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Induction of apostosis following antigen presentation by T cells: Anergy and apoptosis are two separate phenomena
ELSEVIER
Induction of Apoptosis Following Antigen Presentation Anergy and Apoptosis Are Two Separate Phenomena R.G. Hargreaves, and G. Lombardi
N.J. Borthwick,
M.S.G. Montani,
T
WO of the mechanisms involved in peripheral tolerance, referred to as anergy and apoptosis, act by silencing T cells or by removing them from the system altogether, respectively. Anergy has been reported in many systems and often involves the recognition of peptide/MHC complexes in the absence of cell division. This may result from the absence of necessary costimulatory signals’ or through the recognition of partial agonist peptides.’ Apoptosis can be induced in cells expressing Fas (CD9.5) upon encounter with cells expressing Fas-ligand, such as activated T cells, a process known as activation-induced cell death (AICD).3 The relationship between anergy and apoptosis is still a matter of debate with some groups, suggesting that they are overlapping phenomena.4 Therefore, to clarify this relationship, we have used an in vitro system of T cell-mediated antigen presentation.5 Activated human T cells express MHC class II molecules and thereby acquire the capacity to present antigen to CDCpositive T cells. We have previously reported that antigen presentation by T cells leads to the delivery of a partial signal culminating in a non-responsive state.6,7 Here we report that a minority of T cell clones undergo apoptosis in response to antigen presentation by T cells. Cell death could be prevented by inhibiting the interaction of Fas with its ligand; however, the cells that were rescued from death were observed to be anergic. These data suggest that apoptosis and anergy are two distinct outcomes of partial T cell signaling. MATERIALS Monoclonal
AND
E. Piccolella,
P. Carmichael,
by T Cells:
R.I. Lechler, A.N. Akbar,
(HA) peptide 307 to 319 and restricted by HLA-DRB1*0701, were maintained on a 2-weekly cycle of stimulation using peptide-pulsed, autologous PBMC plus rIL-2. Two Stage Cultures
for Tolerance
Induction
T cell clones were washed extensively and resuspended in RPM1 containing 10% Human Serum (HS) and cultured in the presence or absence of peptide HA307-319 (kindly donated by Dr H. Stauss, RPMS). In Fas blocking experiments, the anti-Fas antibody was added at a concentration of 10 FgjrnL for the duration of this incubation period. Following overnight incubation, the cells were washed extensively, and the viable cells plated out into 96-well plates at a concentration of 1 X 10“ cells/well. Peptide-pulsed, X-irradiated (120 Gy) B-LCL’s were added to the triplicate cultures at a concentration of 3 X lo4 cells/well and the plates cultured for 48 hours at 37°C. 3H-TdR (1 @/well) was added and the plates were harvested 18 hours later for counting by liquid scintillation spectrophotometry. Counts are expressed as mean corrected (A) cpm for triplicate cultures. Standard errors were routinely
of Viable Cells
Viable cells were distinguished by their forward angle scatter and 90”side scatter profiles and were counted using a CytoronAbsolute flow cytometer (Ortho Diagnostics Ltd, High Wycombe, Bucks, UK) as described previously.“ Cells undergoing apoptosis were identified morphologically in cytospin preparations stained with May-Grunwald Giemsa (MGG, Merk Ltd, Poole, Dorset, UK). A total of at least 5 fields per sample (>500 cells) were counted by two independent investigators. Apoptosis was also measured by cell cycle analysis using the flow cytometer, apoptotic cells characteristically appearing as a sub-GO/G1 peak after propidium iodide (PI) staining.
METHODS
Antibodies
Anti-CD95 Monoclonal Antibodies (rnAb) M3 was a kind gift from Immunex Research and Development Corporation (Seattle, USA). MR6 IgGl control was donated by Professor Mary Ritter, (Department Immunology, RPMS). Cell Lines and Clones
Epstein Barr virus (EBV)-transformed lymphoblastoid B cell lines (B-LCL’s), obtained from the 10th International Histocompatibility Workshop, were cultured in RPM1 1640 medium supplemented with 10% FCS. Clones 7P.24 and 7P.41, specific for Influenza Haemagglutinin
0041-1315/97/$17.00 PII SO041 -1345(96)00433-2
RESULTS Recognition
AND
DISCUSSION
of Peptide
on T-APC
Can Lead to Apoptosis
Two T cell clones specific for HA307-319 and restricted by DR7 were used in these experiments. Neither of these T cell clones proliferated when cultured with soluble peptide From Department Immunology, RPMS, Hammersmith Hospital, London, UK (PH., P.C., R.L., G.L.); and Department Clinical Immunology, RFHMS, Pond St., London, UK (N.B., A.A.); University of “La Tuscia”, Viterbo, Italy (M.M.); and Department Cellular and Developmental Biology, University “La Sapienza”, Rome, Italy (E.P.)
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1102
Transplantation
Proceedings,
29, 1102-l 104 (1997)
ANERGY AND APOPTOSIS:
TWO SEPARATE PHENOMENA
1103
Table 1. Recognition of Antigen Displayed by T Cells Can Induce Apoptosis Overnight
Condition
Go/G1
7P.41
medium
84.33
7P.41
peptide
(%)
S/GZ/M
(%)
63.42
8.15
88.17
10.61
7P.24
27.01
6.04
Clones
were
HA307-319
cultured
peptide.
with Propidium
overnight
either
Iodide.
Cells were analysed
7~41
(%)
28.97 1.38
in medium
The T cells were then washed
1 a)
1.23
7P.24medium peptide
Apoptotic
14.36
40
69.86 or
with
1 pg/mL
and subsequently
by Flow cytometry
of
stained
and assessed
for
DNA content.
in the absence of added accessory cells. Notably, it was observed that T cells incubated overnight with increasing doses of peptide (0.01 to 100 &mL) suffered a loss in viability. In order to investigate the nature of this cell death, T cell clones were incubated with either medium or 1 pg/rnL of peptide and stained the following day with Propidium Iodide. The results are shown in Table 1. The majority of clone 7P.41 were in GOIGl, with 29% of the cells hypodiploid following overnight incubation with 1 pg/mL of peptide. In contrast, only 27% of clone 7P.24 were in GO/G1 and almost 70% of the cells expressed hypodiploid nuclei, characteristic of apoptotic cells. These results demonstrate that the dramatic reduction in cell recovery in clone 7P.24 following recognition of peptide on T cells is due to apoptosis. PCR and FACS analysis of these clones demonstrated that the levels of Fas appeared to be identical and was constitutively expressed. Fas-ligand mRNA, however, appeared after 3 hours of incubation with peptide and subsided by 12 hours coinciding with a loss in cell viability. Interestingly, the levels of Fas-L mRNA in clone 7P.24 appeared to be higher than those induced in clone 7P.41 upon recognition of peptide presented by T cells. This was further confirmed using Jurkat T cells as targets in a Fas/Fas-ligand mediated killing assay (data not shown). This difference in expression may account for the differential ability of peptide presentation to induce apoptosis in the two T cell clones.
Table 2. Blocking Fas/Fas-L Interactions Prevents T:T Presentation-Induced Apoptosis 7P.24 OvernIght
Medium Medium Medium Peptide Peptide Peptide
Condition
+ anti-Fas + MR6 + anti-Fas + MR6
(Percent
Viable
100 100 100 12.4 94 14.4
mea
I
pepr
, ,
med
I
anti-Fas
E
e d
PePt
‘01
b) 7~24
40-
30-
20-
10-
0-r
mea
I
II
pept
_
med anti-Fas
overnight
condition
Fig 1. Prevention of Apoptosis fails to rescue from anergy induction. T cell clones 7P.41 (panel a) and 7P.24 (panel b) were cultured overnight either in medium (open bars), or with 1 pg/mL of peptide HA307-319 (closed bars) in the presence or absence of 10 pg/mL of monoclonal anti-Fas antibody. T cells were washed the following day and subsequently plated out into 96-well plates at 1 x lo4 cells/well with 3 x 104 B-LCL prepulsed with 1 WglmL of HA307-319 peptide. After 48 hours, plates were pulsed with 1 &i/well of 3H-TdR and the results expressed as mean counts per minute of triplicate cultures.
7P.41 Cells)
(Percent
Viable
Cells)
100 100 100 80 100 89.9
T ceils were cultured as before in the presence or absence of 10 fig/mL of anti-Fas mAb or control antibody MR6 at 10 pg/mL. The cells were then analysed by Flow cytometry to determine the percentage of cell recovery. The results are expressed as percentage input of cells calculated by counting the T cells after the overnight culture divided by the initial number of cells.
Neutralisation from Apoptosis
of Fas/Fas-Ligand
Interactions
Rescues
Induction
In order to determine whether the mechanism involved in the apoptosis observed with clone 7P.24 was CD95mediated, Fas/Fas-L interactions were blocked using anti-Fas mAb, M3, during peptide presentation by T cells. T cells were harvested after overnight incubation and subsequently analyzed for cell viability. As shown in Table 2, the greatest loss of cells following peptide presentation was observed in
1104
clone 7P.24. However, the presence of a CD95 neutralising antibody fully rescued the cells from apoptotic death. A smaller percentage of cells from clone 7P.41 underwent apoptosis following culture with peptide and viability was also completely restored with the anti-CD95 mAb. An isotype-matched control antibody, MR6, had no effect on cell viability with either clone.
HARGREAVES,
BORTHWICK,
MONTANI
ET AL
Taken together, these data suggest that recognition of peptide on T cells can lead to either anergy or apoptosis, perhaps dependent on the levels of Fas-Ligand expressed on the responder T cells. When apoptosis is prevented, the T cells remain unable to respond to professional APC suggesting that anergy can be induced independently of cell death. This supports the argument that anergic cells do exist and may be involved in immunoregulation.
T Cells Rescued from Apoptosis Exhibit the Features of Anergy
Finally, the relationship between anergy and apoptosis was addressed by testing the reactivity of T cells that had been protected from apoptosis by the anti-CD95 mAb. As shown in Figure la) and b), the presence of the anti-CD95 mAb during the overnight culture with peptide, while protected the T cells from the induction of apoptosis, did not affect the induction of non responsive state when the T cells were rechallenged with peptide-pulsed B-LCL. Protection from cell death by blocking Fas/Fas-L interactions yielded T cells with an anergic phenotype since the T cells became hyper-responsive to exogenous IL-2 suggesting that anergy had been induced in the absence of apoptosis (data not shown).
REFERENCES 1. Jenkins MK, Chen C, Jung G, et al: J Immunol 144:16, 1990 2. Sloan-Lancaster J, Shaw AS, Rothbard JB, et al: Cell 79:913, 1994 3. van Parijs L and Abbas AK: Curr Opin Immunol8:355,
1996
4. Bossu P, Singer GG, Andres P, et al: J Immunol 151:7233, 1993 5. Sidhu S, Deacock S, Bal V, et al: J Exp Med 176:875, 1992 6. Lombardi G, Sidhu S, Dodi T, et al: Eur J Immunol 24:523, 1993 7. Lombardi G, Hargreaves 156:2769, 1996
R, Sidhu S, et al: J Immunol
Induction of Apoptosis Following Antigen Presentation Anergy and Apoptosis Are Two Separate Phenomena R.G. Hargreaves, and G. Lombardi
N.J. Borthwick,
M.S.G. Montani,
T
WO of the mechanisms involved in peripheral tolerance, referred to as anergy and apoptosis, act by silencing T cells or by removing them from the system altogether, respectively. Anergy has been reported in many systems and often involves the recognition of peptide/MHC complexes in the absence of cell division. This may result from the absence of necessary costimulatory signals’ or through the recognition of partial agonist peptides.’ Apoptosis can be induced in cells expressing Fas (CD9.5) upon encounter with cells expressing Fas-ligand, such as activated T cells, a process known as activation-induced cell death (AICD).3 The relationship between anergy and apoptosis is still a matter of debate with some groups, suggesting that they are overlapping phenomena.4 Therefore, to clarify this relationship, we have used an in vitro system of T cell-mediated antigen presentation.5 Activated human T cells express MHC class II molecules and thereby acquire the capacity to present antigen to CDCpositive T cells. We have previously reported that antigen presentation by T cells leads to the delivery of a partial signal culminating in a non-responsive state.6,7 Here we report that a minority of T cell clones undergo apoptosis in response to antigen presentation by T cells. Cell death could be prevented by inhibiting the interaction of Fas with its ligand; however, the cells that were rescued from death were observed to be anergic. These data suggest that apoptosis and anergy are two distinct outcomes of partial T cell signaling. MATERIALS Monoclonal
AND
E. Piccolella,
P. Carmichael,
by T Cells:
R.I. Lechler, A.N. Akbar,
(HA) peptide 307 to 319 and restricted by HLA-DRB1*0701, were maintained on a 2-weekly cycle of stimulation using peptide-pulsed, autologous PBMC plus rIL-2. Two Stage Cultures
for Tolerance
Induction
T cell clones were washed extensively and resuspended in RPM1 containing 10% Human Serum (HS) and cultured in the presence or absence of peptide HA307-319 (kindly donated by Dr H. Stauss, RPMS). In Fas blocking experiments, the anti-Fas antibody was added at a concentration of 10 FgjrnL for the duration of this incubation period. Following overnight incubation, the cells were washed extensively, and the viable cells plated out into 96-well plates at a concentration of 1 X 10“ cells/well. Peptide-pulsed, X-irradiated (120 Gy) B-LCL’s were added to the triplicate cultures at a concentration of 3 X lo4 cells/well and the plates cultured for 48 hours at 37°C. 3H-TdR (1 @/well) was added and the plates were harvested 18 hours later for counting by liquid scintillation spectrophotometry. Counts are expressed as mean corrected (A) cpm for triplicate cultures. Standard errors were routinely
of Viable Cells
Viable cells were distinguished by their forward angle scatter and 90”side scatter profiles and were counted using a CytoronAbsolute flow cytometer (Ortho Diagnostics Ltd, High Wycombe, Bucks, UK) as described previously.“ Cells undergoing apoptosis were identified morphologically in cytospin preparations stained with May-Grunwald Giemsa (MGG, Merk Ltd, Poole, Dorset, UK). A total of at least 5 fields per sample (>500 cells) were counted by two independent investigators. Apoptosis was also measured by cell cycle analysis using the flow cytometer, apoptotic cells characteristically appearing as a sub-GO/G1 peak after propidium iodide (PI) staining.
METHODS
Antibodies
Anti-CD95 Monoclonal Antibodies (rnAb) M3 was a kind gift from Immunex Research and Development Corporation (Seattle, USA). MR6 IgGl control was donated by Professor Mary Ritter, (Department Immunology, RPMS). Cell Lines and Clones
Epstein Barr virus (EBV)-transformed lymphoblastoid B cell lines (B-LCL’s), obtained from the 10th International Histocompatibility Workshop, were cultured in RPM1 1640 medium supplemented with 10% FCS. Clones 7P.24 and 7P.41, specific for Influenza Haemagglutinin
0041-1315/97/$17.00 PII SO041 -1345(96)00433-2
RESULTS Recognition
AND
DISCUSSION
of Peptide
on T-APC
Can Lead to Apoptosis
Two T cell clones specific for HA307-319 and restricted by DR7 were used in these experiments. Neither of these T cell clones proliferated when cultured with soluble peptide From Department Immunology, RPMS, Hammersmith Hospital, London, UK (PH., P.C., R.L., G.L.); and Department Clinical Immunology, RFHMS, Pond St., London, UK (N.B., A.A.); University of “La Tuscia”, Viterbo, Italy (M.M.); and Department Cellular and Developmental Biology, University “La Sapienza”, Rome, Italy (E.P.)
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1102
Transplantation
Proceedings,
29, 1102-l 104 (1997)
ANERGY AND APOPTOSIS:
TWO SEPARATE PHENOMENA
1103
Table 1. Recognition of Antigen Displayed by T Cells Can Induce Apoptosis Overnight
Condition
Go/G1
7P.41
medium
84.33
7P.41
peptide
(%)
S/GZ/M
(%)
63.42
8.15
88.17
10.61
7P.24
27.01
6.04
Clones
were
HA307-319
cultured
peptide.
with Propidium
overnight
either
Iodide.
Cells were analysed
7~41
(%)
28.97 1.38
in medium
The T cells were then washed
1 a)
1.23
7P.24medium peptide
Apoptotic
14.36
40
69.86 or
with
1 pg/mL
and subsequently
by Flow cytometry
of
stained
and assessed
for
DNA content.
in the absence of added accessory cells. Notably, it was observed that T cells incubated overnight with increasing doses of peptide (0.01 to 100 &mL) suffered a loss in viability. In order to investigate the nature of this cell death, T cell clones were incubated with either medium or 1 pg/rnL of peptide and stained the following day with Propidium Iodide. The results are shown in Table 1. The majority of clone 7P.41 were in GOIGl, with 29% of the cells hypodiploid following overnight incubation with 1 pg/mL of peptide. In contrast, only 27% of clone 7P.24 were in GO/G1 and almost 70% of the cells expressed hypodiploid nuclei, characteristic of apoptotic cells. These results demonstrate that the dramatic reduction in cell recovery in clone 7P.24 following recognition of peptide on T cells is due to apoptosis. PCR and FACS analysis of these clones demonstrated that the levels of Fas appeared to be identical and was constitutively expressed. Fas-ligand mRNA, however, appeared after 3 hours of incubation with peptide and subsided by 12 hours coinciding with a loss in cell viability. Interestingly, the levels of Fas-L mRNA in clone 7P.24 appeared to be higher than those induced in clone 7P.41 upon recognition of peptide presented by T cells. This was further confirmed using Jurkat T cells as targets in a Fas/Fas-ligand mediated killing assay (data not shown). This difference in expression may account for the differential ability of peptide presentation to induce apoptosis in the two T cell clones.
Table 2. Blocking Fas/Fas-L Interactions Prevents T:T Presentation-Induced Apoptosis 7P.24 OvernIght
Medium Medium Medium Peptide Peptide Peptide
Condition
+ anti-Fas + MR6 + anti-Fas + MR6
(Percent
Viable
100 100 100 12.4 94 14.4
mea
I
pepr
, ,
med
I
anti-Fas
E
e d
PePt
‘01
b) 7~24
40-
30-
20-
10-
0-r
mea
I
II
pept
_
med anti-Fas
overnight
condition
Fig 1. Prevention of Apoptosis fails to rescue from anergy induction. T cell clones 7P.41 (panel a) and 7P.24 (panel b) were cultured overnight either in medium (open bars), or with 1 pg/mL of peptide HA307-319 (closed bars) in the presence or absence of 10 pg/mL of monoclonal anti-Fas antibody. T cells were washed the following day and subsequently plated out into 96-well plates at 1 x lo4 cells/well with 3 x 104 B-LCL prepulsed with 1 WglmL of HA307-319 peptide. After 48 hours, plates were pulsed with 1 &i/well of 3H-TdR and the results expressed as mean counts per minute of triplicate cultures.
7P.41 Cells)
(Percent
Viable
Cells)
100 100 100 80 100 89.9
T ceils were cultured as before in the presence or absence of 10 fig/mL of anti-Fas mAb or control antibody MR6 at 10 pg/mL. The cells were then analysed by Flow cytometry to determine the percentage of cell recovery. The results are expressed as percentage input of cells calculated by counting the T cells after the overnight culture divided by the initial number of cells.
Neutralisation from Apoptosis
of Fas/Fas-Ligand
Interactions
Rescues
Induction
In order to determine whether the mechanism involved in the apoptosis observed with clone 7P.24 was CD95mediated, Fas/Fas-L interactions were blocked using anti-Fas mAb, M3, during peptide presentation by T cells. T cells were harvested after overnight incubation and subsequently analyzed for cell viability. As shown in Table 2, the greatest loss of cells following peptide presentation was observed in
1104
clone 7P.24. However, the presence of a CD95 neutralising antibody fully rescued the cells from apoptotic death. A smaller percentage of cells from clone 7P.41 underwent apoptosis following culture with peptide and viability was also completely restored with the anti-CD95 mAb. An isotype-matched control antibody, MR6, had no effect on cell viability with either clone.
HARGREAVES,
BORTHWICK,
MONTANI
ET AL
Taken together, these data suggest that recognition of peptide on T cells can lead to either anergy or apoptosis, perhaps dependent on the levels of Fas-Ligand expressed on the responder T cells. When apoptosis is prevented, the T cells remain unable to respond to professional APC suggesting that anergy can be induced independently of cell death. This supports the argument that anergic cells do exist and may be involved in immunoregulation.
T Cells Rescued from Apoptosis Exhibit the Features of Anergy
Finally, the relationship between anergy and apoptosis was addressed by testing the reactivity of T cells that had been protected from apoptosis by the anti-CD95 mAb. As shown in Figure la) and b), the presence of the anti-CD95 mAb during the overnight culture with peptide, while protected the T cells from the induction of apoptosis, did not affect the induction of non responsive state when the T cells were rechallenged with peptide-pulsed B-LCL. Protection from cell death by blocking Fas/Fas-L interactions yielded T cells with an anergic phenotype since the T cells became hyper-responsive to exogenous IL-2 suggesting that anergy had been induced in the absence of apoptosis (data not shown).
REFERENCES 1. Jenkins MK, Chen C, Jung G, et al: J Immunol 144:16, 1990 2. Sloan-Lancaster J, Shaw AS, Rothbard JB, et al: Cell 79:913, 1994 3. van Parijs L and Abbas AK: Curr Opin Immunol8:355,
1996
4. Bossu P, Singer GG, Andres P, et al: J Immunol 151:7233, 1993 5. Sidhu S, Deacock S, Bal V, et al: J Exp Med 176:875, 1992 6. Lombardi G, Sidhu S, Dodi T, et al: Eur J Immunol 24:523, 1993 7. Lombardi G, Hargreaves 156:2769, 1996
R, Sidhu S, et al: J Immunol