Figure 3.
Figure 3. BDCA3+ DC exhibit an enhanced ability to cross present antigen delivered to lysosomes. (A) Antigen cross presentation via DEC205 by BDCA3+ DCs and BDCA1+ DCs. Day 1 DCs from HLA-A*0201 donors were fed with anti-DEC205 or control isotype antibody conjugated to Flu-M1 (aa 55–72) at the indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8+ T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8+ T cell expansion was evaluated by gating on CFSElow cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of n > 6. (B) As in A; anti-DEC205 and the control isotype antibodies were conjugated to CMV-pp65 (aa 488–508). Shown is one representative experiment of two. (C) Accumulation of anti-DEC205 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 antibody continuously for 4–6 h at 4 or 37°C. Results were analyzed by flow cytometry. 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1+ DCs. Data shown are the mean MFI ± SD (n = 7 independent experiments). (D) Internalization of anti-DEC205 antibody. Day 1 DCs were incubated with Alexa Fluor 488–labeled anti-DEC205 antibody for 30 min at 4°C. The cells were then washed and cultured at 37°C for the indicated times. At each time point, cells were labeled with an Alexa Fluor 647–labeled anti–human IgG antibody to label remaining surface bound antibody. Cells were then analyzed by flow cytometry. The total DEC205 is Alexa Fluor 488–labeled antibody, whereas the surface DEC205 is the Alexa Fluor 647–labeled anti–human antibody. Shown is one representative experiment of four. (E) Anti-DEC205 antibody trafficking. Day 1 DCs were fed Alexa Fluor 488–labeled anti-DEC205 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes and cell membrane were stained using anti-Lamp1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bars, 7.5 µm. Shown is one representative experiment of five. (F) Detection of the indicated lysosomal proteases by Western blot of day 1 BDCA1+ DCs, BDCA3+ DCs, and mo-DCs. DC subsets were lysed and analyzed by Western blot for lysosomal protease expression. Shown is one representative experiment of three.

BDCA3+ DC exhibit an enhanced ability to cross present antigen delivered to lysosomes. (A) Antigen cross presentation via DEC205 by BDCA3+ DCs and BDCA1+ DCs. Day 1 DCs from HLA-A*0201 donors were fed with anti-DEC205 or control isotype antibody conjugated to Flu-M1 (aa 55–72) at the indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8+ T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8+ T cell expansion was evaluated by gating on CFSElow cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of n > 6. (B) As in A; anti-DEC205 and the control isotype antibodies were conjugated to CMV-pp65 (aa 488–508). Shown is one representative experiment of two. (C) Accumulation of anti-DEC205 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 antibody continuously for 4–6 h at 4 or 37°C. Results were analyzed by flow cytometry. 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1+ DCs. Data shown are the mean MFI ± SD (n = 7 independent experiments). (D) Internalization of anti-DEC205 antibody. Day 1 DCs were incubated with Alexa Fluor 488–labeled anti-DEC205 antibody for 30 min at 4°C. The cells were then washed and cultured at 37°C for the indicated times. At each time point, cells were labeled with an Alexa Fluor 647–labeled anti–human IgG antibody to label remaining surface bound antibody. Cells were then analyzed by flow cytometry. The total DEC205 is Alexa Fluor 488–labeled antibody, whereas the surface DEC205 is the Alexa Fluor 647–labeled anti–human antibody. Shown is one representative experiment of four. (E) Anti-DEC205 antibody trafficking. Day 1 DCs were fed Alexa Fluor 488–labeled anti-DEC205 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes and cell membrane were stained using anti-Lamp1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bars, 7.5 µm. Shown is one representative experiment of five. (F) Detection of the indicated lysosomal proteases by Western blot of day 1 BDCA1+ DCs, BDCA3+ DCs, and mo-DCs. DC subsets were lysed and analyzed by Western blot for lysosomal protease expression. Shown is one representative experiment of three.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal