CD25 Regulatory T Cells Can Develop In Vivo from +CD4 + ...

The Journal of Immunology

Alloantigen-Induced CD25ⴙCD4ⴙ Regulatory T Cells Can Develop In Vivo from CD25ⴚCD4ⴙ Precursors in a Thymus-Independent Process1 Mahzuz Karim, Cherry I. Kingsley, Andrew R. Bushell,2 Birgit S. Sawitzki, and Kathryn J. Wood The capacity of naturally occurring autoreactive CD25ⴙCD4ⴙ regulatory T cells (Treg) to control immune responses both in vivo and in vitro is now well established. It has been demonstrated that these cells undergo positive selection within the thymus and appear to enter the periphery as committed CD25ⴙCD4ⴙ Treg. We have shown previously that CD25ⴙCD4ⴙ Treg with the capacity to prevent skin allograft rejection can be generated by pretreatment with donor alloantigen under the cover of anti-CD4 therapy. Here we demonstrate that this process does not require an intact thymus. Furthermore, generation of these Treg is not dependent on the expansion of CD25ⴙCD4ⴙ thymic emigrants, because depletion of CD25ⴙ cells before pretreatment does not prevent Treg development, and Treg can be generated from CD25ⴚCD4ⴙ precursors. Taken together, these results clearly demonstrate that CD25ⴙCD4ⴙ Treg can be generated in the periphery from CD25ⴚCD4ⴙ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25ⴙCD4ⴙ Treg develop. These observations may have important implications for the design of protocols, both experimental and clinical, for the induction of tolerance to autoantigens or alloantigens in adults with limited thymic function. The Journal of Immunology, 2004, 172: 923–928.

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lucidation of the mechanisms by which the immune system achieves self-tolerance has been a major focus of immunological research for many years. Central deletion of autoreactive T cells by the process of negative selection during intrathymic development is known to play an important role in achieving this (1). However, it is also well recognized that a number of other mechanisms operate in the periphery including deletion, ignorance, anergy, clonal exhaustion, and suppression or regulation. In recent years, increasing attention has been focused on the role of regulatory T cells (Treg)3 in controlling immune responses both in vivo and in vitro, and in particular many studies have attempted to define phenotypic markers that identify Treg populations. The most useful cell surface marker available at present that enriches for cells within the CD4⫹ population that possess regulatory activity is CD25, the ␣ subunit of the IL-2 receptor. It has been demonstrated that normal unmanipulated mice contain CD25⫹CD4⫹ cells that are able to prevent the development of autoimmune disease (2–5), so-called naturally occurring autoreactive CD25⫹CD4⫹ Treg. Studies investigating the mechanisms by which these naturally occurring Treg develop have demonstrated that they undergo a

Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom Received for publication July 2, 2003. Accepted for publication November 4, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was funded by The Wellcome Trust. M.K. and B.S.S. are Wellcome Trust Research Fellows. C.I.K. is a Medical Research Council Graduate Student. K.J.W. holds a Royal Society Wolfson Research Merit Award. 2 Address correspondence and reprint requests to Dr. Andrew R. Bushell, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. E-mail address: [email protected] 3 Abbreviations used in this paper: Treg, regulatory T cells; AIRE, autoimmune regulator; CyC, CyChrome; DST, donor-specific transfusion; GITR, glucocorticoid-induced TNFR-related gene; MST, median survival time.

Copyright © 2004 by The American Association of Immunologists, Inc.

positive selection process within the thymus, involving the presentation of self-peptide by MHC class II-positive thymic cortical epithelium, and leave as committed CD25⫹CD4⫹ Treg (6 – 8). The development of CD25⫹CD4⫹ Treg appears to require high-affinity interactions between the TCR and MHC-peptide complex (6, 8 –10), and indeed this affinity may under other circumstances be sufficient to lead to the deletion by negative selection of autoreactive T cells (9, 11). One factor that may confer resistance to deletion during this process is expression of the glucocorticoidinduced TNFR family-related gene (GITR). It has been postulated that GITR⫹ cells possessing TCR with high affinity for self-Ag are skewed toward a Treg phenotype (12). The concept that development of Treg with high affinity for self-Ag develop in parallel with the thymic ontogeny of non-Treg populations is an attractive one because it provides a mechanism for controlling the activity of T cell populations with high affinity for self-Ag that, unchecked, may have the potential to cause autoimmune disease. Interestingly, it has been shown recently that self-peptides previously only thought to be expressed in the periphery can be expressed in the thymus in a process regulated by the autoimmune regulator (AIRE) gene (13). Thus the TCR repertoire of naturally occurring CD25⫹CD4⫹ Treg has the potential to cover a relatively broad range of autoantigens. In addition to their role in autoimmune disease, CD25⫹CD4⫹ Treg have been shown to play a role in the prevention of allograft rejection. Such Treg have been demonstrated in rodents with longterm surviving cardiac (14 –16) and pancreatic islet (17, 18) allografts. In our laboratory, we have previously described a system in which CD25⫹CD4⫹ Treg may be generated before transplantation by pretreatment with donor alloantigen (19). In this model, CBA (H2k) mice are pretreated with a donor-specific transfusion (DST) of fully allogeneic B10 (H2b) blood administered under the cover of the anti-CD4 Ab YTS177 (177/DST pretreatment). CD25⫹CD4⫹ cells isolated from the spleens of these mice 28 days later are able to prevent the rejection of donor strain, but not third party, skin allografts 0022-1767/04/$02.00

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CD25⫹ REGULATORY T CELLS CAN DEVELOP IN VIVO FROM CD25⫺ PRECURSORS

mediated by CD45RBhighCD4⫹ effector cells. In this system regulation is donor specific, and equivalent numbers of CD25⫺CD4⫹ cells from 177/DST pretreated mice or of CD25⫹CD4⫹ cells from naive animals or animals pretreated with YTS177 or DST alone are unable to regulate in this way, demonstrating the importance of the pretreatment protocol in the development of these alloantigen-induced Treg (19). We have previously postulated three pathways by which 177/ DST may generate CD25⫹CD4⫹ Treg (20). First, alloantigen used for pretreatment may enter the thymus and mediate the positive selection of alloreactive CD25⫹CD4⫹ Treg in a process akin to the development of naturally occurring Treg. Second, alloantigen may act in the periphery to expand populations of naturally occurring autoreactive CD25⫹CD4⫹ Treg generated within the thymus that cross-react with alloantigen. Finally, non-Treg populations (which may be naive CD25⫺ cells or Ag-experienced CD25⫹ cells) may be converted to a Treg phenotype. The aim of the current study was to examine the role of these pathways in Treg generation by alloantigen.

Histological examination Skin grafts were fixed in buffered 10% formalin. Six-micrometer paraffinembedded sections were cut and stained with H&E.

Results

Generation of CD25⫹CD4⫹ Treg by 177/DST is not thymus dependent We first wished to establish whether generation of alloantigeninduced CD25⫹CD4⫹ Treg by the 177/DST protocol might be dependent upon migration of donor Ag into the thymus and subsequent positive selection of donor-reactive CD25⫹CD4⫹ Treg. Therefore, we administered 177/DST to mice that had previously undergone thymectomy and then tested the ability of CD25⫹CD4⫹ cells isolated from the spleens of these animals to regulate rejection of B10 skin allografts mediated by CD45RBhighCD4⫹ effector cells in immunodeficient CBA-Rag⫺/⫺ recipients (Fig. 1a). Although mice reconstituted with CD45RBhighCD4⫹ cells alone acutely rejected their B10 skin allografts (median survival time (MST) ⫽ 16 days, n ⫽ 7), cotransfer of CD25⫹CD4⫹ cells from

Materials and Methods Mice CBA.Ca (CBA, H2k), C57BL/10 (B10, H2b), and CBA-Rag 1⫺/⫺ (CBARag⫺/⫺, H2k, kindly provided by D. Kioussis, Division of Molecular Immunology, National Institute for Medical Research, Mill Hill, London, U.K.) were obtained from and housed in the Biomedical Services Unit, John Radcliffe Hospital (Oxford, U.K.). Sex-matched mice between 6 and 12 wk of age the time of first experimental procedure were used in all experiments.

Reagents and mAbs The following Abs were used for cell purification, flow cytometry, and in vivo administration. The hybridoma TIB120 (anti-MHC class II) was obtained from American Type Culture Collection, (Manassas, VA); YTS169 (anti-CD8) and YTS177.9 (anti-CD4) (21) were kindly provided by H. Waldmann (Sir William Dunn School of Pathology, Oxford, U.K.); PC61 (anti-mouse CD25) (22). Cychrome (CyC)-conjugated RM4-5 (anti-mouse CD4), PE-conjugated 16A (anti-mouse CD45RB), PE-conjugated 7D4 (antimouse CD25), biotinylated 7D4, streptavidin-PE, and streptavidin-allophycocyanin were purchased from BD PharMingen (San Diego, CA).

In vivo 177/DST pretreatment protocol Adult CBA mice received 200 ␮g of the anti-CD4 mAb YTS177 i.v. on days ⫺28 and ⫺27. On day ⫺27, they also received 250 ␮l of B10 blood i.v. Spleens were harvested on day 0 for cell isolation of CD25⫹CD4⫹ cells.

Cell purification CD45RBhighCD4⫹ T cells were isolated from lymph nodes and spleens of naive CBA mice, and CD25⫹CD4⫹ T cells were obtained from spleens of animals pretreated with 177/DST. Populations were purified by negative selection using magnetic beads followed by FACS sorting as previously described (19). On reanalysis, all populations were ⬎95% pure.

Cell adoptive transfer and skin transplantation CBA-Rag⫺/⫺ mice were reconstituted i.v. with 105 CD45RBhighCD4⫹ cells with or without 2 ⫻ 105 CD25⫹CD4⫹ cells. The following day, full-thickness B10 tail skin allografts were transplanted onto graft beds prepared on the flanks of the reconstituted mice. Allografts were monitored and graft survival between groups was compared using the log rank test (23) using software developed and kindly provided by S. Cobbold (Sir William Dunn School of Pathology).

Flow cytometric analysis Flow cytometric analysis was performed as previously described (19). Briefly, single-cell splenocyte suspensions were incubated with fluorescent-labeled Abs for surface staining. The cells were then fixed with 4% formaldehyde, permeabilized with 0.5% saponin, and incubated with Abs for intracellular staining. Statistical comparison of groups was performed using the Student’s t test.

FIGURE 1. Generation of alloantigen-induced Treg is not thymus dependent. a, CBA mice underwent thymectomy and were rested for 2 wk before pretreatment with 200 ␮g of YTS177 on days ⫺28 and ⫺27 together with 250 ␮l of B10 blood on day ⫺27. On day 0, 2 ⫻ 105 CD25⫹CD4⫹ cells from the spleens of these animals were adoptively transferred into CBA-Rag⫺/⫺ recipients together with 105 CD45RBhighCD4⫹ cells from naive animals, and the following day a B10 skin allograft was performed. b, Survival of B10 skin allografts: 䡺, animals reconstituted with CD45RBhighCD4⫹ cells only (MST ⫽ 16 days, n ⫽ 7); f, animals reconstituted with both CD45RBhighCD4⫹ and CD25⫹CD4⫹ cells (MST ⬎100 days, n ⫽ 6; p ⬍ 0.05).

The Journal of Immunology athymic donors led to long-term graft survival in the majority of cases (MST ⬎100 days, n ⫽ 6; p ⬍ 0.05) (Fig. 1b), in common with the CD25⫹CD4⫹ cells from euthymic mice given 177/DST that we have previously described (19). This result clearly demonstrates that the generation of CD25⫹CD4⫹ Treg by the administration of 177/DST is not thymus dependent and can occur in the periphery. The mAb PC61 depletes CD25⫹ cells Having shown that donor alloantigen can generate CD25⫹CD4⫹ Treg in the periphery, we wished to establish whether or not this requires the expansion of naturally occurring autoreactive CD25⫹CD4⫹ thymic emigrants that cross-react with alloantigen. To address this question, we sought to delete CD25⫹ cells from mice before administration of the 177/DST protocol. It has previously been demonstrated that administration of the anti-CD25 mAb PC61 to normal mice leads to the loss of detectable CD25⫹ cells from the periphery (24). However, although this may be due to the deletion of CD25⫹ cells, it is also possible that PC61 instead leads to the modulation of CD25 from the cell surface. To distinguish between these two possibilities, we stained splenocytes for CD25 expression both on the surface and intracellularly using the anti-CD25 Ab 7D4, which recognizes a distinct epitope from PC61 (Fig. 2). In splenocyte preparations from normal CBA mice, 2.38 ⫾ 0.07% of cells expressed CD25 both on the surface and intracellularly, whereas 3.20 ⫾ 0.17% showed intracellular expression only (n ⫽ 2). We predicted that if PC61 treatment led to modulation of CD25 from the cell surface, then PC61-treated animals would show a loss of the population expressing surface CD25, but that the proportion of cells showing only intracellular expression would increase by a corresponding percentage. Conversely, if PC61 depleted CD25⫹ cells, then treated animals would again show a loss of the population expressing surface CD25 but without any corresponding increase in the number of cells showing intracellular expression. Mice that received PC61 did indeed show a loss of cells expressing surface CD25 (0.35 ⫾ 0.07%, n ⫽ 2; p ⬍ 0.05 compared with naive group), but the proportion of cells expressing only intracellular CD25 (3.66 ⫾ 0.09%) was indistinguishable from naive animals ( p ⫽ 0.14), demonstrating that PC61 does indeed deplete CD25⫹ cells.

925 CD25⫹CD4⫹ Treg can be generated by 177/DST in the absence of CD25⫹ thymic emigrants The fact that PC61 depletes CD25⫹ cells allowed us to address the possibility that the generation of alloantigen-induced Treg depends on the peripheral expansion of cross-reactive naturally occurring CD25⫹CD4⫹ Treg generated in thymus. If this were the case, then depletion of these thymic emigrants before administration of 177/ DST should render this protocol ineffective. Therefore, we thymectomized CBA mice to prevent export of new CD25⫹CD4⫹ thymic emigrants, administered PC61 to deplete CD25⫹ cells in the periphery, and then rested the mice for 3 wk to allow Ab clearance (24) before pretreating with 177/DST (Fig. 3a). At the time of 177/DST administration we confirmed by flow cytometric analysis that 95% of peripheral CD25⫹CD4⫹ cells were depleted in these mice (Fig. 3b, p ⬍ 0.05). 177/DST pretreatment of these CD25-depleted animals led to the generation of CD25⫹CD4⫹ T cells that upon adoptive transfer maintained their ability to regulate B10 skin allograft rejection mediated by CD45RBhighCD4⫹ cells in CBA-Rag⫺/⫺ recipients (MST ⬎100 days, n ⫽ 5; p ⬍ 0.05 compared with the group receiving CD45RBhighCD4⫹ cells only) (Fig. 3c), clearly showing that alloantigen-induced CD25⫹CD4⫹ Treg could be generated in the absence of pre-existing CD25⫹ T cells. Treg can be generated from CD25⫺CD4⫹ precursors As an alternative approach to investigating whether alloantigeninduced Treg can be generated in the absence of CD25⫹ precursors, we reconstituted CBA-Rag⫺/⫺ mice with purified CD25⫺CD4⫹ cells alone and then administered 177/DST. To test for the presence of Treg with the capacity to suppress graft rejection mediated by effector cells, 28 days after the 177/DST pretreatment these animals received an infusion of CD45RBhighCD4⫹ cells as effectors followed by a B10 skin allograft the next day (Fig. 4a). Control mice reconstituted with CD25⫺CD4⫹ and CD45RBhighCD4⫹ cells but not given 177/DST pretreatment all rejected their B10 skin grafts acutely (MST ⫽ 9 days, n ⫽ 5), whereas the group that did receive 177/DST all accepted their grafts ⬎100 days (n ⫽ 4; p ⬍ 0.05) (Fig. 4b). This graft acceptance cannot have been due to effects such as deletion of B10reactive cells in the 177/DST group, because these mice clearly contained cells with regulatory capacity, demonstrated by the inability of the CD45RBhighCD4⫹ effector cells to cause rejection.

Discussion

FIGURE 2. PC61 depletes CD25⫹ cells. Spleen cells were taken from naive mice (left) or from mice that had received 1 mg of PC61 i.v. 7 days previously (right) and stained for surface CD25 expression using antiCD25-biotin followed by streptavidin-allophycocyanin. They were then permebilized and stained with CD25-PE. Results are representative of two independent experiments.

Although much attention has been focused on the pathways by which naturally occurring autoreactive CD25⫹CD4⫹ Treg develop, the mechanisms by which alloantigen-induced Treg are generated have been less well characterized. In a large animal model, it has been shown that thymus-dependent mechanisms facilitate the development of allograft tolerance (25–27). Rodent studies have demonstrated that donor alloantigen administered peripherally can, under some circumstances, enter the recipient thymus (28, 29). It has also been shown that direct intrathymic injection of alloantigen can allow the acceptance of allografts through mechanisms other than deletion (30, 31) and, moreover, that this may even be mediated through the generation of CD25⫹CD4⫹ Treg (32). Therefore, it is possible that 177/DST may generate Treg through such an intrathymic mechanism. Although our observation that 177/DST can effectively generate CD25⫹CD4⫹ Treg in athymic mice (Fig. 1) does not exclude the possibility that this pathway is operating, it does clearly demonstrate that it is not essential and that Treg generation can occur in the periphery.

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FIGURE 3. Generation of alloantigen-induced Treg does not require CD25⫹ thymic emigrants. a, CBA mice underwent thymectomy and 2 wk later received 1 mg PC61 i.v. They were then rested for 3 wk before pretreatment with 200 ␮g of YTS177 on days ⫺28 and ⫺27 together with 250 ␮l of B10 blood on day ⫺27. On day 0, 2 ⫻ 105 CD25⫹CD4⫹ cells from the spleens of these animals were adoptively transferred into CBARag⫺/⫺ recipients together with 105 CD45RBhighCD4⫹ cells from naive animals, and the following day a B10 skin allograft was performed. b, CD25 expression by CD4⫹ splenocytes at time of 177/DST pretreatment (day ⫺28). CBA mice underwent thymectomy and 2 wk later received 1 mg of PC61 i.v. Three weeks later, splenocytes were harvested and stained with CD4-CyC and CD25-PE. Histogram plot shows percentage (⫾ SD) of CD4⫹ cells expressing CD25 in naive vs treated mice (n ⫽ 4 in each group, p ⬍ 0.05). c, Survival of B10 skin allografts: 䡺, animals reconstituted with CD45RBhighCD4⫹ cells only (MST ⫽ 14 days, n ⫽ 4); f, animals reconstituted with both CD45RBhighCD4⫹ and CD25⫹CD4⫹ cells (MST ⬎100 days, n ⫽ 5; p ⬍ 0.05).

Because cross-reactivity is regarded as a likely mechanism to account for the high proportion of alloreactive cells in the normal peripheral T cell pool, it is logical to postulate that naturally oc-

FIGURE 4. Treg can be generated from CD25⫺CD4⫹ precursors. a, Control CBA-Rag⫺/⫺ mice were reconstituted with 106 CD25⫺CD4⫹ cells from naive CBA donors on day ⫺35. On day 0, they received 105 CD45RBhighCD4⫹ cells from naive CBA mice and a B10 skin graft the following day. In addition, the 177/DST experimental group received 200 ␮g of YTS177 on days ⫺28 and ⫺27 together with 250 ␮l of B10 blood on day ⫺27. b, Survival of B10 skin allografts: 䡺, control group (MST ⫽ 9 days, n ⫽ 5); f, 177/DST group (MST ⬎100 days, n ⫽ 4; p ⬍ 0.05). c, B10 skin graft from 177/DST group 100 days after transplantation. d, Histology of graft in c.

curring autoreactive CD25⫹CD4⫹ Treg may also show some cross-reactivity with alloantigen, and that 177/DST may therefore simply serve to expand this population. However, while this may be occurring, it is clearly not essential because 177/DST is able to generate CD25⫹CD4⫹ Treg in vivo from CD25⫺ CD4⫹ precursors (Fig. 3). This is supported by the demonstration that 177/DST can induce Treg development in CBA-Rag⫺/⫺ mice containing only CD25⫺CD4⫹ cells (Fig. 4), although in this latter case we have not demonstrated that the Treg generated are CD25⫹. The mechanisms by which 177/DST pretreatment generates CD25⫹CD4⫹ Treg are, at present, unclear and remain an ongoing

The Journal of Immunology focus in our laboratory. It is well recognized that administration of Ag i.v. often leads to the development of tolerance rather than to sensitization, perhaps due to the absence of “danger” signals (33), and it is possible that the coadministration of anti-CD4 Ab facilitates this natural process. For example, previous work in our laboratory has shown that Ag encounter by CD4⫹ cells in the presence of YTS177 perturbs the proximal signaling events that normally occur following T cell activation, resulting in a signaling profile resembling that of anergic T cells (34). In addition, it has recently been demonstrated that T cells that develop in the absence of CD4-MHC class II interactions show the characteristics of Treg in that they up-regulate CD25, express the transcription factor Foxp3, secrete IL-10, and upon adoptive transfer can prevent the development of colitis in vivo (35). In the 177/DST tolerance induction protocol, it is quite possible that alloantigen recognition in the presence of anti-CD4 Ab, which blocks CD4-class II binding, facilitates the generation of Treg in a similar manner. Although CD25 expression serves to enrich for CD4⫹ cells with regulatory activity, it is also a marker of T cell activation (36). Thus bulk CD25⫹CD4⫹ populations are likely to contain both activated and Treg cells. Several candidate markers that may assist in the identification of Treg populations have been identified recently, including GITR (12, 37, 38), the integrin ␣E␤7 (CD103) (37–39), and the transcription factor Foxp3 (40 – 42). The use of such markers may help to establish the relative proportions of Treg and activated CD25⫹CD4⫹ cells generated by the 177/DST pretreatment protocol, and such studies are currently in progress in our laboratory. In summary, it has been shown previously that CD25⫹CD4⫹ Treg may be generated in vitro from CD25⫺ precursors (43). However, to our knowledge this study is the first direct demonstration that this process can also occur in vivo following peripheral Ag administration. This observation may have important implications for the design of protocols to enable the induction of tolerance to alloantigens (in transplantation) or autoantigens (in autoimmune disease) in adults with limited thymic function.

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