Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells

Abstract

Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.

Figure 1

The general caspase inhibitor zVAD blocks anti-CD3–induced T cell activation. (A) Inhibition of PBMC proliferation by zVAD. Purified PBMCs were cultured in medium alone (NS) or stimulated with 1 μg/ml anti-CD3 and 20 U/ml IL-2 (CD3) in the presence of the indicated concentration of zVAD. At each time point, 1 μCi/well of [³H]thymidine was added for the last 6 h of culture, cells were harvested, and incorporated thymidine was counted by liquid scintillation. [³H]Thymidine ([³H]-TdR) incorporation is expressed as mean cpm ± SEM of triplicate cultures. Results are representative of three experiments where full kinetics was carried out. (B) zVAD does not inhibit cell cycle machinery. Fresh PBMCs were placed in RPMI medium, and 100 μM of zVAD was added 1 h before or 1, 2, or 3 d after stimulation in the presence of anti-CD3 antibody and IL-2. Cells were cultured for 4 d at 37°C, and [³H]thymidine was added for the last 6 h of incubation. Thymidine incorporation at day 4 is expressed as cpm ± SEM from triplicate cultures. Two independent experiments gave similar results. (C) Inhibition of blastic transformation by zVAD. Change in PBMCs morphology was monitored by flow cytometric analysis of cellular forward (y-axis) and side (x-axis) scatter. Percentage of resting PBMCs (region R1) is indicated inside dot plots. One representative experiment out of five is shown. (D) zVAD affects the expression of MHC class II. Surface staining was performed for CD69, HLA-DR, and CD25 on PBMCs activated for 4 d with anti-CD3/IL-2, in the presence or absence of 100 μM of zVAD. One representative experiment of three is shown. (E) The effect of zVAD is independent of accessory cells. Purified T lymphocytes isolated from PBMCs by negative selection were preincubated for 1 h in medium alone or with 100 μM zVAD, and cultured for 4 d without stimulation (NS) or in the presence of IL-2, anti-CD3, or both. Proliferation was assessed by [³H]thymidine incorporation as described in A. Results are representative of two independent experiments.

Figure 2

Caspase-3 is cleaved into multiple fragments in stimulated lymphocytes. (A) Proliferation. Purified PBMCs were obtained through Ficoll gradient separation and stimulated with 1 μg/ml of anti-CD3 and 20 U/ml of IL-2 (Anti-CD3) or not (Medium). After 1–4 d at 37°C, thymidine incorporation was determined as in the legend to Fig. 1. (B) Caspase-3 Western blot. Proteins were extracted from the same PBMCs treated in A by cell lysis using a denaturing Laemmli buffer containing 8 M urea. Total proteins extracted from 10⁶ fresh (d0), unstimulated (−), or stimulated cells (+) were separated on SDS-PAGE, and Western blot analysis was performed using a polyclonal rabbit anti–caspase-3 (reference 5). (C) Detection of active caspases by affinity blot. PBMCs stimulated or not for 4 d with anti-CD3, anti-TCR, or PHA in the presence of IL-2 were lysed using a nondenaturing lysis buffer (see Materials and Methods), and lysates were incubated with 5 μM of a biotinylated zVAD peptide, followed by a separation by SDS-PAGE and Western blot analysis using Extra-HRP. (D) Identification of active subunits of caspase-3. PBMCs were activated for 4 d with PHA and IL-2, and cell lysates were incubated with the substrate. Caspase-3 immunoprecipitation was then performed on half of the lysate. Total extract (Lys) or immunoprecipitated caspase-3 (IP) was subjected to Western blot analysis using the anti–caspase-3 antibody (left, WB cas-3) or with Extra-HRP (right, Affinity blot). Results are from one of at least three independent experiments giving similar results. (E) Detection of caspase activity in intact cells. Fresh PBMCs were left untreated (NS) or stimulated (CD3/IL-2) with anti-CD3 antibody and IL-2 for 4 d. A fraction of these activated cells was then incubated for 6 h in anti-Fas–coated plates (CD3/Fas). After a 1-h labeling with the cell-permeable substrate Phiphilux, the cells were analyzed by flow cytometry. Results are from one representative experiment out of three.

Figure 2

Caspase-3 is cleaved into multiple fragments in stimulated lymphocytes. (A) Proliferation. Purified PBMCs were obtained through Ficoll gradient separation and stimulated with 1 μg/ml of anti-CD3 and 20 U/ml of IL-2 (Anti-CD3) or not (Medium). After 1–4 d at 37°C, thymidine incorporation was determined as in the legend to Fig. 1. (B) Caspase-3 Western blot. Proteins were extracted from the same PBMCs treated in A by cell lysis using a denaturing Laemmli buffer containing 8 M urea. Total proteins extracted from 10⁶ fresh (d0), unstimulated (−), or stimulated cells (+) were separated on SDS-PAGE, and Western blot analysis was performed using a polyclonal rabbit anti–caspase-3 (reference 5). (C) Detection of active caspases by affinity blot. PBMCs stimulated or not for 4 d with anti-CD3, anti-TCR, or PHA in the presence of IL-2 were lysed using a nondenaturing lysis buffer (see Materials and Methods), and lysates were incubated with 5 μM of a biotinylated zVAD peptide, followed by a separation by SDS-PAGE and Western blot analysis using Extra-HRP. (D) Identification of active subunits of caspase-3. PBMCs were activated for 4 d with PHA and IL-2, and cell lysates were incubated with the substrate. Caspase-3 immunoprecipitation was then performed on half of the lysate. Total extract (Lys) or immunoprecipitated caspase-3 (IP) was subjected to Western blot analysis using the anti–caspase-3 antibody (left, WB cas-3) or with Extra-HRP (right, Affinity blot). Results are from one of at least three independent experiments giving similar results. (E) Detection of caspase activity in intact cells. Fresh PBMCs were left untreated (NS) or stimulated (CD3/IL-2) with anti-CD3 antibody and IL-2 for 4 d. A fraction of these activated cells was then incubated for 6 h in anti-Fas–coated plates (CD3/Fas). After a 1-h labeling with the cell-permeable substrate Phiphilux, the cells were analyzed by flow cytometry. Results are from one representative experiment out of three.

Figure 2

Caspase-3 is cleaved into multiple fragments in stimulated lymphocytes. (A) Proliferation. Purified PBMCs were obtained through Ficoll gradient separation and stimulated with 1 μg/ml of anti-CD3 and 20 U/ml of IL-2 (Anti-CD3) or not (Medium). After 1–4 d at 37°C, thymidine incorporation was determined as in the legend to Fig. 1. (B) Caspase-3 Western blot. Proteins were extracted from the same PBMCs treated in A by cell lysis using a denaturing Laemmli buffer containing 8 M urea. Total proteins extracted from 10⁶ fresh (d0), unstimulated (−), or stimulated cells (+) were separated on SDS-PAGE, and Western blot analysis was performed using a polyclonal rabbit anti–caspase-3 (reference 5). (C) Detection of active caspases by affinity blot. PBMCs stimulated or not for 4 d with anti-CD3, anti-TCR, or PHA in the presence of IL-2 were lysed using a nondenaturing lysis buffer (see Materials and Methods), and lysates were incubated with 5 μM of a biotinylated zVAD peptide, followed by a separation by SDS-PAGE and Western blot analysis using Extra-HRP. (D) Identification of active subunits of caspase-3. PBMCs were activated for 4 d with PHA and IL-2, and cell lysates were incubated with the substrate. Caspase-3 immunoprecipitation was then performed on half of the lysate. Total extract (Lys) or immunoprecipitated caspase-3 (IP) was subjected to Western blot analysis using the anti–caspase-3 antibody (left, WB cas-3) or with Extra-HRP (right, Affinity blot). Results are from one of at least three independent experiments giving similar results. (E) Detection of caspase activity in intact cells. Fresh PBMCs were left untreated (NS) or stimulated (CD3/IL-2) with anti-CD3 antibody and IL-2 for 4 d. A fraction of these activated cells was then incubated for 6 h in anti-Fas–coated plates (CD3/Fas). After a 1-h labeling with the cell-permeable substrate Phiphilux, the cells were analyzed by flow cytometry. Results are from one representative experiment out of three.

Figure 2

Caspase-3 is cleaved into multiple fragments in stimulated lymphocytes. (A) Proliferation. Purified PBMCs were obtained through Ficoll gradient separation and stimulated with 1 μg/ml of anti-CD3 and 20 U/ml of IL-2 (Anti-CD3) or not (Medium). After 1–4 d at 37°C, thymidine incorporation was determined as in the legend to Fig. 1. (B) Caspase-3 Western blot. Proteins were extracted from the same PBMCs treated in A by cell lysis using a denaturing Laemmli buffer containing 8 M urea. Total proteins extracted from 10⁶ fresh (d0), unstimulated (−), or stimulated cells (+) were separated on SDS-PAGE, and Western blot analysis was performed using a polyclonal rabbit anti–caspase-3 (reference 5). (C) Detection of active caspases by affinity blot. PBMCs stimulated or not for 4 d with anti-CD3, anti-TCR, or PHA in the presence of IL-2 were lysed using a nondenaturing lysis buffer (see Materials and Methods), and lysates were incubated with 5 μM of a biotinylated zVAD peptide, followed by a separation by SDS-PAGE and Western blot analysis using Extra-HRP. (D) Identification of active subunits of caspase-3. PBMCs were activated for 4 d with PHA and IL-2, and cell lysates were incubated with the substrate. Caspase-3 immunoprecipitation was then performed on half of the lysate. Total extract (Lys) or immunoprecipitated caspase-3 (IP) was subjected to Western blot analysis using the anti–caspase-3 antibody (left, WB cas-3) or with Extra-HRP (right, Affinity blot). Results are from one of at least three independent experiments giving similar results. (E) Detection of caspase activity in intact cells. Fresh PBMCs were left untreated (NS) or stimulated (CD3/IL-2) with anti-CD3 antibody and IL-2 for 4 d. A fraction of these activated cells was then incubated for 6 h in anti-Fas–coated plates (CD3/Fas). After a 1-h labeling with the cell-permeable substrate Phiphilux, the cells were analyzed by flow cytometry. Results are from one representative experiment out of three.

Figure 2

Caspase-3 is cleaved into multiple fragments in stimulated lymphocytes. (A) Proliferation. Purified PBMCs were obtained through Ficoll gradient separation and stimulated with 1 μg/ml of anti-CD3 and 20 U/ml of IL-2 (Anti-CD3) or not (Medium). After 1–4 d at 37°C, thymidine incorporation was determined as in the legend to Fig. 1. (B) Caspase-3 Western blot. Proteins were extracted from the same PBMCs treated in A by cell lysis using a denaturing Laemmli buffer containing 8 M urea. Total proteins extracted from 10⁶ fresh (d0), unstimulated (−), or stimulated cells (+) were separated on SDS-PAGE, and Western blot analysis was performed using a polyclonal rabbit anti–caspase-3 (reference 5). (C) Detection of active caspases by affinity blot. PBMCs stimulated or not for 4 d with anti-CD3, anti-TCR, or PHA in the presence of IL-2 were lysed using a nondenaturing lysis buffer (see Materials and Methods), and lysates were incubated with 5 μM of a biotinylated zVAD peptide, followed by a separation by SDS-PAGE and Western blot analysis using Extra-HRP. (D) Identification of active subunits of caspase-3. PBMCs were activated for 4 d with PHA and IL-2, and cell lysates were incubated with the substrate. Caspase-3 immunoprecipitation was then performed on half of the lysate. Total extract (Lys) or immunoprecipitated caspase-3 (IP) was subjected to Western blot analysis using the anti–caspase-3 antibody (left, WB cas-3) or with Extra-HRP (right, Affinity blot). Results are from one of at least three independent experiments giving similar results. (E) Detection of caspase activity in intact cells. Fresh PBMCs were left untreated (NS) or stimulated (CD3/IL-2) with anti-CD3 antibody and IL-2 for 4 d. A fraction of these activated cells was then incubated for 6 h in anti-Fas–coated plates (CD3/Fas). After a 1-h labeling with the cell-permeable substrate Phiphilux, the cells were analyzed by flow cytometry. Results are from one representative experiment out of three.

Figure 5

Caspase-8 is cleaved in activated lymphocytes, whereas caspase-9 remains as a proenzyme. (A) Caspase-8 is processed in proliferating cells. PBMCs were stimulated (+) or not (−) with anti-CD3 antibody and IL-2 for 1–4 d, and total proteins from 10⁶ cells were analyzed by Western blot using an anti–caspase-8 antiserum. The proenzyme and cleaved subunits are indicated on the right by arrows. (B) Caspase-8, but not caspase-9, is activated early after T cell stimulation. Fresh (0) or anti-CD3–stimulated PBMCs (1–48 h) were lysed, and 10 μg of proteins from each sample was separated by SDS-PAGE and analyzed for caspase-3, -8, and -9 processing by Western blot, as described above. Results are representative of two independent experiments.

Figure 5

Caspase-8 is cleaved in activated lymphocytes, whereas caspase-9 remains as a proenzyme. (A) Caspase-8 is processed in proliferating cells. PBMCs were stimulated (+) or not (−) with anti-CD3 antibody and IL-2 for 1–4 d, and total proteins from 10⁶ cells were analyzed by Western blot using an anti–caspase-8 antiserum. The proenzyme and cleaved subunits are indicated on the right by arrows. (B) Caspase-8, but not caspase-9, is activated early after T cell stimulation. Fresh (0) or anti-CD3–stimulated PBMCs (1–48 h) were lysed, and 10 μg of proteins from each sample was separated by SDS-PAGE and analyzed for caspase-3, -8, and -9 processing by Western blot, as described above. Results are representative of two independent experiments.

Figure 3

Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4⁺, CD8⁺, CD45RO⁺, or CD45RA⁺ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19⁺ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.

Figure 3

Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4⁺, CD8⁺, CD45RO⁺, or CD45RA⁺ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19⁺ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.

Figure 3

Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4⁺, CD8⁺, CD45RO⁺, or CD45RA⁺ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19⁺ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.

Figure 4

Selective downstream caspase processing in activated lymphocytes. (A) Caspase-6 and -7 are also processed after TCR triggering. PBMCs were stimulated (+) or not (−) with anti-CD3 antibody and IL-2 for 1–4 d, and total proteins from 10⁶ cells were separated by SDS-PAGE and analyzed by Western blot using anti–caspase-6 or -7 antisera. The proenzymes and cleaved subunits are indicated on the right by arrows. (B) All caspases are not processed in activated cells. Day 4 unstimulated (−) or anti-CD3/IL-2 stimulated (+) PBMCs were analyzed for caspase-1, -2, or -4 processing by Western blot, as in A.

Figure 4

Selective downstream caspase processing in activated lymphocytes. (A) Caspase-6 and -7 are also processed after TCR triggering. PBMCs were stimulated (+) or not (−) with anti-CD3 antibody and IL-2 for 1–4 d, and total proteins from 10⁶ cells were separated by SDS-PAGE and analyzed by Western blot using anti–caspase-6 or -7 antisera. The proenzymes and cleaved subunits are indicated on the right by arrows. (B) All caspases are not processed in activated cells. Day 4 unstimulated (−) or anti-CD3/IL-2 stimulated (+) PBMCs were analyzed for caspase-1, -2, or -4 processing by Western blot, as in A.

Figure 7

Substrate processing during T cell activation is caspase dependent and is different than after apoptosis. (A) Substrate cleavage is caspase dependent. Fresh PBMCs were stimulated for 48 h with anti-CD3 and IL-2 in the presence or absence of 100 μM of zVAD. An aliquot was conserved for quantitation of DNA synthesis by thymidine incorporation, and cells were lysed in denaturing buffer for Western blot analysis of PARP, Wee1, lamin B, and caspase-3 cleavage. Black arrows indicate the full-length proteins, and white arrows designate the cleaved forms. (B) DFF45 cleavage is detected only after apoptosis induction. PBMCs activated for 4 d with anti-CD3 and IL-2 were placed in anti-Fas–coated plates and cultured for 6 h at 37°C. Apoptosis was assessed by AV/PI staining, and the cells were subjected to Western blot analysis using the DFF45 antiserum. Results are from one representative experiment out of three.

Figure 6

Caspase activation results in selective substrate cleavage. Total proteins from 10⁶ unstimulated (−) or anti-CD3–activated (+) PBMCs were obtained at different time points (day 1 to day 4), separated by SDS-PAGE, and analyzed by Western blot using an anti-PARP, DFF45, or caspase-3 antiserum, or an anti-Wee1 mAb. As a control for caspase-mediated cleavage, total proteins from 10⁶ Jurkat cells cultured on uncoated or anti-Fas–coated plates (M3: 20 μg/ml) for 4 h were subjected to the same treatment as PBMCs, and results are shown on the right. The 45-kD strong band observed with the anti-Wee1 antibody throughout the kinetic results from a cross-reactivity observed in some tissues with this antibody, according to the manufacturer. These experiments were performed three times, and gave similar results with different PBMC donors.

Figure 8

Caspase and substrate cleavage occur in nonapoptotic cells. (A) Caspase-3 and PARP processing are detected in AV⁻ cells. PBMCs were stimulated (+) or not (−) with anti-CD3 antibody and IL-2 for 4 d, and stained for 10 min with FITC-conjugated AV. Living (AV⁻) and dying (AV⁺) activated lymphocytes were purified by flow cytometric cell sorting. Proteins from sorted cells were subjected to Western blot analysis for caspase-3 and PARP. Both antibodies were used on the same membrane. One representative experiment of three is shown. (B) Caspase-6 and -7 and the caspase substrates are processed in viable sorted cells. Unstimulated and unsorted PBMCs (lane 1) or activated and AV⁻ sorted cells (lane 2) were lysed in Laemmli buffer. Proteins from 5 × 10⁵ cells were analyzed by Western blot for PARP, DFF45, Wee1, caspase-6, or caspase-7 processing using the appropriate antisera. Arrows on the right indicate the full-length form of each protein. Results are representative of two independent experiments.