Supporting Online Material for

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1 Supporting Online Material for Immunological Reversal of Autoimmune Diabetes Without Hematopoietic Replacement of β Cells Anish Suri,* Boris Calderon, Thomas J. Esparza, Katherine Frederick, Patrice Bittner, Emil R. Unanue* *To whom correspondence should be addressed. (A.S.); (E.R.U.) This PDF file includes: Materials and Methods Figs. S1 to S6 References Published 24 March 2006, Science 311, 1778 (2006) DOI: /science

2 Supporting Online Material Materials and Methods Mice and disease treatment. Female NOD mice (Taconic Farms, Germantown, NY) and male CByB6F1 mice (Jackson Laboratory, Bar Harbor, ME) were maintained under pathogen-free conditions. NOD mice were followed for development of diabetes by blood glucose measurements; two consecutive blood glucose concentrations of >250 mg/dl were indicative of diabetes. For the original protocol presented by Kodama et al (1), diabetic NOD female mice were maintained on daily injections of U of NPH human insulin per 100g of body weight for 7-20 days after which they received an islet transplant under the kidney capsule along with a single injection of CFA (50µl in each hind footpad) and a series of injection of male CByB6F1 spleen cells (twice a week for 40 days). Most recipient mice were transplanted with islets under the kidney capsule although a few mice received 300 islets (Table 1). Islets were isolated from 5-7 week old pre-diabetic NOD female mice (1). CFA (Difco, Detroit, MI) was prepared freshly by mixing with an equal volume of saline (150mM NaCl). On the same day of the transplant, the mice received a single injection of 50µl of CFA into each hind foot pad followed by the first injection of CByB6F1 spleen cells. Splenocytes for treatment of transplanted NOD female mice were derived from male CByB6F1 mice. Whole spleens were homogenized into single cell suspensions and 9x10 6 splenocytes in 500µl volume were injected intravenously into the tail vein biweekly for 40 days. In the second protocol (1), 12 week old pre-diabetic NOD female mice received a single injection of CFA (50µl in each hind footpad) alone or together with intravenous tail vein injections of spleen cells (5x10 5 cells administered twice a week for two weeks) from male F1 GFP mice. The male F1 GFP mice (CByB6F1-GFP) were generated by breeding GFP-expressing male C57BL/6 mice from Jackson Laboratory (C57BL/6- TgH(ACTbEGFP)) with BALB/c female mice. Flow cytometry. To detect for chimerism of CByB6F1 cells, splenocytes or peripheral blood leukocytes from treated NOD female mice were analyzed for the expression of H- 2K d and H-2K b class I MHC molecules. For H-2K d, cells were first stained with the biotinylated SF1-1.1 antibody (BD Pharmingen) followed by secondary streptavidinallophycocyanin. For H-2K b, cells were stained with the phycoerythrin-conjugated AF antibody (BD Pharmingen). To detect whether the sera of treated NOD female mice contained alloantibodies, spleen cells from CByB6F1 mice were stained first with 10µl of sera from treated or control NOD female mice followed by a secondary stain with a Cy5-conjugated goat-anti-mouse IgG antibody. Mixed lymphocyte proliferation assay. 2.5x10 5 splenocytes from treated NOD female mice (responders) were incubated with varying numbers of irradiated (3000 rads) CByB6F1 spleen cells (stimulators). All assays were done in triplicate in a 96-well round

3 bottom plate in a final volume of 200µL of DMEM containing 10% fetal calf serum. Post 72 hours in a 37ºC incubator, each well was pulsed with 0.5µCi thymidine ( 3 H-TdR) for 12 hours and then harvested. Transfer of diabetes by spleen cells. 20x10 6 spleen cells from treated diabetic or prediabetic NOD female mice were transferred intravenously via the tail vein in 0.5 ml volume into NOD.scid recipient mice. Immunofluorescence and immunohistochemistry. Serial sections of 5µm from 10% formalin fixed paraffin embedded pancreas were de-paraffinized in xylene thrice with each incubation lasting 5 minutes, and re-hydrated in 100% ethanol three times for 5 minutes each and then rinsed under running tap water for 5 minutes. The slides were then boiled in 50mM Trizma Base buffer at ph 8.0 for 20 minutes for antigen retrieval process. Slides were then cooled down for 20 minutes and incubated in blocking buffer (10% BSA (Sigma, St. Louis, MO) and 3% Triton x-100 (Sigma)) for 15 minutes at room temperature. Sections were incubated overnight at 4 C with rabbit anti-pdx-1 polyclonal antibody (1:5000) (gift from Dr. Christopher V. E. Wright, Vanderbilt University School of Medicine, Nashville, TN) and guinea pig anti-insulin polyclonal antibody (1:100) (Linco, St. Charles, MO) in blocking buffer. Slides were washed three times for 5 minutes with PBS and then incubated for 1 hour at room temperature with Alexa-488 labeled goat anti-rabbit IgG antibody (1:500) (Invitrogen, Carlsbad, CA) and Texas redconjugated goat anti-guinea pig secondary antibody (1:100) (Jackson ImmunoResearch, West Grove, PA). Slides were then rinsed in PBS. All sections were counterstained with bis-benzamide (Sigma) for 5 minutes, washed in PBS and then quenched to reduce background auto-fluorescence with Sudan Black (Sigma) (Sudan black 0.5% in 70% ethanol) for 2 minutes and finally rinsed in PBS. Slides were placed under a cover-slip with GVA mounting solution (Invitrogen). In other studies, the tissue sections were stained with HistoMouse-SP Kit (AEC, Broad Spectrum, Bulk) (Invitrogen) using guinea pig anti-insulin polyclonal antibody (1:100) (Linco) or Rabbit anti-glucagon polyclonal antibody (1:250) and rabbit anti-somatostatin polyclonal antibody (1:200) (Chemikon Temecula, CA). Immunoblot analysis. Pancreas frozen by submersion in liquid nitrogen was homogenized with a glass tissue grinder in 1 ml of a solution comprising 0.7 ml PBS, 0.1 ml Protease Inhibitor Cocktail (Sigma), 0.1 ml Phosphatase Inhibitor Cocktail 1 (Sigma), and 0.1 ml Phosphatase Inhibitor Cocktail 2 (Sigma). To the homogenate, 1 ml of M- PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL) was added and agitated for 10 minutes. The resulting lysate was centrifuged for 15 minutes at 15,000g to pellet cellular debris. Total protein concentration in the supernatant was measured by BCA (Pierce Biotechnology). For each extract 5 µg of total protein was fractionated on a 12% SDS-PAGE gel and transferred to PVDF membrane. The membrane was subjected to immunoblot analysis with a primary rabbit polyclonal antibody to GFP (1:2500 dilution) (Abcam, Cambridge, MA) and secondary horseradish peroxidase conjugated goat antibody (1:10,000 dilution) to rabbit immunoglobulin G

4 (Abcam). A directly conjugated horseradish peroxidase mouse antibody to GAPDH (1:10,000 dilution) (Abcam) was used as a loading control. The immunoblots were developed using ECL Plus reagents (Amersham Bioscience, Piscataway, NJ). FISH analysis. FISH analysis was performed on paraffin-fixed sections as described (2) with minor modifications. Briefly, de-paraffinized sections were treated with 0.2M HCl for 20 minutes prior to antigen retrieval at 80 C in 1M sodium thiocyanate for 20 minutes. Rinsed slides were then digested in 0.05 mg/ml Proteinase K in TEN buffer (0.05M Tris, 0.01M EDTA, 0.01M NaCl, ph7.8) at 42 C for 3-7 minutes (determined experimentally for each slide lot). Subsequently the slides were treated with 4% formaldehyde in PBS for 10 minutes and then dehydrated through an ethanol series (from 70% to 80% to 100%; 2 minutes each time). Fluorescein isothiocynate (FITC) conjugated-nucleotide probe for the Y-chromosome (Cambio Ltd, Cambridge, UK) was co-denatured on the slides by incubation at 70 C for 10 min and then at 37 C overnight. After stringency washes, the FITC signal was further amplified using the AlexFluor 555 Tyramide Signal Amplification kit (Invitrogen) in conjunction with a rabbit antifluorescein IgG biotin-conjugated antibody (Invitrogen). Nuclei were counterstained with Vectashield (Vector Labs, Burlingame, CA) Mounting Medium containing DAPI (4,6 diamidino-2-phenylindole) and examined on an Olympus BX51 epi-fluorescence microscope.

5 Supporting References and Notes 1. S. Kodama, W. Kuhtreiber, S. Fujimura, E. A. Dale, D. L. Faustman, Science 302, 1223 (Nov 14, 2003). 2 K. L. Johnson, D. K. Zhen, D. W. Bianchi, Biotechniques 29, 1220 (Dec, 2000).

6 Figure S1 A B Figure S1 (A). Analysis of mouse #138, table 1. Immunohistochemistry and immunofluorescence analyses for detection of glucagon, insulin and PDX-1 in the pancreatic islets of mouse #138, which remained normoglycemic post nephrectomy. Note PDX-1 positive cells that were very weak in insulin content. (B) Immunofluorescence staining for PDX-1 and insulin in the pancreatic islets of 4 experimental mice (table 1). These four mice remained normoglycemic post-nephrectomy. Note that almost all islets are positive for PDX-1 but negative for insulin. Bar indicates 20µm.

7 Figure S2 Figure S2. FISH analyses of pancreatic islets of the other 3 mice that remained normoglycemic post nephrectomy (table 1). As with the earlier results of a representative mouse shown in figure 2A, none of the other normoglycemic mice showed the presence of Y- chromosome positive cells in the islets. Islets are indicated by arrows.

8 Figure S3 A

9 B C

10 D Figure S3. Immunological status of mice from table 1 (A) Flow cytometry analysis of peripheral blood leukocytes (PBL) or spleen cells from mouse (#137) searching for CByB6F1 male cells. Indicated are the results of the F1 cells from CbyB6F1 used in the injections. These were not found in #137 or in any of the transplanted mice (table 1). First column to the left indicates the specificities of the antibodies used for FACS analysis. (B) Mixed lymphocyte proliferation assay using irradiated CByB6F1 spleen cells as stimulators and mouse #137 splenocytes as responders. Note proliferation of the responders. Proliferation of the responders in the absence of stimulator cells was 2166 cpm. (C) Reaction of sera from control and experimental mice against CBbyB6F1 splenocytes (table 1). While serum from a control NOD female mouse (thin black line) did not react, those from mice #136D (green), #137 (blue) and #138 (pink) showed allo antibodies against CByB6F1 cells. (D) Attempts to transfer diabetes with spleen cells from experimental mice #35D and #61, of protocol 1 (from table 1).Diabetes developed in all recipient NOD.scid mice.

11 Figure S4 Figure S4. Blood glucose levels of 12-week old pre-diabetic NOD female mice treated with a single injection of CFA along with 4 injections of F1-GFP spleen cells (administered biweekly for 2 weeks). As a control group, 12-week old pre-diabetic NOD females received only a single injection of CFA with no allogeneic F1-GFP cells. Neither group developed diabetes.

12 Figure S5 Figure S5. Transfer of diabetes by spleen cells from pre-diabetic NOD female mice treated as in figure S4. Spleen cells from 3 different donor experimental mice (#s 15, 21 and 29) were transferred into NOD.scid recipient mice. All mice developed diabetes.

13 Figure S6 Figure S6. Examination of diabetic mice. (A) Numbers of residual islets or β cells, as indicated by positive staining for insulin, present in 14 diabetic NOD female mice that were maintained on insulin for at least 2 weeks or 5 NOD female mice that were diabetic for 2 days. For identification of islets and β cells, 10 independent slides (100µm apart) from each mouse were scored. Note that a normal NOD.scid mouse has approximately 174 islets containing 8176 β cells. (B) Immunofluorescence analysis of pancreatic islets of diabetic NOD females to detect for PDX-1 and insulin. Note that while PDX-1 expression on β cells was more frequently detectable, there were a few islets that also contained insulin-positive β cells. Bar indicates 20µm.