Figure 5.
Figure 5. Conditional depletion of recipient IL-17–producing populations exacerbates acute GVHD. (A) B6.WT or B6.iDTR-Rosa26–IL-17 mice (n = 10 per group) were lethally irradiated and received T-cell replete or TCD G-CSF mobilized BALB/c.WT grafts. Saline or DT was administered peritransplant (days −3, −1, and +1). Kaplan-Meier curves are shown with combined data from 2 replicate experiments. ***P = .0007, DT-treated WT (red filled circles) vs B6.iDTR-Rosa26-IL-17 recipients (blue filled squares). (B-C) Efficiency of depletion was determined by flow cytometry (panels Bi and Bii top, saline treated; panels Bi and Bii bottom, DT-treated). Spleen (Bi) and GI tract (Bii) were harvested from mice 2 days after SCT, and the frequency and absolute number of IL-17eYFP+ cells enumerated (n = 3 to 11 per group). Data combined from 2 replicate experiments are shown in panel C. Spleen: ****P < .0001, saline (red bars) vs DT-treated (blue bars) B6.iDTR-Rosa26–IL-17 recipients. Representative dot plots are shown in (B). (D) Representative H&E images of ileum harvested from naïve B6.iDTR-Rosa26–IL-17 mice treated with saline or DT only (no total body irradiation or SCT) or B6.iDTR-Rosa26–IL-17 mice post-alloSCT (day 2) with saline or DT administration. (E) Representative immunofluorescence images showing lysozyme expression (AF555, red) in ileum harvested on day 2 post-alloSCT from saline (top) or DT-treated (bottom) B6.iDTR-Rosa26–IL-17 recipients. DAPI nuclear stain (blue). Scale bar = 20 µm. (F) Semiquantitative histopathology of the ileum on day 2 from saline (red bar) or DT-treated (blue bar) B6.iDTR-Rosa26–IL-17 recipients (n = 8 per group). (G) Lethally irradiated B6.WT (red circles) or B6.IL-17RA−/− (blue squares) mice received 25 × 106 G-CSF immobilized BALB/c.WT grafts and the ileum harvested on day 2. Lysozyme P and Reg 3γ expression (n = 8 to 14 per group) was analyzed by RT-qPCR and normalized to WT expression. Combined data from 3 replicate experiments are shown. *P = .04, WT vs IL-17RA−/−. DAPI, 4,6, diamidino-2-phenylindole; H&E, hematoxylin and eosin; RT-qPCR, real-time quantitative polymerase chain reaction.

Conditional depletion of recipient IL-17–producing populations exacerbates acute GVHD. (A) B6.WT or B6.iDTR-Rosa26–IL-17 mice (n = 10 per group) were lethally irradiated and received T-cell replete or TCD G-CSF mobilized BALB/c.WT grafts. Saline or DT was administered peritransplant (days −3, −1, and +1). Kaplan-Meier curves are shown with combined data from 2 replicate experiments. ***P = .0007, DT-treated WT (red filled circles) vs B6.iDTR-Rosa26-IL-17 recipients (blue filled squares). (B-C) Efficiency of depletion was determined by flow cytometry (panels Bi and Bii top, saline treated; panels Bi and Bii bottom, DT-treated). Spleen (Bi) and GI tract (Bii) were harvested from mice 2 days after SCT, and the frequency and absolute number of IL-17eYFP+ cells enumerated (n = 3 to 11 per group). Data combined from 2 replicate experiments are shown in panel C. Spleen: ****P < .0001, saline (red bars) vs DT-treated (blue bars) B6.iDTR-Rosa26–IL-17 recipients. Representative dot plots are shown in (B). (D) Representative H&E images of ileum harvested from naïve B6.iDTR-Rosa26–IL-17 mice treated with saline or DT only (no total body irradiation or SCT) or B6.iDTR-Rosa26–IL-17 mice post-alloSCT (day 2) with saline or DT administration. (E) Representative immunofluorescence images showing lysozyme expression (AF555, red) in ileum harvested on day 2 post-alloSCT from saline (top) or DT-treated (bottom) B6.iDTR-Rosa26–IL-17 recipients. DAPI nuclear stain (blue). Scale bar = 20 µm. (F) Semiquantitative histopathology of the ileum on day 2 from saline (red bar) or DT-treated (blue bar) B6.iDTR-Rosa26–IL-17 recipients (n = 8 per group). (G) Lethally irradiated B6.WT (red circles) or B6.IL-17RA−/− (blue squares) mice received 25 × 106 G-CSF immobilized BALB/c.WT grafts and the ileum harvested on day 2. Lysozyme P and Reg 3γ expression (n = 8 to 14 per group) was analyzed by RT-qPCR and normalized to WT expression. Combined data from 3 replicate experiments are shown. *P = .04, WT vs IL-17RA−/−. DAPI, 4,6, diamidino-2-phenylindole; H&E, hematoxylin and eosin; RT-qPCR, real-time quantitative polymerase chain reaction.

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