p38α deficiency ameliorates psoriasis development by downregulating STAT3-mediated keratinocyte proliferation and cytokine production.

Psoriasis is characterized by keratinocyte (KC) hyperproliferation and inflammatory cell infiltration, but the mechanisms remain unclear. In an imiquimod-induced mouse psoriasiform model, p38 activity is significantly elevated in KCs and p38α specific deletion in KCs ameliorates skin inflammation. p38α signaling promotes KC proliferation and psoriasis-related proinflammatory gene expression during psoriasis development. Mechanistically, p38α enhances KC proliferation and production of inflammatory cytokines and chemokines by activating STAT3. While p38α signaling in KCs does not affect the expression of IL-23 and IL-17, it substantially amplifies the IL-23/IL-17 pathogenic axis in psoriasis. The therapeutic effect of IL-17 neutralization is associated with decreased p38 and STAT3 activities in KCs and targeting the p38α-STAT3 axis in KCs ameliorates the severity of psoriasis. As IL-17 also highly activates p38 and STAT3 in KCs, our findings reveal a sustained signaling circuit important for psoriasis development, highlighting p38α-STAT3 axis as an important target for psoriasis treatment.

LAYN Serves as a Prognostic Biomarker and Downregulates Tumor-Infiltrating CD8+ T Cell Function in Hepatocellular Carcinoma.

Background: Layilin (LAYN) represents a valuable prognostic biomarker across various tumor types, while also serving as an innovative indicator of dysfunctional or exhausted CD8+ T cells and exhibiting correlation with immune context. However, the immune function and prognostic significance of LAYN in hepatocellular carcinoma (HCC) remain unexplored. Therefore, our objective is to investigate the role of LAYN in CD8+ T cell exhaustion, clinical prognosis, and the tumor microenvironment within HCC. Methods: TIMER or GEPIA databases were used to analyze LAYN expression level and its correlation with immune infiltration in HCC. Bioinformatics analysis was conducted on TCGA and scRNA-seq cohorts. The evaluation of LAYN expression level in fresh specimens was performed through IF, IHC, and ELISA assays. Flow cytometry and mRNA-seq were employed to investigate co-expressed genes of LAYN, the LAYN+CD8+ T cell exhaustion signature and immune function. Cell proliferation ability and killing activity were assessed using CCK8 and CFSE/PI. Results: The expression level of LAYN in HCC tumors was significantly higher compared to peri-tumors. Patients with high levels of LAYN exhibited poorer OS. GO or KEGG analysis confirmed that LAYN was involved in immune response and was positively associated with CD8+ T cell immune infiltration levels. Furthermore, LAYN negatively regulated the immune function of CD8+ T cells, leading to dysfunctional phenotypes characterized by elevated levels of CD39, TIM3 and reduced levels of perforin, TNF-α, Ki-67. CFSE/PI assays demonstrated that LAYN+CD8+ T cells displayed decreased cytotoxic activity. Additionally, there was a positive correlation between LAYN and CD146 levels, which are involved in adhesion and localization processes of CD8+ T cells. Interestingly, blocking LAYN partially restored the exhaustion properties of CD8+ T cells. Conclusion: LAYN exhibits a strong correlation with immune infiltration in the TME and represents a novel biomarker for predicting clinical prognosis in HCC. Moreover, targeting LAYN may hold promise as an effective strategy for HCC immunotherapy.

Tumor infiltrating B lymphocytes (TIBs) associate with poor clinical outcomes, unfavorable therapeutic benefit and immunosuppressive context in metastatic clear cell renal cell carcinoma (mccRCC) patients treated with anti-PD-1 antibody plus Axitinib.

PURPOSE: Immune checkpoint inhibitors (ICIs) plus tyrosine kinase inhibitors (TKIs) has become first-line therapy for metastatic renal cell carcinoma patients. This study aims to investigate the effect of tumor infiltrating B lymphocytes (TIBs) on the combination therapy. METHODS: The retrospective analysis was conducted on the clinical records of 115 metastatic clear cell renal cell carcinoma (mccRCC) patients treated with anti-PD-1 antibody plus Axitinib between March 2020 and June 2023. Observation target: objective response rate (ORR), and overall survival (OS), progression-free survival (PFS) and immune profile. RESULTS: Patients with high TIBs portended lower ORR of the combination therapy (p = 0.033). TIBs was an independent predictor for poorer OS (p = 0.013) and PFS (p = 0.021) in mccRCC patients with combination treatment. TIBs infiltration was associated with more CD4+T (p < 0.001), CD8+T (p < 0.001), M2 macrophages (p = 0.020) and regulatory T cells (Tregs) (p = 0.004). In TIBs high patients, the percentages of PD-1, CTLA-4 and TIM-3 positive rate were significantly increased in CD4+T (p = 0.038, 0.029 and 0.002 respectively) and CD8+T cells (p = 0.006, 0.026 and < 0.001 respectively). CONCLUSIONS: Our study revealed TIBs infiltration predicted adverse outcomes in mccRCC patients treated with anti-PD-1 antibody plus Axitinib. As a corollary, TIBs positively associated with M2 macrophages and Tregs, leading to subsequent multiple immune checkpoints related exhaustion of T cells. Thus, only PD-1 blockade are inadequate to reverse T cells exhaustion effectively in high TIBs mccRCC patients.

miR-612 Enhances RSL3-Induced Ferroptosis of Hepatocellular Carcinoma Cells via Mevalonate Pathway.

Background: MicroRNA-612 (miR-612) has been proven to suppress the formation of invadopodia and inhibit hepatocellular carcinoma (HCC) metastasis by hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA)-mediated lipid reprogramming. However, its biological roles in HCC cell ferroptosis remain unclear. Methods and Results: In this study, we found that HCC cells with high metastatic potential were more resistant to ferroptosis, indicating that ferroptosis is related to HCC metastasis. The levels of lipid reactive oxygen species (ROS) were found to be much lower in HCC cells with high metastatic potential by flow cytometry (FCM). We used HCC cells with miR-612 overexpression/knockout and HADHA overexpression/knockdown to test cell viability after stimulation with RSL3. HCC cells overexpressing miR-612 were more sensitive to ferroptosis, and miR-612 could increase lipid ROS levels. Furthermore, colony formation assays and Transwell assays showed that miR-612 could inhibit the proliferation and metastasis of HCC cells by promoting ferroptosis. We next confirmed that miR-612 influenced HCC cell ferroptosis by regulating HADHA. HADHA could upregulate the expression of key enzymes in the mevalonate (MVA) pathway. HADHA overexpression upregulated the expression of CoQ10 and decreased polyunsaturated fatty acid (PUFA) levels and lipid peroxide abundance. miR-612 also suppressed HCC cell proliferation and metastasis by enhancing RSL3- and lovastatin-induced ferroptosis in vivo. Conclusion: Overall, miR-612 promotes ferroptosis in HCC cells and affects HCC proliferation and metastasis by downregulating CoQ10 and increasing cellular PUFA levels and lipid peroxides via the HADHA-mediated MVA pathway.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

MAPK Phosphatase-1 Deficiency Exacerbates the Severity of Imiquimod-Induced Psoriasiform Skin Disease.

Persistent activation of mitogen-activated protein kinase (MAPK) is believed to be involved in psoriasis pathogenesis. MAPK phosphatase-1 (MKP-1) is an important negative regulator of MAPK activity, but the cellular and molecular mechanisms of MKP-1 in psoriasis development are largely unknown. In this study, we found that the expression of MKP-1 was decreased in the imiquimod (IMQ)-induced psoriasiform mouse skin. MKP-1-deficient (MKP-1-/-) mice were highly susceptible to IMQ-induced skin inflammation, which was associated with increased production of inflammatory cytokines and chemokines. MKP-1 acted on both hematopoietic and non-hematopoietic cells to regulate psoriasis pathogenesis. MKP-1 deficiency in macrophages led to enhanced p38 activation and higher expression of interleukin (IL)-1ß, CXCL2, and S100a8 upon R848 stimulation. Moreover, MKP-1 deficiency in the non-hematopoietic compartments led to an enhanced IL-22 receptor signaling and higher expression of CXCL1 and CXCL2 upon IMQ treatment. Collectively, our data suggest a critical role for MKP-1 in the regulation of skin inflammation.

p38α signaling in Langerhans cells promotes the development of IL-17-producing T cells and psoriasiform skin inflammation.

Dendritic cells (DCs) contribute to psoriasis pathogenesis. In a mouse model of imiquimod-induced psoriasiform skin inflammation, we found that p38α activity in Langerhans cells (LCs), a skin-resident subset of DCs, promoted the generation of T cells that produce IL-17, a proinflammatory cytokine that is implicated in autoimmune disease. Deletion of p38α in LCs, but not in other skin or circulating DC subsets or T cells, decreased T cell-mediated psoriasiform skin inflammation in mice. The activity of p38α in LCs specifically promoted IL-17 production from γδ and CD4+ T cells by increasing the abundance of IL-23 and IL-6, two cytokines that stimulate IL-17 secretion. Inhibition of p38 activity through either pharmacological inhibition or genetic deletion also reduced the severity of established psoriasiform skin inflammation. Together, our findings indicate a critical role for p38α signaling in LCs in promoting inflammatory responses in the skin and suggest that targeting p38α signaling in LCs may offer an effective therapeutic approach to treat psoriasis.