NOD, BALB/c, and C57BL/6 mice of both genders were purchased from Taconic or bred in our animal facility. Only nondiabetic NOD mice were used in this paper. Sprague Dawley rats and C57BL/6 Thy1.1 IgMa
mice were obtained from The Jackson Laboratory. C57BL/6 β7
integrin KO (β7
KO) mice are described previously (16
). OVA TCR transgenic mice (DO11.10) were obtained from Drs. D. Loh and O. Kanagawa (Washington University, School of Medicine, St. Louis, MO), and The Jackson Laboratory. All animals were housed under specific pathogen-free conditions in the Palo Alto Veterans Affairs or Stanford University animal facilities.
Antibodies and Other Reagents.
mAbs used in this paper were the following: anti-CD4 (GK1.5), anti-CD8 (53–6.7.2), anti-α4 (PS/2), anti–LFA-1 (FD441.8), and anti–ICAM-1 (BE29 and YN1.7), grown from hybridomas obtained from the American Type Culture Collection; anti–α4β7 integrin heterodimer (DATK32), anti-β7 (FIB 504), anti–L-selectin (MEL-14), anti-PNAd (MECA79), and anti–MAdCAM-1 (MECA367), grown from hybridomas produced in our laboratory; anti-VCAM-1 (MK2.7, 4B12, 2A11.1 and 6C7.1, Dr. B. Engelhardt, Max Planck Institute, Munster, Germany); anti-CD45R/B220 (RA3–6B2, Dr. R. Coffman, DNAX Research Institute, Palo Alto, CA); anti-OVA TCR (KJ1–26, Dr. J. Kappler, National Jewish Medical and Research Center, Denver, CO); anti–P-selectin (RB40.34), anti–E-selectin (10E9.6), anti–ICAM-2 (3C4), anti-CD44 (IM7), anti-Thy1.1 (OX-7), and anti-IgMa (DS-1), purchased from BD Biosciences; and anti-CD3 (KT3) from Accurate Chemical. Rat anti–human CD44 (9B5; our laboratory) and rat anti–mouse cerebellar antigen (OZ42; Dr. L. Pickford, Coulter Pharmaceutical, Menlo Park, CA) were used as negative control mAbs.
Biotin-anti–rat IgG, FITC-anti–rat IgG, and FITC-anti–rat IgM were purchased from Vector Laboratories. Biotin-anti–rat IgM and peroxidase-streptavidin were purchased from Jackson ImmunoResearch Laboratories, whereas PE-anti–rat IgG came from Biosource International. 3,3′-diaminobenzidine-tetrahydrochloride (DAB) was purchased from Sigma-Aldrich, while 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) mixed isomers, tetramethylrhodamine-5(6)-isothiocyanate mixed isomers (TRITC), and Alexa Fluor 488 were obtained from Molecular Probes.
Morphometric Analysis of BALT.
Mice were killed, and the lungs filled with PBS/OCT (1:1) by intratracheal injection, removed, and frozen in OCT. Frozen sections of the left lung were stained with hematoxylin and eosin; slides that showed a longitudinal section of a main bronchus that extended for at least one half the length of the lung tissue were used for image analysis as described previously (VAS II; Video Systems; reference 17
). In brief, we measured the area of all BALT aggregates and the bronchus on at least 20 nonsequential sections from each mouse. Results for each mouse are presented as the ratio of the BALT area (mm2
) to the bronchus area (mm2
Acetone-fixed frozen sections of lung, peripheral LN (PLN), and PP were sequentially incubated with hybridoma supernatant or purified mAb (1 h), biotin-anti–rat IgG or biotin-anti–rat IgM Ab (30 min), peroxidase-streptavidin (30 min), and DAB solution (10 min). The slides were washed in PBS after each incubation step. Some slides were counterstained with hematoxylin or methylene blue.
Evaluation of Vascular Luminal VCAM-1 Expression.
100 μg anti–VCAM-1 (4B12, 2A11.1, or 6C7.1) or negative control (9B5) mAb was given intravenously to NOD and BALB/c mice (8–15-mo-old). The mice were killed 3 min after injection and perfused immediately with PBS through the right ventricle. Frozen sections of lung, PLN, mesenteric LN (MLN), and PP were incubated sequentially with 2 μg/ml PE-anti–rat IgG, PBS, 10% normal rat serum, and Alexa 488–conjugated anti-PNAd, anti–MAdCAM-1, or control mAb (10 μg/ml). We examined the sections by confocal or immunofluorescence (IF) microscopy, and determined how many HEVs in BALT, PLN, and MLN expressed both PNAd and VCAM-1, only PNAd, or only VCAM-1. Similarly, we determined how many PP HEVs expressed both MAdCAM-1 and VCAM-1, only MAdCAM-1, or only VCAM-1.
T and B Cell Migration into BALT.
5 × 107
lymphocytes from LN and spleen of C57BL/6 Thy1.1 IgMa
mice were transferred intravenously into each NOD host mouse (Thy1.2, IgMb
; >8-mo old). Hosts were killed 2 h later; 106
lymphocytes from PLN, PP, MLN, spleen, and blood suspensions, as well as 106
donor lymphocytes used for the transfer (input cells), were incubated for 30 min at 4°C in 100 μl of 0.5 μg/ml PE–anti-Thy1.1 and 1 μg/ml FITC–anti-IgMa
or isotype- and conjugation-matched negative control mAbs. Two-color flow cytometry was used as described previously to determine the percentage of cells in the lymphocyte scatter gate that expressed Thy1.1 or IgMa
). At least 5 × 104
cells in the lymphocyte gate were analyzed on each sample. Frozen sections of host lungs stained with 2 μg/ml PE–anti-Thy1.1 and 4 μg/ml FITC-anti-IgMa
mAbs were evaluated by confocal microscopy; donor T and B cells in BALT were counted on >30 sections of lung from each mouse. Data are expressed as the ratio of donor T (Thy1.1+
)/donor B (IgMa +
) cells in each tissue divided by the Thy1.1+
In Vivo Blocking of Lymphocyte Homing by Anti–adhesion Molecule mAbs.
Lymphocytes from LN and spleen of 3–4-mo-old NOD mice were labeled by incubating 2 × 107
cells/ml with 0.8 μg/ml TRITC in labeling medium (50% RPMI 1640, 48.5% HBSS, and 1.5% BCS) at 37°C for 15 min (19
). Similarly, rat LN and spleen lymphocytes were labeled with 4 μM CFSE as described previously for TRITC (20
). The cells were centrifuged through BCS, washed, and resuspended in transfer medium (DMEM with 10 mM Hepes, and 1% BCS).
To block endothelial adhesion molecules, each host mouse received 500 μg intravenously of anti–endothelial adhesion molecule or control mAb, followed 30 min later by 5 × 107 TRITC-labeled mouse cells intravenously. To block lymphocyte adhesion molecules, TRITC-labeled mouse cells were treated with 10 μg/ml anti–lymphocyte adhesion molecule or control mAb on ice for 10 min; 5 × 107 mouse cells and 108 CFSE-labeled rat cells were transferred intravenously into each host. The rat cells, which do not react with the anti–mouse lymphocyte adhesion molecule mAbs used in these experiments, served as an internal standard to control for differences between host mice in blood flow to tissues and in the efficiency of the injection. An aliquot of the labeled mouse and rat cell mixture was used to obtain the input mouse/rat cell ratio. To ensure saturating mAb levels in vivo, additional mAb (100 μg anti–endothelial or 250 μg anti–lymphocyte adhesion molecule) was given along with the donor cells. In all experiments, host mice were killed 2 h after transfer. Staining with FITC-anti–rat IgG or IgM of tissues from host mice that received anti–VCAM-1 or anti-PNAd mAb, and staining of lymphocyte suspensions from host mice that were given anti–LFA-1 mAb-coated lymphocytes showed that mAb was present on mouse endothelia or lymphocytes, respectively, at the time of sacrifice (unpublished data).
In experiments involving blocking of endothelial adhesion molecules, flow cytometry was used to determine how many donor cells were in host blood and how many donor cells had migrated into host PLN, PP, MLN, and spleen. In brief, for each cell suspension, we determined the absolute number of donor cells (TRITC+) and total cells in the lymphocyte scatter gate; at least 5 × 104 total gated cells were analyzed for each sample. We calculated donor cells as a percentage of total gated cells in each tissue. The donor cells in BALT were observed by confocal microscopy, and homed cell density was calculated as the number of donor cells/103 μm2 of BALT. At least 30 sections of lung were evaluated per mouse. For each tissue, results for each specific mAb treatment are expressed as percent homing in control mAb-treated host mice.
In experiments involving blocking of lymphocyte adhesion molecules, the numbers of donor mouse (TRITC+) and internal standard rat (CFSE+) cells were evaluated in host PLN, PP, MLN, spleen, and blood by two-color flow cytometry and in BALT by confocal microscopy. The donor mouse/internal standard cell ratios for all tissues were normalized by the input mouse/internal standard cell ratio. For each tissue, results for each specific mAb treatment are expressed as percent homing of control mAb-treated cells.
In preliminary experiments, we compared data in PLN, PP, and MLN obtained by flow cytometry and confocal microscopy and confirmed that there is a good correlation between the two assays (r = 0.97; unpublished data).
Homing of Lymphocytes from β7 KO Mice.
LN and spleen lymphocytes from 3–4-mo-old C57BL/6 β7 KO and C57BL/6 wild-type (WT) mice were labeled with TRITC and CFSE, respectively, and injected intravenously into NOD mice (5 × 107 of each cell type per mouse). Numbers of donor cells in PLN, PP, MLN, spleen, blood, and BALT were determined as described previously. The results are presented as the ratio of β7 KO/WT lymphocytes in each tissue divided by the β7 KO/WT input ratio.
Naive T Cell Homing.
We prepared lymphocyte suspensions of LN and spleen from 6-wk-old DO11.10 OVA TCR transgenic mice and age-matched C57BL/6 Thy1.1 mice. The DO11.10 T cells have a naive phenotype, whereas the C57BL/6 Thy1.1 T cells, which are a mix of naive and memory cells, served as internal standards. 5 × 107 DO11.10 lymphocytes and an equal number of C57BL/6 Thy1.1 lymphocytes were transferred intravenously into each NOD mouse. 2 h later, mice were killed and lymphocyte suspensions from PLN, PP, MLN, spleen, and blood were stained with 2 μg/ml FITC-anti–OVA TCR (KJ1–26) and 0.5 μg/ml PE–anti-Thy1.1 mAbs; donor DO11.10 naive T cells (OVA TCR+) and internal standard C57BL/6 T cells (Thy1.1+) were detected by flow cytometry as described earlier in Materials and Methods. Frozen sections of lung stained with 4 μg/ml FITC-KJ1–26 and 2 μg/ml PE–anti-Thy1.1 mAbs were examined by confocal microscopy for DO11.10 and internal standard T cells in BALT. Data are expressed as the ratio of OVA TCR+ (naive)/Thy1.1+ (internal standard) T cells in BALT, PLN, PP, MLN, and blood normalized for their ratio in the spleen.
To evaluate the roles of PNAd, α4, and VCAM-1 in the homing of total T cells, naive T cells, and B cells to BALT, 5 × 107 lymphocytes from 6-wk-old DO11.10 mice (KJ1–26+ naive T cells, IgMa+ B cells) or 19-mo-old C57BL/6 Thy1.1 mice were transferred intravenously into NOD host mice; as described earlier in Materials and Methods, the host mice were treated with anti-PNAd or anti–VCAM-1 mAb, or the donor cells with anti-α4 mAb, before the transfer. Host mice were killed and their lymphoid tissues were stained with a FITC-conjugated mAb against OVA TCR, IgMa, or Thy 1.1, and evaluated as described earlier in Materials and Methods. The results for the specific mAb treatment are presented as percent homing of the control mAb treatment.
Data are given as mean ± SE for each group. One-way analysis of variance (ANOVA) was used for statistical analysis unless otherwise indicated. P < 0.05 is considered to be statistically significant.