Journal: Nature biotechnology

Article Title: The mouse lymph node as an ectopic transplantation site for multiple tissues

doi: 10.1038/nbt.2379

Figure Lengend Snippet: Direct injection of hepatocytes into a single lymph node (LN) of a C57BL/6 wild type mouse. ( a ) Jejunal LN (yellow dotted oval) not transplanted (top panel) and just after transplantation (bottom panel) with hepatocytes. Primary hepatocytes were mixed with 3% Evans blue dye and Matrigel before injection. ( b ) In vivo optical imaging of animals transplanted at different sites. Primary hepatocytes from luciferase transgenic mice were transplanted in a single jejunal or popliteal LN, in spleen (SP) or intraperitoneally (IP). Signals (blue to red) depict different concentrations of donor hepatocytes at day 1 (top panels) and 1 week (bottom panels) after transplant. ( c ) Distribution of donor hepatocytes in a single jejunal LN. Top left panel, whole mount imaging of a LN one week after injection of donor GFP + hepatocytes. Bright field was merged with fluorescence. Top middle to bottom right panels, immunofluorescent (IF) staining of frozen LN serial sections with mAbs (red) against ER-TR7 (reticular fibroblasts), LYVE-1 (lymphatic vessels), PNAd (high endothelial venules), B220 (B cells) and CD4/CD8 (CD4 T and CD8 T cells) with the presence of GFP + hepatocytes (green). ( d ) IF staining of donor GFP+ hepatocytes (green) in jejunal LN one week after transplantation. Serial sections were stained with mAbs (red) against E-Cadherin (ECad), C-C chemokine receptor type 7 (CCR7) and sphingosine-1-phosphate receptor 1 (S1PR1). Nativel iver sections were stained as control (right panels). In native liver, CCR7 (red) was co-stained with dipeptidyl peptidase-4 (DPPIV) (green). All sections were counterstained with Hoechst 33342 (blue). ( e ) Proliferation of engrafted hepatocytes in LN after partial hepatectomy (PHx). Paraffin sections of injected LN and corresponding native liver one and two weeks after transplantation. Sections were stained for GFP (one and two weeks after transplantation) and BrdU (two weeks after transplantation), and revealed by peroxidase (brown cytoplasmic and brown nuclei staining respectively). Sections were counterstained with hematoxylin. Yellow outlines mark small populations of GFP + hepatocytes. 2 weeks after transplantation, bar graphs show the number of GFP + hepatocytes and % of BrdU + hepatocytes observed per section after immunostaining from animals with or without PHx.* P<0.05, ** P<0.0001. Data (mean + sem) are representative of one experiment with three to five mice per group. The experiment was repeated two times. Scale bar: (a) 1mm, (c–e) 100μm.

Article Snippet: Antibodies specific for the following antigens were purchased for immunohistochemistry: ER-TR7, LYVE1, GFP, Glutamine Synthetase, CCR7, S1PR1 (EDG1), F4/80 (Abcam), PNAd, B220, CD4 CD8 and Gr-1 (BD Biosciences), BrdU (Santa Cruz biotechnology), DPPIV (AbD Serotec), E-Cadherin (Zymed), Keratin 5 (Covance), Keratin 8 (DSHB), and C-peptide and Glucagon (Cell Signaling Technologies).

Techniques: Injection, Transplantation Assay, In Vivo, Optical Imaging, Luciferase, Transgenic Assay, Imaging, Fluorescence, Staining, Control, Immunostaining

Journal: Nature biotechnology

Article Title: The mouse lymph node as an ectopic transplantation site for multiple tissues

doi: 10.1038/nbt.2379

Figure Lengend Snippet: Direct injection of hepatocytes into a single LN of a Fah −/− mouse. ( a ) Macroscopic appearances of non-transplanted and transplanted (hepatized) LN; LN are marked by yellow dotted oval). For non-transplanted axillary and popliteal LN, Evans blue was injected to highlight the presence of the LN.. The representative images of the jejunal, axillary and popliteal hepatized LN were captured at 12, 22, 25 weeks after transplantation respectively. ( b ) Histology of hepatized jejunal LN in rescued Fah −/− mice. Left panels, serial sections of hepatized LN composed of hepatic and lymphatic tissue. Sections are stained with hematoxylin and eosin (H&E)(top left panel), Fah (brown) and counterstained with hematoxylin (blue)(middle left panel). The immunofluorescence shows lymphatic vessels (LYVE1, red) and hepatocytes (GFP + , green)(bottom left panel). Right panels, higher magnification of H&E showing typical cuboidal hepatocytes. Reticular fibroblasts stain with antibodies specific for ER-TR7 (red) while hepatocytes are GFP + , and stain with antibodies specific for DPPIV (red). Glutamine synthetase (GS, red) expression shows unique zonal restriction surrounding terminal hepatic venules. Sections were counterstained with Hoechst 33342 (blue). ( c ) Kaplan-Meier survival curves (top) and body weight (bottom) of Fah −/− mice transplanted in intra- and extra- (blue) abdominal LN vs no treatment. Error bars show standard error. ( d ) Comparison of transplant efficiency in different sites. Left panels, macroscopic appearances of normal and hepatized LN (yellow dotted ovals) in the periportal area. Right top panel, Kaplan-Meier survival curves of Fah −/− mice transplanted into the jejunal vs periportal LN. Right lower panels, average weight of periportal vs jejunal LN and intra- vs extra- abdominal LN. No statistical difference was observed. Error bars show s.e.m. ( e ) Top panels, allogeneic transplantation in LN (LN Tx) and splenic injection (SP Tx). GFP + hepatocytes (C57BL/6 background) were transplanted into Fah −/− mice (129 sv background) by SP Tx and LN Tx and after NTBC removal (lack of NTBC in drinking water induces liver failure in Fah −/− mice). Where indicated, mice were injected with immunosuppressive agents CTLA4-Ig and MR1 on day 0, 2, 4 and 6 after transplantation. The experiment was repeated at least 2 times. Lower panels show one representative LN Tx mouse injected with CTLA4-Ig and MR1. Macroscopic appearance of hepatized jejunal LN (yellow dotted oval). Immunostaining of the hepatized LN for the presence of GFP+ (green) and Fah+ (red) hepatocytes as well as reticular fibroblasts (ER-TR7, red). Sections were counterstained with Hoechst 33342 (blue). Scale bar: (a, d and e, left panels) 5mm, (b, left panels) 1mm and (b, middle right and e middle right panels) 100μm.

Article Snippet: Antibodies specific for the following antigens were purchased for immunohistochemistry: ER-TR7, LYVE1, GFP, Glutamine Synthetase, CCR7, S1PR1 (EDG1), F4/80 (Abcam), PNAd, B220, CD4 CD8 and Gr-1 (BD Biosciences), BrdU (Santa Cruz biotechnology), DPPIV (AbD Serotec), E-Cadherin (Zymed), Keratin 5 (Covance), Keratin 8 (DSHB), and C-peptide and Glucagon (Cell Signaling Technologies).

Techniques: Injection, Transplantation Assay, Staining, Immunofluorescence, Expressing, Comparison, Immunostaining

Journal: Nature biotechnology

Article Title: The mouse lymph node as an ectopic transplantation site for multiple tissues

doi: 10.1038/nbt.2379

Figure Lengend Snippet: Functional ectopic thymus in the jejunal LN. ( a ) Flow cytometric analysis of peripheral blood T cells. Top panels, representative analysis of the gating strategy for CD4 and CD8 T cells in wild type C57BL/6 (WT), BALB/c Nude (Nude), and BALB/c Nude mice into which C57BL/6 GFP + thymic cells were injected into a jejunal LN (LN Tx Nude) mice. The number values assigned to the gates and quadrants represent the percentage of total live cells within that gate or quadrant. All contour plots display 10% probability contours. Lower panels show the percentage of CD3 + , CD3 + CD4 + and CD3 + CD8 + live T cells in the blood of each animal analyzed (each symbol represents one animal); animals were WT, Nude, KC Tx Nude (BALB/c Nude Transplanted under Kidney Capsule) and LN Tx Nude. The thin black line indicates the mean +/− sem. No statistical significance (n.s.) was observed between KC and LN transplantation in each of the 3 panels. ( b ) Left panels, representative flow cytometric analysis of cells present in wild type C57BL/6 (WT) thymus and LN, and LN of BALB/c Nude mice into which thymic cells were injected into the LN (LN Tx Nude). The number values assigned to the gates and quadrants represent the percentage of total live cells within that gate or quadrant. Right panels show (top left) whole mount jejunal LN of a Nude mouse engrafted in LN with GFP + thymic cells. Bright field was merged with fluorescence, Top right shows frozen section with GFP + donor thymic cells. Bottom right shows immunostaining of cytokeratin 5 (K5) (blue), cytokeratin 8 (K8) (red) and counterstaining with Hoechst (white). Bottom left shows WT native thymus stained for K5 (red) and K8 (green). Scale bar: 200μm. ( c ) Flow cytometric analysis of TCR Vβ segment expression in splenocytes from wild type C57BL/6 mice (WT C57BL/6), heterozygous BALB/c Nude mice (Het Nude), or LN transplanted BALB/c Nude mice (LN Tx). Bar graphs show the mean percentage of the particular Vβ receptor. Individual symbols represent data from a single mouse (n=4–5). ( d ) Flow cytometric analysis of regulatory, naïve, central memory, and effector memory T cell subsets in splenocytes. Dot plots and histograms show gating strategy to detect regulatory T cells (FoxP3 + fraction of CD4 + CD25 + T cells) and naïve (CD44 − CD62L + ), central memory (CD44 + CD62L + ), and effector memory (CD44 + CD62L − ) T cells. Graphs show the data from individual mice, labeled as in panel c . The thin black line indicates the mean. ( e ) Top panel, Kaplan-Meier curves showing survival of skin grafts from C57BL/6 or CBA/CaJ donor mice transplanted onto BALB/c Nude recipients that had previously undergone transplantation of C57BL/6 thymic cells into the LN. Middle panel, graph of tumor growth in athymic BALB/c Nude (nu/nu) mice. 300,000 human colon cancer cells were inoculated subcutaneously with matrigel. Tumor sizes were measured once per week using a caliper and calculated as (π/6) × (length (mm) × width 2 (mm 2 )). The experiment was terminated after 8 weeks. Values are mean +/− s.e.m., n=10. Bottom panel, presence (+) or absence (−) of tumor growth after a single subcutaneous injection of 300,000 human colorectal cancer cells into BALB/c Nude recipients that had previously undergone transplantation of C57BL/6 thymic cells into the LN (LN Tx Nude), BALB/c Nude (Nude) recipients or C57BL/6 wild-type (WT) recipients.

Article Snippet: Antibodies specific for the following antigens were purchased for immunohistochemistry: ER-TR7, LYVE1, GFP, Glutamine Synthetase, CCR7, S1PR1 (EDG1), F4/80 (Abcam), PNAd, B220, CD4 CD8 and Gr-1 (BD Biosciences), BrdU (Santa Cruz biotechnology), DPPIV (AbD Serotec), E-Cadherin (Zymed), Keratin 5 (Covance), Keratin 8 (DSHB), and C-peptide and Glucagon (Cell Signaling Technologies).

Techniques: Functional Assay, Injection, Transplantation Assay, Fluorescence, Immunostaining, Staining, Expressing, Labeling

Journal: Nature biotechnology

Article Title: The mouse lymph node as an ectopic transplantation site for multiple tissues

doi: 10.1038/nbt.2379

Figure Lengend Snippet: Ectopic pancreas generation in the jejunal LN after islet transplantation. ( a ) Top panel shows whole mount LN of a streptozotocin-treated diabetic C57BL/6 mouse engrafted with C57BL/6 GFP + pancreatic islets. Bright field was merged with fluorescence. Other panels show immunofluorescence (IF) of LN removed 6 weeks after engraftment. Staining with antibodies specific for ERTR7 (reticular fibroblasts), C-peptide (C-PEP), and glucagon (GLUC) is shown in red and GFP is shown in green and Hoechst counterstain is shown in blue. ( b ) Average blood glucose levels in diabetic recipient mice over the course of 10 weeks after transplantation of islets into the jejunal LN (LN Tx, n=5), under the kidney capsule (KC Tx, n=3), and without transplantation (No Tx, n=6). In 3 of 5 LN Tx mice, a second islet transplant was performed into the jejunal LN one week after the initial transplant. The mice that did not receive a transplant all died after 3 weeks. The data are presented as mean +/− s.e.m. ( c ) Average body weight (top) and blood glucose level (middle) of C57BL/6 wild-type (WT) mice or C57BL/6 LN Tx mice after LPS injection (1 mg/kg). Bottom panel shows average serum concentrations of tumor necrosis factor alpha (TNFα), IL1-β, and IL-6 two hours after LPS injection. All error bars show standard error. All IF image scale bars are 100 μm unless otherwise indicated.

Article Snippet: Antibodies specific for the following antigens were purchased for immunohistochemistry: ER-TR7, LYVE1, GFP, Glutamine Synthetase, CCR7, S1PR1 (EDG1), F4/80 (Abcam), PNAd, B220, CD4 CD8 and Gr-1 (BD Biosciences), BrdU (Santa Cruz biotechnology), DPPIV (AbD Serotec), E-Cadherin (Zymed), Keratin 5 (Covance), Keratin 8 (DSHB), and C-peptide and Glucagon (Cell Signaling Technologies).

Techniques: Transplantation Assay, Fluorescence, Immunofluorescence, Staining, Injection