|
A
|
|---|
|
DBA/2 (H-2d)
|
Culture conditions
|
Antigenic stimulation (SI)
3Do-54.8
|
Background (CPM)
|
|---|
|
4 week old
|
BMMC, GM-CSF primed
|
OVA
|
OVAp
|
IgE-TNP-OVA
|
None
|
| |
WEHI-3
|
0.74
|
1.1
|
.95
|
1,504
|
| |
D11 sup
|
.86
|
.76
|
.58
|
2,045
|
| |
Rec IL-3
|
.75
|
.80
|
.63
|
1,786
|
| |
SCF
|
1.3
|
.67
|
.75
|
1,745
|
| |
WEHI-3+SCF
|
.95
|
.87
|
1.0
|
3,164
|
| |
WEHI-3+IL-4
|
.79
|
.83
|
.87
|
2,568
|
|
3 week old
|
BMMC, GM-CSF primed
| | | | |
| |
WEHI-3
|
7.8*
|
8.5*
|
14.4*
|
2,359
|
| |
SCF
|
10.5*
|
9.6*
|
15.2*
|
2,790
|
| |
WEHI-3+IL-4
|
8.5*
|
7.4*
|
22.3*
|
3,876
|
|
B10.BR (H-2
k
)
| |
Antigenic stimulation (SI)
AD10
|
None
|
|
4 week old
|
BMMC GM-CSF primed
|
PCC
|
PCCP
|
IgE-TNP-PCC
| |
| |
WEHI-3 sup
|
1.4
|
1.3
|
0.8
|
2,140
|
| |
+IL-4
|
1.2
|
1.3
|
.9
|
1,398
|
|
3 week old
|
BMMC, GM-CSF primed
| | | | |
| |
WEHI-3
|
14.4*
|
9.8*
|
18.2*
|
2,575
|
| |
+IL-4
|
15.2*
|
12.1*
|
17.4*
|
2,132
|
|
B
|
|
DBA/2 (H-2
d
)
|
Culture conditions
|
Antigenic stimulation (SI)
3Do-54.8
|
Background (CPM)
| | |
|
Days old of BMMC
|
GM-CSF primed, in WEHI-3 media
|
IgE-TNP-OVA
|
None
| | |
|
Day 22
|
ibid.
|
10.8*
|
1,799
| | |
|
Day 23
|
ibid.
|
12.1*
|
1,325
| | |
|
Day 24
|
ibid.
|
14.5*
|
2,569
| | |
|
Day 25
|
ibid.
|
12.6*
|
1,634
| | |
|
Day 26
|
ibid.
|
3.3*
|
2,788
| | |
|
Day 27
|
ibid.
|
0.86
|
2,961
| | |
- 1A: BMMC (H-2d, or H-2k) were prepared by culturing bone marrow cells in the presence of different sources of IL-3 (20% supernate) or SCF (1 μg/ml) for three or four weeks with weekly decanting of adherent cells. BMMC were primed with GM-CSF (100 U/ml) for 48 hr and some cultures in addition treated with IL-4 at 100 U/ml. BMMC (H-2k) and PCCP-specific AD10 T-cell line were stimulated with pigeon cytochrome c (PCC), PCC peptide (PCCP) or IgE-TNP-PCC complexes at 10 μg/ml for 48 hr and IL-2 production was determined. 1B: For daily assessment of APC function from day 22 to day 27 of the fourth week, nonadherent BMMC were directly obtained from the WEHI-3 supplemented culture wares in the fourth week culture (Day 22 to Day 27) without culture media or plate change. Background CPM was determined in cocultures of differently-treated BMMC plus T cells without antigen stimulation. Stimulation index (SI) was computed as quotients of total CPM of 3H-thymidine uptake of the antigen-stimulated cultures over nonantigen-stimulated controls. Triplicates were set for each group of stimulation and the non-stimulated control, and the means and SE were computed and two-tailed t-test of the antigen-stimulated group compared to background CPM was performed; SE of all observations were calculated within 15% of the computed means. To simplify data, SI with p values < 0.01 was denoted as a * marks in cocultures with competent BMMC stimulation. SI of the group without the mark indicated a failure or loss of antigen presentation by extended cultured mast cells. L-2 secreted in the coculture supernatants was determined by stimulating proliferation and 3H-thymidine uptake of IL-2 dependent NK3 cell line.
