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The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.

Clambey ET, Collins B, Young MH, Eberlein J, David A, Kappler JW, Marrack P - PLoS ONE (2013)

Bottom Line: While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism.Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells.These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Integrated Department of Immunology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. eric.clambey@ucdenver.edu

ABSTRACT
The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

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Experimental setup to investigate the consequence of ectopic expression of dominant negative Ikaros on activated CD8 T cells.(A) Schematic of experimental method to analyze the consequence of forced expression of dominant negative (DN) Ikaros on mature, activated CD8 T cells. Retroviral transduced cells, detected by Thy1.1 expression, are depicted in gray. (B) Thy1.1 expression as measured by flow cytometry on activated, retrovirally transduced CD8 T cells at day 8 in culture with IL-2. Histograms are gated on live, CD8+ events in mock (gray), control RV (blue) or DN Ikaros-transduced cultures (red). (C) Western blot analysis of Ikaros expression in peptide-activated, P14 TCR transgenic T cells transduced with control or DN Ikaros (from two independent cultures, 1 and 2). The smaller Ik6 isoform ∼37 kDa is indicated by asterisk. 3T3 fibroblasts and the JE131 Ikaros-deficient thymoma serve as negative controls for Ikaros expression. (C) Relative abundance of retrovirally transduced CD8 T cells cultured in either IL-15 or IL-2. Data indicate the percentage of the culture that are live, transduced cells (defined as CD8+ Thy1.1+) following culture in IL-15 or IL-2, beginning at day 2. Cells were transduced with either control (filled symbols) or DN Ikaros-expressing retrovirus (open symbols), with data representative of two independent experiments. (E) CD62L expression on live, CD8+ (mock, gray) or CD8+ Thy1.1+ events (using gate in panel B) in control (blue) or DN Ikaros (red) transduced cells cultured in IL-15 (left) or IL-2 (right). (F,G) Relative abundance of retrovirally transduced CD8 T cells cultured in the absence of exogenous cytokines, as measured by the percentage of CD8 T cells that are transduced (F) or the percentage of the culture that are live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) (G). Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Each data point indicates the mean percentage ± SEM) at 2 to 8 days post-activation (cells transduced on day 1 post-activation), with data from four independent experiments and statistically significant differences (p<0.05), determined by paired t test, indicated by asterisk.
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pone-0057435-g002: Experimental setup to investigate the consequence of ectopic expression of dominant negative Ikaros on activated CD8 T cells.(A) Schematic of experimental method to analyze the consequence of forced expression of dominant negative (DN) Ikaros on mature, activated CD8 T cells. Retroviral transduced cells, detected by Thy1.1 expression, are depicted in gray. (B) Thy1.1 expression as measured by flow cytometry on activated, retrovirally transduced CD8 T cells at day 8 in culture with IL-2. Histograms are gated on live, CD8+ events in mock (gray), control RV (blue) or DN Ikaros-transduced cultures (red). (C) Western blot analysis of Ikaros expression in peptide-activated, P14 TCR transgenic T cells transduced with control or DN Ikaros (from two independent cultures, 1 and 2). The smaller Ik6 isoform ∼37 kDa is indicated by asterisk. 3T3 fibroblasts and the JE131 Ikaros-deficient thymoma serve as negative controls for Ikaros expression. (C) Relative abundance of retrovirally transduced CD8 T cells cultured in either IL-15 or IL-2. Data indicate the percentage of the culture that are live, transduced cells (defined as CD8+ Thy1.1+) following culture in IL-15 or IL-2, beginning at day 2. Cells were transduced with either control (filled symbols) or DN Ikaros-expressing retrovirus (open symbols), with data representative of two independent experiments. (E) CD62L expression on live, CD8+ (mock, gray) or CD8+ Thy1.1+ events (using gate in panel B) in control (blue) or DN Ikaros (red) transduced cells cultured in IL-15 (left) or IL-2 (right). (F,G) Relative abundance of retrovirally transduced CD8 T cells cultured in the absence of exogenous cytokines, as measured by the percentage of CD8 T cells that are transduced (F) or the percentage of the culture that are live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) (G). Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Each data point indicates the mean percentage ± SEM) at 2 to 8 days post-activation (cells transduced on day 1 post-activation), with data from four independent experiments and statistically significant differences (p<0.05), determined by paired t test, indicated by asterisk.

Mentions: Given the expression of multiple Ikaros family members within mature CD8 T cells, and the known genetic redundancy within these gene products, we sought to investigate the contribution of the Ikaros gene family to the properties of mature CD8 T cells. To do this, we used retroviral transduction in vitro to express a previously described dominant negative variant of Ikaros (the Ik6 isoform, DN Ikaros) in activated primary CD8 T cells (experimental design depicted in Fig. 2A). Retrovirally-transduced cells were identified by Thy1.1 expression (encoded within the bicistronic retroviruses used here), with DN Ikaros transduced cells routinely having a modestly reduced expression of the Thy1.1 marker gene relative to control retrovirus transduced cells (Fig. 2B). The Ik6 isoform is known to heterodimerize and to inhibit DNA binding of multiple Ikaros family members [4]. Following transduction, mixed cultures of transduced and untransduced CD8 T cells were placed into different cytokine conditions, and the relative abundance and properties of retrovirally-transduced cells (either control or DN Ikaros-transduced) were analyzed. Western blot analysis for Ikaros protein peptide-activated CD8 T cells identified expression of two major species of endogenous Ikaros (∼50–55 kDa, <72 kDa), with DN Ikaros transduced cells also having robust expression of the smaller Ik6 isoform (∼37 kDa, indicated by asterisk) (Fig. 2C).


The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.

Clambey ET, Collins B, Young MH, Eberlein J, David A, Kappler JW, Marrack P - PLoS ONE (2013)

Experimental setup to investigate the consequence of ectopic expression of dominant negative Ikaros on activated CD8 T cells.(A) Schematic of experimental method to analyze the consequence of forced expression of dominant negative (DN) Ikaros on mature, activated CD8 T cells. Retroviral transduced cells, detected by Thy1.1 expression, are depicted in gray. (B) Thy1.1 expression as measured by flow cytometry on activated, retrovirally transduced CD8 T cells at day 8 in culture with IL-2. Histograms are gated on live, CD8+ events in mock (gray), control RV (blue) or DN Ikaros-transduced cultures (red). (C) Western blot analysis of Ikaros expression in peptide-activated, P14 TCR transgenic T cells transduced with control or DN Ikaros (from two independent cultures, 1 and 2). The smaller Ik6 isoform ∼37 kDa is indicated by asterisk. 3T3 fibroblasts and the JE131 Ikaros-deficient thymoma serve as negative controls for Ikaros expression. (C) Relative abundance of retrovirally transduced CD8 T cells cultured in either IL-15 or IL-2. Data indicate the percentage of the culture that are live, transduced cells (defined as CD8+ Thy1.1+) following culture in IL-15 or IL-2, beginning at day 2. Cells were transduced with either control (filled symbols) or DN Ikaros-expressing retrovirus (open symbols), with data representative of two independent experiments. (E) CD62L expression on live, CD8+ (mock, gray) or CD8+ Thy1.1+ events (using gate in panel B) in control (blue) or DN Ikaros (red) transduced cells cultured in IL-15 (left) or IL-2 (right). (F,G) Relative abundance of retrovirally transduced CD8 T cells cultured in the absence of exogenous cytokines, as measured by the percentage of CD8 T cells that are transduced (F) or the percentage of the culture that are live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) (G). Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Each data point indicates the mean percentage ± SEM) at 2 to 8 days post-activation (cells transduced on day 1 post-activation), with data from four independent experiments and statistically significant differences (p<0.05), determined by paired t test, indicated by asterisk.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585008&req=5

pone-0057435-g002: Experimental setup to investigate the consequence of ectopic expression of dominant negative Ikaros on activated CD8 T cells.(A) Schematic of experimental method to analyze the consequence of forced expression of dominant negative (DN) Ikaros on mature, activated CD8 T cells. Retroviral transduced cells, detected by Thy1.1 expression, are depicted in gray. (B) Thy1.1 expression as measured by flow cytometry on activated, retrovirally transduced CD8 T cells at day 8 in culture with IL-2. Histograms are gated on live, CD8+ events in mock (gray), control RV (blue) or DN Ikaros-transduced cultures (red). (C) Western blot analysis of Ikaros expression in peptide-activated, P14 TCR transgenic T cells transduced with control or DN Ikaros (from two independent cultures, 1 and 2). The smaller Ik6 isoform ∼37 kDa is indicated by asterisk. 3T3 fibroblasts and the JE131 Ikaros-deficient thymoma serve as negative controls for Ikaros expression. (C) Relative abundance of retrovirally transduced CD8 T cells cultured in either IL-15 or IL-2. Data indicate the percentage of the culture that are live, transduced cells (defined as CD8+ Thy1.1+) following culture in IL-15 or IL-2, beginning at day 2. Cells were transduced with either control (filled symbols) or DN Ikaros-expressing retrovirus (open symbols), with data representative of two independent experiments. (E) CD62L expression on live, CD8+ (mock, gray) or CD8+ Thy1.1+ events (using gate in panel B) in control (blue) or DN Ikaros (red) transduced cells cultured in IL-15 (left) or IL-2 (right). (F,G) Relative abundance of retrovirally transduced CD8 T cells cultured in the absence of exogenous cytokines, as measured by the percentage of CD8 T cells that are transduced (F) or the percentage of the culture that are live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) (G). Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Each data point indicates the mean percentage ± SEM) at 2 to 8 days post-activation (cells transduced on day 1 post-activation), with data from four independent experiments and statistically significant differences (p<0.05), determined by paired t test, indicated by asterisk.
Mentions: Given the expression of multiple Ikaros family members within mature CD8 T cells, and the known genetic redundancy within these gene products, we sought to investigate the contribution of the Ikaros gene family to the properties of mature CD8 T cells. To do this, we used retroviral transduction in vitro to express a previously described dominant negative variant of Ikaros (the Ik6 isoform, DN Ikaros) in activated primary CD8 T cells (experimental design depicted in Fig. 2A). Retrovirally-transduced cells were identified by Thy1.1 expression (encoded within the bicistronic retroviruses used here), with DN Ikaros transduced cells routinely having a modestly reduced expression of the Thy1.1 marker gene relative to control retrovirus transduced cells (Fig. 2B). The Ik6 isoform is known to heterodimerize and to inhibit DNA binding of multiple Ikaros family members [4]. Following transduction, mixed cultures of transduced and untransduced CD8 T cells were placed into different cytokine conditions, and the relative abundance and properties of retrovirally-transduced cells (either control or DN Ikaros-transduced) were analyzed. Western blot analysis for Ikaros protein peptide-activated CD8 T cells identified expression of two major species of endogenous Ikaros (∼50–55 kDa, <72 kDa), with DN Ikaros transduced cells also having robust expression of the smaller Ik6 isoform (∼37 kDa, indicated by asterisk) (Fig. 2C).

Bottom Line: While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism.Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells.These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Integrated Department of Immunology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. eric.clambey@ucdenver.edu

ABSTRACT
The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

Show MeSH
Related in: MedlinePlus