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Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

Janardhan SV, Marks R, Gajewski TF - PLoS ONE (2014)

Bottom Line: This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression.Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus.Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

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Impaired effector cytokine production is not rescued by TCR bypass; is reflected at mRNA level.A. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Figure 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads or PMA (50 ng/mL) and Ionomycin (500 mg/mL) and analyzed for cytokine production by ELISA. B. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Fig. 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads for the times indicated. IL-4 and IFN-γ mRNA was analyzed by real-time RT-PCR. Values are represented as gene copy number relative to GAPDH (2-(ΔCt)). A significant difference in IFN-γ and IL-4 protein levels was noted between EV and Ras61L transduced cells regardless of method of stimulation (p<0.05).
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pone-0112831-g003: Impaired effector cytokine production is not rescued by TCR bypass; is reflected at mRNA level.A. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Figure 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads or PMA (50 ng/mL) and Ionomycin (500 mg/mL) and analyzed for cytokine production by ELISA. B. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Fig. 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads for the times indicated. IL-4 and IFN-γ mRNA was analyzed by real-time RT-PCR. Values are represented as gene copy number relative to GAPDH (2-(ΔCt)). A significant difference in IFN-γ and IL-4 protein levels was noted between EV and Ras61L transduced cells regardless of method of stimulation (p<0.05).

Mentions: It was conceivable that the presence of constitutive Ras signaling over the course of 4 days of stimulation may have induced a negative feedback loop that resulted in refractory or downregulated proximal TCR signaling, as occurs with T cell anergy. In order to address this hypothesis, Ras61L-transduced CARTg CD4+ T cells were primed under Th1 and Th2 conditions, and then re-stimulated with the pharmacologic agents PMA and Ionomycin to bypass proximal TCR signaling. Cells stimulated in this way still exhibited impaired effector cytokine production relative to vector control cells (Figure 3A), indicating that defective effector cytokine production in cells primed in the presence of constitutive Ras signaling was not merely due to refractory proximal TCR signaling.


Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

Janardhan SV, Marks R, Gajewski TF - PLoS ONE (2014)

Impaired effector cytokine production is not rescued by TCR bypass; is reflected at mRNA level.A. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Figure 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads or PMA (50 ng/mL) and Ionomycin (500 mg/mL) and analyzed for cytokine production by ELISA. B. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Fig. 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads for the times indicated. IL-4 and IFN-γ mRNA was analyzed by real-time RT-PCR. Values are represented as gene copy number relative to GAPDH (2-(ΔCt)). A significant difference in IFN-γ and IL-4 protein levels was noted between EV and Ras61L transduced cells regardless of method of stimulation (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232516&req=5

pone-0112831-g003: Impaired effector cytokine production is not rescued by TCR bypass; is reflected at mRNA level.A. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Figure 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads or PMA (50 ng/mL) and Ionomycin (500 mg/mL) and analyzed for cytokine production by ELISA. B. Purified splenic CARTg CD4+ T cells were transduced and then primed as in Fig. 2A. Cells were then re-stimulated with anti-CD3 and anti-CD28-coated beads for the times indicated. IL-4 and IFN-γ mRNA was analyzed by real-time RT-PCR. Values are represented as gene copy number relative to GAPDH (2-(ΔCt)). A significant difference in IFN-γ and IL-4 protein levels was noted between EV and Ras61L transduced cells regardless of method of stimulation (p<0.05).
Mentions: It was conceivable that the presence of constitutive Ras signaling over the course of 4 days of stimulation may have induced a negative feedback loop that resulted in refractory or downregulated proximal TCR signaling, as occurs with T cell anergy. In order to address this hypothesis, Ras61L-transduced CARTg CD4+ T cells were primed under Th1 and Th2 conditions, and then re-stimulated with the pharmacologic agents PMA and Ionomycin to bypass proximal TCR signaling. Cells stimulated in this way still exhibited impaired effector cytokine production relative to vector control cells (Figure 3A), indicating that defective effector cytokine production in cells primed in the presence of constitutive Ras signaling was not merely due to refractory proximal TCR signaling.

Bottom Line: This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression.Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus.Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

Show MeSH
Related in: MedlinePlus