Recombinant S100A9 proteins or upregulation of S100A9 by treatment of anti-IFNGRα antibodies reduce GVHD by altering donor cell trafficking. (A) Survival rate of mice treated with recombinant murine S100A9 with or without S100A8 proteins (1 μg/injection starting on day 0, 5 times per week for 2 weeks), after allo-HSCT. (B) T cells obtained from C57BL/6 were cocultured with irradiated (2000 rad) whole splenocytes obtained from Balb/c in the presence of αmGR-Nab (10 μg/mL) or isotype control (10 μg/mL). After 6 days of coculture, mRNA was isolated and the expression of S100a9 was determined by real-time PCR. (C) Human PBMCs were stimulated with a CD3/CD28 activator and LPS (100 ng/mL) in the presence of αhGR-Nab (10 μg/mL) or isotype control (10 μg/mL). After 4 days, CD4 and CD8 T cells were sorted by flow cytometry and mRNA was extracted from them. The expression of S100A9 was determined by real-time PCR. (D-F) Xenogeneic cell transplantation was performed as follows. 5 × 10⁶ human PBMCs were transplanted on day 0 into sublethally irradiated (250 cGy at day −1) NSG recipient mice. αhGR-Nab (200 μg/injection) was administered on days 0, 3, 7, and 10. (D) On day 14 after xenogeneic cell transplantation, intestines were harvested, and immunofluorescence staining was performed with anti-human S100A9 (red) and CD3 (green). DAPI was used for counterstaining (blue). (E) The absolute numbers of S100A9⁺CD3⁺ T cells in mm² of the small intestines are shown. (F) Survival rate after xenogeneic cell transplantation. Shown is a pool of 2 independent experiments. The scale bar represents 50 μm. ∗P < .05 and ∗∗∗P < .001. The error bars for panels B-C and F are represent the mean ± standard deviation and mean ± SEM, respectively. mRNA, messenger RNA; PCR, polymerase chain reaction; SEM, standard error of the mean.