Combinatorial vs individual TL1A-Ig and IL-2 treatment before major and minor MHC-mismatched aHSCT. (A-C) Evaluation of recipients after individual TNFRSF25 or high-affinity IL-2 receptor stimulation compared with combined stimulation before conditioning and aHSCT. Mice were administered TL1A-Ig only, IL-2_LD only, or TL1A-Ig+IL-2_LD (see supplemental Figure 4A for details). aHSCT was performed using a B6→BALB/c donor/recipient mouse model. B6-FoxP3^RFP (H2^b) TCD BM (5.5 × 10⁶) cells and an adjusted spleen cell number containing 6.5 × 10⁵ T cells were transplanted into BALB/c (H2^d) recipients after TBI conditioning (7.75 Gy) on day-1. (A) Overall survival (n = 7), (B) GVHD clinical score (n = 7), and (C) representative colon histological images of tissue harvested at day +56. Arrows indicate differences in villi structure, more normal appearance in TL1A-Ig+IL-2_LD vs monotherapy. Combinatorial pretreatment resulted in the best outcomes, with fewer lymphocytic infiltrates in the colonic tissue. (D-G) The combinatorial treatment was examined in a second (low lethality) aHSCT model. MHC-matched, minor antigen–mismatched C3H.SW (H2^b) TCD BM (7 × 10⁶) cells and 2 × 10⁶ CD8⁺ T cells were transplanted into B6 (H2^b) recipients after TBI conditioning (10.5 Gy) on day 0. (D) At day −2, assessing Treg frequencies showed significant CD4⁺FoxP3⁺/CD4⁺ expansion with pretreatment. (E) GVHD clinical scores were less severe in pretreated recipients (n = 4-8). (F) Representative images of mice show appearances at 4 and 8 weeks after aHSCT. Pretreated recipients exhibited less skin and ocular GVHD involvement. (G) Collagen deposition (Masson trichrome stain, blue) in the skin of mice 8 weeks after aHSCT. In treated recipients, note the thin epidermis, scant dermis collagen staining, and the absence of inflammatory cells compared with untreated recipients exhibiting a thickened epidermis, extensive collagen deposition in the dermis, and a modest infiltration of chronic inflammatory cells (original magnification ×10). Heightened collagen and hepatic periportal infiltrates were detected in untreated MHC-matched recipients. (H) Treg depletion immediately before transplant reduces but does not abolish GVHD. BALB/c DT receptor mice were untreated or pretreated with TL1A-Ig+IL-2_LD before transplant with B6 TCD BM spleen cells. DT (1 μg IP) was administered on day −1 before TBI. Depleting resident Tregs reduced overall survival in pretreated as well as in untreated recipients (n = 5-7). Wks, weeks.