- Email: [email protected]
TGF-β: A Critical Modulator of Immune Cell Function
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 84, No. 3, September, pp. 244–250, 1997 Article No. II974409
MOLECULE OF THE MONTH TGF-b: A Critical Modulator of Immune Cell Function John J. Letterio and Anita B. Roberts Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892-5055
INTRODUCTION
An imposing array of secreted signaling peptides orchestrates the host response to stimuli. Although the list of cytokines which make up this array continues to grow, we are only beginning to understand their distinct roles and the interplay between intracellular pathways which mediate their effects. As a subset of this group, the transforming growth factors-b (TGF-b) are distinguished by their ability to influence almost every facet of the immune response, including the growth and differentiation of precursors for multiple hematopoietic lineages, the proliferation and migration of mature immune cells into sites of injury or response, and even the suppression of such responses once they have been established (1). The complimentary autocrine, paracrine, and even endocrine modes of TGF-b activity that underly these effects have been elegantly delineated through extensive in vitro and in vivo analyses of function, including various TGF-b transgenic models and isoform-specific gene knockouts. In this discussion, we will emphasize how dysfunctional TGF-b pathways might contribute to the pathogenesis of immune disorders, explain how these preclinical studies have challenged some basic concepts regarding mechanisms of TGF-b action, and end by speculating on how this knowledge may be brought to bear on important clinical problems. THE TGF-b FAMILY
The prototype of this family, TGF-b1, was isolated and characterized nearly 15 years ago based on its ability to stimulate the anchorage-independent growth of fibroblasts, a correlate of tumorigenicity in vivo (for a review see Ref. 2). The surprising identification of platelets and bone as major sources of this peptide suggested prominent physiological roles. At present, with the advent of gene knockout and transgenic approaches, we are beginning to gain a greater appreciation for the complexity and breadth of the activity of TGF-b1 and its highly homologous isoforms, TGF-bs 2 and 3, as major negative regulators of the growth and differentiation of most cell lineages.
244
0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
Clin 4409
/
a516$$$241
TGF-bs 1, 2, and 3 are localized to human chromosomes 19q13, 1q41, and 14q24, (mouse chromosomes 7, 1, and 12) respectively (2). TGF-b1 is expressed by all immune cells and is the isoform most often acutely regulated in response to a variety of stress and disease signals (1, 3). All TGF-bs are encoded as precursor molecules which are processed proteolytically to the highly conserved (greater than 72%) biologically active carboxy-terminal domain of 112 amino acids, with the unique feature that the remainder of the pre–pro domain (latency-associated protein, LAP) associates noncovalently with the C-terminal fragment to reversibly block its activity (Fig. 1) (4, 5). Latency is an important component of regulation of TGF-b activity and can be conferred not only by the LAP protein, but also by its associated protein LTBP which directs the complex to matrix, and by molecules such as a2-macroglobulin which play a role in clearance (6, 7). In vivo activation of these latent forms of secreted TGF-b is a highly regulated, yet incompletely understood process, mediated by multiple pathways involving proteolytic mechanisms (4–6), redox (8), free radicals including nitric oxide, and binding to molecules such as thrombospondin (9). The recent identification of an active form of TGF-b complexed with IgG, as produced by B cells and plasma cells (see Fig. 1), points to yet another mode of regulation which may be key in the modification of specific immune cell responses (10–13). Signals from TGF-b ligands are mediated by a heterotetrameric receptor complex, distinguished by its intrinsic serine–threonine kinase activity from receptors for other immunoregulatory cytokines that signal through tyrosine kinase-mediated pathways (14, 15). The first downstream mediators of this signal transduction pathway to be identified include, minimally, a set of at least five highly conserved molecules called Smads, each related to the Drosophila protein Mad which acts downstream of the TGF-b family ligand dpp and its receptors (14–16). Steps involved in transduction of signals from TGF-b (or activin) involve direct serine phosphorylation of Smad2 or 3 by the type I receptor subunit, complexation with Smad4, which plays a unique role common to pathways of all TGF-b family ligands, and transloca-
08-02-97 13:02:57
clina
AP: Clin
TGF-b: MODULATOR OF IMMUNE CELL FUNCTION
FIG. 1. TGF-b signaling has multiple levels of regulation. Dysregulated activation of latent TGF-b and altered expression and activity of either its cell surface receptors or its cytoplasmic signaling intermediates contribute to the pathogenesis of a variety of immune diseases. Inactivation of TGF-b signaling (receptors or Smad proteins) in carcinogenesis can result in upregulation of TGF-b expression (53) and consequent paracrine suppression of immune surveillance. Among many mechanisms described for activation of latent TGF-b, one of particular importance for immune cells is the secretion by B-cells and plasma cells of a complex of TGF-b bound to IgG (10, 13). Whether binding to IgG alters the conformation of the latencyassociated peptide (LAP) to expose the receptor-binding epitope of TGF-b (13) or whether the TGF-b/IgG complex is internalized via Fc receptors and activated cytoplasmically is not known (11). TF, transcription factor analogous to the forkhead activin signal transducer-1 (FAST-1) (17).
tion of the complex to the nucleus where it activates expression of target genes by binding to transcription factors (Fig. 1) (14–17). The specific features of this signaling pathway germane to the activation, proliferation, and functional responses of specific immune cell populations remain to be elucidated. EVENTS REGULATED BY TGF-b IN LEUKOCYTE DEVELOPMENT
Nearly all leukocyte lineages are influenced by TGFb, at steps ranging from their production in the marrow compartment to their differentiation and maturation in the periphery (18). For example, the recent discovery of a requirement for TGF-b1 during the in vitro differentiation of functional dendritic cells (DC) identified a new role for TGF-b in regulating this class of antigenpresenting cells (19). Though in vitro differentiation of DC from cultures of CD34/ precursor cells is also dependent on the presence of other cytokines, such as TNF-a, SCF, and GM-CSF, it is quite inefficient with-
AID
Clin 4409
/
a516$$$242
08-02-97 13:02:57
245
out the addition of exogenous TGF-b1, which effectively substitutes for serum or plasma. The mechanism underlying this effect appears to be the protection of DC viability, with the percentage of apoptotic cells reduced by more than 60% at 72 hr in culture (20). The importance of TGF-b1 in DC development has been further emphasized by recent studies in the TGFb1 knockout mouse. The complete absence of epidermal DC or Langerhans cells (LC) is a striking feature of their phenotype (21), which includes generalized activation of most immune cell populations and widespread tissue inflammation (22, 23). Though inflammatory cytokines are known to effect migration of LC from the epidermis, TGF-b1 null mice bred onto a variety of immunodeficient backgrounds (SCID, athymic nude, RAG2-null), or successfully treated with the immunosuppressive agent rapamycin, are also deficient in epidermal LC (21). These data suggest that the absence of LC is a direct consequence of disruption of endogenous TGF-b1 gene expression, rather than an indirect result of the inflammatory process characteristic of the TGF-b1-null mouse. Transplantation of TGF-b1-null marrow into wild-type, lethally irradiated recipients leads to repopulation of recipient skin with donor-derived LC, implying that normal LC progenitors exist in the TGF-b1-null mouse, and that paracrine sources of TGF-b1 are sufficient to support normal LC development, and perhaps even their migration into the epidermis (24). Finally, the fact that TGF-b1-null mice also lack gp40/ lymph node DC (despite normal numbers of CD11c/ DC) suggests that the loss of TGF-b1 may have a broader effect on normal DC development (21). This influence of TGF-b on maturation and differentiation of leukocytes extends to lymphoid lineages as well, as demonstrated by the events regulating the differentiation of thymic precursors. Progression of the CD40CD8lo thymocyte to the CD4/CD8/ doublepositive stage of differentiation is a step that requires entry into S phase, and is inhibited by the addition of TGF-b (25). In fetal thymus organ culture, TGF-b produced by various lines of cortical thymic epithelial cells inhibits thymocyte progression to the doublepositive state (25), and the addition of exogenous TGF-b also blocks the ability of alymphoid thymic lobe cultures to support T cell development of precursor cells obtained from fetal liver (26). Similar studies of isolated human early thymic precursors suggest a role for autocrine sources of TGF-b as well. Autologous, irradiated CD3/CD8/CD40 T cells inhibit growth of the triple-negative (CD2/CD3// TCR0CD40CD80) thymocyte precursors by activating latent TGF-b produced by the precursor cells, an affect abolished by the addition of a neutralizing antiTGF-b antibody (27). So while the CD8/ cells were not the predominant source of TGF-b, they were re-
clina
AP: Clin
246
LETTERIO AND ROBERTS
sponsible for activating TGF-b produced in an autocrine loop by the precursors. Thus, in murine and human systems, TGF-b acts as both a paracrine and an autocrine inhibitory factor, during the generation of thymic T cells. Knowing that such key steps in the development of both antigen-presenting cells and T lymphocytes are controlled by endogenous TGF-b expression, one might predict that loss of the predominant TGF-b isoform, TGF-b1, might lead to considerable disruption in immunological homeostasis. In particular, the loss of negative inhibition of thymic precursors might lead to increased numbers of mature CD4/ or CD8/, singlepositive lymphocytes, which may escape important selection steps regulated by TGF-b1. Indeed, the thymus of a TGF-b1 knockout mouse exhibits decreased cellularity, primarily due to a loss of immature double-positive (CD4/CD8/) precursors, with a predominance of mature single-positive T cells, expressing high levels of MHC class I and CD3. In the periphery, this is accompanied in lymph nodes (and in the spleen to a lesser extent) by increased numbers of CD4/ T cells displaying an activated phenotype (increased H1.2F3 and CD44, with diminished CD45RB expression) (28). It has been speculated that a defect in negative selection may result from the premature exit of these differentiated T cell subsets from the thymus. These TGF-b1null T cells appear to be in a continuous state of proliferation, expressing mRNA for both IL-2 and IL-2 receptors, and producing a spectrum of proinflammatory cytokines, including IL-2, IL-6, and IL-10, which may act as generalized promoters for an autoimmune response, a predominant feature of the phenotype of the TGF-b1 knockout mouse (29, 30). TGF-b1-null mice develop IgG autoantibodies with a spectrum of specificities, with glomerular immune complex deposition and widespread vasculitis as other hallmarks of the phenotype (30). These key features associated with TGF-b1 deficiency in vivo support many of the outcomes or functions predicted for this molecule from in vitro analyses of function. THE DUAL ROLE OF TGF-b IN DISEASE PATHOGENESIS
TGF-b has been implicated in a wide variety of physiological repair processes and in the pathogenesis of various diseases, often playing a direct role in mediating associated immune defects (3, 31). Examples in which dysregulated expression of TGF-b1 by immune cells may drive the disease process include the fibrosis that accompanies chronic inflammation (32), and parasitic diseases in which TGF-b1 secretion blocks activation of macrophages by interferon-g (IFN-g), diminishing their oxidative responses (33). In this regard, it has been demonstrated that the intracellular parasite
AID
Clin 4409
/
a516$$$242
08-02-97 13:02:57
Trypanosoma cruzi not only triggers activation of the TGF-b signaling pathway, but also that it is required for parasite entry into mammalian cells (34). Similarly, macrophage infection by the protozoan parasite Leishmania induces production of biologically active TGFb; systemic administration of neutralizing anti-TGF-b antibodies can halt progression of this disease in genetically susceptible mice (35). The mechanisms of activation of TGF-b in these processes are clearly key areas for future study. This association between expression and activation of TGF-b and susceptibility to infection has also recently been identified as a feature of both murine models of autoimmunity and their human disease counterparts (31). Evaluation of the host defense status of the MRL/lpr mouse revealed that acquired defects in polymorphonuclear (PMN) chemotaxis and phagocytosis are linked to increased susceptibility to both gram-negative and gram-positive infection (10). Most importantly, these defects were directly linked to the elaboration of excessive levels of both the active and latent forms of TGF-b1, and were reversed in mice treated with neutralizing anti-TGF-b antibodies. Although other factors such as concurrent immunosuppressive therapy may be responsible for similar PMN defects in patients with systemic lupus erythematosis (SLE), it is interesting that an analysis of TGF-b1 levels in the plasma of SLE patients revealed similar increases in circulating levels of this cytokine (10). Overproduction of TGF-b in autoimmune disorders may also underlie or promote tissue injury associated with these disorders. Pathogenic autoantibody production resulting in glomerular immune complex deposition is a shared feature of many of these diseases, and it is most significant that fibrotic lesions and glomerulonephritis strikingly similar to this immune complex-mediated injury also develop in transgenic mice which overexpress active TGF-b1 under the control of murine albumin promoter and enhancer sequences (36). Although these observations suggest that TGF-b plays a central role in development of inflammatory and infectious diseases complications, it clearly plays contradictory roles, in that its powerful immunosuppressive activity also contributes to the prevention and recovery from autoimmune disorders. Expression of TGF-b1 by immune cells has been implicated in the recovery phase of the relapsing–remitting course of experimental allergic encephalomyelitis (EAE), the murine model of multiple sclerosis (MS) (37). The ability of systemic TGF-b to suppress symptoms and delay progression, and the appearance of increased endogenous TGF-b1 expression during the recovery phase, points toward a beneficial role for TGF-b production in autoimmune disease (38, 39). Perhaps the strongest evidence for this protective role comes from studies which link disease prevention to the suppressive effects
clina
AP: Clin
TGF-b: MODULATOR OF IMMUNE CELL FUNCTION
generated by oral tolerance (40). In EAE, the oral administration of myelin basic protein leads to development of a T-cell-mediated suppression that can be adoptively transferred, can suppress immune responses in vitro, and can be blocked by neutralizing anti-TGF-b antibodies (41). It has since been demonstrated that oral antigen induces both mucosally derived TH2-like CD4/ T cells (producing active TGF-b, IL-4, and IL-10) and the TGF-b-producing CD8/ T cells originally identified (42, 43). Recent studies have shown that the protective effects of oral antigen are enhanced by systemic anti-IL-12 treatment, which leads to substantially increased TGF-b production and T cell apoptosis in Peyer’s patches (44, 45). Again, perhaps the strongest argument for the beneficial role of TGF-b1 in the maintenance of immunological homeostasis comes from the TGF-b1-null mouse, where loss of the TGF-b1 ligand results in many hallmarks of autoimmune disease, progressive tissue inflammation, and death (23). In addition to these conflicting roles played by TGFb in autoimmune and inflammatory disorders, it also has both direct and indirect effects on immune cell populations in the process of carcinogenesis (46). In both epithelial and lymphoid malignancies, tumor cells commonly acquire resistance to the growth inhibitory effects of TGF-b. The TGF-b receptors, as well as the cytoplasmic signaling intermediates Smads 2 and 4, have been shown to have tumor suppressor activity, since their loss results in increased tumorigenicity (47– 49). Functional inactivation of receptors can occur by a wide variety of mechanisms including truncation resulting from microsatellite instability, or mutation of the kinase domain or the ligand binding site (47, 50). In human T cell lymphomas, receptor mutations resulting in dominant negative behavior have been reported (51, 52), while in murine plasmacytomas, a defect in ligand binding by the receptor has been noted (manuscript in preparation). Transcriptional repression of receptor expression is also observed. Recently, it has been shown that loss of receptor function, as modeled in transgenic mice expressing a dominant-negative type II TGF-b receptor subunit (DNRII), leads directly to secretion of substantial quantities of biologically active TGF-b, as is characteristic of tumor cells resistant to TGF-b (53). Purification of a glioblastoma-derived, T-cell-suppressive factor (later identified as TGF-b2) provided the first evidence of the potential for TGF-b to act as an inhibitor of the mechanisms mediating immune surveillance of cancer (Fig. 2). In depth studies have led to the conclusion that production of TGF-b by a tumor is essential for its progression, preventing destruction by both specific cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (54). Indeed, transduction of a highly immunogenic, UV-induced fibrosarcoma with a TGF-b1 expression vector
AID
Clin 4409
/
a516$$$243
08-02-97 13:02:57
247
FIG. 2. TGF-b-mediated inhibition of cytotoxic T lymphocytes. CTL killing of tumor cells is antigen-specific, and tumor cells bearing class I MHC-associated antigen can trigger CTL activation, causing them to release contents of membrane-associated cytoplasmic granules, including the pore-forming proteins perforin and cytosolin, and lymphotoxins such as TNF-a and IFN-g (A). Tumor cells which lose sensitivity to TGF-b, resulting either from receptor mutation or inactivation, often secrete substantial amounts of active TGF-b. This consequence of malignant progression has been shown to block CTLmediated tumor lysis, leading instead to CTL apoptosis (B). (Apoptotic cells are in dotted outline.)
not only renders it incapable of stimulating primary CTL responses in vitro, but is also sufficient to allow for escape from immune surveillance and progressive growth in vivo (55). Similarly, differentiating agents which dramatically reduce the production of TGF-b by MOPC-315 plasmacytoma cells in BALBc mice, also lead to the development of potent CD8/ T-cell-mediated anti-MOPC-315 CTL activity (56). Thus, a positive correlation between invasiveness, progression, decreased TGF-b sensitivity, and increased TGF-b production has been established, and the latter may contribute to locally impaired immune response to tumor cells. These experiments dramatically demonstrate the therapeutic potential of strategies targeting either tumor cell production or immune cell responsiveness to TGF-b. Approaches based on the inhibition of TGFb production, by the introduction of TGF-b antisense expression vectors into tumor cells, have already proven useful for induction of CTL, both in vitro or in vivo, with the latter leading to rejection of established tumors and T cell memory in an animal model of glioblastoma (57). The development of clinical vaccine trials using TGF-b antisense tumor modification is currently in progress. It is clear that similar approaches to block activity or interrupt endogenous production of TGF-b, whether by tumor cells or activated immune cell populations, may be an important strategy for boosting immune function. While the systemic administration of antiTGF-b neutralizing antibodies has been successful in enhancing CTL and NK cell function in animal models
clina
AP: Clin
248
LETTERIO AND ROBERTS
(58–60), the long-term efficacy and application of such an approach in humans is still questionable. In contrast, for those inflammatory and autoimmune disorders which may benefit from the potent immunosuppressive effects of TGF-b (rheumatoid arthritis, MS, Sjo¨grens disease, graft-vs-host disease, allograft rejection), we must look toward strategies that may enhance production (61), and activation of endogenous TGF-b, and work to identify agents which may target and activate some of the newly identified signaling intermediates. With over a decade of preclinical evaluation pointing toward the critical immunomodulatory properties of TGF-b, it ultimately will become both the target and the tool for fighting human diseases linked to altered immune function. REFERENCES
10. Lowrance, J. H., O’Sullivan, F. X., Caver, T. E., Waegell, W., and Gresham, H. D., Spontaneous elaboration of transforming growth factor-beta suppresses host defense against bacterial infection in autoimmune MRL/lpr mice. J. Exp. Med. 180, 1693– 1703, 1994. 11. Stach, R. M., and Rowley, D. A., A first or dominant immunization. II. Induced immunoglobulin carries transforming growth factor-beta and suppresses cytolytic T cell responses to unrelated alloantigens. J. Exp. Med. 178, 841–852, 1993. 12. Rowley, D. A., Becken, E. T., and Stach, R. M., Autoantibodies produced spontaneously by young 1pr mice carry transforming
Clin 4409
/
a516$$$244
14.
15.
16.
17.
18.
1. McCartney-Francis, N. L., and Wahl, S. M., Transforming growth factor-beta: a matter of life and death. J. Leukocyte Biol. 55, 401–409, 1994. 2. Roberts, A. B., and Sporn, M. B., The transforming growth factors-b. In ‘‘Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors’’ (M. B. Sporn and A. B. Roberts, Eds.), pp. 419–472, Springer Verlag, Berlin, 1990. 3. Roberts, A. B., and Sporn, M. B., Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Factors 8, 1–9, 1993. 4. Munger, J. S., Harpel, J. G., Gleizes, P. E., Mazzieri, R., Nunes, I., and Rifkin, D. B., Latent transforming growth factor-beta: Structural features and mechanisms of activation. Kidney Int. 51, 1376–1382, 1997. 5. Barcellos-Hoff, M. H., Latency and activation in the control of TGF-b. J. Mammary Gland Biol. Neoplasia 1, 351–361, 1997. 6. Nunes, I., Gleizes, P. E., Metz, C. N., and Rifkin, D. B., Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta. J. Cell Biol. 136, 1151–1163, 1997. 7. LaMarre, J., Hayes, M. A., Wollenberg, G. K., Hussaini, I., Hall, S. W., and Gonias, S. L., An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice. J. Clin. Invest. 87, 39–44, 1991. 8. Barcellos-Hoff, M. H., and Dix, T. A., Redox-mediated activation of latent transforming growth factor-beta 1. Mol. Endocrinol. 10, 1077–1083, 1996. 9. Schultz-Cherry, S., Ribeiro, S., Gentry, L., and Murphy-Ullrich, J. E., Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system. J. Biol. Chem. 269, 26775–26782, 1994.
AID
13.
08-02-97 13:02:57
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
growth factor-beta and suppress cytotoxic T lymphocyte responses. J. Exp. Med. 181, 1875–1880, 1995. Caver, T. E., O’Sullivan, F. X., Gold, L. I., and Gresham, H. D., Intracellular demonstration of active TGF-beta1 in B cells and plasma cells of autoimmune mice. IgG-bound TGF-beta1 suppresses neutrophil function and host defense against Staphylococcus aureus infection. J. Clin. Invest. 98, 2496–2506, 1996. Kim, D. H., and Kim, S.-J., Transforming growth factor-b receptors: Role in physiology and disease. J. Biomed. Sci. 3, 143–158, 1996. Attisano, L., and Wrana, J. L., Signal transduction by members of the transforming growth factor-beta superfamily. Cytokine Growth Factor Rev. 7, 327–339, 1996. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague, J., Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 383, 832–836, 1996. Chen, X., Rubock, M. J., and Whitman, M., A transcriptional partner for MAD proteins in TGF-beta signalling. Nature 383, 691–696, 1996. Ruscetti, F., Varesio, L., Ochoa, A., and Ortaldo, J., Pleiotropic effects of transforming growth factor-beta on cells of the immune system. Ann. NY Acad. Sci. 685, 488–500, 1993. Strobl, H., Riedl, E., Scheinecker, C., Bello-Fernandez, C., Pickl, W. F., Rappersberger, K., Majdic, O., and Knapp, W., TGF-beta 1 promotes in vitro development of dendritic cells from CD34/ hemopoietic progenitors. J. Immunol. 157, 1499–1507, 1996. Riedl, E., Strobl, H., Majdic, O., and Knapp, W., TGF-beta 1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis. J. Immunol. 158, 1591–1597, 1997. Borkowski, T. A., Letterio, J. J., Farr, A. G., and Udey, M. C., A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: The skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells. J. Exp. Med. 184, 2417–2422, 1996. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold, R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D., et al., Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359, 693–699, 1992. Kulkarni, A. B., Huh, C. G., Becker, D., Geiser, A., Lyght, M., Flanders, K. C., Roberts, A. B., Sporn, M. B., Ward, J. M., and Karlsson, S., Transforming growth factor-beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90, 770–774, 1993. Borkowski, T. A., Letterio, J. J., Mackall, C. L., Saitoh, A., Wang, X-J., Roop, D. R., Gress, R. E., and Udey, M. C., A role for TGFb1 in Langerhans cell biology: Further characterization of the epidermal Langerhans cell defect in TGF-b1 null mice. J. Clin. Invest., in press, 1997. Takahama, Y., Letterio, J. J., Suzuki, H., Farr, A. G., and Singer, A., Early progression of thymocytes along the CD4/CD8 developmental pathway is regulated by a subset of thymic epithelial cells expressing transforming growth factor-beta. J. Exp. Med. 179, 1495–1506, 1994. Plum, J., De Smedt, M., Leclercq, G., and Vandekerckhove, B., Influence of TGF-beta on murine thymocyte development in fetal thymus organ culture. J. Immunol. 154, 5789–5798, 1995. Mossalayi, M. D., Mentz, F., Ouaaz, F., Dalloul, A. H., Blanc, C., Debre, P., and Ruscetti, F. W., Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism. Blood 85, 3594–3601, 1995. Kulkarni, A. B., Ward, J. M., Geiser, A. G., Letterio, J. J., et al., TGF-b1 Knockout mice: Immune dysregulation and pathology.
clina
AP: Clin
TGF-b: MODULATOR OF IMMUNE CELL FUNCTION
29.
30.
31. 32. 33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
In ‘‘Molecular Biology of Hematopoiesis’’ (N. G. Abraham, R. K. Shadduck, A. S. Levine, and F. Takaku, Eds.), Vol. 3, pp. 749– 757, Intercept Ltd., Andover, Hampshire, 1994. Nakabayashi, T., Letterio, J. J., Kong, N., Ogawa, N., Dang, H., and Talal, N., Upregulation of cytokine mRNA, adhesion molecule proteins and MHC class II proteins in salivary glands of TGF-b1 knockout mice: MHC class II is a factor in the pathogenesis of TGF-b1 knockout mice. J. Immunol. 158, 5527–5535, 1997. Dang, H., Geiser, A. G., Letterio, J. J., Nakabayashi, T., Kong, L., Fernandes, G., and Talal, N., SLE-like autoantibodies and Sjogren’s syndrome-like lymphoproliferation in TGF-beta knockout mice. J. Immunol. 155, 3205–3212, 1995. Wahl, S. M., Transforming growth factor beta: The good, the bad, and the ugly. J. Exp. Med. 180, 1587–1590, 1994. Border, W. A., and Noble, N. A., Transforming growth factor beta in tissue fibrosis. N. Engl. J. Med. 331, 1286–1292, 1994. Tsunawaki, S., Sporn, M., Ding, A., and Nathan, C., Deactivation of macrophages by transforming growth factor-beta. Nature 334, 260–262, 1988. Ming, M., Ewen, M. E., and Pereira, M. E., Trypanosome invasion of mammalian cells requires activation of the TGF-beta signaling pathway. Cell 82, 287–296, 1995. Barral-Netto, M., Barral, A., Brownell, C. E., Skeiky, Y. A., Ellingsworth, L. R., Twardzik, D. R., and Reed, S. G., Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism. Science 257, 545–548, 1992. Kopp, J. B., Factor, V. M., Mozes, M., Nagy, P., Sanderson, N., Bottinger, E. P., Klotman, P. E., and Thorgeirsson, S. S., Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease. Lab. Invest. 74, 991–1003, 1996. Racke, M. K., Dhib-Jalbut, S., Cannella, B., Albert, P. S., Raine, C. S., and McFarlin, D. E., Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1. J. Immunol. 146, 3012–3017, 1991. Racke, M. K., Cannella, B., Albert, P., Sporn, M., Raine, C. S., and McFarlin, D. E., Evidence of endogenous regulatory function of transforming growth factor-beta 1 in experimental allergic encephalomyelitis. Int. Immunol. 4, 615–620, 1992. Racke, M. K., Sriram, S., Carlino, J., Cannella, B., Raine, C. S., and McFarlin, D. E., Long-term treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 2. J. Neuroimmunol. 46, 175–183, 1993. Santos, L. M., al-Sabbagh, A., Londono, A., and Weiner, H. L., Oral tolerance to myelin basic protein induces regulatory TGFbeta-secreting T cells in Peyer’s patches of SJL mice. Cell Immunol. 157, 439–447, 1994. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L., Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor-beta after antigen-specific triggering. Proc. Natl. Acad. Sci. USA 89, 421– 425, 1992. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., and Weiner, H. L., Regulatory T cell clones induced by oral tolerance: Suppression of autoimmune encephalomyelitis. Science 265, 1237– 1240, 1994. Chen, Y., Inobe, J., and Weiner, H. L., Induction of oral tolerance to myelin basic protein in CD8-depleted mice: Both CD4/ and CD8/ cells mediate active suppression. J. Immunol. 155, 910– 916, 1995. Strober, W., Kelsall, B., Fuss, I., Marth, T., Ludviksson, B., Ehrhardt, R., and Neurath, M., Reciprocal IFN-gamma and TGF-
AID
Clin 4409
/
a516$$$244
08-02-97 13:02:57
249
beta responses regulate the occurrence of mucosal inflammation. Immunol. Today 18, 61–64, 1997. 45. Marth, T., Strober, W., and Kelsall, B. L., High dose oral tolerance in ovalbumin TCR-transgenic mice: Systemic neutralization of IL-12 augments TGF-beta secretion and T cell apoptosis. J. Immunol. 157, 2348–2357, 1996. 46. Wakefield, L. M., and Sporn, M. B., Suppression of carcinogenesis: A role for TGF-beta and related molecules in prevention of cancer. Immunol. Ser. 51, 217–243, 1990. 47. Markowitz, S. D., and Roberts, A. B., Tumor suppressor activity of the TGF-beta pathway in human cancers. Cytokine Growth Factor Rev. 7, 93–102, 1996. 48. Hahn, S. A., Schutte, M., Shamsul Hoque, A. T. M., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E., DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271, 350–353, 1996. 49. Eppert, K., Scherer, S. W., Ozcelik, H., Pirone, R., Hoodless, P., Kim, H., Tsui, L. C., Bapat, B., Gallinger, S., Andrulis, I. L., Thomsen, G. H., Wrana, J. L., and Attisano, L., MADR2 maps to 18q21 and encodes a TGF-b-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell 86, 543–552, 1996. 50. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., and Vogelstein, B., Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 268, 1336–1338, 1995. 51. Knaus, P. I., Lindemann, D., DeCoteau, J. F., Perlman, R., Yankelev, H., Hille, M., Kadin, M. E., and Lodish, H. F., A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma. Mol. Cell Biol. 16, 3480–3489, 1996. 52. Kadin, M. E., Cavaille-Coll, M. W., Gertz, R., Massague, J., Cheifetz, S., and George, D., Loss of receptors for transforming growth factor beta in human T-cell malignancies. Proc. Natl. Acad. Sci. USA 91, 6002–6006, 1994. 53. Bo¨ttinger, E. P., Jakubczak, J. L., Roberts, I. S. D., Mumy, M., Hemmati, P., Bagnall, K., Merlino, G., and Wakefield, L. M., Expression of a dominant-negative mutant TGF-b type II receptor in transgenic mice reveals essential roles for TGF-b in regulation of growth and differentiation in the exocrine pancreas. EMBO J. 16(10), 1997, in press. 54. Tada, T., Ohzeki, S., Utsumi, K., Takiuchi, H., Muramatsu, M., Li, X. F., Shimizu, J., Fujiwara, H., and Hamaoka, T., Transforming growth factor-beta-induced inhibition of T cell function. Susceptibility difference in T cells of various phenotypes and functions and its relevance to immunosuppression in the tumorbearing state. J. Immunol. 146, 1077–1082, 1991. 55. Torre-Amione, G., Beauchamp, R. D., Koeppen, H., Park, B. H., Schreiber, H., Moses, H. L., and Rowley, D. A., A highly immunogenic tumor transfected with a murine transforming growth factor type-beta 1 cDNA escapes immune surveillance. Proc. Natl. Acad. Sci. USA 87, 1486–1490, 1990. 56. Weiskirch, L. M., Bar-Dagan, Y., and Mokyr, M. B., Transforming growth factor-beta-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy. Cancer Immunol. Immunother. 38, 215–224, 1994. 57. Fakhrai, H., Dorigo, O., Shawler, D. L., Lin, H., Mercola, D., Black, K. L., Royston, I., and Sobol, R. E., Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy. Proc. Natl. Acad. Sci. USA 93, 2909–2914, 1996.
clina
AP: Clin
250
LETTERIO AND ROBERTS
58. Wojtowicz-Praga, S., Verma, U. M., Wakefield, L., Esteban, J. M., Hartmann, D., and Mazumder, A., Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2. J. Immunother. Emphasis Tumor Immunol. 19, 169–175, 1996. 59. Arteaga, C. L., Hurd, S. D., Winnier, A. R., Johnson, M. D., Fendly, B. M., and Forbes, J. T., Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions
in human breast cancer progression. J. Clin. Invest. 92, 2569– 2576, 1993. 60. Hoefer, M., and Anderer, F. A., Anti-(transforming growth factor-beta) antibodies with predefined specificity inhibit metastasis of highly tumorigenic human xenotransplants in nu/nu mice. Cancer Immunol. Immunother. 41, 302–308, 1995. 61. Qin, L., Ding, Y., and Bromberg, J. S., Gene transfer of transforming growth factor-beta 1 prolongs murine cardiac allograft survival by inhibiting cell-mediated immunity. Hum. Gene Ther. 7, 1981–1988, 1996.
Received May 28, 1997; accepted June 13, 1997
AID
Clin 4409
/
a516$$$244
08-02-97 13:02:57
clina
AP: Clin
Vol. 84, No. 3, September, pp. 244–250, 1997 Article No. II974409
MOLECULE OF THE MONTH TGF-b: A Critical Modulator of Immune Cell Function John J. Letterio and Anita B. Roberts Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892-5055
INTRODUCTION
An imposing array of secreted signaling peptides orchestrates the host response to stimuli. Although the list of cytokines which make up this array continues to grow, we are only beginning to understand their distinct roles and the interplay between intracellular pathways which mediate their effects. As a subset of this group, the transforming growth factors-b (TGF-b) are distinguished by their ability to influence almost every facet of the immune response, including the growth and differentiation of precursors for multiple hematopoietic lineages, the proliferation and migration of mature immune cells into sites of injury or response, and even the suppression of such responses once they have been established (1). The complimentary autocrine, paracrine, and even endocrine modes of TGF-b activity that underly these effects have been elegantly delineated through extensive in vitro and in vivo analyses of function, including various TGF-b transgenic models and isoform-specific gene knockouts. In this discussion, we will emphasize how dysfunctional TGF-b pathways might contribute to the pathogenesis of immune disorders, explain how these preclinical studies have challenged some basic concepts regarding mechanisms of TGF-b action, and end by speculating on how this knowledge may be brought to bear on important clinical problems. THE TGF-b FAMILY
The prototype of this family, TGF-b1, was isolated and characterized nearly 15 years ago based on its ability to stimulate the anchorage-independent growth of fibroblasts, a correlate of tumorigenicity in vivo (for a review see Ref. 2). The surprising identification of platelets and bone as major sources of this peptide suggested prominent physiological roles. At present, with the advent of gene knockout and transgenic approaches, we are beginning to gain a greater appreciation for the complexity and breadth of the activity of TGF-b1 and its highly homologous isoforms, TGF-bs 2 and 3, as major negative regulators of the growth and differentiation of most cell lineages.
244
0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
Clin 4409
/
a516$$$241
TGF-bs 1, 2, and 3 are localized to human chromosomes 19q13, 1q41, and 14q24, (mouse chromosomes 7, 1, and 12) respectively (2). TGF-b1 is expressed by all immune cells and is the isoform most often acutely regulated in response to a variety of stress and disease signals (1, 3). All TGF-bs are encoded as precursor molecules which are processed proteolytically to the highly conserved (greater than 72%) biologically active carboxy-terminal domain of 112 amino acids, with the unique feature that the remainder of the pre–pro domain (latency-associated protein, LAP) associates noncovalently with the C-terminal fragment to reversibly block its activity (Fig. 1) (4, 5). Latency is an important component of regulation of TGF-b activity and can be conferred not only by the LAP protein, but also by its associated protein LTBP which directs the complex to matrix, and by molecules such as a2-macroglobulin which play a role in clearance (6, 7). In vivo activation of these latent forms of secreted TGF-b is a highly regulated, yet incompletely understood process, mediated by multiple pathways involving proteolytic mechanisms (4–6), redox (8), free radicals including nitric oxide, and binding to molecules such as thrombospondin (9). The recent identification of an active form of TGF-b complexed with IgG, as produced by B cells and plasma cells (see Fig. 1), points to yet another mode of regulation which may be key in the modification of specific immune cell responses (10–13). Signals from TGF-b ligands are mediated by a heterotetrameric receptor complex, distinguished by its intrinsic serine–threonine kinase activity from receptors for other immunoregulatory cytokines that signal through tyrosine kinase-mediated pathways (14, 15). The first downstream mediators of this signal transduction pathway to be identified include, minimally, a set of at least five highly conserved molecules called Smads, each related to the Drosophila protein Mad which acts downstream of the TGF-b family ligand dpp and its receptors (14–16). Steps involved in transduction of signals from TGF-b (or activin) involve direct serine phosphorylation of Smad2 or 3 by the type I receptor subunit, complexation with Smad4, which plays a unique role common to pathways of all TGF-b family ligands, and transloca-
08-02-97 13:02:57
clina
AP: Clin
TGF-b: MODULATOR OF IMMUNE CELL FUNCTION
FIG. 1. TGF-b signaling has multiple levels of regulation. Dysregulated activation of latent TGF-b and altered expression and activity of either its cell surface receptors or its cytoplasmic signaling intermediates contribute to the pathogenesis of a variety of immune diseases. Inactivation of TGF-b signaling (receptors or Smad proteins) in carcinogenesis can result in upregulation of TGF-b expression (53) and consequent paracrine suppression of immune surveillance. Among many mechanisms described for activation of latent TGF-b, one of particular importance for immune cells is the secretion by B-cells and plasma cells of a complex of TGF-b bound to IgG (10, 13). Whether binding to IgG alters the conformation of the latencyassociated peptide (LAP) to expose the receptor-binding epitope of TGF-b (13) or whether the TGF-b/IgG complex is internalized via Fc receptors and activated cytoplasmically is not known (11). TF, transcription factor analogous to the forkhead activin signal transducer-1 (FAST-1) (17).
tion of the complex to the nucleus where it activates expression of target genes by binding to transcription factors (Fig. 1) (14–17). The specific features of this signaling pathway germane to the activation, proliferation, and functional responses of specific immune cell populations remain to be elucidated. EVENTS REGULATED BY TGF-b IN LEUKOCYTE DEVELOPMENT
Nearly all leukocyte lineages are influenced by TGFb, at steps ranging from their production in the marrow compartment to their differentiation and maturation in the periphery (18). For example, the recent discovery of a requirement for TGF-b1 during the in vitro differentiation of functional dendritic cells (DC) identified a new role for TGF-b in regulating this class of antigenpresenting cells (19). Though in vitro differentiation of DC from cultures of CD34/ precursor cells is also dependent on the presence of other cytokines, such as TNF-a, SCF, and GM-CSF, it is quite inefficient with-
AID
Clin 4409
/
a516$$$242
08-02-97 13:02:57
245
out the addition of exogenous TGF-b1, which effectively substitutes for serum or plasma. The mechanism underlying this effect appears to be the protection of DC viability, with the percentage of apoptotic cells reduced by more than 60% at 72 hr in culture (20). The importance of TGF-b1 in DC development has been further emphasized by recent studies in the TGFb1 knockout mouse. The complete absence of epidermal DC or Langerhans cells (LC) is a striking feature of their phenotype (21), which includes generalized activation of most immune cell populations and widespread tissue inflammation (22, 23). Though inflammatory cytokines are known to effect migration of LC from the epidermis, TGF-b1 null mice bred onto a variety of immunodeficient backgrounds (SCID, athymic nude, RAG2-null), or successfully treated with the immunosuppressive agent rapamycin, are also deficient in epidermal LC (21). These data suggest that the absence of LC is a direct consequence of disruption of endogenous TGF-b1 gene expression, rather than an indirect result of the inflammatory process characteristic of the TGF-b1-null mouse. Transplantation of TGF-b1-null marrow into wild-type, lethally irradiated recipients leads to repopulation of recipient skin with donor-derived LC, implying that normal LC progenitors exist in the TGF-b1-null mouse, and that paracrine sources of TGF-b1 are sufficient to support normal LC development, and perhaps even their migration into the epidermis (24). Finally, the fact that TGF-b1-null mice also lack gp40/ lymph node DC (despite normal numbers of CD11c/ DC) suggests that the loss of TGF-b1 may have a broader effect on normal DC development (21). This influence of TGF-b on maturation and differentiation of leukocytes extends to lymphoid lineages as well, as demonstrated by the events regulating the differentiation of thymic precursors. Progression of the CD40CD8lo thymocyte to the CD4/CD8/ doublepositive stage of differentiation is a step that requires entry into S phase, and is inhibited by the addition of TGF-b (25). In fetal thymus organ culture, TGF-b produced by various lines of cortical thymic epithelial cells inhibits thymocyte progression to the doublepositive state (25), and the addition of exogenous TGF-b also blocks the ability of alymphoid thymic lobe cultures to support T cell development of precursor cells obtained from fetal liver (26). Similar studies of isolated human early thymic precursors suggest a role for autocrine sources of TGF-b as well. Autologous, irradiated CD3/CD8/CD40 T cells inhibit growth of the triple-negative (CD2/CD3// TCR0CD40CD80) thymocyte precursors by activating latent TGF-b produced by the precursor cells, an affect abolished by the addition of a neutralizing antiTGF-b antibody (27). So while the CD8/ cells were not the predominant source of TGF-b, they were re-
clina
AP: Clin
246
LETTERIO AND ROBERTS
sponsible for activating TGF-b produced in an autocrine loop by the precursors. Thus, in murine and human systems, TGF-b acts as both a paracrine and an autocrine inhibitory factor, during the generation of thymic T cells. Knowing that such key steps in the development of both antigen-presenting cells and T lymphocytes are controlled by endogenous TGF-b expression, one might predict that loss of the predominant TGF-b isoform, TGF-b1, might lead to considerable disruption in immunological homeostasis. In particular, the loss of negative inhibition of thymic precursors might lead to increased numbers of mature CD4/ or CD8/, singlepositive lymphocytes, which may escape important selection steps regulated by TGF-b1. Indeed, the thymus of a TGF-b1 knockout mouse exhibits decreased cellularity, primarily due to a loss of immature double-positive (CD4/CD8/) precursors, with a predominance of mature single-positive T cells, expressing high levels of MHC class I and CD3. In the periphery, this is accompanied in lymph nodes (and in the spleen to a lesser extent) by increased numbers of CD4/ T cells displaying an activated phenotype (increased H1.2F3 and CD44, with diminished CD45RB expression) (28). It has been speculated that a defect in negative selection may result from the premature exit of these differentiated T cell subsets from the thymus. These TGF-b1null T cells appear to be in a continuous state of proliferation, expressing mRNA for both IL-2 and IL-2 receptors, and producing a spectrum of proinflammatory cytokines, including IL-2, IL-6, and IL-10, which may act as generalized promoters for an autoimmune response, a predominant feature of the phenotype of the TGF-b1 knockout mouse (29, 30). TGF-b1-null mice develop IgG autoantibodies with a spectrum of specificities, with glomerular immune complex deposition and widespread vasculitis as other hallmarks of the phenotype (30). These key features associated with TGF-b1 deficiency in vivo support many of the outcomes or functions predicted for this molecule from in vitro analyses of function. THE DUAL ROLE OF TGF-b IN DISEASE PATHOGENESIS
TGF-b has been implicated in a wide variety of physiological repair processes and in the pathogenesis of various diseases, often playing a direct role in mediating associated immune defects (3, 31). Examples in which dysregulated expression of TGF-b1 by immune cells may drive the disease process include the fibrosis that accompanies chronic inflammation (32), and parasitic diseases in which TGF-b1 secretion blocks activation of macrophages by interferon-g (IFN-g), diminishing their oxidative responses (33). In this regard, it has been demonstrated that the intracellular parasite
AID
Clin 4409
/
a516$$$242
08-02-97 13:02:57
Trypanosoma cruzi not only triggers activation of the TGF-b signaling pathway, but also that it is required for parasite entry into mammalian cells (34). Similarly, macrophage infection by the protozoan parasite Leishmania induces production of biologically active TGFb; systemic administration of neutralizing anti-TGF-b antibodies can halt progression of this disease in genetically susceptible mice (35). The mechanisms of activation of TGF-b in these processes are clearly key areas for future study. This association between expression and activation of TGF-b and susceptibility to infection has also recently been identified as a feature of both murine models of autoimmunity and their human disease counterparts (31). Evaluation of the host defense status of the MRL/lpr mouse revealed that acquired defects in polymorphonuclear (PMN) chemotaxis and phagocytosis are linked to increased susceptibility to both gram-negative and gram-positive infection (10). Most importantly, these defects were directly linked to the elaboration of excessive levels of both the active and latent forms of TGF-b1, and were reversed in mice treated with neutralizing anti-TGF-b antibodies. Although other factors such as concurrent immunosuppressive therapy may be responsible for similar PMN defects in patients with systemic lupus erythematosis (SLE), it is interesting that an analysis of TGF-b1 levels in the plasma of SLE patients revealed similar increases in circulating levels of this cytokine (10). Overproduction of TGF-b in autoimmune disorders may also underlie or promote tissue injury associated with these disorders. Pathogenic autoantibody production resulting in glomerular immune complex deposition is a shared feature of many of these diseases, and it is most significant that fibrotic lesions and glomerulonephritis strikingly similar to this immune complex-mediated injury also develop in transgenic mice which overexpress active TGF-b1 under the control of murine albumin promoter and enhancer sequences (36). Although these observations suggest that TGF-b plays a central role in development of inflammatory and infectious diseases complications, it clearly plays contradictory roles, in that its powerful immunosuppressive activity also contributes to the prevention and recovery from autoimmune disorders. Expression of TGF-b1 by immune cells has been implicated in the recovery phase of the relapsing–remitting course of experimental allergic encephalomyelitis (EAE), the murine model of multiple sclerosis (MS) (37). The ability of systemic TGF-b to suppress symptoms and delay progression, and the appearance of increased endogenous TGF-b1 expression during the recovery phase, points toward a beneficial role for TGF-b production in autoimmune disease (38, 39). Perhaps the strongest evidence for this protective role comes from studies which link disease prevention to the suppressive effects
clina
AP: Clin
TGF-b: MODULATOR OF IMMUNE CELL FUNCTION
generated by oral tolerance (40). In EAE, the oral administration of myelin basic protein leads to development of a T-cell-mediated suppression that can be adoptively transferred, can suppress immune responses in vitro, and can be blocked by neutralizing anti-TGF-b antibodies (41). It has since been demonstrated that oral antigen induces both mucosally derived TH2-like CD4/ T cells (producing active TGF-b, IL-4, and IL-10) and the TGF-b-producing CD8/ T cells originally identified (42, 43). Recent studies have shown that the protective effects of oral antigen are enhanced by systemic anti-IL-12 treatment, which leads to substantially increased TGF-b production and T cell apoptosis in Peyer’s patches (44, 45). Again, perhaps the strongest argument for the beneficial role of TGF-b1 in the maintenance of immunological homeostasis comes from the TGF-b1-null mouse, where loss of the TGF-b1 ligand results in many hallmarks of autoimmune disease, progressive tissue inflammation, and death (23). In addition to these conflicting roles played by TGFb in autoimmune and inflammatory disorders, it also has both direct and indirect effects on immune cell populations in the process of carcinogenesis (46). In both epithelial and lymphoid malignancies, tumor cells commonly acquire resistance to the growth inhibitory effects of TGF-b. The TGF-b receptors, as well as the cytoplasmic signaling intermediates Smads 2 and 4, have been shown to have tumor suppressor activity, since their loss results in increased tumorigenicity (47– 49). Functional inactivation of receptors can occur by a wide variety of mechanisms including truncation resulting from microsatellite instability, or mutation of the kinase domain or the ligand binding site (47, 50). In human T cell lymphomas, receptor mutations resulting in dominant negative behavior have been reported (51, 52), while in murine plasmacytomas, a defect in ligand binding by the receptor has been noted (manuscript in preparation). Transcriptional repression of receptor expression is also observed. Recently, it has been shown that loss of receptor function, as modeled in transgenic mice expressing a dominant-negative type II TGF-b receptor subunit (DNRII), leads directly to secretion of substantial quantities of biologically active TGF-b, as is characteristic of tumor cells resistant to TGF-b (53). Purification of a glioblastoma-derived, T-cell-suppressive factor (later identified as TGF-b2) provided the first evidence of the potential for TGF-b to act as an inhibitor of the mechanisms mediating immune surveillance of cancer (Fig. 2). In depth studies have led to the conclusion that production of TGF-b by a tumor is essential for its progression, preventing destruction by both specific cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (54). Indeed, transduction of a highly immunogenic, UV-induced fibrosarcoma with a TGF-b1 expression vector
AID
Clin 4409
/
a516$$$243
08-02-97 13:02:57
247
FIG. 2. TGF-b-mediated inhibition of cytotoxic T lymphocytes. CTL killing of tumor cells is antigen-specific, and tumor cells bearing class I MHC-associated antigen can trigger CTL activation, causing them to release contents of membrane-associated cytoplasmic granules, including the pore-forming proteins perforin and cytosolin, and lymphotoxins such as TNF-a and IFN-g (A). Tumor cells which lose sensitivity to TGF-b, resulting either from receptor mutation or inactivation, often secrete substantial amounts of active TGF-b. This consequence of malignant progression has been shown to block CTLmediated tumor lysis, leading instead to CTL apoptosis (B). (Apoptotic cells are in dotted outline.)
not only renders it incapable of stimulating primary CTL responses in vitro, but is also sufficient to allow for escape from immune surveillance and progressive growth in vivo (55). Similarly, differentiating agents which dramatically reduce the production of TGF-b by MOPC-315 plasmacytoma cells in BALBc mice, also lead to the development of potent CD8/ T-cell-mediated anti-MOPC-315 CTL activity (56). Thus, a positive correlation between invasiveness, progression, decreased TGF-b sensitivity, and increased TGF-b production has been established, and the latter may contribute to locally impaired immune response to tumor cells. These experiments dramatically demonstrate the therapeutic potential of strategies targeting either tumor cell production or immune cell responsiveness to TGF-b. Approaches based on the inhibition of TGFb production, by the introduction of TGF-b antisense expression vectors into tumor cells, have already proven useful for induction of CTL, both in vitro or in vivo, with the latter leading to rejection of established tumors and T cell memory in an animal model of glioblastoma (57). The development of clinical vaccine trials using TGF-b antisense tumor modification is currently in progress. It is clear that similar approaches to block activity or interrupt endogenous production of TGF-b, whether by tumor cells or activated immune cell populations, may be an important strategy for boosting immune function. While the systemic administration of antiTGF-b neutralizing antibodies has been successful in enhancing CTL and NK cell function in animal models
clina
AP: Clin
248
LETTERIO AND ROBERTS
(58–60), the long-term efficacy and application of such an approach in humans is still questionable. In contrast, for those inflammatory and autoimmune disorders which may benefit from the potent immunosuppressive effects of TGF-b (rheumatoid arthritis, MS, Sjo¨grens disease, graft-vs-host disease, allograft rejection), we must look toward strategies that may enhance production (61), and activation of endogenous TGF-b, and work to identify agents which may target and activate some of the newly identified signaling intermediates. With over a decade of preclinical evaluation pointing toward the critical immunomodulatory properties of TGF-b, it ultimately will become both the target and the tool for fighting human diseases linked to altered immune function. REFERENCES
10. Lowrance, J. H., O’Sullivan, F. X., Caver, T. E., Waegell, W., and Gresham, H. D., Spontaneous elaboration of transforming growth factor-beta suppresses host defense against bacterial infection in autoimmune MRL/lpr mice. J. Exp. Med. 180, 1693– 1703, 1994. 11. Stach, R. M., and Rowley, D. A., A first or dominant immunization. II. Induced immunoglobulin carries transforming growth factor-beta and suppresses cytolytic T cell responses to unrelated alloantigens. J. Exp. Med. 178, 841–852, 1993. 12. Rowley, D. A., Becken, E. T., and Stach, R. M., Autoantibodies produced spontaneously by young 1pr mice carry transforming
Clin 4409
/
a516$$$244
14.
15.
16.
17.
18.
1. McCartney-Francis, N. L., and Wahl, S. M., Transforming growth factor-beta: a matter of life and death. J. Leukocyte Biol. 55, 401–409, 1994. 2. Roberts, A. B., and Sporn, M. B., The transforming growth factors-b. In ‘‘Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors’’ (M. B. Sporn and A. B. Roberts, Eds.), pp. 419–472, Springer Verlag, Berlin, 1990. 3. Roberts, A. B., and Sporn, M. B., Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Factors 8, 1–9, 1993. 4. Munger, J. S., Harpel, J. G., Gleizes, P. E., Mazzieri, R., Nunes, I., and Rifkin, D. B., Latent transforming growth factor-beta: Structural features and mechanisms of activation. Kidney Int. 51, 1376–1382, 1997. 5. Barcellos-Hoff, M. H., Latency and activation in the control of TGF-b. J. Mammary Gland Biol. Neoplasia 1, 351–361, 1997. 6. Nunes, I., Gleizes, P. E., Metz, C. N., and Rifkin, D. B., Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta. J. Cell Biol. 136, 1151–1163, 1997. 7. LaMarre, J., Hayes, M. A., Wollenberg, G. K., Hussaini, I., Hall, S. W., and Gonias, S. L., An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice. J. Clin. Invest. 87, 39–44, 1991. 8. Barcellos-Hoff, M. H., and Dix, T. A., Redox-mediated activation of latent transforming growth factor-beta 1. Mol. Endocrinol. 10, 1077–1083, 1996. 9. Schultz-Cherry, S., Ribeiro, S., Gentry, L., and Murphy-Ullrich, J. E., Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system. J. Biol. Chem. 269, 26775–26782, 1994.
AID
13.
08-02-97 13:02:57
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
growth factor-beta and suppress cytotoxic T lymphocyte responses. J. Exp. Med. 181, 1875–1880, 1995. Caver, T. E., O’Sullivan, F. X., Gold, L. I., and Gresham, H. D., Intracellular demonstration of active TGF-beta1 in B cells and plasma cells of autoimmune mice. IgG-bound TGF-beta1 suppresses neutrophil function and host defense against Staphylococcus aureus infection. J. Clin. Invest. 98, 2496–2506, 1996. Kim, D. H., and Kim, S.-J., Transforming growth factor-b receptors: Role in physiology and disease. J. Biomed. Sci. 3, 143–158, 1996. Attisano, L., and Wrana, J. L., Signal transduction by members of the transforming growth factor-beta superfamily. Cytokine Growth Factor Rev. 7, 327–339, 1996. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague, J., Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 383, 832–836, 1996. Chen, X., Rubock, M. J., and Whitman, M., A transcriptional partner for MAD proteins in TGF-beta signalling. Nature 383, 691–696, 1996. Ruscetti, F., Varesio, L., Ochoa, A., and Ortaldo, J., Pleiotropic effects of transforming growth factor-beta on cells of the immune system. Ann. NY Acad. Sci. 685, 488–500, 1993. Strobl, H., Riedl, E., Scheinecker, C., Bello-Fernandez, C., Pickl, W. F., Rappersberger, K., Majdic, O., and Knapp, W., TGF-beta 1 promotes in vitro development of dendritic cells from CD34/ hemopoietic progenitors. J. Immunol. 157, 1499–1507, 1996. Riedl, E., Strobl, H., Majdic, O., and Knapp, W., TGF-beta 1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis. J. Immunol. 158, 1591–1597, 1997. Borkowski, T. A., Letterio, J. J., Farr, A. G., and Udey, M. C., A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: The skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells. J. Exp. Med. 184, 2417–2422, 1996. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold, R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D., et al., Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359, 693–699, 1992. Kulkarni, A. B., Huh, C. G., Becker, D., Geiser, A., Lyght, M., Flanders, K. C., Roberts, A. B., Sporn, M. B., Ward, J. M., and Karlsson, S., Transforming growth factor-beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90, 770–774, 1993. Borkowski, T. A., Letterio, J. J., Mackall, C. L., Saitoh, A., Wang, X-J., Roop, D. R., Gress, R. E., and Udey, M. C., A role for TGFb1 in Langerhans cell biology: Further characterization of the epidermal Langerhans cell defect in TGF-b1 null mice. J. Clin. Invest., in press, 1997. Takahama, Y., Letterio, J. J., Suzuki, H., Farr, A. G., and Singer, A., Early progression of thymocytes along the CD4/CD8 developmental pathway is regulated by a subset of thymic epithelial cells expressing transforming growth factor-beta. J. Exp. Med. 179, 1495–1506, 1994. Plum, J., De Smedt, M., Leclercq, G., and Vandekerckhove, B., Influence of TGF-beta on murine thymocyte development in fetal thymus organ culture. J. Immunol. 154, 5789–5798, 1995. Mossalayi, M. D., Mentz, F., Ouaaz, F., Dalloul, A. H., Blanc, C., Debre, P., and Ruscetti, F. W., Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism. Blood 85, 3594–3601, 1995. Kulkarni, A. B., Ward, J. M., Geiser, A. G., Letterio, J. J., et al., TGF-b1 Knockout mice: Immune dysregulation and pathology.
clina
AP: Clin
TGF-b: MODULATOR OF IMMUNE CELL FUNCTION
29.
30.
31. 32. 33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
In ‘‘Molecular Biology of Hematopoiesis’’ (N. G. Abraham, R. K. Shadduck, A. S. Levine, and F. Takaku, Eds.), Vol. 3, pp. 749– 757, Intercept Ltd., Andover, Hampshire, 1994. Nakabayashi, T., Letterio, J. J., Kong, N., Ogawa, N., Dang, H., and Talal, N., Upregulation of cytokine mRNA, adhesion molecule proteins and MHC class II proteins in salivary glands of TGF-b1 knockout mice: MHC class II is a factor in the pathogenesis of TGF-b1 knockout mice. J. Immunol. 158, 5527–5535, 1997. Dang, H., Geiser, A. G., Letterio, J. J., Nakabayashi, T., Kong, L., Fernandes, G., and Talal, N., SLE-like autoantibodies and Sjogren’s syndrome-like lymphoproliferation in TGF-beta knockout mice. J. Immunol. 155, 3205–3212, 1995. Wahl, S. M., Transforming growth factor beta: The good, the bad, and the ugly. J. Exp. Med. 180, 1587–1590, 1994. Border, W. A., and Noble, N. A., Transforming growth factor beta in tissue fibrosis. N. Engl. J. Med. 331, 1286–1292, 1994. Tsunawaki, S., Sporn, M., Ding, A., and Nathan, C., Deactivation of macrophages by transforming growth factor-beta. Nature 334, 260–262, 1988. Ming, M., Ewen, M. E., and Pereira, M. E., Trypanosome invasion of mammalian cells requires activation of the TGF-beta signaling pathway. Cell 82, 287–296, 1995. Barral-Netto, M., Barral, A., Brownell, C. E., Skeiky, Y. A., Ellingsworth, L. R., Twardzik, D. R., and Reed, S. G., Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism. Science 257, 545–548, 1992. Kopp, J. B., Factor, V. M., Mozes, M., Nagy, P., Sanderson, N., Bottinger, E. P., Klotman, P. E., and Thorgeirsson, S. S., Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease. Lab. Invest. 74, 991–1003, 1996. Racke, M. K., Dhib-Jalbut, S., Cannella, B., Albert, P. S., Raine, C. S., and McFarlin, D. E., Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1. J. Immunol. 146, 3012–3017, 1991. Racke, M. K., Cannella, B., Albert, P., Sporn, M., Raine, C. S., and McFarlin, D. E., Evidence of endogenous regulatory function of transforming growth factor-beta 1 in experimental allergic encephalomyelitis. Int. Immunol. 4, 615–620, 1992. Racke, M. K., Sriram, S., Carlino, J., Cannella, B., Raine, C. S., and McFarlin, D. E., Long-term treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 2. J. Neuroimmunol. 46, 175–183, 1993. Santos, L. M., al-Sabbagh, A., Londono, A., and Weiner, H. L., Oral tolerance to myelin basic protein induces regulatory TGFbeta-secreting T cells in Peyer’s patches of SJL mice. Cell Immunol. 157, 439–447, 1994. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L., Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor-beta after antigen-specific triggering. Proc. Natl. Acad. Sci. USA 89, 421– 425, 1992. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., and Weiner, H. L., Regulatory T cell clones induced by oral tolerance: Suppression of autoimmune encephalomyelitis. Science 265, 1237– 1240, 1994. Chen, Y., Inobe, J., and Weiner, H. L., Induction of oral tolerance to myelin basic protein in CD8-depleted mice: Both CD4/ and CD8/ cells mediate active suppression. J. Immunol. 155, 910– 916, 1995. Strober, W., Kelsall, B., Fuss, I., Marth, T., Ludviksson, B., Ehrhardt, R., and Neurath, M., Reciprocal IFN-gamma and TGF-
AID
Clin 4409
/
a516$$$244
08-02-97 13:02:57
249
beta responses regulate the occurrence of mucosal inflammation. Immunol. Today 18, 61–64, 1997. 45. Marth, T., Strober, W., and Kelsall, B. L., High dose oral tolerance in ovalbumin TCR-transgenic mice: Systemic neutralization of IL-12 augments TGF-beta secretion and T cell apoptosis. J. Immunol. 157, 2348–2357, 1996. 46. Wakefield, L. M., and Sporn, M. B., Suppression of carcinogenesis: A role for TGF-beta and related molecules in prevention of cancer. Immunol. Ser. 51, 217–243, 1990. 47. Markowitz, S. D., and Roberts, A. B., Tumor suppressor activity of the TGF-beta pathway in human cancers. Cytokine Growth Factor Rev. 7, 93–102, 1996. 48. Hahn, S. A., Schutte, M., Shamsul Hoque, A. T. M., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E., DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271, 350–353, 1996. 49. Eppert, K., Scherer, S. W., Ozcelik, H., Pirone, R., Hoodless, P., Kim, H., Tsui, L. C., Bapat, B., Gallinger, S., Andrulis, I. L., Thomsen, G. H., Wrana, J. L., and Attisano, L., MADR2 maps to 18q21 and encodes a TGF-b-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell 86, 543–552, 1996. 50. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., and Vogelstein, B., Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 268, 1336–1338, 1995. 51. Knaus, P. I., Lindemann, D., DeCoteau, J. F., Perlman, R., Yankelev, H., Hille, M., Kadin, M. E., and Lodish, H. F., A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma. Mol. Cell Biol. 16, 3480–3489, 1996. 52. Kadin, M. E., Cavaille-Coll, M. W., Gertz, R., Massague, J., Cheifetz, S., and George, D., Loss of receptors for transforming growth factor beta in human T-cell malignancies. Proc. Natl. Acad. Sci. USA 91, 6002–6006, 1994. 53. Bo¨ttinger, E. P., Jakubczak, J. L., Roberts, I. S. D., Mumy, M., Hemmati, P., Bagnall, K., Merlino, G., and Wakefield, L. M., Expression of a dominant-negative mutant TGF-b type II receptor in transgenic mice reveals essential roles for TGF-b in regulation of growth and differentiation in the exocrine pancreas. EMBO J. 16(10), 1997, in press. 54. Tada, T., Ohzeki, S., Utsumi, K., Takiuchi, H., Muramatsu, M., Li, X. F., Shimizu, J., Fujiwara, H., and Hamaoka, T., Transforming growth factor-beta-induced inhibition of T cell function. Susceptibility difference in T cells of various phenotypes and functions and its relevance to immunosuppression in the tumorbearing state. J. Immunol. 146, 1077–1082, 1991. 55. Torre-Amione, G., Beauchamp, R. D., Koeppen, H., Park, B. H., Schreiber, H., Moses, H. L., and Rowley, D. A., A highly immunogenic tumor transfected with a murine transforming growth factor type-beta 1 cDNA escapes immune surveillance. Proc. Natl. Acad. Sci. USA 87, 1486–1490, 1990. 56. Weiskirch, L. M., Bar-Dagan, Y., and Mokyr, M. B., Transforming growth factor-beta-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy. Cancer Immunol. Immunother. 38, 215–224, 1994. 57. Fakhrai, H., Dorigo, O., Shawler, D. L., Lin, H., Mercola, D., Black, K. L., Royston, I., and Sobol, R. E., Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy. Proc. Natl. Acad. Sci. USA 93, 2909–2914, 1996.
clina
AP: Clin
250
LETTERIO AND ROBERTS
58. Wojtowicz-Praga, S., Verma, U. M., Wakefield, L., Esteban, J. M., Hartmann, D., and Mazumder, A., Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2. J. Immunother. Emphasis Tumor Immunol. 19, 169–175, 1996. 59. Arteaga, C. L., Hurd, S. D., Winnier, A. R., Johnson, M. D., Fendly, B. M., and Forbes, J. T., Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions
in human breast cancer progression. J. Clin. Invest. 92, 2569– 2576, 1993. 60. Hoefer, M., and Anderer, F. A., Anti-(transforming growth factor-beta) antibodies with predefined specificity inhibit metastasis of highly tumorigenic human xenotransplants in nu/nu mice. Cancer Immunol. Immunother. 41, 302–308, 1995. 61. Qin, L., Ding, Y., and Bromberg, J. S., Gene transfer of transforming growth factor-beta 1 prolongs murine cardiac allograft survival by inhibiting cell-mediated immunity. Hum. Gene Ther. 7, 1981–1988, 1996.
Received May 28, 1997; accepted June 13, 1997
AID
Clin 4409
/
a516$$$244
08-02-97 13:02:57
clina
AP: Clin