The GTPase Rab3b/3c-positive recycling vesicles are involved in cross-presentation in dendritic cells

Abstract

Antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. In this study, we examined the effect of siRNA-mediated knockdown of 57 Rab GTPases, the key regulators of membrane trafficking, on antigen cross-presentation. Twelve Rab GTPases were identified to be associated with antigen cross-presentation, and Rab3b/3c was indicated to be colocalized with MHC class I molecules at perinuclear tubular structure. Tracing with fluorescence protein-tagged beta(2)-microglobulin demonstrated that the MHC class I molecules were internalized from the plasma membrane to Rab3b/3c-positive compartments, which were also colocalized with the internalized transferrin. Moreover, depletion of Rab3b/3c strongly reduced the fast phase recycling rate of transferrin receptors. Furthermore, the Rab3b/3c-positive compartments were colocalized with a fraction of Rab27a at a juxtaposition of phagosomes. Together, these data demonstrate that Rab3b/3c-positive recycling vesicles are involved in and may constitute one of the recycling compartments in exogenous antigen cross-presentation.

Fig. 1.

Identification of the cross-presentation–associated Rab GTPases with lentivirus-based siRNA screening. (A) Scheme for identifying cross-presentation–related Rab GTPases by lentiviral-based siRNA. (B) Efficiency of lentivirus infection. DC2.4 cells were transfected with different titers of lentivirus for 24 h and subsequently cultured with puromycin (7 μg/mL) and counted after 7 days. (C) B3Z T-cell responses to different numbers of DC2.4 cells were analyzed after DC2.4 was prepulsed with 2.5 or 1.25 μL of E. coli BL21/pThio-OVA. (D) Antigen presentation ability scores of stably transfected dendritic cells. The antigen presentation ability score was the ratio of the IL-2 value of Rab siRNA stably transfected DC2.4 cells to the controls for each time of the antigen cross-presentation assay. The threshold ratio (0.8–1.2) was determined using 50 negative control data points (filled circles) from all screening processes (dash-dot line). Each open square represents 1 Rab siRNA stably transfected DC2.4 cell and is sorted in order of increasing antigen presentation ability scores. (E) B3Z response to DC2.4 cells containing siRNA against positive-screened Rab genes prepulsed with E. coli BL21/pThio-OVA at different doses.

Fig. 2.

The colocalization between MHC class I molecules and Rab3b/3c at perinuclear tubular vesicles. (A) After being stably transfected with TagRFP-Rab3b and -Rab3c and with EYFP–H2-K^b, DC2.4 cells were plated on coverslips and visualized using confocal microscopy. A stereo 3D rendering of collected confocal images shows the colocalization between Rab3b and Rab3c tubular vesicles (red) and H2-K^b molecules (green). (B) Distribution of Rab3c and EYFP–H2-K^b was analyzed by Western blotting after subcellular fractionation of EYFP–H2-K^b–expressing DC2.4 cells. (C) Colocalization between endogenous Rab3c and H2-K^b was analyzed by staining with rabbit anti-mouse Rab3c in EYFP–H2-K^b–expressing DC2.4 cells. (Scale bar: 10 μm.) These optically merged images are representative of at least 100 transduced cells examined by immunofluorescent confocal microscopy.

Fig. 3.

Transporting the internalized MHC class I molecules to Rab3b/3c-positive vesicles. (A) Stably transfected DC2.4 cells were incubated with media or 10 μg/mL BFA for 4 h. The confocal images revealed that the concentration of MHC class I molecules (green) in Rab3b/3c-positive vesicles (red) is resistant to BFA treatment. (B) Quantitative analysis of the mean fluorescence intensity of EYFP–H2-K^b in Rab3b/3c-positive vesicles with or without BFA treatment. Each symbol represents an individual Rab3b/3c-positive vesicle. Small horizontal lines indicate the mean. (C) β₂-Microglobulin–ZsGreen fusion proteins were incubated with stable transfected TagRFP-Rab3b or TagRFP-Rab3c dendritic cells for 30 min on ice, and cells were then washed and warmed to 37 °C for 1 h. The distribution of TagRFP-Rab3b/3c and β₂-microglobulin–ZsGreen fusion proteins was analyzed by fluorescence intensity measurement after subcellular fractionation. (D) Confocal images of stably transduced DC2.4 cells pulsed with β₂-microglobulin–ZsGreen fusion proteins. These optically merged images are representative of at least 100 transduced cells examined by immunofluorescent confocal microscopy. Yellow indicates colocalization of green and red. (Scale bar: 10 μm.)

Fig. 4.

Rab3c is localized at recycling endosomes and controls a rapid recycling transport step of the transferrin receptor. (A) TagRFP-Rab3c–expressing DC2.4 cells were pulsed with FITC-Tfn and viewed under confocal microscopy. Images revealed the colocalization of FITC-Tfn (green) with Rab3c-positive vesicles (red). (B, C) DC2.4 cells stably silencing Rab3b (B) or Rab3c (C) were incubated with FITC-Tfn for 1 h at 37 °C. Transferrin receptor recycling to the cell surface was measured by quantification of intracellular FITC-Tfn after different chase times. The results are expressed as percentages of initial FITC-transferrin (mean ± SEM, n = 3, >8,000 cells were analyzed by cytometry for each time point).

Fig. 5.

Rab3c was colocalized with a fraction of Rab27a at a juxtaposition of phagosomes. (A) Stably transfected DC2.4 cells were incubated with E. coli BL21/pECFP-OVA for 4 h. Coverslips were mounted on slides and detected by confocal microscopy. Stereo 3D rendering of collected confocal images shows that Rab3c tubular vesicles (red) colocalize with H2-K^b molecules (green) at a juxtaposition of phagosomes. (B) DC2.4 transfected with EYFP-Rab27a and TagRFP-Rab3c lentivirus. After incubation with E. coli BL21/pECFP-OVA, confocal images reveal that Rab3c tubular vesicles (red) colocalize with Rab27a molecules (green) at a juxtaposition of phagosomes. Enlarged images show the phagosomes and concentrated compartments (arrowheads) after engulfment. These optically merged images are representative of at least 100 transduced cells by immunofluorescent confocal microscopy. (Scale bar: 10 μm.)