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Blood, Vol. 113, Issue 26, 6638-6647, June 25, 2009
Lymphoid tissue–specific homing of bone marrow–derived dendritic cells
Blood Creusot et al.
113: 6638
Supplemental materials for: Creusot et al
Immunohistochemistry
Select tissues from CD45.2+ DC-injected mice were also snap frozen in OCT Compound (Tissue-Tek). 6 µm sections were cut, fixed in cold acetone for 10 min, air-dried, wash in PBS 3 × 5min, blocked (PBS, 1% FBS, 3% goat serum) for 1 hour at room temperature, washed in PBS 3 × 10 min, stained with anti–CD45.2-biotin overnight at 4C, washed in PBS 2 × 10 min, stained with SA-Texas Red for 1 hour at room temperature, washed in PBS 2 × 10 min, stained with DAPI for 10 min, mounted in Vectashield and observed by epifluorescence microscopy. Comparative analysis of lymph node gene expression by microarray
Pancreatic (PLN), mesenteric (MLN) and inguinal (ILN) lymph nodes were extracted from 12 week old NOD, NOD.B10, FVB and BALB/c mice (pooled from 3–5 mice), and homogenized in 0.5 ml Trizol reagent (Invitrogen). After addition of 100 µl chloroform, samples were centrifuged for 10 min at 8000 rpm. The aqueous phase was mixed with an equal volume of 70% ethanol and applied to an RNeasy Mini column (Qiagen). Total RNA isolation was then conducted according to the manufacturer’s instructions. cRNA amplification and fluorescence labeling (cyanine 3 for ILN or MLN, and cyanine 5 for PLN) was performed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Both were combined and hybridized onto the Agilent 41K whole mouse genome (60-mer) oligo microarray slide. Ratios PLN/ILN and PLN/MLN were obtained using Feature Extraction and GeneSpring GX7 software (Agilent Technologies). Raw data (file name PLNvsMLN-ILN.xls) are available on http://fathmanlab.stanford.edu/therapy.html, under “Comparison of gene expression between PLN and MLN/ILN.” The gastric (GLN) and pancreaticoduodenal (PDLN) nodes of the PLN) were separately extracted from 4 individual 12 week old NOD mice, and homogenized in 0.5 ml Trizol reagent (Invitrogen). Total RNA was extracted and labeled as described above (cyanine 3 for PDLN and cyanine 5 for GLN). Both were combined and hybridized onto the Agilent 4 × 44K whole mouse genome oligo microarray slide. Ratios GLN/PDLN were obtained using Feature Extraction and GeneSpring GX10 softwares (Agilent Technologies). Raw data (file name GLNvsPDLN.xls) are available on http://fathmanlab.stanford.edu/therapy.html, under “Comparison of gene expression between GLN and PDLN.”
Files in this Data Supplement:
- Table S1. Differential expression of genes involved in adhesion and chemoattraction between PLN and either MLN or ILN from BALB/c, FVB, NOD, and NOD.B10 mice (PDF, 252 KB) -
Data were processed and normalized using the GeneSpring GX7 software. Legend of conditions: B = BALB/c; F = FVB, N = NOD; NB = NOD.B10; pm = PLN vs MLN; pi = PLN vs ILN. Raw data (file “PLNvsMLN-ILN.xls”) are available on the Web site http://fathmanlab.stanford.edu/therapy.html, under “Comparison of gene expression between PLN and MLN/ILN.”
- Table S2. Gene over-expressed in GLN compared to PDLN in NOD mice (PDF, 80.7 KB) -
Data were processed and normalized using Feature Extraction and GeneSpring GX10 software. All the genes listed were increased over 1.5 fold in GLN in all four tested mice (ratio GLN / PDLN shown). Only Nos1 was increased over 2 fold in GLN in all four tested mice. Raw and filtered data (file “GLNvsPDLN.xls”) are available on the Web site http://fathmanlab.stanford.edu/therapy.html, under “Comparison of gene expression between GLN and PDLN.”
- Figure S1. View of the surroundings of the PLN in a NOD mouse (JPG, 70.9 KB)
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Pancreas (1), PDLN (right PLN, two lobes apparent on this particular mouse) (2), GLN (left PLN) (3), caudate lobe of the liver (4), omental tissue connecting two sides of the pancreas (5), spleen (6) and stomach (7).
- Figure S2. Further ex vivo biodistribution analyses (JPG, 408 KB)
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(A) Absolute biodistribution (Relative Light Units in whole tissue) of Luc+ DCs in NOD and FVB mice on day 3 and day 5 after i.v. or i.p. injection. (B) Absolute biodistribution of Luc+ DCs in FVB mice on days 1, 3, 6 and 12 after i.v. or i.p. injection. (C) Relative (left panel) and absolute (right panel) biodistribution of Luc+ DCs in BALB/c mice on days 1, 3, 5 and 8 after i.v. injection. All data show the mean from 3 mice (A–B) or 4 mice (C) ± standard error. See Table 1 for tissue legend (left lobe of the liver was used).
- Figure S3. Homing of BM-DCs at early time points (JPG, 283 KB)
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(A) Quantitation of the signal emitted from Luc+ DCs in the spleen area or in the lungs/liver area after i.v. injection in NOD mice (as shown in Fig. 3A, left panel). Mice were imaged for 5 min under anesthesia. (B,C) Absolute biodistribution (Relative Light Units in whole tissue) of Luc+ DCs in NOD mice at 6, 11 and 26 hours after i.v. (B) or i.p. injection (C). See Table 1 for tissue legend (caudate lobe of the liver was used). Data show the mean from 3 mice ± standard error.
- Figure S4. Intrasplenic injection of DCs (JPG, 237 KB)
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In vivo imaging of the left flank of NOD mice at different time points after intrasplenic injection of 1 × 105 Luc+ DCs (top pictures). Some mice were sacrificed a few hours after intrasplenic injection and 10 min after substrate injection to verify that there was no signal above background outside the spleen, hence no leakage (bottom pictures).
- Figure S5. Differential activation of T cells by DCs in different lymphoid tissues (JPG, 414 KB)
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(A,B) Spleen, PLN, TLN and MLN were collected 3 days after i.v. injection of PBS, 5 × 106 DCs cultured in medium with 1.5% mouse serum (ms-DC) or 5 × 106 DCs cultured in medium with 10% FBS (fbs-DC). Significant T cell activation, as evidence by increase cell number (A) or up-regulation of CD25 (B), was not observed in MLN or with ms-DCs. Data show the mean from 3 mice ± standard error. T-test was used for statistical analyses. (C) Representative FACS dot plots showing the percentage of CD4+ CD25+ T cell, and the mean fluorescence intensity of CD25 expression among those cells, in the spleen, PLN, MLN and LLN of NOD mice, 2 days after i.v. or i.p. injection of CFSE+ DCs. Mean fluorescence intensity of CD25 data are compiled in Fig. 5D.
- Figure S6. BM-DCs in the omentum (JPG, 153 KB)
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(A) Staining of CD45.2+ DCs outside the pancreas on sections obtained from NOD mice that were injected i.p. with 5 × 106 CD45.2+ DCs. Sections were stained with anti–CD45.2-biotin, followed by SA-Texas Red, and counterstained with DAPI (40× magnification). (B) Ex vivo imaging of spleens showing the distribution of Luc+ DCs within the spleen 2 days after i.v. or i.p. injection. Tissues were imaged for 5 min. Data are representative of more than 10 i.v.-injected mice and of a fraction of i.p.-injected mice (the remaining i.p.-injected mice showing little over-background signal in the spleen).
- Figure S7. Activation of BDC2.5 T cells by DCs draining from pancreatic islets (JPG, 504 KB)
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(A) FACS analysis of BDC2.5 T cells before and after purification, showing purity of CD4+ T cells (>92%), successful removal of CD25+ cells and percentage of islet antigen-specific (Vβ4+) T cells. (B) Percentage of CFSE+ BDC2.5 T cells among CD4+ T cells (y axis) in PDLN, GLN and RLN, 20 hours after i.p. or i.v. injection. Data from CLN and MLN (not shown) were similar to RLN. (C,D) Percentage of CD69+ cells among CFSE+ CD4+ T cells (blue histograms, gate and percentage shown on graph) compared to CFSE− CD4+ T cells (red histograms, %CD69+ = 13.54 ±1.64), in PDLN, GLN and RLN on day 1 (C) and day 3 (D) after i.p. or i.v. injection. CD69 was upregulated on a maximum of ~50% of the CFSε T cells (D), corresponding to ~50% expressing the V64 chain of the islet antigen-specific TCR (A). Data from CLN and MLN (not shown) were similar to RLN.
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