5-aminolevulinic acid photodynamic therapy using 560-1200 nm followed by 420-1200 nm broadband light in the treatment of moderate-to-severe acne.

BACKGROUND: Moderate-to-severe acne vulgaris, which is a chronic inflammatory skin disease, seriously impacts millions of people. However, traditional therapies may cause severe adverse reactions that are unacceptable to many patients, thus limiting the further application of these therapies. Novel therapeutic approaches to effectively treat moderate-to-severe acne vulgaris with minimal adverse reactions are urgently needed. In this retrospective study, we investigated the efficacy and adverse reactions of photodynamic therapy (PDT) using 560-1200 nm followed by 420-1200 nm broadband light (BBL). METHODS: Twenty-four patients with moderate-to-severe acne vulgaris were included in the study and all patients expressed a strong desire for beauty. After aminolevulinic acid (ALA) gel applied, the entire face was sequentially irradiated by using BBL with a 560 nm cut-off filter (560-1200 nm), followed by BBL with a 420 nm cut-off filter (420-1200 nm). The clinical efficacy was evaluated by the proportion of patients achieving cured response and excellent response (effective rate), based on the percentage of lesions reduction (treatment rate). The fluorescent images and photographs of acne vulgaris were recorded. Pain and other common local adverse reactions during the treatment were also recorded and evaluated. RESULTS: In patients with moderate acne, the mean treatment rates were 57.74 ± 16.40 (%) and 87.40 ± 8.521 (%) at the 6th week and 12th week of treatment, respectively. In patients with severe acne, the mean treatment rates were 60.95 ± 12.06 (%) and 85.04 ± 9.115 (%) at the 6th week and 12th week of treatment, respectively. At the 6th and 12th weeks of treatment, the effective rates of patients were 20.00 % and 93.33 % in patients with moderate acne, and 0.000 % and 88.89 % in patients with severe acne, respectively. Pain scores were significantly higher in patients with severe acne compared to patients with moderate acne when receiving 560-1200 nm BBL-PDT. Additionally, patients when receiving 420-1200 nm BBL-PDT exhibited significantly higher pain scores than those when receiving 560-1200 nm BBL-PDT. The degree of erythema was more severe in patients with severe acne than in those with moderate acne. The pigmentation was observed in one patient with moderate acne and one patient with severe acne. CONCLUSION: The 560-1200 nm and 420-1200 nm BBL-PDT therapy can effectively treat moderate-to-severe acne vulgaris with tolerable adverse reactions, providing a new option for patients with higher esthetic requirements.

The role of CD4+ T cells in tumor and chronic viral immune responses.

Immunotherapies are mainly aimed to promote a CD8+ T cell response rather than a CD4+ T cell response as cytotoxic T lymphocytes (CTLs) can directly kill target cells. Recently, CD4+ T cells have received more attention due to their diverse roles in tumors and chronic viral infections. In antitumor and antichronic viral responses, CD4+ T cells relay help signals through dendritic cells to indirectly regulate CD8+ T cell response, interact with B cells or macrophages to indirectly modulate humoral immunity or macrophage polarization, and inhibit tumor blood vessel formation. Additionally, CD4+ T cells can also exhibit direct cytotoxicity toward target cells. However, regulatory T cells exhibit immunosuppression and CD4+ T cells become exhausted, which promote tumor progression and chronic viral persistence. Finally, we also outline immunotherapies based on CD4+ T cells, including adoptive cell transfer, vaccines, and immune checkpoint blockade. Overall, this review summarizes diverse roles of CD4+ T cells in the antitumor or protumor and chronic viral responses, and also highlights the immunotherapies based on CD4+ T cells, giving a better understanding of their roles in tumors and chronic viral infections.

Albendazole induces an anti-tumor effect and potentiates PD-L1 blockade immunotherapy.

BACKGROUND: Previously, albendazole (ABZ) has been reported as an anti-parasitic drug rather than anti-tumor drug. Our study aim to investigate whether ABZ also has a potential anti-tumor effect by shaping the tumor immune microenvironment and interrogate whether ABZ could synergize with the PD-L1 blockade. METHODS: C57BL/6 mice (C57) were intravenously injected with B16F10-luciferase (B16-luc) cells to establish a lung metastatic melanoma model and subcutaneously inoculated with B16-luc cells to establish a subcutaneous tumor model. The tumor volume and tumor metastasis loci of the mice were measured by a vernier caliper and in vivo imaging. RNA sequencing was performed to analyze the different genes and pathways of immune cells in the tumors. Flow cytometry and immunofluorescence were used to analyze the different subsets of tumor-infiltrating immune cells. RESULTS: The results suggested that ABZ significantly inhibited lung melanoma metastasis with decreased fluorescence intensity and nodule score and mediated the regression of subcutaneous melanoma in mice with decreased tumor volume. Moreover, RNA sequencing results showed that ABZ regulated the gene expression levels and pathways of immune cells in the tumor microenvironment (TME). Meanwhile, flow cytometry and immunofluorescence showed that the number and percentage of CD8+ T cells, CD4+ T cells, and TH1 cells were enhanced in tumors after ABZ treatment. Furthermore, the combination of ABZ and anti-PD-L1 treatment significantly potentiated anti-tumor efficacy in both lung metastasis and subcutaneous melanoma models and mediated an increase in the percentage of CD8+ T cells, CD4+ T cells, and TH1 cells as compared to the control group. CONCLUSION: ABZ inhibits melanoma growth and metastasis. Moreover, ABZ synergized with PD-L1 blockade mediates tumor regression.

The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes.

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

EZH2 restricts Tcf7 DNA methylation and promotes TFH differentiation during acute viral infection.

Follicular helper T (TFH) cells provide specialized help for B cells to ensure optimal humoral immunity. The histone methyltransferase EZH2, as a chromatin repressor, secures the TFH differentiation by promoting TFH lineage associated gene expression during acute viral infection, including Tcf7 and Bcl6. By using conditional deletion murine system, we observed that EZH2 ablation in CD4+ T cells was accompanied by aberrant accumulation of DNA methyltransferases (DNMTs) DNMT1 and DNMT3B in TFH cells. And the loss of EZH2 promoted aggravation of DNA methylation status at Tcf7 locus. Therefore, our findings suggested that EZH2 plays an important role in maintenance of hypomethylation at Tcf7 locus thus affecting TFH differentiation during acute viral infection.

CD4+ T-cell epitope-based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy.

BACKGROUND: Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy. METHODS: Listeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61-80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing. RESULTS: CD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells. CONCLUSION: CD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

Low-Dose Albendazole Inhibits Epithelial-Mesenchymal Transition of Melanoma Cells by Enhancing Phosphorylated GSK-3ß/Tyr216 Accumulation.

Albendazole (ABZ) is an effective broad-spectrum anthelmintic agent that has been widely used for humans and animals. Previous studies have reported that ABZ exhibits antitumor effects against melanoma and other different cancer types; however, it is unknown whether ABZ exerts the inhibitory effect against melanoma metastasis. In this study, we aimed to investigate the inhibitory effect of ABZ on melanoma cells. Through in vitro studies, we discovered that low-dose ABZ treatment significantly inhibited the migration and invasion, but not the proliferation, of A375 and B16-F10 cells in a dose-dependent manner. Further analysis revealed that ABZ treatment reduced the expression level of snail family transcriptional repressor 1 (Snail) in the cytoplasm and nucleus by decreasing the levels of phosphorylated AKT (pAKT) Ser473/GSK-3ß (pGSK-3ß) Ser9 and increasing pGSK-3ß/Tyr216, resulting in a significant upregulation of E-cadherin and downregulation of N-cadherin and ultimately reversing the epithelial-mesenchymal transition (EMT) process of melanoma cells. In contrast, the continuous activation of AKT via transfected plasmids elevated the protein levels of pAKT Ser473/pGSK-3ß Ser9 and Snail and antagonized the inhibitory action of ABZ. We also confirmed that ABZ treatment effectively inhibited the lung metastasis of melanoma in nude mice in vivo. Subsequent immunohistochemical analysis verified the decreased pAKT Ser473/pGSK-3ß Ser9 and increased pGSK-3ß/Tyr216 levels in ABZ-treated subcutaneous tumors. Therefore, our findings demonstrate that ABZ treatment can suppress the EMT progress of melanoma by increasing the pGSK-3ß/Tyr216-mediated degradation of Snail, which may be used as a potential treatment strategy for metastatic melanoma.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis. Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Bcl6 Preserves the Suppressive Function of Regulatory T Cells During Tumorigenesis.

During tumorigenesis, tumor infiltrating regulatory T (Treg) cells restrict the function of effector T cells in tumor microenvironment and thereby promoting tumor growth. The anti-tumor activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of various types of human cancers. However, the immune suppressive function of Treg cells remains a major hurdle to broader effectiveness of tumor immunotherapy. In this article, we reported that the deletion of Bcl6 specifically in Treg cells led to stunted tumor growth, which was caused by impaired Treg cell responses. Notably, Bcl6 is essential in maintaining the lineage stability of Treg cells in tumor microenvironment. Meanwhile, we found that the absence of follicular regulatory T (Tfr) cells, which is a result of Bcl6 deletion in Foxp3+ cells, was dispensable for tumor control. Importantly, the increased Bcl6 expression in Treg cells is associated with poor prognosis of human colorectal cancer and lymph node metastasis of skin melanoma. Furthermore, Bcl6 deletion in Treg cells exhibits synergistic effects with immune checkpoint blockade therapy. Collectively, these results indicate that Bcl6 actively participates in regulating Treg cell immune responses during tumorigenesis and can be exploited as a therapeutic target of anti-tumor immunity.

The histone methyltransferase EZH2 primes the early differentiation of follicular helper T cells during acute viral infection.

Epigenetic modifications to histones dictate the differentiation of naïve CD4+ T cells into different subsets of effector T helper (TH) cells. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the mechanism regulating the differentiation of TH1, TH2 and regulatory T (Treg) cells. However, whether and how EZH2 regulates follicular helper T (TFH) cell differentiation remain unknown. Using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, we observed abundant EZH2 expression and associated H3K27me3 modifications preferentially in the early committed virus-specific TFH cells compared to those in TH1 cells. Ablation of EZH2 in LCMV-specific CD4+ T cells leads to a selective impairment of early TFH cell fate commitment, but not late TFH differentiation or memory TFH maintenance. Mechanistically, EZH2 specifically stabilizes the chromatin accessibility of a cluster of genes that are important for TFH fate commitment, particularly B cell lymphoma 6 (Bcl6), and thus directs TFH cell commitment. Therefore, we identified the chromatin-modifying enzyme EZH2 as a novel regulator of early TFH differentiation during acute viral infection.

The Transcription Factor T-Bet Is Required for Optimal Type I Follicular Helper T Cell Maintenance During Acute Viral Infection.

Follicular helper T cells (TFH cells), known as the primary "helpers" of the germinal center (GC) reaction, promote the humoral immune response to defend against various pathogens. Under conditions of infection by different types of pathogens, many shared transcription factors (TFs), such as Bcl-6, TCF-1, and Maf, are selectively enriched in pathogen-specific TFH cells, orchestrating TFH cell differentiation and function. In addition, TFH cells also coexpress environmentally associated TFs as their conventional T cell counterparts (such as T-bet, GATA-3, or ROR-γt, which are expressed in Th1, Th2, or Th17 cells, respectively). These features likely indicate both the lineage-specificity and environmental adaption of the TFH cell responses. However, the extent to which the TFH cell response relies on these environmentally specific TFs is not completely understood. Here, we found that T-bet was specifically expressed in Type I TFH cells but not Type II TFH cells. While dispensable for the early fate commitment of TFH cells, T-bet was essential for the maintenance of differentiated TFH cells, promoting their proliferation, and inhibiting their apoptosis during acute viral infection. Microarray analysis showed both similarities and differences in transcriptome dependency on T-bet in TFH and TH1 cells, suggesting the distinctive role of T-bet in TFH cells. Collectively, our findings reveal an important and specific supporting role for T-bet in type I TFH cell response, which can help us gain a deeper understanding of TFH cell subsets.

A Novel Method for Constructing Histological Section Datasets of the Basal Ganglia in Digitized Human Brain.

To investigate the construction of the histological section datasets in the basal ganglia of digitized human brain to provide a reference for the meso-level histological data acquisition. A fresh adult brain from a cadaver with no neurological disease was selected, and tissue blocks of the basal ganglia in the right hemisphere was extracted using the visualization method, followed by pretreatments including gradient dehydrating, gelatin-embedding and setting of calibration points. And then the tissue blocks was cryosectioned into 60-µm-thick coronal sections and the sectional images were captured simultaneously by a digital camera at a fixed position. Two series of sections (one section out of ten) were Nissl-stained with Toluidine blue and immunostained with the calbindin D-28K, respectively. Stained sections were digitized by a high resolution scanner. After alignment and registration, contours of nuclei and different nucleic function divisions in the digital images of stained sections were identified, and then were segmented and labeled using software exploited by ourselves. Datasets of one set of registrated serial sectional images and two sets of registrated histochemically stained images in basal ganglia area were obtained, which provide a histological reference for the neurosurgery and diagnostic imaging. a systematic method of cutting, slicing, staining, data acquisition and image registration of large tissue blocks was established, providing a reference for histological data acquisition on the digital human. Anat Rec, 300:1011-1021, 2017. © 2016 Wiley Periodicals, Inc.