ECP treatment induces host-type Foxp3⁺ Tregs that substantially contribute to but are not solely responsible for improved outcome. (A) Preemptive ECP treatment specifically increases host-type Tregs in C57BL/6→BALB/c. FACS gating strategy for Tregs isolated at day +4 (left), total percentage of Treg, fold increase of host-type Treg in spleen and pLNs based on Tcon group, and absolute numbers of host Foxp3⁺ cells per spleen are shown (right). (B) Proliferation capacity of Treg after BMT. Transplanted mice received BrdU injection (1 mg per mouse per IP) every second day followed by pLN harvest at day +7. No proliferation of host-type Treg was observed in mice transplanted with TCD-BM plus Tcon (left, middle). In mice receiving TCD-BM alone, host and donor-Treg proliferated (right). (C) ECP increases Tregs within 48 hours. Mice with or without ECP treatment were challenged with LPS (10 μg per mouse per IV) 48 hours after injection of ECP-treated cells and Tregs were analyzed in pLNs. Mice treated with ECP had significantly higher proportions of CD4⁺/Foxp3⁺ cells, compared with WT mice challenged with LPS only. Unchallenged mice served as a baseline. (D) CD4⁺ T cells from mice in panel C were evaluated for surface CTLA4 expression. ECP-treated mice had significantly higher levels of CTLA4 expression within the CD4⁺ population (left) and CD4⁺/Foxp3⁺ subpopulation, compared with untreated mice challenged with LPS only. (E) Treg from ECP-treated mice more actively suppressed T-cell proliferation than WT Tregs. [³H]-Thymidine incorporation of WT C57BL/6 responder Tcons (R) to Balb/c stimulators (S) in the presence of WT Tregs or ECP Tregs at different Treg to responder ratios is shown, P = .035. (F) Specific depletion of host Tregs prior to BMT in FVB→Foxp3DTR-C57BL/6. WT-C57BL/6 recipients received FVB Tcon (Tcon, ▲; ECP, ▪), as control for DT toxicity 1 group received 50 μg/kg DT at day −2 and −1 (WT Tcon+DT, ♦; WT ECP+DT, ▼). To specifically deplete host Tregs, C57BL/6-Foxp3DTR recipients were injected with 50 μg/kg DT at day −2 and −1 and transplanted with FVB Tcon (Foxp3DTR Tcon, ○; Foxp3DTR ECP, □). ECP group showed significant survival improvement in comparison with Tcon group, ▪ vs ▲, P = .03. Mice injected with DT showed exacerbated GVHD due to DT toxicity in all groups. Foxp3DTR ECP had no survival benefit in comparison with Foxp3DTR Tcon whereas benefit was maintained in WT ECP + DT vs WT Tcon + DT. (G) Adoptive transfer of host-type Tregs into untreated recipients prior to BMT failed to inhibit donor T-cell proliferation. Recipient mice received 10⁵ sort-purified Tregs (CD4⁺CD25^high) 24 hours prior to BMT. Tregs originated either from WT BALB/c (WT Treg) or from mice treated with ECP (ECP Treg). Recipients were transplanted with luc⁺Tcon and followed by BLI imaging. Data are representative (A,B,C,F,G) of at least 2 individual experiments with 3 to 10 mice per group, or (D) are a composite of 2 experiments with 10 mice per group or are done in triplicates with 3 independent mice (E). Error bars indicate mean ± SEM and significance was assessed by 2-tailed Student t test and log-rank test.