Furthermore we show that the induction of

Furthermore, we show that the induction of TSCM glycine receptors is completely independent from the Wnt signalling pathway. In line with this, the role of Wnt in memory T cell formation has already been called into question by reports about memory T cell formation in CD8+ T cells, in which β-catenin was conditionally knocked out (Driessens et al., 2010; Prlic and Bevan, 2011). Nevertheless, these reports have to be seen with caution, since, in contrast to our study, specifically, TSCM cell formation was not investigated and mice with KO of only β-catenin, which might have favoured a bypassed activity of Wnt signalling by γ-catenin, were used. Moreover, our data offer an unexpected answer to the paradox finding that the Wnt activator TWS119 induces TSCM cells, whereas none of alternative Wnt activators was able to do so, by the discovery of an mTORC1 inhibiting effect of TWS119. Interestingly, this effect finds further confirmation by a recent report, confirming in mouse T cells that TWS119 inhibits mTORC1 (Xiao et al., 2013). At present, it is unclear what the scope of TWS119 off-target effects on other kinases is or whether its inhibition of the mTORC1 kinase requires, as rapamycin, FKBP12. Future biochemical and pharmacological studies will have to address the precise molecular mechanism of mTORC1 inhibition by TWS119. We also showed that mTORC1 inhibition switched the metabolic programme of activated nCD4+ TN cells to an oxidative metabolism dependent on FAO. However, prominently, in the in vitro experiments only a fraction of activated TN cells was arrested in a TN-like state by TWS119 or rapamycin, suggesting that not all TN cells from the phenotypically homogenous CCR7+, CD45RA+ starting population react to mTORC1 inhibition in the same way. Future studies will have to address whether TN cell intrinsic factors can be identified which predispose certain cells to stop differentiation upon mTORC1 inhibition. One such factor might be Krüppel-like-factor 2 (KLF2) which has been shown to maintain the expression of CCR7 and CD62L and has been suggested to be up-regulated upon mTORC1 inhibition (Chi, 2012; van der Windt et al., 2012). Interestingly, only a small fraction of the CCR7+, CD45RA+, nCD4+ TN cell starting pool exhibited a high KLF2 expression, whereas the vast majority showed a low expression of KLF2 (Fig. S5e), suggesting KLF2 as possible discriminator to delineate TN cells with TSCM cell precursor potential. We sought to compare the transcriptome of CD4+ TSCM cells induced by either TWS119 or rapamycin. Indeed, we observed a very highly overlapping gene expression signature shared by rapamycin- and TWS119-induced CD4+ TSCM cells. Among 21,481 interrogated genes, only 565 genes were significantly differentially expressed between them (adj. p<0.05; log2FC >1), further supporting a common pharmacological mechanism of these drugs. It is very interesting that several of the up-regulated genes in the rapamycin treatment group were related with cell metabolism (Supplemental Table 5). Of note, the most highly up-regulated gene with rapamycin induction is NAD(P)H:quinone oxidoreductase (NQO1), which protects cells against oxidative stress and toxic quinones. In line with this, TXNRD1 (encoding the thioredoxin reductase 1) was also up-regulated in rapamycin-induced TSCM cells. This protein could reduce thioredoxins and plays an important role in protection against oxidative stress. High expression of NQO1 and TXNRD1 might be closely related with the increased oxidative phosphorylation and fatty acid oxidation upon rapamycin induction of TSCM, which definitely needs to be addressed further. On the other hand, the most up-regulated gene in TWS119-induced TSCM cells is LAMP3 (CD63). CD63 is barely expressed in naïve T cells but induced upon T cell activation. Crosslinking of CD63 has been shown to deliver a potent co-stimulatory signal to T cells. To our surprise, we noticed a striking induction of interferon responsive gene expression pattern in TWS119-induced TSCM cells (for example, interferon-induced protein with tetratricopeptide repeats 2, IFIT2; Interferon-Induced Protein with Tetratricopeptide Repeats 3, IFIT3; interferon alpha-inducible proteins 6, IFI6; interferon alpha-inducible proteins 27, IFI27). Many of these genes have been shown to be important for antiviral innate immunity. Some of them emerge to play important roles in regulating T cell activation and immune response. For instance, ISG15 protease UBP43 (USP18) regulates T cell activation. USP18 deficient T cells exhibit hyperactivation of NF-κB and NFAT upon TCR triggering and are defective in Th17 differentiation. The roles of many of those genes in regulating T cells immunity remain to be determined in the near future.