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 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.

Fig. 1. Re-stimulation greatly increases PB nTreg expansion, and cell-based aAPCs are more effective than bead-based aAPCs

nTregs were purified from peripheral blood leukapheresis products and expanded using GMP anti-CD3/CD28 mAb-coated beads or an anti-CD3 mAb-loaded cell line (KT64/86). (A) GMP purification schema. (B) Schema showing time course of experiment and ranges for size-based re-stimulation (R0=no re-stimulation, R1= one re-stimulation, etc.). Fold nTreg expansion (average ± SEM); Total (C) or following each stimulation (D). (E) Percentage of cultured cells (CD4-gated) that are CD127⁻Foxp3⁺ after each stimulation. (F) Percent suppression of in vitro, anti-CD3-mediated CD8 T cell proliferation at 1:4 (nTreg:PBMNC) as determined by CFSE dye dilution. (G) nTregs from each stimulation were re-stimulated with PMA and ionomycin for 4 hours in the presence of brefeldin A, and the percentage of cells secreting IL-2 or IFNγ was determined by flow cytometry. (H) Bead-purified PB nTregs re-stimulated 3 or 4 times (black and gray symbols, respectively) with anti-CD3 mAb-loaded KT 64/86 cells were harvested and genomic DNA was purified. Foxp3 TSDR demethylation status was assessed using bisulfite sequencing and is compared to nTreg purity (percentage of CD4+ cells that are CD127-Foxp3+) or percent suppression at a 1:4 ratio of nTregs:PBMNCs. Averages are for three independent experiments, individual symbols in (E) and (F) represent independent experiments. Individual brackets indicate the range of days for each stimulus. * represents p<0.05.

Fig. 2. Sort-purified nTregs maintain Foxp3 and suppressive function after multiple stimulations

PB nTreg were sort-purified (CD4⁺25^hi127⁻) and expanded with anti-CD3 mab-loaded KT64/86 in the presence or absence of rapamycin using 4 or 2 re-stimulations, respectively. (A) Schema showing time course of experiment and time ranges for size-based re-stimulation (R0=no re-stimulation, R1= one re-stimulation, etc.), individual brackets indicate the range of days for each stimulus. (B) Average cell size (± SEM) over time for PB nTreg cultures re-stimulated ± rapamycin. Representative examples (C) and average (D) expansion of nTregs ± rapamycin, respectively. Arrows in (C) on days 25 and 55 mark two distinct phases (plateau and growth phase) seen after first re-stimulation of nTreg cultures grown without rapamycin. Average percent CD127-Foxp3+(CD4-gated) or percent suppression of in vitro T cell proliferation at a 1:4 ratio of nTregs:PBMNCs for cultures expanded in the absence (E and F) or presence (G and H) of rapamycin, respectively. Bars represent average; other symbols represent individual experiments.

Fig. 3. nTreg cultures re-stimulated with KT64/86 cells in the presence of rapamycin do not secrete IL-2 or effector cytokines

PB nTregs were sort-purified and expanded with multiple rounds of stimulation with anti-CD3 mAb-loaded KT 64/86 cells in the presence or absence of rapamycin. The R1 without rapamycin sample corresponds to the d25 time point with high Foxp3 staining. (A) Representative example of cytokine production by Foxp3+ and - cells (CD4−gated). Average (± SEM) % of cells secreting IL-2 (B), IFNγ (C), IL-4 (D), or IL-17 (E). Averages are for three independent experiments.

Fig. 4. PB nTregs expanded over 50 million fold can still ameliorate disease in a xenogeneic model of GVHD, even after freezing and thawing

(A) Summary of purity (percentage of CD4+ cells that are CD127-Foxp3+) and in vitro suppressive function for in vitro expanded nTregs or CD4+25− cells (grown ± TGFß) after a single stimulation with KT64/86 cells. (B) Kaplan-Meier survival curve comparing NOD/Scid/γc^−/− mice receiving human PBMNCs only or co-transferred with nTregs, CD4+25− cells, or CD4+25− cells expanded in TGFß co-transferred at 1:1 (e.g. 30×10⁶ PBMNCs and 30×10⁶ nTregs) (C) Summary of fold expansion, purity (percentage of CD4+ cells that are CD127-Foxp3+), and in vitro suppressive function for nTreg expanded with 4 re-stimulations (R4). (D) Kaplan-Meyer survival curve comparing NOD/Scid/γ^−/− mice receiving human PBMNCs ± fresh nTreg re-stimulated 4 times (R4) co-transferred at 1:1. (E) Average weight (percentage of initial) for mice surviving on a given day for different groups of mice. (*) p ≤ 0.05 for fresh nTregs from days 14-21. (F) Summary of fold expansion, purity (percentage of CD4+ cells that are CD127-Foxp3+), and in vitro suppressive function for expanded nTregs re-stimulated 3 or 4 times (R3 and R4, respectively). (G) Kaplan-Meyer survival curve showing survival of mice receiving human PBMNC ± cryopreserved and thawed R3 or R4 nTregs (HLA-A2⁺) co-transferred at 1:2 (i.e. 15×10⁶ nTregs and 30×10⁶ PBMNCs). n= 10, 8, and 7 for groups PBMNC, R3 nTregs, and R4 nTregs, respectively. (H) Average number (±SEM) of human CD4⁺HLA-A2⁻, CD8⁺HLA-A2⁻ or Total CD4/8A2− cells per μl blood on day 30 for animals in (G).