fig4: IL-10–producing T cells derived in VitD3/Dex prevent the induction of EAE: cells (1–3 × 106 cells per mouse) activated in the presence of neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs as described in Fig. 2 with a combination of VitD3/Dex, in the presence of VitD3 or Dex alone, or as controls under neutral, Th1, or Th2 conditions were injected into BALB/c (A) or CSJLF1/J mice (B and C) as indicated on the plots, 24 h after intracranial injection of OVA/Alum. EAE was then induced as described (thin line, no cells: thick line, cells developed in vitro as described). The experiments each were performed using 5–10 mice per group and all mice included in the groups were used in the analysis. One representative experiment of five is shown (so 40–50 mice per group in total were analyzed in the BALB/c, 25–35 in the CSJLF1/J mice) although the total number of experiments is presented in Table I. (C) Sections of spinal cord were stained with hematoxylin and eosin for light microscopy as described (reference 45). Arrows indicate the white matter of the spinal cord. Original magnification was 100×.

Mentions: CD4+ T cells producing primarily IL-10 would be predictably potent inhibitors of inflammatory responses. However, before they could be considered regulatory T cells, it was necessary to prove this directly in vivo. To demonstrate the regulatory capacity of the Vit/Dex induced IL-10–producing T cells to inhibit inflammatory pathologies, OVA-specific DO11.10 T cells were transferred into mice 3 d before the induction of EAE with MSCH. These cells included Th1 and Th2 cells as well as cells derived under neutral conditions, in the presence of VitD3 alone, or Dex alone as controls, or T cells derived using the combination Vit/Dex, all in the presence of neutralizing mAbs to IL-12, IFN-γ, and IL-4. 1 d before the cell transfer, OVA adsorbed to Alum was injected intracranially to provide a reservoir of antigen for activating the regulatory T cell population. Only cells induced with the combination of Vit/Dex were able to prevent EAE with absolute abrogation of disease onset and absence of clinical signs, which is consistent to what was observed in vitro regarding cytokine production (Fig. 4 A). The EAE incidence was 30–70% in the BALB/c mice where disease was induced in the presence of, no cells, or T cells driven under neutral, Th1, Vit alone, or Dex alone, while in the mice treated with the Vit/Dex T cell group the incidence of EAE was <15% (with only 7% of the mice reaching over grade 1 of the disease; Table I). Th2 cells conferred incomplete and marginally significant protection (Fig. 4 A and Table I). Identical results were obtained when IL-10–producing regulatory T cells were generated with Vit/Dex obtained from DO11.10 RAG−/− mice, and furthermore when derived in the absence of APCs (data not shown). In a more severe model of EAE using a genetically susceptible strain derived from BALB/c × SJL/J (CSJLF1/J mice [45]), the IL-10–producing T regulatory cells induced by Vit/Dex completely prevented death and delayed the onset of the disease (Fig. 4 B). Furthermore, although in the CSJLF1/J mice, the incidence of EAE in the mice injected with Vit/Dex-derived IL-10–producing cells was 64% compared with >90% in the no cells group, importantly, only 21% of the mice reached over grade 3 with all the mice recovering from the disease, compared with control groups where >90% of the mice reached over grade 3 with accompanying mortality (Table I). In contrast, Th2 cells that gave minor protection using the BALB/c model did not confer any protection in the CSJLF1/J mice model (data not shown). In untreated CSJLF1/J mice, intense mononuclear infiltrates in the white matter of the spinal cord correlated with the clinical symptoms of EAE. In contrast, recipients of IL-10–producing T regulatory cells derived in the combination of Vit/Dex were EAE resistant and spinal cords showed reduced inflammatory infiltrates that were restricted to the perivascular spaces of the white matter (Fig. 4 C).

Barrat FJ, Cua DJ, Boonstra A, Richards DF, Crain C, Savelkoul HF, de Waal-Malefyt R, Coffman RL, Hawrylowicz CM, O'Garra A - J. Exp. Med. (2002)

Bottom Line: Furthermore, nuclear factor (NF)-kappaB and activator protein (AP)-1 activities were inhibited in the IL-10-producing cells described here as well as key transcription factors involved in Th1 and Th2 subset differentiation.The regulatory function of these in vitro generated IL-10-producing T cells was demonstrated by their ability to prevent central nervous system inflammation, when targeted to the site of inflammation, and this function was shown to be IL-10 dependent.Generating homogeneous populations of IL-10-producing T cells in vitro will thus facilitate the use of regulatory T cells in immunotherapy.

Affiliation: DNAX Research Institute, Department of Immunology, Palo Alto, CA 94304, USA. fbarrat@dvax.com

Abstract: We show that a combination of the immunosuppressive drugs, vitamin D3 and Dexamethasone, induced human and mouse naive CD4(+) T cells to differentiate in vitro into regulatory T cells. In contrast to the previously described in vitro derived CD4(+) T cells, these cells produced only interleukin (IL)-10, but no IL-5 and interferon (IFN)-gamma, and furthermore retained strong proliferative capacity. The development of these IL-10-producing cells was enhanced by neutralization of the T helper type 1 (Th1)- and Th2-inducing cytokines IL-4, IL-12, and IFN-gamma. These immunosuppressive drugs also induced the development of IL-10-producing T cells in the absence of antigen-presenting cells, with IL-10 acting as a positive autocrine factor for these T cells. Furthermore, nuclear factor (NF)-kappaB and activator protein (AP)-1 activities were inhibited in the IL-10-producing cells described here as well as key transcription factors involved in Th1 and Th2 subset differentiation. The regulatory function of these in vitro generated IL-10-producing T cells was demonstrated by their ability to prevent central nervous system inflammation, when targeted to the site of inflammation, and this function was shown to be IL-10 dependent. Generating homogeneous populations of IL-10-producing T cells in vitro will thus facilitate the use of regulatory T cells in immunotherapy.