Recalling the Biological Significance of Immune Checkpoints on NK Cells: A Chance to Overcome LAG3, PD1, and CTLA4 Inhibitory Pathways by Adoptive NK Cell Transfer?

Immune checkpoint receptors (IC) positively or negatively regulate the activation of the host immune response, preventing unwanted reactions against self-healthy tissues. In recent years the term IC has been mainly used for the inhibitory ICs, which are critical to control Natural Killer (NK) and Cytotoxic CD8(+) T cells due to its high cytotoxic potential. Due to the different nature of the signals that regulate T and NK cell activation, specific ICs have been described that mainly regulate either NK cell or T cell activity. Thus, strategies to modulate NK cell activity are raising as promising tools to treat tumors that do not respond to T cell-based immunotherapies. NK cell activation is mainly regulated by ICs and receptors from the KIR, NKG2 and NCRs families and the contribution of T cell-related ICs is less clear. Recently, NK cells have emerged as contributors to the effect of inhibitors of T cell-related ICs like CTLA4, LAG3 or the PD1/PD-L1 axes in cancer patients, suggesting that these ICs also regulate the activity of NK cells under pathological conditions. Strikingly, in contrast to NK cells from cancer patients, the level of expression of these ICs is low on most subsets of freshly isolated and in vitro activated NK cells from healthy patients, suggesting that they do not control NK cell tolerance and thus, do not act as conventional ICs under non-pathological conditions. The low level of expression of T cell-related ICs in "healthy" NK cells suggest that they should not be restricted to the detrimental effects of these inhibitory mechanisms in the cancer microenvironment. After a brief introduction of the regulatory mechanisms that control NK cell anti-tumoral activity and the conventional ICs controlling NK cell tolerance, we will critically discuss the potential role of T cell-related ICs in the control of NK cell activity under both physiological and pathological (cancer) conditions. This discussion will allow to comprehensively describe the chances and potential limitations of using allogeneic NK cells isolated from a healthy environment to overcome immune subversion by T cell-related ICs and to improve the efficacy of IC inhibitors (ICIs) in a safer way

All about (nk cell-mediated) death in two acts and an unexpected encore: initiation, execution and activation of adaptive immunity

NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences

Fluorogenic Granzyme A Substrates Enable Real‐Time Imaging of Adaptive Immune Cell Activity

6 figures.Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.A. S. acknowledges a PhD fellowship from the Aragon Government. M. A. A. acknowledges funds from the Ministry of Economy and Competitiveness, Spain (IJC2019- 039192-I). This research was supported by CIBER (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU, FEDER (Group B29_20R), Ministry of Science, Innovation and Universities and Agencia Estatal de Investigación, Spain (SAF2017-83120-C2-1-R and PID2020-113963RB-I00). T. K. acknowledges funding from an MRC Career Development Award (MR/S006982/1) and an MRC Centre Grant (MR/N022556/1). E. W. R. acknowledges a Cancer Research UK grant (A_BICR_1920_Roberts). M. V. acknowledges funds from an ERC Consolidator Grant (DYNAFLUORS, 771443) and an ERC PoC Grant (IBDIMAGE, 957535). This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (859908).Peer reviewe

Cell death induced by cytotoxic CD8+ T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens

BackgroundElimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+ T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved.MethodsICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killed in vitro or in vivo by Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XL or a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used. Ex vivo cytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA.ResultsMice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XL in EL4 cells had no effect on induction of Tc cell antitumor response and protection.ConclusionsElimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies

The new strains Brucella inopinata BO1 and Brucella species 83-210 behave biologically like classic infectious Brucella species and cause death in murine models of infection

International audienceThe novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species

Software for data analysis of Brazilian rivers (vRivers)

Software for data analysis of Brazilian rivers obtained from the historical series database of the Agência Nacional de Águas e Saneamento Básico (ANA). https://www.snirh.gov.br/hidroweb/serieshistoricasThe authors acknowledges the TRIATLAS project, which has received funding from the European Union's Horizon 2020 Research and Innovation Program under grant agreement no. 817578 and to the International Joint Laboratory TAPIOCA (IRD-UFPE-UFRPE

Fungal polysaccharides from Inonotus obliquus are agonists for Toll-like receptors and induce macrophage anti-cancer activity

Abstract Fungal polysaccharides can exert immunomodulating activity by triggering pattern recognition receptors (PRRs) on innate immune cells such as macrophages. Here, we evaluate six polysaccharides isolated from the medicinal fungus Inonotus obliquus for their ability to activate mouse and human macrophages. We identify two water-soluble polysaccharides, AcF1 and AcF3, being able to trigger several critical antitumor functions of macrophages. AcF1 and AcF3 activate macrophages to secrete nitric oxide and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Combined with interferon-γ, the fungal polysaccharides trigger high production of IL-12p70, a central cytokine for antitumor immunity, and induce macrophage-mediated inhibition of cancer cell growth in vitro and in vivo. AcF1 and AcF3 are strong agonists of the PRRs Toll-like receptor 2 (TLR2) and TLR4, and weak agonists of Dectin-1. In comparison, two prototypical particulate β-glucans, one isolated from I. obliquus and one from Saccharomyces cerevisiae (zymosan), are agonists for Dectin-1 but not TLR2 or TLR4, and are unable to trigger anti-cancer functions of macrophages. We conclude that the water-soluble polysaccharides AcF1 and AcF3 from I. obliquus have a strong potential for cancer immunotherapy by triggering multiple PRRs and by inducing potent anti-cancer activity of macrophages

NOD2 Modulates Serotonin Transporter and Interacts with TLR2 and TLR4 in Intestinal Epithelial Cells

Background/Aims: Serotonin (5-HT) is a chief modulator of intestinal activity. The effects of 5-HT depend on its extracellular availability, which is mainly controlled by serotonin transporter (SERT), expressed in enterocytes. On the other hand, innate immunity, mediated by Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), is known to control intestinal microbiota and maintain intestinal homeostasis. The dysregulation of the intestinal serotonergic system and innate immunity has been observed in inflammatory bowel diseases (IBD), the incidence of which has severely increased all over the world. The aim of the present study, therefore, was to analyze the effect of NOD2 on intestinal SERT activity and expression, as well as to study the crosstalk of NOD2 with TLR2 and TLR4. Methods: Intestinal epithelial cell line Caco-2/TC7 was used to analyze SERT activity and SERT, NOD2, TLR2 and TLR4 molecular expression by real-time PCR and western blotting. Moreover, intestinal tract (ileum and colon) from mice deficient in TLR2, TLR4 or TLR2/4 receptors was used to test the interdependence of NOD2 with these TLR receptors. Results: NOD2 activation inhibits SERT activity in Caco-2/TC7 cells, mainly due to the decrement of SERT molecular expression, with RIP2/RICK being the intracellular pathway involved in this effect. This inhibitory effect on SERT would yield an increment of extracellular 5-HT availability. In this sense, 5-HT strongly inhibits NOD2 expression. In addition, NOD2 showed greater interdependence with TLR2 than with TLR4. Indeed, NOD2 expression significantly increased in both cells treated with TLR2 agonists and the intestinal tract of Tlr2-/- mice. Conclusions: It may be inferred from our data that NOD2 could play a role in intestinal pathophysiology not only through its inherent innate immune role but also due to its interaction with other receptors as TLR2 and the modulation of the intestinal serotonergic system decreasing SERT activity and expression

Marine Mammal <i>Brucella</i> Reference Strains Are Attenuated in a BALB/c Mouse Model

<div><p>Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of <i>Brucella</i> spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains <i>Brucella pinnipedialis</i> 12890 (harbour seal, <i>Phoca vitulina</i>) and <i>Brucella ceti</i> 12891 (harbour porpoise, <i>Phocoena phocoena</i>) were included in genus <i>Brucella</i> in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with <i>Brucella suis</i> 1330 as a control. Both marine mammal strains were attenuated, however, <i>B</i>. <i>ceti</i> was present at higher levels than <i>B</i>. <i>pinnipedialis</i> in blood, spleen and liver throughout the infection, in addition <i>B</i>. <i>suis</i> and <i>B</i>. <i>ceti</i> were isolated from brains and faeces at times with high levels of bacteraemia. In <i>B</i>. <i>suis</i>-infected mice serum cytokines peaked at day 7. In <i>B</i>. <i>pinnipedialis</i>-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In <i>B</i>. <i>ceti</i>-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected <i>in vitro</i> after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses <i>in vivo</i> were also induced by the three brucellae. The immunological pattern of <i>B</i>. <i>ceti</i> in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for <i>Brucella neotomea</i>.</p></div

Spleen bacterial counts and organ weights.

<p>Bacterial counts for B. <i>pinnipedialis</i> 12890 (open diamonds), <i>B</i>. <i>ceti</i> 12891 (crosses) and <i>B</i>. <i>suis</i> 1330 (black squares) in spleens (a) of BALB/c mice after intraperitoneal (ip) inoculation of 10⁵ colony forming units (CFU) of bacteria. Uninfected control mice received sterile phosphate buffered saline ip (black lines). Four or five mice were euthanized per lot at day 3, 7, 14, 21, 35, 56 and 84 post infection (day 56 and 84; only <i>B</i>. <i>ceti</i> 12891 and <i>B</i>. <i>suis</i> 1330). The number of viable bacteria was determined and the numbers of bacterial counts were logarithmic transformed. Spleen weights (b) were determined in parallel. Results are expressed as mean + one standard deviation. Whether CFU numbers differed significantly between mice infected with <i>B</i>. <i>suis</i> 1330, and <i>B</i>. <i>ceti</i> 12891 or <i>B</i>. <i>pinnipedialis</i> 12890, respectively, at the different times pi, are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150432#pone.0150432.t001" target="_blank">Table 1</a>. Whether spleen weights of the infected mice were significantly different from those of the uninfected control mice, at the different times pi, is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150432#pone.0150432.s005" target="_blank">S1 table</a>.</p