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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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SMCs fail to induce OT-II CD4+ T cell proliferation. Representative plots and relative statistical analysis showing the effect of SMCs on OT-II CD4+ T cell proliferation. IFN-γ-stimulated SMCs were treated with OVA (1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with CFSE-labeled OT-II CD4+ T cells for 72 h. OVA-treated DCs were used as a positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. **P < 0.01 versus OT-II CD4+ T cells alone.
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fig2: SMCs fail to induce OT-II CD4+ T cell proliferation. Representative plots and relative statistical analysis showing the effect of SMCs on OT-II CD4+ T cell proliferation. IFN-γ-stimulated SMCs were treated with OVA (1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with CFSE-labeled OT-II CD4+ T cells for 72 h. OVA-treated DCs were used as a positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. **P < 0.01 versus OT-II CD4+ T cells alone.

Mentions: We next assessed the ability of SMCs to activate OVA-specific transgenic CD4+ T cells. In preliminary experiments by using FITC-OVA we confirmed the uptake of the model antigen by SMCs (data not shown). Using CFSE to track proliferation, we evaluated the number of Tg T cells undergoing proliferation after 72 h of coculture with SMCs or bone marrow derived DCs, used as positive control. The proportion of dividing T cells (expressed as percentage of CFSE− CD4+ cells) was approximately 0.5–1% in both presence and absence of cocultured unstimulated SMCs (Figure 2). Neither stimulation with IFN-γ nor treatment with OVA or OVA323–339 peptide of SMCs affected the proliferation of OT-II CD4+ T cells. In contrast, coculture with OVA-treated DCs significantly (P < 0.01) increased the proportion of dividing OT-II CD4+T cells by around 20% (Figure 2).


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)

SMCs fail to induce OT-II CD4+ T cell proliferation. Representative plots and relative statistical analysis showing the effect of SMCs on OT-II CD4+ T cell proliferation. IFN-γ-stimulated SMCs were treated with OVA (1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with CFSE-labeled OT-II CD4+ T cells for 72 h. OVA-treated DCs were used as a positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. **P < 0.01 versus OT-II CD4+ T cells alone.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: SMCs fail to induce OT-II CD4+ T cell proliferation. Representative plots and relative statistical analysis showing the effect of SMCs on OT-II CD4+ T cell proliferation. IFN-γ-stimulated SMCs were treated with OVA (1 mg/mL) or OVA323–339 peptide (0.5 µg/mL) overnight and then cocultured with CFSE-labeled OT-II CD4+ T cells for 72 h. OVA-treated DCs were used as a positive control. Results are expressed as mean ± SEM from three separate experiments run in triplicate. **P < 0.01 versus OT-II CD4+ T cells alone.
Mentions: We next assessed the ability of SMCs to activate OVA-specific transgenic CD4+ T cells. In preliminary experiments by using FITC-OVA we confirmed the uptake of the model antigen by SMCs (data not shown). Using CFSE to track proliferation, we evaluated the number of Tg T cells undergoing proliferation after 72 h of coculture with SMCs or bone marrow derived DCs, used as positive control. The proportion of dividing T cells (expressed as percentage of CFSE− CD4+ cells) was approximately 0.5–1% in both presence and absence of cocultured unstimulated SMCs (Figure 2). Neither stimulation with IFN-γ nor treatment with OVA or OVA323–339 peptide of SMCs affected the proliferation of OT-II CD4+ T cells. In contrast, coculture with OVA-treated DCs significantly (P < 0.01) increased the proportion of dividing OT-II CD4+T cells by around 20% (Figure 2).

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