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Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.

Maddaluno M, MacRitchie N, Grassia G, Ialenti A, Butcher JP, Garside P, Brewer JM, Maffia P - Biomed Res Int (2014)

Bottom Line: Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II.Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation.Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy ; Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK ; Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

ABSTRACT
In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323-339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

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Effect of SMCs on DO11.10-GFP hybridoma cell activation. IFN-γ-stimulated SMCs were treated with (OVA, 1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with DO11.10-GFP hybridoma cells for 24 h. OVA-treated DCs were used as positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus DO11.10-GFP hybridoma cells alone.
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fig5: Effect of SMCs on DO11.10-GFP hybridoma cell activation. IFN-γ-stimulated SMCs were treated with (OVA, 1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with DO11.10-GFP hybridoma cells for 24 h. OVA-treated DCs were used as positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus DO11.10-GFP hybridoma cells alone.

Mentions: The murine DO11.10-GFP hybridoma was originally obtained by stably transfecting a DO11.10 T cell hybridoma with a construct in which GFP expression is under the control of a nuclear factor of activated T cells (NFAT) regulated promoter [28]. Thus, once activated, hybridoma cells, detectable using the KJ1-26 clonotypic antibody, become GFP-positive. DO11.10 hybridoma cells express the TCR recognizing OVA323–339 peptide in the context of either I-Ad or I-Ab MHC class II [32] without any requirement for costimulation [29]. Coculture with unstimulated SMCs had no effect on GFP expression by DO11.10-GFP hybridoma cells and similar results were obtained after stimulation with IFN-γ and/or treatment of SMCs with OVA or OVA323–339 peptide. On the contrary, DCs treated with OVA, used as positive control, caused a significant (P < 0.001) increase in GFP expression by hybridoma cells (Figure 5). These data confirm that SMCs are unable to present exogenous protein antigens in the context of MHC class II.


Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.

Maddaluno M, MacRitchie N, Grassia G, Ialenti A, Butcher JP, Garside P, Brewer JM, Maffia P - Biomed Res Int (2014)

Effect of SMCs on DO11.10-GFP hybridoma cell activation. IFN-γ-stimulated SMCs were treated with (OVA, 1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with DO11.10-GFP hybridoma cells for 24 h. OVA-treated DCs were used as positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus DO11.10-GFP hybridoma cells alone.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127268&req=5

fig5: Effect of SMCs on DO11.10-GFP hybridoma cell activation. IFN-γ-stimulated SMCs were treated with (OVA, 1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with DO11.10-GFP hybridoma cells for 24 h. OVA-treated DCs were used as positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus DO11.10-GFP hybridoma cells alone.
Mentions: The murine DO11.10-GFP hybridoma was originally obtained by stably transfecting a DO11.10 T cell hybridoma with a construct in which GFP expression is under the control of a nuclear factor of activated T cells (NFAT) regulated promoter [28]. Thus, once activated, hybridoma cells, detectable using the KJ1-26 clonotypic antibody, become GFP-positive. DO11.10 hybridoma cells express the TCR recognizing OVA323–339 peptide in the context of either I-Ad or I-Ab MHC class II [32] without any requirement for costimulation [29]. Coculture with unstimulated SMCs had no effect on GFP expression by DO11.10-GFP hybridoma cells and similar results were obtained after stimulation with IFN-γ and/or treatment of SMCs with OVA or OVA323–339 peptide. On the contrary, DCs treated with OVA, used as positive control, caused a significant (P < 0.001) increase in GFP expression by hybridoma cells (Figure 5). These data confirm that SMCs are unable to present exogenous protein antigens in the context of MHC class II.

Bottom Line: Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II.Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation.Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy ; Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK ; Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

ABSTRACT
In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323-339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

Show MeSH
Related in: MedlinePlus