Eur. J. Immunol. 2015. 45: 1921–1925

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DOI: 10.1002/eji.201545762

Commentary

Transplantation tolerance: Context matters Luis Graca1,2 1 2

Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Lisbon, Portugal Instituto Gulbenkian de Ciˆencias, Oeiras, Portugal

Costimulation blockade has been one of the most studied strategies to achieve immune tolerance, particularly in transplantation. Yet, in spite of the robust nature of the tolerance-inducing potential of costimulation blockade, a comprehensive understanding of the molecular and cellular mechanisms underlying tolerance induction is still missing. Nevertheless, progress has been continuously made. In this issue of the European Journal of Immunology, Chai et al. [Eur. J. Immunol. 2015. 45: 2017–2027] show that transplantation tolerance induced with an anti-CD154 monoclonal antibody relies on the coexistence of several tolerogenic mechanisms rather than one simple regulatory mechanism. These observations highlight the importance of concerted actions involving multiple pathways, namely apoptosis, acquisition of regulatory cells, or inhibition of proliferation, all of which contribute to the induction and maintenance of robust immune tolerance. A better understanding of these distinct tolerogenic pathways may lead to the development of better tolerance-inducing therapeutics.

Keywords: Activation-induced cell death r Costimulation blockade transfusion r Regulatory T cells r Transplantation tolerance

r

CD154

r

Donor-specific

See accompanying article by Chai et al.

Costimulation is a critical event for T-cell activation. As a consequence, it has become an attractive target for therapeutic strategies aiming to dampen pathologic immune responses, namely by using CTLA4-Ig to prevent CD28-signaling in rheumatic diseases; or conversely by enhancing T-cell function, for instance with anti-CTLA-4 mAbs used in cancer therapy. In addition, costimulation blockade, namely with anti-CD154 (CD40 ligand) mAbs, has been shown an effective strategy to achieve immune tolerance in transplantation. Mice or primates treated with nondepleting antiCD154 mAbs at the time of exposure to alloantigens, develop a state of immune unresponsiveness to those foreign antigens, while preserving immune competence toward unrelated antigens [1, 2]. Clinical development of tolerance-inducing therapeutics targeting human CD154 was, however, compromised due to the expression of CD154 on human platelets, leading to concerns of thromboembolic complications [3]. Efforts are still ongoing to develop

reagents able to block CD154 on human T cells without affecting platelets [4]. Other mAbs targeting molecules important for T-cell activation, such as CD3 or CD4, can also lead to immune tolerance. Perhaps the most well-studied example in transplantation is provided by nondepleting anti-CD4 mAbs that are able to induce transplantation tolerance in TCR-transgenic RAG-deficient mice, where all T cells are alloreactive [5]. However, the use of nondepleting antiCD4 or anti-CD154 mAbs to induce transplantation tolerance in wild-type animals requires additional control of CD8+ T cells [6]. Therefore, it has been necessary to use a nondepleting anti-CD8 mAb together with the other mAbs in order to induce tolerance to transplants with multiple mismatches or even to fully MHC mismatched allografts [7, 8].

Death and proliferation Correspondence: Dr. Luis Graca e-mail: [email protected]

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An initial explanation for the tolerogenic effect of costimulation blockade was provided by Bretscher and Cohn through their

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two-signal model for self-nonself discrimination [9]. According to their model, a T-cell requires TCR signaling (signal 1) and costimulation (signal 2) for productive activation. In conditions where signal 1 is available in the absence of signal 2 (such as upon costimulation blockade with anti-CD154 mAb) T cells would die, thus leading to tolerance by elimination of those antigen-specific T cells. Almost three decades after the publication of this theory, two studies directly assessed the requirement for T-cell death in transplantation tolerance as induced by costimulation blockade [10, 11]. Both groups of researchers found that anti-CD154 treatment could not prevent allograft rejection following genetic modification of T cells to maintain high expression of the antiapoptotic gene Bcl-xL, demonstrating that T-cell apoptosis following costimulation blockade was essential for effective induction of transplantation tolerance. In fact, the efficient elimination of alloreactive T cells has been shown to be sufficient to achieve transplantation tolerance (Fig. 1). One strategy to purge the alloreactive T-cell pool is through the induction of mixed hematopoietic chimerism. In animal models of transplantation, but also in exploratory clinical trials, it was found that transplantation of donor hematopoietic precursors can lead to immune tolerance toward a subsequently transplanted solid organ (reviewed in [12]). Tolerance, in mice, has been shown to correlate with the development of hematopoietic micro-chimerism, where donor leukocytes persist in the host’s blood, and also with the elimination of donorspecific T cells [13]. As a consequence, donor-specific lymphocytes become hard to identify in in vitro proliferation assays. Anti-CD154 mAb treatment has been also shown to be effective in facilitating donor hematopoietic stem cell engraftment in mice [14, 15]. This phenomenon appears to be a consequence of the development of immune tolerance to the alloantigens [16, 17]. Most experimental systems studying the induction of immune tolerance are not specifically adapted to the study of T cells involved in indirect allorecognition. The report by Chai et al. specifically addresses the fate of T cells mediating indirect allospecificity—i.e. recognizing allogeneic MHC peptides presented by recipient APCs [18]. To achieve this goal, the researchers followed the fate of TCR transgenic T cells (named TCR75) adoptively transferred into recipient mice that were subsequently tolerized by donor-specific blood transfusion (DST) under the cover of anti-CD154 mAb treatment. The authors found that a major outcome of transplantation tolerance was a severe reduction in the number of allospecific TCR transgenic cells. Such reduction in cell numbers could not be simply explained by inhibition of cell proliferation, as TCR75 cells from antiCD154-treated mice were not arrested in the undivided (CFSEhigh) state. These data are in line with reports suggesting costimulation blockade leads to activation-induced cell death of T cells entering cell division—a time when costimulatory signals are essential for their survival [10, 11]. Nevertheless, many alloreactive T cells survive and yet the transplants are not rejected.

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Regulatory and effector functions Induction of transplantation tolerance exclusively based on the elimination of alloreactive T cells became generally known as “recessive tolerance,” by opposition to “dominant tolerance,” which relies on active regulatory or immune-suppressive mechanisms [6]. Transplantation tolerance based on the induction of mixed chimerism, as described above, is an example of “recessive tolerance.” Two experimental approaches can demonstrate the existence of dominant regulatory mechanisms: one is the resistance of tolerant mice to the adoptive transfer of alloreactive T cells that would lead to rejection if transferred into a lymphopenic host; the other is a phenomenon known as “linked suppression.” Linked suppression is the ability of a tolerant animal to accept a transplant that simultaneously expresses “tolerized” and third-party antigens [19]. The same host would reject transplants expressing thirdparty antigens alone. The explanation for this phenomenon relies on the principle of dominant regulation, which acts on tolerized antigens and, as a consequence, also prevents immune responses to other antigens present in the same cells. In the case of transplantation tolerance induced following CD154-blockade, it quickly became obvious that cell death could not be the only underlying mechanism [18–22]. The existence of dominant regulatory mechanisms, based on CD4+ T cells, was demonstrated in mice through the identification of linked suppression to transplanted antigens [20] and, subsequently, by resistance of the tolerant state to adoptively transferred alloreactive T cells [21]. Furthermore, when those alloreactive T lymphocytes were allowed to coexist with the tolerant cells, tolerance would arise on the transferred alloreactive T cells, as this tolerance was demonstrated to occur following the specific ablation of the first cohort of tolerant cells—a phenomenon known as “infectious tolerance” [21]. Soon after, Foxp3+ Treg cells were implicated as mediators of immune tolerance, as these cells were shown to be induced upon costimulation-blockade in transplanted mice [22, 23]. In addition to Treg-cell induction, clonal anergy of the alloreactive T cells was also documented in similar experiments of murine transplantation [24]. The detailed analysis of the fate of TCR75 cells, which are specific for indirectly presented alloantigens, provides a novel insight on the regulatory mechanisms induced following co-stimulation blockade [18]. Strikingly, induction of Foxp3 expression was not observed on allospecific TCR transgenic cells [18]. Furthermore, TCR75 cells retained the ability to reject allografts when removed from their tolerance-supportive environment and transferred into another host [18]. However, the nature of the endogenous immune regulatory network that maintains the TCR transgenic cells under control remains to be clarified.

Context matters The literature on transplantation tolerance suggests that often the same therapeutic protocol may trigger different

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immunesuppressive mechanisms, depending on the context. A clear example was provided when nondepleting anti-CD4 mAbs were used to induce tolerance to allogeneic bone marrow (BM) in mice [25]. In those experiments, the dose of BM cells could shift the tolerogenic mechanism from deletion of alloreactive cells (in transplants of large numbers of BM cells) to dominant tolerance based on active regulation (when smaller numbers of BM cells were transplanted). In dominant tolerance, the nature of the suppressive cells may also change as a function of context. Transplantation tolerance induced with nondepleting anti-CD4 leads to peripheral Treg-cell induction [5, 26–28], a population that may be stabilized by the addition of TGF-β [29], and cannot be established in the absence of Foxp3 [30]. However, the same tolerogenic protocol can induce robust tolerance, even in a primed immune system, to foreign proteins administered systemically (ovalbumin or factor VIII) [31, 32]. Under those conditions, tolerance can be induced in Foxp3deficient hosts, with the underlying mechanism relying on IL-10producing regulatory cells. Unlike the report by Chai et al. [18], which shows that endogenous Treg cells but not induced Treg cells are important in costimulation blockade-driven tolerance, transplantation tolerance induced following anti-CD154 treatment has been linked to the induction of Foxp3+ Treg cells in previous studies [22]. However, endogenous Treg cells may contribute to the overall transplantation tolerance, being abundant within the tolerated skin graft [18]. One possible explanation for the absence of peripheral induction of Foxp3 in TCR75 cells may be the systemic administration of the antigen as a DST [18]. The parallel with tolerance induction



Figure 1. The fate of antigen-specific CD4+ T cells as immune tolerance is established. Different tolerance-inducing protocols may have distinct impact on antigen-specific T cells. However, effective tolerance induction requires a shift in the balance between proinflammatory and anti-inflammatory/regulatory mechanisms. (A) Productive immune responses, such as the ones leading to transplant rejection, predominantly rely on the proliferation and acquisition of effector functions by alloreactive T cells. (B) There has been a long-standing view that tolerance-inducing protocols act by controlling the size of the effector T-cell pool while, simultaneously, enhancing active regulatory mechanisms. The reduction of the alloreactive cell pool is achieved through the combination of inhibition of T-cell proliferation, their functional inactivation (anergy), and/or by inducing cell death (for instance by reducing available costimulation signals). Abrogation of the acquisition of effector functions by alloreactive T cells, as observed following treatment with anti-CD4 or anti-CD154 mAbs, also contributes to the reduction of the number of T cells that are able to engage in graft rejection. At the same time, an increase in active regulation can take place through peripheral induction of Foxp3+ Treg cells, mediated by activation in presence of TGF-β, or through induction of IL-10-producing Tr1 cells. (C) Chai et al. reports that different T-cell fates, that at a first glance may appear contradictory, can coexist in tolerant mice [18]. Indeed, it is shown some antigen-specific T cells do proliferate and are capable of acquiring effector function as tolerance is established, but other mechanisms, such as cell death and active regulation, limit the proinflammatory potential of those cells within a tolerant mouse. Remarkably, tolerance induction with DST + anti-CD154 did not lead to the induction of antigen-specific Foxp3+ Treg cells, although endogenous Treg cells were shown to play an important role, and it remains unclear whether IL-10-producing Tr1 cells can play an active role in maintaining tolerance [18].  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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with anti-CD4 suggests that while a solid transplant may require a local environment with TGF-β and peripheral Foxp3+ Treg-cell induction, the systemic dissemination of an antigen (such as in i.p. administration of Factor VIII or a DST) might lead to Foxp3independent regulation based on IL-10. The observation that alloreactive T cells from DST + antiCD154 treated mice retain functional competence to reject a transplant, while remaining under control, also demonstrates persistent active regulation [18]. It has been also reported that in tolerance induced with nondepleting anti-CD4 mAbs, alloreactive CD8+ T-cell effector functions can be disarmed by Treg cells, while persisting in tolerant mice [33]. An outstanding issue in transplantation tolerance is how the environment influences T-cell regulation. Several immune regulatory genes, such as Indoleamine 2,3-Dioxygenase and heme oxygenase-1, have been identified in tissues that can contribute to immune suppression [34]. There is also increasing evidence that factors interfering with cell metabolism can have a strong impact on effector versus regulatory responses, namely by modulation of mTOR activity [35]. Therefore, alloreactive effector T cells that survive deletion may be kept in check by immune suppressive mechanisms involving different types of regulatory cells—immune and nonimmune. A better understanding of distinctive immune tolerance mechanisms, and the different rules that lead to their triggering, will be key for the rational development of effective immune modulatory strategies.

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4 Daley, S. R., Cobbold, S. P. and Waldmann, H., Fc-disabled anti-mouse CD40L antibodies retain efficacy in promoting transplantation tolerance. Am. J. Transplant. 2008. 8: 2265–2271. 5 Cobbold, S. P., Castejon, R., Adams, E., Zelenika, D., Graca, L., Humm, S. and Waldmann, H., Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants. J. Immunol. 2004. 172: 6003–6010. 6 Graca, L., Le Moine, A., Cobbold, S. P. and Waldmann, H., Dominant transplantation tolerance. Opinion. Curr. Opin. Immunol. 2003. 15: 499–506. 7 Trambley, J., Bingaman, A. W., Lin, A., Elwood, E. T., Waitze, S. Y., Ha, J., Durham, M. M. et al., Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection. J. Clin. Invest. 1999. 104: 1715–1722. 8 Graca, L., Le Moine, A., Lin, C. Y., Fairchild, P. J., Cobbold, S. P. and Waldmann, H., Donor-specific transplantation tolerance: the paradoxical behavior of CD4+CD25+ T cells. Proc. Natl. Acad. Sci. USA 2004. 101: 10122–10126. 9 Bretscher, P. and Cohn, M., A theory of self-nonself discrimination. Science 1970. 169: 1042–1049. 10 Li, Y., Li, X. C., Zheng, X. X., Wells, A. D., Turka, L. A. and Strom, T. B., Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 1999. 5: 1298–1302. 11 Wells, A. D., Li, X. C., Li, Y., Walsh, M. C., Zheng, X. X., Wu, Z., Nunez, G. et al., Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat. Med. 1999. 5: 1303–1307. 12 Sachs, D. H., Kawai, T. and Sykes, M., Induction of tolerance through mixed chimerism. Cold Spring Harb. Perspect. Med. 2014. 4: a015529. 13 Kurtz, J., Shaffer, J., Lie, A., Anosova, N., Benichou, G. and Sykes, M., Mechanisms of early peripheral CD4 T-cell tolerance induction by antiCD154 monoclonal antibody and allogeneic bone marrow transplantation: evidence for anergy and deletion but not regulatory cells. Blood 2004. 103: 4336–4343. 14 Durham, M. M., Bingaman, A. W., Adams, A. B., Ha, J., Waitze, S. Y., Pearson, T. C. and Larsen, C. P., Cutting edge: administration of anti-CD40 lig-

Ackowledgments: Research in LG laboratory is funded by Fundacao para a Ciencia e Tecnologia FCT/HMSP-ICT/0034/2013.

and and donor bone marrow leads to hemopoietic chimerism and donorspecific tolerance without cytoreductive conditioning. J. Immunol. 2000. 165: 1–4. 15 Wekerle, T., Kurtz, J., Ito, H., Ronquillo, J. V., Dong, V., Zhao, G., Shaffer, J., Sayegh, M. H. and Sykes, M., Allogeneic bone marrow transplantation

Conflict of interest: The author declares no financial or commercial conflict of interest.

with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat. Med. 2000. 6: 464–469. 16 Graca, L., Daley, S., Fairchild, P. J., Cobbold, S. P. and Waldmann, H., Coreceptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning. BMC Immunol. 2006. 7: 9.

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Received: 13/5/2015 Revised: 13/5/2015 Accepted: 27/5/2015 Accepted article online: 28/5/2015

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Transplantation tolerance: context matters.

Costimulation blockade has been one of the most studied strategies to achieve immune tolerance, particularly in transplantation. Yet, in spite of the ...
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