Triple-color immunofluorescence microscopy (IFM) of secondary lymphatic tissues and GVHD target organs. IFM was performed using 3 T-lymphocyte markers (antibody-conjugates): CD4-PE (red), CD8-FITC (green), and the donor-specific T-cell marker Thy1.1-APC (blue). Fresh frozen tissues sampled on 3 time points after transfer of allogeneic splenocytes (3, 4, and 6 days) are shown for mesenteric lymph nodes (mLNs) (panels A-C), spleen (panels E-G), and small bowel (panels I-K). Lymphatic tissues of day 3 (panels A,E) display a predominant infiltration by CD4⁺ donor T cells, which are confined to the corresponding T zones of mLNs and spleen, while target organs of day 3 remain negative for donor T cells. Day 4 represents a dramatic change in that the small bowel mucosa as a GVHD target tissue (panel J) is now infiltrated by donor T lymphocytes, predominantly CD4⁺, while lymphatic organs (panels B,F) shift to an expansion of CD8⁺ donor T cells (compare also Figure 4A). On day 6 all relevant GVHD target tissues are infiltrated by donor T cells, now predominantly CD8⁺, which are frequently in contact with CD4⁺ T cells of donor origin and sporadically with host CD4⁺ T cells (panels K,M-O). CD4⁺ and CD8⁺ T cells of host origin have mostly disappeared in mLNs, spleen, and small bowel of control tissues of Balb/c mice on day 6 after irradiation, which had not received an allogeneic transplantion (panels D,H,L). In contrast, PP samples of this control mouse show numerous CD4⁺ host T cells, which display a distinct membrane staining (panel P). The costaining for Thy1.1 was negative on all of the control tissues.