Massive ex Vivo Expansion of Human Natural Regulatory T Cells (T _regs ) with Minimal Loss of in Vivo Functional Activity

Abstract

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.

Comparison of Regulatory T Cells in Hemodialysis Patients and Healthy Controls

“…Rapamycin promotes the expansion of phenotypically and functionally stable Tregs in GMP-compatible systems while impairing the proliferation of CD4 + CD25 2 T effectors (13,32,42). Thus, in the absence of Rapamycin treatment, the elevated IL-17 production by HD Tregs may be attributed to increased contaminating Th17 cells or a greater propensity for these cells to differentiate into Th17 cells (consistent with the chronic inflammation seen in HD and/or with vitamin D insufficiency/deficiency in ESKD, because this vitamin prevents Th17 differentiation) (43).…”

“…Having established that HD patients have the same numbers of Tregs as HCs with normal expression of maturation and activation markers within subsets, albeit with slight reduction in population II, we next sought to determine whether they could be expanded in vitro under the GMP-compatible conditions (13,32) as required for a program of cell therapy.…”

“…Thus, clinical cell therapy with Tregs is a realistic possibility. Indeed, case series and phase I studies have shown beneficial outcomes in type 1 diabetes mellitus (11) and the prevention or treatment of post-bone marrow transplantation graft versus host disease in humans (9,12,13).…”

Treatment with agonistic DR3 antibody results in expansion of donor Tregs and reduced graft-versus-host disease

“…10 Ex vivo expansion of Tregs has been accomplished by treating purified cells with IL-2 combined with antibodies directed against CD3/CD28. 11,12 Overgrowth of effector T cells has been frequently observed after ex vivo expansion, and rapamycin has been used to reduce it. 13 Isolation and expansion of Tregs requires specialized facilities and can be challenging to obtain sufficient numbers of cells for clinical applications.…”

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

“…Similarly, in hepatitis B virus infection, increased numbers of CD39 + Treg cells correlate with disease progression [15]. Humanised mouse models have provided proof of principle for Treg-cell-based immunotherapies, with Ag-specific Treg cells appearing to be more efficacious than polyclonal Treg cells [16][17][18]. In order for this approach to move towards use in humans, methodology is required for the isolation and expansion of human Ag-specific Treg cells.…”