Isolation of FDC from Human Tonsils by Percoll Gradient.
Tonsils obtained from children undergoing tonsillectomy were cut into small pieces and digested for 12 min at 37°C with an enzyme cocktail in RPMI 1640 medium (GIBCO BRL
, Gaithersburg, MD) containing collagenase IV (1 mg/ml; Sigma Chemical Co.
, St. Louis, MO) and deoxyribonuclease I (50 kU/ml; Sigma Chemical Co.
). The released cells were collected and a new stock of enzyme solution was added to the remaining tissue fragments for another 12 min. The cells, collected after two successive rounds of enzymatic digestion, were pooled and centrifuged through Ficoll-Hypaque (Eurobio, Paris, France) for 20 min at 400 g
to remove red and dead cells. After two washes, cells were layered on a 1.5% BSA (Pentex®
Path-o-cyte 5; Miles Inc., Kankakee, IL) gradient and centrifuged at 10 g
for 10 min at 4°C. The FDC-lymphocyte clusters were recovered from the pellet. This BSA gradient process was repeated two to three times. The resulting cell population contains 15–30% FDC that form tight clusters with lymphocytes (13
Isolation of a Highly Purified Single FDC Suspension by FACS® Sorting of CD14+CD21+ Large Tonsillar Cells.
Since human B cells, T cells, fibroblasts, endothelial cells, and epithelial cells express no or low levels of CD14, and human T cells, fibroblasts, endothelial cells, and epithelial cells express no or low levels of CD21, CD14highCD21high FDC were isolated by FACS® sorting of enriched FDC preparations by Percoll gradient. After cell sorting, the resulting population contained >98% pure single FDC (Fig. ). These highly purified FDCs may have been damaged inasmuch as they displayed cytoplasm losses and were unable to support B cell growth in vitro. However, these cells were used for PCR assays.
Figure 3 Isolation of highly purified FDC by FACS® sorting. (A) Low density tonsillar cells were stained by anti–CD21-PE and anti–CD14FITC (detailed in Materials and Methods). FDCs were sorted according to their CD21++ (more ...) Purification of Tonsillar B Cells, Follicular Mantle B Cells, and GC B Cells.
Briefly, tonsils were finely minced and the resulting cell suspension was subjected to two rounds of T cell depletion using first rosetting with sheep red blood cells, and then depletion with anti-CD3 magnetic beads. The resulting purified cells contained >97% CD19+ B cells and <1% T cells and monocytes. To isolate IgD+CD38− follicular mantle B cells and IgD−CD38+ germinal center B cells, total tonsillar B cells (107/ml) were incubated with anti–IgD-FITC and anti–CD38-PE in PBS containing 2% BSA (PBS–BSA) for 30 min. Cells were washed twice and suspended in PBS at 3 × 106/ml. The two B cell subpopulations were then purified by cell sorting. Two rounds of cell sorting were carried out to obtain >98% purity.
Generating FDC-specific mAb 7D6.
BALB/c mice were immunized with 5 × 106 enriched human tonsillar FDC intraperitoneally three times at 3-wk intervals. The final boost was carried out 3 d before fusion. Using polyethylene glycol 1500 (Boehringer Mannheim GmbH, Mannheim, Germany), 50 × 106 splenic cells were fused with NS1 myeloma cells. Hybridomas were cultured in complete medium supplemented with 20% vol/vol FCS, hypoxanthine and azaserine, oxaloacetic acid, pyruvate, and insulin (OPI; Sigma Chemical Co.). Hybridomas were selected by immunohistological staining of the culture supernatants of the FDC networks on tonsillar tissue sections. Ascites was produced in BALB/c mice, and mAb 7D6 (IgG1) was purified by highpressure liquid chromatography with an anion-exchange column (DEAE 5PW; Waters Chromatography Div., Milford, MA).
Frozen sections from human tonsils, spleen, and thymus were washed in PBS for 5 min. The sections were incubated with mouse IgG1 mAb 7D6, anti-CD21, and anti-CD54, respectively, for 60 min. After washing for 5 min in PBS, the sections were incubated with sheep anti–mouse IgG1 for 30 min in PBS containing 10% human serum, and then with alkaline phosphatase coupled to mouse antibodies specific for alkaline phosphatase (APAAP complexes; Dako, Roskilde, Denmark). After a final washing, alkaline phosphatase was developed by Fast red substrate (Sigma Chemical Co.) which gives a red color. Cytospin preparations of FDC clusters were fixed in acetone for 10 min at 4°C. The slides were washed in PBS and incubated for 1 h with the anti-FDC mAb 7D6. After washing, the cytospin slides were incubated for 30 min with anti–mouse IgG1. The binding of antibody was revealed using APAAP method and developed by Fast red substrate.
cDNA Library Construction and Screening.
RNA was purified from a B lymphoblastic cell line IM9 established from a bone marrow sample of a myeloma patient. This cell line stained weakly (variable;
28% positive cells) with mAb 7D6. cDNA library construction was as described (18
) using the Superscript Reverse Transcriptase cDNA Synthesis Kit (GIBCO BRL
). Doublestranded cDNA was size-fractionated using a Chromaspin-1000 column (Clontech, Palo Alto, CA) and ligated into the BstxI/ NotI-digested pJFE14 expression vector (19
A cDNA clone encoding the 7D6 antigen was isolated by a method similar to that described (18
) except that cell sorting (FACS®
), rather than panning, was used to enrich COS7 cells transiently expressing the 7D6 antigen (20
). After a 1 h incubation with 50 μg/ml isotype IgG1 antibody to block binding to FcγR, COS7 cells were stained with 10 μg/ml biotinylated mAb 7D6. Cells which bound mAb 7D6 were detected with streptavidin–phycoerythrin (Becton Dickinson, Milpitas, CA). Plasmid DNA recovered from sorted cells was transformed into Escherichia coli
DH10B for expansion and then reintroduced into COS7 cells. A cDNA clone (p7D6) with a 4 kb insert was identified which encoded the antigen recognized by mAb 7D6. The sequence of the cDNA insert was determined in part manually as described (18
), and in part on an automated sequencer (Applied Biosystems, Foster City, CA) using Taq Dye Deoxy Terminator cycle sequencing.
Expression of the 7D6 Antigen.
The 7D6 cDNA clone was expressed transiently in COS7 cells (18
). Mouse Ltk−
cells (L cells) stably expressing the 7D6 antigen were generated by cotransfection with a neomycin-resistance plasmid by the calcium phosphate method (GIBCO BRL
). Cells which survived in 1 mg/ml G418 were selected for 7D6 expression by FACS®
and also were positive for CD21 (CALTAG Labs., South San Francisco, CA).
PCR Assay to Detect the Expression of Short and Long CD21 Isoforms.
mRNA was extracted from 104 FDC purified by FACS® sorting according to their high expression of CD21 and CD14 antigens. cDNA was obtained by reverse transcription (Superscript Reverse Transcriptase Kit; GIBCO BRL). PCR assay was performed using a 5′ primer UHCR2-1704 (GGAGAGAGCACCATCCGTTG), a 3′ primer ULCR2-2363 (GGGCAGCGAGTCACAGGAGGAG) (see Fig. ), and a taq polymerase (Perkin-Elmer Corp., Norwalk, CT) in a thermal cycler. The first cycle of denaturation was at 94°C for 3 min, and then 35 cycles including 1 min of denaturation at 94°C, 2 min for primer annealing at 60°C, and 3 min of extension at 72°C. Complete extension was achieved for 10 min at 72°C. PCR products were loaded on a 1% low melting point gel for purification (WIZARD PCR DNA Purification System; Promega Biotec, Madison, WI). These products were ligated and cloned in the PCRtmII vector with TA cloning kit (Invitrogen, San Diego, CA). Plasmids were extracted from individual bacterial colonies and both strands were sequenced on an automated DNA sequencer (Applied Biosystems) using PCR II vector primers (21 M13, and M13RP).
Figure 2 Diagrams of CD21S and CD21L and their detection by PCR assay. This figure is made according to Ahearn and Fearon (30). Boxes represent the short consensus repeats (SCRs). 15 (CD21S) and 16 (CD21L) SCRs are grouped into four long homologous repeats (more ...)