l kynurenine  (MedChemExpress)

Journal: Nature Immunology

Article Title: Metabolic adaptations rewire CD4 + T cells in a subset-specific manner in human critical illness with and without sepsis

doi: 10.1038/s41590-025-02390-6

Figure Lengend Snippet: a , Flow cytometry histograms of kynurenine (KYN) uptake following 5 minute pulse of NHC and CI-Sep CD4 + T reg cells with kynurenine (100 μM), without pre-treatment or pretreated with SLC7A5 inhibitor BCH (10 mM) or L-leucine (LEU, 5 mM) or incubated at 4 °C prior to adding kynurenine. b , c , Glycolytic capacity by puromycin-incorporation assay in CI-Sep CD4 + T N , T EM , and T reg cells from paired PBMC cultures treated with vehicle or KMO inhibitor GSK180 (KMOi, 10 μM) for 3 h ( n = 7) (b) or 24 h ( n = 6) (c). Displayed as percentage of the maximum puromycin incorporation without metabolic inhibitors. d , LC–MS/MS showing kynurenine concentration in HPLM with or without dialyzed FBS (dFBS), fresh or stored at 4 °C for 30 days. e , Glycolytic capacity as in (b) in NHC ( n = 4) and CI-Sep ( n = 4) CD4 + T N , T EM , and T reg cells from paired PBMC cultures treated with vehicle or L-Leucine (500 μM) for 24 h. f , Glycolytic capacity as in (b) in NHC ( n = 5) and CI-Sep ( n = 7) CD4 + T N , T EM , and T reg cells from paired PBMC cultures treated with vehicle or LDHA inhibitor FX-11 (LDHi, 20 μM) for 24 h. g , h Flow cytometric quantification of PD-1 and LAG-3 expression (g) or IFNγ, IL-17A, and IL-2 cytokine expression (h) in NHC ( n = 10) and CI-Sep ( n = 12) CD4 + FOXP3 − T conv cells from PBMC isolated 2 days post-ICU admission stimulated with CD3/CD28/CD2 Ab for 24 h with or without low-dose oligomycin (10 nM) and treated with vehicle or KMO inhibitor GSK180 (10 μM). Shown as percent change in the frequency of PD-1 + , LAG-3 + , IFNγ + , IL-17A + , or IL-2 + cells among T conv cells from stimulated, oligomycin-treated PBMC with vehicle or KMO inhibitor GSK180 relative to stimulated, untreated PBMC. i , FBA as a heatmap showing in silico flux potential (z-scored) for individual metabolic reactions in scRNA-seq data from NHC and CI-NS CD4 + T N , T EM , and T reg cells. j , FBA pairwise comparisons of Cohen’s d effect size of tryptophan metabolism reactions among NHC, CI-NS, and CI-Sep T N , T EM , and T reg cells. k , Scatterplot showing FBA results as in (i) for CI-NS T reg cells versus NHC T reg cells and CI-Sep T reg cells versus NHC T reg cells reported as Cohen’s d values. A linear regression line modeling the association between all reactions is shown in red. Statistical analysis for b,c,g,h was performed using with two-sided Wilcoxon signed-rank test for paired sample analysis; for e,f, using two-tailed permutation testing for paired samples; for j, using two-tailed Mann–Whitney U testing with Benjamini–Hochberg FDR correction; for k, using a two-sided t-test of the regression slope. Data for b,c,e-h are presented as mean ± SEM. Each data point in b,c,e-h represents a biologic or in d represents a technical replicate.

Article Snippet: Cells were then washed once with prewarmed 37 °C HBSS and resuspended in 37 °C HBSS containing l -kynurenine (MedChemExpress, cat. no. HY-104026, 100 μM) for 5 min. Uptake was quenched with 100 μl IC Fixation Buffer (Invitrogen, cat. no. 00-8333) for 30 min at room temperature, washed twice with flow staining buffer and analyzed by flow cytometry. l -kynurenine was detected by its intrinsic spectral properties using a 405-nm excitation (violet laser) and a 450/50-nm emission filter (V1 channel, MACSQuant Analyzer 16).

Techniques: Flow Cytometry, Incubation, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Expressing, Isolation, In Silico, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Metabolic adaptations rewire CD4 + T cells in a subset-specific manner in human critical illness with and without sepsis

doi: 10.1038/s41590-025-02390-6

Figure Lengend Snippet: a , FBA as a heatmap showing in silico flux potential for individual metabolic reactions scaled by standard z -scoring in scRNA-seq data from NHCs and CI-Sep CD4 + T N , T EM and T reg cells. Larger values indicate higher predicted flux. b , Schematic of tryptophan and kynurenine metabolism showing metabolites (black), enzymes (tan) and Recon2 pathway annotations (blue). Panel b created in BioRender. Stier, M. (2025) https://BioRender.com/q07s159 . c – e , FBA pairwise comparison of effect size of tryptophan metabolism reactions for CI-Sep T reg cells versus NHC T reg cells ( c ), CI-Sep T reg cells versus CI-Sep CD4 + T N cells ( d ) and CI-Sep T reg cells versus CI-Sep CD4 + T EM cells ( e ). Effect size reported as Cohen’s d value (standardized mean difference). f , Dot plot of single-cell normalized, log-transformed counts of kynurenine metabolism genes in NHC ( n = 9) and CI-Sep ( n = 19) CD4 + T N , T EM and T reg cells. Values are scaled within each gene. g , Normalized (log(CPM + 1)) scRNA-seq pseudobulked SLC7A5 expression in NHC ( n = 9) and CI-Sep ( n = 19) CD4 + T N , T EM and T reg cells. CPM, counts per million. h , Flow cytometric detection of kynurenine uptake in NHC ( n = 12) and CI-Sep ( n = 18) CD4 + T N , T EM , and T reg cells following a 5-min pulse with l -kynurenine (100 μM). i , AUCell pathway activity analysis of scRNA-seq data for a consensus human pan-tissue AHR gene signature in CI-Sep T reg cells compared to NHC T reg cells (Cohen’s d = 0.522, P = 6.44 × 10 −111 ). Larger values represent higher AHR-associated transcriptional activity. j , Glycolytic capacity by puromycin-incorporation assay in NHC ( n = 14) CD4 + T N , T EM and T reg cells from paired PBMC cultures treated with vehicle or l -kynurenine (KYN; 10 μM) for 24 h displayed as percentage of the maximum puromycin incorporation without metabolic inhibitors. k , Glycolytic capacity by puromycin-incorporation assay in CI-Sep ( n = 6) T reg cells from paired PBMC cultures treated with vehicle or KMO inhibitor (KMOi) GSK180 (10 μM) for 24 h displayed as percentage of the maximum puromycin incorporation without metabolic inhibitors. l , Glycolytic capacity by puromycin-incorporation assay in NHC ( n = 5) T N , T EM and T reg cells and CI-Sep ( n = 7) CD4 + T N , T EM and T reg cells from paired PBMC cultures treated with vehicle or QPRT inhibitor (QPRTi) phthalic acid (100 μM) for 24 h displayed as percentage of the maximum puromycin incorporation without metabolic inhibitors. m , Percentage change in FOXP3 MFI among CD4 + FOXP3 + T reg cells (left) or the percentage change in the frequency of TIGIT + cells among CD4 + FOXP3 + T reg cells (right) from PBMCs isolated from NHC ( n = 10) and CI-Sep ( n = 12) at day 2 post-ICU admission, stimulated with CD3/CD28/CD2 antibodies for 24 h, treated with low-dose oligomycin (10 nM) and with vehicle or GSK180 (10 μM) relative to stimulated, untreated PBMCs. n , Percentage change in the frequency of TNF + cells among T conv cells from NHC ( n = 10) and CI-Sep ( n = 12) donors, stimulated, oligomycin-treated PBMCs with vehicle or KMO inhibitor GSK180 relative to stimulated, untreated PBMCs as in m . Statistical analysis was performed using a two-sided Mann–Whitney U -test with Benjamini–Hochberg FDR correction for multiple testing ( c – e ); a Kruskal–Wallis test followed by Dunn’s post hoc testing ( h ); a two-sided Wilcoxon signed-rank test for paired sample analysis ( j , k , m , n ); and a two-tailed permutation test for paired samples ( l ). Data for h , j – n are presented as mean ± s.e.m. Boxplots show the median with boxes bounded by IQR (25–75th percentile) and whiskers extending to the minimum and maximum values within 1.5 × IQR. Each data point represents an individual donor.

Article Snippet: Cells were then washed once with prewarmed 37 °C HBSS and resuspended in 37 °C HBSS containing l -kynurenine (MedChemExpress, cat. no. HY-104026, 100 μM) for 5 min. Uptake was quenched with 100 μl IC Fixation Buffer (Invitrogen, cat. no. 00-8333) for 30 min at room temperature, washed twice with flow staining buffer and analyzed by flow cytometry. l -kynurenine was detected by its intrinsic spectral properties using a 405-nm excitation (violet laser) and a 450/50-nm emission filter (V1 channel, MACSQuant Analyzer 16).

Techniques: In Silico, Comparison, Transformation Assay, Expressing, Activity Assay, Isolation, MANN-WHITNEY, Two Tailed Test