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recombinant murine il 4 (R&D Systems)

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R&D Systems recombinant murine il 4
Recombinant Murine Il 4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/murine+il/Recombinant+Mouse+IL-4+Protein/pmc13111333-81-53-56
Average 96 stars, based on 617 article reviews

recombinant murine il 4 - by Bioz Stars, 2026-09

96/100 stars

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Concentration Assay:

Article Title: IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells.
Article Snippet: .. Xenogeneic cultures were maintained as described previously.17 After 3 weeks of culture, goat anti–murine IL-7–neutralizing antibody (AB-407NA; R&D Systems) was added to a final concentration of 10 g/mL, and cultures were maintained for 1 additional week without medium changes. ..

Article Title: IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells
Article Snippet: .. 17 After 3 weeks of culture, goat anti–murine IL-7–neutralizing antibody (AB-407-NA; R&D Systems) was added to a final concentration of 10 μg/mL, and cultures were maintained for 1 additional week without medium changes. ..

Enzyme-linked Immunosorbent Assay:

Article Title: IL-21 Exacerbates Autoimmune Myositis by Enhancing the Accumulation of GM-CSF–Producing γδ T Cells in the Muscle
Article Snippet: Serum levels of IL-21 and GM-GSF were analyzed by a human IL-21 ELISA MAX kit and a human GM-GSF ELISA MAX kit, respectively (BioLegend, San Diego, CA). .. Serum levels of human CX3CL1 and murine IL-21 were analyzed by a human CX3CL1/ Fractalkine Quantikine ELISA Kit and a mouse IL-21 DuoSet ELISA, respectively (R&D Systems, Minneapolis, MN). ..

Article Title: MHCII-Mediated Dialog between Group 2 Innate Lymphoid Cells and CD4 + T Cells Potentiates Type 2 Immunity and Promotes Parasitic Helminth Expulsion
Article Snippet: Cytokine concentration was detected by sequential incubation with a biotinylated detection anti-cytokine antibody (IL-4 (BD Bioscience), and IL-5 (BD Bioscience), streptavidin-horseradish peroxidase and an HRP colorimetric reagent. .. Murine IL-13 was detected with Quantikine IL-13 (R&D Systems) or mouse IL-13 ELISA Ready- Set-Go! (eBioscience). .. Human IL-4 ELISA (eBioscience) were performed according to the manufacturer’s protocol.

Recombinant:

Article Title: The RNA helicase DHX15 is a critical regulator of natural killer-cell homeostasis and functions.
Article Snippet: The RNA helicase DHX15 is widely expressed in immune cells and traditionally thought to be an RNA splicing factor or a viral RNA sensor.. However, the role of DHX15 in NK-cell activities has not been studied thus far.. Here, we generated Dhx15-floxed mice and found that conditional deletion of Dhx15 in NK cells (Ncr1Dhx15 mice) resulted in a marked reduction in NK cells in the periphery and that the remaining Dhx15-deleted NK cells failed to acquire a mature phenotype.

Article Title: Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis
Article Snippet: .. Cells were maintained in XVIVO-20 mediumsupplemented with 5% Fetal Bovine Serum (FBS) (ThermoFisherScientific #10270106), 1% PSG (Gibco #10378-016) and recombinant murine SCF 50 ng/mL (PeproTech # 250-03-250UG), murine IL-3 10 ng/mL (PeproTech #313-13-10UG) and murine IL-6 10 ng/mL (R&D Systems #406-ML-025), in a 37°C and 5% CO 2 atmospheric environment. ..

Stem Cell Culture:

Image Search Results

( 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.

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

( 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.

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.

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

( 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.

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.

Techniques: Software, Activation Assay, Standard Deviation

( 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.

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.

Techniques: Activation Assay, Standard Deviation

( 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.

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.

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

( 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.

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.

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

( 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.

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.

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

( 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.

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.

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

( 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.

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.

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

IL-27 impairs early B-cell development in the bone marrow. (A) Experimental scheme for AAV-mediated IL-27 delivery and analysis of splenic and BM B-cell compartments. (B–D) Splenic B-cell analysis 10 weeks after AAV-Ctrl or AAV-IL-27 treatment (n = 2 per group). (B) Frequency of total splenic B cells. (C) CIBERSORTx-based deconvolution of splenic B-cell subsets, including clustering and pseudotime analysis (left) and relative subset distribution (right). (D) Cd93 expression (TPM) in splenic B cells. (E–G) Analysis of splenic and BM B-cell compartments 3 weeks after treatment (n = 4 per group). (E) Representative flow cytometry plots. (F) Frequencies of B220 + CD19 − and B220 − CD19 + populations in the BM and spleen. (G) Relative distribution of B-cell developmental subsets in the BM. (H–I) Kinetics of B-cell populations following IL-27 treatment (n = 2 per group at each time point). (H) Frequency and absolute number of BM B220 + B cells. (I) Proportions of B-cell subsets within the B220 + compartment at the indicated time points. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 impairs early B-cell development in the bone marrow. (A) Experimental scheme for AAV-mediated IL-27 delivery and analysis of splenic and BM B-cell compartments. (B–D) Splenic B-cell analysis 10 weeks after AAV-Ctrl or AAV-IL-27 treatment (n = 2 per group). (B) Frequency of total splenic B cells. (C) CIBERSORTx-based deconvolution of splenic B-cell subsets, including clustering and pseudotime analysis (left) and relative subset distribution (right). (D) Cd93 expression (TPM) in splenic B cells. (E–G) Analysis of splenic and BM B-cell compartments 3 weeks after treatment (n = 4 per group). (E) Representative flow cytometry plots. (F) Frequencies of B220 + CD19 − and B220 − CD19 + populations in the BM and spleen. (G) Relative distribution of B-cell developmental subsets in the BM. (H–I) Kinetics of B-cell populations following IL-27 treatment (n = 2 per group at each time point). (H) Frequency and absolute number of BM B220 + B cells. (I) Proportions of B-cell subsets within the B220 + compartment at the indicated time points. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Cell Analysis, Expressing, Flow Cytometry

Exogenous IL-27 inhibits CLP formation in the bone marrow. (A) qPCR analysis of B-lineage- and myeloid-associated transcription factors in BM cells 5 days after AAV-Ctrl or AAV-IL-27 administration (n = 3 per group). (B) qPCR analysis of CEBPA expression in Reh cells treated with hIL-27 and DAC for 3 days (n = 3 per group). (C–D) Frequencies and absolute numbers of CLPs in WT and CD19 Cre ;IL-27R fl/fl mice (n = 3 per group). (E–F) BM B-cell frequency (E) and proportion of B220 + CD43 hi IgM − B cells (F) in WT and CD19 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for WT Ctrl group; n = 3 for WT IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 4 for CD19 Cre IL-27 group). (G–H) BM B-cell frequency (G) and proportion of B220 + CD43 hi IgM − B cells (H) in CD4 Cre ;IL-27R fl/fl and Lyz2 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for CD4 Cre Ctrl group; n = 3 for CD4 Cre IL-27 group; n = 5 for Lyz2 Cre Ctrl group; n = 6 for Lyz2 Cre IL-27 group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 inhibits CLP formation in the bone marrow. (A) qPCR analysis of B-lineage- and myeloid-associated transcription factors in BM cells 5 days after AAV-Ctrl or AAV-IL-27 administration (n = 3 per group). (B) qPCR analysis of CEBPA expression in Reh cells treated with hIL-27 and DAC for 3 days (n = 3 per group). (C–D) Frequencies and absolute numbers of CLPs in WT and CD19 Cre ;IL-27R fl/fl mice (n = 3 per group). (E–F) BM B-cell frequency (E) and proportion of B220 + CD43 hi IgM − B cells (F) in WT and CD19 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for WT Ctrl group; n = 3 for WT IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 4 for CD19 Cre IL-27 group). (G–H) BM B-cell frequency (G) and proportion of B220 + CD43 hi IgM − B cells (H) in CD4 Cre ;IL-27R fl/fl and Lyz2 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for CD4 Cre Ctrl group; n = 3 for CD4 Cre IL-27 group; n = 5 for Lyz2 Cre Ctrl group; n = 6 for Lyz2 Cre IL-27 group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Expressing

Exogenous IL-27 reshapes immune reconstitution after bone marrow transplantation. (A) Experimental scheme. WT mice underwent BM transplantation followed by AAV-Ctrl or AAV-IL-27 treatment. Splenic and BM immune reconstitution was analysed at 10 weeks after treatment (n = 4 mice for the Ctrl group; n = 5 mice for the IL-27 group). (B) Percentages and absolute numbers of CD45 + splenocytes. (C–F) Frequencies of major immune cell populations within CD45 + splenocytes: myeloid cells (C), macrophages (D), T and B cells (E), and NK cells (F). (G) Frequency of CD4 + T cells among splenic T cells. (H) Proportion of Tregs among CD4 + T cells. (I–J) BM B-cell reconstitution. Frequency of BM B220 + B cells (I) and distribution of B220 + B-cell subsets (J). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 reshapes immune reconstitution after bone marrow transplantation. (A) Experimental scheme. WT mice underwent BM transplantation followed by AAV-Ctrl or AAV-IL-27 treatment. Splenic and BM immune reconstitution was analysed at 10 weeks after treatment (n = 4 mice for the Ctrl group; n = 5 mice for the IL-27 group). (B) Percentages and absolute numbers of CD45 + splenocytes. (C–F) Frequencies of major immune cell populations within CD45 + splenocytes: myeloid cells (C), macrophages (D), T and B cells (E), and NK cells (F). (G) Frequency of CD4 + T cells among splenic T cells. (H) Proportion of Tregs among CD4 + T cells. (I–J) BM B-cell reconstitution. Frequency of BM B220 + B cells (I) and distribution of B220 + B-cell subsets (J). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Transplantation Assay

IL-27 inhibits B-cell development through both direct and indirect mechanisms. (A) Schematic of the BM chimera model. WT (WTR) or IL-27R −/− (KOR) recipients were treated with AAV-Ctrl or AAV-IL-27. Donor BM cells were a 1:1 mix of WT (CD45.1 + ) and IL-27R −/− (CD45.1 − ) cells. Analyses were performed at 5 weeks (B–E; n = 3 per group) or 10 weeks (F–I; n = 5 per group) post-transplantation. (B) Percentage of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (C) Absolute numbers of total CD19 + B cells in spleen and BM of WTR and KOR mice. (D) Absolute numbers of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (E) Percentage of B220 + CD19 − B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in BM of WTR and KOR mice. (F) Absolute numbers of B220 + CD19 - B cells from CD45.1 + or CD45.1 - (IL-27R −/− ) donors in BM of WTR and KOR mice. (G) Frequencies and absolute numbers of BM B220 + B cells derived from CD45.1 + or IL-27R −/− donors in WTR mice. (H) Proportion of CD43 hi IgM − subsets from CD45.1 + or IL-27R −/− donors in BM B220 + B cells in WTR mice. (I–J) Frequencies of CLPs (I) and their donor-specific contributions (J) in BM chimeras. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 inhibits B-cell development through both direct and indirect mechanisms. (A) Schematic of the BM chimera model. WT (WTR) or IL-27R −/− (KOR) recipients were treated with AAV-Ctrl or AAV-IL-27. Donor BM cells were a 1:1 mix of WT (CD45.1 + ) and IL-27R −/− (CD45.1 − ) cells. Analyses were performed at 5 weeks (B–E; n = 3 per group) or 10 weeks (F–I; n = 5 per group) post-transplantation. (B) Percentage of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (C) Absolute numbers of total CD19 + B cells in spleen and BM of WTR and KOR mice. (D) Absolute numbers of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (E) Percentage of B220 + CD19 − B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in BM of WTR and KOR mice. (F) Absolute numbers of B220 + CD19 - B cells from CD45.1 + or CD45.1 - (IL-27R −/− ) donors in BM of WTR and KOR mice. (G) Frequencies and absolute numbers of BM B220 + B cells derived from CD45.1 + or IL-27R −/− donors in WTR mice. (H) Proportion of CD43 hi IgM − subsets from CD45.1 + or IL-27R −/− donors in BM B220 + B cells in WTR mice. (I–J) Frequencies of CLPs (I) and their donor-specific contributions (J) in BM chimeras. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Transplantation Assay, Derivative Assay

Exogenous IL-27 indirectly inhibits BM B-ALL cells. (A) Experimental design. GFP + B-ALL cells were transferred into WT mice, followed by AAV-Ctrl or AAV-IL-27 administration. BM and splenic B-ALL cells were analysed 9–10 days later (n = 3 per group). (B–C) Representative flow cytometry plots and quantification of GFP + B-ALL and GFP − B cells. (D) GSVA analysis of transcriptional changes in BM B-ALL cells isolated from mice in (A). (E) Gene Ontology (GO) enrichment analysis of DEGs between Ctrl and IL-27 groups in (A) (adjusted P < 0.05, |log 2 fold change| > 1). The outer ring indicates enriched GO terms, the middle ring represents relative gene expression changes in the IL-27 group, and the inner ring shows background gene counts, with colour intensity reflecting enrichment significance. Enriched pathways include interferon-γ-mediated signalling, interferon-β responses, adhesion-related processes, protozoan defence responses, calmodulin-dependent kinase signalling, and negative regulation of STAT tyrosine phosphorylation. (F–G) Expression of apoptosis- and cell cycle-related genes in BM B-ALL cells from (A). (H) Experimental design. WT and IL-27R −/− mice transplanted with GFP + B-ALL cells were treated with AAV-Ctrl or AAV-IL-27 (n = 4 for WT Ctrl group; n = 4 for WT IL-27 group; n = 5 for IL-27R −/− Ctrl group; n = 5 for IL-27R −/− IL-27 group). (I–J) Representative flow plots and quantification of BM B-ALL cells. (K–L) Flow cytometry plots and quantification of BM B-ALL cells in CD4 Cre ;IL-27R fl/fl , CD19 Cre ;IL-27R fl/fl , and Lyz2 Cre ;IL-27R fl/fl mice (n = 2 for CD4 Cre Ctrl group; n = 2 for CD4 Cre IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 3 for CD19 Cre IL-27 group; n = 3 for Lyz2 Cre Ctrl group; n = 3 for Lyz2 Cre IL-27 group). (M) Flow cytometry plots and quantification of BM B-ALL cells in Rag1 −/− mice (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 indirectly inhibits BM B-ALL cells. (A) Experimental design. GFP + B-ALL cells were transferred into WT mice, followed by AAV-Ctrl or AAV-IL-27 administration. BM and splenic B-ALL cells were analysed 9–10 days later (n = 3 per group). (B–C) Representative flow cytometry plots and quantification of GFP + B-ALL and GFP − B cells. (D) GSVA analysis of transcriptional changes in BM B-ALL cells isolated from mice in (A). (E) Gene Ontology (GO) enrichment analysis of DEGs between Ctrl and IL-27 groups in (A) (adjusted P < 0.05, |log 2 fold change| > 1). The outer ring indicates enriched GO terms, the middle ring represents relative gene expression changes in the IL-27 group, and the inner ring shows background gene counts, with colour intensity reflecting enrichment significance. Enriched pathways include interferon-γ-mediated signalling, interferon-β responses, adhesion-related processes, protozoan defence responses, calmodulin-dependent kinase signalling, and negative regulation of STAT tyrosine phosphorylation. (F–G) Expression of apoptosis- and cell cycle-related genes in BM B-ALL cells from (A). (H) Experimental design. WT and IL-27R −/− mice transplanted with GFP + B-ALL cells were treated with AAV-Ctrl or AAV-IL-27 (n = 4 for WT Ctrl group; n = 4 for WT IL-27 group; n = 5 for IL-27R −/− Ctrl group; n = 5 for IL-27R −/− IL-27 group). (I–J) Representative flow plots and quantification of BM B-ALL cells. (K–L) Flow cytometry plots and quantification of BM B-ALL cells in CD4 Cre ;IL-27R fl/fl , CD19 Cre ;IL-27R fl/fl , and Lyz2 Cre ;IL-27R fl/fl mice (n = 2 for CD4 Cre Ctrl group; n = 2 for CD4 Cre IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 3 for CD19 Cre IL-27 group; n = 3 for Lyz2 Cre Ctrl group; n = 3 for Lyz2 Cre IL-27 group). (M) Flow cytometry plots and quantification of BM B-ALL cells in Rag1 −/− mice (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Flow Cytometry, Isolation, Gene Expression, Phospho-proteomics, Expressing

IL-27 reshapes the BM niche, reducing B-cell support and leukaemogenesis. (A) Pseudotime analysis of BM B-cell subsets (C0–C10). (B) Expression of representative marker genes across B-cell clusters in (A). (C–G) BM cells were harvested 8 days after AAV-Ctrl or AAV-IL-27 administration for RNA-seq analysis (n = 3 per group). (C) CIBERSORTx analysis showing increased pre-pro-B and cycling pro-B subsets after IL-27 treatment. (D) KEGG pathway enrichment analysis highlighting alterations in adhesion-related pathways. (E–F) Heatmaps of transcription factors (E) and adhesion molecules (F). (G) RNA-seq analysis of genes shown in the figure in BM cells. (H) RNA-seq analysis of genes shown in the figure in human MSCs with or without hIL-27 treatment (n = 2 per group). (I) qPCR analysis of genes shown in the figure in murine MSCs with or without IL-27 (n = 3 per group). (J) RNA-seq analysis of genes shown in the figure in BM Gr-1 + cells 3 weeks after AAV-Ctrl or AAV-IL-27 administration (n = 2 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 reshapes the BM niche, reducing B-cell support and leukaemogenesis. (A) Pseudotime analysis of BM B-cell subsets (C0–C10). (B) Expression of representative marker genes across B-cell clusters in (A). (C–G) BM cells were harvested 8 days after AAV-Ctrl or AAV-IL-27 administration for RNA-seq analysis (n = 3 per group). (C) CIBERSORTx analysis showing increased pre-pro-B and cycling pro-B subsets after IL-27 treatment. (D) KEGG pathway enrichment analysis highlighting alterations in adhesion-related pathways. (E–F) Heatmaps of transcription factors (E) and adhesion molecules (F). (G) RNA-seq analysis of genes shown in the figure in BM cells. (H) RNA-seq analysis of genes shown in the figure in human MSCs with or without hIL-27 treatment (n = 2 per group). (I) qPCR analysis of genes shown in the figure in murine MSCs with or without IL-27 (n = 3 per group). (J) RNA-seq analysis of genes shown in the figure in BM Gr-1 + cells 3 weeks after AAV-Ctrl or AAV-IL-27 administration (n = 2 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Expressing, Marker, RNA Sequencing

IL-27 exhibits therapeutic potential while modulating B-cell compartments. (A–B) Experimental design of combination therapy in WT (A) and Rag1 −/− (B) mice, with corresponding Kaplan–Meier survival analysis. (C–D) Experimental design of IL-27R −/− CAR-T cell therapy combined with AAV-Ctrl or AAV-IL-27 in B-ALL-bearing mice, with Kaplan–Meier survival curves. B-ALL cells were transplanted either prior to chemotherapy (C) or after chemotherapy (D). (E–F) NSG mice transplanted with human G-PBSCs were injected intramuscularly with AAV-Ctrl or hIL-27. 10 days later, frequencies and absolute numbers of B cells in the BM and spleen were analysed (n = 5 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; log-rank test (A–D) and unpaired Student's t-test (E–F).

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 exhibits therapeutic potential while modulating B-cell compartments. (A–B) Experimental design of combination therapy in WT (A) and Rag1 −/− (B) mice, with corresponding Kaplan–Meier survival analysis. (C–D) Experimental design of IL-27R −/− CAR-T cell therapy combined with AAV-Ctrl or AAV-IL-27 in B-ALL-bearing mice, with Kaplan–Meier survival curves. B-ALL cells were transplanted either prior to chemotherapy (C) or after chemotherapy (D). (E–F) NSG mice transplanted with human G-PBSCs were injected intramuscularly with AAV-Ctrl or hIL-27. 10 days later, frequencies and absolute numbers of B cells in the BM and spleen were analysed (n = 5 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; log-rank test (A–D) and unpaired Student's t-test (E–F).

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Injection

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