3. Materials and methods
4.2 Generation of tolDC and PSTreg in the baboon system
4.2.4 Baboon PSTreg showed specific immunosuppressive activity
85
Figure 4.2.9 PSTreg express high amounts of GARP mRNA. GARP mRNA was quantified by RT-PCR in PSTreg and NTreg. NTreg and PSTeff served as controls for PSTreg, NTeff as control for NTreg. GAPDH mRNA was used as endogenous reference gene for each sample. Data represent 5-6 independent experiments from 5-6 baboons. Wilcoxon signed-rank test was used to determine the p values.
RESULTS
86
Figure 4.2.10 Baboon PSTreg showed specific immunosuppressive activity towards CD154 expression on PSTeff and expressed high amounts of IL-10 after 7 h coculture. PS/N Treg/Teff were seeded in 96 well plates (ratio 1:1) and stimulated with CD3/CD28 beads.
After incubation for 7 h, cells were harvested to measure CD154 expression by flow cytometry, an example is shown in (A), and the suppression ratio is shown in (B). Data represent 5 independent experiments from 5 baboons. IL-10 was quantified in the
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2.53
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1.69
NN
NP PP
PN
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0 102 103 104 105
1.69
0 50K 100K 150K 200K 250K
0 102 103 104 105
4.12
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0 102 103 104 105
14.8
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0 102 103 104 105
15.1
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10.7
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5.6
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FSC
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CD154
15.1
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CD154
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CD154
2.66
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FSC
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CD154
4.27
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FSC
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CD154
5.02
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FSC
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CD154
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15.1
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2.66
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4.27
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5.02
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8.02
N
PP PN
P
NP NN
PP: PSTeff+PSTreg PN: PSTeff+NTreg NP: NTeff+PSTreg NN: NTeff+NTreg C: PSTeff/NTeff
A
B
C
RESULTS
87
supernatant by ELISA (C). Data represent 5 independent experiments from 5 baboons.
ANOVA with Bonferroni’s Multiple Comparison Test was used to determine the statistical significance. ***p < 0.001, **p < 0.01, *p < 0.05, NS, non-significant, p > 0.05. Error bars:
s.e.m.
CD25, as an intermediate activation marker of Teff, was tested for expression in PS/NTeff after interaction with PS/NTreg for 4 days (Figure 4.2.11 A, B).
39.73±4.282% of CD25 expression in mean of PSTeff was suppressed by PSTreg, which was significantly higher than in the other groups. NTreg only suppressed 25.63±3.508 % of CD25 expression in PSTeff. PSTreg showed no specific suppressive function of NTeff: PSTreg only suppressed 22.86±3.095% of CD25 expression in NTeff, and there was no significant difference compared to a 20.41±2.769% suppression ratio of CD25 in NTeff interacting with NTreg. IL-10 cytokine accumulated in cocultures of all groups (Figure 4.2.11 C). The culture medium of the PP group contained the highest amount of IL-10 compared to the other groups. Consistently, the IL-10 level in the NP group was not significantly higher than in the NN group. In the PS/NTeff control group (C), the level of IL-10 also increased from 6.64±4.019 pg/ml at 7 h to 86.15±52.625 pg/ml at 96 h.
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<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_001.fcs Event Count: 8390
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<PE-Cy7-A>
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<PE-A>
30.6
<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_001.fcs Event Count: 2564
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<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_002.fcs Event Count: 8493
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<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_002.fcs Event Count: 2600
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<Pacific Blue-A> 99.6
FSC-A, <<Indo-1 (violet)-A> subset Specimen_001_Tube_003.fcs Event Count: 1732
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<APC-A>
0 102 103 104 105
<PerCP-A> 70.7
<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_003.fcs Event Count: 1225
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<PE-Cy7-A>
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<PE-A>
36.7
<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_004.fcs Event Count: 1187
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<Pacific Blue-A> 67.6
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<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_004.fcs Event Count: 2826
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<PE-Cy7-A>
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42
<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_004.fcs Event Count: 1187
0 102 103 104 105
<PE-Cy7-A>
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<Pacific Blue-A> 67.6
Treg/CD3CD4=44.6%
Treg/CD3CD4=49.9%
Treg/CD3CD4=41.9%
Treg/CD3CD4=38.1%
NTreg
PSTreg
PSTreg
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FSC-A, <<FITC-A> subset Specimen_003_Tube_012.fcs Event Count: 3077
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FSC-A, <<FITC-A> subset Specimen_003_Tube_009.fcs Event Count: 3147
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<PE-Cy7-A>
18.7
FSC-A, <<FITC-A> subset Specimen_003_Tube_008.fcs Event Count: 1727
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<PE-Cy7-A>
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FSC-A, <<FITC-A> subset Specimen_003_Tube_007.fcs Event Count: 1740
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14.1
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control
NP
NN
PP
PN
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<PE-Cy7-A>
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19.1
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<PE-Cy7-A>
20.1
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<PE-Cy7-A>
19.2
FSC-A, <<FITC-A> subset Specimen_004_Tube_008.fcs Event Count: 4246
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102 103 104 105
<PE-Cy7-A>
21
NP PP PN NN
Baboon CD25 functional assay 16.11.11
FSC-A, <<Indo-1 (violet)-A> subset Specimen_001_Tube_005.fcs Event Count: 2611
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<APC-A>
0 102 103 104 105
<PerCP-A> 3.64
<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_005.fcs Event Count: 20
0 102 103 104 105
<PE-Cy7-A>
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<Pacific Blue-A> 20
<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_005.fcs Event Count: 95
0 102 103 104 105
<PE-Cy7-A>
0 102 103 104 105
<PE-A>
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<APC-A>
0 102 103 104 105
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<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_006.fcs Event Count: 61
0 102 103 104 105
<PE-Cy7-A>
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<PE-A>
9.84
<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_006.fcs Event Count: 6
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<PE-Cy7-A>
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<Pacific Blue-A> 66.7
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<APC-A>
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<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_007.fcs Event Count: 44
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<PE-A>
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<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_007.fcs Event Count: 7
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<PE-Cy7-A>
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<Pacific Blue-A> 85.7
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<<APC-A>, <<PerCP-A> subset Specimen_001_Tube_008.fcs Event Count: 78
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<PE-Cy7-A>
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<PE-A>
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<<PE-Cy7-A>, <<PE-A> subset Specimen_001_Tube_008.fcs Event Count: 4
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<PE-Cy7-A>
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<Pacific Blue-A> 75
PSTeff PSTeff NTeff NTeff
Treg/CD3CD4=4.21%
Treg/CD3CD4=6.56%
Treg/CD3CD4=13.6%
Treg/CD3CD4=3.85%
0 102 103 104 105
CD4
0 102 103 104 105
CD3
75
0 102 103 104 105
CD25
0 102 103 104 105
CD127
30.6
0 102 103 104 105
CD25
0 102 103 104 105
Foxp3
99.8
0 102 103 104 105
CD4
0 102 103 104 105
CD3
77.8
0 102 103 104 105
CD25
0 102 103 104 105
CD127
30.6
0 102 103 104 105
CD25
0 102 103 104 105
Foxp3
99.6
0 50K 100K 150K 200K 250K
FSC
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CD25
22
0 50K 100K 150K 200K 250K
FSC
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CD25
21.4
0 50K 100K 150K 200K 250K
FSC
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CD25
14.1
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FSC
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CD25
17.9
0 50K 100K 150K 200K 250K
FSC
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CD25
18.6
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FSC
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CD25
18.7
0 50K 100K 150K 200K 250K
0 102 103 104 105
22
0 50K 100K 150K 200K 250K
0 102 103 104 105
21.4
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0 102 103 104 105
14.1
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0 102 103 104 105
17.9
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0 102 103 104 105
18.6
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0 102 103 104 105
18.7
P PP PN
NP NN
N
A
88
Figure 4.2.11 Baboon PSTreg showed specific immunosuppressive activity towards CD25 expression on PSTeff and expressed high amounts of IL-10 after 4 days coculture. After incubation for 4 days, cells were harvested to analyze CD25 expression. An example is shown in (A), suppression ratio is shown in (B). Data represent 4 independent experiments from 4 baboons. IL-10 was quantified in the supernatant after 4 day coculture by ELISA (C). Data represent 4 independent experiments from 4 baboons. ANOVA with Bonferroni’s Multiple Comparison Test was used to determine the statistical significance. ***p < 0.001, **p < 0.01,
*p < 0.05, NS, non-significant, p > 0.05. Error bars: s.e.m.
PP: PSTeff+PSTreg PN: PSTeff+NTreg NP: NTeff+PSTreg NN: NTeff+NTreg C: PS/NTeff
B
C
89
4.3 Immune monitoring of baboons pre and post porcine heart