Limits...
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.

Show MeSH

Related in: MedlinePlus

SMCs fail to induce OT-II CD4+ T cell activation. Expression of CD69, CD25, and CD44 on OT-II CD4+ T cells cocultured with OVA- or OVA323–339 peptide-treated SMCs or OVA-treated DCs (used as positive control). Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus OT-II CD4+ T cells alone at 24 h; °°P < 0.01, °°°P < 0.001 versus OT-II CD4+ T cells alone at 48 h; ##P < 0.01 and ###P < 0.001 versus OT-II CD4+ T cells alone at 72 h.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4127268&req=5

fig3: SMCs fail to induce OT-II CD4+ T cell activation. Expression of CD69, CD25, and CD44 on OT-II CD4+ T cells cocultured with OVA- or OVA323–339 peptide-treated SMCs or OVA-treated DCs (used as positive control). Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus OT-II CD4+ T cells alone at 24 h; °°P < 0.01, °°°P < 0.001 versus OT-II CD4+ T cells alone at 48 h; ##P < 0.01 and ###P < 0.001 versus OT-II CD4+ T cells alone at 72 h.

Mentions: We also examined cell surface expression of activation markers such as CD25, CD44, and CD69 on OT-II CD4+ T cells after coculture with SMCs or bone marrow derived DCs. CD25 and CD69 were detected in approximately 2% of OT-II CD4+ T cells, alone or cocultured for 24, 48, and 72 h with unstimulated SMCs, IFN-γ-stimulated SMCs, or IFN-γ-stimulated SMCs treated with OVA or OVA323–339 peptide. Moreover, the percentage of CD25 and CD69 positive T cells did not change after SMC treatment with OVA or OVA323–339 peptide alone, while a significant (P < 0.001) increase was observed only after coculture with OVA-treated DCs at all of the time points considered (Figure 3). The percentage of CD44 positive OT-II CD4+ T cells was about 7% at all of the time points considered, in both presence and absence of unstimulated SMCs. Stimulation with IFN-γ and/or treatment of SMCs with OVA or OVA323–339 did not affect CD44 expression. A significant (P < 0.01) increase in CD44 positive OT-II CD4+ T cells was observed after 48 and 72 h of coculture with OVA-treated DCs (Figure 3). These data demonstrate that antigen-pulsed aortic murine SMCs are not able to induce antigen-specific T cell activation/proliferation.


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 activation. Expression of CD69, CD25, and CD44 on OT-II CD4+ T cells cocultured with OVA- or OVA323–339 peptide-treated SMCs or OVA-treated DCs (used as positive control). Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus OT-II CD4+ T cells alone at 24 h; °°P < 0.01, °°°P < 0.001 versus OT-II CD4+ T cells alone at 48 h; ##P < 0.01 and ###P < 0.001 versus OT-II CD4+ T cells alone at 72 h.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: SMCs fail to induce OT-II CD4+ T cell activation. Expression of CD69, CD25, and CD44 on OT-II CD4+ T cells cocultured with OVA- or OVA323–339 peptide-treated SMCs or OVA-treated DCs (used as positive control). Results are expressed as mean ± SEM from three separate experiments run in triplicate. ***P < 0.001 versus OT-II CD4+ T cells alone at 24 h; °°P < 0.01, °°°P < 0.001 versus OT-II CD4+ T cells alone at 48 h; ##P < 0.01 and ###P < 0.001 versus OT-II CD4+ T cells alone at 72 h.
Mentions: We also examined cell surface expression of activation markers such as CD25, CD44, and CD69 on OT-II CD4+ T cells after coculture with SMCs or bone marrow derived DCs. CD25 and CD69 were detected in approximately 2% of OT-II CD4+ T cells, alone or cocultured for 24, 48, and 72 h with unstimulated SMCs, IFN-γ-stimulated SMCs, or IFN-γ-stimulated SMCs treated with OVA or OVA323–339 peptide. Moreover, the percentage of CD25 and CD69 positive T cells did not change after SMC treatment with OVA or OVA323–339 peptide alone, while a significant (P < 0.001) increase was observed only after coculture with OVA-treated DCs at all of the time points considered (Figure 3). The percentage of CD44 positive OT-II CD4+ T cells was about 7% at all of the time points considered, in both presence and absence of unstimulated SMCs. Stimulation with IFN-γ and/or treatment of SMCs with OVA or OVA323–339 did not affect CD44 expression. A significant (P < 0.01) increase in CD44 positive OT-II CD4+ T cells was observed after 48 and 72 h of coculture with OVA-treated DCs (Figure 3). These data demonstrate that antigen-pulsed aortic murine SMCs are not able to induce antigen-specific T cell activation/proliferation.

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