L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes.

Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

T lymphocytes induce human cancer cells derived from solid malignant tumors to secrete galectin-9 which facilitates immunosuppression in cooperation with other immune checkpoint proteins.

BACKGROUND: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. METHODS: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. RESULTS: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. CONCLUSION: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.

Crucial involvement of xanthine oxidase in the intracellular signalling networks associated with human myeloid cell function.

Xanthine oxidase (XOD) is an enzyme which plays a central role in purine catabolism by converting hypoxanthine into xanthine and then further into uric acid. Here we report that XOD is activated in THP-1 human myeloid cells in response to pro-inflammatory and growth factor stimulation. This effect occurred following stimulation of THP-1 cells with ligands of plasma membrane associated TLRs 2 and 4, endosomal TLRs 7 and 8 as well as stem cell growth factor (SCF). Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) transcription complexes were found to be responsible for XOD upregulation. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, was also found to be essential for XOD activation. Specific inhibition of XOD by allopurinol and sodium tungstate led to an increase in intracellular AMP levels triggering downregulation of mTOR activation by phosphorylation of its T2446 residue. Taken together, our results demonstrate for the first time that XOD is not only activated by pro-inflammatory stimuli or SCF but also plays an important role in maintaining mTOR-dependent translational control during the biological responses of human myeloid cells.

The HIF-1 transcription complex is essential for translational control of myeloid hematopoietic cell function by maintaining mTOR phosphorylation.

Mammalian myeloid cells are crucial effectors of host innate immune defense. Normal and pathological responses of these cells require adaptation to signaling stress through the hypoxia-inducible factor 1 (HIF-1) transcription complex. Adapted cells activate the mammalian target of rapamycin (mTOR), via S2448 phosphorylation, which induces de novo translation of vital signaling proteins. However, the molecular mechanisms underlying this signaling dogma remain unclear. Here, we demonstrate for the first time that inactivation of HIF-1, by silencing its inducible alpha subunit, significantly decreases mTOR S2448 phosphorylation caused by ligand-dependent activation of human myeloid leukemia cells. This shows that HIF-1 is essential for the activation of mTOR and serves at a crucial juncture of myeloid cell function in both in vitro and in vivo systems.

Gold nanoparticles downregulate interleukin-1ß-induced pro-inflammatory responses.

Interleukin 1 beta (IL-1ß)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1ß-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1ß both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1ß, thus opening potentially novel options for further therapeutic applications.

Dysfunctional mitochondria contain endogenous high-affinity human Toll-like receptor 4 (TLR4) ligands and induce TLR4-mediated inflammatory reactions.

Mitochondria, known to share many common features with prokaryotic cells, accumulate several endogenous ligands of the pattern-recognition Toll-like receptor 4 (TLR4), such as the heat shock proteins (Hsp) 70 and 60. TLR4 specifically recognises and responds to LPS of Gram-negative bacteria and participates in both autoimmune reactions and tissue regeneration due to its ability to recognise endogenous ligands. In the present study we show that mitochondria extracts obtained from hydrogen peroxide-dysfunctionalised cells induce a pro-inflammatory response in human THP-1 myeloid leukaemia cells. This inflammatory response was similar to that caused by LPS and much stronger than that induced by the extracts of normal mitochondria. Such reactions include activation of stress-adaptation hypoxia-inducible factor 1 alpha (HIF-1α) and expression/release of the pro-inflammatory cytokines IL-6 and TNF-α. Pre-treatment of THP-1 myeloid macrophages with TLR4-neutralising antibody before exposure to mitochondria extracts or LPS attenuated the inflammatory responses. Signalling pathways recruited by TLR4 in response to LPS and mitochondria-derived ligands were found to be the same. An in vitro ELISA-based TLR4-ligand binding assay, in which the ligand-binding domain of human TLR4 was immobilised, showed that mitochondria extracts contain endogenous TLR4 ligands. These results were verified in surface plasmon resonance experiments in which the affinity of the ligands derived from dysfunctional mitochondria was comparable with that of LPS and was much higher than that observed for normal mitochondria.