Recent data suggest that vascular endothelial growth factor receptor inhibitor (VEGFRi) can enhance the anti-tumor activity of the anti-programmed cell death-1 (anti-PD-1) antibody in colorectal cancer (CRC) with microsatellite stability (MSS). However, the comparison between this combination and standard third-line VEGFRi treatment is not performed, and reliable biomarkers are still lacking. We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi (combination group, n=54) or VEGFRi alone (VEGFRi group, n=32), and their efficacy and safety were evaluated. We additionally examined the immune characteristics of the MSS CRC tumor microenvironment (TME) through single-cell and spatial transcriptomic data, and an MSS CRC immune cell-related signature (MCICRS) that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort. Compared with VEGFRi alone, the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit (median progression-free survival: 4.4 vs. 2.0 months, P=0.0024; median overall survival: 10.2 vs. 5.2 months, P=0.0038) and a similar adverse event incidence. Through single-cell and spatial transcriptomic analysis, we determined ten MSS CRC-enriched immune cell types and their spatial distribution, including naive CD4⁺ T, regulatory CD4⁺ T, CD4⁺ Th17, exhausted CD8⁺ T, cytotoxic CD8⁺ T, proliferated CD8⁺ T, natural killer (NK) cells, plasma, and classical and intermediate monocytes. Based on a systemic meta-analysis and ten machine learning algorithms, we obtained MCICRS, an independent risk factor for the prognosis of MSS CRC patients. Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation, and hence a significant relation with the superior efficacy of pan-cancer immunotherapy. More importantly, the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort. Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity. MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.
Humans ; Colorectal Neoplasms/drug therapy* ; Male ; Female ; Immunotherapy ; Middle Aged ; Aged ; Tumor Microenvironment/immunology* ; Retrospective Studies ; Microsatellite Instability ; Transcriptome ; Single-Cell Analysis ; Programmed Cell Death 1 Receptor/immunology* ; Gene Expression Profiling ; Immune Checkpoint Inhibitors/therapeutic use* ; Adult ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors*

Objective: To dynamically observe the clinical efficacy of entecavir and the changes of PD-1⁺CXCR5⁺CD4⁺T lymphocytes and sPD-1 levels in peripheral blood of HBeAg-positive chronic hepatitis B virus carriers treated with entecavir, and further explore its clinical significance. Methods: There were 31 cases of chronic hepatitis B virus carriers in the treatment group (A), 32 cases of chronic hepatitis B virus carriers in the treatment group (B), and 15 cases of chronic hepatitis B virus carriers in the non-treatment group (C).Three groups peripheral blood samples and clinical data at 0, 24 and 48 weeks were collected and compared. PD-1⁺CXCR5⁺CD4⁺T lymphocytes were detected by flow cytometry, and the level of sPD-1 was detected by enzyme-linked immunosorbent assay. ANOVA and Spearman correlation analysis were performed on the measurement data among the three groups. Results: At week 0, the serum levels of HBsAg, HBeAg and HBV DNA were significantly higher in groups A and C than group B. PD-1⁺CXCR5⁺CD4⁺T lymphocytes in peripheral blood were significantly higher in group B (4.70%±1.58%) than group A (3.25%±1.01%) and group C (2.77%±0.67%) (F=16.65, P<0.05). There was no significant difference between group A and group C (P>0.05). Peripheral blood sPD-1 in group B [(1 866.62±1 472.70) pg/ml] was significantly higher than group A [(824.86±538.66) pg/ml] and group C [(618.19±602.62) pg/ml] (F=10.95, P<0.05). There was no significant difference between group A and group C (P>0.05). At 48 weeks, the serum HBsAg did not decrease significantly in groups A and C than baseline (P>0.05), but were significantly higher than group B (P<0.05). Serum HBeAg levels were decreased significantly in groups A and B than baseline (P<0.05). <0.05), but group A was significantly higher than group B (P<0.05), and there was no significant difference between group A and group C (P>0.05). Serum HBV DNA level was significantly lower in groups A and B than group C (P<0.05), and there was no significant difference between group A and group B (P>0.05). Peripheral blood PD-1⁺CXCR5⁺CD4⁺T lymphocytes were significantly lower in Group A (1.56%±0.73%) and group B (1.32%±0.43%) than group C (2.64%±0.85%) (P<0.05). Peripheral blood sPD-1 were significantly lower in group A [(289.05±215.86) pg/ml] and group B [(236.01±173.92) pg/ml] than group C [(650.34±598.46) pg/ml] (P<0.05). There was no significant difference between group A and group B. Correlation analysis results: In group A at 48 weeks, the decreased level of PD-1⁺CXCR5⁺CD4⁺T lymphocyte ratio had no correlation with the decreased level of HBsAg and HBV DNA, but was positively correlated with the decreased level of HBeAg (r=0.376, P<0.05). The decreased level of sPD-1 had no correlation with the changes of HBsAg, but was positively correlated with the decreased levels of HBeAg and HBV DNA (r=0.598 and 0.384, P<0.05). In group B at 48 weeks, the decreased levels of PD-1⁺CXCR5⁺CD4⁺T lymphocytes and sPD-1 were positively correlated with the decreased levels of HBsAg, HBeAg, and HBV DNA (P<0.05). Conclusion: Hepatitis B virus replication and expressions in HBeAg-positive chronic hepatitis B virus carriers were significantly inhibited after 48 weeks of antiviral treatment, which is related not only to entecavir treatment, but also to the immunological mechanism involved in sPD-1. Moreover, the inhibition of HBeAg expression is associated with a decrease in the number and/or activity of PD-1⁺CXCR5⁺CD4⁺T lymphocytes.
Antiviral Agents/therapeutic use* ; DNA, Viral ; Guanine/analogs & derivatives* ; Hepatitis B Surface Antigens ; Hepatitis B e Antigens ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic ; Humans ; Programmed Cell Death 1 Receptor ; Receptors, CXCR5/analysis* ; T-Lymphocytes

Objective: To elucidate the expression levels of key immune biomarkers, phosphate and tension homology deleted on chromosome ten (PTEN) and programmed cell death protein1(PD-1),of different immune tolerance pathway in classic Hodgkin's lymphoma (CHL) to further determine their clinical role and prognostic significance. Methods: The clinical features and prognostic factors of 56 CHL patients, who were admitted to the TianJin Medical University Cancer Institute from February 2003 to August 2013, were retrospectively analyzed. PTEN and PD-1 protein expression levels were analyzed by immunohistochemistry, Epstein-Barr virus encoded RNA (EBER) was performed by in situ hybridization assay. Correlations between the expression of biomarkers and clinicopathologic parameters were examined and survival analyses were performed. Results: This cohort of 56 CHL patients included 34 males and 22 females with a median age of 25 years (ranged from 7 to 71 years). In a univariate analysis, age≥45, IPS score >2, EBER positive, high expression of PTEN protein conferred inferior 5-year OS and 5-year PFS; In a multivariate model, age≥45, IPS score >2, EBER positive, high expression of PTEN protein were identified as the independent adverse prognostic factors for CHL. Conclusions: This study suggested for the first time that PTEN was independent prognostic immune biomarkers in CHL, which provided the novel therapeutic strategy of immune therapy for CHL.
Adolescent ; Adult ; Aged ; Child ; Female ; Hodgkin Disease ; Humans ; Male ; Middle Aged ; PTEN Phosphohydrolase/analysis* ; Prognosis ; Programmed Cell Death 1 Receptor/analysis* ; Retrospective Studies ; Young Adult

BACKGROUNDHost immune responses against hepatitis B virus (HBV) induced by antiviral therapy play a crucial role in viral clearance. To further investigate the immune mechanisms underlying the differences between respondents and non-respondents, we analyzed myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), FoxP3(+) regulatory T cells (FoxP3(+) Treg) and programmed death 1 (PD-1) expression in CD4(+)/CD8(+) T cells in chronic hepatitis B patients undergoing pegylated interferon (PegIFN)α-2b treatment.

METHODSPatients received PegIFNα-2b for 24 or 48 weeks, with follow-up at 24 weeks. The frequencies of mDCs, pDCs, FoxP3(+) Treg, and PD-1 expression by CD4(+)/CD8(+) T cells were evaluated by flow cytometry at baseline, weeks 4 and 12, end of treatment, and follow-up (12/24 weeks).

RESULTSIn HBeAg seroconverters (respondents), the mDC relative frequency decreased at week 4 and then rebounded at week 12. The pDC relative frequency decreased consistently. In non-HBeAg seroconverters (non-respondents), both mDC and pDC frequencies decreased slightly. The FoxP3(+) Treg relative frequency decreased during treatment and remained low during follow-up in respondents, while in non-respondents it decreased slightly during therapy but rebounded after discontinuation. In patients with HBeAg < 17.55 PEI-U/ml at week 12 and < 8.52 PEI-U/ml at week 24, the FoxP3(+) Treg frequency decreased during treatment and at follow-up. In respondents, CD4(+)PD-1 and CD8(+)PD-1 levels decreased at week 4 and remained low at week 12. In non-respondents, PD-1 expression decreased at week 4 but rebounded at week 12.

CONCLUSIONSThe results indicate that the dynamic changes in DCs, FoxP3(+) Treg frequency, and PD-1 expression by CD4(+) and CD8(+) T cells exhibit different trends in HBeAg and non-HBeAg seroconversion patients. During PegIFNα-2b treatment of chronic hepatitis B patients, these changes may be of predictive value for HBeAg seroconversion. HBsAg and HBeAg levels are related to FoxP3(+) Treg frequency.

Adult ; Antiviral Agents ; therapeutic use ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; DNA, Viral ; blood ; Female ; Forkhead Transcription Factors ; analysis ; Hepatitis B ; drug therapy ; immunology ; Hepatitis B e Antigens ; blood ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Polyethylene Glycols ; therapeutic use ; Programmed Cell Death 1 Receptor ; analysis ; Recombinant Proteins ; therapeutic use ; T-Lymphocytes ; immunology ; T-Lymphocytes, Regulatory ; immunology

BACKGROUNDOxymatrine has certain antiviral effects in the treatment of chronic hepatitis B (CHB), but its exact mechanism is unclear. The objective of the present study was to explore oxymatrine's antiviral mechanism by studying its effect on the hepatitis B virus (HBV) specific cytotoxic T lymphocyte (CTL) surface programmed death receptor-1 (PD-1) expression in CHB patients.

METHODSSixty-five CHB patients who had HBV DNA(3)10(4) copies/ml, positive HBeAg, positive human leukocyte antigen (HLA)-A2, alanine aminotransferase (ALT) > 2 x upper limit of normal value (ULN) were randomly divided into two groups: treatment group (n = 33), treated with an intravenous infusion of 600 mg oxymatrine in glucose solution once a day for a month, then with a 200 mg oxymatrine oral capsule three times a day, and a 200 mg silibin meglumine tablet three times a day; control group (n = 32) patients were treated only with silibin meglumine tablet, method and dosage were the same as those of treatment group. Three months later, peripheral blood HBV-specific CTL surface PD-1 expression, HBV-specific CTL level, HBV DNA, HBeAg, and results of liver function tests were analyzed and compared.

RESULTSThree months post-treatment, in the treatment group, peripheral blood HBV-specific CTL surface PD-1 expression ((19.42 ± 15.94)%) decreased significantly compared to the pretreatment level ((31.30 ± 24.06)%; P < 0.05), and decreased significantly compared to that of control group three months after treatment ((29.45 ± 21.62)%; P < 0.05). HBV-specific CTL level ((0.42 ± 0.07)%) significantly increased compared with the pretreatment ((0.29 ± 0.15)%; P < 0.01), and the control group posttreatment level was (0.31 ± 0.15)% (P < 0.05). HBV DNA level in 11 cases became negative (HBV DNA < 500 copies/ml, 33.33%), which was higher than that of the control group after treatment (two cases, 6.25%; χ(2) = 7.45, P < 0.01), HBeAg of nine cases turned negative (27.27%), which was higher than that of the control group after treatment (one case, 3.13%; χ(2) = 7.27, P < 0.01).

CONCLUSIONOxymatrine could downregulate peripheral blood HBV-specific CTL surface PD-1 expression in CHB patients, increase HBV-specific CTL level, which may be one of the possible mechanisms by which oxymatrine clears or inhibits HBV in CHB patients.

Adult ; Alkaloids ; therapeutic use ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; drug therapy ; immunology ; Humans ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor ; analysis ; Quinolizines ; therapeutic use ; T-Lymphocytes, Cytotoxic ; chemistry

OBJECTIVETo establish a mouse model for human chronic HBV infection, and to investigate the role of PD-1/PD-L1 signaling pathway in antiviral immunity.

METHODSA mouse model was established by hydrodynamic injection of the plasmid pAAV/HBV1.2-GFP into the tail vein of C57BL/6 mice, HBV markers were assayed at different time points after injection. After intraperitoneal injection of anti-PD-L1 monoclonal antibody, the serum ALT, and HBV DNA in the serum, liver and kidney were assayed.

RESULTSThe chronic HBV infection mouse model were established successfully, serum HBsAg and high load of HBV DNA were detectable 90 days after plasmid injection. After blocking of the PD-1/PD-L1 pathway, the serum ALT level of mice were significantly increased (P < 0.01), and the HBV DNA load in serum (P < 0.01), liver (P < 0.05) and kidney (P < 0.05) were decreased significantly.

CONCLUSIONBlocking the PD-1/PD-L1 signaling pathway can enhance antiviral response in mice with chronic HBV infection.

Animals ; Antigens, Surface ; metabolism ; Apoptosis Regulatory Proteins ; metabolism ; B7-1 Antigen ; metabolism ; B7-H1 Antigen ; DNA, Viral ; analysis ; Disease Models, Animal ; Hepatitis B virus ; metabolism ; Hepatitis B, Chronic ; immunology ; metabolism ; virology ; Male ; Membrane Glycoproteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Peptides ; metabolism ; Programmed Cell Death 1 Receptor ; Signal Transduction

OBJECTIVETo clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.

METHODSThe human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing.

RESULTSThe PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing.

CONCLUSIONThe human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.

Amino Acid Sequence ; Antigens, CD ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Apoptosis Regulatory Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; metabolism ; Humans ; Polymerase Chain Reaction ; Programmed Cell Death 1 Receptor ; Prokaryotic Cells ; metabolism ; Sequence Alignment ; Sequence Analysis, DNA