Figure 2
Figure 2. Uptake of apoptotic cells reduces NF-κB activation and costimulatory molecule expression in host DCs and diminishes MHCII uptake in donor T cells. (A) NF-κB activation in LPS-challenged BM-DCs is reduced in ECP-treated mice. Non-ECP-treated (▪) or ECP-treated mice (▲) were challenged with LPS (10 μg/mouse IV) 48 hours after ECP treatment and NF-κB activation was measured by phospho-flow staining. BM cells from unchallenged nontreated mice served as baseline control (●). MFI, Non-ECP-treated (▪) vs ECP-treated (▲), P = .04. (B) CD80 expression of DCs is reduced after ECP treatment but restored in the presence of danger signals during ECP treatment. Left, DCs in non-ECP-treated (▪) or ECP-treated (▲) mice were activated with LPS (10 μg/mouse IV) 48 hours after ECP treatment and harvested 18 hours after activation. MFI of CD80: (▪) vs (▲) in BM, P < .0001, or pLN: P = .0006. Right, Non-ECP-treated (▪) or ECP-treated (▲) mice received additional LPS (10 μg/mouse IV) at time of ECP treatment 48 hours prior to harvest. CD80 expression is restored in BM and pLNs. Unchallenged BM cells from untreated mice served as baseline control in both experiments (●). (C) DC frequency is reduced after ECP treatment but restored in the presence of additional danger signals during time of ECP treatment. Proportional contributions to cellular content of BM-DCs and pLN DCs from mice in panel B are shown. (D) Expression of costimulatory molecules in host-type DCs (H2d) is reduced at day +3 after BMT in ECP-treated mice. Histograms with MFI of CD40, CD80, and MHCII on host-type DCs (H2d) from representative animals are shown. (E) Trogocytosis is lower in ECP-treated mice. CD4+ T cells were isolated from pLN at day +5 post-BMT from untreated mice (WT), syngeneic BMT (BALB/c CD45.1→BALB/c, CD45.2; Syn) and allogeneic BMT (C57BL/6, H2b→BALB/c, H2d) with Tcon alone (Tcon) or plus ECP treatment (ECP) and analyzed for uptake of host MHCII-IAd (H2d) or donor MHCII-IAb (H2b). pLN: Tcon vs ECP, P = .001. WT represents MHCII expression on CD4+ T cells during steady state in untreated mice. (A-E) One representative experiment with at least 4 mice per group of 2 independent experiments is shown. Error bars indicate mean ± SEM and significance was assessed by 2-tailed Student t test. MFI, mean fluorescence intensity; SEM, standard error of the mean.

Uptake of apoptotic cells reduces NF-κB activation and costimulatory molecule expression in host DCs and diminishes MHCII uptake in donor T cells. (A) NF-κB activation in LPS-challenged BM-DCs is reduced in ECP-treated mice. Non-ECP-treated (▪) or ECP-treated mice (▲) were challenged with LPS (10 μg/mouse IV) 48 hours after ECP treatment and NF-κB activation was measured by phospho-flow staining. BM cells from unchallenged nontreated mice served as baseline control (●). MFI, Non-ECP-treated (▪) vs ECP-treated (▲), P = .04. (B) CD80 expression of DCs is reduced after ECP treatment but restored in the presence of danger signals during ECP treatment. Left, DCs in non-ECP-treated (▪) or ECP-treated (▲) mice were activated with LPS (10 μg/mouse IV) 48 hours after ECP treatment and harvested 18 hours after activation. MFI of CD80: (▪) vs (▲) in BM, P < .0001, or pLN: P = .0006. Right, Non-ECP-treated (▪) or ECP-treated (▲) mice received additional LPS (10 μg/mouse IV) at time of ECP treatment 48 hours prior to harvest. CD80 expression is restored in BM and pLNs. Unchallenged BM cells from untreated mice served as baseline control in both experiments (●). (C) DC frequency is reduced after ECP treatment but restored in the presence of additional danger signals during time of ECP treatment. Proportional contributions to cellular content of BM-DCs and pLN DCs from mice in panel B are shown. (D) Expression of costimulatory molecules in host-type DCs (H2d) is reduced at day +3 after BMT in ECP-treated mice. Histograms with MFI of CD40, CD80, and MHCII on host-type DCs (H2d) from representative animals are shown. (E) Trogocytosis is lower in ECP-treated mice. CD4+ T cells were isolated from pLN at day +5 post-BMT from untreated mice (WT), syngeneic BMT (BALB/c CD45.1→BALB/c, CD45.2; Syn) and allogeneic BMT (C57BL/6, H2b→BALB/c, H2d) with Tcon alone (Tcon) or plus ECP treatment (ECP) and analyzed for uptake of host MHCII-IAd (H2d) or donor MHCII-IAb (H2b). pLN: Tcon vs ECP, P = .001. WT represents MHCII expression on CD4+ T cells during steady state in untreated mice. (A-E) One representative experiment with at least 4 mice per group of 2 independent experiments is shown. Error bars indicate mean ± SEM and significance was assessed by 2-tailed Student t test. MFI, mean fluorescence intensity; SEM, standard error of the mean.

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