A novel multimeric form of FasL modulates the ability of diabetogenic T cells to mediate type 1 diabetes in an adoptive transfer model

Abstract

Activation induced cell death (AICD) via Fas/FasL is the primary homeostatic molecular mechanism employed by the immune system to control activated T-cell responses and promote tolerance to self-antigens. We herein investigated the ability of a novel multimeric form of FasL chimeric with streptavidin (SA-FasL) having potent apoptotic activity to induce apoptosis in diabetogenic T cells and modulate insulin-dependent type 1 diabetes (IDDM) in an adoptive transfer model. Diabetogenic splenocytes from NOD/Lt females were co-cultured in vitro with SA-FasL, SA control protein, or alone without protein, and adoptively transferred into NOD/Lt-Rag1^null recipients for diabetes development. All animals receiving control (Alone: n = 16 or SA: n = 17) cells developed diabetes on average by 6 weeks, whereas animals receiving SA-FasL-treated (n = 25) cells exhibited significantly delayed progression (p < .001) and decreased incidence (70%). This effect was associated with an increase in CD4⁺CD25⁺ T cells and correlated with FoxP3 expression in pancreatic lymph nodes. Extracorporeal treatment of peripheral blood lymphocytes using SA-FasL during disease onset represents a novel approach that may alter the ability of pathogenic T cells to mediate diabetes and have therapeutic utility in clinical management of IDDM.

Introduction

Activation-induced cell death (AICD) via Fas/FasL is an important homeostatic molecular mechanism by which the immune system controls activated T-cell responses, promotes tolerance to self antigens, and prevents autoimmunity. T cells upregulate both Fas and FasL upon activation and become sensitive to autocrine and paracrine apoptosis following repeated reengagement with the challenge antigen (Krammer, 2000, Van Parijs and Abbas, 1998, Ju et al., 1995). The importance of Fas/FasL-mediated apoptosis to tolerance to self antigens is demonstrated in the lpr and gld mice wherein the lack of or low expression of Fas and FasL leads to lymphoproliferative disorders and autoimmunity (Nagata and Suda, 1995). Transgenic replacement of Fas expression in mice restores both AICD and a disease-free state (Wu et al., 1994).

The NOD mouse is a widely used spontaneous model for the study of insulin-dependent type 1 diabetes (IDDM). Although molecular and cellular mechanisms responsible for the development of IDDM in NOD are complex and not fully characterized, activation of autoreactive CD4⁺ and CD8⁺ T cells and their recruitment into pancreatic islets play an important role in the initiation of the disease (Bendelac et al., 1987, Yagi et al., 1992). A series of studies reported resistance to AICD in NOD mice which may contribute to the disease process (Decallonne et al., 2003, Leijon et al., 1994, Arreaza et al., 2003). Although the nature of these defects are not fully understood, over 20 susceptibility loci, termed insulin-dependent diabetes (idd), have been identified (Aoki et al., 2005). Some of these defects translate to dysregulated anti-apoptotic gene expression including Bcl-2 (Garchon et al., 1994) and Bcl-x (Lamhamedi-Cherradi et al., 1998), as well as expression of a less apoptotic allele of FasL (Kayagaki et al., 1997).

Despite these claims, a number of studies have reported that selective induction of apoptosis via AICD in diabetogenic lymphocytes can prevent autoimmune diabetes in NOD mice. In vivo administration of Jo-2, an agonistic antibody to Fas, resulted in diabetes remission. Immunohistochemical analysis of pancreata from treated animals demonstrated apoptosis of infiltrating cells, but not insulin and/or glucagon-producing cells, suggesting that remission was due to the induction of AICD in autoreactive lymphocytes (Dharnidharka et al., 2002). In a second study, lipopolysaccharide (LPS)-activated B cells have been shown to express FasL and induce diabetogenic T cells to undergo apoptosis in vitro (Tian et al., 2001). Transfusion of these LPS-activated B cells into prediabetic mice inhibited spontaneous development of diabetes in greater than 80% of the B-cell treated NOD mice at 52 weeks of age. Furthermore, co-transfer of LPS-activated B cells and diabetogenic splenic T cells prevented the disease in an adoptive transfer model. In addition, both membranous as well as soluble forms of human FasL were shown to engage Fas on naïve and diabetogenic T cells, induce apoptosis, and modulate development of diabetes by eliminating autoreactive diabetogenic lymphocytes and pre-activated CD4⁺CD45RB^low memory T cells (Kim et al., 2000). Of further interest, human soluble FasL was shown to function in vivo to delete potentially autoreactive memory lymphocytes in peripheral blood lymphocytes from mice as well as IDDM patients, complementing membrane FasL in promoting AICD (Kim et al., 2002). Altogether, these studies demonstrate that diabetogenic T cells are sensitive to FasL-mediated apoptosis and as such FasL-based immune intervention may have therapeutic utility for the prevention/treatment of IDDM.

FasL, a type II membranous protein, is expressed on antigen-activated T cells in response to TCR signaling and costimulation (Askenasy et al., 2005). This protein is cleaved off from the cell surface in response to various physiologic stimuli by matrix metalloproteinases (Ju et al., 1995, Tanaka et al., 1998). Traditionally, the membranous form is noted for its ability to induce apoptosis in autoreactive and alloreactive T cells and promote tolerance; in contrast, the soluble form may inhibit apoptosis, initiate inflammatory responses, and promote the active recruitment of neutrophils, thereby accelerating disease or allograft rejection (Ottonello et al., 1999, Seino et al., 1998, Suda et al., 1997). However, this functional difference between membranous and soluble forms of FasL applies to murine FasL more than the human molecule, which appears to have apoptotic function in both forms (Kim et al., 2002, Tanaka et al., 1995).

These diverse functions of soluble and membraneous forms of FasL may be a manifestation of their differential ability to crosslink the Fas receptor upon engagement and the quality/quantity of the ensuing signals (Siegel et al., 2000). We have recently generated a chimeric SA-FasL protein that forms tetramers and higher structures and has potent apoptotic activity on Fas⁺ cells in soluble form. Immunomodulation with biotinylated donor cells decorated with the chimeric molecule resulted in effective elimination of alloreactive T cells, leading to the survival of transplanted islet and cardiac allografts without any sign of general toxicity or chemotactic function on neutrophils (Askenasy et al., 2005, Yolcu et al., 2002, Askenasy et al., 2003). In this study, we tested whether chimeric SA-FasL can be utilized to modulate autoreactive responses by altering the ability of transferred cells to mediate IDDM. Ex vivo treatment of diabetogenic splenocytes with SA-FasL resulted in significant apoptosis of T cells as well as slower progression and lower incidence of disease in an adoptive transfer model. The observed effect was associated with increased percentage and absolute number of CD4⁺CD25⁺ T regulatory cells. The implication of these findings for the use of chimeric FasL to alter autoreactive T cell populations from IDDM patients under extracorporeal conditions as a novel therapeutic intervention is discussed.