Extended Data Fig. 10: Pseudotime analysis and experimental validation.

a–h, WT and Rptor^Il17aCre (R26R^eYFP) mice were immunized with MOG, and YFP⁺ cells were analysed by single-cell transcriptomics analysis at day nine post-immunization. a, Empirical dispersion and mean expression using the single-cell-analysis toolkit Monocle 2, including the genes used for temporal ordering in black; each grey or black dot represents one gene. The red line shows Monocle’s expected dispersion, with more- and less-dispersed genes based on average expression above and below the red line, respectively. b, Pseudotime densities for each individual cluster. For example, cluster 1, associated with the proliferative signature, was in the centre of the pseudotime spectrum, while clusters 2, 3 and 8 (early in pseudotime; predominantly Raptor-deficient cells) and clusters 7 and, to a lesser extent, 4 and 5 (late in pseudotime; predominantly WT cells) were on the opposite end of the spectrum. c, Projection of signature scores for ‘early memory’, ‘late memory’ and ‘T-bet targets’ onto pseudotime trajectory; the keys indicate the relative scores per cell. d, tSNE visualization of Tbx21 and Ifng gene expression. e, Tbx21 and Ifng gene expression during pseudotime; cells that did not express Tbx21 or Ifng were filtered out in their respective graphs. f, Pseudotime assignment for WT and Rptor^Il17aCre cells, coloured by genotype; each dot represents one cell. g, Cd27 and Tcf7 gene expression across pseudotime, coloured by genotype. h, tSNE visualization of Cd27 and Tcf7 expression. i, Flow cytometry analysis of T-bet expression in freshly isolated CD27⁺ and CD27⁻ cells from dLN of Il17aCre (R26R^eYFP) mice at day nine post MOG-immunization. j, Fold change in the percentage of the IL-17⁻ IFN-γ⁺ cells amongst CD27⁺ or CD27⁻ YFP⁺ cells stimulated with MOG plus IL-12 as compared with freshly isolated cells (n = 12). k, CD27⁺ YFP⁺ cells from MOG-immunized WT and Rptor^Il17aCre mice were sorted and transferred into CD45.1⁺ hosts. The following day, CD45.1⁺ host mice were immunized with MOG; four days later, YFP⁺ cells were analysed by flow cytometry for surface CD27 expression (left; a summary plot is at the right: n = 6, WT; n = 5, Rptor^Il17aCre). l, CD27⁺ YFP⁺ cells from MOG-immunized WT and Rptor^Il17aCre mice were stimulated with MOG plus IL-12 for four days, and then CD27 expression was analysed. m, T_H17 cells are functionally and metabolically heterogeneous, and are composed of a subset with stemness features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into T_H1 cells. These two subsets are further distinguished by selective expression of the transcription factors TCF-1 and T-bet, respectively, and discrete levels of CD27 expression. mTORC1 activation drives reprogramming of anabolic metabolism, favouring transcription that is mediated by T-bet rather than TCF-1; consequently, T_H17 transdifferentiation into T_H1-like T_H17 cells occurs. Memory/stem-like T_H17 cells can become reactivated and have the potential to undergo terminal differentiation and acquire T_H1-like phenotypes. Data are means ± s.e.m. and from one experiment (a–h), or are representative of three (i) or five (j–l) independent experiments. Numbers in plots represent frequencies of cells in gates; numbers within histograms represent mean fluorescence intensities. Mann–Whitney U-test (two-sided) was used in panel j to determine statistical significance.

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