Assessment of Roles for Calreticulin in the Cross-Presentation of Soluble and Bead-Associated Antigens
Figure 2
In vitro cross-presentation of a calreticulin-fused soluble antigen.
(A) Gel-filtration chromatogram of E. coli-derived OVA or the OVA-calreticulin (OVA- CRT) fusion protein (left). SDS-PAGE analysis of pooled fractions from left panel; proteins were loaded in equimolar amounts (right) and coomassie stained. (B) Indicated proteins were incubated with BMDC for 3 hours. BMDC were fixed and CFSE labeled OT-I T cells were added. IL-2 levels in supernatants were determined by ELISA (left panel; 24 hour time point). OT-I T cell proliferation was measured at 72 hours in response to 44 µM OVA or OVA-CRT. The solid grey profile indicates the condition where no antigen (no Ag) was added. Data are representative of two independent analyses. (C, D) OVA-CRT and OVA were labeled with allophycocyanin. (C) Labeling intensity was determined by fluorescence imaging of the proteins after separation by SDS-PAGE (inset). Fluorescence intensity was quantified for the indicated proteins. (D) Binding of fluorescent proteins to BMDC was assessed by flow cytometry. BMDC were incubated with labeled proteins on ice before being analyzed by flow cytometry. BMDC not incubated with proteins are depicted as a grey filled. Representative of two independent experiments performed with the same labeled proteins. Mean ± s.e.m. are shown in B.