Figure 2.

CD4⁺ T‐cell proliferation induced by dead‐cell‐associated antigens was impaired by macrophage (Mφ) depletion. (a) C57BL/6 mice received clodronate liposome (CLL) or PBS‐loaded control liposomes (PBSL) intravenously (i.v.) in a 200‐μl final volume. Splenic cells were prepared on days 1, 5 and 12 after CLL injection. The disappearance of splenic phagocyte populations and the kinetics of their repopulation were analysed by flow cytometry. (b) The spleen cryosections were stained with anti‐CD169 and anti‐MARCO antibodies specific for marginal metallophilic Mφ (MMM) and marginal zone Mφ (MZM), respectively, at each time‐point as described in (a). Scale bars, 50 μm. (c) CD45.2 mice received CLL or PBSL i.v. in a 200‐μl final volume followed by injection of CFSE‐labelled dead cells from CD45.1 mice after 5 days. One hour later, splenic cells were prepared and stained with the indicated antibodies. The amount of engulfed dead cells was assessed by FACS. CD45.2⁺ CFSE ⁺ cells were identified as cells that have taken up dead cells, and their distribution among F4/80^hi macrophages was further analysed by CD11b, CD11c and F4/80 staining. Overlays show F4/80^hi CFSE ⁺ cells (blue) from CD45.2⁺ CD11c^{neg to low} CFSE ⁺ cells and live CD11c^{neg to low} CD45.2⁺ cells (yellow) as indicated. (d) CD45.2 mice were injected i.v. with CLL or PBSL in a 200‐μl final volume. eFluor‐450 dye‐labelled CD62L⁻ Vβ5⁺ CD4⁺ CD45.1⁺ OT‐II T cells (1 × 10⁶) were transferred i.v. into CLL‐ or PBSL‐treated mice at different time‐points followed by 4 × 10⁷ ovalbumin (OVA)/cells transferred i.v. after 1 day. Three days later, the proliferation of adoptively transferred CD4⁺ T cells was detected by flow cytometry. (e) Relative proliferation ratio was further analysed using a column graph. Data are shown as the mean ± SD of three to five mice in each group and are representative of three independent experiments. Two‐tailed Student's t‐test was used for data analysis. *P < 0·05, ***P < 0·0001.