Tracking Fate-Mapped Th17 Cells in Adoptive Transfer

CD27⁺ or CD27⁻ fractions of YFP⁺ cells were sorted after enrichment with CD4 beads (Miltenyi) from spleens and lymph nodes of MOG-immunized WT or Rptor^Il17aCre mice. Sorting was performed on a Reflection (iCyt/Sony) cytometer, and sort-purified cells were transferred via retroorbital injection into Rag1^−/− mice. Due to low cell numbers, we used day 1 after transfer as the baseline reading to normalize subsequent analyses at days 14–15, as previously described^{27 (link)}, for donor cell abundance in the spleen and lymph nodes. Similar transfer experiments were performed using CD45.1⁺ hosts, except that 2D2-transgenic mice (expressing MOG antigen-specific T cell receptors^{28 (link)}; crossed onto the Il17aCre (R26R^eYFP) fate-mapping system) were used as donor mice, and CD4 T cell enrichment was performed at the time of analysis, to facilitate flow cytometric detection of the rare population of donor cells.