Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement
The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.
Retinoic Acid Accelerates the Maturation of Human CD4Foxp3 Regulatory T Cells Induced Ex-Vivo with IL-2 and TGF-
Lu, Ling, Wang, Julie, Zheng, Song Guo, Horwitz, David A.
Mouse CD4+ cells activated for 1 week ex-vivo with IL-2 and TGF-b are induced to become Foxp3+CD25+ regulatory T cells (iTregs) that are protective in models of SLE. Similar treatment of human CD4+ cells for one week, however, results in only partially differentiated polyclonal iTregs. Repeated re-stimulation was needed for them to become anergic, express membrane-bound TGF-b, and develop suppressive activity. Since retinoic acid (RA) enhances TGF-b induced iTregs in the intestinal immune system, we reasoned that RA might be able to accelerate the differentiation of human CD4 Foxp3+ iTregs.
Naïve human CD4+ cells prepared by negative selection were suboptimally TCR activated for 6 days with anti-CD3/28 coated beads with IL-2, ± TGF-b1 and all-trans retinoic acid (atRA). The cells were then examined by FACS for surface markers characteristic of natural Foxp3+ Tregs, for intracellular cytokine production, for anergy, and for suppressive activity in vitro and in vivo.
After 6 days of TCR stimulation with IL-2 and TGF-b >50% of CD4+ cells expressed Foxp3, but these cells proliferated robustly upon further stimulation, produced large amounts of IL-2, IFN-g, and TNF-a, and lacked the ability to markedly suppress the proliferation of CFSE-labeled T cells. The addition of atRA to IL-2 and TGF-b increased Foxp3+ cells to 85%, and enhanced DR and membrane-bound TGF-b expression. Moreover, production of IL-2 and IFN-g was markedly reduced, as was their proliferative response to restimulation. Remarkably, these cells had strong in vitro suppressive activity at a dilution of 1 to 32 and this effect was completely TGF-b dependent. To assess in vivo suppressive activity, sublethally irradiated NOD SCID IL-2R g chain-/- mice were injected IV with human PBMC and additional naïve CD4+ cells. These mice spontaneously produced large amounts of human IgG after 1 week, and succumbed to a xenogenic graft-versus-host disease by two weeks. Substitution of CD4+ cells conditioned with IL-2 and TGF-b instead of naïve CD4+ cells offered minimal protection. However, the addition of CD4+ cells conditioned with IL-2, TGF-b and atRA to PBMC resulted in complete suppression of human IgG production, and significantly enhanced the survival of these mice (p<0.01).
These studies suggest that large numbers of potentially therapeutic Foxp3+ iTreg cells can be produced rapidly and expediently from naive T cells of patients with SLE and other chronic immune-mediated diseases. Following transfer back to the donor, the effects of TGF-b should enable these iTreg cells to home to both lymphoid tissues and inflammatory sites and retain their ability to expand. Studies will also be presented to indicate whether the addition of RA to IL-2 and TGF-b has also conferred resistance to proinflammatory cytokines that decrease Treg suppressive effects, and promote their conversion to effector T cells. Studies are also in progress to learn whether the addition of other epigenetic agents to RA can improve even further the generation of Foxp3+ iTregs ex-vivo.
To cite this abstract, please use the following information:
Lu, Ling, Wang, Julie, Zheng, Song Guo, Horwitz, David A.; Retinoic Acid Accelerates the Maturation of Human CD4Foxp3 Regulatory T Cells Induced Ex-Vivo with IL-2 and TGF- [abstract]. Arthritis Rheum 2009;60 Suppl 10 :1076