Calreticulin as an Adjuvant In Vivo to Promote Dendritic Cell Maturation and Enhance Antigen-Specific T Lymphocyte Responses against Melanoma

Figure 1

Expression and purification of recombinant proteins and CRT/39-272 promote DC function in vitro. (a) Affinity-purified OVAp-CRT (lane 1), OVAp (lane 2), and CRT/39-272 (lane 3) were run in three identical 10% SDS-PAGs. One gel was stained with Coomassie blue. Protein molecular mass markers (M) were loaded in the right lane. (b) Protein bands in the two unstained gels were transferred to PVDF membranes for western blotting with mouse anti-CRT monoclonal antibodies (left) and rabbit anti-OVA polyclonal antibodies (right). (c) Immature BMDCs were stimulated with OVAp or OVAp-CRT for 24 h. The generated DCs, DC-OVAp, and DC-OVAp-CRT were stained with PE-conjugated anti-CD40, anti-CD80, and anti-CD86. (d) IL-12 levels in the supernatants of cultured DCs, DC-OVAp, and DC-OVAp-CRT were analyzed by ELISA. (e and f) BMDCs were incubated with CFSE-labeled pan T cells from OT-I mice at a 1 : 10 ratio in the presence of medium, OVAp, or OVAp-CRT for 3 days. The mean fluorescence intensity (MFI) of the CFSE was analyzed (e) and fluorescence-activated cell sorting (FACS) histograms are shown (f). The results were replicated three times with a consistent trend. Data are representative of at least three independent experiments. . .

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