il-2, mouse  (MedChemExpress)

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Experimental schematic for Panels B-M. ( B-D ) Percent IL-2 + of live CD8 + T cells at multiple time points of polyclonal stimulation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose ( B ), or after 72 hours ( C ). Representative histograms after 72 hours of activation ( D ). ( E-G ) Percent IFN-γ + of live CD8 + T cells at multiple time points of polyclonal stimulation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose ( E ), or after 72 hours ( F ). Representative histograms after 72 hours of activation ( G ). ( H-J ) Percent TNF-α + of live CD8 + T cells at multiple time points of polyclonal stimulation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose ( H ), or after 72 hours ( I ). Representative histograms after 72 hours of activation ( J ). ( K-M ) Barplots depicting the percent polyfunctional (IL-2 + IFN-γ + TNF-α + ) of live CD8 + T cells ( K ), GZMB + of live CD8 + T cells ( L ), or PD-1 + of live CD8 + T cells ( M ) after 72 hours of activation in high (10 mM) versus low (1.5 mM) glucose. Abbreviations : Glc = glucose; FMO = Fluorescence Minus One (control condition); IFN-γ = Interferon Gamma; TNF-α = Tumor Necrosis Factor alpha; GZMB = Granzyme B; PD-1 = Programmed Cell Death Protein 1 Statistics : All data points are distinct biological replicates. Data are presented as mean ± standard deviation (B, C, E, F, H, I, K-M). Panels B, E, and H show data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Panel C contains data aggregated from 10 independent experiments with n=34 biological replicates. Panels F, I, and K-M contains data aggregated from 10 independent experiments with n=22 biological replicates. Statistical significance assessed by Student’s t-test (B, C, E, F, H, I, K-M). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Fluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Experimental schematic for panels B-E. ( B-E ) Percent IL-2 + ( B ), IFN-γ + ( C ), TNF-α + ( D ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( E ) of live JAXBoy-OT-I CD8 + T cells after 72 hours of stimulation with SIINFEKL peptide in high (10 mM) versus low (1.5 mM) glucose. ( F ) Experimental schematic for panels G-J. ( G-J ) Percent IL-2 + ( G ), IFN-γ + ( H ), TNF-α + ( I ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( J ) of live CD8 + T cells after 24 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 supplemented with IL-2 and then expanded in IL-2 for an additional 72 hours while maintained continuously in high (10 mM) versus low (1.5 mM) glucose. ( K ) Experimental schematic for panels L-O. ( L-O ) Percent IL-2 + ( L ), IFN-γ + ( M ), TNF-α + ( N ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( O ) of live human CD8 + T cells after 3 days of activation on DynaBeads in 11mM glucose supplemented with hIL-2, followed by 2 days expansion in 10 mM versus 1.5 mM glucose with hIL-2. Abbreviations : Glc = Glucose; hIL-2 = Human Interleukin-2; IFN-γ = Interferon Gamma; PBMCs = Peripheral Blood Mononuclear Cells; TNF-α = Tumor Necrosis Factor Alpha Statistics : All data points are distinct biological replicates. Data are presented as mean ± standard deviation (B-E, G-J). Panels B-E and G-J show data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Data points in L-O are derived from three independent healthy human donors. Statistical significance assessed by Student’s t-test (B-E), two-way ANOVA (G-J), or two-tailed paired t-test (L-O). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Proliferation Index of live CD8 + T cells after 72 hours of polyclonal stimulation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. Proliferation index was calculated using the proliferation modeling tool in FlowJo software. ( B ) Proliferation Index of live CD8 + T cells after 24 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 supplemented with IL-2 and then expanded in IL-2 for an additional 72 hours while maintained continuously in high (10 mM) versus low (1.5 mM) glucose. Proliferation index was calculated using the proliferation modeling tool in FlowJo software. Statistics : All data points are distinct biological replicates. Data are presented as mean ± standard deviation (A and B). Panels A and B contain data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Statistical significance assessed by two-way ANOVA. P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Software, Activation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Experimental schematic for panels B-E. ( B-E ) Percent IL-2 + ( B ), IFN-γ + ( C ), TNF-α + ( D ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( E ) of live CD8 + T cells after 72 hours of activation on plate-bound α-CD3/ α-CD28. Cells were provided glucose at the indicated concentrations and times. (F) Experimental schematic for panel G. (G) Percent IL-2 + of live CD8 + T cells activated on plate-bound α-CD3/ α-CD28 in low (1.5 mM) glucose for the first 48 hours before glucose supplementation to the indicated concentrations for the last 24 hours of activation. (H) Experimental schematic for panel I. (I) Percent IL-2 + of live CD8 + T cells activated on plate-bound α-CD3/ α-CD28 at the indicated glucose concentrations. After 48 hours, a glucose uptake inhibitor (KL-11743) was added for the final 24 hours of activation. Abbreviations : GLUT1inh = Glucose Transporter Inhibitor KL-11743; hrs = hours; IFN-γ = Interferon Gamma; TNF-α = Tumor Necrosis Factor Alpha Statistics : All data points are distinct biological replicates. Data are presented as mean ± standard deviation (B-E, G, I). Panels B-E, G, and I show data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Statistical significance assessed by one-way ANOVA (B-E, G, I). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A-D ) Barplots depict percent IL-2 + ( A ), IFN-γ + ( B ), TNF-α + ( C ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( D ) of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in glucose or galactose at the indicated concentrations. ( E-F ) Oxygen consumption rate (OCR) as measured during the Seahorse XF Cell Mito Stress Test on CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in glucose or galactose at the indicated concentrations ( E ). Barplot depicts baseline OCR ( F ). Seahorse assay medium contained the same sugar source as the activation condition. ( G-H ) Extracellular Acidification Rate (ECAR) as measured during the Seahorse XF Cell Mito Stress Test on CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in glucose or galactose at the indicated concentrations ( G ). Barplot depicts baseline ECAR ( H ). Seahorse assay medium contained the same sugar source as the activation condition. ( I ) Diagram depicting the fates of citrate produced in the mitochondria. ( J-L ) Metabolomics analysis on CD8 + T cells after 72 hours of activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose measured by liquid chromatography-mass spectrometry (LC-MS). Barplots depict ratios of peak areas for citrate to α-ketoglutarate ( J ), α-ketoglutarate to malate ( K ), or citrate to malate ( L ). ( M ) Media citrate levels by LC-MS after culturing CD8 + T cells for 24, 48, and 72 hours on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose, compared to cell-free controls. ( N ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of dichloroacetate or vehicle control. ( O ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of the mitochondrial pyruvate carrier (MPC) inhibitor UK-5099 or vehicle control. ( P ) Ratio of extracellular lactate to pyruvate levels measured in culture media of CD8 + T cells after 24, 48, and 72 hours of activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose, compared to cell-free controls. ( Q-R ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of the mitochondrial citrate carrier inhibitor (SLC25A1) inhibitor CTPI-2 or vehicle control ( Q ). Representative flow cytometry histograms of IL-2 signal after 72 hours of activation with CTPI-2 or vehicle control, including an un-stimulated naïve control ( R ). ( S ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of sodium citrate. ( T-V ) Metabolomics analysis on CD8 + T cells after 72 hours of activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with SLC25A1 inhibition by CTPI-2. Barplots depict ratios of peak areas for citrate to α-ketoglutarate ( T ), α-ketoglutarate to malate ( U ), or citrate to malate ( V ). Abbreviations : Ac-CoA = Acetyl-Coenzyme A; αKG = Alpha-Ketoglutarate; CTPI-2 = Inhibitor of the Mitochondrial Citrate Carrier SLC25A1; DCA = Dichloroacetate; ECAR = Extracellular Acidification Rate; Gal = Galactose; Glc = glucose; IFN-γ = Interferon Gamma; Lac = Lactate; OCR = Oxygen Consumption Rate; Pyr = Pyruvate; TCA cycle = Tricarboxylic Acid cycle; TNF-α = Tumor Necrosis Factor Alpha; UK-5099 = Inhibitor of Mitochondrial Pyruvate Carrier Statistics : Data are presented as mean ± standard deviation (A-D, F, H, J-Q, S-V). Panels A-D contain data aggregated from 5 independent experiments with n=15 biological replicates. Panels E-H contain data aggregated from 3 biological replicates with 5 technical replicates each. Panels J-L contain data aggregated from 2 independent experiments with n=6 biological replicates. Panels M and P contain data aggregated from 4 biological replicates. Panels N-O, S, and T-V show data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Panels Q-R contain data aggregated from 3 independent experiments with n=7 biological replicates. Statistical significance assessed by one-way ANOVA (A-D, F, H), Student’s t-test (J-L), or two-way ANOVA (M-Q, S-V). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Produced, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Flow Cytometry, Inhibition, Standard Deviation

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Proliferation Index of live CD8 + T cells after 72 hours of polyclonal activation by plate-bound α-CD3/ α-CD28 in glucose or galactose at the indicated concentrations. Proliferation index was calculated using the proliferation modeling tool in FlowJo software. ( B ) Metabolomics analysis on CD8 + T cells after 72 hours of activation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose measured by liquid chromatography-mass spectrometry (LC-MS). Barplots depict ratios of peak areas for citrate to succinate. ( C-E ) Percent IFN-γ + ( C ), TNF-α + ( D ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( E ) of live CD8 + T cells after 72 hours of polyclonal activation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of the mitochondrial citrate carrier inhibitor (SLC25A1) inhibitor CTPI-2 or vehicle control. ( F ) Proliferation Index of live CD8 + T cells after 72 hours of polyclonal activation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of the mitochondrial citrate carrier inhibitor (SLC25A1) inhibitor CTPI-2 or vehicle control. ( G-I ) Percent IFN-γ + ( G ), TNF-α + ( H ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( I ) of live CD8 + T cells after 72 hours of polyclonal activation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with varying concentrations of sodium citrate. ( J-M ) Percent IL-2 + ( J ), IFN-γ + ( K ), TNF-α + ( L ), and polyfunctional (IL-2 + IFN-γ + TNF-α + ) ( M ) of live CD8 + T cells after 72 hours of polyclonal activation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose, with addition of 7.5 mM sodium acetate alone or in combination with 100 μM of the mitochondrial citrate carrier inhibitor (SLC25A1) inhibitor CTPI-2. Abbreviations : Glc = Glucose; IFN-γ = Interferon Gamma; TNF-α = Tumor Necrosis Factor alpha; CTPI-2 = Inhibitor of the Mitochondrial Citrate Carrier SLC25A1. Statistics : Data are presented as mean ± standard deviation (A-M). Panel A contains data aggregated from 5 independent experiments with n=15 biological replicates. Panel B contains data aggregated from 2 independent experiments with n=6 biological replicates with 5 technical replicates each. Panels C-F contain data aggregated from 3 independent experiments with n=7 biological replicates. Panels G-I contain data representative of two independent experiments where n=3 biological replicates are shown. Panels J-M contain data representative of two independent experiments where n=5 biological replicates are shown. Statistical significance assessed by one-way ANOVA (A), Student’s t-test (B), or two-way ANOVA (C-M). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Software, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Standard Deviation

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. Exogenous IL-2 is supplemented at 12.5 ng/mL as indicated. ( B ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. Samples are treated with a CD25-blocking antibody or isotype control as indicated. ( C-D ) Flow cytometry histograms of intracellular IL-2 expression by live CD8 + T cells after 72 hours of activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose, where PMA/ Ionomycin and GolgiPlug are added during re-stimulation as indicated. ( E ) IL-2 mRNA expression measured by qPCR quantification in CD8 + cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. IL-2 mRNA was normalized to actin B (ACTB) mRNA levels. ( F ) ELISA for IL-2 secretion into culture medium by CD8 + T cells activated on plate-bound α-CD3/ α-CD28 for 72 hours in high (10 mM) versus low (1.5 mM) glucose. ( G ) Venn diagrams of significantly up-regulated ( left ) or down-regulated ( right ) genes in bulk RNA sequencing from CD8 + T cells after 72 hours of polyclonal activation in galactose versus high (10 mM) glucose compared to low (1.5 mM) versus high (10 mM) glucose. ( H-I ) Gene Ontology (GO) analysis on common significantly differentially genes from analysis of bulk RNA sequencing datasets as in panel G. Up- and down-regulated genes were evaluated separately, considering only genes that were significantly differentially expressed in the same direction for galactose versus high (10 mM) glucose or low (1.5 mM) versus high (10 mM) glucose. Cellular Component (CC) gene signatures were assessed in ( H ), and Panther Pathways were assessed in ( I ). Pathways enriched among up-regulated genes are shown in green, and pathways enriched among down-regulated genes are shown in purple. ( J ) Heat map depicting the expression of cytokine genes from bulk RNA-sequencing of CD8 + T cells after 72 hours of polyclonal activation in the indicated conditions. ( K ) Heatmap of transcriptional programs inferred from bulk RNA-sequencing of CD8 + T cells after 72 hours of polyclonal activation in the indicated conditions. ( L-O ) ImageStream flow cytometry of CD8 + T cells after 72 hours of polyclonal activation in low (1.5 mM) versus high (10 mM) glucose, or high glucose with addition of the SLC25A1 inhibitor CTPI-2 versus vehicle control. Representative images for Nfatc2 ( L ) and Nfatc1 ( N ) are shown, as well as violin plots showing per cell similarity scores for Nfatc2 ( M ) or Nfatc1 ( O ) relative to propidium iodide (PI) nuclear signal. ( P ) Flow cytometry histograms of intracellular IL-2 expression by live CD8 + T cells after 72 hours of activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose, where the calcineurin inhibitor cyclosporin A is added as indicated during restimulation with PMA/ ionomycin. Abbreviations : ACTB = actin B; CsA = Cyclosporin A; ELISA = Enzyme-Linked Immunosorbent Assay; Glc = glucose; GO = Gene Ontology; Gzmb = Granzyme B; NFAT = Nuclear Factor of activated T cells; NFkB = Nuclear Factor Kappa-light-chain-enhancer of Activated B cells; PMA = Phorbol 12-myristate 13-acetate; qPCR = Quantitative Polymerase Chain Reaction; TFs = Transcription Factors Statistics : All data points shown are distinct biological replicates. Barplots are presented as mean ± standard deviation (A-B, E-F). Violin plots depict the median as a horizontal line (M, O). Panels A, B, and L-P show data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Bulk RNA-sequencing data in panels G-K are derived from n=3 biological replicates. Panel E contains data aggregated from 2 independent experiments with n=6 biological replicates. Panel F contains data from 3 biological replicates with n=5 technical replicates, where technical replicates are shown. Statistical significance assessed by two-way ANOVA (A, B), by one-way ANOVA (F, M, O), or by Welch’s corrected two-tailed t-test (E, F). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Blocking Assay, Control, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Proliferation index of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. Exogenous IL-2 is supplemented as indicated. Proliferation index was calculated using the proliferation modeling tool in FlowJo software. ( B ) Proliferation index of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ CD28 in high (10 mM) versus low (1.5 mM) glucose. A CD25-blocking antibody or isotype control are added as indicated. Proliferation index was calculated using the proliferation modeling tool in FlowJo software. (C) Principal component analysis plot of differential gene analysis from bulk RNA sequencing of CD8 + T cells after 72 hours of activation in high (10 mM) glucose, low (1.5 mM) glucose, or galactose. (D) Heatmap highlighting differentially expressed genes from bulk RNA sequencing of CD8 + T cells that participate in calcium intake and handling. Statistics : All data points are distinct biological replicates. Data are presented as mean ± standard deviation (A-B). Panels A-B contain data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Statistical significance assessed by two-way ANOVA (A-B). P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Activation Assay, Software, Blocking Assay, Control, RNA Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Citrate Compartmentalization Controls Calcium-Dependent Cytokine Production in Effector T Cells

doi: 10.64898/2026.06.11.731694

Figure Lengend Snippet: ( A ) Chemical structure of citrate showing three carboxylate groups that can chelate Ca 2+ . ( B ) Blanked absorbance of o-Cresolphthalein bound to calcium with increasing concentrations of citrate or EDTA, across a range of calcium concentrations. (C) Mean Fluorescence Intensity (MFI) of Fluo-4 dye in live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose with addition of the SLC25A1 inhibitor CTPI-2 or vehicle control. ( D-E ) Percent IL-2 + of live CD8 + T cells after 72 hours of polyclonal activation on plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. The calcineurin inhibitor cyclosporin A or sodium citrate are added during re-stimulation with PMA/ ionomycin as indicated. Barplots shown in ( D ) and representative flow cytometry histograms shown in ( E ). ( F ) Dotplots depicting geometric Mean Fluorescence Intensity (gMFI) of mitochondrial aconitase 2 (ACO2) by intracellular flow cytometry staining in live CD8 + T cells across 72 hours of polyclonal activation by plate-bound α-CD3/ α-CD28 in high (10 mM) versus low (1.5 mM) glucose. ( G-I ) Positive and negative correlations between transcription factor programs calculated with decoupleR and selected metabolite levels: citrate ( G ), citrate/ isocitrate ratio ( H ), and citrate/ α-Ketoglutarate ratio ( I ) across cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE). ( J ) Calcium-responsive transcription factor programs ranked by enrichment among all transcription factor programs with significant negative associations between program score and metabolite abundance across cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE). ( K ) Gene-gene correlation network across TCGA tumors (n=9,279 patient tumor samples). Pairwise Spearman correlations were calculated from bulk tumor RNA-sequencing data. Nodes represent genes involved in lipid metabolism, calcium signaling, and T cell function. Edges connect gene pairs exhibiting significant correlations (|ρ| > 0.2, p-value<1E-20). Edge color indicates the direction of the correlation, and edge width is proportional to correlation strength. Node size is proportional to network degree. ( L ) Representative images of Visium human tumor tissue sections overlaid with the calcium response gene signature score or KEGG oxidative phosphorylation gene signature score. Regions with low or high scores are manually annotated for visual clarity. ( M ) Barplots depicting the number of Visium tumor samples with significant cross-correlation (FDR-adjusted p-value < 0.001 and |Moran’s I| > 0.1) between the indicated KEGG pathway and the calcium response signature score. Abbreviations : ACO2 = Aconitase 2; CCLE = Cancer Cell Line Encyclopedia; CsA = Cyclosporin A; EDTA = ethylenediaminetetraacetic acid; FDR = False Discovery Rate; gMFI = Geometric Mean Fluorescence Intensity; HIF1A = Hypoxia-Inducible Factor 1-Alpha; Lac = Lactate; MFI = Mean Fluorescence Intensity; PMA = Phorbol 12-myristate 13-acetate; Pyr = pyruvate; TCGA = The Cancer Genome Atlas; TFs = Transcription Factors Statistics : All data points shown are distinct biological replicates. All plots depict mean ± standard deviation (C, D, F). Panel C contains aggregated from 2 independent experiments with n=6 biological replicates. Panels D-E show data representative of two independent experiments where n=3 biological replicates are shown from a representative experiment. Panel F shows data averaged from four biological replicates. Statistical significance assessed by on-way ANOVA (C) or two-way ANOVA (D). All other statistics are detailed in the Materials and Methods. P-values: not significant (ns) for p>0.05, ∗ for p≤0.05, ∗∗ for p≤0.01, ∗∗∗ for p≤0.001, and ∗∗∗∗ for p≤0.0001.

Article Snippet: Where indicated, JAXBoy-OT-I T cells were alternatively activated with 1 μg/mL of SIINFEKL peptide derived from hen egg white ovalbumin corresponding to amino acids 257-264 (AnaSpec, AS-60193-1) and 12.5 ng/mL of murine IL-2 (Miltenyi Biotec, 130-120-330).

Techniques: Fluorescence, Activation Assay, Control, Flow Cytometry, Staining, RNA Sequencing, Cell Function Assay, Phospho-proteomics, Standard Deviation

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: MPDA contains IL-2 and Syr that serve as a T cell growth factor and an MCT inhibitor, respectively. Upon laser exposure, MPDA generates mPTT to stimulate DC maturation and activate further T cell-mediated anticancer immunity. Syr inhibits lactate excretion, thereby promoting IL-2-mediated T cell activation and proliferation.

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: Activation Assay

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: Characterization of Syr/IL-2@MPDA NPs. (A) Synthetic route of Syr/IL-2@MPDA; (B) TEM image of Syr/IL-2@MPDA; (C) Elemental analysis of Syr/IL-2@MPDA by EDS-HAADF; (D) Number-weighted size distribution of Syr/IL-2@MPDA; (E) UV–vis spectra of Ni-MPDA, Syr/IL-2@MPDA and pure Syr; (F) EE and DL of Syr by MPDA (1 mg) at different Syr inputs; (G) Percentage of loaded His-tagged IL-2 (1 µg) after 1 h of co-incubation with MPDA or Ni-MPDA (1 mg) at 4 °C; Relative release curves of (H) Syr and (I) IL-2 from Syr/IL-2@MPDA in PBS; (J) Intensity-weighted hydrodynamic size and PDI of Syr/IL-2@MPDA for 7 d in PBS containing 10% FBS; (K) Infrared thermography of a 10 mm cuvette containing different concentrations of Syr/IL-2@MPDA solution under laser irradiation (2.0 W/cm 2 ); (L) Photothermal heating curves of the Syr/IL-2@MPDA solution under laser irradiation (2.0 W/cm 2 ) at various concentrations; (M) Photothermal heating curves of the Syr/IL-2@MPDA solution (250 µg/ml) under laser irradiation; (N) Photothermal stability under four cycles of laser irradiation and natural cooling. Error bars represent means ± SD ( n = 3).

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: Incubation, Irradiation

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: Syr/IL-2@MPDA mediated therapy in vitro . (A) Lactic acid content of Hepa 1–6 cells of each group; (B) pH value of Hepa 1–6 cells of each group; (C) Concentration-dependent pH value of Hepa 1–6 cells treated with Syr/IL-2@MPDA; (D) Schematic illustration of experiment for CD8 + T cells activation and proliferation; (E-F) Quantification analysis of CFSE-labeled (E) CD8 + T cells and (F) IFN-γ + TNF-α + cells in CD8 + T cells after different treatments; (G) Flow cytometry histogram of CFSE-labeled CD8 + T cells; (H) Concentration of IFN-γ in the medium of each group; (I) Quantification analysis of tumor cell viability in a co-culture system; (J) Flow cytometry histogram of IFN-γ + TNF-α + cells in CD8 + T cells; (K) Schematic illustration of the experiment for BMDC maturation; (L) quantification analysis of mature DCs (CD80 + CD86 + CD11c + cells) of each group and (M) flow cytometry histogram. Data were expressed as mean ± SD.

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: In Vitro, Concentration Assay, Activation Assay, Labeling, Flow Cytometry, Co-Culture Assay

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: Syr/IL-2@MPDA mediated cancer immunotherapy in vivo . (A) Schematic illustration and timeline of Syr/IL-2@MPDA in a Hepa1–6 tumor model; (B) Illustration of mPTT treatment under infrared thermography monitoring; (C) Tumor volume growth curves of each group (PBS, MPDA, MPDA + Light, Syr@MPDA, IL-2@MPDA, Syr/IL-2@MPDA, and Syr/IL-2@MPDA + Light, n = 5); (D) Tumor histological analysis of each group. Scale bar: 200 µm: (E-H) Corresponding quantification of (E) matured DCs, (F) CD3 + CD8 + T cells, (G) GZMB + CD8 + T cells, and (H) Treg cells in tumors. Data are expressed as mean ± SD.

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: In Vivo

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: Syr/IL-2@MPDA mediated enhanced ICB therapy in vivo . (A) Schematic illustration and timeline of Syr/IL-2@MPDA combined with αPD-L1 in the bilateral Hepa1–6 tumor model; (B) Body weight of each group after different treatments (PBS, αPD-L1, Syr/IL-2@MPDA + L and Syr/IL-2@MPDA + L + αPD-L1, n = 5); (C) Tumor volume, (D) digital image and (E) tumor weight of primary tumor and distant tumor in each group. (F-H) Corresponding quantification of CD3 + CD8 + T cells and Treg cells in (F) tumors and (G) spleen. (H) PD-L1 expression in the tumors of each group was assessed by immunohistochemistry. Scale bar: 100 µm.

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: Syr/IL-2@MPDA-mediated enhanced TAE therapy in vivo . (A) Schematic illustration and timeline of Syr/IL-2@MPDA combined with TAE in the orthotopic N1S1 rat model; (B) T 1 -weighted MR images of N1S1 rats at different times post treatment (PBS, TAE, αPD-L1, Syr/IL-2@MPDA + L + TAE, and Syr/IL-2@MPDA + L + αPD-L1 + TAE, n = 5); (C) Body weight and (D) tumor volume of each group. (E) H&E staining of tumor slices collected from each group at Day 2 post-treatment. Scale bar: 200 µm; (F) Immunofluorescence images of CD8 + T cells in the tumors of each group. Scale bar: 50 µm.

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

doi: 10.1016/j.ajps.2026.101165

Figure Lengend Snippet: Transcriptomic analysis of Syr/IL-2@MPDA + L with TAE. (A) PCA of transcriptomic analysis data; (B) Venn diagram of genes detected in N1S1 tumors treated with PBS and Syr/IL-2@MPDA + L + TAE; (C) Volcano plot of differentially expressed genes in the PBS and Syr/IL-2@MPDA + L + TAE groups; (D) Heatmap of differential genes in the PBS and Syr/IL-2@MPDA + L + TAE groups; (E) KEGG enrichment analysis and (F) GO enrichment analysis of genes related to immune system processes.

Article Snippet: Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress.

Techniques: