Activation-Induced Cell Death As a Key Event on Peripheral Blood Mononuclear Cells from Allergic...
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Activation-Induced Cell Death As a Key Event on Peripheral Blood Mononuclear Cells from Allergic Patient. [Bautista Fortuna Patricia, Cerdán Sánchez Patricia, Gómez Serrano María, Del Monte Monge Alberto, Poquet Aparicio María Teresa, Prats Valcárcel Silvia]** **Alumnos 5º Biología, Facultad de Biología de la Universidad de Alcalá de Henares (Edificio de Ciencias, Campus Universitario: Ctra. Madrid-Barcelona Km 33,600. E-28871 Alcalá de Henares,-Madrid-) To characterize the apoptosis of different T cell populations under AICD conditions in Peripheral Blood Mononuclear Cells (PBMCs) from healthy control and allergic patient. PBMCs were obtained by standard protocols from a poly-allergic patient and a healthy control. The distribution of T, B and NK cell subpopulations and the percentage of spontaneous and induced apoptosis were characterized by four color flow cytometry in FACSCalibur analyzer using fluorocrome-labeled monoclonal antibodies, Annexin-V and 7-ADD. The prevalence of apoptosis was determined after three days of culture under five conditions: without exogenous apoptosis inducers and in the presence of T Cell Expander (TCE) with and without Interleukine- 2 (IL-2). Staurosporine (ST) was used as positive control. Data analysis (Figure 1) and illustrations were performed using FlowJo and Sigma Plot v11, respectively. In all subpopulations studied, we did not find differences in spontaneous apoptosis between healthy control and allergic patient (Figure 2). However, we found a significantly higher percentage of apoptotic T cells in the allergic patient under IL-2 with and without TCE. This pattern of apoptosis was similar in both Tc (Figure 3) and Th (Figure 4) subset. After regarding results, we suggest a synergic effect of TCE and IL-2 in apoptosis by AICD, probably because of IL-2 activity. The Th apoptotic cell profile might be a result of the increased tendency to activation, and consequently to an increased AICD. Thus, a similar apoptosis pattern of Tc lymphocytes suggests they may also have certain relevance in allergic disease. BACKGROUND T lymphocytes constitute a large population of the cellular infiltration in allergic inflammation, and a disregulated immune response seems to be an important pathogenic factor in this process. Increased activation and defects in apoptosis define the allergic disease in some T cell subsets. Activation-induced cell death (AICD) can occur spontaneously and it is influenced by the nature of the initial T-cell activation events. Figure 2. Percentage of apoptotic cells of different peripheral blood mononuclear cell subsets in Medium. The vertical blue bars represent the healthy control data, and the red ones the allergic patient. Figure.1 Flow cytometric analysis of the percentage of different T cell subsets of control and allergic patient under TCE + IL-2 culture conditions. In both cases, Tc (CD3+CD8+) and Th (CD3+CD8-) populations were plotted to determinate the different apoptotic profiles. The total apoptotic cells was circled in blue in the healthy control and in red in the allergic patient. The comparation between these percentages of apoptotic cells in TCE+IL-2 culture and the other conditions is represented in figures 3 and 4. Universida d de Alcalá Cell subset Tc Th B NK % ofapoptotic cells 0 5 10 15 20 25 30 35 C ulture conditions M edium ST TCE TC E + IL-2 IL-2 % ofapoptotic cells 0 20 40 60 80 C ulture conditions M edium ST TCE TC E + IL-2 IL-2 % ofapoptotic cells 0 20 40 60 80 BACKGROUND OBJECTIVE MATERIAL AND METHODS RESULTS CONCLUSIONS 0 200 400 600 800 1000 FSC-H: FSC-Height 0 200 400 600 800 1000 SSC-H: SSC-Height 20.2 0 200 400 600 800 1000 FSC-H: FSC-Height 0 200 400 600 800 1000 SSC-H: SSC-Height 36.3 10 0 10 1 10 2 10 3 10 4 FL4-H: CD3 APC 10 0 10 1 10 2 10 3 10 4 FL2-H: CD8 PE 16 38 38.8 10 0 10 1 10 2 10 3 10 4 FL4-H: CD3 APC 10 0 10 1 10 2 10 3 10 4 FL2-H: CD8 PE 20.8 32.2 40 10 0 10 1 10 2 10 3 10 4 FL1-H: Anexinna V FITC 10 0 10 1 10 2 10 3 10 4 FL3-H: 7AAD 1.79 19 13.8 65.3 Tc 10 0 10 1 10 2 10 3 10 4 FL1-H: Anexinna V FITC 10 0 10 1 10 2 10 3 10 4 FL3-H: 7AAD 0.38 10.7 14.7 74.2 Th 10 0 10 1 10 2 10 3 10 4 FL1-H: Anexinna V FITC 10 0 10 1 10 2 10 3 10 4 FL3-H: 7AAD 1.96 44.6 15 38.5 Tc 10 0 10 1 10 2 10 3 10 4 FL1-H: Anexinna V FITC 10 0 10 1 10 2 10 3 10 4 FL3-H: 7AAD 0.67 28.2 18.9 52.3 Th Lymphocytes Lymphocytes CONTROL ALLERGIC PATIENT a h g f e c d b Figure 3. Percentage of apoptotic cells under different culture conditions of Tc (CD3+CD8+) subset. The vertical blue bars represent the healthy control data, and the red ones the allergic patient. Figure 4. Percentage of apoptotic cells under different culture conditions of Th (CD3+CD8-) subset. The vertical blue bars represent the healthy control data, and the red ones the allergic patient.
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Transcript of Activation-Induced Cell Death As a Key Event on Peripheral Blood Mononuclear Cells from Allergic...
Activation-Induced Cell Death As a Key Event on Peripheral Blood Mononuclear Cells from
Allergic Patient.[Bautista Fortuna Patricia, Cerdán Sánchez Patricia, Gómez Serrano María,
Del Monte Monge Alberto, Poquet Aparicio María Teresa, Prats Valcárcel Silvia]**
**Alumnos 5º Biología, Facultad de Biología de la Universidad de Alcalá de Henares(Edificio de Ciencias, Campus Universitario: Ctra. Madrid-Barcelona Km 33,600. E-28871 Alcalá de Henares,-Madrid-)
To characterize the apoptosis of different T cell populations under AICD conditions in Peripheral Blood Mononuclear Cells (PBMCs) from healthy control and allergic patient.
PBMCs were obtained by standard protocols from a poly-allergic patient and a healthy control. The distribution of T, B and NK cell subpopulations and the percentage of spontaneous and induced apoptosis were characterized by four color flow cytometry in FACSCalibur analyzer using fluorocrome-labeled monoclonal antibodies, Annexin-V and 7-ADD. The prevalence of apoptosis was determined after three days of culture under five conditions: without exogenous apoptosis inducers and in the presence of T Cell Expander (TCE) with and without Interleukine-2 (IL-2). Staurosporine (ST) was used as positive control.
Data analysis (Figure 1) and illustrations were performed using FlowJo and Sigma Plot v11, respectively.
In all subpopulations studied, we did not find differences in spontaneous apoptosis between healthy control and allergic patient (Figure 2). However, we found a significantly higher percentage of apoptotic T cells in the allergic patient under IL-2 with and without TCE. This pattern of apoptosis was similar in both Tc (Figure 3) and Th (Figure 4) subset.
After regarding results, we suggest a synergic effect of TCE and IL-2 in apoptosis by AICD, probably because of IL-2 activity. The Th apoptotic cell profile might be a result of the increased tendency to activation, and consequently to an increased AICD. Thus, a similar apoptosis pattern of Tc lymphocytes suggests they may also have certain relevance in allergic disease.
BACKGROUNDT lymphocytes constitute a large population of the cellular infiltration in allergic inflammation, and a disregulated immune response seems to be an important pathogenic factor in this process. Increased activation and defects in apoptosis define the allergic disease in some T cell subsets. Activation-induced cell death (AICD) can occur spontaneously and it is influenced by the nature of the initial T-cell activation events.
Figure 2. Percentage of apoptotic cells of different peripheral blood mononuclear cell subsets in Medium. The vertical blue bars represent the healthy control data, and the red ones the allergic patient.
Figure.1 Flow cytometric analysis of the percentage of different T cell subsets of control and allergic patient under TCE + IL-2 culture conditions . In both cases, Tc (CD3+CD8+) and Th (CD3+CD8-) populations were plotted to determinate the different apoptotic profiles. The total apoptotic cells was circled in blue in the healthy control and in red in the allergic patient. The comparation between these percentages of apoptotic cells in TCE+IL-2 culture and the other conditions is represented in figures 3 and 4.
Universidad de Alcalá
Cell subset
Tc Th B NK
% o
f ap
opto
tic c
ells
0
5
10
15
20
25
30
35
Culture conditions
Medium ST TCE TCE + IL-2 IL-2
% o
f ap
opto
tic c
ells
0
20
40
60
80
Culture conditions
Medium ST TCE TCE + IL-2 IL-2
% o
f ap
opto
tic c
ells
0
20
40
60
80
BACKGROUND
OBJECTIVE
MATERIAL AND METHODS
RESULTS
CONCLUSIONS
0 200 400 600 800 1000FSC-H: FSC-Height
0
200
400
600
800
1000
SS
C-H
: SS
C-H
eigh
t
20.2
0 200 400 600 800 1000FSC-H: FSC-Height
0
200
400
600
800
1000
SS
C-H
: SS
C-H
eigh
t
36.3
100 101 102 103 104
FL4-H: CD3 APC
100
101
102
103
104
FL2
-H: C
D8
PE
16
3838.8
100 101 102 103 104
FL4-H: CD3 APC
100
101
102
103
104
FL2
-H: C
D8
PE
20.8
32.240
100 101 102 103 104
FL1-H: Anexinna V FITC
100
101
102
103
104
FL3
-H: 7
AA
D
1.79 19
13.865.3
Tc
100 101 102 103 104
FL1-H: Anexinna V FITC
100
101
102
103
104
FL3
-H: 7
AA
D
0.38 10.7
14.774.2
Th
100 101 102 103 104
FL1-H: Anexinna V FITC
100
101
102
103
104
FL3
-H: 7
AA
D
1.96 44.6
1538.5
Tc
100 101 102 103 104
FL1-H: Anexinna V FITC
100
101
102
103
104
FL3
-H: 7
AA
D
0.67 28.2
18.952.3
Th
Lymphocytes
Lymphocytes
CONTROL ALLERGICPATIENT
a
hg
f
e
c d
b
Figure 3. Percentage of apoptotic cells under different culture conditions of Tc (CD3+CD8+) subset. The vertical blue bars represent the healthy control data, and the red ones the allergic patient.
Figure 4. Percentage of apoptotic cells under different culture conditions of Th (CD3+CD8-) subset. The vertical blue bars represent the healthy control data, and the red ones the allergic patient.
