Limits...
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.

Show MeSH

Related in: MedlinePlus

DN Ikaros expression in activated CD8 T cells confers a pronounced advantage for cells cultured in IL-12.(A) Relative abundance of retroviral transduced cells between day 4 to 8 of culture in IL-12, as measured by Thy1.1 expression, in cultures transduced with control (top) or DN Ikaros (bottom) expressing retrovirus. Histograms depict Thy1.1 expression within live (7AAD negative), CD8+ cells as measured by flow cytometry. (B) Data indicate the percentage of CD8 T cells that are transduced from day 2 to 8. (C) Percentage of live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) between day 2 and 8 of culture in 5 ng/mL of mIL-12. Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Data depict the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from five independent experiments, with seven independent cultures, two of which used sorted cells. (D) Data indicate the fold change in number of live, transduced cells (defined as CD8+ Thy1.1+ cells) following culture in mIL-12 (5 ng/mL), relative to the starting number of transduced cells at day 2 of the culture. Data depict mean ± SEM from two independent experiments. (E) Relative abundance of retroviral transduced cells between day 2 to 8 of culture in IL-12 in the presence of neutralizing IL-2 antibodies in cultures transduced with control or DN Ikaros retrovirus. Data indicate percentage of CD8 T cells that are transduced (Thy1.1+) between day 2 and 8 of culture in 5 ng/mL of mIL-12, where cultures were incubated either with an isotype control or an anti-IL2 antibody. Each data point indicates the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from two independent experiments. (F) DN Ikaros expression in activated CD8 T cells does not profoundly alter the relative ability of CD8 T cells to respond to IL-12, as measured by IFN-γ production following IL-12/IL-18 synergistic induction of IFN-γ. Activated CD8 T cells, transduced with DN Ikaros were cultured in IL-2 for 6 days and assayed for IFN-γ production by intracellular cytokine staining. Data depict mean of 2–3 replicates per condition. As a positive control, cells were stimulated with cognate peptide (SIINFEKL at 5 µM). Statistically significant differences (p<0.05) are indicated by asterisk, calculated by paired t-test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585008&req=5

pone-0057435-g004: DN Ikaros expression in activated CD8 T cells confers a pronounced advantage for cells cultured in IL-12.(A) Relative abundance of retroviral transduced cells between day 4 to 8 of culture in IL-12, as measured by Thy1.1 expression, in cultures transduced with control (top) or DN Ikaros (bottom) expressing retrovirus. Histograms depict Thy1.1 expression within live (7AAD negative), CD8+ cells as measured by flow cytometry. (B) Data indicate the percentage of CD8 T cells that are transduced from day 2 to 8. (C) Percentage of live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) between day 2 and 8 of culture in 5 ng/mL of mIL-12. Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Data depict the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from five independent experiments, with seven independent cultures, two of which used sorted cells. (D) Data indicate the fold change in number of live, transduced cells (defined as CD8+ Thy1.1+ cells) following culture in mIL-12 (5 ng/mL), relative to the starting number of transduced cells at day 2 of the culture. Data depict mean ± SEM from two independent experiments. (E) Relative abundance of retroviral transduced cells between day 2 to 8 of culture in IL-12 in the presence of neutralizing IL-2 antibodies in cultures transduced with control or DN Ikaros retrovirus. Data indicate percentage of CD8 T cells that are transduced (Thy1.1+) between day 2 and 8 of culture in 5 ng/mL of mIL-12, where cultures were incubated either with an isotype control or an anti-IL2 antibody. Each data point indicates the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from two independent experiments. (F) DN Ikaros expression in activated CD8 T cells does not profoundly alter the relative ability of CD8 T cells to respond to IL-12, as measured by IFN-γ production following IL-12/IL-18 synergistic induction of IFN-γ. Activated CD8 T cells, transduced with DN Ikaros were cultured in IL-2 for 6 days and assayed for IFN-γ production by intracellular cytokine staining. Data depict mean of 2–3 replicates per condition. As a positive control, cells were stimulated with cognate peptide (SIINFEKL at 5 µM). Statistically significant differences (p<0.05) are indicated by asterisk, calculated by paired t-test.

Mentions: Next we tested whether expression of DN Ikaros might confer enhanced survival or proliferation on activated CD8 T cells in the presence of other cytokines (IL-4, IFN-γ or IL-12) that influence CD8 T cell proliferation or differentiation [34], [35], [36], [37]. Dominant negative Ikaros expression in activated CD8 T cells did not significantly change the dynamics of cell growth or survival in response to either IL-4, which induced robust proliferation of activated CD8 T cells, or IFN-γ, in which CD8 T cells exhibited little proliferation (data not shown). When activated CD8 T cells were cultured in IL-12, control cultures showed limited proliferation and a high amount of cell death (Fig. 4A, top panel). Unexpectedly, DN Ikaros-transduced cells demonstrated a striking competitive advantage relative to non-transduced cells in IL-12 cultures, as revealed by the progressive increase in the percentage of DN Ikaros-transduced cells within these cultures over time (identified by Thy1.1 expression, a surrogate marker of retroviral transduction, Fig. 4A–B). By day 8 of culture, DN Ikaros-transduced cultures routinely contained at least 80% DN Ikaros-transduced cells and there was an overall increase in the percentage of viable cells within the culture relative control-transduced cultures (Fig. 4A–B). Notably, the advantage of DN Ikaros transduced cells relative to control cells was more pronounced in IL-12 cultures than that observed following cytokine withdrawal (Fig. 4C compared to Fig. 2G). IL-12 cultures of either untransduced or vector-transduced cells were characterized by limited cell division and a high rate of cell death, with a three-fold increase in the number of transduced cells at day 4, followed by attrition (Fig. 4D). Based on this, DN Ikaros-transduced cells had a profoundly delayed rate of attrition relative to control or non-transduced cells (Fig. 4C–D).


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)

DN Ikaros expression in activated CD8 T cells confers a pronounced advantage for cells cultured in IL-12.(A) Relative abundance of retroviral transduced cells between day 4 to 8 of culture in IL-12, as measured by Thy1.1 expression, in cultures transduced with control (top) or DN Ikaros (bottom) expressing retrovirus. Histograms depict Thy1.1 expression within live (7AAD negative), CD8+ cells as measured by flow cytometry. (B) Data indicate the percentage of CD8 T cells that are transduced from day 2 to 8. (C) Percentage of live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) between day 2 and 8 of culture in 5 ng/mL of mIL-12. Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Data depict the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from five independent experiments, with seven independent cultures, two of which used sorted cells. (D) Data indicate the fold change in number of live, transduced cells (defined as CD8+ Thy1.1+ cells) following culture in mIL-12 (5 ng/mL), relative to the starting number of transduced cells at day 2 of the culture. Data depict mean ± SEM from two independent experiments. (E) Relative abundance of retroviral transduced cells between day 2 to 8 of culture in IL-12 in the presence of neutralizing IL-2 antibodies in cultures transduced with control or DN Ikaros retrovirus. Data indicate percentage of CD8 T cells that are transduced (Thy1.1+) between day 2 and 8 of culture in 5 ng/mL of mIL-12, where cultures were incubated either with an isotype control or an anti-IL2 antibody. Each data point indicates the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from two independent experiments. (F) DN Ikaros expression in activated CD8 T cells does not profoundly alter the relative ability of CD8 T cells to respond to IL-12, as measured by IFN-γ production following IL-12/IL-18 synergistic induction of IFN-γ. Activated CD8 T cells, transduced with DN Ikaros were cultured in IL-2 for 6 days and assayed for IFN-γ production by intracellular cytokine staining. Data depict mean of 2–3 replicates per condition. As a positive control, cells were stimulated with cognate peptide (SIINFEKL at 5 µM). Statistically significant differences (p<0.05) are indicated by asterisk, calculated by paired t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057435-g004: DN Ikaros expression in activated CD8 T cells confers a pronounced advantage for cells cultured in IL-12.(A) Relative abundance of retroviral transduced cells between day 4 to 8 of culture in IL-12, as measured by Thy1.1 expression, in cultures transduced with control (top) or DN Ikaros (bottom) expressing retrovirus. Histograms depict Thy1.1 expression within live (7AAD negative), CD8+ cells as measured by flow cytometry. (B) Data indicate the percentage of CD8 T cells that are transduced from day 2 to 8. (C) Percentage of live, transduced cells (defined as 7AAD negative, CD8+ Thy1.1+ events) between day 2 and 8 of culture in 5 ng/mL of mIL-12. Cells were transduced with either control (gray filled circles) or DN Ikaros-expressing retrovirus (open triangles). Data depict the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from five independent experiments, with seven independent cultures, two of which used sorted cells. (D) Data indicate the fold change in number of live, transduced cells (defined as CD8+ Thy1.1+ cells) following culture in mIL-12 (5 ng/mL), relative to the starting number of transduced cells at day 2 of the culture. Data depict mean ± SEM from two independent experiments. (E) Relative abundance of retroviral transduced cells between day 2 to 8 of culture in IL-12 in the presence of neutralizing IL-2 antibodies in cultures transduced with control or DN Ikaros retrovirus. Data indicate percentage of CD8 T cells that are transduced (Thy1.1+) between day 2 and 8 of culture in 5 ng/mL of mIL-12, where cultures were incubated either with an isotype control or an anti-IL2 antibody. Each data point indicates the mean percentage of live, transduced cells (+/− SEM) within cultures at 2 to 8 days post-activation (cells transduced on day 1 post-activation). Data are from two independent experiments. (F) DN Ikaros expression in activated CD8 T cells does not profoundly alter the relative ability of CD8 T cells to respond to IL-12, as measured by IFN-γ production following IL-12/IL-18 synergistic induction of IFN-γ. Activated CD8 T cells, transduced with DN Ikaros were cultured in IL-2 for 6 days and assayed for IFN-γ production by intracellular cytokine staining. Data depict mean of 2–3 replicates per condition. As a positive control, cells were stimulated with cognate peptide (SIINFEKL at 5 µM). Statistically significant differences (p<0.05) are indicated by asterisk, calculated by paired t-test.
Mentions: Next we tested whether expression of DN Ikaros might confer enhanced survival or proliferation on activated CD8 T cells in the presence of other cytokines (IL-4, IFN-γ or IL-12) that influence CD8 T cell proliferation or differentiation [34], [35], [36], [37]. Dominant negative Ikaros expression in activated CD8 T cells did not significantly change the dynamics of cell growth or survival in response to either IL-4, which induced robust proliferation of activated CD8 T cells, or IFN-γ, in which CD8 T cells exhibited little proliferation (data not shown). When activated CD8 T cells were cultured in IL-12, control cultures showed limited proliferation and a high amount of cell death (Fig. 4A, top panel). Unexpectedly, DN Ikaros-transduced cells demonstrated a striking competitive advantage relative to non-transduced cells in IL-12 cultures, as revealed by the progressive increase in the percentage of DN Ikaros-transduced cells within these cultures over time (identified by Thy1.1 expression, a surrogate marker of retroviral transduction, Fig. 4A–B). By day 8 of culture, DN Ikaros-transduced cultures routinely contained at least 80% DN Ikaros-transduced cells and there was an overall increase in the percentage of viable cells within the culture relative control-transduced cultures (Fig. 4A–B). Notably, the advantage of DN Ikaros transduced cells relative to control cells was more pronounced in IL-12 cultures than that observed following cytokine withdrawal (Fig. 4C compared to Fig. 2G). IL-12 cultures of either untransduced or vector-transduced cells were characterized by limited cell division and a high rate of cell death, with a three-fold increase in the number of transduced cells at day 4, followed by attrition (Fig. 4D). Based on this, DN Ikaros-transduced cells had a profoundly delayed rate of attrition relative to control or non-transduced cells (Fig. 4C–D).

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