Immune analysis of fibrolamellar hepatocellular carcinoma.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 324-324 ◽  
Author(s):  
Mehmet Akce ◽  
Luis M Vence ◽  
Jorge M Blando ◽  
Padmanee Sharma ◽  
Manal Hassan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
Surgical Resection ◽  
Unmet Need ◽  
Immune Monitoring ◽  
Cancer Center ◽  
Flow Cytometry Analysis ◽  
Fibrolamellar Hepatocellular Carcinoma ◽  
Programmed Death

324 Background: Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare variant of hepatocellular carcinoma (HCC) with about 200 annual cases reported globally. Surgical resection is the main treatment. Although FLHCC develops in the absence of cirrhosis surrounding hepatic parenchyma may have mononuclear cell and lymphocyte infiltrates. Immunotherapy by targeting programmed death 1 (PD-1) has emerged as a potential therapy for HCC and multiple clinical trials are being conducted, however little is known about immunological features of FLHCC. We aimed to analyze tumor microenvironment by immunohistochemistry and flow cytometry. Methods: Surgical samples of two FLHCC cases, 23 year old woman (case 1) and 25 year old man (case 2) who underwent neoadjuvant chemotherapy with 5 Fluorouracil + Interferon followed by surgical resection were analyzed. Expression of CD4, CD8, PD-1, programmed death ligand 1(PD-L1), CD45RO, CD 57, CD68, OX 40, inducible costimulator (ICOS), FoxP3, Gr-B was evaluated by immunohistochemistry. Flow cytometry analysis of CD8, CD4 effector (Teff) and regulatory (Treg) T cells were performed. Immune monitoring studies were conducted by the Immunotherapy Platform at MD Anderson Cancer Center. Results: By immunohistochemistry,expression of PD-1 and PDL-1 in the tumor sampleswas asfollows:PD-1 (5.9%) and PDL-1 (4.1%) of total cells in case 1 versus PD-1 (6.3%) and PDL-1 (3.5%) in case 2. FoxP3 expression was 0.3% and 1%, whereas ICOS expression was 0.2% and 1% in case 1 and 2, respectively. By flow cytometry analysis, case 1 presented the following frequencies: CD8+CTLA4+ (40%), CD8+PD1+ (40%) of total CD8 T cells, CD4+TeffCTLA4+ (55%), CD4+TeffPD1+ (30%), CD4+TeffICOS+ (15%) of total CD4 Teff cells and CD4+TregCTLA+ (90%), CD4+TregPD1+ (30%), CD4+TregICOS+ (70%) of total CD4 Tregs. Similarly, in case 2 the percentages were CD8+CTLA-4+ (20%), CD8+PD1+ (40%), CD4+TeffCTLA4+ (25%), CD4+TeffPD1+ (65%), CD4+TeffICOS+ (30%), CD4+TregCTLA+ (90%), CD4+TregPD1+ (70%), CD4+TregICOS+ (80%). Conclusions: PD-1, PDL-1, CTLA-4 and ICOS expression in FLHCC might render support to consideration of potential utilization of antibodies against PD-1, PDL-1 and CTLA-4 for treatment for a devastating cancer with unmet need for treatment.

scholarly journals Cisplatin Increased the Expression of PD-1 and PD-L1 by YAP1 in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Liyuan Hao ◽  
Yinglin Guo ◽  
Qing Peng ◽  
Zhiqin Zhang ◽  
Shenghao Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
Cancer Cells ◽  
Flow Cytometry Analysis ◽  
Chemotherapy Drugs ◽  
The World ◽  
Hcc Cells

Abstract Hepatocellular carcinoma (HCC) was one of the most malignant cancers in the world. Cisplatin (DDP) was one of the main chemotherapy drugs for HCC, but the mechanism of DDP treatment for HCC remains unclear. In this presentation, we found that DDP inhibited the growth of HCC cells and promoted the expression of PD-1 and its ligand PD-L1 in cancer cells. Meanwhile, flow cytometry analysis revealed that DDP enhanced PD-1-CD8+ T cells expression and decreased PD-1+CD8+ T cells expression. ELISA analysis suggested that DDP decreased TGF-β expression in vivo. Therefore, the study indicated that DDP enhanced PD-1 and PD-L1 expression and inhibited the growth of HCC.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals Identification of Immune-Related Prognostic mRNA and lncRNA in Patients with Hepatocellular Carcinoma

2022 ◽  
Vol 2022 ◽  
pp. 1-16
Author(s):  
Dan Chen ◽  
Xiaoting Li ◽  
Hui Li ◽  
Kai Wang ◽  
Xianghua Tian
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
Regression Analysis ◽  
Survival Analysis ◽  
Immune Cells ◽  
Cox Regression ◽  
Differentially Expressed ◽  
Cox Regression Analysis ◽  
Immune Related Genes

Background. As the most common hepatic malignancy, hepatocellular carcinoma (HCC) has a high incidence; therefore, in this paper, the immune-related genes were sought as biomarkers in liver cancer. Methods. In this study, a differential expression analysis of lncRNA and mRNA in The Cancer Genome Atlas (TCGA) dataset between the HCC group and the normal control group was performed. Enrichment analysis was used to screen immune-related differentially expressed genes. Cox regression analysis and survival analysis were used to determine prognostic genes of HCC, whose expression was detected by molecular experiments. Finally, important immune cells were identified by immune cell infiltration and detected by flow cytometry. Results. Compared with the normal group, 1613 differentially expressed mRNAs (DEmRs) and 1237 differentially expressed lncRNAs (DElncRs) were found in HCC. Among them, 143 immune-related DEmRs and 39 immune-related DElncRs were screened out. These genes were mainly related to MAPK cascade, PI3K-AKT signaling pathway, and TGF-beta. Through Cox regression analysis and survival analysis, MMP9, SPP1, HAGLR, LINC02202, and RP11-598F7.3 were finally determined as the potential diagnostic biomarkers for HCC. The gene expression was verified by RT-qPCR and western blot. In addition, CD4 + memory resting T cells and CD8 + T cells were identified as protective factors for overall survival of HCC, and they were found highly expressed in HCC through flow cytometry. Conclusion. The study explored the dysregulation mechanism and potential biomarkers of immune-related genes and further identified the influence of immune cells on the prognosis of HCC, providing a theoretical basis for the prognosis prediction and immunotherapy in HCC patients.

scholarly journals B7-H4 Inhibits the Development of Primary Sjögren’s Syndrome by Regulating Treg Differentiation in NOD/Ltj Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xu Zheng ◽  
Qikai Wang ◽  
Xiang Yuan ◽  
Yingbo Zhou ◽  
Hui Chu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Salivary Glands ◽  
Inflammatory Cytokines ◽  
Mrna Levels ◽  
Lymphocyte Infiltration ◽  
Flow Cytometry Analysis ◽  
Treg Differentiation

Background. This study is aimed at exploring the role of B7-H4 in the pathogenesis of primary Sjögren’s syndrome (pSS) in NOD/Ltj mouse. Methods. B7-H4 expression in salivary glands was examined by IHC, and lymphocyte infiltration was showed by H&E. Next, anti-B7-H4 mAb or immunoglobulin isotype was injected into NOD/Ltj mice. Cytokine levels were measured by quantitative RT-PCR, and immunoglobulins were measured by ELISA. T cell subsets were analyzed by flow cytometry. Last, we treated NOD/Ltj mice with B7-H4Ig and control Ig. CD4+Foxp3+ T cells were assessed by immunohistochemistry. Two-tailed Student’s t-tests were used to detect the statistical difference in various measures between the two groups. Results. B7-H4 expression was remarkably reduced in salivary glands of NOD/Ltj mice at 15 weeks compared with the NOD/Ltj mice at 8 weeks. Anti-B7-H4 mAb treatment increased lymphocyte infiltration in salivary glands. Inflammatory cytokines including IL-12, IL-18, IL-1α, TNF-α, IFN-α, and BAFF were upregulated markedly in anti-B7-H4 mAb-treated mice compared to IgG isotype-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb-treated mice had lower levels of CD4+Foxp3+/CD4+ T cells in spleen. Moreover, Foxp3 mRNA levels of salivary glands were diminished in anti-B7-H4 mAb-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb inhibited CD4+Foxp3+/CD4+ T cell production, while B7-H4Ig would promote naïve CD4+ T into Treg differentiation. Administration with B7-H4Ig displayed significantly decreased lymphocyte infiltration in salivary glands and low levels of total IgM and IgG in serum. Analysis of inflammatory cytokines in salivary glands after B7-H4Ig treatment revealed that the mRNA levels of IL-12, IL-6, IL-18, IL-1α, TNF-α, and IFN-α were significantly downregulated in B7-H4Ig-treated mice compared to control Ig treatment. B7-H4Ig-treated mice had significantly higher levels of CD4+Foxp3+/CD4+ T cells in spleen. IHC in salivary gland revealed that CD4+Foxp3+ T cells of B7-H4Ig treatment mouse were more than control Ig treatment. Conclusions. Our findings implicate that B7-H4 has a protective role for salivary gland epithelial cells (SGECs) and therapeutic potential in the treatment of pSS.

scholarly journals Expression and clinical significance of LAG-3, FGL1, PD-L1 and CD8+T cells in hepatocellular carcinoma using Multiplex Quantitative Analysis

2020 ◽  
Author(s):  
Mengzhou Guo ◽  
Feifei Yuan ◽  
Feng Qi ◽  
Jialei Sun ◽  
Qianwen Rao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Prognostic Significance ◽  
Normal Liver ◽  
Patient Stratification ◽  
Programmed Death ◽  
Programmed Death 1 ◽  
Low Levels

Abstract Background: fibrinogen-like protein 1 (FGL1) - Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown.Methods: The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8+T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied.Results: We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3+cells but not PD-L1. CD8+ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3+cells and low levels of CD8+ T cells were correlated with poor disease outcome. Moreover, LAG-3+cells deteriorated patient stratification based on the abundance of CD8+ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC+) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC-). Furthermore, PD-L1 TC- in combination with high densities of LAG-3+cells showed the worst prognosis, and PD-L1 TC+ patients with low densities of LAG-3+cells had the best prognosis.Conclusions: LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3+cells and CD8+ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

scholarly journals Combination immunotherapy induces distinct T-cell repertoire responses when administered to patients with different malignancies

2020 ◽  
Vol 8 (1) ◽  
pp. e000368
Author(s):  
Jason Cham ◽  
Li Zhang ◽  
Serena Kwek ◽  
Alan Paciorek ◽  
Tao He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Metastatic Melanoma ◽  
Flow Cytometry Analysis ◽  
Cell Repertoire ◽  
Gm Csf ◽  
Cell Counts ◽  
Melanoma Patients ◽  
Circulating T Cells

BackgroundCTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.MethodsWe used next-generation sequencing of T-cell receptors (TCR) to compare the circulating T cells of melanoma and mCRPC patients receiving the same treatment with ipilimumab and GM-CSF by Wilcoxon rank sum test. Flow cytometry was utilized to investigate specific T-cell populations. TCR sequencing results were correlated with each T-cell subpopulation by Spearman’s rank correlation coefficient. Of note, 14 metastatic melanoma patients had samples available for TCR sequencing and 21 had samples available for flow cytometry analysis; 37 mCRPC patients had samples available for sequencing of whom 22 have TCR data available at both timepoints; 20 of these patients had samples available for flow cytometry analysis and 16 had data available at both timepoints.ResultsWhile melanoma and mCRPC patients had similar pretreatment circulating T-cell counts, treatment induces greater expansion of circulating T cells in melanoma patients. Metastatic melanoma patients have a higher proportion of clones that increased more than fourfold after the treatment compared with mCRPC patients (18.9% vs 11.0%, p=0.017). Additionally, melanoma patients compared with mCRPC patients had a higher ratio of convergent frequency (1.22 vs 0.60, p=0.012). Decreases in clonality induced by treatment are associated with baseline CD8+ T-cell counts in both patient groups, but are more pronounced in the melanoma patients (r=−0.81, p<0.001 vs r=−0.59, p=0.02).Trial registration numbersNCT00064129;NCT01363206.

scholarly journals Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

2020 ◽  
Vol 21 (11) ◽  
pp. 4180
Author(s):  
Jae Wook Jung ◽  
Jin Hong Chun ◽  
Jung Seok Lee ◽  
Si Won Kim ◽  
Ae Rin Lee ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Viral Infection ◽  
T Cell Subsets ◽  
Olive Flounder ◽  
Flow Cytometry Analysis ◽  
Color Flow

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

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