Generation of regulatory T cells by T cell receptor gene transfer

Introduction

Tregs play a crucial role in maintaining immune tolerance during normal homeostasis as well as controlling and resolving active immune responses [1]. Several different groups of regulatory T cells have been identified, which play varying roles in the maintenance of physiological immune tolerance [2]. The most intensely studied of these are the FoxP3+ Tregs, which will be the focus of the remainder of this review. Until recently it was thought that FoxP3+ Tregs were generated exclusively in the thymus, hence their common description as natural Tregs. However, it has now been demonstrated that a proportion of FoxP3+ Tregs are generated in the periphery from conventional CD4+ T cells, termed adaptive Tregs [3]. FoxP3+ Tregs exert regulation via a number of different mechanisms, which at present remain poorly defined. The known mediators of these mechanisms can broadly be split into the contact-dependent mechanisms, including membrane-bound TGFβ [4], CTLA-4 [5, 6] and intra-cellular/peri-cellular adenosine compound generation [7, 8]; and the contact-independent cytokine mediated mechanisms, which include the effects of IL-10 [9] and TGFβ [10].

TCR gene transfer into Tregs

Like conventional T cells, Tregs require stimulation via TCR interaction with a cognate peptide: MHC complex in order to exert suppression [11]; therefore they would be malleable to specificity re-direction by TCR gene transfer. Tregs are capable of potently suppressing T cell responses at naïve, effector and memory stages. In addition they have also been demonstrated to act on various other immune cells, including B cells [12], DCs [5], and monocytes [13]. Many aspects of Treg-mediated suppression make them ideal candidates for Ag targeted therapy of immuno-pathology. Firstly, although Tregs require Ag specific stimulation via the TCR, they suppress in an Ag non-specific manner [11]. This phenomenon, termed linked suppression, means that a Treg of one specificity can suppress a conventional T cell of an unrelated specificity provided the cognate Ag for both is expressed on the same antigen-presenting cell (APC). Utilizing this phenomenon of linked suppression along with an intelligent Ag targeting, it would be possible to direct suppression toward the organ or tissue which is affected regardless of whether the causative epitope, or indeed any of the epitopes additionally involved by antigenic spreading, have been identified. Secondly, Treg-mediated tolerance against one peptide specificity can be transferred to other related specificities [14]. This process, referred to as infectious tolerance, is mediated by modulation of dendritic cells (DC) and de novo induction of adaptive Tregs, and would allow for the generation of long lasting multi-epitope mediated tolerance regardless of the limits of the Ag specificity and persistence of the transferred Tregs. Thirdly, Tregs are an endogenous immune control mechanism, present in all healthy individuals. It is clear that all normal inflammatory protective immune responses are elicited in the presence of Tregs, indicating that re-establishing tolerance via Treg adoptive transfer would not preclude future protective immune responses. This is supported by skin graft models, in which Treg-induced tolerance to allo-grafted skin on the flank was not affected by the rejection of a distinct skin allograft on the contra-lateral flank [15]. If these properties of Tregs could be effectively harnessed they could provide the therapeutic panacea for clinical immune pathology, namely a long lasting Ag-specific control without the complications of a general immune suppression.

Tregs in autoimmunity

There are numerous examples of the use of Tregs to prevent murine models of autoimmunity. However, to the best of our knowledge there are only three examples of reversion of ongoing autoimmunity using Tregs. Intriguingly, two of those studies were carried out using Ag specific Tregs [16, 17]. The third was carried out in a model where the Treg niche was empty before adoptive transfer of the Tregs [18]. It is postulated that the reconstitution of this niche may have created a situation whereby Ag-specific expansion of the Tregs was favored – hence providing the level of Ag specificity required to reverse the ongoing disease. It is compelling that in the former study non-obese diabetic mice were reverted from ongoing autoimmunity using a Treg population specific for a single pancreatic Ag [17]. In this elegant study the authors demonstrated that a transgenic monoclonal Treg population was capable of reverting disease by controlling a multiple epitope T cell responses against peptides derived from an entire organ. This work is a clear indication that Ag specificity is required to revert ongoing autoimmunity, and that Tregs specific to a single disease-related Ag may be sufficient to control a complex and advanced immune response. The importance of Ag specificity in autoimmunity, therefore, is of clear importance: autoimmunity is rarely a predictable disease and typically presents after the establishment of a strong immune response and considerable damage.

Tregs in transplantation

Hematopoietic stem cell transplantation is an effective treatment for a number of hematological diseases, but is accompanied by the potential for development of graft versus host disease (GvHD). Several murine studies have demonstrated the efficacy of adoptive Treg transfer in curing GvHD. In addition to this, whilst the adoptive transfer of Tregs in murine models could prevent GvHD, they did not impact on the advantageous graft versus leukemia (GvL) response. The need for Ag-specific targeting in the prevention of GvHD is less clear than is seen in an autoimmune setting. This is probably due to two factors. Firstly, the Tregs are being adoptively transferred into irradiated and hence lymphopenic hosts, and the subsequent expansion of the Tregs to fill their niche may allow for the preferential expansion of allo-Ag specific Tregs. Secondly, and directly related to the first point, there is likely to be a larger proportion of allo-Ag specific Tregs than auto-Ag specific Tregs in the autoimmune situation. Interestingly in GvHD, Ag-specific Tregs offer only marginal improvement in protection upon adoptive transfer when compared with polyclonal Tregs [19, 20]. These promising pre-clinical studies using polyclonal Tregs have encouraged two separate groups to begin early clinical trials in adoptive Treg transfer in the treatment of human GvHD. However, the potential efficacy of polyclonal Tregs in the treatment of GvHD does not negate the need to examine the potential of Ag-targeted Tregs to treat this disease. Indeed, it should be noted that in all three of the murine studies quoted here, very high number of Tregs were transferred to induce tolerance (around 1:1 Treg:conventional T cell). It is likely that if these Tregs were Ag-specific the number could be reduced substantially. In addition, work is ongoing to identify the distinguishing factors between the development of GvHD and GvL; any advancement in our understanding of this difference will likely allow for targeted Treg therapy to prevent GvHD without impacting on GvL.

There is a clear correlation in the clinical setting between solid organ transplant tolerance and Treg levels. In contrast to autoimmune and GvHD settings, prevention of solid organ rejection by polyclonal Treg transfer in murine models has not been demonstrated. However, numerous studies have demonstrated that the transfer of Tregs from previously tolerized mice is sufficient to prevent rejection of solid organs [21]. More recently, it has been additionally demonstrated that Tregs expanded in vitro against allo-antigen are capable of mediating prevention of rejection [15, 22]. These latter studies also highlighted the importance of Tregs directed against indirect allo-Ag in preventing chronic rejection. Both strategies demonstrate the need for Ag specificity targeted against the most appropriate Ag to induce Treg-mediated tolerance.

FoxP3⁺ Tregs: Potential for antigen specific therapy

It is clear that Ag specificity will be an important factor in successfully translating the promising pre-clinical data into a clinical setting. There are many obstacles to generating Ag-specific Tregs, mainly related to the physiological nature of Tregs as a small population of poorly responsive (in vitro) T cells. Whilst in vitro protocols to expand Ag specific Tregs have advanced in recent years [23] they still represent at present a labor intensive, expensive, and flawed process. Identification of a functional Treg population is at present an imperfect process. Whilst the transcription FoxP3 is generally considered as the only reliable Treg marker, as an intracellular protein it is of no use in identifying functional Tregs. For this reason Tregs are generally identified by a constellation of surface markers, mainly CD4 and CD25. However, CD4+CD25+ population also includes a contaminating fraction of activated conventional T cells. Expansion of this bulk population—whether Ag-specific or polyclonal—leads to an outgrowth of this contaminating conventional T cells population [24]. The more expansion required, the more outgrowth of these cells is seen. The numerous rounds of stimulation required to achieve a sufficient numbers of Ag-specific Tregs for effective treatment in a human setting, if at all possible, would undoubtedly lead to substantial outgrowth of this contaminating population. This contamination population of conventional T cells could potentially be a risk in exacerbating disease. Numerous surface markers have been added to CD4+CD25+ in identifying Tregs (GITR, CD127, CD39, FR4, HLA-DR CD45RA) [25-29] and although many of them allow for higher purity Treg sorting, each additional parameter leads to a decrease in the proportion of Tregs obtained. This is a practical problem when dealing with a population of cells already limited by their paucity.

We are currently examining TCR gene transfer into bead-sorted CD4+CD25+ Tregs. We have been consistently able to express a TCR of known specificity in 60-90% of polyclonally activated Tregs after a single round of activation and transduction. These Tregs demonstrate in vitro Ag-dependent linked suppression of a naïve TCR transgenic CD8+ T cells up-to 30 fold greater than that seen in absence of Ag. With appropriate modifications, including exploration of alternative Treg sorting strategies and optimizing (i.e., reducing) proliferation and transduction protocols, this approach could be used to generate large populations of Ag-specific highly pure Tregs. Other advantages of this approach include the ability to select TCR from outside the normal Treg TCR repertoire. It may be possible to use higher affinity TCR generated in the conventional T cell repertoire or indeed generate high affinity TCR using the allo-restricted strategy [30]. However, it is also important to acknowledge that safety issues must be addressed before the routine use of TCR gene transfer into Tregs. Briefly, those risks primarily involve the danger of development of malignancy caused by insertional mutagenesis and the potential for the creation of unknown specificity TCR from mis-pairing of the endogenous and introduced TCR. The risk of insertional mutagenesis is greatly decreased in mature T cells compared to hematopoietic stem cells, but it remains an important consideration before proceeding with any form of stable gene insertion. The second issue is the generation of novel specificity TCR through mis-pairing of the introduced TCR alpha or beta chains with the corresponding alpha and beta chains endogenously expressed in each T cell. These novel TCR have not been thymically educated and may potentially be strongly self-reactive. It is unclear what the effect of any self-reactive TCR generated through mis-pairing might have in Tregs. It is possible that TCR mis-pairing may be less of an issue in Tregs which are proposed to have a bias in TCR selection towards self specific Ag:MHC complexes. However, it cannot be ruled out that Tregs with a TCR affinity greater than that normally selected during Treg TCR selection may mediate inappropriate suppression. There is considerable effort being employed in addressing these issues in conventional T cells and any advance in TCR gene therapy in that setting will almost certainly be applicable Tregs (see King et al. in this issue for more in-depth review of these issues).

Genetically induced Treg-like T cells

As well as the use of naturally occurring Tregs to treat immune-pathology, there have also been a number of studies using genetically modified “Treg-like” cells. It is well documented that ectopic expression of the regulatory transcription factor FoxP3 induces Treg-like function in conventional murine T cells [31]. Similar to the study described earlier in NOD mice, two studies demonstrated the transduction of FoxP3 into pancreatic islet specific transgenic CD4+ and CD8+ T cells was capable of ameliorating diabetes in NOD mice [32, 33]. FoxP3 expression in polyclonal T cells had no affect in these models. Similar findings have been demonstrated in GvHD settings [34] and solid organ transplantation [35]. Transfer of this concept into a human setting is hindered by subtle differences in the expression and function of FoxP3 in human T cells. Human conventional T cells have been shown to transiently up-regulate FoxP3 subsequent to activation, without the acquisition of any regulatory function. In addition, the ectopic expression of FoxP3 does not consistently instill the same level of regulatory function in human T cells [36]. However, a recent study has demonstrated that lenti-viral mediated expression of FoxP3 in human T cells under a constitutive (i.e., activation state independent) promoter produces consistently efficient regulatory like phenotype [37].

We are currently examining the potential of co-transfer of TCR genes along with the FoxP3 transcription factor using a single tri-cistronic vector to generate functionally suppressive T cells. Subsequent to transduction these cells show limited proliferation and little or no IFNγ and IL-2 secretion. Whilst in our hands the level of Ag-dependent suppression elicited by these T cells is less marked than TCR expressing CD4+CD25+ Tregs, it has proven a reliable method of generating large numbers of homogenous TCR expressing Treg-like T cells. It should also be noted that although the in vitro linked suppression assay is a useful indicator of suppressive ability it is not always completely predictive of the level of suppression in vivo. It will be interesting to compare these two types of regulatory T cells in vivo.

Conclusion

There is now a substantial body of evidence in pre-clinical models for the efficacy of Tregs in the treatment of immuno-pathology. However, as yet there are only two ongoing human clinical trials, both in a GvHD setting. Many unanswered questions and obstacles stand in the way of utilizing Tregs to their maximal effect. Not least amongst these is the question of how to generate sufficiently large populations of Ag-specific regulatory T cells. Here we have highlighted the importance of Ag specificity and proposed that TCR gene transfer into polyclonally expanded Tregs as well as artificially generating FoxP3+ TCR expressing T cells may provide an efficient way of generating large populations of Ag specific Tregs. Studies are ongoing as to the efficacy of each of these approaches in models of auto-immunity and GvHD, and from initial indications we expect these methods to show considerable efficacy in the treatment of immune mediated pathology.

Acknowledgements

The authors would like to thank Mario Perro for his constructive discussions.

References

1. Sakaguchi S, Yamaguchi T, Nomura T, and Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133:775-787.

2. Shevach EM. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity. 2006;25:195-201.

3. Coombes JL, Siddiqui KR, rancibia-Carcamo CV, Hall J, Sun CM, Belkaid Y, and Powrie F. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med. 2007;204:1757-1764.

4. Nakamura K, Kitani A, Fuss I, Pedersen A, Harada N, Nawata H, and Strober W. TGF-beta 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol. 2004;172:834-842.

5. Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, Belladonna ML, Fioretti MC, Alegre ML, and Puccetti P. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol. 2003;4:1206-1212.

6. Read S, Malmstrom V, and Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med. 2000;192:295-302.

7. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, Kuchroo VK, Strom TB, and Robson SC. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007;204:1257-1265.

8. Bopp T, Becker C, Klein M, Klein-Hessling S, Palmetshofer A, Serfling E, Heib V, Becker M, Kubach J, Schmitt S, Stoll S, Schild H, Staege MS, Stassen M, Jonuleit H, and Schmitt E. Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression. J Exp Med. 2007;204:1303-1310.

9. Asseman C, Read S, and Powrie F. Colitogenic Th1 cells are present in the antigen-experienced T cell pool in normal mice: control by CD4+ regulatory T cells and IL-10. J Immunol. 2003;171:971-978.

10. Powrie F, Carlino J, Leach MW, Mauze S, and Coffman RL. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells. J Exp Med. 1996;183:2669-2674.

11. Thornton AM and Shevach EM. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol. 2000;164:183-190.

12. Lim HW, Hillsamer P, Banham AH, and Kim CH. Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells. J Immunol. 2005;175:4180-4183.

13. Tiemessen MM, Jagger AL, Evans HG, van Herwijnen MJ, John S, and Taams LS. CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proc. Natl Acad Sci U S A. 2007;104:19446-19451.

14. Waldmann H, Adams E, Fairchild P, and Cobbold S. Infectious tolerance and the long-term acceptance of transplanted tissue. Immunol Rev. 2006;212:301-313.

15. Golshayan D, Jiang S, Tsang J, Garin MI, Mottet C, and Lechler RI. In vitro-expanded donor alloantigen-specific CD4+CD25+ regulatory T cells promote experimental transplantation tolerance. Blood. 2007;109:827-835.

16. Tarbell KV, Petit L, Zuo X, Toy P, Luo X, Mqadmi A, Yang H, Suthanthiran M, Mojsov S, and Steinman RM. Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. J Exp Med. 2007;204:191-201.

17. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, and Bluestone JA. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 2004;199:1455-1465.

18. Mottet C, Uhlig HH, and Powrie F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol. 2003;170:3939-3943.

19. Yamazaki S, Patel M, Harper A, Bonito A, Fukuyama H, Pack M, Tarbell KV, Talmor M, Ravetch JV, Inaba K, and Steinman RM. Effective expansion of alloantigen-specific Foxp3+ CD25+ CD4+ regulatory T cells by dendritic cells during the mixed leukocyte reaction. Proc Natl Acad Sci U S A. 2006;103:2758-2763.

20. Trenado A, Sudres M, Tang Q, Maury S, Charlotte F, Gregoire S, Bonyhadi M, Klatzmann D, Salomon BL, and Cohen JL. Ex vivo-expanded CD4+CD25+ immunoregulatory T cells prevent graft-versus-host-disease by inhibiting activation/differentiation of pathogenic T cells. J Immunol. 2006;176:1266-1273.

21. Wood KJ and Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol. 2003;3:199-210.

22. Joffre O, Santolaria T, Calise D, Al ST, Hudrisier D, Romagnoli P, and van Meerwijk JP. Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes. Nat Med. 2008;14:88-92.

23. Allan SE, Broady R, Gregori S, Himmel ME, Locke N, Roncarolo MG, Bacchetta R, and Levings MK. CD4+ T-regulatory cells: toward therapy for human diseases. Immunol Rev. 2008;223:391-421.

24. Tang Q and Bluestone JA. Regulatory T-cell physiology and application to treat autoimmunity. Immunol Rev. 2006;212:217-237.

25. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, and Sakaguchi S. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 2002;3:135-142.

26. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, Kelleher A, and Fazekas de St GB. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med. 2006;203:1693-1700.

27. Yamaguchi T, Hirota K, Nagahama K, Ohkawa K, Takahashi T, Nomura T, and Sakaguchi S. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity. 2007;27:145-159.

28. Baecher-Allan C, Wolf E, and Hafler DA. MHC class II expression identifies functionally distinct human regulatory T cells. J Immunol. 2006;176:4622-4631.

29. Hoffmann P, Eder R, Boeld TJ, Doser K, Piseshka B, Andreesen R, and Edinger M. Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood. 2006;108:4260-4267.

30. Sadovnikova E and Stauss HJ. Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: reagents for tumor immunotherapy. Proc Natl Acad Sci U S A. 1996;93:13114-13118.

31. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, and Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity. 2005;22:329-341.

32. Jaeckel E, von BH, and Manns MP. Antigen-specific FoxP3-transduced T-cells can control established type 1 diabetes. Diabetes. 2005;54:306-310.

33. Peng J, Dicker B, Du W, Tang F, Nguyen P, Geiger T, Wong FS, and Wen L. Converting antigen-specific diabetogenic CD4 and CD8 T cells to TGF-beta producing non-pathogenic regulatory cells following FoxP3 transduction. J Autoimmun. 2007;28:188-200.

34. Albert MH, Liu Y, Anasetti C, and Yu XZ. Antigen-dependent suppression of alloresponses by Foxp3-induced regulatory T cells in transplantation. Eur J Immunol. 2005;35:2598-2607.

35. Chai JG, Xue SA, Coe D, Addey C, Bartok I, Scott D, Simpson E, Stauss HJ, Hori S, Sakaguchi S, and Dyson J. Regulatory T cells, derived from naive CD4+. Transplantation. 2005;79:1310-1316.

36. Gavin MA, Torgerson TR, Houston E, DeRoos P, Ho WY, Stray-Pedersen A, Ocheltree EL, Greenberg PD, Ochs HD, and Rudensky AY. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc Natl Acad Sci U S A. 2006;103:6659-6664.

37. Allan SE, Alstad AN, Merindol N, Crellin NK, Amendola M, Bacchetta R, Naldini L, Roncarolo MG, Soudeyns H, and Levings MK. Generation of potent and stable human CD4+ T regulatory cells by activation-independent expression of FOXP3. Mol Ther. 2008;16:194-202.