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Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

Cyropyrin-associated periodic syndromes (CAPS) are clinically distinct syndromes that encompass a phenotypic spectrum yet are caused by alterations in the same gene, NLRP3. Many CAPS cases and other NLRP3-autoinflammatory diseases (NLRP3-AIDs) are directly attributed to protein-coding alterations in NLRP3 and the subsequent dysregulation of the NLRP3 inflammasome leading to IL-1β-mediated inflammatory states. Here, we used bioinformatics tools, computational modeling, and computational assessments to explore the proteomic consequences of NLRP3 mutations, which potentially drive NLRP3 inflammasome dysregulation. We analyzed 177 mutations derived from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), and the non-hereditary chronic infantile neurologic cutaneous and articular syndrome, also known as neonatal-onset multisystem inflammatory disease (CINCA/NOMID), as well as other NLRP3-AIDs. We found an inverse relationship between clinical severity and the severity of predicted structure changes resulting from mutations in NLRP3. Bioinformatics tools and computational modeling revealed that NLRP3 mutations that are predicted to be structurally severely-disruptive localize around the ATP binding pocket and that specific proteo-structural changes to the ATP binding pocket lead to enhanced ATP binding affinity by altering hydrogen-bond and charge interactions. Furthermore, we demonstrated that NLRP3 mutations that are predicted to be structurally mildly- or moderately-disruptive affect protein-protein interactions, such as NLRP3-ASC binding and NLRP3-NLRP3 multimerization, enhancing inflammasome formation and complex stability. Taken together, we provide evidence that proteo-structural mechanisms can explain multiple mechanisms of inflammasome activation in NLRP3-AID.

Given the presence of engineered nanomaterials in consumers’ products and their application in nanomedicine, nanosafety assessment is becoming increasingly important. In particular, immunosafety aspects are being actively investigated. In nanomaterial immunosafety testing strategies, it is important to consider that nanomaterials and nanoparticles are very easy to become contaminated with endotoxin, which is a widespread contaminant coming from the Gram-negative bacterial cell membrane. Because of the potent inflammatory activity of endotoxin, contaminated nanomaterials can show inflammatory/toxic effects due to endotoxin, which may mask or misidentify the real biological effects (or lack thereof) of nanomaterials. Therefore, before running immunosafety assays, either in vitro or in vivo, the presence of endotoxin in nanomaterials must be evaluated. This calls for using appropriate assays with proper controls, because many nanomaterials interfere at various levels with the commercially available endotoxin detection methods. This also underlines the need to develop robust and bespoke strategies for endotoxin evaluation in nanomaterials.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the posttranscriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies. [ABSTRACT FROM AUTHOR]