Preclinical models revealed that the immune system can mediate rejection of established tumors, but direct evidence in humans has been limited to largely immunogenic tumors such as melanoma. The recent success of immune checkpoint inhibitors and adoptive T cell transfer immunotherapy in clinical trials has instilled new hope for the use of T-cell immunotherapy in the treatment of cancer. Interleukin-15 (IL-15), a potent immunostimulatory cytokine, both potentiates host T and NK-cell immune responses and promotes the generation of long-lived memory T cells with superior functional capacity with potential use in adoptive T-cell transfer protocols. IL-15 has been recently tested in the clinic and showed dramatic effects at the level of responding NK and CD8+ memory T cells. The recent advances in the knowledge of IL-15-dependent regulation of T-cell responses, gene expression and metabolic adaptation have important implications for the use of IL-15 in T cell-based immunotherapy of cancer.

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Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

In mice, the ability of naive T (T N) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8 + T N cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3 + T N cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3 + T N cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3 + T N cells were transcriptionally equivalent to murine CXCR3 + T N cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8 + T cells.

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The diversity of the naïve T cell repertoire drives the replenishment potential and capacity of memory T cells to respond to immune challenges. Attrition of the immune system is associated with an increased prevalence of pathologies in aged individuals, but whether stem cell memory T lymphocytes (T SCM ) contribute to such attrition is still unclear. Using single cells RNA sequencing and high-dimensional flow cytometry, we demonstrate that T SCM heterogeneity results from differential engagement of Wnt signaling. In humans, aging is associated with the coupled loss of Wnt/β-catenin signature in CD4 T SCM and systemic increase in the levels of Dickkopf-related protein 1, a natural inhibitor of the Wnt/β-catenin pathway. Functional assays support recent thymic emigrants as the precursors of CD4 T SCM . Our data thus hint that reversing T SCM defects by metabolic targeting of the Wnt/β-catenin pathway may be a viable approach to restore and preserve immune homeostasis in the context of immunological history.

The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2 1 cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation. 2086Introduction TOR (target of rapamycin) integrates environmental cues, including amino acid and nutrient availability, energy stores and growth factor signaling, and subsequently directs cell growth and proliferation in yeast and mammals. Mammalian (m)TOR exists in two complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [1]. mTORC1 comprises mTOR, Raptor, mLST8 and PRAS40 while the functionally distinct mTORC2 contains mTOR, mLST8 and the unique regulatory proteins Rictor, mSIN1 and PROTOR (reviewed in [2]). mTORC1 controls transcription and translation in response to nutrient and amino acid levels, growth factors and cytokines through phosphorylation of the p70 S6 kinase (p70S6K) and the initiation factor 4E-binding protein 1 (4EBP1), and is strongly sensitive to inhibition by the naturally occurring compound rapamycin (RAPA). While the function of mTORC2 remains largely uncharacterized, it has been shown to regulate aspects of the actin cytoskeleton, and to directly phosphorylate Akt (Ser473), PKC-a (Ser657) and SGK1 (Ser422). While mTORC2 is insensitive to transient RAPA administration, prolonged RAPA treatment hinders mTORC2 assembly, thus blocking mTORC2 function in some cells [2].In T cells, mTOR appears to regulate several aspects of lymphocyte biology and to lie at the crossroads of T-cell proliferation and tolerance [3,4]. T-cell-specific deletion of Frap1 (encoding for mTOR) hinders Th1 and Th2 differentiation in response to polarizing cytokines favoring differentiation of Foxp3 1 cells [5]. In contrast, deletion of Rictor abrogates Th1and Th2 cell differentiation without, however, diverting...

Human T memory stem (TSCM) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long‐lived T‐cell memory and are thus considered an attractive population to be used in adoptive transfer‐based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T‐cell receptor stimulation curbs human effector CD8+ T‐cell differentiation and allows the generation of CD45RO–CD45RA+CCR7+CD27+CD95+ ‐phenotype cells from highly purified naïve T‐cell precursors, resembling naturally‐occurring human TSCM. These cells proliferate extensively in vitro and in vivo, express low amounts of effector‐associated genes and transcription factors and undergo considerable self‐renewal in response to IL‐15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly‐activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long‐lived memory T cells with potential application in adoptive cell transfer immunotherapy.