Sequential Monitoring and Stability of Ex Vivo–Expanded Autologous and Nonautologous Regulatory T Cells Following Infusion in Nonhuman Primates

Abstract

Ex vivo-expanded cynomolgus monkey CD4+CD25+CD127− regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-Specific Demethylation Region (TSDR), and potently suppressed T cell proliferation through 3 rounds of expansion. When CFSE- or VPD450-labeled autologous (auto) and non-autologous (non-auto) expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 post-infusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively-transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these non-transplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of non-human primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells

“…This offers several advantages, including advanced preparation of therapeutic cells, and could evolve the clinical feasibility of Treg-based cellular therapy. A potential limitation of the use of third-party Tregs for cellular therapy is their shorter survival time compared to autologous or donor Tregs after adoptive transfer (60,61). Further studies would be necessary to compare the survival of allogeneic thymic Tregs with that of autologous Tregs.…”

Monocytic myeloid-derived suppressor cells generated from rhesus macaque bone marrow enrich for regulatory T cells

“…After 4 d, the cells were harvested and surfaced stained with cross-reactive anti-human mAbs: CD3, CD4, CD25 and CD127, as detailed in Table 1. After washing, the cells were stained intracellularly for forkhead box p3 (Foxp3), as described [18]. …”

Adoptive Cell Therapy with Tregs to Improve Transplant Outcomes: the Promise and the Stumbling Blocks

“…Also, the ability of Treg to suppress alloreactive effector T cell proliferation [87, 83] and their pharmacokinetics in blood and trafficking to host lymphoid tissue following adoptive transfer after lymphodepletion [88, 89] have been studied. In one study by Singh et al, rhesus macaque autologous Treg were eliminated from the peripheral blood in two phases where their half-life was 32.4 ± 11.3 hours in the first phase and 120.4 ± 19.7 hours in the second phase.…”