Immunophenotype and antitumor activity of cytokine-induced killer cells from patients with hepatocellular carcinoma.

BACKGROUND: Cytokine-induced killer (CIK) cells are heterogeneous lymphocytes from human peripheral blood mononucleated cells (PBMCs) co-cultured with several cytokines. The main purpose of this study is to evaluate the functional characteristics and anticancer ability of CIK cells from hepatocarcinoma (HCC) patients. METHODS: CIK cells were activated ex-vivo and expanded from PBMCs from HCC patients. The immunophenotype and the ex-vivo killing ability of CIK cells were evaluated. Human CIK cells were intravenously injected into NOD/SCID mice to evaluate the in vivo anticancer ability. RESULTS: More than 70% of CIK cells were CD3+CD8+, and 15%-30% were CD3+CD56+. These cells expressed an increased number of activated natural killer (NK) receptors, such as DNAM1 and NKG2D, and expressed low-immune checkpoint molecules, including PD-1, CTLA-4, and LAG-3. Among the chemokine receptors expressed by CIKs, CXCR3 and CD62L were elevated in CD8+ T cells, representing the trafficking ability to inflamed tumor sites. CIK cells possess the ex-vivo anticancer activity to different cell lines. To demonstrate in vivo antitumor ability, human CIK cells could significantly suppress the tumor of J7 bearing NOD/SCID mice. Furthermore, human immune cells could be detected in the peripheral blood and on the tumors after CIK injection. CONCLUSIONS: This study revealed that CIK cells from HCC patients possess cytotoxic properties, and express increased levels of effector NK receptors and chemokine molecules and lower levels of suppressive checkpoint receptors. CIK cells can suppress human HCC ex-vivo and in vivo. Future clinical trials of human CIK cell therapy for HCC are warranted.

China's practice to prevent and control COVID-19 in the context of large population movement.

BACKGROUND: The emerging infectious disease, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a serious threat in China and worldwide. Challenged by this serious situation, China has taken many measures to contain its transmission. This study aims to systematically review and record these special and effective practices, in hope of benefiting for fighting against the ongoing worldwide pandemic. METHODS: The measures taken by the governments was tracked and sorted on a daily basis from the websites of governmental authorities (e.g. National Health Commission of the People's Republic of China). And the measures were reviewed and summarized by categorizations, figures and tables, showing an ever-changing process of combating with an emerging infectious disease. The population shift levels, daily local new diagnosed cases, daily mortality and daily local new cured cases were used for measuring the effect of the measures. RESULTS: The practices could be categorized into active case surveillance, rapid case diagnosis and management, strict follow-up and quarantine of persons with close contacts, and issuance of guidance to help the public understand and adhere to control measures, plus prompt and effective high-level policy decision, complete activation of the public health system, and full involvement of the society. Along with the measures, the population shift levels, daily local new diagnosed cases, and mortality were decreased, and the daily local new cured cases were increased in China. CONCLUSIONS: China's practices are effective in controlling transmission of SARS-CoV-2. Considering newly occurred situations (e.g. imported cases, work resumption), the control measures may be adjusted.

Metabolic profiling of tyrosine kinase inhibitor nintedanib using metabolomics.

Nintedanib is a promising tyrosine kinase inhibitor for clinically treating idiopathic pulmonary fibrosis (IPF). Some clinical cases reported that nintedanib treatment can cause hepatotoxicity and myocardial toxicity. U. S. FDA warns the potential drug-drug interaction when it is co-administrated with other drugs. In order to understand the potential toxicity of nintedanib and avoid drug-drug interaction, the metabolism of nintedanib was systematically investigated in human liver microsomes and mice using metabolomics approach, and the toxicity of metabolites was predicted by ADMET lab. Nineteen metabolites were detected in vivo and in vitro metabolism, and 8 of them were undescribed. Calculated partition coefficients (Clog P) were used to distinguish the isomers of nintedanib metabolites in this study. The major metabolic pathways of nintedanib majorly included hydroxylation, demethylation, glucuronidation, and acetylation reactions. The ADMET prediction indicated that nintedanib was a substrate of the cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). And nintedanib and most of its metabolites might possess potential hepatotoxicity and cardiotoxicity. This study provided a global view of nintedanib metabolism, which could be used to understand the mechanism of adverse effects related to nintedanib and its potential drug-drug interaction.

Functional Change of Effector Tumor-Infiltrating CCR5+CD38+HLA-DR+CD8+ T Cells in Glioma Microenvironment.

Human glioma facilitates an impaired anti-tumor immunity response, including defects in circulation of T lymphocytes. The level of CD8+ T-cell activation acts as an immune regulator associated with disease progression. However, little is known about the characteristics of peripheral and tumor-infiltrating CD8+ T cells in patients with glioma. In this study, we examined the level of CD8+ T-cell activation in a group of 143 patients with glioma and determined that peripheral CD3+ T cells decreased in accordance with disease severity. The patients' peripheral CD8+ T-cell populations were similar to that of healthy donors, and a small amount of CD8+ tumor-infiltrating lymphocytes was identified in glioma tissues. An increase in activated CD8+ T cells, characterized as CD38+HLA-DR+, and their association with disease progression were identified in the patients' peripheral blood and glioma, and shown to display enriched CCR5+ and TNFR2+ expression levels. Ex vivo examination of CD38+HLA-DR+CD8+ T cells indicated that this subset of cells displayed stronger secretion of IFN-γ and IL-2 before and after a 6-h stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) relative to healthy CD38+HLA-DR+CD8+ T cells, indicating the functional feasibility of CD38+HLA-DR+CD8+ T cells. Higher CCL5 protein and mRNA levels were identified in glioma tissues, which was consistent with the immunohistochemistry results revealing both CCL5 and CD38+HLA-DR+CD8+ T cell expression. Patients' CCR5+CD38+HLA-DR+CD8+ T cells were further validated and shown to display increases in CD45RA+CCR7- and T-bet+ accompanied by substantial CD107-a, IFN-γ, and Granzyme B levels in response to glioma cells.

PPARα Mediates the Hepatoprotective Effects of Nutmeg.

Nutmeg is a Traditional Chinese Medicine used to treat gastrointestinal diseases. Some reports have indicated that nutmeg has hepatoprotective activity. In this study, a thioacetamide (TAA)-induced acute liver injury model in mice was used to explore the mechanism of the protective effects of nutmeg extract (NME), including its major bioactive component myrislignan. The results indicated that NME could effectively protect TAA-induced liver damage as assessed by recovery of increased serumtransaminases, decrease in hepatic oxidative stress, and lower hepatic inflammation. Metabolomics analysis further revealed that treatment with NME led to the recovery of a series of lipids including lysophosphatidylcholines that were decreased and a lowering of acylcarnitines that were increased in mouse plasma and liver after TAA exposure. Gene expression analysis demonstrated that the hepatoprotective effect of NME was achieved by modulation of the peroxisome proliferator-activated receptor alpha (PPARα) as well as the decrease in oxidative stress. NME could not protect from TAA-induced liver injury in Ppara-null mice, suggesting that its protective effect was dependent on PPARα. Myrislignan, a representative neolignan in nutmeg, showed potent protective activity against TAA-induced liver toxicity. These data demonstrate that nutmeg alleviates TAA-induced liver injury through the modulation of PPARα and that the lignan compounds in nutmeg such as myrislignan partly contributed to this action.

Memory Regulatory T cells Increase Only In Inflammatory Phase of Chronic Hepatitis B Infection and Related to Galectin-9/Tim-3 interaction.

CD4+Foxp3+ regulatory T cells (Tregs) are the main immune suppressors with subpopulation of inflamed-tissue related memory Tregs (mTregs) and non-related resting Treg (rTregs). Previously, Treg was proposed to be the cause of chronicity of hepatitis B virus (HBV) infection but with controversies. We then investigated the role of mTregs in distinct immune phases of chronic HBV infection, especially the non-inflammatory versus inflammatory phases. It was found mTregs but not rTregs increased only in the inflammatory phase and correlated with serum alanine aminotransferase (ALT) level. These mTregs accumulated in the inflamed liver, expressed significantly higher Tim-3, CCR4, CCR5 and fewer CCR7, and possessed potent suppressive function. These mTregs mainly originated from natural Tregs because of high Helios expression. Hierarchical clustering analysis showed higher frequency of mTreg was concordant with higher serum ALT and galectin-9 levels. Furthermore, galectin-9 could expand mTregs through galectin-9/Tim-3 interaction. In conclusion, increased mTregs are found only in inflammatory phase of chronic HBV infection. Galectin-9, associated with liver inflammation, contributes to the expansion of mTregs through galectin-9/Tim-3 interaction. Therefore, this expansion of mTregs only reflects as an immune regulatory mechanism to limit the on-going liver damages rather than the cause of chronicity of HBV infection.

CD4⁺ T cells expressing latency-associated peptide and Foxp3 are an activated subgroup of regulatory T cells enriched in patients with colorectal cancer.

Latency-associated peptide (LAP) - expressing regulatory T cells (Tregs) are important for immunological self-tolerance and immune homeostasis. In order to investigate the role of LAP in human CD4⁺Foxp3⁺ Tregs, we designed a cross-sectional study that involved 42 colorectal cancer (CRC) patients. The phenotypes, cytokine-release patterns, and suppressive ability of Tregs isolated from peripheral blood and tumor tissues were analyzed. We found that the population of LAP-positive CD4⁺Foxp3⁺ Tregs significantly increased in peripheral blood and cancer tissues of CRC patients as compared to that in the peripheral blood and tissues of healthy subjects. Both LAP⁺ and LAP⁻ Tregs had a similar effector/memory phenotype. However, LAP⁺ Tregs expressed more effector molecules, including tumor necrosis factor receptor II, granzyme B, perforin, Ki67, and CCR5, than their LAP⁻ negative counterparts. The in vitro immunosuppressive activity of LAP⁺ Tregs, exerted via a transforming growth factor-ß-mediated mechanism, was more potent than that of LAP⁻ Tregs. Furthermore, the enrichment of LAP⁺ Treg population in peripheral blood mononuclear cells (PBMCs) of CRC patients correlated with cancer metastases. In conclusion, we found that LAP⁺ Foxp3⁺ CD4⁺ Treg cells represented an activated subgroup of Tregs having more potent regulatory activity in CRC patients. The increased frequency of LAP⁺ Tregs in PBMCs of CRC patients suggests their potential role in controlling immune response to cancer and presents LAP as a marker of tumor-specific Tregs in CRC patients.

Activated but not resting regulatory T cells accumulated in tumor microenvironment and correlated with tumor progression in patients with colorectal cancer.

Activated T regulatory (T(reg)) cells are potent suppressors that mediate immune tolerance. We investigated the relationship between activated T(reg) cells and the progression of human colon cancer. We designed a cross-sectional study of CD4(+) Foxp3(+) T cells from peripheral blood, primary tumor and nontumor colon tissue of 42 patients with colon cancer and correlated the percentages of different subgroups of T(reg) cells with colon cancer stage. The phenotypes, cytokine-release patterns and suppression ability of these T(reg) cells were analyzed. We found that T(reg) cells increased significantly in both peripheral blood and cancer tissue. In addition, the T(reg) cells expressed significantly lower levels of CCR7, CD62L and CD45RA in comparison to normal volunteers. Further dividing T(reg) cells into subgroups based on Foxp3 and CD45RA expression revealed that both activated T(reg) cells (Foxp3(hi) CD45RA(-)) and nonsuppressive T(reg) cells (Foxp3(lo) CD45RA(-)), but not resting T(reg) cells (Foxp3(low) CD45RA(+)), increased in the peripheral blood and cancer tissue of patients with colon cancer. Only the activated T(reg) cells expressed significantly higher levels of tumor necrosis factor receptor 2 and cytotoxic T-cell antigen-4. Activated T(reg) cells, however, secreted significantly lower levels of effector cytokines (interleukin-2, tumor necrosis factor-α and interferon-γ) than did resting T(reg) cells and nonsuppressive cells upon ex vivo stimulation. Activated, but not resting, T(reg) cells in cancer tissue correlated with tumor metastases. In summary, we confirmed that activated T(reg) cells are a distinct subgroup with effector memory phenotype and fully functional regulatory activity against human colorectal cancer immunity.

LAP+CD4+ T cells are suppressors accumulated in the tumor sites and associated with the progression of colorectal cancer.

PURPOSE: Suppressor T cells are one of the determinants of colorectal cancer (CRC) clinical outcome. LAP(+)CD4(+) T cell is a recently identified subset of suppressor T cells. This study was designed to investigate their clinical relevance in patients with CRC. EXPERIMENTAL DESIGN: Sixty patients with CRC and 24 healthy donors (HD) were enrolled in this study. The percentages of LAP(+)CD4(+) T cells in peripheral blood and tumor tissue were measured. The phenotype and functional relevance of LAP(+)CD4(+) T cells were analyzed subsequently. RESULTS: The percentages of LAP(+)CD4(+) T cells in peripheral blood of patients with CRC were significantly higher than HD (HD vs. CRC: 3.1% ± 0.78% vs. 8.8% ± 5.8%, P < 0.0001) and in tumor tissue when compared with nontumor tissue (nontumor vs. tumor: 3.2% ± 1.1% vs. 9.5% ± 5.5%, P = 0.0002). In addition, LAP(+)CD4(+) T cells with effector memory (EM) phenotype were more likely to accumulate in the tumor sites than in peripheral blood. These LAP(+)CD4(+) T cells produced significantly higher levels of IFN-γ, IL-17 and comparatively lower IL-2 and very few IL-10. LAP(+)CD4(+) T cells could suppress the proliferation of LAP(-)CD4(+) T cells that were partially mediated by TGF-ß. Furthermore, these LAP(+)CD4(+) T cells accumulated in tumor site and increased further in the peripheral blood in patients with metastasis. CONCLUSIONS: LAP(+)CD4(+) T cells as a suppressor subset could accumulate in the tumor microenvironment and circulated more in the peripheral blood with tumor progression in patients with CRC.