated T cells comprise less than 10% of total T cells, we purposely set the ratio of naive splenic T cells to Con A-activated T cells as 10:1. CD25 was used as a marker for T cell activation in the current studies. The enhanced expression of CD25 correlates with the phenotypes CD43hi, CD44hi and CD62Llo (data not shown), the molecules that are also T cell activation markers besides CD25 [7–9]. After 6 h of incubation, the percentage of antibody-specific cell death was determined separately in activated (CD3+ CD25+) and resting (CD3+ CD25–) T cells using annexin V staining. The result was normalized with the starting percentages of each group and is shown in Fig. 1. We found that TAB4 induced significantly higher apoptosis in activated T cells as opposed to resting T cells (at 3 and 10 lg/ ml). This effect is TAB4 specific as no apoptosis was observed in cultures treated with control Ig. Although PSGL-1 is expressed on most murine hematopoietic cells, TAB4 does not induce significant apoptosis above background on resting B cells, CD11b+ cells, LPSactivated B cells and macrophages (Table 1). These results thus demonstrate that TAB4 preferentially induces apoptosis of activated T cells, even when they are tenfold outnumbered by resting T cells.
Fig. 1. TAB4 antibody induces apoptosis of activated T cells. Con A-activated mouse splenocytes (104 cells) were co-cultured with freshly prepared splenocytes (1.2105 cells) in the presence of TAB4 or hamster Ig (HIg) at the indicated concentrations. After 6 h of incubation, apoptosis of activated T cells (CD25+) or resting cells (CD25–) was analyzed separately. Each bar represents the value of (the percentage of annexin V-positive cells)/(the percentage of the respective cells in the starting mixture) 100%. The data shown are representative of three mice per group from four independent experiments. The asterisk indicates a significant difference (p
