Background: Natural Treg (nTreg) must migrate from graft to draining lymph node (LN) to prolong islet allograft survival, and we previously showed that the transcription factor T-bet regulates this migratory step. T-bet directly activates the transcription of IFNγ and CXCR3. IFNγ is also a mediator of Th1 inflammation and stimulates lymphocyte recruitment into inflamed sites, such as an allograft. However, the role of IFNγ in modulating Treg migration into and beyond the graft site is not known. We hypothesized that IFNγ is important for afferent lymphatic migration and is a key factor missing in T-bet-/- nTreg.

Methods: Quantitative RT-PCR was used to evaluate IFNγ expression; a footpad migration assay measured Treg migration from peripheral tissue to draining LN; and an ear pinna assay quantified in vivo migration of Treg from tissue to lymphatic vessels using immunohistochemistry. LN egress was assessed by i.v. injection of CFSE-labeled wild type (WT) or T-bet-/- T cells into WT C57BL/6 mice, followed by in vivo equilibration and then treatment with anti-CD62L or control mAb to block T cell entry into LN.

Results: As expected, IFNγ expression was reduced in T-bet-/- nTreg compared to WT nTreg under both resting and activated conditions. To investigate if IFNγ expression regulated nTreg afferent lymphatic migration, WT nTreg were treated with anti-IFNγ or control IgG, injected s.c. into the footpad, and migration to the draining popliteal LN quantified by flow cytometry. Anti-IFNγ significantly decreased nTreg migration from footpad to popliteal draining LN. To exclude the possibility that T-bet-/- nTreg did migrate into draining LN appropriately, but then exited the LN faster, LN egress was assessed using the LN equilibration assay, and LN egress activity was unchanged compared to WT. CFSE-labeled WT nTreg mixed with anti-IFNγ were injected into ear pinnae, and their distance to lymphatics was measured after 18-24 hours by immunofluorescence. This assay showed significantly fewer anti-IFNγ treated WT nTreg in proximity to or within lymphatic vessels compared to nTreg treated with control IgG.

Conclusions: Down regulation of IFNγ expression in T-bet-/- nTreg contributed to impaired afferent lymphatic migration and thus early graft loss. This suggests that IFNγ is necessary for optimal nTreg migration and ultimately immunosuppressive capacity. Careful consideration must be given when altering Th1 cytokines not to impair Treg function.

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