protein concentration  (R&D Systems)

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: LAG3 expression on NK cells is elevated in HIV-infected individuals and correlated with HIV disease progression. ( A ) Flow cytometry gating strategy used for identification of NK cells and subsets. ( B ) Representative flow cytometry plots indicating the expression of LAG3 on NK cells in the HC and PLWH groups. Fluorescence minus one (FMO) control was applied to establish the gating threshold. ( C ) Comparison of the percentages (left) and MFI (right) of LAG3 on total NK cells from the HC ( n = 25) and PLWH ( n = 52) groups. ( D ) Comparison of the percentages (left) and MFI (right) of LAG3 on total NK cells from the HIV ART− ( n = 22) and HIV ART+ ( n = 30) groups. All 30 ART-treated individuals were on ART for more than 2 years and had undetectable plasma HIV RNA (viral load; VL). ( E and F ) Spearman correlation analysis of absolute CD4 + T cell count (cells/µL) and CD4/CD8 ratio with LAG3 expression (left: percentage; right: MFI) on NK cells ( n = 52).

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: Expressing, Infection, Biomarker Discovery, Flow Cytometry, Fluorescence, Control, Comparison, Clinical Proteomics, Cell Counting

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: High LAG3 expression inhibits NK cell activation and interferon-gamma (IFN-γ) production, and LAG3-Fc protein significantly enhances NK cell function. ( A–C ) Analysis of the correlation between NKG2C, HLA-DR, and CD38 expression and LAG3 expression (left: percentage; right: MFI) on NK cells from HIV groups ( n = 16). ( D ) Paired comparisons of Ki67 in LAG3+ and LAG3− NK cells from HIV groups ( n = 15). ( E ) Comparison of IFN-γ production by NK cells from the HIV-infected ( n = 6) and HC ( n = 6) groups. ( F ) Paired comparisons were performed between IFN-γ producing LAG3+ and LAG3− NK cells following 24 h stimulation with the cytokine mixture including IL-12 (10 ng/mL), IL-15 (50 ng/mL), and IL-18 (100 ng/mL) for stimulated groups ( n = 29). For unstimulated groups, peripheral blood mononuclear cells (PBMCs) were cultured without the cytokine mixture. ( G and H ) Paired comparisons of CD69, HLA-DR, NKG2C, CD38, and Ki67 expression on NK cells from HIV groups (CD69: n = 10, HLA-DR: n = 8, NKG2C: n = 8, CD38: n = 10, Ki67: n = 10) after treatment with 2 µg/mL LAG3-Fc or IgG-Fc protein (as a negative control). ( I ) Paired comparison of the production of IFN-γ in NK cells from HIV groups ( n = 10) after treatment with 2 µg/mL LAG3-Fc or IgG-Fc protein. ( J ) Paired comparison of cytotoxicity of NK cells against K562 targets in HC group ( n = 6) following 24 h treatment with 2 µg/mL LAG3-Fc or IgG-Fc protein.

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: Expressing, Activation Assay, Cell Function Assay, Comparison, Infection, Cell Culture, Negative Control

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: LAG3 activation significantly inhibits IFN-γ production and Ki67 expression in NK cells. ( A and B ) Paired comparisons of the IFN-γ production and Ki67 expression in NK cells after treatment with 10 µg/mL LAG3 antibody or IgG control from the HC and HIV groups (IFN-γ: HC, n = 8, HIV, n = 7; Ki67: HC, n = 7, HIV, n = 11). ( C ) Paired comparisons of IFNG and MKI67 TPM expression in NK cells after the above treatment in cells from the HC group ( n = 5).

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: Activation Assay, Expressing, Control

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: Transcriptome sequencing showed that LAG3 inhibits glycolysis-related gene expression in NK cells. ( A ) Heatmap of transcriptome sequencing data (anti-IgG: n = 5, anti-LAG3: n = 5); blue indicates downregulated gene expression (row Z -score < 0), and red indicates upregulated gene expression (row Z -score > 0). ( B ) Volcanic map of all identified genes and DEGs. ( C ) Gene subnetworks and top network key drivers of downregulated DEGs. KDA genes are indicated in red, initial genes in blue, and all other genes in gray. ( D and E ) KEGG pathway and GO biological process enrichment analyses of downregulated DEGs. ( F and G ) GSEA of enriched gene sets in the anti-LAG3 and anti-IgG groups. ( H ) Heatmap of differentially expressed glycolysis-related genes. ( I ) Paired comparisons of glycolysis-related gene expression values in NK cells from the anti-LAG3 ( n = 5) and anti-IgG ( n = 5) groups.

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: Sequencing, Gene Expression

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: In vitro experiments demonstrated that LAG3 inhibits glycolysis in NK cells via the PI3K/AKT/mTOR signaling pathway, thereby affecting NK cell function. ( A ) Schematic diagram of the PI3K/AKT signaling pathway regulating glycolysis in NK cells. ( B ) Paired comparisons of Glut-1 expression on NK cells from the HIV group ( n = 12) after treatment with 10 µg/mL LAG3 antibody or IgG control. ( C ) Paired comparisons of AKT, mTOR, and S6 phosphorylation after treatment with the above antibody in NK cells from the HIV group ( n = 7). ( D ) Paired comparisons of IFN-γ production by NK cells from the HC ( n = 6) and HIV ( n = 6) groups after 24 h stimulation with 2-DG, glucose, and the cytokine mixture. ( E ) Paired comparisons of IFN-γ–producing LAG3+ and LAG3− NK cells from the HC ( n = 8) and HIV ( n = 6) groups after 24 h stimulation with glucose and the above cytokine mixture.

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: In Vitro, Cell Function Assay, Expressing, Control, Phospho-proteomics

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: Transcriptome sequencing and in vitro analyses showed that activated LAG3 suppresses the STAT1/IFNG pathway by upregulating PSAT1 expression. ( A ) Gene subnetworks and top network key drivers of upregulated DEGs. ( B and C ) Paired comparisons of PSAT1 and STAT1 TPM expression in NK cells from the anti-LAG3 ( n = 5) and anti-IgG ( n = 5) groups. ( D ) Correlation analysis of PSAT1 and STAT1 TPM expression in NK cells ( n = 10). ( E ) Paired comparisons of total STAT1 phosphorylation after treatment with 10 µg/mL LAG3 antibody or IgG control in NK cells from the HC ( n = 3) and HIV ( n = 5) groups.

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: Sequencing, In Vitro, Expressing, Phospho-proteomics, Control

Journal: mBio

Article Title: The inhibitory receptor LAG3 affects NK cell IFN-γ production through glycolysis and the PSAT1/STAT1/IFNG pathway

doi: 10.1128/mbio.00230-25

Figure Lengend Snippet: Schematic illustration of the mechanism of LAG3 inhibition of IFN-γ production by NK cells. During HIV infection, the expression of LAG3 on NK cells is increased, thereby activating the cells, which in turn inhibits the PI3K/AKT/mTOR signaling pathway regulating glycolysis, thus limiting NK cell IFN-γ production through a reduction in glycolysis. Simultaneously, LAG3 suppresses the STAT1/IFNG pathway by upregulating PSAT1 expression, which in turn suppresses IFN-γ production in NK cells.

Article Snippet: Recombinant human LAG3 Fc Chimera Protein or IgG Fc protein (R&D Systems) were added to PBMCs and incubated for 24 h, and the expression levels of CD69, HLA-DR, NKG2C, CD38, and Ki67 were evaluated as described above.

Techniques: Inhibition, Infection, Expressing