Large Scale Selective Ex Vivo Expansion of CD4⁺CD25⁺FOXP3⁺ Regulatory T Cells from Peripheral Blood.

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. However, the frequencies of Treg in peripheral blood are very low. Here we attempted the ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. CD4⁺ T cells from peripheral blood of healthy donors or patients with chronic graft-versus-host disease (GVHD) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-b. After one week of culture, expanding cells were once detached from the plate and subjected to the fresh medium including rhIL-2 and rhTGF-b but not MAbs. After 2-weeks of culture, phenotypic and functional analyses were performed. Mixed lymphocyte reaction was done using CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Xenogeneic -GVHD in NOD-Scid mice was induced by the injection of human T cells expressing luciferace transgene, followed by in vivo bioluminescence imaging (BLI) analysis using a CCD camera.

Our expansion procedure with TGF-b yielded 45-83% purity of Foxp3⁺CD25⁺CD4⁺Treg co-expressing CTLA-4, CD54 and GITR, while 8-42% purity without TGF-b(p<0.001). These cell populations also displayed CD45RO⁺CD45RA⁻CD26^high+ memory phenotype. An average expansion rate of Treg was 62,200 fold (25,500–97,900) in healthy donors during the culture periods (n=5). Thus, we obtained an average of 4.7x10⁸ Treg from the initial number of 5x10⁵ CD4⁺ T cells in peripheral blood. Additionally, from peripheral CD4⁺ T cells in patients with chronic GVHD, Treg could be expanded equivalently to healthy donors. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled T cells to autologous and allogeneic DC (Figure 1). The ex vivo expanded Treg-rich populations had the inhibitory effect on xeno-reactive T cells expressing luciferase transgene in a xenogeneic GVHD model (Figure 2). Our procedure has allowed efficient ex vivo expansion of Treg-rich populations from a small volume of peripheral blood, and will be applicable to clinical use.

Figure 1.

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MLR inhibited by expanded Treg

Figure 1.

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MLR inhibited by expanded Treg

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Figure 2.

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Xenogeneic GVHD diminished by expanded Treg.

Figure 2.

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Xenogeneic GVHD diminished by expanded Treg.

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