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Homing to suppress: address codes for Treg migration
Opinion
TRENDS in Immunology
Vol.26 No.12 December 2005
Homing to suppress: address codes for Treg migration Jochen Huehn and Alf Hamann Experimental Rheumatology, Charite´ University Medicine Berlin, c/o DRFZ, Schumannstr. 21/22, 10117 Berlin, Germany
Compelling evidence suggests that diverse types of immune reactions can be suppressed by CD25CCD4C regulatory T cells (Tregs). Although increasing knowledge has accumulated concerning the generation and functional properties of Tregs, relatively little attention has been paid to another key question: where does immune regulation by Tregs take place in vivo? Tregs can inhibit both the priming and the effector phase of an immune response, so suppression might occur both within lymphoid tissues and at peripheral sites during immune reactions. This leads to the hypothesis that appropriate localization is indispensable for in vivo Treg function and that the migratory behavior of Treg subsets influences their in vivo suppressive capacity. Current data suggest a division of labor between subpopulations of Tregs, which is mainly based on specialized homing patterns.
Tregs - a Brief introduction The molecular mechanisms governing Treg development and function are currently a hot topic because of their importance in controlling host responses to tumors, preventing transplant rejection and inhibiting the development of autoimmunity and allergy [1,2]. For some time, CD4C Tregs were identified solely by the constitutive expression of CD25, but the existence of CD25– Tregs, together with the induced expression of CD25 on recently activated conventional T cells, somewhat limited the results obtained by this phenotypic characterization. Recent studies have shown that the forkhead box transcription factor Foxp3 is highly expressed in Tregs and regulates their development and function [3]. Foxp3 now serves as the best marker to identify Tregs and, excitingly, both Foxp3-transduced T cells and CD25CCD4C Tregs have been shown to exhibit therapeutical potential [4,5]. To use Tregs for an efficient treatment of autoimmune disorders and to prevent transplant rejections, immunologists have made an enormous effort during recent years to discover the principles underlying the development and generation of Tregs, their antigen-specificity and their suppressor mechanisms [1,2]. However, another important aspect within this context has gained less attention so far; that is, at which sites does immune suppression by Tregs takes place in vivo? Here, by focusing on the migration behavior Corresponding author: Huehn, J. ([email protected]). Available online 21 October 2005
of Tregs, we will describe that not only the suppressor potential but also appropriate localization determines the in vivo suppressive capacity of Tregs. We therefore propose that Treg subsets, which differ in homing patterns, will also display different in vivo suppressive capabilities. Occurrence of Tregs in vivo: lymphoid organs versus peripheral and inflamed sites CD25CCD4C Tregs were first identified in lymphoid tissues, including thymus, spleen and lymph nodes (LNs), as well as in peripheral blood and constitute w5– 10% of total CD4C T cells [6–8]. More recent immunohistological studies have shown that under steady-state conditions Foxp3CCD25CCD4C Tregs were evenly distributed in the T-cell areas of murine and human lymphoid tissues [9–11]. Tregs were barely detectable in B cell follicles, however, they were found to change their chemotactic behavior upon activation, which would enable migration into germinal centers and the suppression of antibody production [10]. In the transfer colitis model, CD25CCD4C Tregs cotransferred with pathogenic CD45RBhighCD4CT cells migrated into both mesenteric LNs, as well as the colon, suggesting that suppression might take place at both sites in this model [5]. Interestingly, several recent publications studying adhesion molecule expression or chemokine responsiveness of Treg subsets suggest that Tregs are indeed able both to recirculate through lymphoid tissues and to enter inflamed sites [10,12–15]. Particularly under pathological conditions, Tregs were identified within various peripheral sites, including inflamed organs, tumors and infectious sites (Table 1). Thus, although parabiosis experiments (where two congenic mice were joined laterally to allow bloodflow between them and cells of the individual mice can be kept apart by use of the congenic marker) have shown that under homeostatic conditions Tregs display a reduced recirculation rate in comparison to conventional T cells, which might be explained by the differential expression of regulator of G-protein signaling (RGS) molecules [16], the prevalence of Tregs at sites of ongoing immune responses clearly demonstrates their capacity to migrate into sites of inflammation. It has been suggested by D’Ambrosio et al. that the expression of specific chemokine receptors (CCRs) on Tregs could guide them to different types of chemokinesecreting antigen-presenting cells and subsets of effector
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Table 1. Tregs have been identified within various peripheral sites Tissue or site Inflamed pancreas Inflamed mucosa Inflamed joint Inflamed skin Inflamed brain Allotransplant Tumor Pneumocystis carinii-infected lung Leishmania major-infected skin Schistosoma mansoni-induced granuloma Litomosoides sigmodontis-infected thoracic cavity Viral lesions
Species Murine Murine Human Murine Human Murine Human Murine Murine Murine Human Murine Murine Murine
Refs [25,32] [5] [36] [28] [37,38] [28,29] [39,40] [41] [31,42] [9,43] [18,44] [45] [46,47] [48]
Murine
[49]
Murine
[50]
cells, enabling direct cell–cell contact, which is required for efficient suppression [17]. Indeed, CCL22-producing tumor cells and environmental macrophages have been shown recently, in a human cancer study, to recruit Foxp3CCD25CCD4C Tregs selectively [18]. Tumor Tregs preferentially accumulated in tumors and ascites but rarely entered draining LNs in later cancer stages. Such an accumulation of Tregs within tumors predicted reduced survival of the patients, indicating an efficient suppression of anti-tumor responses directly within the tumor. Where does suppression take place? Evidence for a division of labor It is not completely understood at which stages and by which mechanisms Tregs suppress immune reactions in vivo. The presence of Tregs in lymphoid organs, as well as peripheral sites, would enable both suppression of the priming phase, taking place within lymphoid tissues, and control of later stages of the response, directly at the effector site. However, do the same cells do these jobs in such different environments? Increasing evidence points towards a division of labor between subsets of Tregs, where the competence to migrate into either lymphoid or inflammatory sites is a distinctive determinant of their function. The first experimental evidence for this division of labor between Treg subsets came from studies comparing the in vivo suppressive potential of CD62Lhigh and CD62Llow CD25CCD4C Treg subsets. CD62Lhigh Tregs were superior to their CD62Llow counterparts in preventing the development of autoimmunity or in suppressing graft-versus-host disease (GvHD) [14,19,20]. CD62Lhigh and CD62Llow Treg subsets have been reported in several studies to harbor a similar in vitro suppressive capacity [14,21], therefore, the high in vivo regulatory capacity of the CD62Lhigh subset in these models most probably reflects differences in homing properties, rather than suppressor potential per se. Interestingly, in all models where CD62LhighCD25CCD4C Tregs showed high suppressive capacity, adoptive Treg transfer was performed before the onset of disease. This indicates that in such www.sciencedirect.com
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cases, where the induction of an immune reaction within the LNs has to be prevented, only those Tregs that can efficiently enter these lymphoid sites can control the development of effector cells and, thereby, the induction of autoimmunity [22]. Further experimental evidence for the LN being a crucial site for tolerance induction was obtained in an allotransplantation model, where blockade of migration of Foxp3CCD25CCD4CCD62Lhigh Tregs into LNs using anti-CD62L mAbs resulted in graft rejection [23]. In this model, progressive expansion of Tregs could only be observed within the LNs but not in other anatomic sites, indicating that alloantigen-specific Tregs developed and/or expanded in the LNs of tolerized animals, and are necessary for the induction and maintenance of tolerance. Although these data were supported by studies in autoimmune diabetes models, where failure of extensive expansion of Tregs in pancreatic LNs correlated with accelerated diabetes progression [24,25], a more recent study has extended these findings by showing that development of autoimmune diabetes not only depends on the absolute number of Tregs within the pancreatic LN but also on the delicate balance between effector cells and Tregs [26]. Recently, it has been shown that the integrin aEb7 subdivides the Treg compartment into aE-negative ðaK E CD C K and aC 25CÞ and aE-positive Tregs (aC E CD25 E CD25 ), which largely differ in their in vivo suppressive capacity and in the expression of adhesion molecules and chemoC kine receptors [27,28]. aK E CD25 Tregs displayed a naivelike phenotype and efficiently migrated into LNs fitting to their high CD62L expression and their high responsiveness towards CCR7 ligands. Interestingly, these cells lacked suppressive capacity under acute inflammatory conditions but turned out to be most potent at suppressing naive CD4C T-cell proliferation within the LN [28,29]. By contrast, aC E Tregs showed increased frequencies of E/P selectin ligand expression combined with a substantial downregulation of CD62L and high responsiveness towards several inflammatory chemokines. Correspondingly, aC E Tregs displayed only a rather poor capacity to migrate into LNs but, by contrast, efficiently entered inflamed sites. Strikingly, this inflammation-seeking capacity was combined with high suppressive potential in various inflammation models [28,29]. In summary, these findings strengthen the functional relevance of the appropriate localization of Tregs for their in vivo suppressive capacity and lead us to propose a model of ‘division of labor’ for the function of Tregs, which is mainly based on their differential migration behavior (Box 1 and Figure 1). Functional and molecular evidence for the importance of the appropriate Treg localization Experimental evidence for the importance of the appropriate Treg localization first came from Schwarz and colleagues, who analyzed the suppressive capacity of UVinduced, hapten-specific Tregs in a contact hypersensitivity model [30]. Interestingly, the UV-induced Tregs expressed high levels of CD62L but not the ligands for the skin-homing receptors E- and P-selectin. In addition, they were only capable of controlling the induction phase
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Vol.26 No.12 December 2005
Box 1. ‘Division of labor’ model based on differential migration behavior of Treg subsets To maintain a tolerant state, the presence of Tregs in lymphoid organs or at the effector site might be required depending on the stage of the immune response. During the priming phase, Tregs with a naive-like phenotype suppress the induction, expansion and differentiation of autoreactive effector cells within lymphoid tissues. In case of inflammation, Tregs become activated and differentiate into an effector/memory-like stage, characterized by the expression of chemokine receptors and adhesion molecules required for migration into inflamed tissues. These effector/memory-like Tregs enter the inflamed site and serve to limit either peripheral expansion, cytokine secretion or cytolytic function of effector cells at later stages of the response. Under these circumstances, self-limitation and resolution of the inflammatory reaction might be their key function. Interestingly, the conversion of naive-like Tregs into effector/memory-like Tregs has recently been shown to occur upon stimulation both in vitro and in vivo (J. Huehn, unpublished results). This might be an important issue for the therapeutic application of inflammation-seeking Tregs.
of the skin inflammation but showed no suppressive capacity during the effector phase. Strikingly, if these hapten-specific Tregs were injected directly into the effector site they could even suppress the challenge reaction showing that they were, in principle, able to inhibit the effector phase [30]. The assumption that the efficient recruitment of Foxp3C Tregs to effector sites is required to achieve tolerance was strengthened in further models. In an allotransplantation model both CCR4 and one of its ligands, CCL22, were upregulated in tolerized allografts, and tolerance induction could not be achieved in CCR4K/K recipients [31]. CCR5, another inflammatory chemokine receptor, has been demonstrated to be expressed on a Treg subset, which preferentially infiltrates extra-lymphoid sites and sites of inflammation [32]. Interestingly, using a GvHD model, Wysocki et al. have shown that Tregs lacking expression of CCR5 were far less effective in preventing lethality in this model [33]. Whereas the accumulation of Tregs within lymphoid tissues during
(a) Inflamed site
Effector cells
Molecules on effector/memory-like, inflammation seekingTregs:
Treg Blood vessel Activated endothelium
• CCR2, CCR4, CCR5, CCR6 • E/P-selectin ligands • β1-integrin • LFA-1high
(b) Lymphoid organs
Antigen-loaded dendritic cells
Molecules on naive-like, recirculating Tregs: • CCR7 • CCR4 • L-selectin • LFA-1
Naive T cell
Treg
High endothelial venules
TRENDS in Immunology
Figure 1. Treg compartmentalization determines suppressive activity in vivo. Peripheral antigen, transported from the inflamed site (a) to the local draining lymph node (b) by professional antigen-presenting cells, leads to expansion and differentiation of naive T cells (yellow). This priming step is under the control of naive-like Tregs (blue), which express high levels of L-selectin (CD62L) and CCR7. This enables efficient migration of these recirculating Tregs into lymphoid organs via the high endothelial venules expressing ligands for L-selectin and presenting CCR7 ligands on their surface. The chemokine receptor CCR4 might have a role in the interaction between naive-like Tregs and antigen-presenting cells within lymph tissues. (a) By contrast, effector/memory-like Tregs (green) display an inflammation-seeking phenotype by the expression of diverse CCRs, E/P-selectin ligands, b1-integrins and high levels of LFA-1. This enables an efficient entry into inflamed sites because it is expected that activated endothelia will express high levels of E-selectin, P-selectin, ICAM-1 and VCAM-1, and will present inflammatory chemokines on their surface. Thereby, these inflammation-seeking Tregs are predestinated to control the inflammatory action (red lightening arrow) of effector cells at peripheral sites. www.sciencedirect.com
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TRENDS in Immunology
the first week after transplantation was not dependent on CCR5, the lack of function of CCR5K/K Tregs correlated with impaired accumulation of these cells in the target organs liver, lung and spleen, more than one week after transplant. A crucial role for CCR2-expressing Tregs has been shown recently in a model of collagen-induced arthritis. Whereas blockade of CCR2 using monoclonal antibodies during the disease initiation phase markedly improved the signs of arthritis by preventing accumulation of proinflammatory effector cells, blockade during the later phase of arthritis progression significantly aggravated clinical and histological signs of arthritis. The authors postulated that this later effect was most probably due to the interference with the proper in vivo localization of CCR2CCD25CCD4C Tregs, which appeared to be essential for the control of the inflammatory response [34]. This study clearly shows that anti-inflammatory therapies targeting recruitment mechanisms might also influence the migration of beneficial Tregs. By contrast, in the adoptive transfer colitis model, Treg deficiency in b7-integrin expression did not hamper suppressive capacity, which indicates that Treg migration into the inflamed gut is not required in this model [35]. The b7-integrin is not crucial for entry into mesenteric LNs and, therefore, its lack would not necessarily affect control of the induction phase of the inflammatory immune response by Tregs. However, it could be specu=K lated that bK 7 Tregs would be far less efficient than their wildtype counterparts if they have been transferred adoptively after the onset of colitis to control the effector phase and to treat ongoing autoimmunity. Indeed, treatment of ongoing autoimmunity, which was first described in the adoptive transfer colitis model, was accompanied by the migration of CD25CCD4C Tregs, not only into mesenteric LNs but also into the colon [5]. Further proof for the importance of the appropriate Treg localization for their in vivo suppressive capacity came from a study on the role of selectin ligands on Tregs in a skin inflammation model. As mentioned previously, inflammation-seeking aC E Tregs efficiently reduced the inflammatory response [29]. However, aC E Tregs from fucosyltransferaseVII-deficient (FucTVIIK/K), which lack E/P-selectin ligands and fail to migrate into inflamed sites, could not suppress the skin inflammation. Lack of suppression by Tregs deficient in E/P-selectin ligands clearly demonstrated that immigration into inflamed sites is a prerequisite for the resolution of inflammatory reactions in vivo because these selectin ligands merely regulate entry into inflamed tissues [29]. Conclusions Compartmentalization is an important feature of the immune system. To what extent regulatory pathways require a proper localization of cell subsets to specific sites is an emerging field of interest. In this regard, it is noteworthy that the pool of Tregs consists of rather heterogeneous subsets differing in homing receptor expression and chemokine responsiveness. Whereas some Treg subsets appear to be specialized to inhibit the initiation of the immune response within lymphoid www.sciencedirect.com
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tissues, other subsets, which display the capacity to enter inflamed sites, have a pivotal role in the control of established immune reactions (Figure 1). Such a division of labor between Treg subsets might constitute a robust ‘fail-safe’ system, consisting of one line of defense involved in prevention of the development of autoimmunity and a second line becoming active once immune reactions have gone out of control (Box 1). Indeed, an increasing body of data suggests that the migratory behavior of Tregs crucially influences their suppressive activity in vivo. This has important implications for clinical aspects, related both to tumor therapy where the attraction of Tregs might limit the success of immunotherapy, and to approaches aiming to induce or transfer Tregs for the treatment of inflammatory diseases and in transplantation. Future therapeutic strategies based on Tregs have to take into account that Tregs do not merely require potent suppressor mechanisms but they also need appropriate trafficking properties that enable contact with their target cells. Acknowledgements We thank Stefan Martin (Department of Dermatology, Freiburg, Germany) for critical reading of the manuscript and Luise Fehlig for graphics. This work was supported by the Deutsche Forschungsgemeinschaft (SFB650).
References 1 Sakaguchi, S. (2005) Naturally arising Foxp3-expressing CD25C CD4C regulatory T cells in immunological tolerance to self and nonself. Nat. Immunol. 6, 345–352 2 von Boehmer, H. (2005) Mechanisms of suppression by suppressor T cells. Nat. Immunol. 6, 338–344 3 Fontenot, J.D. and Rudensky, A.Y. (2005) A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6, 331–337 4 Jaeckel, E. et al. (2005) Antigen-specific FoxP3-transduced T-cells can control established Type 1 diabetes. Diabetes 54, 306–310 5 Mottet, C. et al. (2003) Cutting edge: Cure of colitis by CD4CCD25C regulatory T cells. J. Immunol. 170, 3939–3943 6 Sakaguchi, S. et al. (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155, 1151–1164 7 Itoh, M. et al. (1999) Thymus and autoimmunity: production of CD25CCD4C naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162, 5317–5326 8 Stephens, L.A. et al. (2001) Human CD4(C)CD25(C) thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur. J. Immunol. 31, 1247–1254 9 Hontsu, S. et al. (2004) Visualization of naturally occurring Foxp3C regulatory T cells in normal and tumor-bearing mice. Int. Immunopharmacol. 4, 1785–1793 10 Lim, H.W. et al. (2004) Regulatory T cells can migrate to follicles upon T cell activation and suppress GC-Th cells and GC-Th cell-driven B cell responses. J. Clin. Invest. 114, 1640–1649 11 Roncador, G. et al. (2005) Analysis of FOXP3 protein expression in human CD4(C)CD25(C) regulatory T cells at the single-cell level. Eur. J. Immunol. 35, 1681–1691 12 Iellem, A. et al. (2001) Unique chemotactic response profile and specific expression of chemokine receptors 4 and CCR8 by CD4(C) CD25(C) regulatory T cells. J. Exp. Med. 194, 847–853 13 Colantonio, L. et al. (2002) Skin-homing CLAC T cells and regulatory CD25C T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309. Eur. J. Immunol. 32, 3506–3514
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14 Szanya, V. et al. (2002) The subpopulation of CD4CCD25C splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J. Immunol. 169, 2461–2465 15 Iellem, A. et al. (2003) Skin-versus gut-skewed homing receptor expression and intrinsic CCR4 expression on human peripheral blood CD4CCD25C suppressor T cells. Eur. J. Immunol. 33, 1488–1496 16 Agenes, F. et al. (2005) Differential expression of regulator of G-protein signalling transcripts and in vivo migration of CD4C naive and regulatory T cells. Immunology 115, 179–188 17 D’Ambrosio, D. et al. (2003) Special attractions for suppressor T cells. Trends Immunol. 24, 122–126 18 Curiel, T.J. et al. (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10, 942–949 19 Taylor, P.A. et al. (2004) L-Selectinhi but not the L-selectinlo CD4C 25C T-regulatory cells are potent inhibitors of GVHD and BM graft rejection. Blood 104, 3804–3812 20 Ermann, J. et al. (2005) Only the CD62LC subpopulation of CD4C CD25C regulatory T cells protects from lethal acute GVHD. Blood 105, 2220–2226 21 Thornton, A.M. and Shevach, E.M. (2000) Suppressor effector function of CD4CCD25C immunoregulatory T cells is antigen nonspecific. J. Immunol. 164, 183–190 22 Fisson, S. et al. (2003) Continuous activation of autoreactive CD4C CD25C regulatory T cells in the steady state. J. Exp. Med. 198, 737–746 23 Ochando, J.C. et al. (2005) Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3C regulatory T cells. J. Immunol. 174, 6993–7005 24 Salomon, B. et al. (2000) B7/CD28 costimulation is essential for the homeostasis of the CD4CCD25C immunoregulatory T cells that control autoimmune diabetes. Immunity 12, 431–440 25 Green, E.A. et al. (2002) Pancreatic lymph node-derived CD4(C) CD25(C) Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity 16, 183–191 26 Bour-Jordan, H. et al. (2004) Costimulation controls diabetes by altering the balance of pathogenic and regulatory T cells. J. Clin. Invest. 114, 979–987 27 Lehmann, J. et al. (2002) Expression of the integrin alphaEbeta7 identifies unique subsets of CD25C as well as CD25- regulatory T cells. Proc. Natl. Acad. Sci. U. S. A. 99, 13031–13036 28 Huehn, J. et al. (2004) Developmental stage, phenotype and migration distinguish naive- and effector/memory-like CD4C regulatory T cells. J. Exp. Med. 199, 303–313 29 Siegmund, K. et al. Migration matters: regulatory T cell compartmentalization determines suppressive activity in vivo. Blood DOI: 10. 1182/blood-2005- 05-1864 (www.bloodjournal.org) 30 Schwarz, A. et al. (2004) Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity. J. Immunol. 172, 1036–1043 31 Lee, I. et al. (2005) Recruitment of Foxp3C T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor. J. Exp. Med. 201, 1037–1044 32 Herman, A.E. et al. (2004) CD4CCD25C T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J. Exp. Med. 199, 1479–1489
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33 Wysocki, C.A. et al. Critical role for CCR5 in the function of donor CD4C CD25C regulatory T cells during acute graft-versus-host disease. Blood DOI: 10.1182/blood-2005-04-1632 (www.bloodjournal.org) 34 Bruhl, H. et al. (2004) Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2C T cells. J. Immunol. 172, 890–898 35 Denning, T.L. et al. (2005) Cutting edge: CD4CCD25C regulatory T cells impaired for intestinal homing can prevent colitis. J. Immunol. 174, 7487–7491 36 Maul, J. et al. (2005) Peripheral and intestinal regulatory CD4C CD25(high) T cells in inflammatory bowel disease. Gastroenterology 128, 1868–1878 37 Cao, D. et al. (2003) Isolation and functional characterization of regulatory CD25brightCD4C T cells from the target organ of patients with rheumatoid arthritis. Eur. J. Immunol. 33, 215–223 38 van Amelsfort, J.M. et al. (2004) CD4(C)CD25(C) regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum. 50, 2775–2785 39 Lecart, S. et al. (2001) Phenotypic characterization of human CD4C regulatory T cells obtained from cutaneous dinitrochlorobenzeneinduced delayed type hypersensitivity reactions. J. Invest. Dermatol. 117, 318–325 40 Sugiyama, H. et al. (2005) Dysfunctional blood and target tissue CD4CCD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation. J. Immunol. 174, 164–173 41 Kleinewietfeld, M. et al. (2005) CCR6 expression defines regulatory effector/memory-like cells within the CD25(C)CD4C T-cell subset. Blood 105, 2877–2886 42 Graca, L. et al. (2002) Identification of regulatory T cells in tolerated allografts. J. Exp. Med. 195, 1641–1646 43 Yu, P. et al. (2005) Intratumor depletion of CD4C cells unmasks tumor immunogenicity leading to the rejection of late-stage tumors. J. Exp. Med. 201, 779–791 44 Woo, E.Y. et al. (2002) Cutting edge: Regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J. Immunol. 168, 4272–4276 45 Hori, S. et al. (2002) CD25CCD4C regulatory T cells suppress CD4C T cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice. Eur. J. Immunol. 32, 1282–1291 46 Belkaid, Y. et al. (2002) CD4CCD25C regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502–507 47 Suffia, I. et al. (2005) A role for CD103 in the retention of CD4C CD25C Treg and control of Leishmania major infection. J. Immunol. 174, 5444–5455 48 Hesse, M. et al. (2004) The pathogenesis of schistosomiasis is controlled by cooperating IL-10-producing innate effector and regulatory T cells. J. Immunol. 172, 3157–3166 49 Taylor, M.D. et al. (2005) Removal of regulatory T cell activity reverses hyporesponsiveness and leads to filarial parasite clearance in vivo. J. Immunol. 174, 4924–4933 50 Suvas, S. et al. (2004) CD4CCD25C regulatory T cells control the severity of viral immunoinflammatory lesions. J. Immunol. 172, 4123–4132
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TRENDS in Immunology
Vol.26 No.12 December 2005
Homing to suppress: address codes for Treg migration Jochen Huehn and Alf Hamann Experimental Rheumatology, Charite´ University Medicine Berlin, c/o DRFZ, Schumannstr. 21/22, 10117 Berlin, Germany
Compelling evidence suggests that diverse types of immune reactions can be suppressed by CD25CCD4C regulatory T cells (Tregs). Although increasing knowledge has accumulated concerning the generation and functional properties of Tregs, relatively little attention has been paid to another key question: where does immune regulation by Tregs take place in vivo? Tregs can inhibit both the priming and the effector phase of an immune response, so suppression might occur both within lymphoid tissues and at peripheral sites during immune reactions. This leads to the hypothesis that appropriate localization is indispensable for in vivo Treg function and that the migratory behavior of Treg subsets influences their in vivo suppressive capacity. Current data suggest a division of labor between subpopulations of Tregs, which is mainly based on specialized homing patterns.
Tregs - a Brief introduction The molecular mechanisms governing Treg development and function are currently a hot topic because of their importance in controlling host responses to tumors, preventing transplant rejection and inhibiting the development of autoimmunity and allergy [1,2]. For some time, CD4C Tregs were identified solely by the constitutive expression of CD25, but the existence of CD25– Tregs, together with the induced expression of CD25 on recently activated conventional T cells, somewhat limited the results obtained by this phenotypic characterization. Recent studies have shown that the forkhead box transcription factor Foxp3 is highly expressed in Tregs and regulates their development and function [3]. Foxp3 now serves as the best marker to identify Tregs and, excitingly, both Foxp3-transduced T cells and CD25CCD4C Tregs have been shown to exhibit therapeutical potential [4,5]. To use Tregs for an efficient treatment of autoimmune disorders and to prevent transplant rejections, immunologists have made an enormous effort during recent years to discover the principles underlying the development and generation of Tregs, their antigen-specificity and their suppressor mechanisms [1,2]. However, another important aspect within this context has gained less attention so far; that is, at which sites does immune suppression by Tregs takes place in vivo? Here, by focusing on the migration behavior Corresponding author: Huehn, J. ([email protected]). Available online 21 October 2005
of Tregs, we will describe that not only the suppressor potential but also appropriate localization determines the in vivo suppressive capacity of Tregs. We therefore propose that Treg subsets, which differ in homing patterns, will also display different in vivo suppressive capabilities. Occurrence of Tregs in vivo: lymphoid organs versus peripheral and inflamed sites CD25CCD4C Tregs were first identified in lymphoid tissues, including thymus, spleen and lymph nodes (LNs), as well as in peripheral blood and constitute w5– 10% of total CD4C T cells [6–8]. More recent immunohistological studies have shown that under steady-state conditions Foxp3CCD25CCD4C Tregs were evenly distributed in the T-cell areas of murine and human lymphoid tissues [9–11]. Tregs were barely detectable in B cell follicles, however, they were found to change their chemotactic behavior upon activation, which would enable migration into germinal centers and the suppression of antibody production [10]. In the transfer colitis model, CD25CCD4C Tregs cotransferred with pathogenic CD45RBhighCD4CT cells migrated into both mesenteric LNs, as well as the colon, suggesting that suppression might take place at both sites in this model [5]. Interestingly, several recent publications studying adhesion molecule expression or chemokine responsiveness of Treg subsets suggest that Tregs are indeed able both to recirculate through lymphoid tissues and to enter inflamed sites [10,12–15]. Particularly under pathological conditions, Tregs were identified within various peripheral sites, including inflamed organs, tumors and infectious sites (Table 1). Thus, although parabiosis experiments (where two congenic mice were joined laterally to allow bloodflow between them and cells of the individual mice can be kept apart by use of the congenic marker) have shown that under homeostatic conditions Tregs display a reduced recirculation rate in comparison to conventional T cells, which might be explained by the differential expression of regulator of G-protein signaling (RGS) molecules [16], the prevalence of Tregs at sites of ongoing immune responses clearly demonstrates their capacity to migrate into sites of inflammation. It has been suggested by D’Ambrosio et al. that the expression of specific chemokine receptors (CCRs) on Tregs could guide them to different types of chemokinesecreting antigen-presenting cells and subsets of effector
www.sciencedirect.com 1471-4906/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2005.10.001
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Table 1. Tregs have been identified within various peripheral sites Tissue or site Inflamed pancreas Inflamed mucosa Inflamed joint Inflamed skin Inflamed brain Allotransplant Tumor Pneumocystis carinii-infected lung Leishmania major-infected skin Schistosoma mansoni-induced granuloma Litomosoides sigmodontis-infected thoracic cavity Viral lesions
Species Murine Murine Human Murine Human Murine Human Murine Murine Murine Human Murine Murine Murine
Refs [25,32] [5] [36] [28] [37,38] [28,29] [39,40] [41] [31,42] [9,43] [18,44] [45] [46,47] [48]
Murine
[49]
Murine
[50]
cells, enabling direct cell–cell contact, which is required for efficient suppression [17]. Indeed, CCL22-producing tumor cells and environmental macrophages have been shown recently, in a human cancer study, to recruit Foxp3CCD25CCD4C Tregs selectively [18]. Tumor Tregs preferentially accumulated in tumors and ascites but rarely entered draining LNs in later cancer stages. Such an accumulation of Tregs within tumors predicted reduced survival of the patients, indicating an efficient suppression of anti-tumor responses directly within the tumor. Where does suppression take place? Evidence for a division of labor It is not completely understood at which stages and by which mechanisms Tregs suppress immune reactions in vivo. The presence of Tregs in lymphoid organs, as well as peripheral sites, would enable both suppression of the priming phase, taking place within lymphoid tissues, and control of later stages of the response, directly at the effector site. However, do the same cells do these jobs in such different environments? Increasing evidence points towards a division of labor between subsets of Tregs, where the competence to migrate into either lymphoid or inflammatory sites is a distinctive determinant of their function. The first experimental evidence for this division of labor between Treg subsets came from studies comparing the in vivo suppressive potential of CD62Lhigh and CD62Llow CD25CCD4C Treg subsets. CD62Lhigh Tregs were superior to their CD62Llow counterparts in preventing the development of autoimmunity or in suppressing graft-versus-host disease (GvHD) [14,19,20]. CD62Lhigh and CD62Llow Treg subsets have been reported in several studies to harbor a similar in vitro suppressive capacity [14,21], therefore, the high in vivo regulatory capacity of the CD62Lhigh subset in these models most probably reflects differences in homing properties, rather than suppressor potential per se. Interestingly, in all models where CD62LhighCD25CCD4C Tregs showed high suppressive capacity, adoptive Treg transfer was performed before the onset of disease. This indicates that in such www.sciencedirect.com
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cases, where the induction of an immune reaction within the LNs has to be prevented, only those Tregs that can efficiently enter these lymphoid sites can control the development of effector cells and, thereby, the induction of autoimmunity [22]. Further experimental evidence for the LN being a crucial site for tolerance induction was obtained in an allotransplantation model, where blockade of migration of Foxp3CCD25CCD4CCD62Lhigh Tregs into LNs using anti-CD62L mAbs resulted in graft rejection [23]. In this model, progressive expansion of Tregs could only be observed within the LNs but not in other anatomic sites, indicating that alloantigen-specific Tregs developed and/or expanded in the LNs of tolerized animals, and are necessary for the induction and maintenance of tolerance. Although these data were supported by studies in autoimmune diabetes models, where failure of extensive expansion of Tregs in pancreatic LNs correlated with accelerated diabetes progression [24,25], a more recent study has extended these findings by showing that development of autoimmune diabetes not only depends on the absolute number of Tregs within the pancreatic LN but also on the delicate balance between effector cells and Tregs [26]. Recently, it has been shown that the integrin aEb7 subdivides the Treg compartment into aE-negative ðaK E CD C K and aC 25CÞ and aE-positive Tregs (aC E CD25 E CD25 ), which largely differ in their in vivo suppressive capacity and in the expression of adhesion molecules and chemoC kine receptors [27,28]. aK E CD25 Tregs displayed a naivelike phenotype and efficiently migrated into LNs fitting to their high CD62L expression and their high responsiveness towards CCR7 ligands. Interestingly, these cells lacked suppressive capacity under acute inflammatory conditions but turned out to be most potent at suppressing naive CD4C T-cell proliferation within the LN [28,29]. By contrast, aC E Tregs showed increased frequencies of E/P selectin ligand expression combined with a substantial downregulation of CD62L and high responsiveness towards several inflammatory chemokines. Correspondingly, aC E Tregs displayed only a rather poor capacity to migrate into LNs but, by contrast, efficiently entered inflamed sites. Strikingly, this inflammation-seeking capacity was combined with high suppressive potential in various inflammation models [28,29]. In summary, these findings strengthen the functional relevance of the appropriate localization of Tregs for their in vivo suppressive capacity and lead us to propose a model of ‘division of labor’ for the function of Tregs, which is mainly based on their differential migration behavior (Box 1 and Figure 1). Functional and molecular evidence for the importance of the appropriate Treg localization Experimental evidence for the importance of the appropriate Treg localization first came from Schwarz and colleagues, who analyzed the suppressive capacity of UVinduced, hapten-specific Tregs in a contact hypersensitivity model [30]. Interestingly, the UV-induced Tregs expressed high levels of CD62L but not the ligands for the skin-homing receptors E- and P-selectin. In addition, they were only capable of controlling the induction phase
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Box 1. ‘Division of labor’ model based on differential migration behavior of Treg subsets To maintain a tolerant state, the presence of Tregs in lymphoid organs or at the effector site might be required depending on the stage of the immune response. During the priming phase, Tregs with a naive-like phenotype suppress the induction, expansion and differentiation of autoreactive effector cells within lymphoid tissues. In case of inflammation, Tregs become activated and differentiate into an effector/memory-like stage, characterized by the expression of chemokine receptors and adhesion molecules required for migration into inflamed tissues. These effector/memory-like Tregs enter the inflamed site and serve to limit either peripheral expansion, cytokine secretion or cytolytic function of effector cells at later stages of the response. Under these circumstances, self-limitation and resolution of the inflammatory reaction might be their key function. Interestingly, the conversion of naive-like Tregs into effector/memory-like Tregs has recently been shown to occur upon stimulation both in vitro and in vivo (J. Huehn, unpublished results). This might be an important issue for the therapeutic application of inflammation-seeking Tregs.
of the skin inflammation but showed no suppressive capacity during the effector phase. Strikingly, if these hapten-specific Tregs were injected directly into the effector site they could even suppress the challenge reaction showing that they were, in principle, able to inhibit the effector phase [30]. The assumption that the efficient recruitment of Foxp3C Tregs to effector sites is required to achieve tolerance was strengthened in further models. In an allotransplantation model both CCR4 and one of its ligands, CCL22, were upregulated in tolerized allografts, and tolerance induction could not be achieved in CCR4K/K recipients [31]. CCR5, another inflammatory chemokine receptor, has been demonstrated to be expressed on a Treg subset, which preferentially infiltrates extra-lymphoid sites and sites of inflammation [32]. Interestingly, using a GvHD model, Wysocki et al. have shown that Tregs lacking expression of CCR5 were far less effective in preventing lethality in this model [33]. Whereas the accumulation of Tregs within lymphoid tissues during
(a) Inflamed site
Effector cells
Molecules on effector/memory-like, inflammation seekingTregs:
Treg Blood vessel Activated endothelium
• CCR2, CCR4, CCR5, CCR6 • E/P-selectin ligands • β1-integrin • LFA-1high
(b) Lymphoid organs
Antigen-loaded dendritic cells
Molecules on naive-like, recirculating Tregs: • CCR7 • CCR4 • L-selectin • LFA-1
Naive T cell
Treg
High endothelial venules
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Figure 1. Treg compartmentalization determines suppressive activity in vivo. Peripheral antigen, transported from the inflamed site (a) to the local draining lymph node (b) by professional antigen-presenting cells, leads to expansion and differentiation of naive T cells (yellow). This priming step is under the control of naive-like Tregs (blue), which express high levels of L-selectin (CD62L) and CCR7. This enables efficient migration of these recirculating Tregs into lymphoid organs via the high endothelial venules expressing ligands for L-selectin and presenting CCR7 ligands on their surface. The chemokine receptor CCR4 might have a role in the interaction between naive-like Tregs and antigen-presenting cells within lymph tissues. (a) By contrast, effector/memory-like Tregs (green) display an inflammation-seeking phenotype by the expression of diverse CCRs, E/P-selectin ligands, b1-integrins and high levels of LFA-1. This enables an efficient entry into inflamed sites because it is expected that activated endothelia will express high levels of E-selectin, P-selectin, ICAM-1 and VCAM-1, and will present inflammatory chemokines on their surface. Thereby, these inflammation-seeking Tregs are predestinated to control the inflammatory action (red lightening arrow) of effector cells at peripheral sites. www.sciencedirect.com
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the first week after transplantation was not dependent on CCR5, the lack of function of CCR5K/K Tregs correlated with impaired accumulation of these cells in the target organs liver, lung and spleen, more than one week after transplant. A crucial role for CCR2-expressing Tregs has been shown recently in a model of collagen-induced arthritis. Whereas blockade of CCR2 using monoclonal antibodies during the disease initiation phase markedly improved the signs of arthritis by preventing accumulation of proinflammatory effector cells, blockade during the later phase of arthritis progression significantly aggravated clinical and histological signs of arthritis. The authors postulated that this later effect was most probably due to the interference with the proper in vivo localization of CCR2CCD25CCD4C Tregs, which appeared to be essential for the control of the inflammatory response [34]. This study clearly shows that anti-inflammatory therapies targeting recruitment mechanisms might also influence the migration of beneficial Tregs. By contrast, in the adoptive transfer colitis model, Treg deficiency in b7-integrin expression did not hamper suppressive capacity, which indicates that Treg migration into the inflamed gut is not required in this model [35]. The b7-integrin is not crucial for entry into mesenteric LNs and, therefore, its lack would not necessarily affect control of the induction phase of the inflammatory immune response by Tregs. However, it could be specu=K lated that bK 7 Tregs would be far less efficient than their wildtype counterparts if they have been transferred adoptively after the onset of colitis to control the effector phase and to treat ongoing autoimmunity. Indeed, treatment of ongoing autoimmunity, which was first described in the adoptive transfer colitis model, was accompanied by the migration of CD25CCD4C Tregs, not only into mesenteric LNs but also into the colon [5]. Further proof for the importance of the appropriate Treg localization for their in vivo suppressive capacity came from a study on the role of selectin ligands on Tregs in a skin inflammation model. As mentioned previously, inflammation-seeking aC E Tregs efficiently reduced the inflammatory response [29]. However, aC E Tregs from fucosyltransferaseVII-deficient (FucTVIIK/K), which lack E/P-selectin ligands and fail to migrate into inflamed sites, could not suppress the skin inflammation. Lack of suppression by Tregs deficient in E/P-selectin ligands clearly demonstrated that immigration into inflamed sites is a prerequisite for the resolution of inflammatory reactions in vivo because these selectin ligands merely regulate entry into inflamed tissues [29]. Conclusions Compartmentalization is an important feature of the immune system. To what extent regulatory pathways require a proper localization of cell subsets to specific sites is an emerging field of interest. In this regard, it is noteworthy that the pool of Tregs consists of rather heterogeneous subsets differing in homing receptor expression and chemokine responsiveness. Whereas some Treg subsets appear to be specialized to inhibit the initiation of the immune response within lymphoid www.sciencedirect.com
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tissues, other subsets, which display the capacity to enter inflamed sites, have a pivotal role in the control of established immune reactions (Figure 1). Such a division of labor between Treg subsets might constitute a robust ‘fail-safe’ system, consisting of one line of defense involved in prevention of the development of autoimmunity and a second line becoming active once immune reactions have gone out of control (Box 1). Indeed, an increasing body of data suggests that the migratory behavior of Tregs crucially influences their suppressive activity in vivo. This has important implications for clinical aspects, related both to tumor therapy where the attraction of Tregs might limit the success of immunotherapy, and to approaches aiming to induce or transfer Tregs for the treatment of inflammatory diseases and in transplantation. Future therapeutic strategies based on Tregs have to take into account that Tregs do not merely require potent suppressor mechanisms but they also need appropriate trafficking properties that enable contact with their target cells. Acknowledgements We thank Stefan Martin (Department of Dermatology, Freiburg, Germany) for critical reading of the manuscript and Luise Fehlig for graphics. This work was supported by the Deutsche Forschungsgemeinschaft (SFB650).
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