MATERIALS AND METHODS
Islet transplantation
Pancreatic islets were isolated by collagenase digestion followed by density gradient
separation and then hand-picking, as described previously1. 600 islets were transplanted
under the renal capsule of mice, which were rendered diabetic with 225 mg/kg of
streptozotocin (STZ, Sigma-Aldrich, St. Louis, MO administered i.p.). Rejection of islet
allografts was defined by blood glucose levels of >250 mg/dl for at least 2 consecutive
days.
Trafficking studies
Allogeneic islets isolated from DTR-GFP-DC mice (on a BALB/C background, H-2d)
were transplanted under the kidney capsule of STZ-induced diabetic C57BL/6 mice (H2b). At 3 hours and at days 1, 7 and 30 post transplantation, islet allografts, spleen, bone
marrow (BM) and draining (DLN) and nondraining lymph nodes (NDLN) were
recovered. DLN are located on top of the kidneys beneath the left lower lobe of the liver.
DC were isolated by tissue homogenization, and the viability of recovered cells was
assessed by trypan blue exclusion. Fractions of recovered tissue were used in
immunohistological analysis, and were stained for CD11c PE (BD Pharmingen, San
Diego, CA) and assessed for GFP expression. dDC were identified as CD11c+GFP+ cells,
while rDC were identified as CD11c+GFP-cells.
Immunofluorescent microscopy
4 µm frozen sections were incubated in 100 µg/ml avidin D (Vector Laboratories,
Burlingame, CA) to block endogenous biotin. Sections were washed and excess avidin
was bound by adding 10 µg/ml d-biotin (Sigma Chemical Co., St. Louis, MO).
1
Monoclonal antibody to CD11c (Pharmingen) was applied for 60 minutes. Sections were
washed and incubated sequentially, first with biotinylated mouse anti-hamster IgG
(1:100) (Vector Laboratories), and after washing with PE-streptavidin (1:50) (Biomeda,
Foster City, CA), each for 30 minutes. For Ki-67 staining, rabbit anti-Ki67 (Novacastra)
diluted at 1/1000 was used overnight and incubated sequentially with biotinylated mouse
anti-rabbit at 1/100 and after washing with PE-streptavidin 1/50.
Flow cytometry
Rat anti-mouse CD11c APC, CD80 (B7-1) PE, CD86 (B7-2) PE, H-2d FITC, CD4 FITC,
CD25 PE, CD45 FITC, CD45.1 FITC, CD45.2 FITC, CD184 (CXCR4) PE, and CD195
(CCR5) PE were purchased from BD Pharmingen, and anti-mouse CD197 (CCR7) PE
and Foxp3 APC were purchased from eBiosciences (San Diego, CA). Cells recovered
from transplanted organs and peripheral lymphoid tissues were subjected to FACS
analysis, and were run on a FACSCalibur™ (Becton Dickinson, San Jose, CA). Data was
analyzed using FloJo software version 6.3.2 (Treestar, Ashland, OR). The level of
maturation of dDC was determined by FACS analysis by examining the expression of
CD80, 86 and Class II. The temporal expression of chemokine receptors on DC was
studied using CXCR4, CCR5 and CCR7 antibodies. Foxp3 analysis was performed
following overnight permeabilization of cells extracted from spleens and DLN of islet
transplanted mice using commercially available antibodies and gating on CD4+CD25+
cells. For the tracking of congenic strain cells, CD45.1 and CD45.2 FITC antibodies were
used.
Depletion of DC using diphtheria toxin (DT) or CCL21
2
We established 2 groups of DC-depleting experiments. In the first group we injected 250
ng DT i.p. into BALB/C GFP-DTR-DT mice and determined the magnitude of DC
depletion in the spleen and pancreas at 6 and 48 hours after injection by examining the
presence of CD11c+GFP+ cells by FACS analysis. We then isolated islets from DTtreated mice to perform allogeneic transplantation in STZ-induced diabetic C57BL/6
mice. In the second group of experiments, given that CCR7 is highly expressed on dDC,
recombinant murine CCL21/6Ckine (CCR7 ligand) (R&D Systems, Minneapolis, MN)
was used to increase the efflux of DC from islets. Islets were plated in 6-well plates at a
concentration of 600 islets per well and were cultured in 2 ml of RPMI (Cambrex,
Walkersville, MD) containing 10% FCS and 1% penicillin/streptomycin with either 200
or 800 ng/ml CCL21. Islets were incubated for 24 hours at 37° C and 5% CO2, the
medium was carefully aspirated, and islets were washed three times with PBS. Collected
islets were either transplanted or treated with collagenase and homogenized to be
subjected to FACS analysis, and the aspirated medium and PBS was studied to evaluate
CD45+CD11c+ cells by FACS.
DC isolation, culture, and adoptive transfer
BALB/C DC were generated as previously described from murine bone marrow2. Briefly,
the femurs and tibiae of mice are flushed, and bone marrow cells are cultured in Petri
dishes with 10 ml of RPMI supplemented with 10% FCS, 1% penicillin-streptomycin,
and 50 m 2-mercaptoethanol (R10 medium) and containing 20 ng/ml recombinant
murine granulocyte monocyte-colony stimulating factor (rmGM-CSF, R&D Systems). At
day 3, 10 ml of fresh R10 medium containing 20 ng/ml rmGM-CSF is added to Petri
dishes. At days 6 and 8, 10 ml of medium was collected, centrifuged, resuspended in 10
3
ml of fresh medium with 10 ng/ml rmGM-CSF, and added back to Petri dishes. To
induce oxidative stress, DC were incubated for 30 minutes at 37° C and 5% CO2 in the
presence of 200 µm of H2O2 as previously performed using other cell types3, and DC
maturity was assessed by FACS. As an add-back experiment for depletion studies, DC
were isolated from BALB/C spleens using anti-CD11c magnetic beads (Miltenyi Biotec),
and 5x105 of these BALB/C DC were adoptively transferred by tail vein injection in the
transplanted C57BL/6 mice immediately following islet transplantation as previously
described3-5.
References
1.
Abdi R, Smith RN, Makhlouf L, et al. The role of CC chemokine receptor 5
(CCR5) in islet allograft rejection. Diabetes. 2002;51:2489-2495.
2.
Sallusto F, Schaerli P, Loetscher P, et al. Rapid and coordinated switch in
chemokine receptor expression during dendritic cell maturation. Eur J Immunol.
1998;28:2760-2769.
3.
Farber CM, Liebes LF, Kanganis DN, Silber R. Human B lymphocytes show
greater susceptibility to H2O2 toxicity than T lymphocytes. J Immunol. 1984;132:25432546.
4.
Wang Z, Castellaneta A, De Creus A, Shufesky WJ, Morelli AE, Thomson AW.
Heart, but not skin, allografts from donors lacking Flt3 ligand exhibit markedly
prolonged survival time. J Immunol. 2004;172:5924-5930.
5.
Marriott I, Hammond TG, Thomas EK, Bost KL. Salmonella efficiently enter and
survive within cultured CD11c+ dendritic cells initiating cytokine expression. Eur J
Immunol. 1999;29:1107-1115.
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