Natural Tr1-like cells do not confer long-term tolerogenic memory

IL-10-producing Tr1 cells promote tolerance but their contributions to tolerogenic memory are unclear. Using 10BiT mice that carry a Foxp3-eGFP reporter and stably express CD90.1 following IL-10 production, we characterized the spatiotemporal dynamics of Tr1 cells in a house dust mite model of allergic airway inflammation. CD90.1+Foxp3-IL-10+ Tr1 cells arise from memory cells and rejoin the tissue-resident memory T-cell pool after cessation of IL-10 production. Persistent antigenic stimulation is necessary to sustain IL-10 production and Irf1 and Batf expression distinguishes CD90.1+Foxp3-IL-10+ Tr1 cells from CD90.1+Foxp3-IL-10- ‘former’ Tr1. Depletion of Tr1-like cells after primary sensitization exacerbates allergic airway inflammation. However, neither transfer nor depletion of former Tr1 cells influences either Tr1 numbers or the inflammatory response during subsequent allergen memory re-challenge weeks later. Together these data suggest that naturally-arising Tr1 cells do not necessarily give rise to more Tr1 upon allergen re-challenge or contribute to tolerogenic memory. This phenotypic instability may limit efforts to re-establish tolerance by expanding Tr1 in vivo.