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Transforming growth factor beta blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation.

Chen CH, Seguin-Devaux C, Burke NA, Oriss TB, Watkins SC, Clipstone N, Ray A - J. Exp. Med. (2003)

Bottom Line: Here we show that TGF-beta inhibits T cell differentiation at a more proximal step.We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation.Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

View Article: PubMed Central - PubMed

Affiliation: Vion Pharmaceuticals, Incorporated, New Haven, CT 06511, USA.

ABSTRACT
Transforming growth factor (TGF)-beta inhibits T cell proliferation and differentiation. TGF-beta has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-beta inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

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TGF-β blocks Ca2+ influx in activated CD4+ T cells. (A) Fluo-3 AM loaded CD4+ T cells were incubated with anti-CD3 and anti-CD28 for 15 min on ice. After measuring the basal mean fluorescence intensity (mean F0) for 80 s, cells were stimulated by cross-linking with rabbit anti–mouse antibody in a Hepes-buffered saline solution containing IL-2 (50 U/ml) with or without TGF-β. Ca2+ flux was monitored by measuring mean fluorescence intensity (F) for the next 10 min. (B) CD4+ T cells were added to the warmed chambered anti-CD3– and anti-CD28–coated cover-slip containing culture medium and images were collected every 10 s. The cells are colored to show the relative intensities of the 340 nm (green) and 380 nm (red) signal. At early time points (for example in the 1 min and 40 s time point shown here), most cells appear red/orange reflecting the higher 380 nm component to the signal. However, at later time points (in this example the 8 min 30 s images are shown), while there is little change in the ratio in cells treated with TGF-β, the control cells show a clear change in the ratio, and most of the cells have a significant increase in the 340 component (bar = 50 microns). (C) Graphical representation of the results. The measured values represent the ratio for 15 cells in each experimental condition.
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fig7: TGF-β blocks Ca2+ influx in activated CD4+ T cells. (A) Fluo-3 AM loaded CD4+ T cells were incubated with anti-CD3 and anti-CD28 for 15 min on ice. After measuring the basal mean fluorescence intensity (mean F0) for 80 s, cells were stimulated by cross-linking with rabbit anti–mouse antibody in a Hepes-buffered saline solution containing IL-2 (50 U/ml) with or without TGF-β. Ca2+ flux was monitored by measuring mean fluorescence intensity (F) for the next 10 min. (B) CD4+ T cells were added to the warmed chambered anti-CD3– and anti-CD28–coated cover-slip containing culture medium and images were collected every 10 s. The cells are colored to show the relative intensities of the 340 nm (green) and 380 nm (red) signal. At early time points (for example in the 1 min and 40 s time point shown here), most cells appear red/orange reflecting the higher 380 nm component to the signal. However, at later time points (in this example the 8 min 30 s images are shown), while there is little change in the ratio in cells treated with TGF-β, the control cells show a clear change in the ratio, and most of the cells have a significant increase in the 340 component (bar = 50 microns). (C) Graphical representation of the results. The measured values represent the ratio for 15 cells in each experimental condition.

Mentions: To determine the effects of TGF-β on Ca2+ flux in activated CD4+ T cells, we used both flow cytometry and real-time imaging techniques using the Ca2+-binding ratiometric dye Fura-2M. In the flow cytometry method, cells were loaded with fluo-3 AM and incubated with anti-CD3 and anti-CD28. Cells were activated by cross-linking the bound antibodies and the intracellular Ca2+ level was monitored by flow cytometry. As shown in Fig. 7 A, in the absence of TGF-β, the typical biphasic response was noted with an initial rapid increase in [Ca2+]i that was followed by a sustained low level of [Ca2+]i. Addition of TGF-β caused the initial increase in [Ca2+]i which dropped to basal levels by 4 min. We have also monitored [Ca2+]i in the cells using a ratiometric technique that is being increasingly used to track T cell dynamics at several levels in vitro (50). In this technique, Fura-2M–loaded CD4+ T cells were added to anti-CD3 and anti-CD28–coated chambered glass coverslips mounted in a Harvard Systems culture chamber maintained at 37oC and containing medium with or without TGF-β. Images were collected every 10 s using motorized excitation filters at 340 and 380 nm and emission at 515 nm. Images at two time points are shown in Fig. 7 B. As shown in Fig. 7 C, there was an initial small change in the ratio in both control and TGF-β–treated cells which returned to baseline by ∼150 s. However, the subsequent steady, dramatic increase in the ratio observed in the control cells due to increased calcium levels in the cells was not observed in the presence of TGF-β.


Transforming growth factor beta blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation.

Chen CH, Seguin-Devaux C, Burke NA, Oriss TB, Watkins SC, Clipstone N, Ray A - J. Exp. Med. (2003)

TGF-β blocks Ca2+ influx in activated CD4+ T cells. (A) Fluo-3 AM loaded CD4+ T cells were incubated with anti-CD3 and anti-CD28 for 15 min on ice. After measuring the basal mean fluorescence intensity (mean F0) for 80 s, cells were stimulated by cross-linking with rabbit anti–mouse antibody in a Hepes-buffered saline solution containing IL-2 (50 U/ml) with or without TGF-β. Ca2+ flux was monitored by measuring mean fluorescence intensity (F) for the next 10 min. (B) CD4+ T cells were added to the warmed chambered anti-CD3– and anti-CD28–coated cover-slip containing culture medium and images were collected every 10 s. The cells are colored to show the relative intensities of the 340 nm (green) and 380 nm (red) signal. At early time points (for example in the 1 min and 40 s time point shown here), most cells appear red/orange reflecting the higher 380 nm component to the signal. However, at later time points (in this example the 8 min 30 s images are shown), while there is little change in the ratio in cells treated with TGF-β, the control cells show a clear change in the ratio, and most of the cells have a significant increase in the 340 component (bar = 50 microns). (C) Graphical representation of the results. The measured values represent the ratio for 15 cells in each experimental condition.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193945&req=5

fig7: TGF-β blocks Ca2+ influx in activated CD4+ T cells. (A) Fluo-3 AM loaded CD4+ T cells were incubated with anti-CD3 and anti-CD28 for 15 min on ice. After measuring the basal mean fluorescence intensity (mean F0) for 80 s, cells were stimulated by cross-linking with rabbit anti–mouse antibody in a Hepes-buffered saline solution containing IL-2 (50 U/ml) with or without TGF-β. Ca2+ flux was monitored by measuring mean fluorescence intensity (F) for the next 10 min. (B) CD4+ T cells were added to the warmed chambered anti-CD3– and anti-CD28–coated cover-slip containing culture medium and images were collected every 10 s. The cells are colored to show the relative intensities of the 340 nm (green) and 380 nm (red) signal. At early time points (for example in the 1 min and 40 s time point shown here), most cells appear red/orange reflecting the higher 380 nm component to the signal. However, at later time points (in this example the 8 min 30 s images are shown), while there is little change in the ratio in cells treated with TGF-β, the control cells show a clear change in the ratio, and most of the cells have a significant increase in the 340 component (bar = 50 microns). (C) Graphical representation of the results. The measured values represent the ratio for 15 cells in each experimental condition.
Mentions: To determine the effects of TGF-β on Ca2+ flux in activated CD4+ T cells, we used both flow cytometry and real-time imaging techniques using the Ca2+-binding ratiometric dye Fura-2M. In the flow cytometry method, cells were loaded with fluo-3 AM and incubated with anti-CD3 and anti-CD28. Cells were activated by cross-linking the bound antibodies and the intracellular Ca2+ level was monitored by flow cytometry. As shown in Fig. 7 A, in the absence of TGF-β, the typical biphasic response was noted with an initial rapid increase in [Ca2+]i that was followed by a sustained low level of [Ca2+]i. Addition of TGF-β caused the initial increase in [Ca2+]i which dropped to basal levels by 4 min. We have also monitored [Ca2+]i in the cells using a ratiometric technique that is being increasingly used to track T cell dynamics at several levels in vitro (50). In this technique, Fura-2M–loaded CD4+ T cells were added to anti-CD3 and anti-CD28–coated chambered glass coverslips mounted in a Harvard Systems culture chamber maintained at 37oC and containing medium with or without TGF-β. Images were collected every 10 s using motorized excitation filters at 340 and 380 nm and emission at 515 nm. Images at two time points are shown in Fig. 7 B. As shown in Fig. 7 C, there was an initial small change in the ratio in both control and TGF-β–treated cells which returned to baseline by ∼150 s. However, the subsequent steady, dramatic increase in the ratio observed in the control cells due to increased calcium levels in the cells was not observed in the presence of TGF-β.

Bottom Line: Here we show that TGF-beta inhibits T cell differentiation at a more proximal step.We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation.Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

View Article: PubMed Central - PubMed

Affiliation: Vion Pharmaceuticals, Incorporated, New Haven, CT 06511, USA.

ABSTRACT
Transforming growth factor (TGF)-beta inhibits T cell proliferation and differentiation. TGF-beta has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-beta inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

Show MeSH
Related in: MedlinePlus