Figure 1.
S100a9-overexpressing T cells separate GVHD from GVL, increasing T-cell retention in the spleen. (A) WT or Ifngr1−/− CD4+CD25− T cells obtained from C57BL/6 were cocultured with irradiated (2000 rad) whole splenocytes obtained from Balb/c as alloantigen stimulators. After 6 days of coculture, total RNA was purified from CD4+CD25+ cells sorted by flow cytometry. RNA profiling analyses were performed using the Mouse Genome 430 2.0 array. Shown are up- (blue dots) or down-(red dots) regulated genes in Ifngr1−/− T cells. (B) Expression of S100a9 mRNA and S100A9 protein was determined using real-time PCR and western blotting, respectively. (C) Allo-HSCT was performed as follows; 5 × 106 TCD-BM (CD45.1+ WT) and 5 × 105 T cells (CD45.2+ WT, Ifngr1−/−, or S100a9-Tg) obtained from B6 mice were transplanted on day 0 into lethally irradiated (900 cGy on day −1) Balb/c allogeneic recipient mice. The TCD-BM only group serves as no GVHD control. The mice were monitored for survival. A pool of 3 independent experiments is shown. (D) In vivo BLI was performed to track T cells after allo-HSCT. The representative BLI images are obtained from dissected mice transplanted with WT (upper) and S100a9-overexpressing T cells (lower) on day 11 after allo-HSCT. Photon flux (photons/s) was measured from the spleen, GI tract, and the rest of the whole body. The ratio of signal intensities (photons/s per cm2 per sr) from the spleen and the rest of the body were compared (right panel). (E) The percent donor T cells in the spleens of recipient mice transplanted with WT or S100a9-overexpressing T cells was measured on day 21 after allo-HSCT. The donor T cells were determined by H2-Kd− and CD45.2+. (F-H) Allo-HSCT was performed as follows; luciferase/RFP-expressing A20 leukemia cells (1 × 105) were injected on the day of TCD-BM cell infusion (day 0), followed by delayed donor lymphocyte infusion (DLI; 2 × 106 T cells) on day 11. The leukemia burden was measured weekly using BLI. A pool of 2 independent experiments. (F) Survival rate and (G) leukemia burden. Representative images of each group from days 11 to 31 are shown. (H) Representative images of the small intestine of each group on day 21 after allo-HSCT. (i, TCD-BM only; ii, TCD-BM+A20+WT T cells; and iii, TCD-BM+A20+S100a9-overexpressing T cells). TCD-BM only group has no significant lesions. The arrows indicate crypt apoptosis, luminal debris, and crypt dropout. The scale bar represents 100 μm. Overall histological grades on day 21 after allo-HSCT (right panel). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. All error bars are presented as mean ± standard deviation. BLI, bioluminescence imaging; GI, gastrointestinal; mRNA, messenger RNA; PCR, polymerase chain reaction; TCD, T-cell depleted.

S100a9-overexpressing T cells separate GVHD from GVL, increasing T-cell retention in the spleen. (A) WT or Ifngr1−/− CD4+CD25− T cells obtained from C57BL/6 were cocultured with irradiated (2000 rad) whole splenocytes obtained from Balb/c as alloantigen stimulators. After 6 days of coculture, total RNA was purified from CD4+CD25+ cells sorted by flow cytometry. RNA profiling analyses were performed using the Mouse Genome 430 2.0 array. Shown are up- (blue dots) or down-(red dots) regulated genes in Ifngr1−/− T cells. (B) Expression of S100a9 mRNA and S100A9 protein was determined using real-time PCR and western blotting, respectively. (C) Allo-HSCT was performed as follows; 5 × 106 TCD-BM (CD45.1+ WT) and 5 × 105 T cells (CD45.2+ WT, Ifngr1−/−, or S100a9-Tg) obtained from B6 mice were transplanted on day 0 into lethally irradiated (900 cGy on day −1) Balb/c allogeneic recipient mice. The TCD-BM only group serves as no GVHD control. The mice were monitored for survival. A pool of 3 independent experiments is shown. (D) In vivo BLI was performed to track T cells after allo-HSCT. The representative BLI images are obtained from dissected mice transplanted with WT (upper) and S100a9-overexpressing T cells (lower) on day 11 after allo-HSCT. Photon flux (photons/s) was measured from the spleen, GI tract, and the rest of the whole body. The ratio of signal intensities (photons/s per cm2 per sr) from the spleen and the rest of the body were compared (right panel). (E) The percent donor T cells in the spleens of recipient mice transplanted with WT or S100a9-overexpressing T cells was measured on day 21 after allo-HSCT. The donor T cells were determined by H2-Kd− and CD45.2+. (F-H) Allo-HSCT was performed as follows; luciferase/RFP-expressing A20 leukemia cells (1 × 105) were injected on the day of TCD-BM cell infusion (day 0), followed by delayed donor lymphocyte infusion (DLI; 2 × 106 T cells) on day 11. The leukemia burden was measured weekly using BLI. A pool of 2 independent experiments. (F) Survival rate and (G) leukemia burden. Representative images of each group from days 11 to 31 are shown. (H) Representative images of the small intestine of each group on day 21 after allo-HSCT. (i, TCD-BM only; ii, TCD-BM+A20+WT T cells; and iii, TCD-BM+A20+S100a9-overexpressing T cells). TCD-BM only group has no significant lesions. The arrows indicate crypt apoptosis, luminal debris, and crypt dropout. The scale bar represents 100 μm. Overall histological grades on day 21 after allo-HSCT (right panel). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. All error bars are presented as mean ± standard deviation. BLI, bioluminescence imaging; GI, gastrointestinal; mRNA, messenger RNA; PCR, polymerase chain reaction; TCD, T-cell depleted.

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