Th1 cell differentiation is promoted by glutamate.

<p>(A) CD4⁺ T cells were cultivated on irradiated splenocytes in the presence of 10 µg/mL anti-IL-4 alone or with 10 ng/mL IL-12 for 4 days, with or without 100 µM MK801 or 100 µM glutamate, each of which was reloaded after 2 days of polarization. Cells were washed and restimulated with PMA/ionomycin/BFA for 4 h and analyzed by flow cytometry. Flow cytometry analyses were quantified and expressed as mean ± s.e.m. (n = 3, one-way ANOVA test, *p<0.05). (B) CD4⁺ T cells were cultivated on irradiated splenocytes in the presence 10 µg/mL anti-IL-4 for 4 days with or without 100 µM glutamate, and cells were washed and restimulated with PMA/ionomycin/BFA for 4 h and analyzed by flow cytometry. The histogram represents TBet expressing cells.</p

Astrocytes alone are sufficient to induce Th1 cell production.

<p>(A) CD4⁺ T cells were cultivated for 4 days on no feeder cells or on a monolayer of astrocytes. (B) CD4⁺ T cells were cultivated for 4 days on no feeder cells, on a monolayer of astrocytes, or on irradiated splenocyte feeder cells with 10 µg/mL anti-IL-4. (C) CD4⁺ T cells were cultivated for 4 days on no feeder cells, on a monolayer of astrocytes, or on irradiated splenocyte feeder cells with 10 µg/mL anti-IL-4 and 10 ng/mL IL-12. (A–D) IFNγ-producing, Foxp3 expressing and IL-17A-producing CD4⁺ T cells were analyzed by flow cytometry after restimulation with ionomycin/PMA/BFA for 4 h. Flow cytometry analyses were quantified and expressed as mean ± s.e.m. (for the data in D: n = 4–8, Mann-Whitney test, *p<0.05 compared with cells incubated without anti-IL-12). (E, F) CD4⁺ T cells were cultivated for 4 days on a monolayer of astrocytes or on irradiated splenocyte feeder cells with 10 µg/mL anti-IL-4, with or without 5 ng/mL TGFβ. Foxp3-expressing cells were analyzed by flow cytometry. Flow cytometry analyses were quantified and expressed as mean ± s.e.m. (n = 4, Mann-Whitney test, *p<0.05 compared with cells incubated without TGFβ).</p

Th1 cell differentiation is promoted by CD4⁺ T cell activation.

<p>CD4⁺ T cells were cultivated on astrocytes in the presence of 10 µg/mL anti-IL-4 alone (anti-IL-4), with 10 µg/mL anti-IL-4 and 10 ng/mL IL-12 (Th1), or with 10 µg/mL anti-IL-4 and 2.5 ng/mL TGFβ (Treg) for 4 days. Cells were stained with Hoechst and fixed and analyzed using an ArrayScan VTI automated fluorescent microscope. (A) astrocytes alone with nuclei traced (a 20-fold magnification is shown in the small box), (B) astrocytes co-cultured with CD4⁺ T cells with nuclei traced (a 20-fold magnification is shown in the small box), (C) quantitation of percent of CD4⁺ T cells associated per astrocyte (mean ± s.e.m.; n = 3), (D) scatter plot showing the cells identified as astrocytes (red) and CD4⁺ T cells (blue). (E) CD4⁺ T cells were cultivated on astrocytes in the presence of 10 µg/mL anti-IL-4 and 10 ng/mL IL-12 for 4 days, with or without 10 µg/mL anti-CD4 (GK1.5). Cells were washed, and restimulated with PMA/ionomycin/BFA for 4 h and analyzed by flow cytometry. The histogram represents IFNγ-expressing CD4⁺ T cells (n = 2, unpaired t-test *p<0.05). (F) CD4⁺ T cells were cultivated on astrocytes in the presence of 10 µg/mL anti-IL-4 and with 10 ng/mL IL-12 for 4 days, with or without 10 µg/mL anti-CD4 (GK1.5). Cells were stained with Hoechst and fixed and analyzed with an ArrayScan VTI automated fluorescent microscope. Bars represent mean ± s.e.m. (n = 3).</p

Cross-talk between Th1 cells and astrocytes is necessary to promote maximal Th1 differentiation.

<p>(A) CD4⁺ T cells were polarized toward Th1 cells for 4 days in conditioned medium from unstimulated or LPS-stimulated astrocytes supplemented with 10 ng/mL IL-12 (Th1) or 5 ng/mL TGFβ (Treg). Cells were washed and stimulated for 4 h with PMA/ionomycin/BFA and IFNγ-producing CD4⁺ T and Foxp3 expressing CD4⁺ T cells were analyzed by flow cytometry. Flow cytometry analyses were quantified and expressed as mean ± s.e.m. (n = 2). (B–C) Different quantities of CD4⁺ T cells were cultivated on an astrocyte monolayer and polarized toward (B) Th1, or (C) Treg cells for 4 days in medium supplemented with 10 ng/mL IL-12 (Th1), or 5 ng/mL TGFβ (Treg). Cells were washed, stimulated for 4 h with PMA/ionomycin/BFA, and IFNγ-producing and Foxp3-expressing CD4⁺ T cells were analyzed by flow cytometry. Flow cytometry analyses were quantified and expressed as mean ± s.e.m. (n = 3, , one-way ANOVA test, *p<0.05 compared to 5×10⁵ cells).</p

Preactivation of astrocytes reduces Th1 differentiation.

<p>CD4⁺ T cells were cultivated for 4 days on a monolayer of astrocytes that were pre-activated with LPS for 18 h, with or without 10 µg/mL anti-IL-4 and 10 ng/mL IL-12 where indicated. IFNγ-producing (A) and Foxp3 expressing (B) CD4⁺ T cells were analyzed by flow cytometry after restimulation with ionomycin/PMA/BFA for 4 h. Flow cytometry analyses were quantified and expressed as mean ± s.e.m. (for the data in A: n = 3, one-way ANOVA test, *p<0.05; for the data in B: n = 2–4).</p

Th1 differentiation is not dependent on GDNF, BDNF or NGF.

<p>(A) CD4⁺ T cells were polarized toward Th1 cells for 3 days cultured on astrocytes or with irradiated feeder cells in medium supplemented with 10 ng/mL IL-12 (Th1). Cells were washed and stimulated for 4 hr with PMA/ionomycin and CD4⁺ T cells were analyzed for CFSE staining by flow cytometry gated on IFNγ⁺CD4⁺ T cells. Data are representative of 2 independent experiments. The rate of cell division was higher in cells incubated in Th1-polarizing conditions than undifferentiated cells. (B) CD4⁺ T cells cultivated on astrocytes or irradiated splenocytes were stimulated with 10 ng/mL GDNF, 10 ng/mL BDNF, 100 ng/mL NGF, or a combination as indicated, in the presence or not of Th1 polarizing conditions for 4 days. Cells were washed and stimulated with PMA/ionomycin/BFA for 4 h and IFNγ-producing cells were analyzed by flow cytometry. Plots are representative of 2 independent experiments.</p

FAIR LINCS Data and Metadata powered by the CEDAR Framework

The Library of Integrated Network-based Signatures (LINCS) program generates a wide variety of cell-based perturbation-response signatures using diverse assay technologies. For example, LINCS includes large-scale transcriptional profiling of genetic and small molecule perturbations, and various proteomics and imaging datasets. We have developed data processing pipelines, and supporting informatics infrastructure to access, standardize and harmonize, register and publish LINCS datasets and metadata from all Data and Signature Generating Centers (DSGC’s). Metadata standards specifications provide a foundation for harmonizing and integrating LINCS data. Here we introduce a CEDAR-based LINCS Community Metadata Environment, to support end-to-end metadata management framework that supports authoring, curation, validation, management, and sharing of LINCS metadata, while building upon the existing LINCS metadata standards and data-release workflows. Following this initial validation, our goal is to create reusable metadata modules with user friendly templates for each of the LINCS metadata categories and to make our suite of tools compatible with the CEDAR metadata technologies. This should further simplify metadata handling in the LINCS consortium and facilitate a global metadata repository at CEDAR. As other projects apply the same approach, many more datasets will become cross-searchable and can be linked optimizing the metadata pathway from submission to discovery