Umbilical cord blood regulatory T-cell expansion and functional effects of tumor necrosis factor receptor family members OX40 and 4-1BB expressed on artificial antigen-presenting cells

Abstract

IntroductionAcute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality after allogeneic bone marrow transplantation (BMT) and occurs as a result of the activation, proliferation, and effector cell function of donor T cells that, along with the release of proinflammatory cytokines, result in tissue damage. 1 Recently, a subset of CD4 ϩ T cells with potent suppressor activity, CD4 ϩ 25 ϩ regulatory T cells (Tregs), 2 have been shown to inhibit alloreactive T-cell activation and effector cell function 3,4 and GVHD lethality in murine models. [5][6][7] Thus, Tregs are attractive therapeutic tools for preventing GVHD in humans. [8][9][10][11] When administered at the time of BMT, typically Tregs must be given at approximately the same number of donor T cells (1:1 ratio) to be highly effective at preventing GVHD. [5][6][7] This poses a technical problem since Tregs are present in low frequency in the peripheral blood (ϳ 1% are CD4 ϩ CD25 br cells) 12 and contaminating CD4 ϩ 25 Ϫ T cells out-compete Tregs for expansion and result in loss of Treg suppressor cell function. 11,13 Therefore, many laboratories have been dedicated to developing new approaches for the isolation and expansion of human Tregs from adult peripheral blood without loss of suppressor cell function.Tregs express multiple tumor necrosis factor receptor (TNFR) family members on their cell surface, including glucocorticoidinduced tumor necrosis factor receptor (GITR), OX40 (CD134), and 4-1BB (CD137). 14,15 While it is generally accepted that TNFR expressed on naive T cell functions contributes to T-cell survival (reviewed in Watts 16 ), the exact role of TNFR signaling in Treg generation, expansion, and suppression is unclear. Shimizu and coworkers have shown that stimulation of GITR abrogates Treg suppressor function, 17 although in other studies, McHugh et al showed that loss of suppression depends on whether or not TCR triggering and IL-2 are present before GITR engagement. 15 We have shown that blockade of CD40L signaling on Tregs increases their suppressive function and tolerogenic capacity. [18][19][20] Since signaling via CD40L and GITR can reduce suppressor cell function, neither would seem advantageous for human Treg expansion cultures, so we focused on OX40 and 4-1BB. While OX40 signals do not appear to increase in vitro anti-CD3 mAb/ APC-driven Treg proliferation, 21-23 a recent report indicates that OX40 signals can increase endogenous Treg survival in vivo, 24 although other studies indicated that OX40 signals provided during ex vivo Treg expansion inhibited rodent Treg suppressor function and foxP3 expression. 21,22,24 In humans, OX40L-mediated signaling has been reported to inhibit the generation of IL-10-producing T-regulatory type 1 cells from naive and memory CD4 ϩ T cells and to shut down both IL-10 production and suppressor cell function. 25 4-1BB also is expressed on resting Tregs and up-regulated within 2 days after in vitro anti-CD3 mAb plus IL-2 stimulation. 26 In vitro anti-CD3 mAb or antigen-induce...

Direct Expansion of Human Allospecific FoxP3+CD4+ Regulatory T Cells with Allogeneic B Cells for Therapeutic Application

“…One study reported that mouse third-party Treg cells, when used in combination with rapamycin, prevented rejection of allogeneic bone marrow while rapamycin alone failed to do so (63), suggesting that the third-party Treg cells can survive and function in MHC-incompatible recipient hosts. In a more recent study, human third-party Treg cells were shown to be able to protect hosts from lethality in xenogeneic GVHD models (49). Purified third-party Treg cells, in the absence of contaminating APCs, may be very poorly immunogenic with respect to inducing cytolytic or humoral immune responses that might lead to their destruction.…”

“…However, because of previous barriers in expanding human Ag-specific Treg cells, there has been interest that anti-CD3/CD28-expanded polyclonal Treg cells might be the only approach to generate large numbers of Treg cells for therapeutic applications (43,45,46). Over the last few years, great effort has been made to improve anti-CD3/CD28-based expansion of human polyclonal Treg cells with the goal of improving the yield and purity of the expanded cells (45,(47)(48)(49)(50)(51). Our findings provide an approach to selectively expand alloantigen-specific Treg cells that can serve as a springboard to future development of alloantigen-specific Treg cell-based therapy in humans.…”

Therapeutic Treg expansion in mice by TNFRSF25 prevents allergic lung inflammation

“…*P < 0.05; **P < 0.01; ***P < 0.001. (32)(33)(34)(35). We attempted to induce Treg proliferation in vitro using many different combinations of anti-CD3 and anti-CD28 antibodies, recombinant IL-2, TGF-β, and retinoic acid with or without TNFR25 agonistic antibody; in all cases, TNFR25 stimulation failed to enhance Treg proliferation in vitro, which indicated that additional signals were required (data not shown and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI42933DS1).…”

Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein

“…14 The effect of Tregs on NK cells (NK cell isolation kit, MACS; Miltenyi Biotec) or PBMNCs was tested after labeling with 5-carboxyfluorescein diacetate succinimide ester (CFSE) to assess proliferation induced by anti-CD3 mAb-coated beads (Dynal) or cytokines as indicated. We used the same suppression assay to evaluate patient PBMNCs collected at day 14 with healthy donor NK cells.…”