Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: CD4 + T cells apoptosis is increased and correlated with ER stress. A – D The rate of apoptosis of CD4 + T cells was measured by the ratio of Annexin V-positive and PI-positive/negative CD4 + T cells. E – F The expression level of bax, bcl2, and caspase 3 were examined by Western blotting. G – H The expression level of GRP78and CHOP, the marker of ER stress was measured by western blotting. I – K The microstructure images of ER of CD4 + T cells in WT, WT + CLP, and CLP + 4-PBA mice were observed with electron microscopy. Green arrows represent normal-sized ER. Yellow arrows represent dilation and vesiculation of the ER. Densitometric quantification for expression of protein was normalized to ACTIN protein level. Means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. * P < 0.05.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Expressing, Western Blot, Marker, Electron Microscopy

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: The role of mTOR in ER stress-induced CD4 + T cells apoptosis. A – C Proteins of mTOR pathway, including mTOR, P -mTOR, downstream effectors p70s6k, p-p70s6k were examined by Western blotting. D – E The expression level of GRP78and CHOP, the marker of ER stress was measured by western blotting. F – H The expression level of bax, bcl2, and caspase 3 were examined by Western blotting. Means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Western Blot, Expressing, Marker

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: Deficient autophagy is observed under ER stress in sepsis and the role of mTOR on it. (A-E) With flow cytometry, the rates apoptosis of CD4 + T cells were detected in WT + CLP, mTOR KO + CLP, TSC1 KO + CLP, TSC1 KO + CLP + 4-PBA. (F, H). The expression level of LC3I/LC3II and P62, the markers of autophagy process were measured by western blotting. G , I – K Ultrastructural features of CD4 + T cells were investigated using transmission electron microscopy (TEM). In WT group, CD4 + T cells had normal morphologies, revealing baseline autophagy status. WT + CLP mice displayed increased autophagic vacuolization but no significant increase in autolysosome frequency. Large autolysosomes containing abundant contents were seen. More autophagic vacuolization and more autolysosomes were showed in mTOR KO + CLP. Autophagosomes and autolysosomes were fewer in TSC1 KO + CLP mice. Autophagosomes were double-membrane vacuoles containing cytosol or organelles (red arrow). Autolysosomes were single-membrane structures containing digested cytoplasmic components (blue arrow). Means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Flow Cytometry, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Membrane

Journal: Inflammation Research

Article Title: mTOR pathway mediates the endoplasmic reticulum stress -apoptosis of CD4+ T cell through inhibiting autophagy flux in sepsis

doi: 10.1007/s00011-025-02114-4

Figure Lengend Snippet: mTOR deletion activates autophagy to alleviate ER stress-induced apoptosis. (A-F) The proportion of apoptosis of CD4 + T cells were detected in WT + CLP, mTOR KO + CLP, CLP + Rap, mTOR KO + CLP + Baf, CLP + Baf by flow cytometry analysis. Densitometric quantification for expression of protein was normalized to ACTIN protein level. G – I The expression level of GRP78, CHOP, bax, and bcl2, the marker of ER stress and apoptosis were measured by western blotting. Data was presented as means ± standard deviations (SDs) of four mice per group are shown. It was deemed statistically significant when P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The splenocyte suspension was incubated with CD4 + antibody (CD4 + T cells isolation Kit, mouse Miltenyi, 130–104-454), washed, and passed through a separation column for negative screening, and finally centrifuged to obtain CD4 + T cells.

Techniques: Flow Cytometry, Expressing, Marker, Western Blot

Journal: PLOS One

Article Title: Evidence of an allostatic response by intestinal tissues following induction of joint inflammation

doi: 10.1371/journal.pone.0338053

Figure Lengend Snippet: (A) At time of sacrifice, the diameters of knee joints were measured as described in the Method from rats without treatment (Pre-Treat), after IAI of PBS and 3 days (3d), 7 days (7d) and 14 days (14d) post-CFA. CFA-IAI knees showed significant swelling at all three time points, when compared to pre-treatment or PBS-IAI (*** = p < 0.001). (B,C) Abundance of bikunin species in synovial fluids ( B ) and sera ( C ) were determined by western blotting as described in the Methods and Supporting Information . Note that for pre-treatment (Pre-Treat) and PBS groups, the measurements at 0d and 14d, respectively, were combined since no significant differences were observed between those two groups (general linear model 0d v. 3d v. 14d PBS serum p = 0.226). For A-C * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Coronal formalin fixed paraffin embedded (FFPE) sections of pretreatment and 14d IAI-CFA knee joints were stained with H&E ( D & G ), anti-CD68 ( E & H ) or anti-CD4 ( F & I ). The CFA-induced synovial hyperplasia enriched in collagenous ECM and infiltrated by CD68 + macrophages is indicated by black arrows and the swollen patellar tendon by (*). M = meniscus, Fc = femoral condyles, TP = tibial plateau, S = synovium, PT = patellar tendon.

Article Snippet: Sections of intestinal Swiss rolls were stained using the Alcian Blue Kit (Vector Labs, H-3501), anti-CD68 (1:100), anti-CD4 (1:2000), anti-CD8 (Abcam, Ab237709 , 1:500) or anti-Ki67 (Invitrogen, MA5–14520; 1:100), Invitrogen, MA5–14520) or the following biotinylated lectins (all from Vector Labs), Griffonia Bandeiraea Simplidifolia Lectin I, (GSL I, B-1105–2), Maackia Amurensis Lectin II (MAL II, B-1265–1) and Ulex Europaeus Agglutinin I (UEA, B-1065–2).

Techniques: Western Blot, Formalin-fixed Paraffin-Embedded, Staining

Journal: PLOS One

Article Title: Evidence of an allostatic response by intestinal tissues following induction of joint inflammation

doi: 10.1371/journal.pone.0338053

Figure Lengend Snippet: Adjacent FFPE-sections from proximal and distal regions of the ileum and colon were stained with ( A ) anti-CD68 to localize macrophages or ( B ) anti-CD4 and ( C ) anti-CD8 to localize T-cells. Black arrows indicate regions of increased reactivity post CFA. ( D ) DEGs typically associated with intestinal macrophages or T cells caused by CFA-IAI are summarized in the heat map. PI = Proximal Ileum, DI = Distal Ileum, PC = Proximal Colon, DC = Distal Colon.

Article Snippet: Sections of intestinal Swiss rolls were stained using the Alcian Blue Kit (Vector Labs, H-3501), anti-CD68 (1:100), anti-CD4 (1:2000), anti-CD8 (Abcam, Ab237709 , 1:500) or anti-Ki67 (Invitrogen, MA5–14520; 1:100), Invitrogen, MA5–14520) or the following biotinylated lectins (all from Vector Labs), Griffonia Bandeiraea Simplidifolia Lectin I, (GSL I, B-1105–2), Maackia Amurensis Lectin II (MAL II, B-1265–1) and Ulex Europaeus Agglutinin I (UEA, B-1065–2).

Techniques: Staining

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: Metabolomics of circulating human memory CD4 + T effector and T regulatory cells reveals distinct metabolic profiles, enriched in phenylalanine and arginine metabolic pathways (A and B) Intermediate gate depicting memory (CD45RA − ) and naive (CD45RA + ) CD3 + CD4 + T cells (A) and final sorting gate (B) of circulating memory CD4 + T effector and T regulatory cells. (C) Venn diagram representing intracellular metabolites ( n = 295) detected by untargeted mass spectrometry metabolomics and lipidomics in memory CD4 + Teff (red) and Treg (blue) cells from healthy individuals. (D) PCA model of memory CD4 + Teff and Treg cells, based on all (shared and unique) metabolites. Data were logarithmic transformed and pareto scaled (log x Par). (E) Pie charts representing biochemical composition of all (shared and unique) metabolites detected in Teff and Treg cells, ordered by abundance. (F) Metabolic pathways analysis in memory CD4 + Teff and Treg cells. Significant (−log 10 ( p value) > 1.3) and corresponding pathways ( n = 25) are shown for either Teff, Treg, or both. Over-representation analysis was performed by IMPaLA including shared and unique metabolites. Pathways related to Phe and Arg metabolism are highlighted in bold. (G) PCA model of memory CD4 + Teff and Treg cells of shared metabolites only ( n = 133). Data were log x Par. (H) Heatmap (left) and bar graph of fold changes (Log 2 FC) (right) of identified shared metabolites in memory CD4 + Teff vs. Treg cells. Data were logarithmic transformed and unit variance scaled. ∗ p < 0.05. Metabolites related to Phe and Arg metabolism are highlighted in bold. (A–H) Analysis done in n = 6 different healthy donors. (E) Metabolites are organized in main biochemical classes according to Human Metabolome Database (HMDB v.2022). Examples of metabolites are shown in major classes (>5%). See also and and , , , , , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Mass Spectrometry, Transformation Assay

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: High level of L-phenylalanine enhances activation-induced glycolysis but inhibits OXPHOS, while arginine enhances activation-induced glycolysis and OXPHOS in human CD4 + T and memory CD4 + T cells (A) Representative glycolytic proton efflux rate (glycoPER) graph of Seahorse glycolytic rate assay (left); quantification of inducible and compensatory glycolysis (right) of CD4 + T cells treated in full medium (containing 1.149 mM Arg) with additional 0.1 (blue) and 1 mM (red) Arg supplementation or vehicle (gray) for 72 h with/without acute CD2, CD3, and CD28 activation. (B) Representative oxygen consumption rate (OCR) graph of Seahorse Mito Stress test (left); quantification of maximum respiratory capacity (right) of CD4 + T cells treated with Arg as in (A). (C) Representative glycoPER graph of Seahorse glycolytic rate assay (left); quantification of inducible and compensatory glycolysis (right) of CD4 + T cells treated in full medium (containing 90.9 μM of Phe) additionally supplemented Phe at concentrations of 0.1 (violet) and 1 mM (green) or vehicle (gray) for 72 h, with/without acute CD2, CD3, and CD28 activation. (D) Representative OCR graph of Seahorse Mito Stress test (left); quantification of maximum respiratory capacity (right) of CD4 + T cells treated with Phe as in (C). (E) Quantification of induced glycolysis, compensatory glycolysis, and maximum respiratory capacity of memory CD4 + T cells treated with Phe as in (C) and (D). (A–D) Data are representative of three independent experiments in three different donors or (E) in one donor. Data were analyzed by one-way ANOVA with Fisher LSD test. Bar graphs represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Arg, L-arginine; Phe, L-phenylalanine; Rot/AA, rotenone/antimycin A; 2DG, 2-deoxyglucose; FCCP, carbonyl cyanide- p -trifluoromethoxyphenylhydrazone. All Seahorse measurements were normalized to total protein concentration.

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Protein Concentration

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: L-phenylalanine inhibits proliferation of human memory CD4 + T cells by induction of interleukin 4 induced gene 1 enzyme (A) Proliferation of memory CD4 + T cells incubated in full medium (containing 90.9 μM of Phe) supplemented with vehicle or 1 mM Phe and activated with CD2, CD3, and CD28 antibody-coated beads for 72 h. n = 3 different subjects. (B) Frequency of live memory CD4 + T cells in the same experiments as in (A). (C) Expression of IL4I1 mRNA in memory CD4 + T cells following similar treatment as in (A). Data from 6 independent experiments in 6 different subjects. (D) IL4I1 mRNA expression (left) and representative WB image of IL4I1 (right) in siRNA knockdown experiments in memory CD4 + T cells. Data show 3 independent experiments in 6 different subjects. One outlier was identified using Grubbs’ test with α = 0.05. One donor was included in two experiments. (E) Proliferation of control siRNA (Ctrl)- and IL4I1 siRNA-treated memory CD4 + T cells from 2 independent experiments in 5 different subjects. One donor was included in both experiments. (F) Viability of control siRNA (Ctrl)- and IL4I1 siRNA-treated memory CD4 + T cells in the same experiments as in (E). (G) Expression of IL4I1 mRNA in in vitro -differentiated human Th1, Th2, Th17, and Treg cells in siRNA knockdown experiments following similar treatment as in (D) ( n = 3 different donors). (H) Proliferation of control siRNA (Ctrl)- and IL4I1 siRNA-treated Th1, Th2, Th17, and Treg cells, incubated in full medium (containing 90.9 μM of Phe) with 1 mM additional Phe and treated with CD2, CD3, and CD28 activation antibody-coated beads for 48 h before flow cytometry ( n = 3 different donors). (A–H) Each dot represents one donor. (E and F) Bar graph shows fold change as compared to activated vehicle-treated cells. Paired t test was used in (A), (B), (C), (G), and (H); Wilcoxon test was used in (D)–(F). All data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also , , and and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Incubation, Expressing, Knockdown, Control, In Vitro, Activation Assay, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: L-phenylalanine inhibits Th2 cell proliferation and mTOR and STAT6 phosphorylation as well as expression of type 2 transcription factors, cytokines, activation, and pathogenicity markers (A) Phe uptake into Th2 cells. In vitro -differentiated Th2 cells from 3 different donors were incubated in indicated conditions for 6 h, and intracellular Phe was colorimetrically quantified in lysates. (B and C) Proliferation (B) and viability (C) of in vitro differentiated Th2 cells subjected to high doses of additional Phe. Bar graphs show fold changes compared to vehicle-treated, activated cells. n = 3 different donors. (D and E) IL4I1 mRNA expression (D) and representative WB image of IL4I1 protein expression (E) in in vitro -differentiated Th2 cells following incubation in increasing doses of Phe with/without concurrent activation. mRNA ( n = 6–8 different donors) and protein expression ( n = 3 different donors). (F) Volcano plot of differentially expressed genes (DEGs, raw p value < 0.05) between activated Th2 cells treated with Phe (1mM) vs. vehicle for 24 h, obtained by RNA-seq analysis ( n = 5 different donors). Genes related to STAT6/mTOR/AMPK signaling, critical for T cell activity, are highlighted in boxes. (G and H) Significantly enriched downregulated (G) and upregulated (H) GO processes in Th2 cells following treatment as in (F). STRING analysis was conducted with significantly changed DEGs (raw p value < 0.05), and relevant enriched pathways are presented. (I–M) Representative WB image (I and K) and quantification (J, L, and M) of phosphorylation of STAT6 and mTOR, respectively, in in vitro -differentiated Th2 cells ( n = 3 different donors) treated with CD2, CD3, and CD28 activation antibodies with/without additional supplementation of 1 mM of Phe for indicated time points. (N) Heatmap of mRNA expression of critical transcription factors, cytokines, and activation markers in activated in vitro -differentiated Th2 cells treated with increasing doses of Phe. mRNA expression was determined using RT-qPCR. n = 6–8 different donors. Data are analyzed using one-way ANOVA with Dunnett’s correction. Z scores were determined and plotted as heatmap with different genes mentioned as rows. Data are row normalized. (O) Frequency of activated IL4 + Th2 cells with/without additional supplementation of 1 mM of Phe ( n = 4 different donors). (P) Frequency of activated CD3 + CD4 + CCR4 + GATA3 + CD161 + Th2 cells following incubation with/without supplementation of 1 mM Phe for 24 h ( n = 3 different donors). (A–D, J, L, M, N, O, and P) Each dot represents one donor. (A–C, J, L, M, O, and P) Paired t test was used for analysis. Bars represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also and , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Phospho-proteomics, Expressing, Activation Assay, In Vitro, Incubation, RNA Sequencing, Activity Assay, Quantitative RT-PCR

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: Low intracellular L-phenylalanine levels in pathogenic memory CD4 + T effector cell populations in severe allergic patients (A–C) Representative flow cytometry dot plots of Th2a cells (A-left) and ILC1, ILC2, and ILC3 (C-left). Number of Th2a cells (A-right); memory CRTH2 + Treg cells and PD1 + Treg cells (B); and ILC1, ILC2, and ILC3 (C-right) in controls ( n = 9) and patients with mild ( n = 7) and severe ( n = 11) allergy (Cohort A). (D) Differentially expressed proteins in serum of controls ( n = 10) and mild ( n = 9) and severe ( n = 10) allergic patients (Cohort A) assessed with PEA technology and presented as NPX. (E) Heatmaps with hierarchical clustering analysis of all metabolites measured in memory CD4 + Teff ( n = 195, left) and Treg ( n = 233, right) cells in controls ( n = 6) and allergic ( n = 11) subjects. A, controls; B, mild allergy; C, severe allergy (from Cohort A). (F) Normalized abundance of Phe in Teff cells in group 1 (control, n = 1; severe allergy, n = 4) and 2 (control, n = 5; mild allergy, n = 1, severe allergy, n = 6) (from Cohort A). (G) t-distributed stochastic neighbor embedding (tSNE) plot of unbiased 2-dimensional flow cytometric analysis of memory CD4 + Teff cells (CD3 + CD4 + CD45RA − CD127 + CD25 − ) from patients with severe allergy (subset of cohort A, n = 9) identifying seven subpopulations based on CD161 and PD-1. (H) Pearson correlation of normalized abundance of intracellular Phe, in memory CD4 + Teff cells from patients with severe allergy ( n = 9), with counts of CD161 + populations within memory CD4 + Teff cells (from Cohort A). Mann-Whitney U test (A–C), one-way ANOVA with Fisher’s LSD test (D), and unpaired t test (F) were used to compare differences among groups. Graphs represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Clinical characteristics of Cohort A are shown in and . NPX, normalized protein expression; Pop, population. See also and , , , , , , , , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Control, MANN-WHITNEY, Expressing

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: Decreased expression of large amino acid transporters in Th2 cells of allergic patients correlates with elevated serum levels of L-phenylalanine (A–C) Top significantly enriched pathways (A) and upregulated (B) and downregulated (C) metabolic networks within differentially expressed genes (DEG, p < 0.05) in allergic asthma patients compared to controls (control n = 15, allergic asthma n = 37) from GEO: GSE75011 (Cohort B). Black line represents ratio of genes in experiment over complete pathway set. (D) Phe metabolism and transport pathway heatmap showing fold change (Log 2 FC) of DEGs in allergic asthma patients ( n = 37) compared to controls ( n = 15) from GEO: GSE75011 (Cohort B). ∗ p < 0.05. Pathway curated and adapted from GSEA and MSigDB Database . (E) Phe metabolism and transport schematic highlighting DEGs in Th2 cells of allergic asthma and controls (GEO: GSE75011 ) (Cohort B). Significantly upregulated (red) and downregulated (blue) genes; detected but not significantly different (black); not detected in original dataset (black and underlined). Adapted from KEGG pathway. (F) Serum Phe concentration in controls ( n = 8) and mild ( n = 30) and severe ( n = 37) allergic patients (Cohort D) quantified by targeted metabolomics. Kruskal-Wallis test was used for analysis. (G) SLC7A5 (LAT1), SLC7A8 (LAT2), and SLC3A2 (CD98; LAT3) mRNA expression in in vitro -differentiated Th2 cells treated with additional Phe with/without CD2, CD3, and CD28 activation for 24 h. Following incubation, mRNA expression was determined using RT-qPCR ( n = 6–8 different donors). (H and I) Representative WB image of LAT1 expression (H) and LAT1 protein quantification in 8 different donors (I) in in vitro -differentiated Th2 cells treated with additional Phe with/without CD2, CD3, and CD28 activation for 24 h. Expression of LAT1 presented as relative ratio normalized to β-actin. (J) Phe uptake into in vitro -differentiated Th2 cells was quantified colorimetrically. Cells were incubated in full medium with/without SLC7A5 inhibitor (KYT0353) and activation of CD2, CD3, and CD28 for 6 h. Data were analyzed using paired t test ( n = 3 different donors). (K) Spearman correlation of serum Phe concentration and relative LAT1 expression in CD4 + T cells in allergic asthma patients (left) and controls (right) (Cohort E). (G–I) Data are analyzed by one-way ANOVA with Dunnett’s correction. (F–J) Bars represent mean ± SEM. Each dot represents one donor. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also and and , , , , , , , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Control, Concentration Assay, In Vitro, Activation Assay, Incubation, Quantitative RT-PCR

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: Metabolomics of circulating human memory CD4 + T effector and T regulatory cells reveals distinct metabolic profiles, enriched in phenylalanine and arginine metabolic pathways (A and B) Intermediate gate depicting memory (CD45RA − ) and naive (CD45RA + ) CD3 + CD4 + T cells (A) and final sorting gate (B) of circulating memory CD4 + T effector and T regulatory cells. (C) Venn diagram representing intracellular metabolites ( n = 295) detected by untargeted mass spectrometry metabolomics and lipidomics in memory CD4 + Teff (red) and Treg (blue) cells from healthy individuals. (D) PCA model of memory CD4 + Teff and Treg cells, based on all (shared and unique) metabolites. Data were logarithmic transformed and pareto scaled (log x Par). (E) Pie charts representing biochemical composition of all (shared and unique) metabolites detected in Teff and Treg cells, ordered by abundance. (F) Metabolic pathways analysis in memory CD4 + Teff and Treg cells. Significant (−log 10 ( p value) > 1.3) and corresponding pathways ( n = 25) are shown for either Teff, Treg, or both. Over-representation analysis was performed by IMPaLA including shared and unique metabolites. Pathways related to Phe and Arg metabolism are highlighted in bold. (G) PCA model of memory CD4 + Teff and Treg cells of shared metabolites only ( n = 133). Data were log x Par. (H) Heatmap (left) and bar graph of fold changes (Log 2 FC) (right) of identified shared metabolites in memory CD4 + Teff vs. Treg cells. Data were logarithmic transformed and unit variance scaled. ∗ p < 0.05. Metabolites related to Phe and Arg metabolism are highlighted in bold. (A–H) Analysis done in n = 6 different healthy donors. (E) Metabolites are organized in main biochemical classes according to Human Metabolome Database (HMDB v.2022). Examples of metabolites are shown in major classes (>5%). See also and and , , , , , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Mass Spectrometry, Transformation Assay

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: High level of L-phenylalanine enhances activation-induced glycolysis but inhibits OXPHOS, while arginine enhances activation-induced glycolysis and OXPHOS in human CD4 + T and memory CD4 + T cells (A) Representative glycolytic proton efflux rate (glycoPER) graph of Seahorse glycolytic rate assay (left); quantification of inducible and compensatory glycolysis (right) of CD4 + T cells treated in full medium (containing 1.149 mM Arg) with additional 0.1 (blue) and 1 mM (red) Arg supplementation or vehicle (gray) for 72 h with/without acute CD2, CD3, and CD28 activation. (B) Representative oxygen consumption rate (OCR) graph of Seahorse Mito Stress test (left); quantification of maximum respiratory capacity (right) of CD4 + T cells treated with Arg as in (A). (C) Representative glycoPER graph of Seahorse glycolytic rate assay (left); quantification of inducible and compensatory glycolysis (right) of CD4 + T cells treated in full medium (containing 90.9 μM of Phe) additionally supplemented Phe at concentrations of 0.1 (violet) and 1 mM (green) or vehicle (gray) for 72 h, with/without acute CD2, CD3, and CD28 activation. (D) Representative OCR graph of Seahorse Mito Stress test (left); quantification of maximum respiratory capacity (right) of CD4 + T cells treated with Phe as in (C). (E) Quantification of induced glycolysis, compensatory glycolysis, and maximum respiratory capacity of memory CD4 + T cells treated with Phe as in (C) and (D). (A–D) Data are representative of three independent experiments in three different donors or (E) in one donor. Data were analyzed by one-way ANOVA with Fisher LSD test. Bar graphs represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Arg, L-arginine; Phe, L-phenylalanine; Rot/AA, rotenone/antimycin A; 2DG, 2-deoxyglucose; FCCP, carbonyl cyanide- p -trifluoromethoxyphenylhydrazone. All Seahorse measurements were normalized to total protein concentration.

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Protein Concentration

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: L-phenylalanine inhibits proliferation of human memory CD4 + T cells by induction of interleukin 4 induced gene 1 enzyme (A) Proliferation of memory CD4 + T cells incubated in full medium (containing 90.9 μM of Phe) supplemented with vehicle or 1 mM Phe and activated with CD2, CD3, and CD28 antibody-coated beads for 72 h. n = 3 different subjects. (B) Frequency of live memory CD4 + T cells in the same experiments as in (A). (C) Expression of IL4I1 mRNA in memory CD4 + T cells following similar treatment as in (A). Data from 6 independent experiments in 6 different subjects. (D) IL4I1 mRNA expression (left) and representative WB image of IL4I1 (right) in siRNA knockdown experiments in memory CD4 + T cells. Data show 3 independent experiments in 6 different subjects. One outlier was identified using Grubbs’ test with α = 0.05. One donor was included in two experiments. (E) Proliferation of control siRNA (Ctrl)- and IL4I1 siRNA-treated memory CD4 + T cells from 2 independent experiments in 5 different subjects. One donor was included in both experiments. (F) Viability of control siRNA (Ctrl)- and IL4I1 siRNA-treated memory CD4 + T cells in the same experiments as in (E). (G) Expression of IL4I1 mRNA in in vitro -differentiated human Th1, Th2, Th17, and Treg cells in siRNA knockdown experiments following similar treatment as in (D) ( n = 3 different donors). (H) Proliferation of control siRNA (Ctrl)- and IL4I1 siRNA-treated Th1, Th2, Th17, and Treg cells, incubated in full medium (containing 90.9 μM of Phe) with 1 mM additional Phe and treated with CD2, CD3, and CD28 activation antibody-coated beads for 48 h before flow cytometry ( n = 3 different donors). (A–H) Each dot represents one donor. (E and F) Bar graph shows fold change as compared to activated vehicle-treated cells. Paired t test was used in (A), (B), (C), (G), and (H); Wilcoxon test was used in (D)–(F). All data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also , , and and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Incubation, Expressing, Knockdown, Control, In Vitro, Activation Assay, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: L-phenylalanine inhibits Th2 cell proliferation and mTOR and STAT6 phosphorylation as well as expression of type 2 transcription factors, cytokines, activation, and pathogenicity markers (A) Phe uptake into Th2 cells. In vitro -differentiated Th2 cells from 3 different donors were incubated in indicated conditions for 6 h, and intracellular Phe was colorimetrically quantified in lysates. (B and C) Proliferation (B) and viability (C) of in vitro differentiated Th2 cells subjected to high doses of additional Phe. Bar graphs show fold changes compared to vehicle-treated, activated cells. n = 3 different donors. (D and E) IL4I1 mRNA expression (D) and representative WB image of IL4I1 protein expression (E) in in vitro -differentiated Th2 cells following incubation in increasing doses of Phe with/without concurrent activation. mRNA ( n = 6–8 different donors) and protein expression ( n = 3 different donors). (F) Volcano plot of differentially expressed genes (DEGs, raw p value < 0.05) between activated Th2 cells treated with Phe (1mM) vs. vehicle for 24 h, obtained by RNA-seq analysis ( n = 5 different donors). Genes related to STAT6/mTOR/AMPK signaling, critical for T cell activity, are highlighted in boxes. (G and H) Significantly enriched downregulated (G) and upregulated (H) GO processes in Th2 cells following treatment as in (F). STRING analysis was conducted with significantly changed DEGs (raw p value < 0.05), and relevant enriched pathways are presented. (I–M) Representative WB image (I and K) and quantification (J, L, and M) of phosphorylation of STAT6 and mTOR, respectively, in in vitro -differentiated Th2 cells ( n = 3 different donors) treated with CD2, CD3, and CD28 activation antibodies with/without additional supplementation of 1 mM of Phe for indicated time points. (N) Heatmap of mRNA expression of critical transcription factors, cytokines, and activation markers in activated in vitro -differentiated Th2 cells treated with increasing doses of Phe. mRNA expression was determined using RT-qPCR. n = 6–8 different donors. Data are analyzed using one-way ANOVA with Dunnett’s correction. Z scores were determined and plotted as heatmap with different genes mentioned as rows. Data are row normalized. (O) Frequency of activated IL4 + Th2 cells with/without additional supplementation of 1 mM of Phe ( n = 4 different donors). (P) Frequency of activated CD3 + CD4 + CCR4 + GATA3 + CD161 + Th2 cells following incubation with/without supplementation of 1 mM Phe for 24 h ( n = 3 different donors). (A–D, J, L, M, N, O, and P) Each dot represents one donor. (A–C, J, L, M, O, and P) Paired t test was used for analysis. Bars represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also and , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Phospho-proteomics, Expressing, Activation Assay, In Vitro, Incubation, RNA Sequencing, Activity Assay, Quantitative RT-PCR

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: Low intracellular L-phenylalanine levels in pathogenic memory CD4 + T effector cell populations in severe allergic patients (A–C) Representative flow cytometry dot plots of Th2a cells (A-left) and ILC1, ILC2, and ILC3 (C-left). Number of Th2a cells (A-right); memory CRTH2 + Treg cells and PD1 + Treg cells (B); and ILC1, ILC2, and ILC3 (C-right) in controls ( n = 9) and patients with mild ( n = 7) and severe ( n = 11) allergy (Cohort A). (D) Differentially expressed proteins in serum of controls ( n = 10) and mild ( n = 9) and severe ( n = 10) allergic patients (Cohort A) assessed with PEA technology and presented as NPX. (E) Heatmaps with hierarchical clustering analysis of all metabolites measured in memory CD4 + Teff ( n = 195, left) and Treg ( n = 233, right) cells in controls ( n = 6) and allergic ( n = 11) subjects. A, controls; B, mild allergy; C, severe allergy (from Cohort A). (F) Normalized abundance of Phe in Teff cells in group 1 (control, n = 1; severe allergy, n = 4) and 2 (control, n = 5; mild allergy, n = 1, severe allergy, n = 6) (from Cohort A). (G) t-distributed stochastic neighbor embedding (tSNE) plot of unbiased 2-dimensional flow cytometric analysis of memory CD4 + Teff cells (CD3 + CD4 + CD45RA − CD127 + CD25 − ) from patients with severe allergy (subset of cohort A, n = 9) identifying seven subpopulations based on CD161 and PD-1. (H) Pearson correlation of normalized abundance of intracellular Phe, in memory CD4 + Teff cells from patients with severe allergy ( n = 9), with counts of CD161 + populations within memory CD4 + Teff cells (from Cohort A). Mann-Whitney U test (A–C), one-way ANOVA with Fisher’s LSD test (D), and unpaired t test (F) were used to compare differences among groups. Graphs represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Clinical characteristics of Cohort A are shown in and . NPX, normalized protein expression; Pop, population. See also and , , , , , , , , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Control, MANN-WHITNEY, Expressing

Journal: Cell Reports Medicine

Article Title: L-Phenylalanine is a metabolic checkpoint of human Th2 cells

doi: 10.1016/j.xcrm.2025.102466

Figure Lengend Snippet: Decreased expression of large amino acid transporters in Th2 cells of allergic patients correlates with elevated serum levels of L-phenylalanine (A–C) Top significantly enriched pathways (A) and upregulated (B) and downregulated (C) metabolic networks within differentially expressed genes (DEG, p < 0.05) in allergic asthma patients compared to controls (control n = 15, allergic asthma n = 37) from GEO: GSE75011 (Cohort B). Black line represents ratio of genes in experiment over complete pathway set. (D) Phe metabolism and transport pathway heatmap showing fold change (Log 2 FC) of DEGs in allergic asthma patients ( n = 37) compared to controls ( n = 15) from GEO: GSE75011 (Cohort B). ∗ p < 0.05. Pathway curated and adapted from GSEA and MSigDB Database . (E) Phe metabolism and transport schematic highlighting DEGs in Th2 cells of allergic asthma and controls (GEO: GSE75011 ) (Cohort B). Significantly upregulated (red) and downregulated (blue) genes; detected but not significantly different (black); not detected in original dataset (black and underlined). Adapted from KEGG pathway. (F) Serum Phe concentration in controls ( n = 8) and mild ( n = 30) and severe ( n = 37) allergic patients (Cohort D) quantified by targeted metabolomics. Kruskal-Wallis test was used for analysis. (G) SLC7A5 (LAT1), SLC7A8 (LAT2), and SLC3A2 (CD98; LAT3) mRNA expression in in vitro -differentiated Th2 cells treated with additional Phe with/without CD2, CD3, and CD28 activation for 24 h. Following incubation, mRNA expression was determined using RT-qPCR ( n = 6–8 different donors). (H and I) Representative WB image of LAT1 expression (H) and LAT1 protein quantification in 8 different donors (I) in in vitro -differentiated Th2 cells treated with additional Phe with/without CD2, CD3, and CD28 activation for 24 h. Expression of LAT1 presented as relative ratio normalized to β-actin. (J) Phe uptake into in vitro -differentiated Th2 cells was quantified colorimetrically. Cells were incubated in full medium with/without SLC7A5 inhibitor (KYT0353) and activation of CD2, CD3, and CD28 for 6 h. Data were analyzed using paired t test ( n = 3 different donors). (K) Spearman correlation of serum Phe concentration and relative LAT1 expression in CD4 + T cells in allergic asthma patients (left) and controls (right) (Cohort E). (G–I) Data are analyzed by one-way ANOVA with Dunnett’s correction. (F–J) Bars represent mean ± SEM. Each dot represents one donor. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also and and , , , , , , , , and .

Article Snippet: Next, total CD3 + CD4 + T cells or memory CD3 + CD4 + T cells were isolated using the human CD4 + T cell Isolation Kit or Memory CD4 + T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), for respective cell types, according to manufacturer instructions on the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Control, Concentration Assay, In Vitro, Activation Assay, Incubation, Quantitative RT-PCR

Journal: World Journal of Diabetes

Article Title: Oral Akkermansia muciniphila may ameliorates immune dysregulation in a murine model of streptozotocin-induced type 1 diabetes

doi: 10.4239/wjd.v16.i12.111771

Figure Lengend Snippet: Analysis of the regulatory T cells phenotype, spleen cytokine profile and intestinal mucosa immunohistochemistry (30-fold) in each group of mice. A: Flow gating strategy; B: Columnar diagram of cluster of differentiation (CD) 4 + forkhead box P3 (FoxP3 + ) regulatory T (Treg) cells expression in the spleens of the mice in each group ( n = 8); C: Spleen CD4 + /CD8 + cell ratio histogram for each group ( n = 8); D: Histogram of transforming growth factor-beta (TGF-β) expression in the spleens of the mice in each group ( n = 10); E: Correlation analysis between CD4 + FoxP3 + Tregs and TGF-β ( n = 20); F: Columnar diagram of tumor necrosis factor-alpha expression in the spleens of the mice in each group ( n = 6-7); G: Spleen interferon-gamma expression histogram for each group of mice ( n = 6-7); H: Histogram of interleukin-4 expression in the spleens of the mice in each group ( n = 6-7); I: Nuclear factor kappa-B (NF-κB) p65 positive expression score for the mouse colon (30 times); J: Histogram of NF-κB p65 comprehensive positive intensity histochemistry score for the colon in each group ( n = 4); K: Signal transducer and activator of transcription 1 positive expression score in the mouse colon (30 times); L: Histochemical score of colon signal transducer and activator of transcription. a P < 0.05 vs negative control group. b P < 0.01 vs negative control group. c P < 0.001 vs negative control group. d P < 0.05 vs streptozotocin-induced type 1 diabetes mellitus group. e P < 0.01 vs streptozotocin-induced type 1 diabetes mellitus group. f P < 0.001 vs streptozotocin-induced type 1 diabetes mellitus group. NC group: Negative control group; STZ group: Streptozotocin-induced type 1 diabetes mellitus group; A. muciniphila group: Akkermansia muciniphila intervention group; FSC-A: Forward scatter area; SSC-A: Side scatter area; FSC-H: Forward scatter height; CD: Cluster of differentiation; FoxP3: Forkhead box P3; Treg: Regulatory T; TGF-β: Transforming growth factor-beta; TNF-α: Tumor necrosis factor-alpha; IFN-γ: Interferon-gamma; IL-4: Interleukin-4; NF-κB: Nuclear factor kappa-B; STAT1: Signal transducer and activator of transcription 1.

Article Snippet: The following resources were used: STZ (Sigma, United States), sodium citrate buffer (Shanghai Zeta Company, China), a hematoxylin-eosin staining kit (Wuhan Sevier Biological Company, China), 4% paraformaldehyde fixative (Wuhan Sevier Biological Company, China), an anti-insulin mouse mAb (Wuhan Sevier Biological Company, China), an anti-mouse CD4-fluorescein isothiocyanate antibody (Hangzhou Lianke Biological Company, China), an anti-mouse CD8- APC antibody (Hangzhou Lianke Biological Company, China), an anti-mouse forkhead box P3 (FoxP3)-PE antibody (Hangzhou Lianke Biological Company, China), a purified anti-mouse CD16/32 antibody (Hangzhou Lianke Biological Company, China), a mouse IgG1 isotype control-PE antibody (Hangzhou Lianke Biological Company, China), mouse fluorescence-activated cell sorting concentrate (4 ×) and diluent (Hangzhou Lianke Biological Company, China), rabbit anti-mouse STAT1 polyclonal antibodies (Wuhan Sevier Biotechnology Company, China), rabbit anti-mouse NF-κB p65 polyclonal antibodies (Wuhan Sevier Biotechnology Company, China), a goat anti-mouse immunoglobulin secondary antibody (Wuhan Sevier Biotechnology Company, China), enzyme-linked immunosorbent assay (ELISA) kits (for TNF-α, IFN-γ, IL-4, IL-10, TGF-β, ZO-1 and zonulin; Shanghai Enzymatic Immunobiology Company, China), a DNA extraction kit (Kaijie, Germany), and a double-stranded DNA determination kit (Sigma, United States).

Techniques: Immunohistochemistry, Expressing, Negative Control