primary human hepatocytes (phhs)  (Thermo Fisher)

Journal: JHEP Reports

Article Title: The dual role of TIGIT in regulatory and effector T cells in chronic liver disease

doi: 10.1016/j.jhepr.2025.101405

Figure Lengend Snippet: Hepatic recruitment and residency markers are upregulated on TIGIT + Tregs. TIGIT + FoxP3 + cells reside around hepatocytes in AIH livers and the TIGIT ligand-CD155 is expressed on inflamed hepatocytes. Ex vivo Tregs from patient peripheral blood and explant livers were phenotyped using flow cytometry. Details of samples used for flow cytometry experiments are in unless stated otherwise. Each data point represents values for an individual patient. (A) Representative histogram (CXCR3 and CD69) or contour plot (CD29/VLA-4) showing the expression of hepatic recruitment and residency markers by live CD3 + CD4 + CD25 + CD127 low TIGIT + Tregs as determined by flow cytometry. In the histograms, grey represents isotype, black represents positive stain. (B) Paired analysis comparing expression of the hepatic recruitment markers CXCR3, VLA-4, and tissue residency marker CD69 by blood and liver TIGIT + and TIGIT - Tregs using flow cytometry. Statistical analysis was performed using mixed-effects analysis, with the Geisser–Greenhouse correction and Šídák’s multiple comparisons test, with individual variances for each comparison. p values are displayed for statistically significant comparisons only ( p <0.05). (C) Immunohistochemistry staining of AIH explant liver sections and non-cirrhotic donor liver sections for TIGIT (brown). (C–E) The right panels represent a magnified view of the area shown on the left (red box). (D) Immunohistochemistry staining for CD155 (brown) on AIH and non-cirrhotic donor explant liver. (E) Representative confocal micrograph showing immunofluorescence staining for TIGIT (red) and FoxP3 (green) in an AIH explant section. Blue = DAPI. (F–G) Quantification of (F) CD155 intensity and (G) TIGIT frequency from immunohistochemistry staining of AIH explant liver and non-cirrhotic donor liver using eight randomly selected fields of view analysed using QuPath. Error bars represent standard error of mean (SEM). Statistical analysis was performed using an unpaired t test. p values are displayed for each statistical comparison made. AIH, autoimmune hepatitis; CDB, chronic liver disease blood; CLD, chronic liver diseases; DiL, disease liver; DoL, donor liver; HCB, healthy control blood; TIGIT, T cell immunoreceptor with Ig and ITIM domains; Tregs, regulatory T cells.

Article Snippet: Primary human hepatocytes (PHHs; Thermo Fisher Scientific, Waltham, Massachusettes, USA) were thawed in thawing media (#CM7500, Thermo Fisher Scientific, Waltham, Massachusettes, USA), washed, and plated in a collagen-coated 96-well flat-bottomed plate in hepatocyte plating media (#CM3000, Thermo Fisher Scientific, Waltham, Massachusettes, USA).

Techniques: Ex Vivo, Flow Cytometry, Expressing, Staining, Marker, Comparison, Immunohistochemistry, Immunofluorescence, Control

Journal: JHEP Reports

Article Title: The dual role of TIGIT in regulatory and effector T cells in chronic liver disease

doi: 10.1016/j.jhepr.2025.101405

Figure Lengend Snippet: TIGIT interactions on TIGIT-expressing CD8 + T cells inhibit hepatocyte apoptosis. (A) Representative histogram of granzyme B and perforin expression, gated on live CD3 + CD8 + TIGIT + T cells. Grey represents isotype, black represents positive stain. (B) Paired comparison of granzyme B and perforin expression by TIGIT + and TIGIT - CD8 + T cells. Statistical tests were conducted using mixed-effects analysis, with the Geisser–Greenhouse correction and Tukey’s multiple comparisons test, with individual variances computed for each comparison. Details of samples used for flow cytometry are in . p values are displayed for statistically significant comparisons only ( p <0.05). (C) PBMCs from AIH patient blood were used for flow cytometry staining for naive T cell markers, CD45RA and CCR7, gated on live CD3 + CD8 + TIGIT + or TIGIT - T cells. (D) Paired comparison of antigen-experienced marker CD40L expression on TIGIT + and TIGIT - CD8 + T cells. Statistical analysis was performed using a mixed-effects analysis, with the Geisser–Greenhouse correction and Šídák’s multiple comparisons test, with individual variances for each comparison. (E) ICC staining of PHHs in co-culture with either TIGIT - or TIGIT + (magenta) labelled CD8 + T cells (CellTracker™ Red/CTR, orange). T cells were isolated from blood derived from patients with AIH using FACS and rested overnight before labelling and co-culture with PHHs. (F) Hepatocyte cell death after 24 h in co-culture with TIGIT - or TIGIT + CD8 + T cells or TIGIT - or TIGIT + Tconv cells in the presence or absence of a TIGIT neutralising antibody (monoclonal; 5 μg/ml) Data were collected from two individual biological repeats. Statistical analysis was performed using one-way ANOVA, with p values shown for statistically significant comparisons only ( p <0.05). Error bars represent the standard error of the mean (SEM). (G) Time-lapse images showing hepatocyte cell death. Images were taken every 30 min of PHH and TIGIT - CD8 + T cells labelled with CellTrace Red (CTR) in culture. The red arrow points towards an immune cell, and the white arrow points towards an apoptosing hepatocyte. The grey image shows the acquisition of phase gradient. (H) Representative images of ICC staining showing the expression of granzyme B (white) on either TIGIT - CD8 + T cells or TIGIT + CD8 T cells (CTR, magenta) and a magnified image of the interaction between a TIGIT - CD8 + T cell (CTR, magenta) and hepatocytes (CellTracker™ Green/CTG, yellow) or the lack of interaction between a TIGIT + CD8 + T cell and hepatocytes. AIH, autoimmune hepatitis; AIHB, AIH patient blood; CDB, chronic liver disease blood; DiL, disease liver; DoL, donor liver; HCB, healthy control blood; ICC, immunocytochemistry; PBMCs, peripheral blood mononuclear cells; PHHs, primary human hepatocytes; Tconv, conventional T cells; TIGIT, T cell immunoreceptor with Ig and ITIM domains.

Article Snippet: Primary human hepatocytes (PHHs; Thermo Fisher Scientific, Waltham, Massachusettes, USA) were thawed in thawing media (#CM7500, Thermo Fisher Scientific, Waltham, Massachusettes, USA), washed, and plated in a collagen-coated 96-well flat-bottomed plate in hepatocyte plating media (#CM3000, Thermo Fisher Scientific, Waltham, Massachusettes, USA).

Techniques: Expressing, Staining, Comparison, Flow Cytometry, Marker, Co-Culture Assay, Isolation, Derivative Assay, Control, Immunocytochemistry

Journal: Journal of Lipid Research

Article Title: Impaired ApoB secretion triggers enhanced secretion of ApoE to maintain triglyceride homeostasis in hepatoma cells

doi: 10.1016/j.jlr.2025.100795

Figure Lengend Snippet: Effects of MTPi and DGAT1/2 depletion on ApoE secretion in PHHs. A: Phase contrast microscopy of primary human hepatocytes (PHHs). Scale bar, 50 μm. B: RT-qPCR determination of APOE and APOB mRNA levels in Huh-7.5 cells versus PHHs. ∗∗∗ P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). C: Relative abundances of ApoE and ApoB in supernatants and lysates of PHHs treated with MTPi (2.5 μM) or DMSO. Immunoblots are shown below. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, two-tailed Student's t test). D: Effects of neuraminidase and O -glycosidase treatments on the post-translational modification of ApoE. Relative abundances of ApoE and ApoB in supernatants of PHHs treated as indicated. Immunoblots are shown below. Upper and lower arrowheads show sialylated and desialylated ApoE. ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). E: RT-qPCR determination of APOE , ST6GAL1 , and ST6GALNAC6 mRNA levels in PHHs treated with MTPi. (n = 3, two-tailed Student's t test). F: Immunoblots of ApoE in supernatants and lysates from Huh-7.5 cells treated with neuraminidase as indicated. Upper and lower arrowheads show sialylated and desialylated ApoE. Relative abundance of ApoE is shown on right. ∗ P < 0.05, ∗∗∗ P < 0.001 (n = 4, two-way ANOVA with Sidak’s multiple comparisons test). G: Relative abundances of DGAT1 and DGAT2 mRNAs in Huh-7.5 cells versus PHHs (left). DGAT1 and DGAT2 mRNA levels in PHHs transfected with indicated siRNAs are shown on right. ∗ P < 0.05, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). H: Relative abundance of ApoE in supernatants of PHHs transfected with indicated siRNAs (left panel). Immunoblots are shown below. ∗ P < 0.05 (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). Relative cell numbers of PHHs transfected with indicated siRNAs as estimated by total RNA content are shown on right. I: Scheme showing the sialylation of secreted ApoE in response to disabled ApoB secretion in PHHs.

Article Snippet: Primary human hepatocytes (PHHs) were purchased from PhoenixBio and maintained in DMEM supplemented with 10% FBS, 20 mM HEPES (Thermo Fisher Scientific), 44 mM sodium bicarbonate, antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin), 15 μg/ml L-proline (Wako), 0.25 μg/ml insulin, 50 nM dexamethasone (Sigma-Aldrich), 5 ng/ml epidermal growth factor (Millipore), 0.1 mM L-ascorbic acid (Wako), and 2% dimethyl sulfoxide (Sigma-Aldrich).

Techniques: Microscopy, Quantitative RT-PCR, Western Blot, Control, Two Tailed Test, Modification, Transfection