Allogeneic Environment and Tissue Damage Provide Critical Stimuli for Regulatory T Cell Expansion and Survival In Vivo after Hematopoietic Cell Transplantation.

CD4+CD25+ regulatory T cells (Treg) mediate alloresponses in murine models of bone marrow transplantation (BMT), leading to protection from graft-versus-host disease (GvHD). However, in vivo migration and tissue localization of Treg during this inflammatory response remain unclear. We previously demonstrated co-localization of Treg with effector T cells (Tcon) with initial expansion in secondary lymphoid organs prior to migration into inflamed tissues in a major MHC-mismatched BMT model. To explore the stimuli for Treg proliferation, we evaluated the role of the allogeneic environment by transferring FVB donor luciferase-expressing (luc⁺) Treg into lethally-irradiated syngeneic recipients. Unlike the allogeneic irradiated setting where Treg expand in the presence or absence of Tcon, adoptively transferred luc⁺ Treg were not detected in secondary lymphoid organs of syngeneic lethally-irradiated BMT recipients by in vivo bioluminescence imaging (BLI). Syngeneic luc⁺ Tcon also had significantly different in vivo dynamics, with a 4 day delay and only moderate expansion in lymph nodes. Proliferation was not detected in the spleen, unlike their allogeneic Tcon counterparts, nor in the bone marrow compartments, as seen in lymphopenic models. To assess whether irradiation induced the observed in vivo dynamics of Treg in the allogeneic setting, we transferred FVB luc⁺ Treg or luc⁺ Tcon into unirradiated Balb/c Rag2^−/−gamma chain (γC) ^−/− recipients, which lack T, B, and NK cells. After adoptive transfer into Rag2^−/−γC^−/− recipients, robust Tcon proliferation was observed in secondary lymphoid organs and the bone marrow compartments; however, Treg expansion was weak, and specific localization to lymphoid or nonlymphoid tissues was not observed. Treg were stimulated to localize to and expand in secondary lymphoid organs by the co-transfer of Tcon in unirradiated Rag2^−/− (γC) ^−/− or by conditioning Rag2^−/− (γC) ^−/− recipients with irradiation. Exogenous IL2 administration two weeks following luc+ Treg transfer into unirradiated Rag2^−/− (γC) ^−/− recipients similarly led to localization and expansion of Treg in secondary lymphoid organs. These studies indicate the critical role of proinflammatory cytokines, such as IL2, generated either by irradiation-induced tissue damage or donor Tcon, in the expansion and localization of Treg. Differences between Tcon and Treg expansion in syngeneic or unconditioned allogeneic Rag2^−/− γC^−/− hosts suggest an important role of conditioning with irradiation alone or in concert with the allogeneic environment, in providing distinct signals for Tcon versus Treg activation, proliferation, and localization.