Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Elevated hepatic and serum ALCAM were observed in patients with AIH. (A) Representative immunohistochemistry images of ALCAM in liver biopsies from HC (n=3) and patients with AIH (n=6). (B) Representative immunofluorescence staining for CD4, CD6 and ALCAM in interface hepatitis lesion of liver sections from patients with AIH (n=3). (C) Concentration of serum ALCAM in HC (n=28) and AIH (n=86) was measured by ELISA assay. Individual correlation between clinical indicators and serum ALCAM was calculated in patients with AIH (n=86). (D) Correlation between the number of hepatic CD6 + cells and paired serum ALCAM concentration was calculated (n=27). ***p < 0.001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: ALCAM promoted CD6 high CD4 + T cells trans-endothelial migration in vitro . (A, B) Human CD4 + T cells were magnetically isolated from PBMC of healthy donors and stimulated with αCD3/28 for 3 days in a flat-bottom 96-well plate, the expression of CD6 and cell proliferation was detected with flow cytometry. (C) After stimulation, the expression of cytokines, surface markers and transcription factors was compared between the CD6 high and CD6 low subsets. (D) Pre-activated CD4 + T cells were placed on a transwell chamber with 5μm pore for 24 hours in the presence of rhALCAM (3ug/ml) or vehicle (PBS). The expression of CD6 and CD69 was measured by flow cytometry. Experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration, In Vitro, Isolation, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Schematic diagram for this study. Upregulated ALCAM on hepatocytes promoted the trans-endothelial migration of pathogenic CD6 high CD4 + T cells, which further aggravated hepatic inflammation of patients with AIH. This study revealed a putative therapeutic approach for patients with AIH.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration

Journal: Journal of Biological Chemistry

Article Title: dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

doi: 10.1074/jbc.m607530200

Figure Lengend Snippet: FIGURE 5. Dlk1 expression synergizes the effect of LPS-induced cytokines in hMSC cells and stimulates the B cell proliferation. A, control and hMSC-dlk1 cells were stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated and analyzed for the expression of the indicated genes by real time PCR. Expression of each target gene was normalized to -actin. Data are shown as means S.D. of three independent experiments. *, p 0.05; **, p 0.005 versus control cells (hMSC-TERT) induced with LPS. B, CD19-purified human peripheral blood B lymphocytes were cultured in CM obtained from hMSC-TERT (control cells) or hMSC-dlk1 cells as described under “Experimental Procedures” in the absence and the presence of 2.5 or 10 g/ml CD40L. Cell proliferation was measured with XTT-based assay. Stimulation in the cell proliferation was represented as fold induction over nonstimulated cells cultured in the corresponding CM. The bars are the means S.D. of five independent experiments. *, p 0.05; **, p 0.005 versus cultured cells in control/CM.

Article Snippet: Cells were then resuspended at 0.5 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated).

Techniques: Expressing, Control, Isolation, Real-time Polymerase Chain Reaction, Purification, Cell Culture, XTT Assay

Journal: iScience

Article Title: Chimeric antigen receptor NK-92 cell function is modulated by HLA class I expression of target cells

doi: 10.1016/j.isci.2025.112523

Figure Lengend Snippet: HLA-I surface expression of target cells modulates secretion of TNFα and IFNγ by CD276-specific CAR NK-92 cells (A) Secretion of TNFα (left y axis, black) and IFNγ (right y axis, blue) after co-incubation of CD276-directed CAR NK-92 cells with the indicated target cell lines as measured by ELISA (E:T ratio 1:1; given are individual data points plus superimposed means ± SE; n = 4). (B–E) HLA-I-directed siRNA treatment increases TNFα and IFNγ secretion for target cells expressing high levels of HLA-I, whereas no changes are observed for siRNA-treated cell lines with low levels of HLA-I. Values (means from n = 2–3 experiments per cell line) were normalized to those obtained for cells treated with non-targeting siRNA control. Absolute cytokine levels are given in . Numbers indicate p values as calculated by paired t-test.

Article Snippet: For IFNγ treatment, around 600,000-900,000 target cells (depending on cell line) were incubated with Recombinant Human IFNγ (R&D Systems, #10067-IF-025) for 48 or 72 hours using an IFNγ concentration of 100 ng/ml and respective volumes of PBS as negative control.

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: iScience

Article Title: Chimeric antigen receptor NK-92 cell function is modulated by HLA class I expression of target cells

doi: 10.1016/j.isci.2025.112523

Figure Lengend Snippet: Breast cancer and non-transformed, normal cells express HER2 on their surface and are lysed by HER2-specific CAR NK-92 cells (A) Representative histograms of HER2-associated fluorescence intensity and respective IgG isotype controls of the lowest (U-87MG, blue and dark gray) and highest (MDA-MB-453, red and light gray) expressing tumor cell lines. (B and C) HER2 surface expression (B) and HER2-specific CAR NK-92-mediated (E:T = 2.5:1, black, and 0.5:1, blue) lysis of MDA-MB-231, MDA-MB-453, and MCF7 breast cancer and U-87MG glioblastoma cells (C) as measured by flow cytometry and calcein release assay, respectively. (D and E) HER2 surface expression (D) and HER2-specific CAR NK-92-mediated (E:T = 2.5:1) lysis of non-transformed SVGA, hCMEC/D3 and HFF cells (E) as analyzed in (B and C). (F) Secretion of TNFα (left y axis, black) and IFNγ (right y axis, blue) after co-incubation of HER2-directed CAR NK-92 cells and the indicated target cell lines as measured by ELISA (E:T ratio 1:1. Data in (b-f) represent individual data points with superimposed means ± SE (n = 4–6). (G) Representative histograms of HER2-associated fluorescence intensity and respective IgG isotype controls of MDA-MB-453 cells after HER2-directed (red and light gray) or non-targeting control (blue and dark gray) siRNA treatment. (H) Mean HER2 surface expression after HER2 siRNA treatment normalized to the respective non-targeting siRNA controls (n = 2–3 experiments per cell line). (I) Lysis rates by HER2-directed CAR NK-92 cells (E:T ratio 2.5:1) of MDA-MB-231, MDA-MB-453, MCF7 and SVGA cells treated with non-targeting or HER2-directed siRNA ( n = 3). Numbers indicate p values as calculated by paired t-test.

Article Snippet: For IFNγ treatment, around 600,000-900,000 target cells (depending on cell line) were incubated with Recombinant Human IFNγ (R&D Systems, #10067-IF-025) for 48 or 72 hours using an IFNγ concentration of 100 ng/ml and respective volumes of PBS as negative control.

Techniques: Transformation Assay, Fluorescence, Expressing, Lysis, Flow Cytometry, Release Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: iScience

Article Title: Chimeric antigen receptor NK-92 cell function is modulated by HLA class I expression of target cells

doi: 10.1016/j.isci.2025.112523

Figure Lengend Snippet: IFNγ upregulates HLA-I expression on target cell lines, but, nevertheless, increases cytotoxicity of HER2-specific CAR NK-92 cells (A) Representative histograms of HLA-I-associated fluorescence intensity and respective IgG isotype controls of MDA-MB-453 cells incubated with IFNγ (blue and dark gray) or without IFNγ (red and light gray). (B and C) IFNγ incubation upregulates mean (n = 2–3 per cell line) HLA-I surface expression normalized to vehicle control (B) and increases specific lysis (C) by HER2-directed CAR NK-92 cells (E:T ratio 2.5:1) for MDA-MB-453, MDA-MB-231, MCF7, and SVGA cells. (D) Representative histograms of HLA-I-associated fluorescence intensity and respective IgG isotype controls of MDA-MB-453 target cells after IFNγ incubation (blue and dark gray), or IFNγ incubation with additional HLA-I directed siRNA treatment (red and light gray). (E–G) HLA-I downregulation by siRNA during IFNγ incubation reduced mean (n = 2–3 per cell line) HLA-I surface expression (E) and increased relative (F) and absolute (G) specific lysis by HER2-directed CAR NK-92 cells (E:T ratio 2.5:1) for MDA-MB-453, MCF7 and SVGA, but not MDA-MB-231 cells. Numbers indicate p values as calculated by paired t-test. Absolute values for each experiment are shown in .

Article Snippet: For IFNγ treatment, around 600,000-900,000 target cells (depending on cell line) were incubated with Recombinant Human IFNγ (R&D Systems, #10067-IF-025) for 48 or 72 hours using an IFNγ concentration of 100 ng/ml and respective volumes of PBS as negative control.

Techniques: Expressing, Fluorescence, Incubation, Control, Lysis