ATMs activate MHC II–restricted antigen-dependent CD4+ T-cell proliferation and Th1 polarization in vitro. SVCs were prepared from eWAT of lean (ND) and obese (HFD) mice and cultured
overnight. Adherent cells were pulsed with the indicated amount of OVA peptide (323–339) or full OVA protein for 2 h before
CFSE-labeled CD4+ cells from OT-II mice were added. After 5 days, CFSE dilution was determined by flow cytometry to quantify proliferating
CD4+ cells in cocultures. Cytokines were measured by ELISA. A: Representative histograms demonstrate antigen-induced proliferation of CFSE-labeled OT-II CD4+ T cells in cocultures containing OVA peptide-pulsed SVCs from eWAT of lean (ND) and obese (HFD) mice. B: Quantitation of OT-II T-cell proliferation in cultures containing ND and HFD SVCs and increasing concentrations of OVA peptide.
C: CD4+ T-cell proliferation in cultures containing OVA peptide-pulsed HFD SVCs in the presence (αMHC II) or absence (IgG) of MHC
II–neutralizing antibodies. D: OT-II CD4+ T-cell proliferation induced by FACS-purified ATMs (F4/80+ CD11b+) and non-ATMs (F4/80− CD11b−) from HFD mice pulsed with OVA peptide. E: OT-II T-cell proliferation induced by ATMs from HFD mice pulsed with whole OVA protein. F: IFN-γ, IL-4, and IL-10 production in cocultures containing ND and HFD SVCs loaded with the indicated amounts of OVA peptide.
G: IL-17 and IL-2 production in cocultures containing HFD SVCs pulsed with OVA peptide. nd = not detected. H: IFN-γ and IL-2 production in cocultures containing FACS-purified ATMs and non-ATMs from HFD mice loaded with OVA peptide
before addition of CFSE-labeled OT-II CD4+ cells. B–H: Data are means ± SEM (n = 3–4 wells) and are representative of two to three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.