BackgroundDespite their revolutionary success in cancer treatment over the last decades, immunotherapies encounter limitations in certain tumor types and patients. The efficacy of immunotherapies depends on tumor antigen-specific CD8 T-cell viability and functionality within the immunosuppressive tumor microenvironment, where oxygen levels are often low. Hypoxia can reduce CD8 T-cell fitness in several ways and CD8 T cells are mostly excluded from hypoxic tumor regions. Given the challenges to achieve durable reduction of hypoxia in the clinic, ameliorating CD8 T-cell survival and effector function in hypoxic condition could improve tumor response to immunotherapies.MethodsActivated CD8 T cells were exposed to hypoxia and metformin and analyzed by fluorescence-activated cell sorting for cell proliferation, apoptosis and phenotype. In vivo, metformin was administered to mice bearing hypoxic tumors and receiving either adoptive cell therapy with tumor-specific CD8 T cells, or immune checkpoint inhibitors; tumor growth was followed over time and CD8 T-cell infiltration, survival and localization in normoxic or hypoxic tumor regions were assessed by flow cytometry and immunofluorescence. Tumor oxygenation and hypoxia were measured by electron paramagnetic resonance and pimonidazole staining, respectively.ResultsWe found that the antidiabetic drug metformin directly improved CD8 T-cell fitness in hypoxia, both in vitro and in vivo. Metformin rescued murine and human CD8 T cells from hypoxia-induced apoptosis and increased their proliferation and cytokine production, while blunting the upregulation of programmed cell death protein 1 and lymphocyte-activation gene 3. This appeared to result from a reduced production of reactive oxygen species, due to the inhibition of mitochondrial complex I. Differently from what others reported, metformin did not reduce tumor hypoxia, but rather increased CD8 T-cell infiltration and survival in hypoxic tumor areas, and synergized with cyclophosphamide to enhance tumor response to adoptive cell therapy or immune checkpoint blockade in different tumor models.ConclusionsThis study describes a novel mechanism of action of metformin and presents a promising strategy to achieve immune rejection in hypoxic and immunosuppressive tumors, which would otherwise be resistant to immunotherapy.
When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP:TGF-β1 complexes induced more frequent immune-mediated rejections of CT26 and MC38 murine tumors than anti-PD-1 alone. In both types of tumors, the activity of anti-GARP:TGF-β1 mAbs resulted from blocking active TGF-β1 production and immunosuppression by GARP-expressing regulatory T cells. In CT26 tumors, combined GARP:TGF-β1/PD-1 blockade did not augment the infiltration of T cells, but did increase the effector functions of already present anti-tumor T cells. Here we show that, in contrast, in MC38, combined GARP:TGF-β1/PD-1 blockade increased infiltration of T cells, as a result of increased extravasation of T cells from blood vessels. Unexpectedly, combined GARP:TGF-β1/PD-1 blockade also increased the density of GARP+ blood vessels covered by pericytes in MC38, but not in CT26 tumors. This appears to occur because anti-GARP:TGF-β1, by blocking TGF-β1 signals, favors the proliferation of and expression of adhesion molecules such as E-selectin by blood endothelial cells. The resulting densification of intratumoral blood vasculature probably contributes to increased T cell infiltration and to the therapeutic efficacy of GARP:TGF-β1/PD-1 blockade in MC38. We conclude from these distinct observations in MC38 and CT26, that the combined blockades of GARP:TGF-β1 and PD-1 can exert anti-tumor activity via multiple mechanisms, including the densification and normalization of intratumoral blood vasculature, the increase of T cell infiltration into the tumor and the increase of the effector functions of intratumoral tumor-specific T cells. This may prove important for the selection of cancer patients who could benefit from combined GARP:TGF-β1/PD-1 blockade in the clinics.
Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.
Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy and tumor progression. Histological follow-up by anatomo-pathologists reveals that 2/3 of surgically-removed thyroids do not present malignant lesions. Continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains thus central to better understand the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E is specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E dependent TC. Finally, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages a PTC mouse model and the relevance of these observations in human thyroid tissues.
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