Decreased Expression of Programmed Death Ligand-L1 by Seven in Absentia Homolog 2 in Cholangiocarcinoma Enhances T-Cell-Mediated Antitumor Activity.

N6-methyladenosine (m6A) has been reported as an important mechanism of post-transcriptional regulation. Programmed death ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. In addition, seven in absentia homolog 2 (Siah2), a RING E3 ubiquitin ligase, has been involved in tumorigenesis and cancer progression. However, the role of m6A-METTL14-Siah2-PD-L1 axis in immunotherapy remains to be elucidated. In this study, we showed that METTL14, a component of the m6A methyltransferase complex, induced Siah2 expression in cholangiocarcinoma (CCA). METTL14 was shown to enrich m6A modifications in the 3'UTR region of the Siah2 mRNA, thereby promoting its degradation in an YTHDF2-dependent manner. Furthermore, co-immunoprecipitation experiments demonstrated that Siah2 interacted with PD-L1 by promoting its K63-linked ubiquitination. We also observed that in vitro and in vivo Siah2 knockdown inhibited T cells expansion and cytotoxicity by sustaining tumor cell PD-L1 expression. The METTL14-Siah2-PD-L1-regulating axis was further confirmed in human CCA specimens. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with low Siah2 levels were more sensitive to anti-PD1 immunotherapy. Taken together, our results evidenced a new regulatory mechanism of Siah2 by METTL14-induced mRNA epigenetic modification and the potential role of Siah2 in cancer immunotherapy.

Corrigendum: Decreased expression of programmed death ligand-L1 by seven in absentia homolog 2 in cholangiocarcinoma enhances T-cell-mediated antitumor activity.

[This corrects the article DOI: 10.3389/fimmu.2022.845193.].

[Genetic diversity of Pseudosciaena polyactis in Zhoushan based on mitochondrial DNA D-loop region sequences].

Pseudosciaena polyactis is an economically important species of marine fish in China that is currently declining due to overexploitation, environmental pollution and related factors. Research in to the genetic structure of Pseudosciaena polyactis populations plays a key role in protecting and promoting sustainable utilization. We collected 53 individuals of Pseudosciaena polyactis from Zhoushan, Zhejiang and sequenced and amplified the mitochondrial DNA (mtDNA) D-loop region using Polymerase Chain Reactions (PCR). The sequence length of the 53 individuals ranged from 795 to 801 bp. The sequences were analyzed by Clustal X1.83, MEGA3.1 and DnaSP4.0. The results showed that the average base content of T, C, A, G was 30.3%, 23.1%, 32.3% and 14.3%, respectively and there were 93 transition or transversion sites, including 53 single nucleotide mutation sites and 40 parsimony informative sites, which accounted for 11.6% of the length of the analyzed sequences. In total, we identified 52 haplotypes and found haplotype diversity (hd) of 0.9993, average number of nucleotide differences were 9.73875 (k), and nucleotide diversity (Π) of 0.01233. The average genetic distance of haplotypes was 0.012, and the average transition/transversion was 4.305. Based on mitochondrial DNA D-loop region sequences, these results indicate that the genetic diversity of the Pseudosciaena polyactis population in Zhoushan is currently at a medium level.

[IgVH mutation status in patients with chronic lymphocytic leukemia].

OBJECTIVE: To evaluate the frequency and mutation status of IgVH gene expression in patients with chronic lymphocytic leukemia (CLL) in China. METHODS: IgVH mutation was detected by multiplex PCR and directly sequencing in 29 CLL patients. IgH somatic hypermutation and mutation site were analysed by IMGT/V-QUEST. RESULTS: Of 29 CLL patients, 21 had IgVH mutation (72%). The most frequently expressed VH gene family was found to be VH3 (55%) followed by VH4 (38%), VH2 (3.5%) and VH7 (3.5%), with no expression of VH1, VH5 and VH6. CONCLUSIONS: The expression frequency of IgVH gene families in Chinese CLL patients is significantly different from that in Western CLL patients, suggesting the involvement of ethnic and/or environmental factors in CLL development, which might partly explain the different incidence of CLL between China and Western countries.

[Expression of CLLU1 in patients with chronic lymphocytic leukemia and its prognostic significance].

OBJECTIVE: To investigate CLLU1 expression and its relationship with clinical stage, expression of CD38 and ZAP-70, and chromosome abnormalities in patients with chronic lymphoid leukemia (CLL). METHODS: Fifty CLL patients were studied. Semiquantitative RT-PCR was performed to detect CLLU1 expression levels; three-color flow cytometry to detect the ZAP-70 and CD38 expression, interphase fluorescence in situ hybridization (FISH) to detect chromosomal aberrations. RESULTS: The expression of CLLU1 mRNA was detected in 26 of the 50 cases (52%), of them 7 cases (26.92%) in Binet A and 19 (73. 08%) in Binet B + C. The expression levels of CLLU1 were significantly higher in Binet stage B + C than in Binet stage A (P < 0. 01). Among 24 CD38+ CLL cases, 17 (70. 83%) expressed high CLLU1 mRNA, whereas in 26 CD38- CLL patients only 9 (34.62%) were CLLU1 positive. The expression of CLLU1 in CD38+ CLL was significantly higher than that in CD38- CLL (P < 0.05). However, no significant difference of CLLU1 expression was found between ZAP-70+ and ZAP-70- patients (P > 0. 05), and between chromosomal aberrations (P > 0. 05). CONCLUSIONS: CLLU1 expression was significantly associated with clinical stage and CD38 expression, and might be an important prognostic factor in CLL patients.

[Application of a four antibody (cMPO/cCD79aalpha/cCD3/CD45) combination to the diagnosis of acute leukemia expressing cross-lineage antigens].

OBJECTIVE: To explore the diagnostic value of intracellular antibody combination in acute leukemia (AL) expressing cross-lineage cell-surface antigens. METHODS: Flow cytometric immunophenotyping using intracellular antibody combination (cMPO/cCD79alpha/cCD3/CD45) was performed additionally in 60 patients who expressed cross-lineage antigens from 269 previously untreated adult AL. RESULTS: Fifty-four of 269 previously untreated adult AL patients who expressed only one kind of intracellular antigen were diagnosed as cross-lineage AL, the percentage of cross-lineage AL in T cell acute lymphoblastic leukemia (T-ALL), B-ALL and acute myeloid leukemia (AML) was 28.6%, 43.6% and 13.4%, respectively. The positive rate of CD7, CD19, CD5 and CD20 in cross-lineage AML was 65.4%, 15.4%, 11.5%, and 7.7%, respectively. The positive rate of CD13, CD33 and CD15 in cross-lineage ALL was 89.3%, 21.4% and 3.6%, respectively. Six (2.3%) patients expressed two-lineage intracellular antigens were diagnosed as biphenotypic AL: 2 of T/B type and 4 B/M (B/myeloid) type. CONCLUSION: Intracellular antibodies possess lineage specificity and four-color combination flow cytometric immunophenotyping can provide fast and multi-parameter data. To ensure accuracy of the results, CD45/SSC gating and normal cells as internal reference should be used in the immunophenotyping of abnormal cells.

[Multiple myeloma cell line U266 apoptosis induced by velcade].

To investigate the effect of velcade on multiple myeloma cell line U266 apoptosis and its mechanism, cell viability was estimated by trypan blue dye exclusion. Annexin-V, mitochondrial transmembrane potential (delta psi m) and reactive oxygen species (ROS) labeled by DCFHDA were examined by flow cytometry, the expression of bcl-2 mRNA was detected by semi-quantitative RT-PCR. The results showed that the velcade inhibited the growth of U266 cells and reduced cell viability accompanied by appearance of morphologic characteristics of apoptosis. Velcade at 50 nmol/L increased Annexin V positivity and fluorescence intensity of DCF because of ROS generation while it decreased the delta psi m of U266 cells. Expression of anti-apoptotic gene bcl-2 mRNA also decreased. It is concluded that velcade inhibited the growth and reduce cell viability of U266 cells. Velcade can induce U266 cells apoptosis by intrinsic cell apoptotic pathway.

[Detection of IgVH mutation status in patients with chronic lymphocytic leukemia by multiplex PCR].

IgVH mutation status is one of the most important independent prognostic factor of chronic lymphocytic leukemia (CLL). In order to evaluate IgVH mutation status in patients with CLL, IgVH mutation was detected by multiplex PCR in 9 CLL patients and purified PCR amplification products were directly sequenced, IgH somatic hypermutation and mutation site were analysed by IMGT/V-QUEST. The results showed that 5 patients had mutated IgVH, and IgVHs were IGHV3-11*03, IGHV3-9*01, IGHV3-23*01, IGHV4-59*01, IGHV4-34*02, respectively; whereas 4 others had unmutated IgVH, these IgVHs were IGHV3-53*01, IGHV3-23*03, IGHV3-33*05 and IGHV3-7*01. It is concluded that multiplex PCR is a rapid and easy method to detect IgVH mutation status, and it solves the limitations and pitfalls of routine PCR, and it is worth being extensively used both in clinic and scientific researches.