Abstract
CD4+CD25+ regulatory T-cells (Treg) have the potential to suppress aberrant immune responses and to regulate peripheral T cell homeostasis. In a murine allogeneic bone marrow transplantation (BMT) model, our laboratory has previously shown that Treg suppress graft-versus-host-disease (GVHD) without abrogating the beneficial graft-versus-tumor immunological effect. However, the mechanisms of immuno-regulation, in particular, the allorecognition properties of Treg, their effects on and interaction with other immune cells, and the sites of suppressive activity, remain unclear. In the current study, we investigate the in vivo trafficking of Treg to better understand how localization may affect their regulatory function. Interpretation of previous studies of Treg localization has been limited by several factors: the need to sacrifice mice does not permit tracking the same set of cells over time; data collected at several arbitrary times on selected tissues do not permit accurate determination of primary and secondary sites of migration, activation, proliferation, and suppression; and CD25 expression on activated CD4+CD25- T cells are difficult to distinguish from Treg. We have developed and characterized a transgenic mouse which constitutively expresses the luciferase gene in all hematologic cells. Treg (1x106 cells) from the spleen and lymph nodes of luc+ transgenic FVB/N (H-2q) mice were co-transplanted into lethally-irradiated (800cGy) Balb/c (H-2d) host along with 5x106 wild-type FVB/N T-cell depleted bone marrow (TCD-BM) cells and 2x106 whole splenocytes, the latter containing approximately 30% T cells (Tconv) which induce GVHD. Upon exposure to the substrate luciferin, the luciferase-expressing Treg emit light which can penetrate through living tissues and is captured by sensitive CCD camera detectors. Bioluminescence imaging (BLI) was performed at various time points established previously by our studies of BLI in GVHD. Within the first 48 hours, Treg localized to the cervical lymph nodes (LN) and the spleen. By day 3, signal is detected in other LN (axillary, mesenteric, inguinal) as well as Peyer’s patches and liver. Signal intensity, measured by photons/second/mouse, significantly increased and peaked on day 4, consistent with the migration and proliferation of Treg to and at these secondary lymphoid organs, respectively. Skin infiltration of Treg is noted on day 6, congruent with a decreased intensity in the spleen, liver, and lymph nodes. A similar pattern of early migration and proliferation of effector immune cells is noted in the GVHD control group, which is transplanted with wild-type FVB/N TCD-BM and luc+ FVB/N whole splenocytes. However, with the GVHD group, the signal intensity continues to increase at all sites. Continued BLI of the Treg group up to day 45 demonstrates persistent strong signal in lymphoid organs and skin sites. Clinically, the Treg group has no significant evidence of GVHD. Chimerism studies on day 45 show complete donor origin, however, lymphoid reconstitution of CD4+ and CD8+ T cells is delayed in the GVHD control group and enhanced in the recipients transplanted with Treg. The above results indicate that in vivo, Treg proliferate and survive long-term. In addition, they co-localize with effector immune cells to secondary lymphoid tissues to positively impact clinical outcomes and lymphoid reconstitution following major-MHC mismatched BMT.
Identification and expansion of highly suppressive CD8+FoxP3+ regulatory T cells after experimental allogeneic bone marrow transplantation