We therefore set out to investigate whether mTORC

We, therefore, set out to investigate whether mTORC1 inhibitors like rapamycin would be relevant for the generation of human TSCM Linsitinib and whether a cross-talk between mTOR and Wnt signalling would exist. Moreover, since current knowledge on the generation and characterization of TSCM cells remains limited to CD8+ TSCM cells, apart from their phenotypic definition, CD4+ TSCM cells remain uninvestigated. The characterization of CD4+ TSCM cells seems to be of great importance all the more, as the role of CD4+ T cells as broad orchestrators of the immune response receives growing attention in anti-tumour immunotherapy (Kamphorst and Ahmed, 2013; Muranski and Restifo, 2009). In the present study, therefore, focus was put on the induction and characterization of CD4+ TSCM cells, nevertheless testing the relevance of our findings on TSCM cell induction also for CD8+ TSCM cells. Here, we revealed the inhibition of mTORC1 with simultaneously active mTORC2 signalling as the molecular mechanism inducing TSCM cells and that TSCM cell induction takes place in complete independence from Wnt signalling. We furthermore present insights into the transcriptomes of naturally occurring and pharmacologically induced CD4+ TSCM cells, the in vivo survival and repopulation capacity of pharmacologically induced CD4+ TSCM cells and the metabolic regulation of CD4+ TSCM cell generation. Taken together, our findings are of direct relevance for the design of improved anti-tumour immunotherapies.
Materials & Methods
Results
Discussion The identification of the signalling pathways, underlying TSCM cell formation, allows their targeted induction and paves the way for the design of novel immunotherapeutic approaches. Here, we show the emergence of a T cell population with phenotypic, transcriptional, functional and metabolic hallmarks of naturally occurring TSCM cells upon in vitro inhibition of mTORC1 during priming of human TN cells. These findings emphasize the potential relevance of the signalling network of mTOR kinase in immunotherapy and of mTOR modulating pharmacological agents. Interestingly, we show that mTORC1 inhibition with drugs like rapamycin mediates an immunostimulatory effect by the induction of TSCM cells, although these drugs are generally used because of their immunosuppressive function (Cobbold, 2013; Ferrer et al., 2011). Thus, these observations indicate that there are distinct conditions which trigger either a preferential immunostimulatory or an immunosuppressive rapamycin effect. Among a variety of different molecular mechanisms, rapamycin has been suggested to fulfil its immunosuppressive function by prevention of full T cell activation (Loewith et al., 2002; Thomson et al., 2009). This effect can be circumvented by strong stimulation of the TCR and co-stimulatory receptors (Slavik et al., 2004). Similarly, in the in vitro experiments the high degree of activation of TN cells by anti-CD3/CD28 beads in a 1:1 bead/cell ratio and 300IU/ml IL-2 might have favoured an immunostimulatory rapamycin effect. Furthermore, rapamycin has been shown to increase the antigen-specific T cell response to a pathogen (short-term persistence of the antigen), but to fail in doing so in response to a graft (long-term persistence of the antigen) (Ferrer et al., 2010). These findings strongly suggest that the period of antigen persistence also regulates the immunological outcome of rapamycin. Thus, the rather short periods of TN cell activation (14days and 4days) in our in vitro experiments might have tipped the balance towards an immunostimulatory rapamycin effect. The used concentration of rapamycin also emerges as an important factor for mediating either an immunostimulatory or an immunosuppressive drug effect. For the in vitro induction of TSCM cells, rapamycin was used in 100nM (90ng/ml), since a rather high concentration of 40–100ng/ml rapamycin, administered during the contraction phase, has been shown to qualitatively improve antigen-specific memory T cells in a mouse model of CD8+ T cell response to acute viral infection (Araki et al., 2009). In contrast, 8–12ng/ml rapamycin blood levels are intended to induce immunosuppression after transplantation (Baan et al., 2005). Together, this suggests that a low rapamycin concentration preferentially results in an immunosuppressive effect, whereas a high rapamycin concentration triggers an immunostimulatory one. Also the immunomodulatory actions of rapamycin might be regulated by the interplay between the two mTOR complexes. Whereas TSCM cell induction, as shown here, follows mTORC1 inhibition without additional inhibition of mTORC2, formation of immunosuppressive regulatory T cells is favoured in additional absence of mTORC2 signalling (Chi, 2012). Thus, immunomodulation by rapamycin appears to be a fine-tuned, highly multidimensional process.