Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion.

BACKGROUND AIMS: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. RESULTS: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. DISCUSSION: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.

Author Correction: Human in vitro-induced regulatory T cells display Dlgh1 dependent and PKC-θ restrained suppressive activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34⁺ hematopoietic stem cells and peripheral blood T-cells. CD34⁺ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

Human in vitro-induced regulatory T cells display Dlgh1dependent and PKC-θ restrained suppressive activity.

In vitro induced human regulatory T cells (iTregs) have demonstrated in vivo therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human iTregs generated in vitro from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4+CD25- effector T cells (Teff). Comparison with expanded cord blood-derived CD4+CD25hi tTreg and expanded Teffs from the same donors indicate that iTreg are intermediate between expanded CD4+CD25hi tTregs and Teffs, whereas modulation of suppressive activities by PKC-θ and Dlgh1 signaling pathways are shared.

Optimization of cGMP purification and expansion of umbilical cord blood-derived T-regulatory cells in support of first-in-human clinical trials.

BACKGROUND AIMS: 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 (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. RESULTS: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with 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 aAPCs 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, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases.

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.

Massive ex vivo expansion of human natural regulatory T cells (T(regs)) with minimal loss of in vivo functional activity.

Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4(+)CD25(+) regulatory T cells (nT(regs)) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nT(regs) has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nT(regs) could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nT(regs) in UCB units is limited, and average yield after expansion was only 1 × 10(9) nT(regs). Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nT(regs). PB nT(regs) were purified under GMP conditions and expanded 80-fold to yield 19 × 10(9) cells using anti-CD3 antibody-loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ~3000-fold and yield to >600 × 10(9) cells while maintaining Foxp3 expression and suppressor function. nT(reg) expansion was ~50 million-fold when flow sort-purified nT(regs) were restimulated four times with aAPCs. Indeed, cryopreserved donor nT(regs) restimulated 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 nT(regs) could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

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.

Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)-coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor beta (TGF-beta) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xeno-geneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1BBL.