Ex vivo expansion of human Tregs specific for alloantigens presented directly or indirectly

Abstract

IntroductionRegulatory T cells (Tregs) play a dominant role in transplantation tolerance, as shown by adoptive cell transfer for the prevention of organ graft rejection and GVHD in experimental models. [1][2][3][4][5][6] Translating Treg therapies to humans requires cell isolation and purification, because transfer of the accompanying effector T cells would exacerbate rather than ameliorate human disease. Naturally occurring Treg populations comprise 1%-3% of the human T-cell repertoire. 7 Tregs are characterized by the constitutive expression of CD4, the IL-2 receptor ␣ chain (CD25), and high levels of a nuclear transcription factor, forkhead box p3 (Foxp3), which is critical for their development and suppressive function. [8][9][10] Tregs express little or no IL-7 receptor ␣ chain (CD127). 11 Therefore, the constitutive expression of CD25 and low expression of CD127 have been used for identifying and purifying Tregs. 12,13 Immunomagnetic cell-separation technology has been adapted for this application. Adoptive transfer of freshly isolated human Tregs has prevented GVHD in patients treated with allogeneic hematopoietic stem cell transplantations partially depleted of conventional T cells. 14 However, T cell-replete grafts are more commonly used, and prevention of GVHD after T cell-replete transplantations require the transfer of a higher number of Tregs that can only be obtained through ex vivo expansion.Tregs can be expanded by more than 1000-fold by anti-CD3 and anti-CD28 Ab-coated beads, but loss of regulatory function and acquisition of effector functions have been a concern with this technology. The addition of rapamycin to artificial APCs has apparently solved this problems, because Tregs maintained Foxp3 expression and in vivo suppressive function in immunodeficient mice. 15 Because in vivo Treg protection from GVHD requires alloantigen-specific (allo-specific) engagement of the TCR, 16 adoptive immunotherapy with allo-specific Tregs offers more advantages than polyspecific Tregs: selective rather than broad immunosuppression, ability to control the conditions for antigen presentation, and economy of scale because they are a fraction of the entire Treg population. Antigen-specific Tregs expanded in response to dendritic cells (DCs) presenting a single self-peptide 17 and suppressed experimental autoimmune diabetes more efficiently than polyclonal Tregs. 18 Human allospecific Tregs could also expand ex vivo, 19 but only by 10-to 20-fold. 18 Because Tregs constitute 1%-3% of human blood T cells, and effective suppression requires a Treg:effector T cell ratio of 1:1, we surmise that therapeutic applications will require expanding allo-specific Tregs by over 100-fold. 20 There is a need to improve on expansion protocols before allo-specific Tregs can be brought to the clinic. Sagoo et al 21 have recently adopted a 2-step process for the large-scale expansion of allo-specific Tregs: primary Treg activation by DCs presenting the specific alloantigen directly, followed by sorting Tregs for expression of the a...

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

“…However, the expansion of thymus derived Treg cells and the generation of polyclonal in vitro-induced Treg (iTreg) cells from naive cells have the disadvantage that these cells are expanded/generated from an existing pool, and have a broad rather than a selective reactivity towards alloantigens. Thus, the use of allospecific Treg cells has the advantage of avoiding broad immune suppression as suggested by Anasetti's group [33]. Moreover, the polyclonal expansion obtained by simply using CD3/CD28 beads risks the expansion of other possible auto-reactive cells.…”

Large-scale in vitro expansion of human regulatory T cells with potent xenoantigen-specific suppression

“…Selection and use of antigen-specific Treg has been proposed to improve Treg therapeutic efficiency and lower the risk for unwanted nonspecific immune suppression caused by transferring large numbers of polyclonal Treg (Koenecke et al 2009;Hall et al 2009;Peters et al 2008;Veerapathran et al 2011). Recent studies have shown that ex vivo expanded alloantigen specific Treg obtained enhanced suppressive capacity in allogeneic responses in vitro (Peters et al 2008;Golshayan et al 2007) and were more potent than polyclonal Treg in protecting against alloimmune-mediated injury of human skin grafts in a humanized mouse model (Yamano et al 2011;Sagoo et al 2011).…”

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15

“…This finding may also explain the observation that a simultaneous influx of both Ag-specific effector T cells and Tregs is observed after s.c. purified protein derivate injection (69). Expansion of alloantigen-specific Tregs through stimulation with other cell types like PBMCs (18,19) and B cells (10,20) has been documented previously. Our results with PBMCs as allogeneic stimulator cells for nTreg expansion are in line with previous publications, and relatively high stimulator/Treg ratios in combination with IL-2 and IL-15 are needed.…”

“…Results from animal studies indicated that DCs can be used to expand alloreactive Tregs (27)(28)(29). Human immature DCs can stimulate the expansion of Tregs (19), but some degree of maturation leading to semimature DCs (30) yielded better results. In all protocols, there is a need for exogenous IL-2 to obtain optimal Treg expansion, but the requirements for exogenous cytokines may be different when mature monocyte-derived DCs (moDCs) are used as stimulator cells (31).…”

“…Addition of rapamycin to this cell culture is used to diminish outgrowth of the contaminating effector T cells (16,17). Alloantigen-specific Tregs with a much greater specificity can be generated by expansion in the presence of allogeneic PBMCs (18,19) or B cells (10,20). The potential benefit of these alloantigen-specific Tregs is targeted suppression rather than general immunosuppression and increased suppressive potency (9,(21)(22)(23)(24)(25)(26).…”