Donor T-cell infiltration and ovary injury after allogeneic SCT. (A-D) Unirradiated B6D2F1 female mice were intravenously injected with 8 × 10⁷ splenocytes from syngeneic (Syn; n = 25) or allogeneic (Allo; n = 26) B6 donors. Clinical GVHD scores (means ± SE; A) and survivals (B) are shown. (C-D) Macroscopic images of the ovaries (C) and estimated ovarian volumes (D) on day +35 after SCT are shown (n = 8/group). (E-L) Unirradiated B6 (syngeneic) or B6D2F1 (allogeneic) mice were injected with 3 × 10⁷ purified EGFP⁺ T cells and 5 × 10⁷ wild-type B6 TCD. (E) The small intestine, liver, and ovary harvested on day +14 were stained with hematoxylin and eosin (H&E; upper) and EGFP (lower; brown). Original magnification ×20. (F-G) Donor T cells infiltrated in the livers (F) and ovaries (G) were enumerated by flow cytometry and shown as means ± SE (n = 3-5/group). (H) Double-label immunofluorescent staining with CD3 (green) and EGFP (red) of the ovary. (I) Representative dot plot of EGFP⁺ cells from ovarian follicles (n = 5). (J) EGFP⁺ CD3⁺ T cells per an ovarian follicle were enumerated on the ovarian sections (n = 6/group). (K) PAS staining. Areas in the white squares are magnified and shown in the right side of original images. Arrowheads indicate infiltrating lymphocytes beyond the disrupted basement membranes of the ovarian follicles. (L) Immunofluorescent staining with cleaved-caspase 3 (green) and EGFP (red). Area in the white rectangle is magnified and shown in the right side of original image. Original magnification ×40. Data are representative of 2 similar experiments and shown as means ± SE. (M) Unirradiated B6D2F1 mice were injected with 3 × 10⁷ purified T cells from wild-type B6 plus 5 × 10⁷ TCD splenocytes from EGFP⁺ mice. Double-label immunofluorescent staining with cleaved-caspase3 (green) and EGFP (red) with counter staining with DAPI (blue) is shown. Scale bar, 1 cm (C), 50 µm (E, H, K, L, and M). *P < .05, **P < .01, ***P < .005.