cytokine neutralization  (Bio X Cell)

Journal: Science Advances

Article Title: Sphingolipid biosynthesis is essential for metabolic rewiring during T H 17 cell differentiation

doi: 10.1126/sciadv.adk1045

Figure Lengend Snippet: ( A ) Naïve CD4 + T cells from WT or KO mice were differentiated into T H 17 cells (as described in Materials and Methods) for 4 days, and intracellular cytokines were scored by flow cytometry. The left panel is a representative flow figure showing frequency of IL-17A + and IFN-γ + cells, and the right panel shows the cumulative data of the same. n = 5 biologically independent samples, representative of five experiments. ( B ) iMFI of IL-17A. iMFI = (MFI)( P ) where, MFI is the median fluorescence intensity of cytokine-positive cells, and P is the percentage of cytokine-positive cells. ( C ) WT and KO naïve T cells were differentiated under T H 17 conditions for 4 days and reactivated with anti-CD3 for 24 hours, and the culture supernatant was analyzed for IL-17A by bioplex assay. n = 3 biologically independent samples. ( D ) WT and KO naïve T cells were differentiated into T H 17 cells for 3 days and RNA quantified by reverse transcription qPCR. n = 3 to 5 biologically independent samples. ( E and F ) WT and KO naïve CD4 + T cells were differentiated into T H 17 cells with IL-1β + IL-6 + IL-23 (E) or TGF-β1 + IL1-β + IL-6 + IL-23 (F) for 4 days and analyzed for frequency of IL-17A + and IFN-γ + cells by flow cytometry. The left panel is a representative flow figure, and the right panel shows the cumulative data for the same. n = 3 biologically independent samples. Each dot represents an individual mouse. All data presented as means ± SEM: ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Antibodies for activation and cytokine neutralization were procured from Bio X Cell, USA.

Techniques: Flow Cytometry, Fluorescence, BioPlex Assay, Reverse Transcription

Journal: Science Advances

Article Title: Sphingolipid biosynthesis is essential for metabolic rewiring during T H 17 cell differentiation

doi: 10.1126/sciadv.adk1045

Figure Lengend Snippet: ( A ) WT and KO naïve cells were differentiated under T H 17 conditions for 12 hours and scored for phospho-Akt(S473) by flow cytometry. The left panel is a representative flow figure, and the right panel shows cumulative p-Akt MFI. n = 3 biologically independent samples. ( B ) GSEA for positive regulation of ROS genes between WT and KO T H 17 cells. ( C ) WT and KO naïve cells were differentiated under T H 17 conditions for 12 hours and intracellular ROS measured using DCFDA by flow cytometry. The left panel is a representative flow figure, and the right panel shows the cumulative data of DCFDA MFI. n = 3 biologically independent samples. ( D ) WT and KO naïve cells were differentiated under T H 17 conditions for 4 days with or without NAC and scored for intracellular cytokine. The left panel is a representative flow figure, and the right panel shows the cumulative data of the same. n = 4 to 6 biologically independent samples. ( E ) WT and KO naïve cells were differentiated under T H 17 conditions with or without NAC for 12 hours and scored for frequency of phospho-S6 + cells by flow cytometry. n = 5 biologically independent samples. ( F to I ) GSEA for (F) mTOR signaling genes, (G) c-Myc target genes, (H) HIF-1α target genes, and (I) glycolytic genes KO and (WT and KO + NAC) T H 17 cells. n = 3 biologically independent samples. ( J ) Hk2 mRNA quantification by qPCR of T H 17 under the indicated differentiation conditions. n = 3 biologically independent samples. ( K ) WT and KO naïve T cells were differentiated under T H 17 conditions with or without NAC for 3 days and ECAR was measured in equal number of viable cells by Seahorse analyzer. n = 3 biologically independent samples. Each dot represents an individual mouse. All data presented as means ± SEM: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Antibodies for activation and cytokine neutralization were procured from Bio X Cell, USA.

Techniques: Flow Cytometry

Journal: medRxiv

Article Title: IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4 + T cells via p-Akt signaling

doi: 10.1101/2023.03.29.23287939

Figure Lengend Snippet: Representative figures from 20 T cell cytokines were analyzed in RA patients’ plasma (n=15) and healthy controls (n=10) and are represented as graphical plots. Inflammatory cytokines including TNF-α, IL- 17A, IL-17E, IL-17F, IFN-γ and IL-33 were significantly elevated in RA patients as compared to HC (A). In addition, Th17 driving cytokines such as IL-6, IL-1β and IL-21 were also significantly higher in RA patients with respect to healthy controls (B). Amongst the anti- inflammatory cytokines, only IL-13 and IL-10 was elevated in RA patients while IL-4 and IL- 5 did not show any difference between the two groups (C). RA patients also displayed higher expression of cytokines playing diverse roles in regulating and maintaining RA pathogenesis such as IL-9, IL-27, IL-15 and IL-2 in contrast to healthy controls (D). Mann-Whitney U Test was performed to compare between the two groups, p<0.05 was considered statistically significant (*), p<0.01 was considered to be very significant (**), p<0.001 was considered to highly significant (***), p<0.0001 was considered extremely significant (****) ns, not significant. Error bar represents SEM.

Article Snippet: In brief, PBMCs derived from RA patients were seeded in flat-bottomed plates at 1×10 6 cells/ml density with neutralization antibodies for the cytokines, IL-21(eBioscience, San Diego, CA, USA), IL-23 p19 (eBioscience, San Diego, CA, USA) and or IL-23 p40 (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature.

Techniques: Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4 + T cells via p-Akt signaling

doi: 10.1101/2023.03.29.23287939

Figure Lengend Snippet: Intracellular staining of CD4 + T cells derived from RA PBMCs (n=8) displayed higher inflammatory cytokine expression than healthy controls (n=8). Representative flow cytometry plots show significantly elevated frequency of PBMC-derived CD4 + T cells expressing inflammatory cytokines such as TNF-α, IFN-γ, IL-17 and GMCSF (A, C, E, G & I) and cumulative graphical representation (B, D, F, H & J). Representative flow cytometry plots show non-significant expression of IL-10 in healthy controls and RA patients (I) and cumulative graphical representation (J). RA CD4 + T cell population co-express multiple inflammatory cytokines including, TNF-α, IFN-γ and GMCSF (K & L) where GMCSF + and IL-17 + cells are gated on IFN-γ-TNF-α + population. Mann-Whitney U Test was performed to compare between the two groups, p<0.05 was considered statistically significant (*), p<0.01 was considered to be very significant (**), p<0.001 was considered to highly significant (***), p<0.0001 was considered extremely significant (****) ns, not significant. Error bar represents SEM.

Article Snippet: In brief, PBMCs derived from RA patients were seeded in flat-bottomed plates at 1×10 6 cells/ml density with neutralization antibodies for the cytokines, IL-21(eBioscience, San Diego, CA, USA), IL-23 p19 (eBioscience, San Diego, CA, USA) and or IL-23 p40 (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature.

Techniques: Staining, Derivative Assay, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: medRxiv

Article Title: IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4 + T cells via p-Akt signaling

doi: 10.1101/2023.03.29.23287939

Figure Lengend Snippet: Intracellular staining of inflammatory cytokines, IL-21 and both subunits of IL-23 displayed significantly higher expression in RA CD4 + T cells (n=6) upon PMA/Ionomycin stimulation as compared to healthy controls (n=6). Representative flow cytometry plots show significant elevation in IL-21, IL- 23p19 and IL-23p40 expression in CD4 + T cells derived from RA and HC PBMCs (A, C and E) and found statistically significant in the corresponding graphical represenatation (B, D and F). Mann-Whitney U Test was performed to compare between the two groups, p<0.05 was considered statistically significant (*), p<0.01 was considered to be very significant (**), p<0.001 was considered to highly significant (***), p<0.0001 was considered extremely significant (****) ns, not significant. Error bar represents SEM.

Article Snippet: In brief, PBMCs derived from RA patients were seeded in flat-bottomed plates at 1×10 6 cells/ml density with neutralization antibodies for the cytokines, IL-21(eBioscience, San Diego, CA, USA), IL-23 p19 (eBioscience, San Diego, CA, USA) and or IL-23 p40 (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature.

Techniques: Staining, Expressing, Flow Cytometry, Derivative Assay, MANN-WHITNEY

Journal: medRxiv

Article Title: IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4 + T cells via p-Akt signaling

doi: 10.1101/2023.03.29.23287939

Figure Lengend Snippet: Modulation of inflammatory cytokines in RA CD4 + T cells by αIL-21 and αIL-23. Representative figures showing modulation of inflammatory cytokines in RA PBMC derived CD4 + T cells (n=8) with αIL-21 and αIL-23 inhibition along with PMA/Ionomycin stimulation. Stimulated cells selectively inhibited with αIL-21 show significant decrease of cytokines, TNF-α, IL-17 and IFN-γ with respect to only PMA/Ionomycin treated as depicted in the representative flow cytometry plots (A, E and I) and in cumulative graphical representation (B, F and J). Significant reduction of TNF-α and IL-17 expression was also observed with αIL-23 inhibition in RA CD4+ T cells as represented in flow cytometry plots (A and E) and cumulative graphs (C and G). IFN-γ expression did not show significant difference with αIL-23 treatment (I and K). Expression of TNF-α, IL-17 and IFN-γ reduced significantly with combined treatment of αIL-21 and αIL-23 as depicted in the representative flow cytometry plots (A, E and I) along with cumulative graphical representation (D, H and L). Paired T Test was performed to compare between the two groups, p<0.05 was considered statistically significant (*), p<0.01 was considered to be very significant (**), p<0.001 was considered to highly significant (***), p<0.0001 was considered extremely significant (****) ns, not significant. Error bar represents SEM.

Article Snippet: In brief, PBMCs derived from RA patients were seeded in flat-bottomed plates at 1×10 6 cells/ml density with neutralization antibodies for the cytokines, IL-21(eBioscience, San Diego, CA, USA), IL-23 p19 (eBioscience, San Diego, CA, USA) and or IL-23 p40 (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature.

Techniques: Derivative Assay, Inhibition, Flow Cytometry, Expressing

Journal: medRxiv

Article Title: IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4 + T cells via p-Akt signaling

doi: 10.1101/2023.03.29.23287939

Figure Lengend Snippet: Negatively isolated CD4 + T cells derived from PBMCs were activated with αCD3/28 stimulation, polarizing cytokines and neutralizing antibodies for 10 days, characterized for Th17 phenotype and examined for altered expression of cytokines and RANKL with αIL-21 and αIL-23 treatment upon PMA/Ionomycin stimulation. Post 10-day polarization, CD4 + T cell population showed significant expression of IL-17, signature cytokine for Th17 subset as compared to the unstimulated cells represented in flow cytometry plots (A) as well as the graphical plots (B). In addition, our representative figures display significant expression of Th17 phenotype’s master transcription factor, RorγT (C and D). Mann-Whitney U Test was performed to compare between the two groups. Treatment of these cells with both, αIL-21 and αIL-23 showed significant decrease in IL-17 expression, independently as well as in combination as is represented in flow cytometry plots (E) and graphical plots (F, G and H). In addition, RANKL expression on CCR6 + cells also decreased significantly with both αIL-21 and αIL-23 treatment as represented in flow cytometry plots (I) and graphical plots (J, K and L). Paired T Test was performed to compare between the two groups, p<0.05 was considered statistically significant (*), p<0.01 was considered to be very significant (**), p<0.001 was considered to highly significant (***), p<0.0001 was considered extremely significant (****) ns, not significant. Error bar represents SEM.

Article Snippet: In brief, PBMCs derived from RA patients were seeded in flat-bottomed plates at 1×10 6 cells/ml density with neutralization antibodies for the cytokines, IL-21(eBioscience, San Diego, CA, USA), IL-23 p19 (eBioscience, San Diego, CA, USA) and or IL-23 p40 (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature.

Techniques: Isolation, Derivative Assay, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: medRxiv

Article Title: IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4 + T cells via p-Akt signaling

doi: 10.1101/2023.03.29.23287939

Figure Lengend Snippet: RA CD4 + T cells were stimulated with αCD3/28 along with αIL-21 and αIL-23 treatment for 24 hours and examined for alteration in pAkt1 expression and later, analyzed for altered expression of cytokines and RANKL with addition of Akt1/2 kinase inhibitor. Representative figures showing significant decrease in pAkt1 expression with αIL-21 and αIL- 23 treatment, independently as well as in combination as shown in flow cytometry plots (A) and graphical plots (B, C and D). Addition of 5µM pAkt1/2 inhibitor to these cells during αCD3 stimulation displayed significant decrease in TNF-α, IFN-γ, IL-17 and RANKL expression as represented in flow cytometry plots (E, G, I and K) and graphical plots (F, H, J and L). Paired T Test was performed to compare between the two groups, p<0.05 was considered statistically significant (*), p<0.01 was considered to be very significant (**), p<0.001 was considered to highly significant (***), p<0.0001 was considered extremely significant (****) ns, not significant. Error bar represents SEM.

Article Snippet: In brief, PBMCs derived from RA patients were seeded in flat-bottomed plates at 1×10 6 cells/ml density with neutralization antibodies for the cytokines, IL-21(eBioscience, San Diego, CA, USA), IL-23 p19 (eBioscience, San Diego, CA, USA) and or IL-23 p40 (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature.

Techniques: Expressing, Flow Cytometry

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: CBA/J mice ( n = 6 mice/group/timepoint) were administered isotype ( a – d ), anti-CD8 ( e , f ), or anti-CD4 ( g , h ) antibodies. Mice then received either C. neoformans Δ sgl1 (white symbols) or PBS (black symbols). After 30 days, mice were challenged with C. neoformans WT (day 0). On days −15, −1, 7, 15, and 24, mice were assessed for T cell-derived cytokines (IFNγ, IL-17A) via intracellular cytokine staining. Graphed data represents the mean ± SD from 2 independent experiments ( n = 3 mice/group/timepoint for each biological replicate). Representative flow cytometry plots for days −1 and 7 for each group are shown, which were gated from live, CD45 + singlets (gating scheme shown in Supplementary Fig. ). Significance was determined by a two-way ANOVA using Šídák’s multiple comparisons test for P value adjustment, and significance is denoted as * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.001.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Derivative Assay, Staining, Flow Cytometry

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: CBA/J mice ( n = 8–10 mice/group) were neutralized of specific cytokines and/or cell types (as indicated in the figure legends) prior to vaccination with C. neoformans Δ sgl1 (white symbols) or PBS controls (black symbols), and neutralizations continued for the entirety of the experiment at noted intervals. a , b . Vaccinated and unvaccinated mice were challenged with C. neoformans WT strain and assessed for survival during specific cytokine neutralization ( a ) or cytokine neutralization during either CD4 or CD8 T cell deficiency ( b ). Survival significance was determined by the Mantel–Cox log-rank test, and denoted on each graph: a . # P < 0.001 for Δ sgl1 /Isotype → WT vs. Δ sgl1 /anti-IL-17A → WT; b . % P < 0.001 for Δ sgl1 /Isotype → WT vs. Δ sgl1 /anti-CD4 + anti-IL-17A → WT or Δ sgl1 /anti-CD8 + anti-IL-17A → WT.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Neutralization

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: C57BL/6 (black symbols) or TLR2 −/− (white symbols) were administered C. neoformans Δ sgl1 and assessed for T cell-derived cytokines via intracellular cytokine stimulation on days −30 (uninfected; a ), −27 ( b ), and −23 ( c ). 30 days after C. neoformans Δ sgl1 administration, mice were challenged with C. neoformans WT and assessed for T cell-derived cytokines via intracellular cytokine stimulation on days −1 (unchallenged; d ), 3 ( e ), and 7 ( f ). At all timepoints, mice were assessed for the total number of γδ T, CD4 + , and CD8 + T cells as well as the number of IFNγ- or IL-17A-producing subsets of these cells. Graphed data represent the mean ± SD and are representative of 1–2 independent experiments ( n = 3–6 mice/timepoint/group). Significance was determined by a two-way ANOVA using Šídák’s multiple comparisons test for P value adjustment, and denoted as * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Derivative Assay

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: Upon intranasal administration of C. neoformans Δ sgl1 , the yeast cells travel down the bronchioles into the alveoli (1) encountering resident alveolar macrophages (AM), dendritic cells (DCs), and γδ T cells. Both sterylglucosides (SGs) and the glucuronoxylomannan (GXM)-rich capsule of C. neoformans Δ sgl1 can be found on the surface of yeast cells or/and in the extracellular environment. γδ T cells recognize and respond directly to C. neoformans Δ sgl1 via a toll-like receptor 2 (TLR2)-directed mechanism (2) producing the host-protective cytokines IFNγ and IL-17A leading to a pro-inflammatory lung cytokine environment with augmented leukocyte recruitment to the lungs (3) including neutrophils and monocytes that mediate early host control by limiting C. neoformans Δ sgl1 replication. Antigen-presenting cells (APCs) become activated, phagocytose the yeast, and travel to the lung-draining lymph node to prime naive CD4 + or CD8 + T cells when CD4 + T cells are absent (4). The naive T cells differentiate into protectively polarized IFNγ- and IL-17A-producing T cells and travel to the lungs (5) resulting in the pulmonary clearance of the mutant. While most recruited leukocytes die upon resolution of the inflammatory response, a small percentage of αβ and γδ T cells become host-protective memory T cells (6) that rapidly respond upon a subsequent WT challenge, promptly producing both IFNγ and IL-17A and conferring strong vaccine protection. Graphical illustration created with BioRender.com.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Mutagenesis