Figure 1.
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.
Figure 2.
Active caspase-3 in antigen activated T cells is expressed in live proliferating cells.
OT1 spleen cells were stimulated in vitro for 48 hours with varying amounts of antigen. Apoptotic control cells were treated with 1 µg/ml staurospaurine. Cells were then stained with anti-CD8 antibody and fixed and permeabilized before TUNEL and caspase-3 staining. Cells were mounted on slides and examinated by fluorescence microscopy. Images show TUNEL staining (green), and active caspase-3 (red) in CD8 labeled T cells (blue). The images are representative of 3 repeated examinations for each differentially stimulated culture. Arrows shown identify cells with positive staining for both caspase-3 and TUNEL.
Figure 3.
Rapidly proliferating CD8+ T cells have an apoptotic like phenotype in vivo.
C57BL6/J mice were injected with 104 OT1 cells and infected with 104 LM-OVA. At various time points mice were sacrificed and splenic cells were stained and analyzed by FACS. Scatterplots show OVA-tetramer binding versus (A) active caspase, (B) PhiPhiLux cleavage by caspase-3 (PPL), (C) Annexin V binding and (d) PD-1, in cells over days 3, 4 and 7 of LM-OVA infection. The % of OVA-tetramer+ CD8+ cells expressing a high level of (E▴) caspase-3, (F•) PPL cleavage, (G Δ) Annexin V binding, and (H ○) PD-1 are shown over the course of an acute OVA specific CD8+ T cell response (▪). *The decrease in apoptotic staining between day 3 and 7 shows significant linear correlation (by Pearson analysis) between active caspase-3 antibody staining and (E) PPL cleavage activity, (F) annexin V binding and (G) PD-1 expression (P<0.05). **Change in active caspase-3 staining is inversely proportional to an increase in OVA-tetramer specific cells (P<0.001, n = 3 mice/timepoint).
Figure 4.
Actively proliferating cells in vivo co-express markers for apoptosis and proliferation with little apparent cell death.
C57BL/6 mice were parked with 104 OT1 cells and challenged with 104 LM-OVA. Splenic cells were obtained from infected C57BL/6 mice at various time points after infection and stained for CD8 and OVA-tatramer binding as well (A) proliferation marker Ki67 vs. active caspase-3. (B) Cells were also co-stained for DNA cleavage (TUNEL) vs. active caspase-3 expression.
Figure 5.
The majority of active caspase-3hi cells do not progress to cell death during early antigen induced proliferation in vivo.
Mice were parked with 104 CD45.1+ OT1 spleen cells and challenged with 104 LM-OVA (iv). Mice were sacrificed at day 5 post LM-OVA infection and CD8+ T cells were magnetically isolated by negative selection. Cells were stained with anti-CD45.1 antibody and PhiPhiLux. (A) Cells were sorted into CD45.1+ caspase-3 activity high and CD45.1+ caspase-3 activity low fractions. (B) Sorted cells were then plated at low cell densities (∼12.5 cells/well) and examined for viability during proliferation over several days using TMRE staining. (C) The total number and number of dead cells were counted directly in at least 6 replicate wells and the changes observed over 72 hours in the plates. (D) Cells were also maintained at higher concentrations (∼100 000/well) and stained for DNA cleavage (TUNEL) after 1 day in culture.
Figure 6.
The activation of caspase-3 occurs in early activated cells before the emergence of fully differentiated effector cells.
C57BL/6 mice were initially parked with 104 OT1 spleen cells. After infection with 104 LM-OVA, mice were sacrificed at various time points and spleens were harvested. Single cell suspensions were co-stained for CD8+, OVA-Tetramer, CD62L, IL7Rα and active caspase-3. (A) Scatter plots show the relative staining of OVA-tetramer+ CD8+ cells for activation markers and active caspase-3. Data is representative for at least 3 mice per time point. (B) OVA-tetramer+ CD8+ T cells were examined for active intracellular active caspase-3 and surface CD62L between days 0–15 of LM-OVA infection. (data is representative for 3 mice per timepoint).
Figure 7.
Caspase 3 and CD62L expression is progressively lost as proliferation proceeds.
104 OT1 splenocytes were stained with CFSE and injected in C57BL/6 recipient mice concurrently with LM-OVA challenge. After 3, 4 and 5 days, mice were sacrificed (3 per time point) and spleen cells stained for expression markers. (A) Scatterplots show expression of active caspase-3 versus CFSE and CD62L in OVA-tetramer+ CD8+ T cells after 4 days in vivo. (B) OVA specific CD8+ T cells were gated for division number based on CFSE dilution. Cells show a significant, coordinate decrease in the expression of active caspase-3 and CD62L/IL7Rα as cell proliferation proceeds (***P<0.005, *P<0.05, n = 3 per timepoint).
Figure 8.
Elevated active caspase-3 is found primarily in secondary lymphoid tissues early in T cell activation.
Mice parked with 104 OT1 cells were challenged with 104 LM-OVA. At various time points, mice were perfused and organs removed. Single cell suspensions were stained and analyzed by FACS. (A) Scatterplots show OVA-tetramer binding versus active caspase-3 expression in CD8+ T cells. Plots are representative of 3 mice analyzed. (B) The % of OVA-tetramer+ cells in each organ (upper right) and the change in the level of active caspase-3 expression in OVA specific CD8+ T cells (lower right) from each organ is shown. There is a significantly higher proportion of active caspase-3 high cells in the spleen when compared to PBL, lungs or brain at day 4 (*P<0.005, n = 2 per timepoint).
Figure 9.
ST-OVA infection induces a lower and protracted increase in the expression of active caspase-3 consistent with delayed and muted antigen induced CD8+ T cell proliferation.
B6129F1 mice that resist infection with ST were parked with 104 OT1 cells and challenged with either 103 LM-OVA or 103 ST-OVA iv and sacrificed at various time points after infection. Single cell suspensions were obtained from spleens and stained with various markers indicated in the figure. Relative expression of various markers on OVA-tetramer+ CD8+ T cells was evaluated (A). Cells were also co-stained for DNA cleavage (TUNEL) vs. active caspase-3 expression (B). (n≥3 per timepoint).
Figure 10.
Caspase-3 is induced by antigen, not inflammation in vivo.
B6129F1 mice were parked with 104 OT1 spleen cells, then infected with ST-OVA or LM-OVA (103, iv) and sacrificed at several time points between days 4 and 60 post-infection. Splenic suspensions were analyzed for (A) bacterial load by CFU, (B) bacterial OVA mRNA expression by qRT-PCR, and (C) the relative induction of 40 cytokines/chemokines by cytokine proteome array (day 6). (D) CFSE labeled OT1 cells were injected into B6129F1 mice that were challenged with LM-OVA or ST-OVA, and the proliferation and caspase 3 expression of OVA-specific evaluated at day 5 after infection. Scatter plots show data obtained from gated OVA specific CD8+ T cells. Data shown is representative for at least 3 mice per timepoint.
Figure 11.
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.