Dll1/Dll4 Notch ligands are critically active during the earliest posttransplant period and can be therapeutically targeted to prevent Scl-cGVHD. B10.D2→BALB/c chimeras were generated by transplanting B10.D2-Thy1.2 TCD BM (light green circle) vs TCD BM plus 12 × 10⁶ B10.D2-Thy1.1 WT splenocytes. T-cell recipients were treated with 4 peritransplant doses of isotype control (red circle), anti-Dll1 (purple-rimmed circle), anti-Dll4 (purple circle), or anti-Dll1 plus anti-Dll4 (blue circle) antibodies (5 mg/kg i.p. on days 0, 3, 7, and 10). (A-B) Biweekly systemic and cutaneous GVHD scores, showing that Dll4 is the dominant Notch ligand in the Scl-cGVHD model and that its targeting induces almost complete disease protection. *P < .01 (1-way ANOVA) for TCD BM and all groups receiving anti-Dll4 vs groups receiving isotype control or anti-Dll1 alone (cumulative data from 2 experiments). (C-D) B10.D2→BALB/c mice were generated as described above. T-cell recipients were treated with 4 doses of isotype control (red circle) or anti-Dll1/4 (blue circle) antibodies at days 0 to 10 vs anti-Dll1/Dll4 at later time points (days 10-20, purple-rimmed circle; days 40-50, purple circle). (C) Systemic GVHD scores were monitored longitudinally. Note lack of GVHD protection with delayed administration of anti-Dll1/4. (D) Histopathological analysis of dermal thickness in mice depicted in panel C. *P < .01 (1-way ANOVA). Note Scl-cGVHD protection with early anti-Dll1/Dll4 administration and trend toward benefit with delayed anti-Dll1/Dll4 treatment (P = .07). (E-F) B10.D2→BALB/c chimeras were generated as described above. In one cohort, anti-Dll1/Dll4 treatment was delayed by 48 hours (purple circle). Note loss of protection from systemic (E) and cutaneous (F) GVHD with delayed anti-Dll1/4 administration. *P < .01 (1-way ANOVA) for TCD BM and group receiving early anti-Dll1/4 vs groups receiving isotype control or delayed anti-Dll1/Dll4 (n = 12, cumulative data from 3 experiments).