Tr1 cells: From discovery to their clinical application

Seminars in Immunology 18 (2006) 120–127

Review

Tr1 cells: From discovery to their clinical application Manuela Battaglia a,b , Silvia Gregori a , Rosa Bacchetta a , Maria-Grazia Roncarolo a,c,∗ a

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milano 20132, Italy b Immunology of Diabetes Unit, San Raffaele Scientific Institute, Milano, Italy c Vita Salute San Raffaele University, Milano, Italy

Abstract Peripheral tolerance is mediated by multiple mechanisms such as anergy and/or active suppression of effector T cells by T regulatory (Tr) cells. Among the CD4+ Tr cells, T regulatory type 1 cells (Tr1) have been shown to down-modulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-␤. Tr1 cells maintain peripheral tolerance, control autoimmunity, and prevent allograft rejection and graft versus host disease (GvHD). Cellular therapy with ex vivo generated Tr1 cells has been proven to be effective in several preclinical models of T cell-mediated pathologies and therefore, represents a promising approach for clinical application. This review will summarize the new findings on Tr1 cells, the recent development of methods for their ex vivo expansion, and their potential clinical relevance as cellular therapy. © 2006 Elsevier Ltd. All rights reserved. Keywords: Tolerance; IL-10; T regulatory type 1 cells

1. Introduction The concept of a dominant form of immunological tolerance involving a specialized population of “suppressor” T cells that act to terminate conventional immune responses and to prevent autoimmune pathology was proposed more than 30 years ago [1]. Early studies hypothesized a suppressor cell cascade involving multiple suppressor factors, anti-idiotypic T cell networks, “suppressor-inducer”’, and “contra-suppressor”’ cells [2]. However, the mechanisms responsible for these suppressive phenomena were never characterized at the molecular and biochemical level primarily because of the difficulty in isolating suppressor T cells at the single cell level. Moreover, key findings of those studies could not be reproduced and the field of suppressor T cell biology was largely discredited [3]. In the last few years modern technologies and new experimental approaches consented a rebirth of suppressor cells (now called T regulatory cells), which are, at present, considered as one of the central players in immune regulation.

∗ Corresponding author at: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milano 20132, Italy. Tel.: +39 02 2643 4703; fax: +39 02 2643 4668. E-mail addresses: [email protected] (M. Battaglia), [email protected] (S. Gregori), [email protected] (R. Bacchetta), [email protected] (M.-G. Roncarolo).

1044-5323/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2006.01.007

Many cell subsets with regulatory activity have been described including CD4+ CD25+ FOXP3+ , CD4+ Th3, CD8+ CD28− , TCR+ CD4− CD8− Tr cells, and NK T cells. For several years, our laboratory had studied the role of IL-10 in the induction of peripheral tolerance and, through these studies, we identified the IL-10-producing CD4+ T regulatory type 1 (Tr1) cells. This review describes the “history” of Tr1 cells from their discovery to the recent progresses in their characterization and to their potential clinical relevance. 2. Discovery of IL-10 and Tr1 cells IL-10 was first described as a soluble factor produced by mouse Th2 cells able to inhibit activation of and cytokine production by Th1 cells, and was therefore originally named cytokine synthesis inhibitory factor (CSIF) [4]. Shortly after mouse cDNA IL-10 was cloned, human IL-10 was isolated from a T cell clone derived from a severe combined immunodeficient (SCID) patient who developed long-term tolerance to stem-cell allograft [5]. Since then, an enormous amount of work has been performed to further characterize this cytokine. IL-10 is now considered a soluble factor that plays a central role in controlling inflammatory processes, suppressing T cell responses, and maintaining immunological tolerance (reviewed in [6]). IL-10 down-regulates the expression of MHC class II, co-stimulatory and adhesion molecules [7–9], inhibits the release of inflammatory cytokines, and modulates the stimulatory capacity of

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dendritic cells (DC) and other antigen presenting cells (APC) [10]. Furthermore, IL-10 inhibits cytokine production by T cells and monocytes/macrophages, and induces long-lasting antigenspecific anergy in both CD4+ and CD8+ T cells [11–13]. Interestingly, the same SCID patients who were instrumental for the cloning of human IL-10 were also essential for the first description of Tr1 cells. CD4+ host-reactive T cell clones isolated from human SCID chimera successfully transplanted with HLA-mismatched hematopoietic stem cells, produced very high levels of IL-10 and IL-5 in the absence of IL-4 and IL-2 after antigen-specific stimulation in vitro. Their presence correlated with the absence of graft versus host disease (GvHD) and with long-term graft tolerance without the need of immunosuppression [14]. Subsequently, in 1998 another study performed by our group [13] demonstrated that ex vivo activation of human or mouse CD4+ T cells in the presence of high doses of exogenous IL-10 resulted in the generation of T cell clones with a cytokine production profile distinct from that of Th1 or Th2 cells but superimposable to that of host-reactive T cell clones isolated from the SCID patients. These T cell clones produced significant amounts of IL-10, TGF-␤, and IL-5 but low amounts of IFN-␥ and IL-2, and no IL-4. Functionally, these T cell clones inhibited antigen-specific activation of autologous T cells via IL-10 and TGF-␤ production. In a murine model of inflammatory bowel diseases (IBD) in SCID mice, cotransfer of pathogenic CD4+ CD45RBhigh T cells together with IL-10-producing murine T cell clones prevented the induction of disease [13]. In addition, transfer of OVA-specific Tr1 cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses [15]. These studies allowed the characterization at the single cell level of these T cells generated either ex vivo in the presence of IL-10 or in vivo in SCID patients [14], and were termed Tr1 cells [13]. 3. Biological features of Tr1 cells Since their discovery, it has been evident that the cytokine production profile of Tr1 cells was their key trait. Tr1 cells, upon activation via the TCR, produce high amounts of IL-10 but are distinct from Th2 cells since they do not produce IL-4, and produce very low levels of IL-2, which are both potent T cell growth factors. Human Tr1 cells also produce IFN-␥, although at levels that are at least 1 log lower than those produced by Th1 cells [16]. On the contrary, murine Tr1 cells rarely produce IFN-␥. TGF-␤ and IL-5, two anti-inflammatory cytokines, are also produced by Tr1 cells in some experimental settings. However, it is still unclear whether their secretion should be included as part of the definition of Tr1 cells. Many reports indeed describe a role for both IL-10 and TGF-␤ for the suppressive effects mediated by Tr1 cells, whereas others describe an exclusive role for IL-10. We propose that the suppressive effects mediated by IL-10-secreting Tr cells (i.e. IL-10+ IL-4− ) that are distinct from classical Th2 cells (i.e. IL-10+ IL-4+ ), should be attributed to Tr1 cells regardless of production of TGF-␤, IL-5, and IFN-␥. Tr1 cells have a very low proliferative capacity upon TCR activation, although they can be expanded ex vivo in the pres-

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ence of cytokines such as IL-2 and IL-15. Their anergic phenotype is partially due to autocrine production of IL-10, since anti-IL-10 mAbs partially restores their proliferative responses [16]. Despite their low in vitro proliferative capacity, Tr1 cell clones express normal levels of activation markers such as CD25, CD40L, CD69, HLA-DR, and CTLA-4 following TCRmediated stimulation, whereas they constitutively express high levels of the IL-2R␤ and ␥ chains independently on their activation status [16]. Tr1 cells suppress both Th1- and Th2-mediated immune responses mainly through IL-10 and TGF-␤ production [13,15]. Nevertheless, a mechanism implicating direct cell–cell contact with the target cells has also been postulated [17]. Importantly, Tr1 cells are inducible, antigen-specific, and need to be activated via their TCR in order to exert their suppressive functions. Although Tr1 cells must encounter their antigen to exert these effects, once activated they suppress in an antigen non-specific manner. Presumably, this bystander suppression is due to the release of immunosuppressive cytokines such as IL-10 and TGF␤ [13]. In the past years, we and others performed extensive studies on Tr1 cells with the aim to identify specific cell marker/s to distinguish Tr1 cells from the other CD4+ T cell subsets. Particularly, there were big expectations around the phenotypical characterization of Tr1 cells in order to discriminate them from the naturally occurring Tr cells that constitutively express the IL-2R ␣ chain (i.e. the CD4+ CD25+ Tr cells, object of other reviews in this same issue). Cobbold et al., by comparing gene expression between murine Tr1 cell clones and CD4+ CD25+ Tr cells, identified the selective expression of the repressor of GATA-3 (ROG) in Tr1 cells but not in CD4+ CD25+ Tr cells [18]. However, ROG is not specific for Tr1 cells since it is expressed also in activated Th cells. Yessel and collaborators generated monoclonal antibodies by immunizing Balb/c mice with a skinderived human Tr1 cell clone differentiated in vivo. Two mAbs recognizing a cell surface molecules of approximately 120 kDa highly reacted with the Tr1 cell clone. However, the molecule recognized by the 2 mAbs was strongly up-regulated also on CD4+ cord blood T cells incubated with IL-7 (see abstract at http://assocbs2.igh.cnrs.fr/journeescbs2/2005/index.htm). Human Tr1 cells in the resting phase express both Th1associated, CXCR3 and CCR5, and Th2-associated CCR3, CCR4 and CCR8 chemokine receptors. The latter receptor is expressed at higher levels compared to Th2 cells. Importantly, upon activation, Tr1 cells migrate preferentially in response to I-309, a ligand for CCR8 [19]. It has been shown that the transcription factor FOXP3 is constitutively expressed by the CD4+ CD25+ Tr cells and controls their differentiation [20]. These data have prompted us and other investigators to determine whether Tr1 cells express FOXP3. Results from these studies proved that Tr1 cells do not constitutively express FOXP3, which in turn can be up-regulated upon activation, similarly to what occurs in CD4+ CD25− T cells [21,22] and Bacchetta, unpublished data). Therefore, up to date Tr1 cells are still orphan of a specific marker that could significantly improve their isolation, and further characterization.

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4. Differentiation of Tr1 cells Tr1 cells are inducible cells and for this reason, similar to Th1 and Th2 cells, they arise from naive precursors and can be differentiated both ex vivo and in vivo. IL-10 is considered the driving force for Tr1 cell generation, as shown by experiments in which antigen-specific murine Tr1 cells can be induced ex vivo by repeated TCR stimulation in the presence of high doses of IL-10 [13]. Similarly, murine T cells stimulated with anti-CD3 plus anti-CD28 mAbs in the presence of vitamin D3, dexamethasone, and anti-IFN-␥, -IL-4 and -IL-12 mAbs differentiate into Tr1 cells. Neutralization of IL-10 in this culture significantly inhibits the development of IL-10-producing T cells [23]. IL-10 is therefore not only responsible for the regulatory function of murine Tr1 cells but it is also fundamental for their differentiation. However, for human Tr1 cells it is now evident that in many experimental settings IL-10 is necessary but probably not sufficient for their differentiation. In a system where murine fibroblasts expressing high levels of hCD32, hCD58, and hCD80 are used in combination with anti-CD3 mAb to activate na¨ıve human CD4+ T cells, addition of exogenous IL-10 results in a relatively small increase in IL-10-producing T cells. IFN-␣, a crucial cytokine for clearing viral infections and increasing IL-10 production by T cells, synergies with IL-10 in this in vitro system to promote differentiation of human CD4+ Tr1 cells [24]. Furthermore, using the same artificial APC Wakkach et al. demonstrated that costimulation via CD2 alone, in the absence of costimulations through CD28- or LFA-1, induced T cell anergy in an IL-10-independent pathway along with the differentiation of Ag-specific Tr1 cells [25]. Atkinson and co-workers reported that simultaneous crosslinking of CD46, which is a complement regulatory protein expressed by most human cells, and CD3 induces IL-10producing Tr cells, which express granzyme B. These cells can suppress bystander T cells through IL-10 secretion and a granzyme B/perforin-dependent mechanism [26,27]. CD46 also functions on human cells as a cellular receptor for multiple viral and bacterial pathogens. One of these CD46-binding microorganisms is Streptococcus pyogenes, a human pathogen causing multiple diseases, that, upon interaction with CD46 on human CD4+ T cells, directly generates T cells with a Tr1-like regulatory phenotype [28]. One important question regarding the differentiation of Tr1 cells is whether there is a distinct subtype or activation status of APC that promotes the differentiation of Tr1 cells rather than effector T cells. DC are professional APC that in the presence of inflammatory signals become mature and promote the activation of effector T cells. On the contrary, immature DC (iDC) have been demonstrated to induce tolerance, mainly through deletion of antigen-specific effector T cells [29,30] and induction of Tr cells [31,32]. Repetitive ex vivo stimulation of human CD4+ T cells isolated from cord blood with allogeneic iDC leads to generation of a population of IL-10-producing Tr cells, which resemble the natural occurring CD4+ CD25+ Tr cells [33]. However, we recently demonstrated that repetitive stimulation of na¨ıve CD4+ T cells isolated from peripheral blood with allo-

geneic iDC results in the generation of Tr cells, which produce IL-10 and are functionally similar to Tr1 cells [22]. Furthermore, in the mouse, it has been shown that a population of DC expressing high levels of CD45RBhigh have an immature-like phenotype, secrete IL-10, and induce Tr1 cells ex vivo and in vivo [34]. In addition to the ex vivo generation, several in vivo protocols have been shown to induce Tr1 cells. Treatment of mice with a killed Mycobacterium vaccae suspension gives rise to allergen-specific Tr1 cells that confer protection against airway inflammation [35]. Treatment with filamentous hemagglutinin from Bordetella pertussis enhances IL-10 production from macrophages and DC, which in turn promotes the induction of Tr1 cell [36]. Moreover, mice immunised with cholera toxin in the presence of antigen give raise to antigen-specific Tr1 cells [37,38]. We recently showed that an in vivo pharmacological treatment with rapamycin + IL-10 is able to prevent allograft rejection in a mouse model of islet transplantation. This treatment not only prevents acute allograft rejection but also leads to active long-term tolerance via induction of antigen-specific Tr1 cells [63]. It remains to be defined whether this therapy is efficacious also in humans for the in vivo differentiation of antigen-specific Tr1 cells. 5. Evidences of Tr1 cells in humans The first suggestion that human Tr1 cells are involved in maintaining peripheral tolerance in vivo came from our studies in SCID patients successfully transplanted with HLAmismatched allogeneic stem cells, as already discussed [14]. In addition, high spontaneous IL-10 production by recipient PBMC before bone marrow transplantation has been associated with a subsequent low incidence of GvHD and transplantrelated mortality [39,40]. Recently, a high frequency of donor cells producing IL-10 in response to recipient alloantigens was found to correlate with absence of acute GvHD after bone marrow transplantation, while low frequency was strongly associated with severe GvHD [41]. In addition, the presence of the IL-10 promoter polymorphism associated with high transcription levels of IL-10 has been shown to be an independent protective factor for severe acute GvHD [42,43]. Studies in patients who spontaneously developed tolerance to kidney or liver allograft revealed the presence of CD4+ T cells which suppress naive T cell responses via production of IL-10 or TGF-␤ [44]. Together, these data indicate that Tr1 cells can naturally regulate immune responses and induce tolerance to alloantigens in vivo in the settings of bone marrow and solid organ transplantation. There are several indications that Tr1 cells can also play a role in modulating responses to self antigens. Kitani et al. isolated self-MHC-reactive Tr1 cell clones that inhibited proliferation of primary CD4+ T cells and tetanus toxoid-specific T cell clones ex vivo and this inhibition was mediated by both IL-10 and TGF-␤ [45]. A decreased frequency of IL-10-producing CD4+ T cells was also observed in the inflamed synovium and peripheral blood of patients with rheumatoid arthritis [46]. Human

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autoantigen-specific Tr1 cells were identified in PBMC from patients with autoimmune hemolytic anemia after ex vivo stimulation with a major red blood cell autoantigen, the RhD protein [47]. Desmoglein 3-specific Tr1 cells have been also isolated from healthy individuals that express the HLA class II alleles associated with pemphigus vulgaris, which is a severe autoimmune bullous skin disorder [48]. Furthermore, autoreactive T cells with a Th1-like phenotype were found in patients with autoimmune type 1 diabetes, while non diabetic HLA-matched controls had a Tr1-like anti-islets peptides immune response in vitro [49]. All these data indicate that Tr1 cells play a central role in maintaining self-tolerance and hence preventing autoimmune diseases. Tr1 cells are also important in down-regulating immune responses toward allergens such as nickel [50], insect venom [51], and cat allergen [52]. Furthermore, we recently isolated Tr1 cell clones specific for gliadin, the immunogenic element of gluten, from the intestinal mucosa of celiac patients in remission (i.e. under gluten-free diet). These Tr1 cell clones were anergic, produced IL-10 and TGF-␤, and had a strong inhibitory capacity on gliadin-specific T cell response in vitro (reviewed in [53] and C. Gianfrani manuscript in preparation). Papadakis et al. also demonstrated that circulating CCR9+ CD4+ T cells from peripheral blood of normal donors have characteristics of mucosal T cells in terms of an activated phenotype, proliferative response to anti-CD2 stimulation, a Th1 or Tr1 cytokine profile, and support for Ig production by cocultured B cells [54]. Interestingly, CCR9+ CD4+ T cells have been suggested to play an important role in small bowel immunity [55], bowel inflammatory disease, and also Crohn’s and celiac disease [56]. It is tempting to speculate that CCR9+ CD4+ Th1 and Tr1 cells are continuously generated in the small intestinal mucosal immune system and play an important role in effector and regulatory functions in the intestine. Overall these studies demonstrate that Tr1 cells are beneficial for the induction of tolerance to self, allo, and non-harmful foreign antigens, such as allergens and food antigens. However, Tr1 cells specific for infectious agents or tumor antigens may interfere with the host’s immune response and thus be detrimental. Presumably, it is advantageous for pathogens to evolve strategies to enhance the differentiation of Tr1 cells, which would then limit the protective immune response and allow long-term infection of the host [36]. Hemagglutinin from B. pertussis inhibits IL-12 and enhances IL-10 production from DC in the lung and bronchial lymph nodes [57]. In chronic helminthes infections, where patients have relatively little sign of dermatitis despite the presence of small worms in the skin, antigen-specific Tr1 cells can be isolated. These cells were able to inhibit proliferation of effector T cell clones [58]. Similarly to infectious agents, several studies report the existence of Tr1 cells specific for tumor antigens. It has been shown that myeloma cells prime DC towards a state that favors the generation of T cells with a Tr1 rather than an effector phenotype [59]. Exposure to cyclooxygenase-2-overexpressing glioma induces mature DC to overexpress IL-10 and decreased IL-12p70 production. These DC induce a Tr1 cell response,

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which is characterized by robust secretion of IL-10 and TGF-␤ with negligible IL-4 secretion by CD4+ T cells, and an inhibitory effect on responder T cells [60]. In addition, Hodgkin lymphoma infiltrating CD4+ lymphocytes (HLILs), unlike their PBMC counterpart, have been found to be anergic, and suppress cell proliferation in vitro. Furthermore, HLILs contain large populations of both IL-10-secreting Tr1 and CD4+ CD25+ Tr cells. Thus, HLILs are highly enriched in Tr cells, which create a profoundly immunosuppressive environment that provides an ineffective immune clearance of cancer cells [61]. It is now evident that the balance between effector T cells and Tr1 cells need to be precisely tuned. 6. Clinical applications with Tr1 cells It is now clear that Tr1 cells are an essential tool through which the immune system can actively control immune responses. Therefore, therapeutical ex vivo or in vivo induction of Tr1 cells might be highly advantageous in several T cell-mediated diseases. Much progress has already been made in animal models, which proved that cellular therapy with Tr cells is a feasible approach (reviewed in [62]). Although clinical translation from mouse to humans has been clearly difficult, there are increasing evidence that Tr1 cells can be envisaged as immunomodulatory therapy in the clinic. HLA-haploidentical transplantation offers a valuable source of hematopoietic stem cells (HSC) to most of the patients in the need of a bone marrow transplant when matched donors are unavailable. A megadose of highly purified CD34+ HSC depleted of mature T cells is crucial for promoting engraftment without GvHD. However, T cell-depleted transplants are at high risk of recurrent life-threatening infections, and of disease relapse due to the absence of graft versus leukemia (GvL) effect. To overcome these limitations, we are currently performing in our institute a clinical trial in which donor cells anergized ex vivo in the presence of IL-10 are used as post-transplant cellular therapy in hematological cancer patients undergoing HLAhaploidentical HSC transplantation. We indeed demonstrated that donor PBMC cultured ex vivo with irradiated host PBMC in the presence of IL-10 for 10 days become anergic towards the host antigens while they preserve the ability to proliferate in response to third party and nominal antigens. Importantly, these IL-10-anergized T cells are highly enriched in alloantigenspecific Tr1 cell precursors (Bacchetta et al. unpublished data) (Fig. 1). Thus, in our clinical trial, PBMC are collected from both the donor prior to mobilization and the host prior conditioning. Subsequently, megadose of highly purified T cell depleted CD34+ HSC (>5 × 106 kg−1 ) are infused in the myeloablated host. Once there are signs of neutrophyl engraftment, donor PBMC are thawed and cultured ex vivo in the presence of irradiated host PBMC and IL-10. After 10 days of culture, the IL-10-anergized donor T cells are infused in the host with the ultimate goal to provide immune-reconstitution with donor T cells that are anergic towards host antigens and contain precursors of host-specific Tr1 cells. The administered cells should

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Fig. 1. IL-10-anergized cells. PBMC of donor origin are cultured ex vivo in the presence of irradiated PBMC of host origin in the presence or absence of IL-10 (i.e. primary mixed lymphocyte reaction, MLR) (upper left panel). Thymidine incorporation assay performed 4 days after primary MLR proves that IL-10 down-regulates T cell proliferation (lower left panel). After 10 days of culture, cells are collected and re-stimulated in a secondary MLR with the original stimulator (SA ) or with a third party donor (SB ). Thymidine incorporation assay performed 3 days after culture demonstrates that cells cultured during primary MLR in the absence of IL-10 proliferate efficiently to a second stimulation with the same stimulator (SA ) and to third party stimulated cells (SB ). On the contrary, cells cultured during primary MLR in the presence of IL-10 are highly anergic in response to the original stimulator (SA ) but respond efficiently to third party antigens (SB ) (lower right panel). To prove that the IL-10-anergized cells are highly enriched in Tr1 cells, donor PBMC cultured for 10 days in the presence of IL-10 are cloned and repetitively stimulated through the TCR. The previously anergic cells regain some ability to proliferate and approximately 5–10% of these anergic cells acquire a unique cytokine production profile (i.e. IL-10+ , TGF-␤+ , IL-4− , IL-2low/− ) that is exclusive for Tr1 cells (upper right panel).

therefore include both memory and na¨ıve T cells able to respond to infectious agents and presumably to provide a GvL effect with a reduced risk of inducing GvHD in comparison to untreated donor T cells, and T cells with the ability to differentiate in fully

competent Tr1 cells (Fig. 2). This trial is currently ongoing in the context of HLA-aploidentical bone marrow transplantation but it has the potential to be extended to allogeneic unrelated bone marrow transplants and to solid organ transplantations. Results

Fig. 2. Clinical protocol with IL-10-anergized cells. PBMC are collected from the HLA-haploidentical donor prior to mobilization and from the host prior conditioning. Stem cells are harvested after donor mobilization and purified CD34+ cells are infused into the recipient who underwent myelo-ablation. After the first signs of neutrophyl reconstitution, donor PBMC are cultured with irradiated host PBMC in the presence of IL-10 (see Fig. 1). After 10 days of culture, the IL-10 anergized donor cells are infused in the patients to provide donor T cells that are: anergic towards donor-antigens, potentially reactive towards cancer cells and infectious agents, and highly enriched in host-specific Tr1 cells precursors. This treatment should contribute to the host immune-reconstitution in the absence of GvHD.

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will be evaluated together with data generated by other groups dedicated to promote transplantation tolerance through cellular therapy, such as RISET: an integrated project to which we and other 20 European partners belong (www.risetfp6.org). Our database search revealed several reports proposing clinical trial with Tr1 cells. Here are listed some of them with the specific website in which more information can be found. It should be emphasized that these information were found in the web and are not peer reviewed reports. However, they provide evidence on how much interest Tr1 cells are evoking as a potential clinical tool for the induction of antigen-specific tolerance. TxCell is a biotechnology company specialized in antiinflammatory immunotherapy. TxCell is working on the durable treatment of chronic inflammations of the immune system whereas current treatments only have a stopgap activity or are symptomatic treatments. The company technology relies on using Tr1 cells for debilitating diseases caused by the dysfunction of the immune system, such as Crohn’s disease, multiple sclerosis, psoriasis, asthma, or rheumatoid arthritis. The company has just raised funds for phase I-II clinical trials to demonstrate the clinical effectiveness of Tr1 cells (http://weblog.aeliosfinance.com/2004/10/txcell raises e.html). Londei and colleagues were awarded from the arthritis research foundation to study the modulation of chronic arthritis via the generation of Tr1 cells and the comparison with the activity of regulatory B cells (http://www.ich.ucl.ac.uk/ publications/research review02/36grants donations1.html). Atkinson and co-workers after the description of Tr1 cells induced ex vivo by triggering CD46 and CD3, propose to use laboratory-grown Tr1 cells for treatments of autoimmune diseases such as lupus and rheumatoid arthritis and for organ rejection following transplantation (http://record.wustl.edu/2003/131-03/discovery.html). Finally, the European vascular genomics network (EVGN) aims to promote tolerance and accelerate the transfer of scientific knowledge to improve the diagnosis and treatment of cardiovascular diseases, especially atherosclerosis. One of the therapeutic approaches envisaged by EVGN to block the formation and development of atherosclerotic plaques involves an anti-plaque “vaccination” that exploit the existence of Tr cells (CD4+ CD25+ and Tr1 cells) and mediators like IL-10, which inhibit the formation of atheroma plaques (http://www.evgn.org/). Although promising, there are still open questions regarding the clinical usage of Tr1 cells for the cure of T cell-mediated diseases. It is still unknown, for example, their migratory ability in vivo, their life-span, their toxicity, and most importantly their ability to promote tumor development. Results from all the ongoing proof of concepts trials and the proposed phase I/II clinical trials will prove fundamental for the future success of Tr1 cells in the clinic. References [1] Gershon RK, Kondo K. Infectious immunological tolerance. Immunology 1971;21(6):903–14. [2] Dorf ME, Benacerraf B. Suppressor cells and immunoregulation. Annu Rev Immunol 1984;2:127–57.

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