In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines

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.

Figure 1.

VitD3/Dex enhances the development of IL-10–producing cells. Purified OVA-specific naive CD4⁺ T cells were activated using OVA_323–339 peptide and APCs in the presence of VitD3 and/or Dex, IL-10 (10 ng/ml), or in neutral (10 ng/ml of IL-2) or Th2 conditions (10 ng/ml of IL-4 and 10 μg/ml of anti–IL-12 mAb). After three rounds of stimulation, cells were characterized for cytokine production by immunoassay (A) and by intracellular flow cytometric analysis (B). Identical results were obtained when T cells were isolated from DO11.10 RAG^−/−. Representative results of more than five experiments are shown.

Figure 1.

VitD3/Dex enhances the development of IL-10–producing cells. Purified OVA-specific naive CD4⁺ T cells were activated using OVA_323–339 peptide and APCs in the presence of VitD3 and/or Dex, IL-10 (10 ng/ml), or in neutral (10 ng/ml of IL-2) or Th2 conditions (10 ng/ml of IL-4 and 10 μg/ml of anti–IL-12 mAb). After three rounds of stimulation, cells were characterized for cytokine production by immunoassay (A) and by intracellular flow cytometric analysis (B). Identical results were obtained when T cells were isolated from DO11.10 RAG^−/−. Representative results of more than five experiments are shown.

Figure 2.

VitD3/Dex induces the development of T cells producing IL-10 only and no inflammatory cytokines upon neutralization of Th1 and Th2 polarizing cytokines. (A) Purified OVA-specific naive CD4⁺ T cells were activated under the same conditions as in Fig. 1; except that neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs were also added. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained when T cells were isolated from DO11.10 RAG^−/−. Representative results of more than five experiments are shown. (B) Purified naive CD4⁺ T cells from DO11.10 RAG^−/− mice were activated using anti-CD3 and anti-CD28 stimulation under neutral or VitD3/Dex conditions. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained using DO11.10 or BALB/c mice. Representative results of three experiments are shown.

Figure 2.

VitD3/Dex induces the development of T cells producing IL-10 only and no inflammatory cytokines upon neutralization of Th1 and Th2 polarizing cytokines. (A) Purified OVA-specific naive CD4⁺ T cells were activated under the same conditions as in Fig. 1; except that neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs were also added. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained when T cells were isolated from DO11.10 RAG^−/−. Representative results of more than five experiments are shown. (B) Purified naive CD4⁺ T cells from DO11.10 RAG^−/− mice were activated using anti-CD3 and anti-CD28 stimulation under neutral or VitD3/Dex conditions. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained using DO11.10 or BALB/c mice. Representative results of three experiments are shown.

Figure 3.

VitD3/Dex induces the development of human T cells producing IL-10 and no IL-4, IL-5, or IFN-γ. Purified human CD4⁺CD45RA⁺ were stimulated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 in the presence of neutralizing anti–IL-4, anti–IFN-γ, and anti–IL-12 mAbs. After four rounds of stimulation, cells were characterized for cytokine production by immunoassay (A) as well as by intracellular flow cytometric analysis (B). Representative results of four experiments are shown.

Figure 3.

VitD3/Dex induces the development of human T cells producing IL-10 and no IL-4, IL-5, or IFN-γ. Purified human CD4⁺CD45RA⁺ were stimulated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 in the presence of neutralizing anti–IL-4, anti–IFN-γ, and anti–IL-12 mAbs. After four rounds of stimulation, cells were characterized for cytokine production by immunoassay (A) as well as by intracellular flow cytometric analysis (B). Representative results of four experiments are shown.

Figure 4.

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×.

Figure 4.

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×.

Figure 4.

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×.

Figure 5.

IL-10 is a positive autocrine factor to enhance the development of IL-10–producing T cells induced by Vit/Dex. (A) Cells were activated as in Fig. 2 in the combination of Vit/Dex, plus neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs in the absence or presence of either anti–IL-10R (10 μg/ml) or anti–TGF-β (10 μg/ml). Additionally, T cells were stimulated using anti-CD3 and anti-CD28 with Vit/Dex in the absence (B) or presence of neutralizing anti–IFN-γ and anti–IL-4 mAbs (C), in the presence or absence of anti–IL-10R mAbs. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Representative results of five experiments are shown.

Figure 5.

IL-10 is a positive autocrine factor to enhance the development of IL-10–producing T cells induced by Vit/Dex. (A) Cells were activated as in Fig. 2 in the combination of Vit/Dex, plus neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs in the absence or presence of either anti–IL-10R (10 μg/ml) or anti–TGF-β (10 μg/ml). Additionally, T cells were stimulated using anti-CD3 and anti-CD28 with Vit/Dex in the absence (B) or presence of neutralizing anti–IFN-γ and anti–IL-4 mAbs (C), in the presence or absence of anti–IL-10R mAbs. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Representative results of five experiments are shown.

Figure 5.

IL-10 is a positive autocrine factor to enhance the development of IL-10–producing T cells induced by Vit/Dex. (A) Cells were activated as in Fig. 2 in the combination of Vit/Dex, plus neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs in the absence or presence of either anti–IL-10R (10 μg/ml) or anti–TGF-β (10 μg/ml). Additionally, T cells were stimulated using anti-CD3 and anti-CD28 with Vit/Dex in the absence (B) or presence of neutralizing anti–IFN-γ and anti–IL-4 mAbs (C), in the presence or absence of anti–IL-10R mAbs. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Representative results of five experiments are shown.

Figure 6.

Prevention of EAE by IL-10–producing T cells derived in VitD3/Dex is abrogated by administration of anti-IL-10R mAbs in vivo and requires antigenic stimulation in the CNS. (A) Cells 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 were injected into BALB/c mice 24 h after intracranial injection of OVA (thin line, no cells: thick line, cells). Anti–IL-10R mAbs or isotype control (1 mg per mouse) was delivered to the mice 1 h before administration of regulatory T cells as well as once a week during the course of the experiment. (B) Alternatively, cells activated with a combination of VitD3/Dex also were injected into CSJLF1/J mice, after intracranial injection of Alum alone (No OVA, left panel); after injection of OVA, intraperitoneally (i.p.; 10 μg in Alum, middle panel) or after intracranial (i.c.) injection of OVA (right panel). As control, OVA was also injected intraperitoneally at a higher dose (1 mg in Alum) but the experiments gave similar results. 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 three is shown.

Figure 6.

Prevention of EAE by IL-10–producing T cells derived in VitD3/Dex is abrogated by administration of anti-IL-10R mAbs in vivo and requires antigenic stimulation in the CNS. (A) Cells 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 were injected into BALB/c mice 24 h after intracranial injection of OVA (thin line, no cells: thick line, cells). Anti–IL-10R mAbs or isotype control (1 mg per mouse) was delivered to the mice 1 h before administration of regulatory T cells as well as once a week during the course of the experiment. (B) Alternatively, cells activated with a combination of VitD3/Dex also were injected into CSJLF1/J mice, after intracranial injection of Alum alone (No OVA, left panel); after injection of OVA, intraperitoneally (i.p.; 10 μg in Alum, middle panel) or after intracranial (i.c.) injection of OVA (right panel). As control, OVA was also injected intraperitoneally at a higher dose (1 mg in Alum) but the experiments gave similar results. 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 three is shown.

Figure 7.

Transcription factors involved in Th1 and Th2 differentiation are downregulated in IL-10–producing T cells. (A) Expression level of T-bet, erm, GATA-3, and c-maf was assessed by Taqman in T cells developed under neutral (Neut), VitD3 alone, Dex alone, and Vit/Dex in the presence of neutralizing antibodies for IL-4, IL-12, and IFN-γ using APC/OVA stimulation as described in Fig. 2 A or anti-CD3/anti-CD28 as described in Fig. 5 C. (B) NF-κB and AP-1 activities were evaluated using 1–2 μg of nuclear extracts from cells developed under neutral conditions or Vit/Dex as described in Fig. 2 A. One representative experiment of three is shown.

Figure 7.

Transcription factors involved in Th1 and Th2 differentiation are downregulated in IL-10–producing T cells. (A) Expression level of T-bet, erm, GATA-3, and c-maf was assessed by Taqman in T cells developed under neutral (Neut), VitD3 alone, Dex alone, and Vit/Dex in the presence of neutralizing antibodies for IL-4, IL-12, and IFN-γ using APC/OVA stimulation as described in Fig. 2 A or anti-CD3/anti-CD28 as described in Fig. 5 C. (B) NF-κB and AP-1 activities were evaluated using 1–2 μg of nuclear extracts from cells developed under neutral conditions or Vit/Dex as described in Fig. 2 A. One representative experiment of three is shown.