DC process and present allopeptides derived from internalized exosomes. (A) Processing of exosomal alloAgs by BMDCs occurred within vesicles at low pH. Following culture of BMDCs (B10) with allogeneic exosomes (BALB/c), mature (CD86⁺) DCs exhibited the highest levels of IA^b-IEα_52-68 on the cell surface recognized by the Y-Ae mAb. NH₄Cl inhibited reversibly the formation of IA^b-IEα_52-68 on DCs. NH₄Cl did not reduce binding of IEα_52-68 to IA^b (not shown). Numbers indicate percentage of cells. (B) BMDCs (B10) were pulsed with graded concentrations of BALB/c exosomes or with B10 or C3H exosomes. Then, decreasing numbers of DCs were added to 10⁵ BEα20.6 T cells specific for IA^b-IEα_52-68. T-cell activation was evaluated by IL-2 secretion assessed by the HT-1 cell proliferation assay. (C) Recognition of IA^b-IEα_52-68 by T cells was inhibited with Y-Ae mAb. IEα_52-68 was used as positive control and the OVA-derived peptide SIINFEKL as irrelevant control. (D) BMDCs increased their capacity to present exosomal allopeptides to 1H3.1 TCRtg CD4⁺ T cells after activation via CD40. 1H3.1 T-cell proliferation was evaluated after 72 hours by [³H] TdR incorporation. Each condition was tested in triplicate and displayed as their means ± SD. Data are representative of 3 independent experiments.