Myeloid-derived suppressor cells (MDSC) accumulate in the spleen and tumor bed during tumor growth. They contribute to the immune tolerance of cancer notably by inhibiting the function of CD8(+) T ...cells. Thus, their elimination may hamper tumor growth by enhancing antitumor T-cell functions. We have previously reported that some anticancer agents relied on T cell-dependent anticancer responses to achieve maximal efficacy. However, the effect of anticancer agents on MDSC has remained largely unexplored. In this study, we observed that gemcitabine and 5-fluorouracil (5FU) were selectively cytotoxic on MDSC. In vivo, the treatment of tumor-bearing mice with 5FU led to a major decrease in the number of MDSC in the spleens and tumor beds of animals whereas no significant effect on T cells, natural killer cells, dendritic cells, or B cells was noted. Interestingly, 5FU showed a stronger efficacy over gemcitabine to deplete MDSC and selectively induced MDSC apoptotic cell death in vitro and in vivo. The elimination of MDSC by 5FU increased IFN-gamma production by tumor-specific CD8(+) T cells infiltrating the tumor and promoted T cell-dependent antitumor responses in vivo. Altogether, these findings suggest that the antitumor effect of 5FU is mediated, at least in part, by its selective cytotoxic action on MDSC.
The advancement of knowledge on tumor biology over the past decades has demonstrated a close link between tumor cells and cells of the immune system. In this context, cytokines have a major role ...because they act as intermediaries in the communication into the tumor bed. Cytokines play an important role in the homeostasis of innate and adaptive immunity. In particular, they participate in the differentiation of CD4 T lymphocytes. These cells play essential functions in the anti-tumor immune response but can also be corrupted by tumors. The differentiation of naïve CD4 T cells depends on the cytokine environment in which they are activated. Additionally, at the tumor site, their activity can also be modulated according to the cytokines of the tumor microenvironment. Thus, polarized CD4 T lymphocytes can see their phenotype evolve, demonstrating functional plasticity. Knowledge of the impact of these cytokines on the functions of CD4 T cells is currently a source of innovation, for therapeutic purposes. In this review, we discuss the impact of the major cytokines present in tumors on CD4 T cells. In addition, we summarize the main therapeutic strategies that can modulate the CD4 response through their impact on cytokine production.
Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in ...tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.
The Nod-like receptor NLRP3 is involved in the formation of NLRP3. Up to now, the immunological functions of NLRP3 independently of inflammasome is unclear. In this dataset containing 6 samples (TH0, ...TH2 cells at day 3 and day 6 in wild type or Nlrp3 deficient cells), we show that NLRP3 expression in CD4+ T cells supports a T helper 2 (TH2) transcriptional program in a cell-intrinsic manner (raw and normalized data are accessible on Gene Expression Omnibus database under the number GSE54561, http://www.dtd.nlm.nih.gov/geo/query/acc.cgi?acc=GSE54561). Indeed, NLRP3 positively-regulated TH2 program independently of inflammasome formation. These data indicated that TH2 specific genes such as cMaf or Il4 were not induced in Nlrp3 deficient cells. These results demonstrate the capacity of NLRP3 to act as a key transcription factor in TH2 differentiation.
Bleomycin (BLM) is an anticancer drug currently used for the treatment of testis cancer and Hodgkin lymphoma. This drug triggers cancer cell death via its capacity to generate radical oxygen species ...(ROS). However, the putative contribution of anticancer immune responses to the efficacy of BLM has not been evaluated. We make here the observation that BLM induces immunogenic cell death. In particular, BLM is able to induce ROS-mediated reticulum stress and autophagy, which result in the surface exposure of chaperones, including calreticulin and ERp57, and liberation of HMBG1 and ATP. BLM induces anti-tumor immunity which relies on calreticulin, CD8(+) T cells and interferon-γ. We also find that, in addition to its capacity to trigger immunogenic cell death, BLM induces expansion of Foxp3+ regulatory T (Treg) cells via its capacity to induce transforming growth factor beta (TGFβ) secretion by tumor cells. Accordingly, Treg cells or TGFβ depletion dramatically potentiates the antitumor effect of BLM. We conclude that BLM induces both anti-tumor CD8(+) T cell response and a counteracting Treg proliferation. In the future, TGFβ or Treg inhibition during BLM treatment could greatly enhance BLM anti-tumor efficacy.
Dacarbazine (DTIC) is a cytotoxic drug widely used for melanoma treatment. However, the putative contribution of anticancer immune responses in the efficacy of DTIC has not been evaluated. By testing ...how DTIC affects host immune responses to cancer in a mouse model of melanoma, we unexpectedly found that both natural killer (NK) and CD8+ T cells were indispensable for DTIC therapeutic effect. Although DTIC did not directly affect immune cells, it triggered the upregulation of NKG2D ligands on tumor cells, leading to NK cell activation and IFNγ secretion in mice and humans. NK cell–derived IFNγ subsequently favored upregulation of major histocompatibility complex class I molecules on tumor cells, rendering them sensitive to cytotoxic CD8+ T cells. Accordingly, DTIC markedly enhanced cytotoxic T lymphocyte antigen 4 inhibition efficacy in vivo in an NK-dependent manner. These results underscore the immunogenic properties of DTIC and provide a rationale to combine DTIC with immunotherapeutic agents that relieve immunosuppression in vivo.
The TH9 subset of helper T cells was initially shown to contribute to the induction of autoimmune and allergic diseases, but subsequent evidence has suggested that these cells also exert antitumor ...activities. However, the molecular events that account for their effector properties are elusive. Here we found that the transcription factor IRF1 enhanced the effector function of TH9 cells and dictated their anticancer properties. Under TH9-skewing conditions, interleukin 1β (IL-1β) induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to the promoters of Il9 and Il21 and enhanced secretion of the cytokines IL-9 and IL-21 from TH9 cells. Furthermore, IL-1β-induced TH9 cells exerted potent anticancer functions in an IRF1- and IL-21-dependent manner. Our findings thus identify IRF1 as a target for controlling the function of TH9 cells.
Working on transcription factors requires studying interactions between protein and DNA. After identification of putative binding-sequences and motifs, Electrophoretic Mobility Shift Assay (EMSA) ...experiment is classically used to determine specific interactions of proteins and nucleic acids. This lengthy process is rather heavy-handed because of radioisotopically labeled DNA and autoradiographic visualization that are required for the experiments.Liquid luminescent DNA precipitation assay provides rapid, reliable and quantitative results concerning protein-DNA interactions. This protein-DNA binding assay is based on solution hybridization between Digoxigenin-labeled (DIG) DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to Glutathione Sepharose 4B beads (Figure 1), without electrophoresis (Toshiharu et al., 2008). Digoxigenin is a steroid found in plants. It is increasingly used as a label for nonradioactive detection of nucleic acids and proteins.Figure 1. Representation of liquid chemiluminescent DNA pull-down assay. A Glutathione S-transferase (GST)-fused NLRP3 (GST-NLRP3) bound to Glutathione Sepharose 4B beads is incubated with a DIG-labeled double-stranded DNA fragment containing putative NLRP3 Binding Site (NBS) in protein-DNA binding buffer. After extensive washing, protein-DNA binding on beads is detected using anti-DIG antibody conjugated to alkaline phosphatase, which is measured by a chemiluminescent reaction using a luminometer Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo 3.3.1.13, 7 decan}-4-yl) phenyl phosphate(CSPD).Here, we described how we used this technique to demonstrate the interaction between NLRP3 protein and its DNA binding site (Bruchard et al., 2015).
Protein-protein interaction experiments, such as co-immunoprecipitation (IP) assays, classically require tremendous amount of cells. This becomes a problem when your work focuses on rare cell ...populations (e.g., lymphocyte subtypes). O-link Bioscience has developed Proximity Ligation Assay (PLA) reagents and procedures to alleviate and solve this kind of issue. Moreover PLA experiments are read out using fluorescence or bright field microscopy, providing additional information on intracellular interactions localization significantly bettering classical IP procedures.PLA reagents are made of complementary small oligonucleotides “minus” and “plus” probes which specifically recognize host species from the primary antibodies (Abs) targeting the two proteins you are interested in. Experiments have to be designed with primary Abs from different species (rabbit, mouse or goat) as PLA probes “minus” or “plus” react against a specific host species of the primary antibody (e.g. “plus” anti-rabbit with “minus” anti-mouse or “plus” anti-mouse with “minus” anti-rabbit combos are allowed if mouse and rabbit primary Abs are used). When the two PLA probes are close enough (40 nm) a ligation occurs upon ligase incubation generating a circle DNA. These circle-forming DNAs are next amplified thanks to a polymerase and complementary fluorescent nucleotides, being incorporated at this step. Each luminescent spot is thereafter considered being an interaction site between the two proteins (Figure 1).Figure 1. Schematic representation of Duolink® experiment. Primary Abs from different host species were used i.e., Mouse (Ms) anti-Nlrp3 and Rabbit (Rb) anti-IRF4. When protein-protein interaction occurs PLA probes allow incorporation of fluorescent oligonucleotides which are analyzed by microscopy.PLA experiments have been performed on differentiated T cells from mice. T cells were grown on cover slip coated with poly-L-Lysine. Please be aware that for T cells and other non-adherent cells, experiments and staining are also possible in 500 µl microtubes until the last washing step before mounting cover slip on slide. This microtube method saves cytokines reagents and allows T cells to grow with usual methods but centrifugation repetition for washing steps is hazardous. Primary Abs incubation also requires also a specific setup if microtube method is chosen.
Here, we characterize a subset of ILC3s that express Neuropilin1 (NRP1) and are present in lymphoid tissues, but not in the peripheral blood or skin. NRP1+ group 3 innate lymphoid cells (ILC3s) ...display in vitro lymphoid tissue inducer (LTi) activity. In agreement with this, NRP1+ ILC3s are mainly located in proximity to high endothelial venules (HEVs) and express cell surface molecules involved in lymphocyte migration in secondary lymphoid tissues via HEVs. NRP1 was also expressed on mouse fetal LTi cells, indicating that NRP1 is a conserved marker for LTi cells. Human NRP1+ ILC3s are primed cells because they express CD45RO and produce higher amounts of cytokines than NRP1− cells, which express CD45RA. The NRP1 ligand vascular endothelial growth factor A (VEGF-A) served as a chemotactic factor for NRP1+ ILC3s. NRP1+ ILC3s are present in lung tissues from smokers and patients with chronic obstructive pulmonary disease, suggesting a role in angiogenesis and/or the initiation of ectopic pulmonary lymphoid aggregates.
Display omitted
•NRP1+ ILC3s are present in lymphoid tissues, but not in the peripheral blood or skin•NRP1+ ILC3s express CD45RO and produce higher amounts of cytokines than NRP1− ILC3s•NRP1 is a marker for human ILC3s with LTi phenotype and in vitro LTi activity•NRP1+ ILC3s are present in lung tissues from smokers and COPD patients
Shikhagaie et al. find that NRP1 expressing human ILC3s are LTi-like cells, which are present in fetal tissues and adult lymphoid tissues, but not in peripheral blood or skin. NRP1+ ILC3s cells are primed and migrate in response to VEGF-A. In addition, their presence in the lungs of smokers and COPD patients provides insight into the formation of ectopic lymphoid aggregates.
PDF
