High sensitivity of host Helios⁺/Neuropilin-1⁺ Treg to pretransplant conditioning hampers development of OX40^bright/integrin-7⁺ regulatory cells in acute gastrointestinal GvHD

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4(+)/CD25(hi)/Foxp3(+) Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-beta 7, LAG3, TGF beta/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios(+)/Nrp1(+) Treg, shifting the balance toward Helios(-)/Nrp1(-) Treg in the host. Upregulation of integrin-beta 7 and OX40 occurred in GI-aGvHD-dependent manner in Helios(+)/Nrp1(+) cells but not in Helios(-)/Nrp1(-) Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-beta 7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1(+)/Helios(+ )Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.