Supplemental Figures (Figure S).
A
70
B
WT Bim-/-
60
500 400
50 40
300
30
200
20 100
10 0
0
10
20
30
0
40
Days after STZ injection
C
100
D
WT Bim-/-
WT
Bim-/-
WT
Bim-/-
600 500
75
400 50
300 200
25
100 0
0
5
10
15
20
25
30
Days after CY injection
0
Figure S1. Resistance of Bim-/- B6 and NOD.B6 mice to type 1 diabetes. Five-to-six week old B6 (A-B) (n=13) and NOD.B6 mice (C-D) (n=9) were injected with low-dose STZ and CY, respectively, as described in Methods. The accumulated diabetes incidences (A and C) and blood glucose leves at 4 weeks (B and D) are shown. Results are compiled from two separate experiments. The differences between WT and Bim-/- mice are statistically significant for all panels (p<0.001).
1
Figure S2. Reduced insulitis in Bim-/- mice. Mice (n=13) were treated as in Fig. S1A, and sacrificed 40 days after the first STZ injection. Pancreata were sectioned, stained with HE, and scored as described in Methods. Results are compiled from two separate experiments. The difference in insulitis score between WT and Bim-/- mice is statistically significant (p<0.02).
2
A
70
WT Bim-/-
60 50 40 30 20 10 0
0
10
20
30
40
50
Days after STZ injection
B 500 400 300 200 100 0
WT
Bim-/-
Figure S3. Reduced diabetogenic activity of Bim-/- T cells. Rag1-/- B6 mice that do not have lymphoid cells were injected with WT (n=10) or Bim-/- T cells (n=11) as described in Methods. They were then treated with a low dose STZ to induce diabetes. The accumulated diabetes incidences (A) and blood glucose leves at 4 weeks (B) are shown. Results are compiled from two separate experiments. The differences between WT and Bim-/- mice are statistically significant for both panels (p<0.01).
3
400
WT Bim-/-
300
200
**
100
0
8000
Control
10 µg/ml Ins
50 µg/ml Ins
** 25 µg/ml GAD65
WT Bim-/-
6000
4000
**
2000
0
** Control
10 µg/ml Ins
50 µg/ml Ins
25 µg/ml GAD65
Figure S4. Reduced responses of Bim-/- NOD T cells to pancreatic antigens. Splenocytes of NOD.B6 mice (n=6) were cutured with or without insulin (ins) and GAD65 as indicated. Culture supernatants were collected 40 hrs later, and cytokine levels were determined by ELISA. Data are representative of two experiments. *p<0.01.
4
600
EL-4 + Bim EL-4
400
* *
200
0
18h
24h
40h
48h
Hours
Figure S5. Bim gene transfer in EL-4 T cells enhances IL-2 production. A recombinant Bim retrovirus was generated by inserting the murine Bim cDNA into the BglII site of the MigR1 vector and by transfecting 293T cells with the recombinant MigR1 plasmid (1-3). EL-4 T cells were infected with either the Bim retrovirus (EL-4 + Bim) or a control retrovirus that carries no Bim cDNA (EL-4). Two days later, cells were stimulated with anti-CD3 and anti-CD28 as described in Figure 3, and IL-2 concentrations in the supernatants were determined by ELISA at the indicated times. *, p<0.01.
Bim-/-
WT 0
12
0
12
Hrs
p27
β-actin
Figure S6. Bim-/- T cells have elevated levels of p27. T cells were prepared and stimulated with anti-CD3 and anti-CD28 as described in Figure 3. Whole cell lysates were prepared at the indicated times, fractionated by SDS-PAGE, and blotted with antibodies to p27 and β-actin.
5
Figure S7. Increased IP3R−Bcl-2 association in Bim-/- T cells. WT and Bim-/- T cells were cultured with (+) or without (-) anti-CD3 and anti-CD28 as described in Figure 3. After 30 minutes, cells were collected and lysed in NP-40 lysis buffer. Co-immunoprecipitation was performed using anti-IP3R. The resultant IP products (upper panel) and the whole cell lysates (lower panel) were fractionated by SDS-PAGE, and blotted with anti-Bcl-2. The numbers below the upper panel denote the relative intensities of the immunoprecipitated Bcl-2 normalized against the input Bcl-2 shown in the lower panel.
6
Supplemental References 1. 2.
3.
Sun, H., Gong, S., Carmody, R.J., Hilliard, A., Li, L., Sun, J., Kong, L., Xu, L., Hilliard, B., Hu, S., et al. 2008. TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis. Cell 133:415-426. Pear, W.S., Miller, J.P., Xu, L., Pui, J.C., Soffer, B., Quackenbush, R.C., Pendergast, A.M., Bronson, R., Aster, J.C., Scott, M.L., et al. 1998. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92:3780-3792. Pui, J.C., Allman, D., Xu, L., DeRocco, S., Karnell, F.G., Bakkour, S., Lee, J.Y., Kadesch, T., Hardy, R.R., Aster, J.C., et al. 1999. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11:299-308.
7
