sFig 1

1
sFig 1
sFig. 1. Apoptosis of UVB-irradiated NIT-1 cells.
We treated NIT-1 cells by UVB-irradiation as
described elsewhere. The cells were incubated in
the incubator for 0, 2, 4, 6 h. The apoptosis was
measured by flow cytometry after Annexin-FITC and
7-AAD staining.
2
sFig 2
sFig 2. T cell proliferation stimulated by
insulin B9-23. NOD spleen cells were stained with
CFSE (5 ?M). CFSE-labeled spleen cells (2 ? 106)
were stimulated with or without insulin B9-23
(12.5 ?M) for 3 days. Thereafter, the cells were
stained with anti-CD4-PE. The cell divisions in
CD4 and CD4- T cell populations were analyzed by
flow cytometry.
3
sFig 3
A
B
sFig 3. UVB-NIT1 treatment induces beta-cell
antigen-specific regulatory T cells. A. We
treated 10-week old NOD mice with 3 weekly
transfusions of UVB-NIT1 or PBS (2 mice in each
group). Thereafter, we challenged all the mice
with intraperitoneal injection of NIT1 lysates
and adjuvant once a week for 2 weeks. The
following week, splenocytes were prepared, and
CD4CD25 T cells were isolated from the pooled
splenocytes of two animals from each treatment
group. Purified CD4 T cells (1x106) from naïve
8-week old NOD mice were cultured with 2x105 of
purified CD4CD25 T cells from UVB-NIT1-treated
or PBS-treated mice in a U-bottom 96-well plate.
At the same time, The purified CD11c splenic
dendritic cells (1x105) and 12.5uM insulin B9-23
peptide were added to each well. The cells were
cultured for 5 days, then, 3H-thymidine
(1uCi/well) was added to each well for additional
16h. The cell proliferation was determined by
scintillation counting) B. The pancreatic lymph
nodes were collected from each animals from the
above experiment. The lymph node cells were
prepared. The lymph node cells (5x105) were
stimulated with 12.5uM insulin B9-23 peptide for
5 days, the proliferation of T cells stimulated
by insulin B9-23 was determined using
3H-thymidine incorporation assay as described
above. Triplicate wells were used for all the
cultures.
4
sFig 4
sFig 4. The cytokine production by T cells in
response to NIT-1 lysate stimulation. The spleen
cells (1 ? 106 ) from the mice for the experiment
shown in sFig 3 were stimulated with insulin
B9-23 (12.5 ?M) or 50 ?g/ml of NIT-1 lysates in
200 ?l culture medium for 5 days. The cytokines,
IL-4, IL-10 and IFN-? were measured by Luminex.
The similar cytokine-producing patterns were
obtained by the stimulations of insulin B9-23 and
NIT-1 lysates.
5
Supplemental table 1
The comparison of MFI by flow cytometry and Index
by RIA assay for anti-ß cell Abs
UVB-NIT1-treated group
PBS-treated group
Animal MFI RIA (Index)
Animal MFI RIA
(Index)
1 1.7 2.5
1 6.1
26.3 2
1.9 5.9
2 8.7
66.7 3 1.9
5.5 3
4.4 15.3
The samples with RIA indexgt9.8 in this
experiment are designated to be positive (For
mouse IAA assay by RIA, see reference Yu L,
Eisenbarth G, Bonifacio E, Thomas J, Atkinson M,
Wasserfall C. Ann N Y Acad Sci. 2003
Nov10051-12 )