The role of cGAMP via the STING pathway in modulating germinal center responses and CD4 T cell differentiation.

Germinal center (GC) responses are essential for establishing protective, long-lasting immunity through the differentiation of GC B cells (BGC) and plasma cells (BPC), along with the generation of antigen-specific antibodies. Among the various pathways influencing immune responses, the STING (Stimulator of Interferon Genes) pathway has emerged as significant, especially in innate immunity, and extends its influence to adaptive responses. In this study, we examined how the STING ligand cGAMP can modulate these key elements of the adaptive immune response, particularly in enhancing GC reactions and the differentiation of BGC, BPC, and follicular helper T cells (TFH). Employing in vivo models, we evaluated various antigens and the administration of cGAMP in Alum adjuvant, investigating the differentiation of BGC, BPC, and TFH cells, along with the production of antigen-specific antibodies. cGAMP enhances the differentiation of BGC and BPC, leading to increased antigen-specific antibody production. This effect is shown to be type I Interferon-dependent, with a substantial reduction in BPC frequency upon interferon (IFN)-ß blockade. Additionally, cGAMP's influence on TFH differentiation varies over time, which may be critical for refining vaccine strategies. The findings elucidate a complex, antigen-specific influence of cGAMP on T and B cell responses, providing insights that could optimize vaccine efficacy.

Cytokines in Follicular Helper T Cell Biology in Physiologic and Pathologic Conditions.

Follicular helper T cells (Tfh) play a crucial role in generating high-affinity antibodies (Abs) and establishing immunological memory. Cytokines, among other functional molecules produced by Tfh, are central to germinal center (GC) reactions. This review focuses on the role of cytokines, including IL-21 and IL-4, in regulating B cell responses within the GC, such as differentiation, affinity maturation, and plasma cell development. Additionally, this review explores the impact of other cytokines like CXCL13, IL-10, IL-9, and IL-2 on GC responses and their potential involvement in autoimmune diseases, allergies, and cancer. This review highlights contributions of Tfh-derived cytokines to both protective immunity and immunopathology across a spectrum of diseases. A deeper understanding of Tfh cytokine biology holds promise for insights into biomedical conditions.

Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers.

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

The Chromatin Regulator Mll1 Supports T Follicular Helper Cell Differentiation by Controlling Expression of Bcl6, LEF-1, and TCF-1.

T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage-defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation.

Single-cell and spatial sequencing application in pathology.

Traditionally, diagnostic pathology uses histology representing structural alterations in a disease's cells and tissues. In many cases, however, it is supplemented by other morphology-based methods such as immunohistochemistry and fluorescent in situ hybridization. Single-cell RNA sequencing (scRNA-seq) is one of the strategies that may help tackle the heterogeneous cells in a disease, but it does not usually provide histologic information. Spatial sequencing is designed to assign cell types, subtypes, or states according to the mRNA expression on a histological section by RNA sequencing. It can provide mRNA expressions not only of diseased cells, such as cancer cells but also of stromal cells, such as immune cells, fibroblasts, and vascular cells. In this review, we studied current methods of spatial transcriptome sequencing based on their technical backgrounds, tissue preparation, and analytic procedures. With the pathology examples, useful recommendations for pathologists who are just getting started to use spatial sequencing analysis in research are provided here. In addition, leveraging spatial sequencing by integration with scRNA-seq is reviewed. With the advantages of simultaneous histologic and single-cell information, spatial sequencing may give a molecular basis for pathological diagnosis, improve our understanding of diseases, and have potential clinical applications in prognostics and diagnostic pathology.

Bcl6-Mediated Transcriptional Regulation of Follicular Helper T cells (TFH).

Follicular helper T cells (TFH) are essential B cell-help providers in the formation of germinal centers (GCs), affinity maturation of GC B cells, differentiation of high-affinity antibody-producing plasma cells, and production of memory B cells. The transcription factor (TF) B cell lymphoma 6 (Bcl6) is at the center of gene regulation in TFH biology, including differentiation and function, but how Bcl6 does this, and what additional TFs contribute, remain complex questions. This review focuses on advances in our understanding of Bcl6-mediated gene regulation of TFH functions, and the modulation of TFH by other TFs. These advances may have important implications in deciphering how repressor TFs can regulate many immunological cell types. An improved understanding of TFH biology will likely provide insights into biomedically relevant diseases.

Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.

T follicular helper (TFH) cells are a distinct type of CD4+ T cells that are essential for most antibody and B lymphocyte responses. TFH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl-6 controls TFH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits.

Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf.

Ezh2 is an histone methyltransferase (HMT) that catalyzes H3K27me3 and functions in TH1, TH2, and Treg cells primarily via HMT activity. Here we show that Ezh2 ablation impairs T follicular helper (TFH) cell differentiation and activation of the TFH transcription program. In TFH cells, most Ezh2-occupied genomic sites, including the Bcl6 promoter, are associated with H3K27ac rather than H3K27me3. Mechanistically, Ezh2 is recruited by Tcf1 to directly activate Bcl6 transcription, with this function requiring Ezh2 phosphorylation at Ser21. Meanwhile, Ezh2 deploys H3K27me3 to repress Cdkn2a expression in TFH cells, where aberrantly upregulated p19Arf, a Cdkn2a protein product, triggers TFH cell apoptosis and antagonizes Bcl6 function via protein-protein interaction. Either forced expression of Bcl6 or genetic ablation of p19Arf in Ezh2-deficient cells improves TFH cell differentiation and helper function. Thus, Ezh2 orchestrates TFH-lineage specification and function maturation by integrating phosphorylation-dependent transcriptional activation and HMT-dependent gene repression.

The prognosis in cases of hepatocellular carcinoma after hepatectomy: young patients versus older patients.

BACKGROUNDS/AIMS: Hepatocellular carcinoma (HCC) is uncommon in young adults and the prognosis of these patients is still unclear. In this retrospective study, we compared the clinicopathological characteristics and outcomes of young patients with HCC with those of older patients with HCC. METHODS: We retrospectively reviewed the clinicopathological characteristics of a total of 1,124 patients with HCC who underwent hepatectomy at our institution between 2006 and 2010. Patients ≤40 years of age at the time of HCC diagnosis were classified in the younger group. RESULTS: One hundred and three patients (9.2%) were classified in the younger group. whereas, 1021 patients were classified in the older group. The incidences of hepatitis B virus infection, alpha-fetoprotein (AFP) levels, and indocyanine green retention test were all higher in younger patients than in older patients (p<0.05). Disease-free survival and overall survival were longer in older patients than in younger patients, without statistical significance. In younger patients, increased levels of protein induced by vitamin K antagonist-II (PIVKA-II) and alkaline phosphatase, portal vein tumor thrombosis, and intrahepatic metastasis were all predisposing factors for tumor recurrence after hepatectomy. CONCLUSIONS: Although the AFP levels were higher in younger patients with HCC than in older patients with HCC, disease-free survival and overall survival after liver resection were not significantly different between the two groups.

Effective suppression of pro-inflammatory molecules by DHCA via IKK-NF-κB pathway, in vitro and in vivo.

BACKGROUND AND PURPOSE: Dehydrodiconiferyl alcohol (DHCA), a lignan compound isolated from Cucurbita moschata, has previously been shown to contain anti-adipogenic and antilipogenic effects on 3T3-L1 cells and mouse embryonic fibroblasts. As some of phytochemicals derived from natural plants show anti-inflammatory or antioxidative activities, we determined whether DHCA affects the production of pro-inflammatory mediators and also investigated its underlying mechanisms. EXPERIMENTAL APPROACH: Raw264.7, a murine macrophage cell line, and primary murine macrophages derived from bone marrow cells were treated with LPS in the presence of DHCA. Furthermore, cells were treated with LPS and palmitate in the presence of DHCA to examine its effect on inflammasomes. The production of various pro-inflammatory mediators was examined and the underlying mechanisms investigated using a variety of molecular biological techniques. To test whether DHCA exhibits anti-inflammatory effects in vivo, mouse dextran sodium sulfate (DSS)-induced colitis model was used. KEY RESULTS: DHCA reduced the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß and CCL2) and mediators (iNOS, COX-2 and ROS) by down-regulating the activity of I-κB kinase and, subsequently, the DNA binding activity of NF-κB. Moreover, DHCA effectively suppressed the palmitate-mediated activation of inflammasomes, which resulted in decreased production of IL-1ß. DHCA also showed therapeutic effects in the mouse DSS-induced colitis model by suppressing the production of TNF-α and IL-1ß and thus preventing weight loss and colon shrinkage. CONCLUSIONS AND IMPLICATIONS: Our data suggest that DHCA is a novel phytochemical that by regulating key molecules involved in inflammation and oxidative stress might exert a broad range of anti-inflammatory activities.

Ameliorating effects of HX106N, a water-soluble botanical formulation, on Aß25-35-induced memory impairment and oxidative stress in mice.

It is well-established that amyloid ß (Aß)-induced oxidative stress plays a crucial role in Alzheimer's disease (AD) and its cognitive deficits. HX106N is a water-soluble extract prepared from a mixture of the plants Dimocarpus longan, Liriope platyphylla, Salvia miltiorrhiza, and Gastrodia elata. These ingredients are traditionally used in various plant-based medicines for the treatment of neurological disease. In this study, we examined the effects of HX106N on memory impairment and oxidative stress caused by the intracerebroventricular injection of Aß25-35 peptide in mice. For one week prior to Aß25-35 peptide injection and 8 d after, mice were given oral HX106N. HX106N treatment reversed the Aß25-35-mediated decrease in alternation percentage and latency time in the Y-maze and passive avoidance tests. Mice treated with HX106N showed decreased levels of thiobarbituric acid reactive substances (TBARS), a lipid peroxidation marker. Quantitative reverse transcription polymerase chain reaction (RT-PCR) demonstrated that HX106 treatment increased levels of heme oxygenase-1 (HO-1) in the hippocampus of Aß25-35-injected mice, while having little effect on the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. In the murine hippocampal neuronal cell line HT22, HX106N was found to upregulate HO-1 expression at the RNA and protein levels as well as to protect cells from glutamate-induced oxidative stress. Taken together, our data suggest that HX106N may potentially act as a preventive and/or therapeutic agent for AD.

Suppressive effects of PG201, an antiarthritic botanical formulation, on lipopolysaccharide-induced inflammatory mediators in Raw264.7 cells.

PG201, an ethanol extract from a mixture of 12 herbs, has strong antiarthritic activity. To understand the molecular mechanisms underlying its anti-inflammatory effects, PG201-mediated suppression of inflammatory mediators was studied in Raw264.7, a mouse macrophage cell line. PG201 decreased the expression of interleukin (IL)-1ß, IL-6 and CC chemokine ligand-2, but not tumor necrosis factor-α, at the protein and mRNA levels in lipopolysaccharide-stimulated Raw264.7 cells. Results from a gel retardation assay indicated that PG201 substantially reduced the DNA-binding activity of the activator protein-1 and cyclic adenosine monophosphate-responsive element-binding protein transcription factors, but not nuclear factor-κB. Western blot and Northern blot analyses showed that PG201 reduced inducible nitric oxide synthase and cytosolic phospholipase A(2) (cPLA(2)) protein expression, but did not affect mRNA expression, ultimately resulting in decreased nitric oxide and prostaglandin E(2). The protein expression of cPLA(2) was decreased by PG201 in the presence of cycloheximide, an inhibitor of translation, suggesting that PG201 may facilitate the degradation of cPLA(2). Taken together, these results suggest that PG201 selectively affects the expression of proteins that play key roles in the inflammatory response at transcriptional and post-translational levels.

Treatment of patients with clinically lymph node-negative squamous cell carcinoma of the oral cavity.

OBJECTIVE: To evaluate treatment outcome and to determine optimal treatment strategy for patients with clinically lymph node-negative (N0) oral cavity squamous cell carcinoma (SCC). METHODS: Two hundred and twenty-seven patients with oral cavity SCC received radiotherapy with curative intent. We retrospectively analyzed 69 patients with clinically N0 disease. Forty-three patients were treated with surgery followed by radiotherapy (S+EBRT) and 26 with radiotherapy alone (EBRT). The median doses administered were 63.0 Gy for S+EBRT and 70.2 Gy for EBRT. RESULTS: The rates of occult metastasis were 60% for T1, 69% for T2, 100% for T3 and 39% for T4, respectively, among patients who underwent neck dissection. A contralateral occult metastasis occurred only in two patients. The median follow-up was 39 months (range, 6-170 months). The 5-year overall survival (OS), disease-free survival (DFS), local control (LC) and regional control (RC) rates for all patients were 56, 50, 66 and 79%, respectively. The 5-year OS, DFS, LC and RC rates were 67/39% (P < 0.01), 66/24% (P < 0.01), 87/30% (P < 0.01) and 73/89% (P = 0.11) for S+EBRT/EBRT, respectively. CONCLUSIONS: The risk for occult neck metastasis is high in patients with oral cavity SCC; therefore, elective neck treatment should be considered. Excellent RC for subclinical disease can be achieved with radiotherapy alone. However, external beam radiotherapy alone to primary tumor resulted in poor LC and combined treatment with surgery and radiotherapy appeared to be a better treatment strategy.