Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

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 reveal 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.

CXCR3 Identifies Human Naive CD8+ T Cells with Enhanced Effector Differentiation Potential.

In mice, the ability of naive T (TN) 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+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN 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+ TN 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+ TN cells were transcriptionally equivalent to murine CXCR3+ TN 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.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

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.

Generating stem-like memory T cells with antioxidants for adoptive cell transfer immunotherapy of cancer.

Among the multiple factors that are responsible for the success of adoptive cell transfer (ACT) immunotherapy for cancer, the differentiation status of the in vitro expanded T cell product at the time of transfer seems to play a major role. In particular, less differentiated memory CD8+ T cells endowed with self-renewing capacity and multipotency exert the most potent antitumor activity. To this aim, expansion protocols that generate sufficient numbers of tumor-specific CD8+ T cells with superior capacity to persist in vivo following ACT are needed. We describe a procedure for the differentiation of TCF-1+ stem-like CD8+ memory T cells from peripheral blood naïve precursors that takes advantage of the use of antioxidants, in particular N-acetylcysteine (NAC), in combination with T cell receptor stimulation and proinflammatory cytokines. We additionally describe how to conduct in vitro assays to test the stem-like features of the generated cells at the phenotypic, functional and metabolic level. Balancing the oxidative metabolism by the addition of antioxidants during in vitro manipulation of CD8+ T cells results in the generation of cell products with potent antitumor characteristics following ACT.

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity.

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.

GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity.

GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14+ monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity.

The oncolytic virus dl922-947 reduces IL-8/CXCL8 and MCP-1/CCL2 expression and impairs angiogenesis and macrophage infiltration in anaplastic thyroid carcinoma.

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.

PARP inhibitor olaparib increases the oncolytic activity of dl922-947 in in vitro and in vivo model of anaplastic thyroid carcinoma.

PARP inhibitors are mostly effective as anticancer drugs in association with DNA damaging agents. We have previously shown that the oncolytic adenovirus dl922-947 induces extensive DNA damage, therefore we hypothesized a synergistic antitumoral effect of the PARP inhibitor olaparib in association with dl922-947. Anaplastic thyroid carcinoma was chosen as model since it is a particularly aggressive tumor and, because of its localized growth, it is suitable for intratumoral treatment with oncolytic viruses. Here, we show that dl922-947 infection induces PARP activation, and we confirm in vitro and in vivo that PARP inhibition increases dl922-947 replication and oncolytic activity. In vitro, the combination with olaparib exacerbates the appearance of cell death markers, such as Annexin V positivity, caspase 3 cleavage, cytochrome C release and propidium iodide permeability. In vivo, we also observed a better viral distribution upon PARP inhibition. Changes in CD31 levels suggest a direct effect of olaparib on tumor vascularization and on the viral distribution within the tumor mass. The observation that PARP inhibition enhances the effects of dl922-947 is highly promising not only for the treatment of anaplastic thyroid carcinoma but, in general, for the treatment of other tumors that could benefit from the use of oncolytic viruses.