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Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo.

McComb S, Mulligan R, Sad S - PLoS ONE (2010)

Bottom Line: Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3(hi) and caspase-3(low) CD8(+) T cells.The expression of active caspase-3 peaked before effector phenotype (CD62L(low)) CD8(+) T cells emerged, and was undetectable in effector-phenotype cells.Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, NRC-Institute for Biological Sciences, University of Ottawa, Ottawa, Canada.

ABSTRACT

Background: CD8(+) T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8(+) T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8(+) T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8(+) T cell responses has yet to be examined.

Methods and findings: We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8(+) T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8(+) T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3(hi) and caspase-3(low) CD8(+) T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L(low)) CD8(+) T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8(+) cells remained active caspase-3(low) throughout the contraction phase.

Conclusions: Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8(+) T cells. Furthermore, the contraction of CD8(+) T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.

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Significant increase in active caspase-3 during T cell activation correlates directly with an increase in proliferation.A single cell suspension of OT1 splenocytes, some of which were first CFSE stained, were placed in culture with varying concentrations of SIINFEKL peptide for 48 hours and analyzed by intracellular flow cytometry (n≥3). Cells were stained with anti-CD8 antibody and OVA-tetramer, then fixed and permeabilized before staining with anti- active caspase-3 antibody and Ki67. Graphs show the mean fluorescence intensity (MFI) of CD8+ OVA-tetramer+ gated cells. (A) MFI of active caspase-3 versus Ki67 show a high amount of linear correlation in their expression levels (P<0.05). There is a significant increase in the expression of both caspase-3 and Ki67 from 10−8 to 10−2 µg/mL OVA (P<0.005). (B) MFI of CFSE versus active caspase-3, inverse correlation was found to be significant (P<0.01). (C) MFI of TUNEL stain versus active caspase-3, inverse correlation was found to be significant (P<0.01). (D) Scatterplots show the relative expression of active caspase-3 versus Ki67 for gated CD8+ cells in splenocyte cultures treated for 48 hours with varying amounts of SIINFEKL peptide as shown. Data shown is representative for two repeated experiments performed in triplicate.
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pone-0015328-g001: Significant increase in active caspase-3 during T cell activation correlates directly with an increase in proliferation.A single cell suspension of OT1 splenocytes, some of which were first CFSE stained, were placed in culture with varying concentrations of SIINFEKL peptide for 48 hours and analyzed by intracellular flow cytometry (n≥3). Cells were stained with anti-CD8 antibody and OVA-tetramer, then fixed and permeabilized before staining with anti- active caspase-3 antibody and Ki67. Graphs show the mean fluorescence intensity (MFI) of CD8+ OVA-tetramer+ gated cells. (A) MFI of active caspase-3 versus Ki67 show a high amount of linear correlation in their expression levels (P<0.05). There is a significant increase in the expression of both caspase-3 and Ki67 from 10−8 to 10−2 µg/mL OVA (P<0.005). (B) MFI of CFSE versus active caspase-3, inverse correlation was found to be significant (P<0.01). (C) MFI of TUNEL stain versus active caspase-3, inverse correlation was found to be significant (P<0.01). (D) Scatterplots show the relative expression of active caspase-3 versus Ki67 for gated CD8+ cells in splenocyte cultures treated for 48 hours with varying amounts of SIINFEKL peptide as shown. Data shown is representative for two repeated experiments performed in triplicate.

Mentions: OVA-specific CD8+ TCR transgenic mouse (OT-1) splenocytes were placed in culture with various concentrations of OVA peptide (SIINFEKL). OVA specific CD8+ T cells began proliferating within 24 hours of initial stimulation. Intracellular staining was performed using fluorescently labeled antibodies. Those OT-1 CD8+ T cells that were stimulated with an amount of antigen greater then about 0.1 nM (10−4 µg/ml) showed active proliferation, as identified by high expression of the active cell cycle marker Ki67 (Fig. 1A). The proliferative capacity of these cells was further confirmed by the loss of CFSE staining after activation (Fig. 1B). Coordinated with an increase in proliferation, we observed a significant increase in the level of active caspase-3 in the CD8+ population as the antigen levels increased from 10−8 to 10−2 µg/ml (P<0.005, Fig. 1). Co-staining revealed direct correlation between caspase-3 cleavage and cell proliferation (Ki67hi) within the CD8+ population (Fig 1D).


Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo.

McComb S, Mulligan R, Sad S - PLoS ONE (2010)

Significant increase in active caspase-3 during T cell activation correlates directly with an increase in proliferation.A single cell suspension of OT1 splenocytes, some of which were first CFSE stained, were placed in culture with varying concentrations of SIINFEKL peptide for 48 hours and analyzed by intracellular flow cytometry (n≥3). Cells were stained with anti-CD8 antibody and OVA-tetramer, then fixed and permeabilized before staining with anti- active caspase-3 antibody and Ki67. Graphs show the mean fluorescence intensity (MFI) of CD8+ OVA-tetramer+ gated cells. (A) MFI of active caspase-3 versus Ki67 show a high amount of linear correlation in their expression levels (P<0.05). There is a significant increase in the expression of both caspase-3 and Ki67 from 10−8 to 10−2 µg/mL OVA (P<0.005). (B) MFI of CFSE versus active caspase-3, inverse correlation was found to be significant (P<0.01). (C) MFI of TUNEL stain versus active caspase-3, inverse correlation was found to be significant (P<0.01). (D) Scatterplots show the relative expression of active caspase-3 versus Ki67 for gated CD8+ cells in splenocyte cultures treated for 48 hours with varying amounts of SIINFEKL peptide as shown. Data shown is representative for two repeated experiments performed in triplicate.
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Related In: Results  -  Collection

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pone-0015328-g001: Significant increase in active caspase-3 during T cell activation correlates directly with an increase in proliferation.A single cell suspension of OT1 splenocytes, some of which were first CFSE stained, were placed in culture with varying concentrations of SIINFEKL peptide for 48 hours and analyzed by intracellular flow cytometry (n≥3). Cells were stained with anti-CD8 antibody and OVA-tetramer, then fixed and permeabilized before staining with anti- active caspase-3 antibody and Ki67. Graphs show the mean fluorescence intensity (MFI) of CD8+ OVA-tetramer+ gated cells. (A) MFI of active caspase-3 versus Ki67 show a high amount of linear correlation in their expression levels (P<0.05). There is a significant increase in the expression of both caspase-3 and Ki67 from 10−8 to 10−2 µg/mL OVA (P<0.005). (B) MFI of CFSE versus active caspase-3, inverse correlation was found to be significant (P<0.01). (C) MFI of TUNEL stain versus active caspase-3, inverse correlation was found to be significant (P<0.01). (D) Scatterplots show the relative expression of active caspase-3 versus Ki67 for gated CD8+ cells in splenocyte cultures treated for 48 hours with varying amounts of SIINFEKL peptide as shown. Data shown is representative for two repeated experiments performed in triplicate.
Mentions: OVA-specific CD8+ TCR transgenic mouse (OT-1) splenocytes were placed in culture with various concentrations of OVA peptide (SIINFEKL). OVA specific CD8+ T cells began proliferating within 24 hours of initial stimulation. Intracellular staining was performed using fluorescently labeled antibodies. Those OT-1 CD8+ T cells that were stimulated with an amount of antigen greater then about 0.1 nM (10−4 µg/ml) showed active proliferation, as identified by high expression of the active cell cycle marker Ki67 (Fig. 1A). The proliferative capacity of these cells was further confirmed by the loss of CFSE staining after activation (Fig. 1B). Coordinated with an increase in proliferation, we observed a significant increase in the level of active caspase-3 in the CD8+ population as the antigen levels increased from 10−8 to 10−2 µg/ml (P<0.005, Fig. 1). Co-staining revealed direct correlation between caspase-3 cleavage and cell proliferation (Ki67hi) within the CD8+ population (Fig 1D).

Bottom Line: Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3(hi) and caspase-3(low) CD8(+) T cells.The expression of active caspase-3 peaked before effector phenotype (CD62L(low)) CD8(+) T cells emerged, and was undetectable in effector-phenotype cells.Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, NRC-Institute for Biological Sciences, University of Ottawa, Ottawa, Canada.

ABSTRACT

Background: CD8(+) T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8(+) T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8(+) T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8(+) T cell responses has yet to be examined.

Methods and findings: We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8(+) T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8(+) T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3(hi) and caspase-3(low) CD8(+) T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L(low)) CD8(+) T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8(+) cells remained active caspase-3(low) throughout the contraction phase.

Conclusions: Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8(+) T cells. Furthermore, the contraction of CD8(+) T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.

Show MeSH
Related in: MedlinePlus