T cell memory relies on the generation of antigen-specific progenitors with stem-like properties.However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells.We used a systematic approach guided by single-cell RNA sequencing data to map the organizational structure of the human CD8 + memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically, and transcriptionally distinct stem-like CD8 + memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage.Collectively, these data revealed the existence of parallel differentiation programs in the human CD8 + memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines. 3 MAIN TEXTAntigen recognition by CD8 + naive T cells initiates a program of clonal expansion and effector differentiation that leads to the clearance of infected or malignant cells and the subsequent formation of heterogeneous memory populations that confer durable immunity 1 . These memory populations are thought to be organized in a developmental hierarchy, according to which stem cell memory T (TSCM) cells self-renew and generate long-lived central memory T (TCM) cells and short-lived effector memory T (TEM) cells 2-6 . However, the mechanisms that underlie the enhanced multipotency of TSCM cells relative to TCM cells have not been clearly defined in molecular terms 7 .Memory T cell differentiation can become corrupted under conditions of persistent antigenic stimulation, as observed during chronic viral infections and progressive malignancies, which promote a state of T cell exhaustion, characterized by an orderly loss of effector functions, impaired proliferation, and the upregulation of inhibitory receptors 8 . This dynamic process occurs over a period of weeks after the initial priming event 9,10 and involves the genome-wide accumulation of epigenetic modifications 11,12 . Recent studies have shown that exhausted T (TEX) cell populations are developmentally and functionally heterogeneous, incorporating stem-like progenitors that express T cell factor 1 (TCF1) which give rise to highly differentiated TEX cells that are constitutively dysfunctional and lack TCF1 [13][14][15][16] . Importantly, the therapeutic benefits of immune checkpoint blockade in the context of chronic viral infections and various cancers are thought to operate via these TCF1 + progenitors, which appear susceptible to interventions that specifically target the inhibitory receptor programmed death-1 (PD-1) 13,15,17-20 .

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.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human solid tumor and current treatments are ineffective in increasing patients' survival. Thus, the development of new therapeutic approaches for ATC is needed. We have previously shown that the oncolytic adenovirus dl922-947 induces ATC cell death in vitro and tumor regression in vivo. However, the impact of dl922-947 on the pro-tumorigenic ATC microenvironment is still unknown. Since viruses are able to regulate cytokine and chemokine production from infected cells, we sought to investigate whether dl922-947 virotherapy has such effect on ATC cells, thereby modulating ATC microenvironment. dl922-947 decreased IL-8/CXCL8 and MCP-1/CCL2 production by the ATC cell lines 8505-c and BHT101-5. These results correlated with dl922-947-mediated reduction of NF-κB p65 binding to IL8 promoter in 8505-c and BHT101-5 cells and CCL2 promoter in 8505-c cells. IL-8 stimulates cancer cell proliferation, survival and invasion, and also angiogenesis. dl922-947-mediated reduction of IL-8 impaired ATC cell motility in vitro and ATC-induced angiogenesis in vitro and in vivo. We also show that dl922-947-mediated reduction of the monocyte-attracting chemokine CCL2 decreased monocyte chemotaxis in vitro and tumor macrophage density in vivo. Interestingly, dl922-947 treatment induced the switch of tumor macrophages toward a pro-inflammatory M1 phenotype, likely by increasing the expression of the pro-inflammatory cytokine interferon-γ. Altogether, we demonstrate that dl922-947 treatment re-shape the pro-tumorigenic ATC microenvironment by modulating cancer-cell intrinsic factors and the immune response. An in-depth knowledge of dl922-947-mediated effects on ATC microenvironment may help to refine ATC virotherapy in the context of cancer immunotherapy.

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.