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Indoleamine 2,3-dioxygenase (IDO) downregulates the cell surface expression of the CD4 molecule.

Huang G, Zeng Y, Liang P, Zhou C, Zhao S, Huang X, Wu L, He X - Int J Mol Sci (2012)

Bottom Line: The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP.Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells.Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Medicine Center, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China; E-Mails: zhcr1234@126.com (C.Z.); zhaosy68@126.com (S.Z.) ; Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China; E-Mails: tzengyy@jnu.edu.cn (Y.Z.); huangxiuyan@gmail.com (X.H.); thexh@jnu.edu.cn (X.H.).

ABSTRACT
Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

No MeSH data available.


Related in: MedlinePlus

IDO downregulates CD4 protein in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP for 60 h. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A) and the cell surface CD4 expression (B,C) on untreated, pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Panel A and panel B illustrate the results from two-color (PE and GFP) channel analysis, whereas panel C depicts the quantitative analysis of CD4 expressing levels in either noninfected pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Data are shown as mean ± SD, representative of three similar experiments; (D) Expression of cell surface CD4 by Western blot analysis. The plasmatic membrane proteins prepared from 6 × 105 noninfected cells or cell infected with either pAd-EGFP or pAd-IDOEGFP for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto a PVDF membrane. CD4 was detected by using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL (upper line). In parallel, the cell lysates (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. β-Actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300. ★p < 0.01, compared with Control and pAd-EGFP respectively.
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f2-ijms-13-10863: IDO downregulates CD4 protein in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP for 60 h. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A) and the cell surface CD4 expression (B,C) on untreated, pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Panel A and panel B illustrate the results from two-color (PE and GFP) channel analysis, whereas panel C depicts the quantitative analysis of CD4 expressing levels in either noninfected pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Data are shown as mean ± SD, representative of three similar experiments; (D) Expression of cell surface CD4 by Western blot analysis. The plasmatic membrane proteins prepared from 6 × 105 noninfected cells or cell infected with either pAd-EGFP or pAd-IDOEGFP for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto a PVDF membrane. CD4 was detected by using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL (upper line). In parallel, the cell lysates (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. β-Actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300. ★p < 0.01, compared with Control and pAd-EGFP respectively.

Mentions: The effect of IDOEGFP on the cell surface CD4 expression was evaluated in MT-2 cells at 60 h post infection. The CD4 expression was monitored by staining CD4 with PE-labeled antibody followed by flow cytometry analysis. As illustrated in Figure 2B,C, the expression of IDO in MT-2 cells resulted in up to 2.2-fold downregulation of cell surface CD4 compared with those of nonviral infected and mock adenoviral infected cells (mean fluorescence intensity (MFI) of 166.3 ± 1.4 vs. 373.9 ± 48.9, p < 0.01 compared with nonviral infected control for nonviral infected cells; and 166.3 ± 1.4 vs. 383.7 ± 5.3, p < 0.01 compared with mock infected control for mock adenoviral cells). To rule out the possibility that adenovirus infection and EGFP might affect CD4 expression, MT-2 cells were infected with mock recombinant adenovirus (pAd-EGFP) and the CD4 expression was analyzed. There is no significant difference in the cell surface CD4 expression between pAd-EGFP infected and non-infected MT-2 cells (MFI of 373.9 ± 48.9 vs. 383.7 ± 5.3, Figure 2B,C). These results suggested that the IDOEGFP expression itself specifically reduced the cell surface CD4 antigen in infected MT-2 cells. To further demonstrate that IDO downregulates the expression of CD4 on the cell surface, both the plasmatic membrane proteins and the cell lysate (lysed in RIPA buffer) extracted from infected or non-infected 6 × 105 MT-2 cells were loaded onto SDS-PAGE gel, followed by Western blot with purified mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL, or with rabbit polyclonal anti-β-actin antibody (1:300 dilution). The results revealed that the expression of the cell surface CD4 molecule in MT-2 cells infected with pAd-IDOEGFP was reduced when compared with that in mock cells or with cells infected with pAd-EGFP (Figure 2D, the upper panel) while similar amounts of β-actin were detected in mock, pAd-EGFP or pAd-IDOEGFP infected MT-2 cells (Figure 2D, the lower panel).


Indoleamine 2,3-dioxygenase (IDO) downregulates the cell surface expression of the CD4 molecule.

Huang G, Zeng Y, Liang P, Zhou C, Zhao S, Huang X, Wu L, He X - Int J Mol Sci (2012)

IDO downregulates CD4 protein in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP for 60 h. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A) and the cell surface CD4 expression (B,C) on untreated, pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Panel A and panel B illustrate the results from two-color (PE and GFP) channel analysis, whereas panel C depicts the quantitative analysis of CD4 expressing levels in either noninfected pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Data are shown as mean ± SD, representative of three similar experiments; (D) Expression of cell surface CD4 by Western blot analysis. The plasmatic membrane proteins prepared from 6 × 105 noninfected cells or cell infected with either pAd-EGFP or pAd-IDOEGFP for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto a PVDF membrane. CD4 was detected by using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL (upper line). In parallel, the cell lysates (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. β-Actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300. ★p < 0.01, compared with Control and pAd-EGFP respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472717&req=5

f2-ijms-13-10863: IDO downregulates CD4 protein in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP for 60 h. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A) and the cell surface CD4 expression (B,C) on untreated, pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Panel A and panel B illustrate the results from two-color (PE and GFP) channel analysis, whereas panel C depicts the quantitative analysis of CD4 expressing levels in either noninfected pAd-EGFP or pAd-IDOEGFP infected MT-2 cells. Data are shown as mean ± SD, representative of three similar experiments; (D) Expression of cell surface CD4 by Western blot analysis. The plasmatic membrane proteins prepared from 6 × 105 noninfected cells or cell infected with either pAd-EGFP or pAd-IDOEGFP for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto a PVDF membrane. CD4 was detected by using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL (upper line). In parallel, the cell lysates (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. β-Actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300. ★p < 0.01, compared with Control and pAd-EGFP respectively.
Mentions: The effect of IDOEGFP on the cell surface CD4 expression was evaluated in MT-2 cells at 60 h post infection. The CD4 expression was monitored by staining CD4 with PE-labeled antibody followed by flow cytometry analysis. As illustrated in Figure 2B,C, the expression of IDO in MT-2 cells resulted in up to 2.2-fold downregulation of cell surface CD4 compared with those of nonviral infected and mock adenoviral infected cells (mean fluorescence intensity (MFI) of 166.3 ± 1.4 vs. 373.9 ± 48.9, p < 0.01 compared with nonviral infected control for nonviral infected cells; and 166.3 ± 1.4 vs. 383.7 ± 5.3, p < 0.01 compared with mock infected control for mock adenoviral cells). To rule out the possibility that adenovirus infection and EGFP might affect CD4 expression, MT-2 cells were infected with mock recombinant adenovirus (pAd-EGFP) and the CD4 expression was analyzed. There is no significant difference in the cell surface CD4 expression between pAd-EGFP infected and non-infected MT-2 cells (MFI of 373.9 ± 48.9 vs. 383.7 ± 5.3, Figure 2B,C). These results suggested that the IDOEGFP expression itself specifically reduced the cell surface CD4 antigen in infected MT-2 cells. To further demonstrate that IDO downregulates the expression of CD4 on the cell surface, both the plasmatic membrane proteins and the cell lysate (lysed in RIPA buffer) extracted from infected or non-infected 6 × 105 MT-2 cells were loaded onto SDS-PAGE gel, followed by Western blot with purified mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL, or with rabbit polyclonal anti-β-actin antibody (1:300 dilution). The results revealed that the expression of the cell surface CD4 molecule in MT-2 cells infected with pAd-IDOEGFP was reduced when compared with that in mock cells or with cells infected with pAd-EGFP (Figure 2D, the upper panel) while similar amounts of β-actin were detected in mock, pAd-EGFP or pAd-IDOEGFP infected MT-2 cells (Figure 2D, the lower panel).

Bottom Line: The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP.Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells.Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Medicine Center, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China; E-Mails: zhcr1234@126.com (C.Z.); zhaosy68@126.com (S.Z.) ; Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China; E-Mails: tzengyy@jnu.edu.cn (Y.Z.); huangxiuyan@gmail.com (X.H.); thexh@jnu.edu.cn (X.H.).

ABSTRACT
Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

No MeSH data available.


Related in: MedlinePlus