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Cleavage of Anti-Apoptotic Bcl-2 Family
Members after TCR Stimulation Contributes
to the Decision between T Cell Activation and
Apoptosis
Alan D. Guerrero, Robert L. Welschhans, Min Chen and Jin
Wang
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2013; 190:168-173; Prepublished online 30
November 2012;
doi: 10.4049/jimmunol.1201610
http://www.jimmunol.org/content/190/1/168
The Journal of Immunology
Cleavage of Anti-Apoptotic Bcl-2 Family Members after TCR
Stimulation Contributes to the Decision between T Cell
Activation and Apoptosis
Alan D. Guerrero, Robert L. Welschhans, Min Chen, and Jin Wang
T
he clearance of significantly expanded T cells after an immune response is critical for the maintenance of lymphocyte homeostasis and immune tolerance (1–4). At
the peak of immune responses, activated T cells may undergo
activation-induced cell death (AICD) that involves the induction
of Fas-mediated apoptosis through autocrine secretion of Fas ligand (5, 6). Genetic disruption of the interaction between the
death receptor Fas and Fas ligand results in the accumulation of
lymphoid cells in systemic autoimmune and lymphoproliferative
disease (7–12). Mutations in Fas or Fas ligand have also been
shown to cause defective AICD of T cells in the autoimmune
lymphoproliferative syndrome in humans (2). AICD is important for limiting the scope of Ag-specific T cell activation and
protecting against the development of autoimmunity (2).
Compound deficiencies of Fas and a proapoptotic Bcl-2 family
member, Bim, have been shown to induce more severe lymphocyte
accumulation and autoimmune manifestations in mice (13–15). This
suggests that death receptor–mediated extrinsic apoptosis and mitochondrion-dependent intrinsic apoptosis pathways are both important for the regulation of lymphocyte homeostasis and immune
tolerance. It has been shown that loss of Bim causes defective AICD
in T cells (16). An activating mutation of NRAS can cause the
development of autoimmune lymphoproliferative syndrome with
downregulation of Bim and T cell expansion (17). Therefore, defects
in the intrinsic apoptosis pathway may lead to decreased AICD in
human autoimmune diseases.
Department of Pathology and Immunology, Baylor College of Medicine, Houston,
TX 77030
Received for publication June 11, 2012. Accepted for publication October 24, 2012.
This work was supported by National Institutes of Health Grants R01GM087710 and
R01AI074949 (to J.W.) and R01DK083164 (to M.C.).
Address correspondence and reprint requests to Dr. Jin Wang, Department of Pathology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030. E-mail address: [email protected]
Abbreviations used in this article: AICD, activation-induced cell death; PI, propidium
iodide; siRNA, small interfering RNA.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201610
Anti-apoptotic Bcl-2 family proteins, such as Bcl-2 and Bcl-xL,
maintain the integrity of the mitochondrial outer membrane by
inhibiting proapoptotic Bax and Bak (18, 19). During apoptosis,
Bcl-2 and Bcl-xL can be cleaved by caspases and lose their antiapoptotic functions (20–22). Bcl-2 and Bcl-xL with mutations of
the cleavage sites are more efficient in inhibiting apoptosis than
their wild-type counterparts, suggesting that caspase-induced cleavage indeed inactivates the anti-apoptotic functions of Bcl-2 and
Bcl-xL (20–22). Notably, the loss of mitochondrial membrane
potential (DCm) during cell death is inhibited when caspase-9, or
caspase-3 and caspase-7, are deficient (23–27). This suggests an
important role for caspases in feedback disruption of mitochondria
to promote apoptosis. We have found that dimerization of caspase9 induces the activation of a downstream caspase cascade through
caspase-3 (28) and triggers a feedback loop that promotes mitochondrial disruption by cleavage of anti-apoptotic Bcl-2 family
proteins (22). However, whether caspase-mediated cleavage of
anti-apoptotic molecules plays a physiological role in the execution of apoptosis in T cells is not known.
Although engagement of the TCR on prestimulated T cells can
trigger AICD, this usually does not lead to apoptosis of all T cells.
A portion of the T cells can survive and become further activated.
What decides whether a T cell lives or dies in response to TCR reengagement is not clear. We detected cleavage of Bcl-2 or Bcl-xL
and simultaneous upregulation of Bcl-xL in T cells after TCR reengagement. Although cleavage of Bcl-2 and Bcl-xL was detected
in apoptotic T cells, upregulation of Bcl-xL was found in surviving
T cells. Our data suggest that cleavage of anti-apoptotic Bcl-2 or
Bcl-xL can tip the balance between cell activation and cell death
after T cells re-encounter the Ag.
Materials and Methods
T cell culture and flow cytometry
Splenocytes from wild-type or lpr mice on the C57BL/6 background were
cultured in the presence of 5 mg/ml Con A in RPMI complete medium for
48 h, followed by culture with 100 U/ml IL-2 for 48 h. For small interfering RNA (siRNA) silencing, mouse splenocytes were stimulated with 5
mg/ml Con A for 48 h, followed with 100 U/ml IL-2 for 48 h, with the
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Engagement of the TCR induces activation-induced cell death (AICD) of T cells that have been previously stimulated. However,
a portion of these T cells can survive and undergo further activation. The molecular mechanism that decides whether a T cell will
live or die after TCR re-engagement is unclear. We found that cross-linking of TCR in preactivated primary mouse T cells led to
the cleavage of anti-apoptotic Bcl-2 and Bcl-xL in dying cells. Cleavage-resistant Bcl-2 and Bcl-xL were more efficient than their
wild-type counterparts in the inhibition of apoptosis in primary mouse T cells and in the H9 T cell line after TCR cross-linking.
In contrast, the surviving T cells after TCR re-engagement displayed upregulation of Bcl-xL, and knockdown of Bcl-xL promoted
AICD. This indicates that caspase-mediated cleavage of anti-apoptotic Bcl-2 or Bcl-xL facilitates AICD in T cells, whereas upregulation of Bcl-xL promotes T cell survival and allows further T cell activation. Our data suggest that cleavage of anti-apoptotic Bcl-2
and Bcl-xL contributes to the decision between T cell activation and apoptosis after TCR re-engagement. The Journal of
Immunology, 2013, 190: 168–173.
The Journal of Immunology
FIGURE 1. Engagement of TCR on preactivated
T cells leads to both cell activation and apoptosis. (A)
T cells stimulated with Con A and IL-2 were cultured
for 24 h in tissue culture plates with or without 1 mg/
ml anti-CD3 coating. The cells were stained with 10
mg/ml PI and analyzed by flow cytometry. Forward
scattering (FSC) versus side scattering (SSC) or FSC
versus PI staining was plotted. (B) T cells without
(control) or with anti-CD3 stimulation as in (A) were
stained with various cell surface markers and analyzed
by flow cytometry. Intracellular staining of IFN-g was
also performed. The expression of different surface
markers and intracellular IFN-g on live CD4 + or
CD8 + T cells was plotted. (C) T cells stimulated
with anti-CD3 were incubated with DEVD-FITC,
followed by staining with PE–annexin V. (D) T cells
stimulated with anti-CD3 and incubated with DEVDFITC were stained with PE–anti-Fas or PE–anti-Fas
ligand, followed by flow cytometry analyses. Cells
positive or negative for DEVD staining were gated,
and the expression of Fas or Fas ligand was plotted.
(E) T cells as in (A) were cultured on plates coated
with different concentrations of anti-CD3 for 24 h.
Percentages of cell death were plotted. (F) T cells as
in (A) were cultured with different concentrations of
anti-CD3 for 6 h in tissue culture plates. The cells
were incubated with DEVD-FITC and analyzed by
flow cytometry.
Western blotting
Cells were lysed in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl,
1 mM EDTA, 1% Triton X-100, 13 protease inhibitor mixture from Roche,
2 mM zVAD-fmk) and used for Western blotting as described (22, 28). The
following primary Abs were used for Western blotting: mouse mAbs to
Bcl-2 (BD Biosciences, San Jose, CA) and anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal Ab to Bcl-xL (Cell
Signaling Technology, Danvers, MA). The blots were then incubated with
HRP-conjugated secondary Abs (Southern Biotech, Birmingham, AL),
followed by development with the SuperSignal West Dura substrate
(Thermo Scientific, Waltham, MA).
Transduction of T cells
cDNA for wild-type or cleavage-resistant Bcl-2 or Bcl-xL was fused to HA
and cloned into a lentiviral plasmid, pTRIP, under the control of a CMVpromoter and followed by IRES-GFP (30). Recombinant pseudotyped
lentiviral vector was generated by cotransfecting pTRIP vectors with
pCMV-d R8.9 encoding gag and pol, and pMD.G encoding env into
293T cells as described (31). The viral supernatants from 24 to 72 h after
transfection were centrifuged at 140,000 3 g for 2 h, and viral pellets were
resuspended in RPMI complete medium and titrated using NIH3T3 cells as
described (30). Mouse T cells after stimulation with 5 mg/ml Con A were
exposed to five multiplicities of infection of the lentiviral vectors and 8 mg/
ml polybrene, followed by centrifugation at 1000 3 g for 45 min at room
temperature. After culture in the presence of 100 U/ml IL-2 at 37˚C for
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presence of 1 mM Acell bcl-xL or nontargeting siRNA in Acell siRNA
delivery media (Dharmacon) throughout the culture. For anti-CD3–induced cell death, T cells (2.5 3 105/ml) were cultured in 96-well flatbottom plates with or without coating with 1 mg/ml anti-CD3 (2C11; BD
Biosciences) for 24 h. The percentage of cell loss was quantitated by flow
cytometry as described (22, 28). T cells were also stained with FITC–antiCD4, cychrome–anti-CD8, and PE-conjugated Abs to CD25, Fas, or
ICAM (BD Biosciences), followed by flow cytometry. Intracellular staining for IFN-g was performed as described (29). For Western blot analyses,
T cells were cultured in 24-well plates with or without anti-CD3 coating
(2 3 106 cell/well) for 8 or 24 h in the presence of 10 mM MG132 during
the last 4 h of culture. The cells were lysed for Western blot analyses. In
some experiments, T cells after 8-h culture on anti-CD3 plates were
stained with FITC–annexin V, followed by sorting for annexin V+ dying
cells or annexin V2 live cells. The sorted cells were used for Western blot
analyses.
Mouse T cells stimulated with Con A and IL-2 as above were cultured
with or without anti-CD3 stimulation for 24 h. Live cells were isolated by
Ficoll-gradient separation and washed to remove IL-2. The cells were then
cultured in the absence or presence of 100 U/ml for 24 h. IL-2 withdrawal–
induced cell death was determined. Cell lysates were also prepared for
Western blot analyses of Bcl-2 and Bcl-xL.
Human H9 T cells were transfected with HA-tagged wild-type or
cleavage-resistant Bcl-2 or Bcl-xL as described (22). The cells were cultured in 96-well plates coated with 10 mg/ml anti-CD3 (UCHT1; BioLegend) for 24 h, followed by Western blot or cell death assays.
169
170
FIGURE 2. Cleavage of Bcl-2/Bcl-xL and AICD in wild-type and Fasdeficient T cells. (A) T cells derived from wild-type (WT) or Fas-deficient
lpr mice were cultured with or without anti-CD3 for 24 h. Anti-CD3–
induced cell death was quantitated by PI exclusion. **p , 0.01 (n = 3). (B)
T cells as in (A) were cultured with or without anti-CD3 for 8 h, followed
by cell lysis and Western blot analyses of Bcl-2 and Bcl-xL.
12 h, T cells were washed and cultured in fresh medium containing 100 U/ml
IL-2 for another 36 h. T cells were then added to tissue culture plates with
or without anti-CD3 coating and cultured for 24 h. The loss of GFP+
transduced T cells was determined by flow cytometry.
Cross-linking of TCR on prestimulated T cells leads to both
T cell apoptosis and T cell activation
It is well established that stimulation of T cells sensitizes T cells
to apoptosis (32, 33). Although re-engagement of TCR on preactivated T cells led to significant induction of cell death (Fig.
1A), a portion of these T cells survived as shown by the exclusion
of the staining with propidium iodide (PI) after re-engagement of
the TCR (Fig. 1A). These surviving T cells displayed increased
cell size by forward scattering (Fig. 1A). They also retained surface expression of Fas, but showed increased CD25 and ICAM-1
after anti-CD3 stimulation (Fig. 1B). In addition, anti-CD3 also
induced the expression of IFN-g in the surviving CD4+ and CD8+
FIGURE 3. Cleavage of Bcl-2 and Bcl-xL during AICD in H9 cells. (A)
H9 cells were cultured on anti-CD3–coated plates for 24 h or left untreated. Cells were lysed for Western blotting to detect Bcl-2 and Bcl-xL.
(B) Western blotting for H9 cells stably expressing wild-type or cleavageresistant Bcl-2–HA or Bcl-xL–HA. (C and D) H9 cells as in (B) were
cultured on anti-CD3–coated plates for 24 h. The cells were used for
Western blot by probing with anti-HA (C) or used for quantitation of cell
death (D). Arrows in (A) and (C) denote caspase-processed fragments.
Mean 6 SDs from triplicate wells are displayed. **p , 0.01 (n = 3).
T cells (Fig. 1B). These data suggest that restimulation of T cells
via the TCR not only triggers apoptosis in some T cells but also
induces activation in the surviving T cells with increased expression of cytokines, cytokine receptors, and adhesion molecules
that may allow better responses to cytokines and interactions with
other cell types. However, what intracellular signaling events
might decide whether a T cell undergoes apoptosis or survives
and becomes further activated after TCR re-engagement is not
clear.
We next compared the surviving and dying cells after TCR
cross-linking. As expected, the dying cells positive for annexin V
staining also displayed caspase activation by cleavage of DEVDFITC (Fig. 1C). T cells with or without caspase activation expressed similar levels of Fas or Fas ligand after TCR cross-linking
(Fig. 1D), suggesting that cell death or cell activation is not determined by differential expression of these apoptosis molecules
on the cell surface. We also observed that increased concentrations of anti-CD3 led to more cell death (Fig. 1E) and caspase
activation (Fig. 1F). This suggests that stronger intensity of TCR
cross-linking favors cell death over cell activation, whereas weaker
initial TCR stimulation may lead to T cell activation without triggering cell death.
Cleavage of Bcl-2 and Bcl-xL after TCR cross-linking in
primary T cells
We have previously observed cleavage of Bcl-2 and Bcl-xL in H9
T cell lines after chemically induced dimerization of a transfected
caspase-9 (22). Cleavage-resistant Bcl-2 and Bcl-xL also provide
resistance to etoposide-induced cell death in H9 cells (22).
However, whether cleavage of Bcl-2 and Bcl-xL plays a role in
FIGURE 4. Cleavage-resistant Bcl-2 and Bcl-xL inhibit AICD in
T cells. Inhibition of anti-CD3–induced cell death in primary mouse
T cells by cleavage-resistant Bcl-2 or Bcl-xL. (A) Primary mouse T cells
were transduced with retroviral vector, or the vector expressing wild-type
(WT) or cleavage-resistant Bcl-2-HA or Bcl-xL-HA. The transduced cells
were used for Western blot analyses. (B) The cells transduced as in (A)
were cultured for 24 h on plates with or without anti-CD3 coating. The loss
of GFP+ transduced cells after anti-CD3 stimulation was quantitated. *p ,
0.05, **p , 0.01 (n = 3).
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Results
CLEAVAGE OF Bcl-2 AND Bcl-xL IN AICD
The Journal of Immunology
171
primary T cell apoptosis is unknown. It is well established that Fas
ligand–induced apoptosis through Fas mediates AICD in T cells in
an autocrine fashion. As expected, TCR cross-linking induced
apoptosis in wild-type mouse T cells stimulated with Con A and
IL-2, whereas Fas-deficient lpr T cells were resistant to such
AICD (Fig. 2A). Bcl-2 and Bcl-xL were also processed in wildtype T cells after 8 h of stimulation with anti-CD3 (Fig. 2B).
However, the processing of Bcl-2 and Bcl-xL was lacking in Fasdeficient T cells (Fig. 2B), indicating a critical role for Fas-mediated
caspase activation in the cleavage of Bcl-2 and Bcl-xL. However,
these experiments do not distinguish whether the processing of
Bcl-2 and Bcl-xL contributes to cell death or is merely a consequence of cell death.
Inhibition of AICD in H9 T cells after TCR cross-linking by
Bcl-2D/A or Bcl-xLD/A
We detected cleavage of Bcl-2 and Bcl-xL in H9 T cells after TCR
cross-linking (Fig. 3A). To investigate whether cleavage of Bcl-2
and Bcl-xL contributed to the induction of cell death in H9 cells,
we expressed wild-type and cleavage-resistant Bcl-2 and Bcl-xL
(22) with aspartate to alanine substitution at the caspase cleavage
sites (Bcl-2D/A or Bcl-xLD/A) in H9 cells (Fig. 3B). As expected,
TCR cross-linking led to the cleavage of wild-type but not
cleavage-resistant Bcl-2 or Bcl-xL (Fig. 3C). TCR cross-linking
also efficiently induced AICD in H9 cells (Fig. 3D). Bcl-2D/A or
Bcl-xLD/A, but not wild-type Bcl-2 or Bcl-xL, significantly inhibited AICD in H9 cells (Fig. 3D). This is consistent with the
possibility that cleavage of Bcl-2 and Bcl-xL facilitates the induction of AICD in H9 T cells.
Cleavage of Bcl-2/Bcl-xL and upregulation of Bcl-xL occur in
apoptotic and activated T cells, respectively
We detected cleaved forms of Bcl-2 and Bcl-xL after stimulation
of T cells for 8 h (Fig. 5A). After 24 h of anti-CD3 stimulation,
however, the processed Bcl-2 and Bcl-xL were decreased (Fig.
5A). Full-length Bcl-2 was decreased, whereas full-length Bcl-xL
was significantly increased (Fig. 5A). It is possible that the processing of Bcl-2 and Bcl-xL contributes to rapid T cell apoptosis,
resulting in the degradation of cleaved Bcl-2 and Bcl-xL in apoptotic T cells. In contrast, TCR cross-linking may induce upregulation of Bcl-xL in the surviving cells. It is possible that cleavage
of Bcl-2 and Bcl-xL induces apoptosis, whereas upregulation of
Bcl-xL promotes cell survival to allow further cell activation. To
test this possibility, we sorted live and apoptotic cells after TCR
cross-linking. After TCR ligation, live and dying T cells were
distinguished by staining with FITC–annexin V. We detected
cleavage of Bcl-2 and Bcl-xL in dying T cells (Fig. 5B). In contrast, the surviving T cells showed no processing of Bcl-2 or BclxL (Fig. 5B). This supports the conclusion that cleavage of Bcl-2
and Bcl-xL contributes to cell death. We also observed upregu-
Inhibition of AICD in primary T cells after TCR cross-linking
by Bcl-2D/A or Bcl-xLD/A
To determine the effects of Bcl-2 and Bcl-xL on AICD in primary
T cells, we generated retroviral vectors expressing either wild-type
or cleavage-resistant Bcl-2 or Bcl-xL followed by a GFP reporter
gene (Fig. 4A). We observed that Bcl-2D/A and Bcl-xLD/A were
more efficient than their wild-type counterparts in the inhibition
of AICD in T cells (Fig. 4B). This supports the conclusion that
cleavage of Bcl-2 and Bcl-xL facilitates the induction of AICD in
primary T cells.
FIGURE 6. T cells surviving anti-CD3 stimulation are susceptible to
IL-2 withdrawal–induced cell death. (A) Mouse T cells stimulated with
Con A and IL-2 were cultured with or without anti-CD3 stimulation. Live
cells were isolated and cultured in the absence or presence of IL-2 for 24 h.
Percentage of cell loss induced by IL-2 withdrawal was quantitated in
T cells with or without anti-CD3 pretreatment. (B) T cells with or without
anti-CD3 pretreatment were cultured in the presence or absence of IL-2
as in (A) and used for Western blot analyses.
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FIGURE 5. Cleavage of Bcl-2 and Bcl-xL is correlated with cell death, whereas upregulation of these molecules is correlated with T cell activation. (A)
Mouse T cells stimulated with Con A and IL-2 were cultured with or without anti-CD3 for 8 or 24 h. The cells were lysed for Western blot analyses. (B)
Mouse T cells stimulated with Con A and IL-2 were cultured on anti-CD3–coated plates for 8 h. The cells were stained with FITC–annexin V. Cells positive
or negative for annexin V staining were sorted (left panel). The cells were lysed and used for Western blot analyses (right panel). The intensities of protein
bands were determined using ImageJ. The ratios of intensities of full-length Bcl-2 versus tubulin: 0.895 (no anti-CD3), 0.966 (anti-CD3 live), and 0.650
(anti-CD3 dying); ratios for full-length Bcl-xL versus tubulin: 0.151 (no anti-CD3), 0.696 (anti-CD3, live), and 0.526 (anti-CD3, dying). (C) Mouse T cells
were cultured in siRNA delivery medium in the presence of Acell bcl-xL or nontargeting control siRNA (Dharmacon) and stimulated with Con A and IL-2.
The cells were then cultured with or without anti-CD3 stimulation. The cells were lysed for Western blot after stimulation for 8 h (upper panel). The ratios
of full-length Bcl-xL versus tubulin band intensities: 1.354 (control, no anti-CD3), 4.386 (control, anti-CD3), 0.208 (Bcl-xL siRNA, no anti-CD3), and
0.733 (Bcl-xL siRNA, anti-CD3). Percentages of cell death after anti-CD3 stimulation for 24 h were also quantitated (lower panel). **p , 0.01.
172
lation of Bcl-xL in the sorted live cells after anti-CD3 stimulation
(Fig. 5B). To determine whether upregulation of Bcl-xL contributes to T cell survival after TCR cross-linking, we silenced bcl-xL
in T cells using the Acell siRNA delivery method (Dharmacon)
with ∼80% knockdown of Bcl-xL protein expression (Fig. 5C,
upper panel). We found that suppression of Bcl-xL led to significant increases of AICD in mouse primary T cells (Fig. 5C, lower
panel). This suggests that the upregulation of Bcl-xL indeed leads
to better T cell survival after TCR cross-linking.
Surviving T cells after TCR re-engagement are susceptible to
IL-2 withdrawal–induced cell death
Discussion
Our results suggest that caspase-mediated cleavage of Bcl-2 and
Bcl-xL can tip the balance between life and death in T cells
reactivated through their TCR. Cross-linking of the TCR led to
both cleavage and upregulation of Bcl-2 and Bcl-xL in activated
primary T cells. Western blot analyses of sorted live and apoptotic
T cells after TCR cross-linking showed the cleavage of Bcl-2 and
Bcl-xL in apoptotic cells. Upregulation of Bcl-xL, but not cleavage
of Bcl-2 or Bcl-xL, was found in live cells. Retroviral transduction
of T cells showed that cleavage-resistant Bcl-2 and Bcl-xL were
efficient in inhibiting AICD in T cells. Our data suggest that reengagement of the TCR leads to a certain level of initial caspase
activation, resulting in cleavage of Bcl-2 and Bcl-xL and amplification of mitochondrial disruption to promote cell death.
Fas plays an important role in activation-induced cell death in
T cells (5). Recent studies suggest mitochondrion-dependent cell
death regulated by Bim is also involved in inducing AICD in
T cells (16). As expected, Fas-deficient T cells were resistant to
AICD. Cleavage of Bcl-2 and Bcl-xL was not detectable in Fasdeficient T cells. We have previously observed that caspase-3 is the
major effector caspase for the processing of Bcl-2 and Bcl-xL (22).
The activation of mitochondrion-dependent apoptosis by cleavage
of Bcl-2 and Bcl-xL is not merely the consequence of apoptosis,
but rather is likely to contribute to AICD in T cells. Consistent with
this possibility, cleavage-resistant Bcl-2 and Bcl-xL both showed
better protection of the H9 T cell line or primary T cells against
AICD. This supports our conclusion that cleavage of Bcl-2 and
Bcl-xL helps to tip the balance between life and death for T cells
re-encountering Ags. We found that more cells displayed caspase
activation and underwent cell death with increased concentrations
of anti-CD3 (Fig. 1C, 1D). This suggests that stimulation over a
certain threshold will favor the induction of cell death. We propose
that a certain level of initial caspase activation leads to cleavage of
Bcl-2 and Bcl-xL, which leads to amplification of mitochondrial
disruption, more caspase activation, and cell death.
AICD is likely to take place at the peak of immune responses
when T cells are activated and Ags are still abundant. AICD is
therefore potentially important for limiting the scope of Ag-specific
immune responses. This may help prevent uncontrolled T cell
expansion and the induction of inflammatory and autoimmune
responses. In addition to triggering cell death in activated T cells,
TCR engagement also upregulated Bcl-xL in T cells that survived.
siRNA silencing experiments suggest that upregulation of Bcl-xL is
critical for suppressing mitochondrion-dependent apoptosis to
protect T cells from AICD (Fig. 5C). This may allow some T cells
to survive longer in the presence of Ag stimulation.
After the clearance of Ags, lack of active immune responses
likely causes the decrease in T cell stimulation and cytokine
production. T cells that have survived the restimulation from TCR
may subsequently undergo cell death due to lack of cytokines.
Different from AICD, IL-2 withdrawal did not trigger the cleavage
of anti-apoptotic Bcl-2 family members, but rather caused the
downregulation of Bcl-xL. The contribution of cleavage of Bcl-2
or Bcl-xL to AICD suggests that different cell death pathways
interact to make the decision between life and death for T cells
during immune responses. This may help to fine-tune the duration
and magnitude of immune responses to various bacterial or viral
infections that have different Ags and infectious cycles.
Acknowledgments
We thank Gustavo Amarante-Mendes for discussions.
Disclosures
The authors have no financial conflicts of interest.
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