Graft-versus-host disease (GVHD) is a frequent and severe complication following hematopoietic cell transplantation. Natural CD4+25+ regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs could be purified and expanded in vitro using GMP reagents; however, the initial number of nTregs in UCB units is limited, and average yield after expansion was only 1×109 nTregs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PB nTregs were purified under GMP conditions and expanded 80-fold to yield 19×109 cells using anti-CD3 antibody loaded, cell-based artificial antigen presenting cells (aAPCs) that expressed the high affinity Fc receptor and CD86. A single re-stimulation increased expansion to ~3,000-fold and yield to >600×109 cells, while maintaining FoxP3 expression and suppressor function. nTreg expansion was ~50 million-fold when flow-sort purified nTregs were re-stimulated four times with aAPCs. Indeed, cryopreserved donor nTregs re-stimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune deficient mice. The capability to efficiently produce donor cell banks of functional nTregs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

Adoptive transfer of thymus-derived natural regulatory T-cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft-versus-host disease (GVHD). TGFβ induces Foxp3 expression and suppressive function in stimulated murine CD4+25- T cells, and these induced Treg (iTregs), like nTreg, suppress auto- and allo-reactivity in vivo. However, while TGFβ induces Foxp3 expression in stimulated human T-cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFβ-dependent Foxp3 expression and induces a potent suppressor function in naïve (CD4+25-45RA+) T cells. Rapa/TGFβ iTregs are anergic, express CD25 at levels higher than expanded nTregs, and few cells secrete IL-2, IFNγ or IL-17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFβ induces suppressive function even in the presence of memory CD4+ T-cells. A single apheresis unit of blood yields an average ~240×109 (range ~70–560×109) iTregs from CD4+25- T-cells in ≤ 2 weeks of culture. Most importantly, Rapa/TGFβ iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.

Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.

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...

Background Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft versus host disease (GVHD). We previously completed a “first-in-human” clinical trial using in vitro expanded umbilical cord blood (UCB) derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. Methods To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPC) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. Results aAPC increased UCB tTreg expansion >8-fold over CD3/28 stimulation. Re-stimulation with aAPC increased UCB tTreg expansion an additional 20–30 fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following cGMP validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared to that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. Discussion Expansion culture with cGMP aAPC and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, since HLA matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.