Evidence of immunogenic cancer cell death induced by honey-processed Astragalus polysaccharides in vitro and in vivo.

Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which improves immune activity. This pharmacological activity of honey-processed Astragalus polysaccharide (HP-APS) might be due to structural changes during the honey roasting process. Previously, we have prepared and characterized HP-APS and preliminarily found its anti-inflammatory effects. However, whether the pharmacodynamic activity of HP-APS induces tumor cell apoptosis and the mechanisms responsible for the immunogenic death (ICD) have not been elucidated. Here, A549, MC38 and B16 cells were used to evaluate the cells viability, apoptosis and cell cycles, respectively. Cellular immunogenic cell death-related molecules calreticulin (CRT), Heat Shock Proteins (HSP)70, major histocompatibility complex I (MHC-I), and co-stimulator molecules CD80/CD86 were determined by flow cytometry. The extracellular ATP release was also detected. B16-OVA and MC38-OVA cells were treated with HP-APS and co-cultivated with OT1 mouse of CD3+T cells for assessment of proliferation, in mice model, and the establishment of C57BL/B6 mouse model bearing B16 cells for assessment of HP-APS the regulation of immune activity in vivo. Our results showed that HP-APS has an inhibitory effect on tumor cell proliferation, which induces tumor cell apoptosis, preventing cells-transforming from G1 phase to S phase in cell cycles. Furthermore, HP-APS could effectively increase the expression of HSP70, CRT, MHC-I, CD86, CD80 and ATP release. T cell proliferation index is significantly improved. CD3 cell proliferation in OT1 mice was significantly increased from the 4th generation to the 5th generation. Moreover, the results have also shown that HP-APS could inhibit tumor growth by increasing immune cell infiltration in the tumor tissues. In the mouse melanoma model with HP-APS treatment, the tumor weight and volume were significantly reduced, and the growth of melanoma was inhibited. CD8+ T is significantly increased. The ratio of CD4+ T and CD8+ T cells numbers are also significantly increased in mouse spleen, but it is less than PD-1 alone treatment separately. Altogether, these findings suggest that HP-APS exerts anti-tumor effects, and that its underlying mechanisms might be associated with the expression of immunogenicity cell death related molecules and the immunomodulatory effects of immune cells.

Application of pathways activity profiling to urine metabolomics for screening Qi-tonifying biomarkers and metabolic pathways of honey-processed Astragalus.

Honey-processed Astragalus, a widely used Qi-tonifying and immunomodulating herb in traditional Chinese medicine, has strengthened the tonic effects and achieved fewer side effects compared with astragali radix in clinical application. Here, we focus on Qi-tonifying biomarkers and pathways of honey-processed Astragalus using urine metabolomics that provide the basis for building the linkage between metabolites in rat urine and its symptoms. The spleen Qi deficiency model group, normal group, astragali radix group, and honey-processed Astragalus group were implemented to evaluate Qi-tonifying effects. Twelve potential biomarkers were screened by multivariate statistical analysis by using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Furthermore, pathways activity profiling showed unique pathways that are primarily involved in tryptophan metabolism, tricarboxylic acids cycle, and methionine metabolism. The results demonstrated that metabolomics coupled with pathway activity profiling were promising tools. It might serve as a novel methodological clue to systematically dissect the underlying Qi-tonifying mechanism of honey-processed Astragalus.

Structure Characterization of Honey-Processed Astragalus Polysaccharides and Its Anti-Inflammatory Activity In Vitro.

Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which strengthens the tonic effect. Astragalus polysaccharide (APS), perform its immunomodulatory effects by relying on the tonic effect of Radix Astragalus, therefore, the improved pharmacological activity of honey-processed Astragalus polysaccharide (HAPS) might be due to structural changes during processing. The molecular weights of HAPS and APS were 1,695,788 Da, 2,047,756 Da, respectively, as determined by high performance gel filtration chromatography combined with evaporative light scattering detection (HPGFC-ELSD). The monosaccharide composition was determined by ultra-performance liquid chromatogram quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) after pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The results showed that the essential components were mannose, glucose, xylose, arabinose, glucuronic acid and rhamnose, is molar ratios of 0.06:28.34:0.58:0.24:0.33:0.21 and 0.27:12.83:1.63:0.71:1.04:0.56, respectively. FT-IR and NMR analysis of HAPS results showed the presence of uronic acid and acetyl groups. The anti-inflammatory activities of HAPS were more effective than those of APS according to the NO contents and the expression of IFN-γ, IL-1ß, IL-22 and TNF-α in lipopolysaccharide (LPS)-induced RAW264.7 cells. This findings suggest that the anti-inflammatory and bioactivity improvement might be associated with molecular structure changes, bearing on the potential immunomodulatory action.

Androgen Signaling Contributes to Sex Differences in Cancer by Inhibiting NF-κB Activation in T Cells and Suppressing Antitumor Immunity.

Sex is known to be an important factor in the incidence, progression, and outcome of cancer. A better understanding of the underlying mechanisms could help improve cancer prevention and treatment. Here, we demonstrated a crucial role of antitumor immunity in the sex differences in cancer. Consistent with observations in human cancers, male mice showed accelerated tumor progression compared with females, but these differences were not observed in immunodeficient mice. Androgen signaling suppressed T-cell immunity against cancer in males. Mechanistically, androgen-activated androgen receptor upregulated expression of USP18, which inhibited TAK1 phosphorylation and the subsequent activation of NF-κB in antitumor T cells. Reduction of testosterone synthesis by surgical castration or using the small-molecular inhibitor abiraterone significantly enhanced the antitumor activity of T cells in male mice and improved the efficacy of anti-PD-1 immunotherapy. Together, this study revealed a novel mechanism contributing to sex differences in cancer. These results indicate that inhibition of androgen signaling is a promising approach to improve the efficacy of immunotherapy in males. SIGNIFICANCE: Androgen signaling induces immunosuppression in cancer by blocking T-cell activity through upregulation of USP18 and subsequent inhibition of NF-κB activity, providing a targetable axis to improve antitumor immunity in males.

Defined tumor antigen-specific T cells potentiate personalized TCR-T cell therapy and prediction of immunotherapy response.

Personalized immunotherapy targeting tumor-specific antigens (TSAs) could generate efficient and safe antitumor immune response without damaging normal tissues. Although neoantigen vaccines have shown therapeutic effect in clinic trials, precise prediction of neoantigens from tumor mutations is still challenging. The host antitumor immune response selects and activates T cells recognizing tumor antigens. Hence, T cells engineered with T-cell receptors (TCRs) from these naturally occurring tumor antigen-specific T (Tas) cells in a patient will target personal TSAs in his/her tumor. To establish such a personalized TCR-T cell therapy, we comprehensively characterized T cells in tumor and its adjacent tissues by single-cell mRNA sequencing (scRNA-seq), TCR sequencing (TCR-seq) and in vitro neoantigen stimulation. Compared to bystander T cells circulating among tissues, Tas cells were characterized by tumor enrichment, tumor-specific clonal expansion and neoantigen specificity. We found that CXCL13 is a unique marker for both CD4+ and CD8+ Tas cells. Importantly, TCR-T cells expressing TCRs from Tas cells showed significant therapeutic effects on autologous patient-derived xenograft (PDX) tumors. Intratumoral Tas cell levels measured by CXCL13 expression precisely predicted the response to immune checkpoint blockade, indicating a critical role of Tas cells in the antitumor immunity. We further identified CD200 and ENTPD1 as surface markers for CD4+ and CD8+ Tas cells respectively, which enabled the isolation of Tas cells from tumor by Fluorescence Activating Cell Sorter (FACS) sorting. Overall, our results suggest that TCR-T cells engineered with Tas TCRs are a promising agent for personalized immunotherapy, and intratumoral Tas cell levels determine the response to immunotherapy.

Dual Vascular Endothelial Growth Factor Receptor and Fibroblast Growth Factor Receptor Inhibition Elicits Antitumor Immunity and Enhances Programmed Cell Death-1 Checkpoint Blockade in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Combining anti-angiogenic therapy with immune checkpoint blockade with anti-programmed cell death-1 (PD-1) antibodies is a promising treatment for hepatocellular carcinoma (HCC). Tyrosine kinase inhibitors are well-known anti-angiogenic agents and offer potential for combination with anti-PD-1 antibodies. This study investigated the possible underlying immunomodulatory mechanisms of combined therapy. METHODS: HCC tissue samples for RNA-sequencing (RNA-seq) were obtained from patients with differential prognoses following anti-PD-1 treatment. Recombinant basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) were used to stimulate T cells following lenvatinib or sorafenib treatment, respectively. T cell function was analyzed by flow cytometry and lactate dehydrogenase assay. In vivo experiments were conducted in murine H22 and Hepa 1-6 competent models of HCC. Local immune infiltration in the tumor microenvironment (TME) was assessed using multicolor flow cytometry. Gene regulation was evaluated by RNA-seq. Microvascular density was measured by immunohistochemistry, and PD-1 ligand (PD-L1) induction was quantified by western blot. RESULTS: The baseline expression of VEGF and fibroblast growth factor (FGF) in patients with progressive disease was significantly higher than in patients achieving stable disease following anti-PD-1 treatment. VEGFA and bFGF significantly upregulated the expression of PD-1, cytotoxic T-lymphocyte-associated protein-4, and Tim-3 on T cells, while inhibiting the secretion of interferon gamma (IFNG) and granzyme B and suppressing T cell cytotoxicity. This immunosuppressive effect was reverted by lenvatinib but not sorafenib. Furthermore, dual lenvatinib/anti-PD-1 antibody therapy led to better antitumor effects than either sorafenib or fibroblast growth factor receptor (FGFR) inhibitor (BGJ398) in H22 murine models of HCC. Combined lenvatinib/anti-PD-1 treatment also led to long-term immune memory formation, while synergistically modulating the TME and enhancing the cytotoxic effect of T cells. Finally, lenvatinib inhibited PD-L1 expression on human umbilical vein endothelial cells, which improved the function of T cells. CONCLUSIONS: Inhibition of vascular endothelial growth factor receptor and FGFR augmented the efficacy of anti-PD-1 antibodies. Combined lenvatinib/anti-PD-1 treatment appears to exert antitumor activity by synergistically modulating effector T cell function in the TME and by mutually regulating tumor vessel normalization.

CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment. METHODS: We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells. RESULTS: The CLL-1 CAR-T cells specifically lysed CLL-1+ cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1+ myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression. CONCLUSIONS: CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

[Cloning and expression of human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells].

OBJECTIVE: To clone and express human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells. METHODS: Primers were designed based on the coding sequence for the human Oct4 cDNA. The fragment containing Oct4 gene, amplified by reverse transcription PCR (RT-PCR) from human HeLa cell cDNA as the template, was subcloned into pET-22b(+) and pcDNA3.1(+) vectors to construct the gene recombinant prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)-his-Oct4, respectively. Thereafter, the double enzymes digestion and the target gene sequencing were performed for identification. The prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)- his-Oct4 from the positive clones were respectively transformed into E.coli BL21 (DE3) and the embryo HEK293T cells for protein expression. RESULTS: DNA fragments (1100 bp) containing the coding region of Oct4 gene were obtained from RT-PCR. The recombinant expression plasmids were confirmed by enzymes digestion identification and sequencing analysis, proving both of the expression vectors were successfully constructed. SDS-PAGE analysis showed that the molecular mass of OCT4 expressed in the host E.coli BL21 (DE3) was 38 000, which was consistent with the theoretical prediction. Western blotting confirmed the expression of pcDNA3.1(+)-his-Oct4 transfected into HEK293T. CONCLUSION: Human Oct4 cDNA was cloned and its protein was successfully expressed in E.coli and HEK293T cells.