American Society of Hematology

Fig. 4.

$Fig. 4. Internalization of apoptotic cells by spleen DCs in vitro: role of serum factors. / (A) PKH67 immunobead-sorted splenic CD11c+ DCs (purity = 96%) were cocultured with PKH26 BALB/c apoptotic splenocytes under different conditions and results were analyzed by flow cytometry. After 2 hours of coculture 33% ± 14% (4 experiments) splenic DCs internalized/bound PKH26 apoptotic cells, a phenomenon blocked by cytochalasin D (Cyto D), EDTA, or at 4°C. (B-C) In 4 experiments, 31% ± 4% IAb+ splenic DCs associated with BALB/c (H2Dd+, PKH26+) apoptotic splenocytes (top right quadrant in panel B). Interestingly, a fraction of these were labeled by anti-H2Dd FITC mAb (top right quadrant in panel C). Because H2Dd is only expressed by the BALB/c apoptotic splenocytes and anti-H2Dd FITC cannot penetrate the DC membrane, this result demonstrates that some apoptotic cells are attached to the DC surface without being internalized. (D-E) The fact that PKH26+ apoptotic cells associated with CD8α− DCs were not reached by anti-H2Dd FITC suggests that they were internalized by DCs. By contrast, apoptotic cells associated with CD8α+ DCs were labeled by anti-H2Dd FITC mAb (E, top right quadrant), a fact that indicates that apoptotic cells were still attached to the DC surface instead of being internalized. Results in panels C to E were gated on IAb+ DCs of panel B (region 1 = R1 in panel B). Numbers inside quadrants indicate percentages of cells. Results are representative of 4 independent experiments. (F-H) Role of heat-labile serum factors in uptake of apoptotic cells by splenic DCs. (F-G) PKH26 splenic (B10) DCs were cocultured with PKH67 apoptotic (BALB/c) splenocytes under different conditions and uptake was assessed by 2-color flow cytometry. (H) Similar experiments were performed with in vitro–generated immunomagnetic bead-sorted immature (B10) BM DCs cocultured with apoptotic (BALB/c) cells. Uptake of apoptotic cells (percents) in the vertical axis of panels F to H represent the percentages of DCs that have captured apoptotic cells.$

Internalization of apoptotic cells by spleen DCs in vitro: role of serum factors.

(A) PKH67 immunobead-sorted splenic CD11c⁺ DCs (purity = 96%) were cocultured with PKH26 BALB/c apoptotic splenocytes under different conditions and results were analyzed by flow cytometry. After 2 hours of coculture 33% ± 14% (4 experiments) splenic DCs internalized/bound PKH26 apoptotic cells, a phenomenon blocked by cytochalasin D (Cyto D), EDTA, or at 4°C. (B-C) In 4 experiments, 31% ± 4% IA^b+ splenic DCs associated with BALB/c (H2D^d+, PKH26⁺) apoptotic splenocytes (top right quadrant in panel B). Interestingly, a fraction of these were labeled by anti-H2D^d FITC mAb (top right quadrant in panel C). Because H2D^d is only expressed by the BALB/c apoptotic splenocytes and anti-H2D^d FITC cannot penetrate the DC membrane, this result demonstrates that some apoptotic cells are attached to the DC surface without being internalized. (D-E) The fact that PKH26⁺ apoptotic cells associated with CD8α⁻ DCs were not reached by anti-H2D^d FITC suggests that they were internalized by DCs. By contrast, apoptotic cells associated with CD8α⁺ DCs were labeled by anti-H2D^d FITC mAb (E, top right quadrant), a fact that indicates that apoptotic cells were still attached to the DC surface instead of being internalized. Results in panels C to E were gated on IA^b+ DCs of panel B (region 1 = R1 in panel B). Numbers inside quadrants indicate percentages of cells. Results are representative of 4 independent experiments. (F-H) Role of heat-labile serum factors in uptake of apoptotic cells by splenic DCs. (F-G) PKH26 splenic (B10) DCs were cocultured with PKH67 apoptotic (BALB/c) splenocytes under different conditions and uptake was assessed by 2-color flow cytometry. (H) Similar experiments were performed with in vitro–generated immunomagnetic bead-sorted immature (B10) BM DCs cocultured with apoptotic (BALB/c) cells. Uptake of apoptotic cells (percents) in the vertical axis of panels F to H represent the percentages of DCs that have captured apoptotic cells.

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