Effect of OPN on migration of donor-derived CD8⁺ T cells. C3H.SW CD8⁺ T cells (CD45.2⁺CD8⁺) were injected into lethally irradiated B6/SJL recipient mice (CD45.1⁺) accompanied by C3H.SW TCD BM (CD45.2⁺) to induce GVHD. The alloreactive donor-derived CD8⁺ T cells (CD45.2⁺CD8⁺) were recovered from the spleens and lymph nodes of the GVHD recipients at day 14 after transplantation. (A) Mobility of naive donor CD8⁺ T cells and alloreactive donor CD8⁺ T cells in response to OPN protein were analyzed in the absence or presence of anti-OPN Ab. Percentages of migrated CD8⁺ T cells were determined. (B) The alloreactive donor CD8⁺ T cells (2 × 10⁶) were adoptively transferred into lethally irradiated secondary B6/SJL recipient mice accompanied by C3H.SW TCD BM (5 × 10⁶). Survival and weight loss of the secondary B6/SJL recipients were monitored (n = 12). Symbols are as follows: (▴), TCD BM alone; and (□), TCD BM plus alloreactive CD8⁺ T cells. (C) The adoptive transferred mice were treated with anti-OPN Ab or control IgG (200 μg/mouse) and were killed 24 hours after adoptive transfer. Absolute numbers and percentages of donor-derived CD8⁺ T cells (CD45.2⁺CD8⁺) in their livers and intestines were determined. (D) mRNAs of chemokines and adhesion molecules in livers and intestines of GVHD recipients treated with anti-OPN Ab or control IgG were measured by quantitative real-time PCR at day 7 after transplantation. Results were normalized to the gene expression in naive B6/SJL mice. (A,C-D) Data are mean ± SEM of 6 mice for each group, respectively. (E) At day 14 after transplantation, donor-derived CD8⁺ T cells (CD45.2⁺CD8⁺) were recovered from GVHD recipients treated with anti-OPN Ab or control IgG, and the levels of CXCR3 and CCR5 molecules on these T cells were measured with flow cytometry. Gray shadow and solid line indicated the immunofluorescence intensity of cells as a control and the test of Abs, respectively. The results are representative of 3 independent experiments. *P < .05.