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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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Caspase-3 activation does not occur during rapid homeostatic proliferation in vivo.Rag1-/- mice were injected with 104 CFSE stained OT1 cells. WT mice were injected with 105 CFSE stained OT1 cells concurrent with PBS, LM or LM-OVA (104, iv). Four days later mice were sacrificed and spleen cell suspensions stained with anti-CD8 antibody, OVA-tetramer, followed by intracellular staining with anti-caspase-3 antibody. Scatterplots show data obtained from gated OVA specific CD8+ T cells and is representative of 3 mice per group.
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pone-0015328-g011: Caspase-3 activation does not occur during rapid homeostatic proliferation in vivo.Rag1-/- mice were injected with 104 CFSE stained OT1 cells. WT mice were injected with 105 CFSE stained OT1 cells concurrent with PBS, LM or LM-OVA (104, iv). Four days later mice were sacrificed and spleen cell suspensions stained with anti-CD8 antibody, OVA-tetramer, followed by intracellular staining with anti-caspase-3 antibody. Scatterplots show data obtained from gated OVA specific CD8+ T cells and is representative of 3 mice per group.

Mentions: Having noted that antigen stimulated proliferating CD8+ T cells express caspase-3, we determined whether non-antigen induced homeostatic CD8+ T cells proliferation is also associated with caspase-3 activation. The transfer of OT-1 CD8+ T cells to lymphopenic hosts (Rag1-deficient recipient mice) results in a relatively rapid proliferation of transferred CD8+ T cells in the absence of antigen. We transferred 105 CFSE stained OT-1 CD8+ T cells to WT and Rag1-deficient mice. Groups of WT mice were also challenged with LM or LM-OVA. Spleens were removed at day 5 post-infection and cells were stained to evaluate the donor CD8 population and the expression of active caspase-3. In Rag-1-deficient hosts, transferred CD8+ T cells had undergone significant homeostatic proliferation as revealed by CFSE dilution, however, there was no apparent upregulation of caspase-3 activity (Fig. 11). Conversely, in the WT mice, only those mice challenged with LM-OVA showed proliferation associated with significant activation of caspase-3. Wild-type LM (without OVA) induced low level non-specific proliferation of transferred cells but failed to induce the expression of caspase-3 on OVA-specific CD8+ T cells. These results further indicate that caspase 3 is specifically induced by antigen presentation and not upregulated during non-specific or homeostatic proliferation.


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)

Caspase-3 activation does not occur during rapid homeostatic proliferation in vivo.Rag1-/- mice were injected with 104 CFSE stained OT1 cells. WT mice were injected with 105 CFSE stained OT1 cells concurrent with PBS, LM or LM-OVA (104, iv). Four days later mice were sacrificed and spleen cell suspensions stained with anti-CD8 antibody, OVA-tetramer, followed by intracellular staining with anti-caspase-3 antibody. Scatterplots show data obtained from gated OVA specific CD8+ T cells and is representative of 3 mice per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008739&req=5

pone-0015328-g011: Caspase-3 activation does not occur during rapid homeostatic proliferation in vivo.Rag1-/- mice were injected with 104 CFSE stained OT1 cells. WT mice were injected with 105 CFSE stained OT1 cells concurrent with PBS, LM or LM-OVA (104, iv). Four days later mice were sacrificed and spleen cell suspensions stained with anti-CD8 antibody, OVA-tetramer, followed by intracellular staining with anti-caspase-3 antibody. Scatterplots show data obtained from gated OVA specific CD8+ T cells and is representative of 3 mice per group.
Mentions: Having noted that antigen stimulated proliferating CD8+ T cells express caspase-3, we determined whether non-antigen induced homeostatic CD8+ T cells proliferation is also associated with caspase-3 activation. The transfer of OT-1 CD8+ T cells to lymphopenic hosts (Rag1-deficient recipient mice) results in a relatively rapid proliferation of transferred CD8+ T cells in the absence of antigen. We transferred 105 CFSE stained OT-1 CD8+ T cells to WT and Rag1-deficient mice. Groups of WT mice were also challenged with LM or LM-OVA. Spleens were removed at day 5 post-infection and cells were stained to evaluate the donor CD8 population and the expression of active caspase-3. In Rag-1-deficient hosts, transferred CD8+ T cells had undergone significant homeostatic proliferation as revealed by CFSE dilution, however, there was no apparent upregulation of caspase-3 activity (Fig. 11). Conversely, in the WT mice, only those mice challenged with LM-OVA showed proliferation associated with significant activation of caspase-3. Wild-type LM (without OVA) induced low level non-specific proliferation of transferred cells but failed to induce the expression of caspase-3 on OVA-specific CD8+ T cells. These results further indicate that caspase 3 is specifically induced by antigen presentation and not upregulated during non-specific or homeostatic proliferation.

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