STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma

Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8⁺ T cells.

Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. Among these, the accumulation of adenosine (Ado) contributes to tumor progression and represents a promising immunotherapeutic target. Ado has been shown to impair T cell effector function, but the role and mechanisms employed by Ado/Ado receptors (AdoRs) in modulating human peripheral and tumor-infiltrating lymphocyte (TIL) function are still puzzling. CD8 <sup>+</sup> T cell cytokine production following stimulation was quantified by intracellular staining and flow cytometry. The cytotoxic capacity of tumor infiltrating lymphocytes (TILs) was quantified by the chromium release assay following co-culture with autologous or anti-CD3-loaded tumor cell lines. The CD8 <sup>+</sup> T cell metabolic fitness was evaluated by the seahorse assay and by the quantification of 2-NBDG uptake and CD71/CD98 upregulation upon stimulation. The expression of AdoRs was assessed by RNA flow cytometry, a recently developed technology that we validated by semiquantitative RT-PCR (qRT-PCR), while the impact on T cell function was evaluated by the use of selective antagonists and agonists. The influence of Ado/AdoR on the PKA and mTOR pathways was evaluated by phosphoflow staining of p-CREB and p-S6, respectively, and validated by western blot. Here, we demonstrate that Ado signaling through the A2A receptor (A2AR) in human peripheral CD8 <sup>+</sup> T cells and TILs is responsible for the higher sensitivity to Ado-mediated suppression of T central memory cells. We confirmed that Ado is able to impair peripheral and tumor-expanded T cell effector functions, and we show for the first time its impact on metabolic fitness. The Ado-mediated immunosuppressive effects are mediated by increased PKA activation that results in impairment of the mTORC1 pathway. Our findings unveil A2AR/PKA/mTORC1 as the main Ado signaling pathway impairing the immune competence of peripheral T cells and TILs. Thus, p-CREB and p-S6 may represent useful pharmacodynamic and efficacy biomarkers of immunotherapies targeting Ado. The effect of Ado on T cell metabolic fitness reinforces the importance of the adenosinergic pathway as a target for next-generation immunotherapy

Interleukin-7 Influences FOXP3+CD4+ Regulatory T Cells Peripheral Homeostasis

Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance

LAG-3 and PD-1+LAG-3 inhibition promote anti-tumor immune responses in human autologous melanoma/T cell co-cultures.

Despite the success of immunotherapy using checkpoint blockade, many patients with solid tumors remain refractory to these treatments. In human cancer, the experimental options to investigate the specific effects of antibodies blocking inhibitory receptors are limited and it is still unclear which cell types are involved. We addressed the question whether the direct interaction between T cells and tumor cells can be enforced through blocking a set of inhibitory receptors including PD-1, TIM-3, BTLA and LAG-3, blocked either individually or in dual combinations with the anti-PD-1 antibody, and to determine the condition that induces maximal T cell function preventing tumor cell proliferation. Using short-term Melan-A-specific or autologous re-stimulations, checkpoint blockade did not consistently increase cytokine production by tumor-derived expanded T cells. We next set up a 5-day co-culture assay with autologous melanoma cell lines and expanded tumor infiltrating T cells, originating from tumor specimens obtained from 6 different patients. Amongst all combos tested, we observed that blockade of LAG-3 alone, and more strongly when combined with PD-1 blockade, enforced T cell responses and tumor cell growth control. The combination of anti-LAG-3 plus anti-PD-1 acted through CD8 T cells and led to increased IFNγ production and cytotoxic capacity. Our results show that LAG-3 and PD-1 are regulating the direct interaction between tumor cells and autologous T cells, suggesting that therapy effects may be promoted by enhanced access of the corresponding blocking reagents to the tumor microenvironment

Netting Neutrophils Activate Autoreactive B Cells in Lupus.

Lupus erythematosus (LE) patients develop autoantibodies that form circulating immune complexes (ICs) with extracellular self-nucleic acids. These ICs are deposited into peripheral tissues, where they trigger detrimental organ inflammation. Recent evidence suggests that ICs contain LL37-DNA complexes derived from neutrophil extracellular traps (NETs) and that LE patients develop pathogenic autoantibodies against these structures, including Abs to LL37. However, the mechanism that leads to the generation of these Abs is unknown. In this study, we show that NETs directly trigger Ab production by human memory B cells. This occurs via LL37-DNA complexes present in NETs, which have the unique ability to gain access to endosomal compartments of B cells and to trigger TLR9 activation. In LE patients, NET-derived LL37-DNA complexes trigger polyclonal B cell activation via TLR9, but also specifically expand self-reactive memory B cells producing anti-LL37 Abs in an Ag-dependent manner. These findings suggest a unique link between neutrophils and B cells in which NETs trigger a concerted activation of TLR9 and BCR leading to anti-NET autoantibody production in lupus

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma

Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8+ T cells

Background: Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. Among these, the accumulation of adenosine (Ado) contributes to tumor progression and represents a promising immunotherapeutic target. Ado has been shown to impair T cell effector function, but the role and mechanisms employed by Ado/Ado receptors (AdoRs) in modulating human peripheral and tumor-infiltrating lymphocyte (TIL) function are still puzzling. Methods: CD8+ T cell cytokine production following stimulation was quantified by intracellular staining and flow cytometry. The cytotoxic capacity of tumor infiltrating lymphocytes (TILs) was quantified by the chromium release assay following co-culture with autologous or anti-CD3-loaded tumor cell lines. The CD8+ T cell metabolic fitness was evaluated by the seahorse assay and by the quantification of 2-NBDG uptake and CD71/CD98 upregulation upon stimulation. The expression of AdoRs was assessed by RNA flow cytometry, a recently developed technology that we validated by semiquantitative RT-PCR (qRT-PCR), while the impact on T cell function was evaluated by the use of selective antagonists and agonists. The influence of Ado/AdoR on the PKA and mTOR pathways was evaluated by phosphoflow staining of p-CREB and p-S6, respectively, and validated by western blot. Results: Here, we demonstrate that Ado signaling through the A2A receptor (A2AR) in human peripheral CD8+ T cells and TILs is responsible for the higher sensitivity to Ado-mediated suppression of T central memory cells. We confirmed that Ado is able to impair peripheral and tumor-expanded T cell effector functions, and we show for the first time its impact on metabolic fitness. The Ado-mediated immunosuppressive effects are mediated by increased PKA activation that results in impairment of the mTORC1 pathway. Conclusions: Our findings unveil A2AR/PKA/mTORC1 as the main Ado signaling pathway impairing the immune competence of peripheral T cells and TILs. Thus, p-CREB and p-S6 may represent useful pharmacodynamic and efficacy biomarkers of immunotherapies targeting Ado. The effect of Ado on T cell metabolic fitness reinforces the importance of the adenosinergic pathway as a target for next-generation immunotherapy

Native/citrullinated LL37-specific T-cells help autoantibody production in Systemic Lupus Erythematosus.

LL37 exerts a dual pathogenic role in psoriasis. Bound to self-DNA/RNA, LL37 licenses autoreactivity by stimulating plasmacytoid dendritic cells-(pDCs)-Type I interferon (IFN-I) and acts as autoantigen for pathogenic Th17-cells. In systemic lupus erythematosus (SLE), LL37 also triggers IFN-I in pDCs and is target of pathogenic autoantibodies. However, whether LL37 activates T-cells in SLE and how the latter differ from psoriasis LL37-specific T-cells is unknown. Here we found that 45% SLE patients had circulating T-cells strongly responding to LL37, which correlate with anti-LL37 antibodies/disease activity. In contrast to psoriatic Th17-cells, these LL37-specific SLE T-cells displayed a T-follicular helper-(T <sub>FH</sub> )-like phenotype, with CXCR5/Bcl-6 and IL-21 expression, implicating a role in stimulation of pathogenic autoantibodies. Accordingly, SLE LL37-specific T-cells promoted B-cell secretion of pathogenic anti-LL37 antibodies in vitro. Importantly, we identified abundant citrullinated LL37 (cit-LL37) in SLE tissues (skin and kidney) and observed very pronounced reactivity of LL37-specific SLE T-cells to cit-LL37, compared to native-LL37, which was much more occasional in psoriasis. Thus, in SLE, we identified LL37-specific T-cells with a distinct functional specialization and antigenic specificity. This suggests that autoantigenic specificity is independent from the nature of the autoantigen, but rather relies on the disease-specific milieu driving T-cell subset polarization and autoantigen modifications

Oxidation of HCl over TiO2-supported RuO2: A density functional theory study

With density functional theory (DFT) calculations, we studied the oxidation of HCl with oxygen producing Cl2 and water on the TiO2(110)-supported RuO2(110). This so-called Sumitomo-a novel Deacon-process proceeds via a one-dimensional Langmuir?Hinshelwood mechanism, in which the recombination of two adjacent chlorine atoms on the surface of the catalyst constitutes the rate-determining step. Very important for industrial application is that substantial Ru resources can be saved in the production of the Sumitomo catalyst. According to our DFT calculations already 1 ML of RuO2(110) supported on TiO2(110) suffices to maintain practically the full activity of bulk-RuO2 in the HCl oxidation reaction. The calculated electron density differences of the TiO2(110)-supported 1 ML RuO2 system in comparison with bulk RuO2(110) are localized at the internal interface, leaving the electronic structure of the topmost undercoordinated Ru sites (active sites) unaffected by the support. This explains naturally the invariant activity of supported 1 ML RuO2(110) in comparison with bulk RuO2(110). The stoichiometric TiO2(110) is not active at all in the HCl oxidation reaction. However, if the undercoordinated Ti surface atoms are substituted by Ru then the resulting 1/2 ML RuO2-TiO2(110) catalyst is active with an activation barrier that is 58 kJ/mol higher than for bulk-RuO2(110)