Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Effect of hypoxia-preconditioned MSCs (MSCs HYP , MSCs HYP→APO , MSCs HYP+APO ) on the proliferation of aGVHD patients derived T-cell. The bar graph represents (A) the percentage of CD3 + CFSE + proliferating T-cell (n=5). (B) the ratio of CD4 + /CD8 + T cell (n=5) in the direct co-culture of MSCs and T-cell. Data shown as Mean±S.D.; Statistical analysis: Tukey’s multiple comparisons test; **≤0.01; ****≤0.0001. Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Derivative Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Dot plots showing the proliferation of CD3 + T-cell in the direct co-culture of aGVHD patients derived T-cell and (A) Untreated (Control). (B) BM-MSCs HYP . (C) BM-MSCs HYP→APO . (D) BM-MSCs HYP+APO . (E) WJ-MSCs HYP . (F) WJ-MSCs HYP→APO . (G) WJ-MSCs HYP+APO . Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Co-Culture Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Effect of hypoxia-preconditioned MSCs (MSCs HYP , MSCs HYP→APO , MSCs HYP+APO ) on the induction of Tregs and polarization of helper T cell from pro-inflammatory (Th1, Th17) to anti-inflammatory (Th2) phenotype. The bar graph represents (A) the induction of CD3 + CD4 + CD25 + FOXP3 + Tregs (n=5). (B) the ratio of Th1/Th2 (n=5). (C) Th1/Th17 ratio (n=5) in the direct co-culture of MSCs and T-cell. Data shown as Mean±S.D.; Statistical analysis: Tukey’s multiple comparisons test; ***≤0.001; ****≤0.0001. Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal stem cells; HYP: Hypoxia-preconditioned; APO: Apoptosis, Tregs: Regulatory helper T-cell, Th: Helper T cell .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Co-Culture Assay

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Dot plots showing the percentage of CD3 + CD4 + CD25 + FOXP3 + in the direct co-culture of aGVHD patients derived CD3 + T-cell and (A) Untreated (Control). (B) BM-MSCs HYP . (C) BM-MSCs HYP→APO . (D) BM-MSCs HYP+APO . (E) WJ-MSCs HYP . (F) WJ-MSCs HYP→APO . (G) WJ-MSCs HYP+APO . Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Co-Culture Assay, Derivative Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Key role of NOS2 and the canonical cGMP-mediated NO signaling pathway in the MDSC-promoted Th17 differentiation of TILs from OvCa patients and human naive and memory CD4 + Th cells. (A) Induction of IL-17A, Rorc (encoding RORγt), FoxP3 (indicating de novo differentiation of FoxP3 + T reg cells from naive precursors), and T-bet gene expression in anti-CD3/CD28–expanded naive CD4 + T cells by tumor–isolated MDSCs (mean ± SD from six patients), as compared with control CD11b + cells (mean ± SD from three healthy donors). (B) IL-17A production levels and percentages of IL-17A + cells (mean ± SD from n donors), and representative intracellular staining (IL-17A vs. IFN-γ, right) in naive CD4 + T cells ( n = 6 healthy donors) or OvCa-infiltrating CD4 + TILs ( n = 3 patients) stimulated with anti-CD3/CD28 antibodies in the presence of cancer-isolated MDSCs or control CD11b + cells. (C) IL-17A production by naive versus memory CD4 + T cells (mean ± SD from n = 4 healthy donors) stimulated with either anti-CD3/CD28 antibodies or TNF-matured allogeneic DCs in the presence of MDSCs or control CD11b + cells. (D and E) Percentage of IL-17A + or IFN-γ + CD4 + T cells (D) and IL-17A secretion (E) in anti-CD3/CD28/MDSC–expanded naive CD4 + T cells (D) and CD4 + TILs (E) in the presence of specific inhibitors of NOS (L-NMMA), COX2 (celecoxib), IDO-, ARG-, or IL-10. The data (mean ± SD) from one representative experiment (performed in replicates: D, triplicate cultures; E, quadruplicate cultures). The results were confirmed in three independent experiments using different patients/healthy donors. (F) Representative staining of IL-17A + or IFN-γ + CD4 + T cells in co-cultures of anti-CD3/CD28–expanded CD4 + TILs and MDSCs in the presence of specific inhibitors of COX2 (celecoxib) and NOS (L-NMMA). The results were confirmed in three independent experiments using different patients. (G) Relative gene expression of IL-17A and Rorc, induced in anti-CD3/CD28–expanded naive CD4 + T cells cultured in the presence of MDSCs in the presence of a specific inhibitor of NOS (L-NMMA). The graphs present the mean ± SD from one representative experiment (triplicate cultures) of two (using different patients/healthy donors). (H) Relative gene expression of NOS2 and IL-17A in 7 d ex vivo anti-CD3/CD28–expanded cultures of OvCa ascites cells from 10 OvCa patients ( n = 10; r 2 = 0.7692; P < 0.001). (I) IL-17A production in anti-CD3/CD28–expanded cultures of naive or memory CD4 + T cells in the presence of MDSCs, with or without specific inhibitors of NOS2 (1400W) or cGMP (ODQ). Data (mean ± SD) from one representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different patients/healthy donors. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: Expressing, Isolation, Staining, Cell Culture, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Exogenous NO supports the cytokine-driven induction of Th17 function in memory Th cells and promotes the de novo induction of Th17 cells from naive precursors. (A) NO 2 − levels (mean ± SD from four patients) in co-cultures of CD4 + TILs with tumor-isolated MDSCs (as compared with blood CD11b + cells) and in the presence of NOS inhibitors ADMA and l -NMMA. (B) Expression of IL-1β, IL-6, TGF-β 1 (spontaneous expression, left), and IL-23 (stimulation with CD40L, right) in MDSCs. Data (mean ± SD) from three experiments involving MDSCs from three different patients. (C) Induction of IL-17A or IFN-γ production by anti-CD3/CD28–stimulated bulk CD4 + T cells from healthy donors, cultured in the absence or presence of Th1 (200 U/ml rhIL-12, 200 ng/ml αIL-4-Ab), Th17 (20 ng/ml rhIL-1β, 50 ng/ml rhIL-6, 10 ng/ml rhIL-23), and T reg cell (5 ng/ml TGF-β 1 , 10 nM 9-cis retinoic acid) -driving cytokines, and physiological concentrations of exogenous NO donor (DETA-NONOate). IL-17A was undetectable in Th1- and T reg cell–driving conditions. Percentage of FoxP3 + cells in control cultures were as follows: (−), 10.4%; Th1, 12.7%; Th17, 5.2%; and T reg cell, 41.2%. The graphs present the mean ± SD from one representative experiment (triplicate cultures) of two (healthy donors). (D) Suppression of CD4 + T cells differentiating in Th1-, T reg -, and Th17-driving conditions by high concentrations (>100 µM) of DETA-NONOate. Proliferation of CFSE-labeled anti-CD3/CD28–stimulated bulk CD4 + T cells cultured in the absence or presence of Th1, Th17, and T reg cell–driving cytokines and supplemented with increasing concentrations of DETA-NONOate. The graph presents the mean ± SD from one representative experiment (triplicate cultures) of two (different healthy donors). (E and F) Relative gene expression of IL-17A (log scale) and Rorc (log scale), and secretion of IL-17A by naive (E) or naive and memory (F) CD4 + T cells, expanded with anti-CD3/CD28 antibodies in the absence or presence of Th17-driving cytokines and NO donor (DETA-NONOate). The graphs present the mean ± SD from one representative experiment (E, triplicate cultures; F, quadruplicate cultures). The results were confirmed in three independent experiments using cells of different healthy donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: Isolation, Expressing, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Endogenous T cell–expressed NOS2 and T cell-produced NO are required for de novo Th17 differentiation from naive precursors and induction of the Th17 phenotype in memory cells. (A) Comparative induction of NOS2 (left) and IL-17A (right) gene expression in naive and memory CD4 + T cells (mean ± SD from three healthy donors) stimulated with anti-CD3/CD28 antibodies in the absence or presence of Th17-driving cytokines. (B) Dose-dependent induction of NOS2 gene expression in naive CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the presence of increasing concentrations of NO donor (DETA-NONOate) and Th17-driving cytokines. The graph presents the mean ± SD from one representative experiment (performed with triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (C) Dose-dependent induction of NOS2 gene expression in bulk CD4 + T cells, stimulated with anti-CD3/CD28 antibodies and Th17-driving cytokines (high: 20 ng/ml IL-1β, 50 ng/ml IL-6, 10 ng/ml IL-23; low: 25× dilution). The graph presents the mean ± SD from one representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (D) Induction of intracellular NOS2 protein (left and middle, representative data; right, mean ± SD from n = 3 healthy donors) in CD3 + -gated bulk CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the presence of Th17-driving cytokines and NO donor (DETA-NONOate). Bar, 10 µm. Data in the right panel is represented as the fold change of the mean fluorescence intensity (MFI) over the isotype control. (E) Induction of intracellular NO (DAF-FM staining; representative experiment, left; mean ± SD from n = 3 healthy donors, right) in CD3 + -gated bulk CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the presence of Th17-driving cytokines and NO donor (DETA-NONOate), or the presence of general NOS inhibitor (L-NMMA) or NOS2-specific inhibitor (1400W). Data in the right panel is expressed as the fold increase of DAF-FM MFI over CD4 + T cells cultured in the absence of Th17-driving cytokines and NO donor. When not otherwise indicated, statistically significant differences compared with the absence of NO inhibitors are shown. (F) IL-17A secretion by naive CD4 + T cells stimulated with Th17-driving cytokines in the presence of a general NOS inhibitor (L-NMMA) or NOS2-specific inhibitor (1400W). The graph presents the mean ± SD from one representative experiment (quadruplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (G) Induction of NOS2 (left, mean ± SD from four healthy donors) gene expression correlated with the IL-17A production (right, mean ± SD from three healthy donors) in bulk CD4 + T cells by the individual Th17-inducing factors IL-1β, IL-6, IL-23, and/or TGF-β 1 . *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: Produced, Expressing, Fluorescence, Staining, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

doi: 10.1084/jem.20121277

Figure Lengend Snippet: Endogenous NOS2 and persistent cGMP signaling are required for the NO-assisted de novo induction of Th17 cells and for the stability of human in vivo-developed Th17 cells from cancer patients. (A) Relative gene expression of IL-17A, IL-17F, and IL-23R in bulk CD4 + T cells, expanded with anti-CD3/CD28 in the absence or presence of Th17-driving cytokines and general NOS inhibitor (L-NMMA). The graphs present the mean ± SD from a representative experiment (triplicate cultures) of two (using different patients/healthy donors), that both yielded similar results. (B) Regulation of (left) surface IL-23R expression on naive and memory CD4 + T cells (mean ± SD from four healthy donors) activated with anti-CD3/CD28 in the presence of NOS inhibitor (L-NMMA) or NO donor (DETA-NONOate). (right) Relative gene expression of IL-23R and IL-2R (right) in naive CD4 + T cells in the presence of increasing concentrations of NO donor and Th17-driving cytokines. Statistically significant differences compared with the absence of DETA-NONOate and Th17-driving cytokines are indicated. The graphs present the mean ± SD from a representative experiment (performed with triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (C) IL-17A production by naive CD4 + T cells stimulated with anti-CD3/CD28 antibodies in the absence or presence of cGMP inhibitor (ODQ, left) or supplemented with cGMP analogue (Br-cGMP, right) in the absence or presence of Th17-driving cytokines. The graphs present the mean ± SD from a representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different healthy donors. (D) IL-17A (left) or IFN-γ (right) production by OvCa-isolated CD4 + TILs (mean ± SD from five patients), expanded with anti-CD3/CD28 antibodies and restimulated in the absence or presence of NOS inhibitor (L-NMMA) or cGMP inhibitor (ODQ) for 48 h (statistically significant differences compared with the absence of inhibitors are indicated). (E) IL-17A production by in vitro–generated Th17 cells (generated in 8 d cultures of CD4 + T cells stimulated with anti-CD3/CD28 in the presence of Th17-driving cytokines), pretreated or not for 48 h with NOS inhibitor (L-NMMA) or cGMP inhibitor (ODQ). The data are shown as mean ± SD from 4 healthy donors. Statistically significant differences compared with condition in the absence of inhibitors are indicated. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: T cells were stimulated with anti-CD3/CD28 Dynabeads (2.0 µl/ml; Invitrogen) in the presence or absence of allogeneic OvCa-isolated MDSCs or control CD11b + cells, pretreated or not with inhibitors, and/or in the presence of the Th17-inducing cytokine cocktail: 20 ng/ml IL-1β, 10 ng/ml IL-23, 50 ng/ml IL-6, and/or 10 ng/ml TGF-β 1 .

Techniques: In Vivo, Expressing, Isolation, In Vitro, Generated