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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

The expression of human CD4 mRNA in IDO expressing C8166 cells. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP, and at 60 h post transfection, the total RNA was extracted and the cDNA was synthesized as described in the Materials and Methods section. Panel A illustrate the total RNA from untreated (control), pAd-EGFP or pAd-IDOEGFP infected C8166 cells, extracted as described in methods and subjected to electrophoresis through a native 1.5% agarose gel; Panel B displays melting curves of RT-PCR product generated using GeneAmp Ster One Plus SDS software.
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f4-ijms-13-10863: The expression of human CD4 mRNA in IDO expressing C8166 cells. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP, and at 60 h post transfection, the total RNA was extracted and the cDNA was synthesized as described in the Materials and Methods section. Panel A illustrate the total RNA from untreated (control), pAd-EGFP or pAd-IDOEGFP infected C8166 cells, extracted as described in methods and subjected to electrophoresis through a native 1.5% agarose gel; Panel B displays melting curves of RT-PCR product generated using GeneAmp Ster One Plus SDS software.

Mentions: The agarose gel electrophoresis of extracted RNA from each cell group show that the 28S, 18S, 5.8S (5S) rRNA bands were clearly visible (Figure 4A). The PCR efficiencies of CD4 gene, β-actin gene and GAPDH gene were 96%, 97.5% and 98% respectively using the method of standard curves of serial dilution of cDNA and a strong linear correlation (R2 values = 0.995) was established. The melting curves in Figure 3 show the specificity of amplification for different cell groups (Figure 4B). The primer pairs for human CD4 gene were designed as described in the Materials and Methods section. There are at least five variants of CD4 mRNA and the primer pairs are common to all the variants. However, we could not perform RT-PCR using variant analysis for the CD4 molecule, because there were no specific primer sets for each variant. The CD4 expression in the transcription level was investigated by real time PCR. We found that there was no significant difference in CD4 expression in pAd-IDOEGFP infected cells compared with either non-infected, or pAd-EGFP infected cells (Table 1). These results indicated that IDO did not affect CD4 expression at the level of mRNA.


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)

The expression of human CD4 mRNA in IDO expressing C8166 cells. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP, and at 60 h post transfection, the total RNA was extracted and the cDNA was synthesized as described in the Materials and Methods section. Panel A illustrate the total RNA from untreated (control), pAd-EGFP or pAd-IDOEGFP infected C8166 cells, extracted as described in methods and subjected to electrophoresis through a native 1.5% agarose gel; Panel B displays melting curves of RT-PCR product generated using GeneAmp Ster One Plus SDS software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijms-13-10863: The expression of human CD4 mRNA in IDO expressing C8166 cells. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP, and at 60 h post transfection, the total RNA was extracted and the cDNA was synthesized as described in the Materials and Methods section. Panel A illustrate the total RNA from untreated (control), pAd-EGFP or pAd-IDOEGFP infected C8166 cells, extracted as described in methods and subjected to electrophoresis through a native 1.5% agarose gel; Panel B displays melting curves of RT-PCR product generated using GeneAmp Ster One Plus SDS software.
Mentions: The agarose gel electrophoresis of extracted RNA from each cell group show that the 28S, 18S, 5.8S (5S) rRNA bands were clearly visible (Figure 4A). The PCR efficiencies of CD4 gene, β-actin gene and GAPDH gene were 96%, 97.5% and 98% respectively using the method of standard curves of serial dilution of cDNA and a strong linear correlation (R2 values = 0.995) was established. The melting curves in Figure 3 show the specificity of amplification for different cell groups (Figure 4B). The primer pairs for human CD4 gene were designed as described in the Materials and Methods section. There are at least five variants of CD4 mRNA and the primer pairs are common to all the variants. However, we could not perform RT-PCR using variant analysis for the CD4 molecule, because there were no specific primer sets for each variant. The CD4 expression in the transcription level was investigated by real time PCR. We found that there was no significant difference in CD4 expression in pAd-IDOEGFP infected cells compared with either non-infected, or pAd-EGFP infected cells (Table 1). These results indicated that IDO did not affect CD4 expression at the level of mRNA.

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