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Analysis of GzmbCre as a Model System for Gene Deletion in the Natural Killer Cell Lineage.

Xu Y, Evaristo C, Alegre ML, Gurbuxani S, Kee BL - PLoS ONE (2015)

Bottom Line: We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor.As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation.We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

View Article: PubMed Central - PubMed

Affiliation: Committee on Molecular Pathogenesis and Molecular Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The analysis of gene function in mature and activated natural killer cells has been hampered by the lack of model systems for Cre-mediated recombination in these cells. Here we have investigated the utility of GzmbCre for recombination of loxp sequences in these cells predicated on the observation that Gzmb mRNA is highly expressed in mature and activated natural killer cells. Using two different reporter strains we determined that gene function could be investigated in mature natural killer cells after GzmbCre mediated recombination in vitro in conditions that lead to natural killer cell activation such as in the cytokine combination of interleukin 2 and interleukin 12. We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor. In vivo and in vitro activation of IKKβ in natural killer cells revealed that constitutive activation of this pathway leads to natural killer cell hyper-activation and altered morphology. As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation. We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

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Constitutive IKKβ activation leads to NK cell hyper-activation and altered morphology.(A) GFP- mNK cells from the spleen of GzmbCre;Rosa26IKKbca mice were isolated by cell sorting and cultured in IL2 or IL2 + IL12 for 5 days prior to analysis of GFP expression by flow cytometry. One representative experiment of 3 is shown. (B) FSC and SSC analysis of the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. (C) Expression of CD25, CD69, and KLRG1 on the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. The shaded grey histogram represents the isotype control staining for GFP+ cells. (D) Giemsa stain for GFP- and (E) GFP+ cells 5 days after transfer of cells from IL2 + IL12 cultures to IL2 alone. One of 3 similar experiments is shown; 100 pixels = 10 μm, 400X.
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pone.0125211.g007: Constitutive IKKβ activation leads to NK cell hyper-activation and altered morphology.(A) GFP- mNK cells from the spleen of GzmbCre;Rosa26IKKbca mice were isolated by cell sorting and cultured in IL2 or IL2 + IL12 for 5 days prior to analysis of GFP expression by flow cytometry. One representative experiment of 3 is shown. (B) FSC and SSC analysis of the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. (C) Expression of CD25, CD69, and KLRG1 on the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. The shaded grey histogram represents the isotype control staining for GFP+ cells. (D) Giemsa stain for GFP- and (E) GFP+ cells 5 days after transfer of cells from IL2 + IL12 cultures to IL2 alone. One of 3 similar experiments is shown; 100 pixels = 10 μm, 400X.

Mentions: To gain further insight into the consequences of IKKβ activation in mNK cells, we examined mNK cells after in vitro recombination of the Rosa26IKKbca transgene. As we observed with the Rosa26 reporter alleles, in vitro culture of GFP-GzmbCre;Rosa26IKKbca mNK cells in IL2 promoted weak recombination of the GzmbCre;Rosa26IKKbca allele (Fig 7A). However, a substantial frequency of GFP-GzmbCre;Rosa26IKKbca mNK cells became GFP+ after a 5 day culture in IL2 + IL12 (Fig 7A). IL2 + IL12 causes NK cell activation, however, the IKKβ-CA expressing mNK cells cultured under these conditions were even larger and more granular than their GFP- counterparts (Fig 7B), indicating that these cells were hyperactivated. Indeed, the IKKβ-CA expressing cells also had very high surface expression of the activation markers CD25, CD69, and KLRG1 (Fig 7C). When transferred from IL2 + IL12 to IL2 alone, to allow the cells to return to conditions that are not activating, the GFP+ cells became irregular in appearance and adherent to the plastic dish making quantification difficult. On Giemsa stained preparations, the GFP+ cells were larger and showed large coarse granules when compared to the GFP- cells (Fig 7D and 7E). These data led us to conclude that constitutive activation of IKKβ resulted in NK cell hyperactivation as measured by increased size and granularity and the high level of expression of activation markers, changes in adhesive properties, and altered morphologic characteristics. Therefore, while activation of NFκB is necessary for NK cell function, constitutive activation of NFκB could lead to chronic activation as well as functional and morphologic changes that could prevent their accumulation.


Analysis of GzmbCre as a Model System for Gene Deletion in the Natural Killer Cell Lineage.

Xu Y, Evaristo C, Alegre ML, Gurbuxani S, Kee BL - PLoS ONE (2015)

Constitutive IKKβ activation leads to NK cell hyper-activation and altered morphology.(A) GFP- mNK cells from the spleen of GzmbCre;Rosa26IKKbca mice were isolated by cell sorting and cultured in IL2 or IL2 + IL12 for 5 days prior to analysis of GFP expression by flow cytometry. One representative experiment of 3 is shown. (B) FSC and SSC analysis of the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. (C) Expression of CD25, CD69, and KLRG1 on the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. The shaded grey histogram represents the isotype control staining for GFP+ cells. (D) Giemsa stain for GFP- and (E) GFP+ cells 5 days after transfer of cells from IL2 + IL12 cultures to IL2 alone. One of 3 similar experiments is shown; 100 pixels = 10 μm, 400X.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414598&req=5

pone.0125211.g007: Constitutive IKKβ activation leads to NK cell hyper-activation and altered morphology.(A) GFP- mNK cells from the spleen of GzmbCre;Rosa26IKKbca mice were isolated by cell sorting and cultured in IL2 or IL2 + IL12 for 5 days prior to analysis of GFP expression by flow cytometry. One representative experiment of 3 is shown. (B) FSC and SSC analysis of the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. (C) Expression of CD25, CD69, and KLRG1 on the GFP+ (black) and GFP- (grey) cells from the day 5 IL2 + IL12 cultures. The shaded grey histogram represents the isotype control staining for GFP+ cells. (D) Giemsa stain for GFP- and (E) GFP+ cells 5 days after transfer of cells from IL2 + IL12 cultures to IL2 alone. One of 3 similar experiments is shown; 100 pixels = 10 μm, 400X.
Mentions: To gain further insight into the consequences of IKKβ activation in mNK cells, we examined mNK cells after in vitro recombination of the Rosa26IKKbca transgene. As we observed with the Rosa26 reporter alleles, in vitro culture of GFP-GzmbCre;Rosa26IKKbca mNK cells in IL2 promoted weak recombination of the GzmbCre;Rosa26IKKbca allele (Fig 7A). However, a substantial frequency of GFP-GzmbCre;Rosa26IKKbca mNK cells became GFP+ after a 5 day culture in IL2 + IL12 (Fig 7A). IL2 + IL12 causes NK cell activation, however, the IKKβ-CA expressing mNK cells cultured under these conditions were even larger and more granular than their GFP- counterparts (Fig 7B), indicating that these cells were hyperactivated. Indeed, the IKKβ-CA expressing cells also had very high surface expression of the activation markers CD25, CD69, and KLRG1 (Fig 7C). When transferred from IL2 + IL12 to IL2 alone, to allow the cells to return to conditions that are not activating, the GFP+ cells became irregular in appearance and adherent to the plastic dish making quantification difficult. On Giemsa stained preparations, the GFP+ cells were larger and showed large coarse granules when compared to the GFP- cells (Fig 7D and 7E). These data led us to conclude that constitutive activation of IKKβ resulted in NK cell hyperactivation as measured by increased size and granularity and the high level of expression of activation markers, changes in adhesive properties, and altered morphologic characteristics. Therefore, while activation of NFκB is necessary for NK cell function, constitutive activation of NFκB could lead to chronic activation as well as functional and morphologic changes that could prevent their accumulation.

Bottom Line: We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor.As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation.We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

View Article: PubMed Central - PubMed

Affiliation: Committee on Molecular Pathogenesis and Molecular Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The analysis of gene function in mature and activated natural killer cells has been hampered by the lack of model systems for Cre-mediated recombination in these cells. Here we have investigated the utility of GzmbCre for recombination of loxp sequences in these cells predicated on the observation that Gzmb mRNA is highly expressed in mature and activated natural killer cells. Using two different reporter strains we determined that gene function could be investigated in mature natural killer cells after GzmbCre mediated recombination in vitro in conditions that lead to natural killer cell activation such as in the cytokine combination of interleukin 2 and interleukin 12. We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor. In vivo and in vitro activation of IKKβ in natural killer cells revealed that constitutive activation of this pathway leads to natural killer cell hyper-activation and altered morphology. As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation. We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

Show MeSH