Background: Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. Methods: We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. Results: For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53^L22Q,W23S, a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53^L22Q,W23S in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. Conclusions: While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.

Serum vitamin D levels negatively correlate with obesity and associated disorders such as non-alcoholic steatohepatitis (NASH). However, the mechanisms linking low vitamin D (VD) status to disease progression are not completely understood. In this study, we analyzed the effect of VD treatment on NASH in mice. C57BL6/J mice were fed a high-fat/high-sugar diet (HFSD) containing low amounts of VD for 16 weeks to induce obesity, NASH and liver fibrosis. The effects of preventive and interventional VD treatment were studied on the level of liver histology and hepatic/intestinal gene expression. Interestingly, preventive and to a lesser extent also interventional VD treatment resulted in improvements of liver histology. This included a significant decrease of steatosis, a trend towards lower non-alcoholic fatty liver disease (NAFLD) activity score and a slight non-significant decrease of fibrosis in the preventive treatment group. In line with these changes, preventive VD treatment reduced the hepatic expression of lipogenic, inflammatory and pro-fibrotic genes. Notably, these beneficial effects occurred in conjunction with a reduction of intestinal inflammation. Together, our observations suggest that timely initiation of VD supplementation (preventive vs. interventional) is a critical determinant of treatment outcome in NASH. In the applied animal model, the improvements of liver histology occurred in conjunction with reduced inflammation in the gut, suggesting a potential relevance of vitamin D as a therapeutic agent acting on the gut–liver axis.

BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8 ⁺ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8 ⁺ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8 ⁺ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient ( Ldlr ^−/− ) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8 ⁺ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8 ⁺ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8 ⁺ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8 ⁺ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8 ⁺ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1 , to be induced by CD8 ⁺ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8 ⁺ T cells to control the SMC phenotype in atherosclerosis. CD8 ⁺ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8 ⁺ T cells could thus be explored as therapeutic target cells during lesion progression.

Zinc (Zn²⁺) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn²⁺ storage and release. To visualize Zn²⁺ storage in human and mouse platelets, the Zn²⁺ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d^−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn²⁺ release upon activation. Platelets from Nbeal2^−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn²⁺ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn²⁺ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2^−/− and Unc13d^−/− mice, and the impairment could be partially restored by extracellular Zn²⁺. Altogether, we conclude that the release of ionic Zn²⁺ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn²⁺ in platelet-dependent fibrin formation.

Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.