Blocking the access to all SLOs prevents aGHVD. Conditioned Balb/c recipients (H-2^d) received transplants of allogeneic luc⁺ T cells and wild type FVB/N (H-2^q) bone marrow cells. To prevent donor T-cell entry to SLOs either Balb/c or splenectomized Balb/c (no spleen, SplEx) aHCT recipients were intravenously injected daily with αCD62L αMAdCAM-1 or isotype control antibodies. (A) In vivo BLI for the first 6 days after aHCT demonstrated effective blocking of nodal sites by combined αCD62L/αMAdCAM-1 treatment. However, alloreactive cells proliferated in the spleen before aGVHD target infiltration (top panel). Splenectomy plus isotype control antibody treatment lead to compensatory alloreactive cell expansion in remaining accessible sites (second from bottom). However, combined αCD62L/αMAdCAM-1_SplEx treatment resulted in less intense BLI signals (middle), that projected predominantly to the bone marrow compartments (sternum, femura and pelvis). By day 4 abdominal sites were targeted in all groups except for the αCD62L/αMAdCAM-1_SplEx group. Shown is one representative animal (of 5) from each group (reproduced in 3 independent experiments). (B) The total body photon emission of αCD62L/αMAdCAM-1_SplEx recipients is reduced in contrast to all other groups (P < .015). This indicates compensatory proliferation in recipients with partially blocked SLO T-cell entry. Means plus or minus SD are shown in the bar graphs. (C) In higher magnification (day 3), BLI reveals the compensatory sites of alloreactive proliferation. The spleen region was positive in the αCD62L/αMAdCAM-1 group, whereas PP and LN regions were positive in the splenectomy plus isotype antibody group. Less intense signals projected to the BM compartment in the αCD62L/αMAdCAM-1_SplEx group. (D) Blocking the access to SLO by αCD62L/αMAdCAM-1_SplEx was effective as measured by ex vivo imaging (top left) and immune fluorescence microscopy (Thy1.1⁺ donor T cells in blue, MHC II⁺ host APCs in red, isotype control right panels). Histopathologic evaluation supported these findings, by showing a grade 2 intestinal aGVHD in isotype controls (bottom right) in comparison to unaffected GIT from the αCD62L/αMAdCAM-1_SplEx group (bottom left). (E) All animals from the αCD62L/αMAdCAM-1_SplEx group survived the aHCT without any signs of acute or chronic GVHD (pooled data from 3 independent experiments, P < .001). In contrast, αCD62L/αMAdCAM-1 treatment, isotype treatment or isotype treatment with splenectomy all resulted in acute GVHD-related mortality (≥ 90%) within 70 days after aHCT. Control groups: BM only, more than 90% survival, no aGVHD; BM and splenocytes, no antibodies: 0% survival, severe aGVHD; radiation only: 0% survival.