ARTHRITIS & RHEUMATISM Vol. 50, No. 6, June 2004, pp 1721–1733 DOI 10.1002/art.20315 © 2004, American College of Rheumatology REVIEW Is Systemic Sclerosis an Antigen-Driven T Cell Disease? Lazaros I. Sakkas1 and Chris D. Platsoucas2 sion of E-selectin (18–20), TGF␤ (21), and endothelin 1 (22,23). Monocyte activation is also reported, as documented by increased production in the peripheral blood of superoxide anion (24), IL-6 (25), IL-1, and TGF␤ (26). Other cells that are found to be activated in SSc include eosinophils (27), mast cells (18), and T cells (28–31). Disease-associated autoantibodies such as anti– topoisomerase I (anti–topo I) antibodies (1), and hypergammaglobulinemia in general, have been observed in patients with SSc, and their production requires both T and B cells (32). However, the mechanism or mechanisms responsible for the initiation of the disease have not been identified. We recently reported the presence of oligoclonal T cells in skin biopsy specimens from patients with SSc (33). The only mechanism that can explain these results is proliferation and clonal expansion of particular clones of T cells in response to specific antigen(s). These results bring the role of T cells in the pathogenesis of SSc to the forefront of the various mechanisms that may contribute to the pathogenesis of the disease. SSc may be a specific antigen–driven T cell disease. Introduction Systemic sclerosis (SSc) is a chronic disease characterized by extensive fibrosis, arterial fibrointimal proliferation, and autoantibody production (1). The disease affects women more often than men and, at least the extensive form, is relatively rare (2). The etiology of SSc is unknown, although certain environmental factors that can cause sclerodermatous changes have been reported (1). The disease may lead to considerable morbidity and mortality (3–5), and the available treatment is largely ineffective. The pathologic hallmark of SSc is excessive collagen deposition and microvascular injury. However, the mechanisms that lead to these changes remain largely unknown. An early skin mononuclear cell infiltrate consisting primarily of T cells and macrophages has been identified (6–9). The degree of mononuclear cell infiltration in the skin of patients with SSc has been shown to correlate well with both the degree and progression of skin thickening (6). The proportions of dermal mast cells are also increased (8). In patients with SSc, fibroblast activation has been documented by the excess production of collagen, fibronectin, and proteoglycans (10), protooncogene c-myc (1), interleukin-1 (IL-1) (11), and adhesion molecules such as intercellular adhesion molecule 1 (12). IL-4 production by Th2 type T cells induces both the production of transforming growth factor ␤ (TGF␤), a fibroblast mitogen (13–15), as well as the production of collagen by fibroblasts (16,17). Endothelial cell activation is evident, as suggested by the expres- T cells in SSc Several lines of evidence suggest that T cells are important in the pathogenesis of SSc. These are listed in Table 1 and are discussed here and in subsequent sections of this review. In sequential skin biopsy specimens from patients with SSc, mononuclear cell infiltrates consisting of T cells and macrophages appear in the skin early (7,34), before any microscopic evidence of fibrosis (7). T cells appear in perivascular areas along with macrophages (Figure 1). T cells with memory phenotype were also found in lung biopsy specimens from patients with SSc and lung involvement (36). Increased numbers of T cells bearing activation markers, such as IL-2 receptor (IL-2R) (28), HLA–DR, and CD29 (28–31), and elevated levels of cytokines such as IL-2 (37–40), IL-4 (37–39), IL-6 (37–39), and IL-17 (41), which indicate T cell activation, were found in the Supported in part by NIH grant R01-AR-48042. 1 Lazaros I. Sakkas, MD, PhD: Temple University School of Medicine, Philadelphia, Pennsylvania, and Thessaly University School of Medicine, Larisa, Greece; 2Chris D. Platsoucas, PhD: Temple University School of Medicine, Philadelphia, Pennsylvania. Address correspondence and reprint requests to Chris D. Platsoucas, PhD, L. H. Carnell Professor and Chairman, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail: email@example.com. Submitted for publication March 26, 2003; accepted in revised form March 5, 2004. 1721 1722 Table 1. Evidence of T cell involvement in systemic sclerosis 1. T cells infiltrate skin early, before any evidence of fibrosis 2. Frequency of activated T cells in blood and skin lesions is increased 3. T cells are Th2 type, producing interleukin-4, which A. Induces fibroblast collagen production B. Induces the production of transforming growth factor ␤, a fibroblast mitogen 4. T cells are necessary for antibody production 5. Graft-versus-host disease, a disease mediated by T cells, has many similarities to systemic sclerosis 6. Treatments directed against T cells ameliorate systemic sclerosis 7. T cells infiltrating skin lesions contain antigen-driven oligoclonal T cells peripheral blood of patients with SSc. Increased protooncogene expression (42) and an increased frequency of hypoxanthine guanine phosphoribosyltransferase gene–mutated T cells (43), both of which are suggestive of T cell activation, were observed in patients with SSc. Increased levels of soluble IL-2R were also found in suction blister fluid from the skin of patients with SSc (44). T cells were found to be necessary for the production of anti–topo I autoantibodies in SSc (32), which, together with other autoantibodies, is a characteristic feature of patients with SSc. Additional evidence for T cell involvement in SSc comes from the sharing of clinical and pathologic characteristics (45,46), including the serologic profile (47), between graft-versus-host disease (GVHD) and SSc. GVHD is mediated by activated T cells and can manifest SAKKAS AND PLATSOUCAS as scleroderma-like disease with an increased frequency of anti–topo I antibodies and an association with particular HLA alleles (47). The various treatment modalities for patients with SSc have also shed light on the role of T cells in the pathogenesis of SSc. Treatments directed against activated T cells, such as cyclosporin A (48,49) and tacrolimus (49) or depletion of T cells (50), have a beneficial effect on skin tightness in patients with SSc. The skin softening that has been observed in certain patients as a result of the treatment was often associated with a reduction in the amount of immature (type III) collagen (51). These treatment approaches against activated T cells were more effective in patients with a short disease duration, in whom inflammation is more intense (48,49). T cell receptors (TCRs) in SSc It is not known whether T cells in SSc are activated nonspecifically (i.e., by cytokines or chemokines) or specifically (i.e., by antigens). T cells use their antigen receptors (TCRs) to recognize, in general, antigenic peptides in association with HLA (classes I and II) and initiate an immune response. Two different TCRs have been reported, the ␣/␤ TCR and the ␥/␦ TCR, which are expressed in a small (usually ⬍10%) proportion of peripheral blood T cells. All 4 TCR chains are highly polymorphic immunoglobulin-like transmembrane proteins. The variable region of the ␣-chain gene consists of V␣ and J␣ gene segments, and the variable Figure 1. Skin lesion in a patient with systemic sclerosis showing the presence of T cells in perivascular areas. Staining was performed using the avidin–biotin complex immunoperoxidase method and an anti-CD3 monoclonal antibody, as described elsewhere (35). (Original magnification ⫻ 100 in A; ⫻ 400 in B.) ROLE OF T CELLS IN PATHOGENESIS OF SSc region of the ␤-chain gene consists of V␤, D␤, and J␤ segments. Approximately 42 V␣ and at least 65 V␤ functional gene segments (52,53) are grouped into 32 V␣ and 26 V␤ families. Enormous diversity of TCRs is generated by combinatorial rearrangement of different VDJ gene segments, imprecise VDJ joining, addition of template-independent nucleotides (N-region diversity) at the VDJ junctions, and use of 2 or more D␤ segments. By analogy to immunoglobulin hypervariable regions, the corresponding V␣ and V␤ regions are called complementarity-determining region 1 (CDR1), CDR2, and CDR3. CDR1 and CDR2 are encoded by V␣ and V␤ gene segments, and CDR3 makes contact with the peptide (for review, see ref. 54). The population of T cells comprises a very large number of different T cell clones, each of which is defined by the expression of a different TCR. The maximum theoretical number of different ␤-chain TCR transcripts has been calculated to be ⬃1012 (54). Although the actual number of ␤-chain TCR transcripts in the T cell repertoire is very likely considerably smaller, the possibility of randomly finding multiple identical copies (ⱖ2) of a single ␤-chain TCR transcript in an independent sample of T cells is negligible. T cells may be activated and proliferate in either a nonspecific manner or in response to a specific antigen. Nonspecific T cell activation and proliferation may take place in vivo in response to chemokines, cytokines, or mitogens and would result in a large, heterogeneous, polyclonal population of T cells that express unique (different) TCRs when compared with each other. Activation in response to superantigens is also possible and will also result in polyclonal activation of T cells that will use a restricted number of V␤ genes and unique CDR3. In contrast, specific-antigen activation and proliferation will result in clonal expansion of clones of only those T cells that recognize this specific antigen through their TCRs. Such a specific antigen–driven T cell clonal expansion will be identified by the presence of multiple identical TCR transcripts, strongly suggesting the presence of monoclonal or oligoclonal populations of T cells. To determine whether skin biopsy specimens from patients with SSc contain an antigen-driven oligoclonal population of T cells, we amplified, using the nonpalindromic polymerase chain reaction (PCR) method, ␤-chain TCR transcripts from biopsy specimens of involved skin of patients with SSc (33). The nonpalindromic PCR method was developed in our laboratory specifically for the amplification of transcripts with an unknown or variable 5⬘ end, such as the TCRs and immunoglobulins (55–58). The amplified transcripts 1723 were cloned and sequenced. Sequence analysis revealed the presence of high proportions of identical ␤-chain TCR transcripts, ranging from 43% to 90% of those sequenced, in skin biopsy specimens from 5 patients with SSc. In 1 patient for whom different skin biopsy specimens were available (obtained at different times [0, 8, and 13 months earlier] and from 3 different skin regions), identical clonally expanded ␤-chain transcripts were identified (33). These results demonstrate the presence of oligoclonal or, in certain cases, monoclonal T cells in biopsy specimens of involved skin from patients with SSc. It is very likely that these T cells have undergone specific antigen–driven proliferation and clonal expansion in vivo in response to an as yet unidentified antigen. These results suggest that SSc is an antigen-driven T cell disease. It remains to be determined whether ␤-chain TCR transcripts that we have identified in skin biopsy specimens (33) are expressed on T cells of fetal origin. Also, it remains to be determined whether these TCR transcripts recognize self antigen(s) or alloantigen(s). Identification of these clonally expanded TCR transcripts may facilitate the identification of the antigen(s) that they recognize. This, in turn, may permit the development of new therapeutic modalities for the treatment of SSc. Similarly, in bronchoalveolar lavage (BAL) fluid from patients with SSc, restricted TCR junctional region lengths suggestive of oligoclonal expansion were detected in CD8⫹ T cells (59). Restricted use of TCR V␤ genes was also observed in CD4⫺,CD8⫺ T cells in patients with SSc (60). Apart from ␣/␤ TCR⫹ T cells, ␥/␦ TCR⫹ T cells were found to display evidence of activation and oligoclonal expansion. An increased proportion of peripheral blood V␦1 T cells exhibited activation antigens (61), and junctional region lengths were found to be skewed, which was suggestive of oligoclonal expansion of ␥/␦ TCR⫹ T cells (62). Non-germline ␦-chain TCR rearrangements have also been reported (63) and suggest the presence of oligoclonal ␥/␦ TCR⫹ T cells. Previous studies have shown an association of particular HLA alleles with SSc (64–74), which supports the concept that an antigen-driven T cell response is important in the pathogenesis of SSc. It appears that HLA associations in SSc may be more involved with disease susceptibility, because of a substantially stronger HLA association with autoantibody profiles than with the disease itself (64–74). 1724 T cell activation and cytokine production in SSc leading to fibrosis Activation and proliferation of the relevant T cell clones in patients with SSc in response to stimulation by specific SSc antigen(s) (peptide/major histocompatibility complex [MHC] epitopes) involve ligation of the TCRs of these T cells with the appropriate peptide/MHC complexes. In the presence of the appropriate costimulatory signals (75), a productive T cell response will take place that is characterized by T cell proliferation, cytokine production, and differentiation of precursor T cells to effector T cells. We propose that this T cell response is a very significant component in the pathogenesis of SSc. The production of T cell–derived cytokines is part of this important process. Among their other functions, these cytokines are responsible for 1) inducing the production and regulation of additional cytokines by other cells of the immune system, 2) inducing the differentiation of precursor cells of the immune system to effector cells, and 3) generating, alone or in combination with other mechanisms, pathologic conditions such as fibrosis. These events are presumed to take place very early in the pathogenesis of the disease. Based on the type of cytokines that they secrete, two polar and reciprocal T cell subsets have been recognized: the Th1 cells that produce proinflammatory cytokines such as IL-2 and interferon-␥ (IFN␥), and the Th2 cells that produce antiinflammatory cytokines such as IL-4 and IL-5 (76–78). In patients with SSc, Th2 cells are activated and produce large amounts of IL-4, a profibrotic cytokine. Plasma IL-4 protein levels are increased in many patients with SSc (37–39,79). We have demonstrated the presence of increased levels of alternately spliced IL-4 (IL-4␦) transcripts in peripheral blood mononuclear cells from patients with SSc (39). Other investigators reported that CD8 T cells from the BAL fluid of patients with SSc produce predominantly IL-4, whereas healthy controls produce IFN␥ (80). IL-4 enhances the production of collagen, fibronectin, glycosaminoglycan, and proteoglycan synthesis in vitro (16,17,81,82). The major IL-4 protein producers are CD4⫹ T cells (83). Overexpression of the IL-4 gene in pancreatic Langerhans’ cells in transgenic mice results in local fibrosis (84). Furthermore, administration of anti– IL-4 monoclonal antibody prevents GVHD in mice, with a concomitant decrease in hepatic fibrosis (85), and markedly reduces hepatic fibrosis in Schistosomainfected mice (86). Finally, IL-4 induces production of the fibrogenic cytokine TGF␤ (13–15) and enhances SAKKAS AND PLATSOUCAS expression of vascular cell adhesion molecule 1 in endothelial cells (87). In contrast, IFN␥ inhibits collagen synthesis by fibroblasts in vitro (82,88) and in vivo in mice (89). IFN␥ may also have a favorable effect on patients with SSc (90–93). The importance of Th1/Th2 cytokines in fibrosis is supported by the finding that MRL/lpr mice lacking the IFN␥ receptor gene exhibit a syndrome that resembles human SSc (94). IL-17, a T cell cytokine that can be produced by both Th1 and Th2 cells (95), is overexpressed in peripheral blood and skin of patients with SSc (41). IL-17 enhances the proliferation of fibroblasts (41), induces IL-6 production by fibroblasts and endothelial cells (96), induces IL-1 production, and enhances the expression of adhesion molecules on endothelial cells (41). In addition to IL-4, the major fibrogenic cytokine in SSc is TGF␤. IL-4 promotes the production of TGF␤ (13–15), which is expressed on mononuclear cell infiltrates, endothelial cells, and fibroblasts in the early stages of skin fibrosis (97,98). There are at least 3 TGF␤ isoforms, TGF␤1, TGF␤2, and TGF␤3. TGF␤, upon binding to its receptor, activates transcription factors called Smads, some of which are stimulatory and others inhibitory (99). SSc skin fibroblasts may produce amounts of TGF␤ similar to those secreted by normal skin fibroblasts but express higher levels of TGF␤ receptor type I (TGF␤RI) and TGF␤RII (100,101). TGF␤1 can cause fibroblast activation and hyperplasia and increased synthesis of collagen and fibronectin (102,103). Also, it inhibits matrix metalloproteinases (MMPs) (104). Blockage of TGF␤1 by antibodies abolishes the up-regulated transcription of the collagen gene (101). TGF␤1 stimulates the production of IL-1 and platelet-derived growth factor (PDGF) by monocytes (105) and the production of connective tissue growth factor (CTGF) by fibroblasts (106). T cells, monocytes, endothelial cells, and fibroblasts produce IL-6, and it is up-regulated by IL-1 (107), IL-17 (96), or PDGF (108) in SSc fibroblasts. Elevated serum levels of IL-6 were observed in patients with SSc (109). IL-6 induces collagen production by fibroblasts and promotes Th2 differentiation (110). CTGF is increased in the serum of patients with SSc (111). It is also expressed during the late sclerotic skin stages of SSc (112). CTGF is a fibroblast mitogen that increases collagen production (91) and is produced by fibroblasts in response to TGF␤ (111–113). It is our opinion that antigen-driven T cell activation and clonal expansion in SSc is the primary event in the pathogenesis of the disease, and that it is mediated ROLE OF T CELLS IN PATHOGENESIS OF SSc at large through T cell–derived cytokines. These cytokines are produced either directly by activated T cells, or their expression is regulated directly or indirectly by T cells and/or T cell–derived cytokines. In addition, certain other cytokines are produced and/or regulated mostly by other cell types (non–T cells) and appear to be secondary to the antigen-driven T cell activation and proliferation. These other cytokines are found in SSc lesions or in the serum of patients with SSc, as the result of the chronic inflammation that has been initiated and is propagated by the antigen-driven T cells (which is the primary event in the pathogenesis of the disease). Certain of these other cytokines are discussed below. IL-1 produced by monocytes, B cells, T cells, and SSc fibroblasts participates in mononuclear cell chemotaxis. It enhances collagen synthesis by fibroblasts either directly or through fibroblast production of IL-6 and PDGF (107). However, IL-1 in synovial fibroblasts stimulates the release of MMPs (114). PDGF is produced by platelets, macrophages, fibroblasts, and endothelial cells. It is increased in SSc Figure 2. Schematic paradigm of T cell involvement in fibrosis of systemic sclerosis. T cells produce interleukin-4 (IL-4), which promotes fibroblast collagen production and macrophage (Mo) production of transforming growth factor ␤ (TGF␤), which in turn induces fibroblast collagen synthesis and proliferation. TGF␤ also induces connective tissue growth factor (CTGF) production by fibroblasts. T cells also produce IL-17, which promotes macrophage production of tumor necrosis factor ␣ (TNF␣) and IL-1. IL-1 in turn induces fibroblast production of collagen, IL-6, and platelet-derived growth factor (PDGF). IL-17 and TNF␣ on endothelial cells (EC) promote the recruitment of inflammatory cells into perivascular areas. MMP ⫽ matrix metalloproteinase. 1725 Figure 3. Simplified diagram of T cell involvement in microvascular injury in systemic sclerosis. Activated T cells produce IL-4 and IL-17. IL-4 in the blood vessel wall induces fibrosis and causes fibroblast hyperplasia through the production of TGF␤. IL-17 stimulates endothelial cell production of adhesion molecules IL-1 and IL-6 through induction of TNF␣. Endothelial cell activation contributes to inflammatory cell influx into the perivascular areas. See Figure 2 for definitions. (97) and can be up-regulated in endothelial cells by complement activation (115) or hypoxia (116). PDGF is a fibroblast mitogen and stimulates collagen synthesis (102). Induction of fibroblast collagen production by TGF␤ may be mediated through up-regulation of PDGF receptors (97). PDGF can also promote IL-6 production by fibroblasts (108). Elevated serum levels of monocyte chemoattractant protein 1, macrophage inflammatory protein 1 (117), and tumor necrosis factor ␣ (TNF␣) (118) were also found in SSc. These molecules participate in mononuclear cell chemotaxis to SSc lesions. Endothelin-1, produced by endothelial cells in patients with SSc, is a potent vasoconstrictor and can induce collagen synthesis and inhibit MMP expression (119). Activated T cells and the cytokines that they produce are sufficient to explain all 3 main characteristics of SSc, as follows: 1) Generation of fibrosis, which is illustrated in Figure 2. As previously discussed, IL-4 is produced by CD4⫹ T cells (16,17,80–83) and promotes the production of collagen by fibroblasts (16,17) and the production of TGF␤ by monocyte/macrophages (13–15). TGF␤ also induces fibroblasts to proliferate and produce collagen (13–15) and to produce CTGF (111–113). CTGF also significantly increases the synthesis of collagen and fibronectin and promotes substantial matrix remodeling of fibroblast-populated 3-dimensional collagen lattices (113). In addition, T lymphocytes produce IL-17, which induces TNF␣ and IL-1 production by monocyte/macro- 1726 Figure 4. Paradigm of likely T cell interactions with B cells. T cells are necessary for anti–topoisomerase I (anti–topo I) autoantibody production and very likely for the production of other antibodies (abs). Antiendothelial cell and antifibroblast antibodies appear to induce IL-1 production after binding to the respective cells. See Figure 2 for other definitions. phages (26,41,96). IL-1 induces production of collagen, IL-6, and PDGF by fibroblasts and endothelial cells (96). IL-17 also induces the expression of IL-1 and adhesion molecules on endothelial cells (41). 2) Microvascular fibrointimal proliferation, which is illustrated in Figure 3. Activated T cells produce IL-4 (16,17,80–83) and IL-17 (41). IL-4 induces fibrosis (16,17) and causes fibroblast hyperplasia by enhancing TGF␤ production (13–15). TGF␤ induces fibroblasts to proliferate and secrete collagen (13–15) and also to produce CTGF (111–113) (see above). IL-17 induces the expression on endothelial cells of adhesion molecules, IL-1, and IL-6. Activation of adhesion molecules of the endothelial cell layer allows inflammatory mononuclear cells to cross the endothelial cell layer into the perivascular space, thereby propagating the disease. 3) Autoantibody production, which is shown diagrammatically in Figure 4. A substantial number of autoantibodies have been detected in the serum of patients with SSc (for review, see refs. 1 and 120) and include anti–topo I (32,68), anticentromere (121), antifibrillarin, and anti–RNA polymerases I, II, and III (1,120). Production of antiendothelial (122) and antifibroblast antibodies (123) has also been reported. The latter antibodies appear to induce IL-1 production after binding to the respective cells. In addition to B cells, T cells also are required for anti–topo I autoantibody SAKKAS AND PLATSOUCAS production (32) and very likely for the production of the other autoantibodies. Other diseases with a predominance of Th2 immune response include helminthic diseases and allergic diseases. Helminthic diseases, such as Schistosoma infection, are characterized by intense local fibrosis (86). However, allergic diseases may lack overt fibrosis. One explanation may be that allergen-reactive Th2 cells in individuals who are atopic may be present in the circulation temporarily during seasonal allergen exposure (77). Nevertheless, airway fibrosis beneath the subepithelial basement membrane still occurs in human atopic asthma as well as in a murine model of atopic asthma (124). It should be noted that the Th1/Th2 immune response pattern is an oversimplification, and that a local cytokine field is a summation of the cytokine fields produced by a variety of individual cells (125). In individuals who are atopic, a local field may be derived from many basophils, which are not present in SSc. Putative T cell antigens in SSc Several lines of evidence suggest that the activation of T cells in SSc is antigen driven, yet the antigen or antigens that drive this T cell clonal expansion (33) are not known. However, several putative SSc antigens have emerged, including the following: HLA of fetal or maternal origin. The presence of small proportions (usually ⬍5%) of fetal cells in the peripheral blood and the skin of women with SSc who were previously pregnant has been well documented (126–133). Additionally, small proportions of maternal cells have been identified in the peripheral blood of women with SSc who were not previously pregnant or in men with SSc (131,133). The presence of fetal cells or maternal cells in these patients with SSc is believed to be the result of microchimerism (126–133), which may persist in both patients with SSc and normal donors (130,131,133). These fetal or maternal cells belong to a number of different hemopoietic cell lineages (126– 133). The sources of the HLA-disparate cells in both men and women may include cells from the fetus (only women who have been pregnant), cells from the mother, cells from a twin sibling, cells from a blood transfusion, or cells from a transplant (the last 4 may be the source of engraftment in both men and women). The mechanisms of engraftment in microchimerism are not fully understood, and it is not known whether regulatory cells that induce/regulate tolerance of the microchimerism are involved. Interestingly, the genotype HLA– DQA1*0501, which is associated with SSc (69), has been ROLE OF T CELLS IN PATHOGENESIS OF SSc found to be associated with the persistence of microchimerism in women with SSc (70). Other investigators were unable to demonstrate such an association (134). However, not all investigators reported increased microchimerism in SSc (135,136). It has been proposed that SSc in previously pregnant women is caused by chronic fetal anti-maternal GVHD that is manifested as SSc; this may be designated as maternal SSc (126–133). In other words, SSc in previously pregnant women is initiated by activation of T cells of fetal origin in response to maternal antigens or unknown antigenic stimuli, or because of breaking of the tolerance (129–133) that controls microchimerism (the coexistence of maternal and fetal cells), resulting in induction of chronic fetal anti-maternal GVHD (126– 133). In a similar manner, T cells of maternal origin may be activated in response to offspring antigens or to unknown antigenic stimuli, or because of breaking of tolerance (127–135), resulting in induction of chronic maternal anti-offspring GVHD, which is manifested as SSc in women who have not been previously pregnant or in men; this may be designated as offspring SSc. Therefore, alloantigens may be putative SSc antigens with an alloresponse of fetal T cells to maternal alloantigens or of maternal T cells to offspring alloantigens possibly responsible for the initiation of SSc. This concept is consistent with the clinical and pathologic characteristics (45,46) and the serologic profile (47) that are shared between patients with GVHD and patients with SSc, including fibrosis, microvascular fibrointimal proliferation, and production of autoantibodies (47). However, additional studies are needed to prove this concept. Scaletti et al reported that 39 of 202 T cell clones derived from skin biopsy specimens or peripheral blood from 3 women with SSc, and 11 of 312 T cell clones derived from the peripheral blood of 3 healthy women, proliferated in response to maternal MHC antigens (137). All 6 women had a male offspring. In situ hybridization studies using appropriate fluorescence probes specific for the Y chromosome revealed that 7 of the maternal MHC-responding T cell clones obtained from the patients with SSc and 1 of the T cell clones obtained from healthy donors exhibited the Y chromosome, demonstrating that these T cell clones are indeed derived from the male offspring. The T cell clones derived from male offspring T cells of women with SSc were of the Th2 type and produced, in response to maternal MHC antigens, substantially higher levels of IL-4 than did all of the remaining T cell clones derived from the same women. In the remaining T cell clones, the Y chromosome was not detectable, although they 1727 responded by proliferation to maternal or allogeneic MHC antigens (137). These results demonstrate that male offspring T cells that are present in small proportions in the skin or in the peripheral blood of women with SSc are able to respond, under appropriate conditions, to maternal MHC antigens and produce Th2 cytokines (137). The long-term presence of these male offspring T cells in women with SSc supports the notion that long-term microchimerism, manifested as chronic GVHD, may play an important role in the pathogenesis of SSc (137). DNA topo I and other autoantigens. DNA topo I elicits both autoantibody responses (32,68) and cellular responses in patients with SSc (137–139). Collaboration of T and B cells is required for autoantibody responses to DNA topoisomerase (32). Anti–topo I antibodies are characteristic of the diffuse type of SSc (1,120). The possibility that an autoantigen elicits both an autoantibody response and a cellular response is supported by the findings of a substantially stronger HLA association with autoantibody profiles than with the disease itself (64–68,140). Kuwana et al reported that HLA–DQ and DR genes together control the anti–topo I antibody response in patients with SSc (68). In that study, all patients with anti–topo I antibody response were HLA– DQB1*0601 positive or DQB1*0301 positive, whereas these alleles were found in only 44% of the anti–topo I antibody–negative patients (P ⬍ 0.00001) and in 58% of the healthy control subjects (P ⬍ 0.00001). Peripheral blood mononuclear cells from 25 of 26 patients (96%) with SSc who had anti–topo I antibody exhibited substantial T cell proliferative responses to human recombinant topo I (138). These responses were evident after only 3 days in culture with topo I. In contrast, only 4 of 10 patients (40%) with SSc who were anti–topo I antibody negative, and 13 of 21 normal donors (62%) exhibited such a proliferative response (138). These responses required a 7-day incubation in culture with topo I. All patients with SSc and normal donors who were positive for either DRB1*1501,2 (DR15), DRB1* 1101,3,4 (DR11), or DRB1*07 (DR7) exhibited T cell proliferative responses to topo I. This response was mediated by CD4⫹ T cells, required antigen-presenting T cells, and was restricted by HLA–DR and to some extent by HLA–DQ. Limiting dilution analysis was used to estimate the frequency of T cell responses to topo I, which was found to be in the range 1/9,277–1/24,853 (138). T cells specific for DNA topo I in patients with SSc used highly restricted CDR3 regions of the ␤-chain TCR transcripts (139). However, identical CDR3 se- 1728 quences were used by DNA topo I–specific T cells derived from normal donors (139). In another study, substantial differences were not found in the phenotypic and functional properties of topo I–reactive T cells from a patient with SSc and her healthy identical twin (141). These results suggest that these topo I T cell clones are members of the normal T cell repertoire. This T cell response is restricted by class II MHC alleles and is not associated with the presence or absence of SSc or the presence of anti–topo I antibody. The anti–topo I antibody and the cellular responses may be autoimmune in nature and may take place in patients with SSc because of the extensive dysregulation of the immune system caused by the disease. If such is the case, this would decrease the likelihood that topo I is a putative antigen responsible for the initiation of SSc. Other putative autoantigens that elicit autoantibody responses in patients with SSc include fibrillarin (142), centromere (121), RNA polymerases I, II, and III (1,120), and other nucleolar autoantigens (143). Antifibrillarin antibodies are found in 33% of men with SSc and in 14% of women with SSc and are associated with the HLA–DQB1*0604 and DRB1*1302 haplotypes (142). Antibodies to CENP-E, a centromere kinesin-like protein, are found mostly in a rather infrequent form of SSc known as the CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias), and their presence is associated with the HLA–DQB1*05 alleles (121). Certain HLA– DRB1 (DRB1*0101,*0405, and *01302) or DQB1 (DQB1*0501) alleles were found to be associated with high serum anticentromere titers (140). Cytomegalovirus (CMV). CMV has been suggested as the cause of SSc, because increased levels of anti-CMV antibodies were found in patients with SSc, and extensive similarities in the pathology between CMV vasculopathy and SSc vascular changes have been demonstrated (144–146). A molecular mimicry has been identified between a peptide sequence of the UL70 protein of CMV (DDGYF) and topo I (residues 122– 126) (145,146). Molecular mimicry is defined as the sharing of common epitopes between microorganisms (such as viruses) and host proteins (for review, see ref. 147). T cells that recognize viral determinants may recognize, by molecular mimicry, host determinants that are shared with the virus, even long after the virus is no longer present. This may result in the development of autoimmune disease. Molecular mimicry may play an important role in the pathogenesis of SSc. Retroviruses. Retroviruses have been long suspected as putative antigens in SSc (148). An increased SAKKAS AND PLATSOUCAS frequency of cancer in first-degree relatives of patients with SSc (149) could be attributed to environmental factors such as retroviruses. A molecular mimicry (complete identity of 6 sequential amino acid residues) has been identified between topo I and retroviral protein p30gag of type C mammalian retroviruses (150). T cells in animal models of SSc The tight-skin (TSK) mouse is perhaps the best animal model for SSc. In the TSK mouse, Th2 T cells infiltrate skin lesions and produce IL-4 (151). In this animal model, fibrosis can be prevented by null mutation of the IL-4 gene (151), by anti–IL-4 treatment (152), or by introduction of an IL-12–encoding plasmid (153). In vivo administration of intravenous immunoglobulin decreased splenocyte secretion of IL-4 and TGF␤ and abrogated fibrosis (154). T cells infiltrating skin lesions use restricted TCR V␤ gene segments (155). However, cutaneous thickening can still occur in TSK mice that are T cell and B cell deficient (156). In a mouse model of SSc induced by local injections of bleomycin, TGF␤1 was detected early in skin lesions (157). Concluding remarks and future perspectives Current evidence suggests that T cells, B cells, macrophages, fibroblasts, endothelial cells, eosinophils, and mast cells are activated in SSc. The presence of monoclonal/oligoclonal expansions of T cells infiltrating the skin lesions of patients with SSc (33) strongly suggests that these T cells have undergone in situ antigen-driven proliferation and clonal expansion in response to an as yet unidentified antigen(s). This antigen may be a self antigen or an alloantigen. In other words, antigen-driven T cell activation in SSc is a primary event and is not secondary to the nonspecific action of cytokines secreted by other cell types. These findings suggest that T cells are very important in the pathogenesis of the disease and make T cells targets of immunotherapy and other treatment modalities in SSc. A new therapy may be redirection of T cell cytokines toward Th1. 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