Comparative study of regulatory T cells expanded ex vivo from cord blood and adult peripheral blood

Abstract

In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers.

© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Figures

Figure 1

Schematic overview of expansion strategies and regulatory T cell (Treg) expansion after primary and secondary stimulation. (a) Adult peripheral blood (APB) and cord blood (CB) Tregs were expanded in either two cycles with polyclonal stimulation or two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. We therefore generated four types of expanded Tregs: APB Treg (PP-PP), APB Treg (HBP-PP), CB Treg (PP-PP) and CB Treg (HBP-PP). (b, c) APB and CB Tregs were expanded as described above. Treg numbers were determined and compared to initial Treg numbers. Data represent the average expansion ± standard deviation (SD) of six independent experiments.

Figure 2

Phenotypic characterization of freshly isolated and expanded regulatory T cells (Tregs). (a) The phenotype of freshly isolated adult peripheral blood (APB) CD4+ CD25− T cells and Tregs from APB and cord blood (CB) was analysed by flow cytometry. (b) CD4+ CD25− T cells were activated with anti-CD3/anti-CD28 microbeads and recombinant human interleukin-2 (rhIL-2) for two cycles. APB and CB Tregs were expanded according to the protocol described in Fig. 1(a). Cell surface expression of CD4/CD25, CD127, CTLA4, CD39, LAG3 and HLA-DR and intracellular expression of FoxP3 were analysed on activated CD4+ CD25− T cells and expanded Tregs. Histograms show the percentage of expression of these markers (bold line) in the gated populations compared with the appropriate isotype control (thin line). Data are representative of three independent experiments.

Figure 3

Reverse transcription-PCR analysis of GARP and cytokine gene expression in expanded regulatory T cells (Tregs) and stimulation assay to analyse T-cell anergy. Adult peripheral blood (APB) and cord blood (CB) Tregs were expanded according to the protocol described in Fig. 1(a). Total RNA was isolated, reverse-transcribed and used for quantitative analysis. (a) Gene expression of GARP, transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) of freshly isolated APB CD4+ CD25− T cells and Tregs from APB and CB was displayed as relative expression normalized to β-actin. Gene expression of GARP (b), IL-2 (c), interferon-γ (IFN-γ) (d), IL-10 (e) and TGF-β (f) of activated CD4+ CD25− T cells and expanded Tregs from APB and CB was displayed as relative expression normalized to β-actin. Values are given as the mean ± standard deviation (SD) of three to five independent experiments. **P < 0·01 compared with activated CD4+ CD25− T cells. (g) CFSE-labelled CD4+ CD25− T cells and expanded Tregs from APB and CB were stimulated with anti-CD3/anti-CD28 antibodies in the absence or presence of exogenous IL-2. Proliferation was determined by CFSE dilution on Day 4.

Figure 4

Suppressive capacity of regulatory T cells (Tregs) expanded from adult peripheral blood (APB) and cord blood (CB). The APB and CB Tregs were expanded according to the protocol described in Fig. 1(a). Progressive CFSE dilution was used as a read-out of responder cell proliferation. CFSE-labelled CD4+ CD25− responder T (Tresp) cells were stimulated with anti-CD3/anti-CD28 antibodies and recombinant human interleukin-2 (rhIL-2) or HLA mismatched dendritic cells presenting the target alloantigen (same as or different form the B cells used for Treg expansion). Third-party expanded Tregs from APB and CB were added to these cultures at the indicated Treg : Tresp ratios (1 : 1, 1 : 4, 1 : 16 and 1 : 64). Proliferation was determined by CFSE dilution of Tresp cells on Day 4. The inhibition rate was calculated as followed: (1 – percentage of proliferated Tresp with expanded Treg/percentage of proliferated Tresp with no Treg) ×100%. Data represent the average inhibition rate of three independent experiments.

Figure 5

Adoptive transfer of ex vivo expanded regulatory T cells (Tregs) from cord blood (CB) and adult peripheral blood (APB) into mice with acute graft-versus-host disease (aGVHD). CB and APB Tregs were expanded ex vivo with two cycles of polyclonal stimulation: CB Treg (PP-PP) and APB Treg (PP-PP). CB Tregs were also expanded with polyclonal stimulation in the first cycle and xenogenic mouse B-cell stimulation in the second cycle or with xenogenic mouse B-cell stimulation in the first cycle and polyclonal stimulation in the second cycle: CB Treg (PP-MBP) and CB Treg (MBP-PP). In the aGVHD mouse model, 1 × 106 of the variously expanded Tregs were adoptively transferred into lethally irradiated recipient DBA/2 (H-2Kd) mice after injection with 3 × 107 bone marrow cells and 1 × 107 spleen cells from donor C57BL/6 mice (H-2Kb). The survival (a), body weight (b) and clinical GVHD score (c) were assessed. The GVHD score and body weight were determined from the surviving mice. (d) The mononuclear cells from the liver and lymph node of aGVHD mice were isolated by density gradient centrifugation using Ficoll–Hypaque on Day 1 or Day 8 after adoptive transfer of ex vivo expanded Tregs. The human molecules of CD4, CD25 and FoxP3 were determined by flow cytometry. Data represent the average percentage of human CD4+ CD25+ T cells ± standard deviation (SD) of six independent experiments.