7 research outputs found
Th1 cell differentiation is promoted by glutamate.
<p>(A) CD4<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> T cells were cultivated for 4 days on no feeder cells or on a monolayer of astrocytes. (B) CD4<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> T cell activation.
<p>CD4<sup>+</sup> 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<sup>+</sup> T cells with nuclei traced (a 20-fold magnification is shown in the small box), (C) quantitation of percent of CD4<sup>+</sup> T cells associated per astrocyte (mean ± s.e.m.; n = 3), (D) scatter plot showing the cells identified as astrocytes (red) and CD4<sup>+</sup> T cells (blue). (E) CD4<sup>+</sup> 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<sup>+</sup> T cells (n = 2, unpaired t-test *p<0.05). (F) CD4<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> T and Foxp3 expressing CD4<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> 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 5×10<sup>5</sup> cells).</p
Preactivation of astrocytes reduces Th1 differentiation.
<p>CD4<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> T cells were analyzed for CFSE staining by flow cytometry gated on IFNγ<sup>+</sup>CD4<sup>+</sup> 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<sup>+</sup> 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
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