This paper describes an antibody (mAb 7D6) that specifically recognizes human follicular dendritic cells (FDCs). By expression cloning, a cDNA clone encoding for the long human CR2/ CD21 isoform (CD21L) that contains an additional exon (10a) was isolated. We demonstrated that FDCs selectively express CD21L, while B cells selectively express the short CR2/CD21 lacking exon 10a (CD21S). By screening mouse Ltk cells transfected with the CD21L cDNA, we further showed that the other two anti–human FDC mAbs DRC-1 and KiM4 also recognize CD21L. Thus, CD21L represents the first characterized human FDC-specific molecule, which may confer unique functions of FDCs in germinal center development.

Secondary lymphoid organs such as lymph nodes, spleen, Peyer's patches, and tonsils represent the major sites where immune responses to antigens occur. These lymphoid organs are divided into a T cell compartment (T cell zone or extrafollicular area) and a B cell compartment (B lymphoid follicle). While T cell zones contain interdigitating dendritic cells that play key roles in presenting antigen to naive T cells (1), B cell follicles contain follicular dendritic cells (FDC) that have the capacity to retain native antigen–antibody immune complexes for long periods of time (2, 3). During T cell–dependent humoral (antibody) immune responses, antigen-specific B cells undergo rapid clonal expansion within the FDC networks of B cell follicles, leading to the formation of germinal centers (GC) (4). During the course of clonal expansion, somatic hypermutation in immunoglobulin variable region genes (5, 6), and isotype switch in immunoglobulin constant region genes occur (7). After antigen-driven affinity selection (8), high affinity germinal center B cells will differentiate into either plasma cells or memory B cells (911). The functions of FDC in the GC reaction were proposed because of their selective localization and their ability to retain immune complexes. Both in vivo and in vitro experiments have suggested that FDC play important roles in GC B cell proliferation, survival, and differentiation (1215). However, little is known at the molecular level of how FDCs contribute to GC development. Several monoclonal antibodies have been generated against FDCs (16, 17); however, the nature and function of the antigens recognized by these monoclonal antibodies are unknown. Here, we describe an antibody (mAb 7D6) that specifically recognizes human FDC. By expression cloning, using mAb 7D6, a cDNA clone encoding for the long isoform of CD21L (CD21L) that contains an additional exon (10a) was isolated. We show that FDC selectively express CD21L, while B cells selectively express the short CD21 (CD21S) lacking exon 10a. By screening mouse L cells transfected with the CD21L cDNA, we further demonstrate that the other two anti–human FDC mAbs, DRC-1 and KiM4, also recognize CD21.

Materials And Methods

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. 3). 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.

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 IgDCD38+ 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).

Immunoenzymatic Staining.

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.

Poly(A)+ 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. 2), 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).

Results And Discussion

mAb 7D6 Selectively Stains FDC.

mAb 7D6 was selected because it specifically stains FDC networks on tonsillar and splenic sections (Fig. 1, A and C). The reactivity on FDC networks was further confirmed by staining of isolated FDCs (Fig. 1, G and H). 7D6 antibody did not give any specific staining on sections from fetal thymus (Fig. 1 E) or fetal liver (not shown). There was no positive staining of mAb 7D6 on total cell suspensions of bone marrow and peripheral blood by FACS® analysis (not shown).

7D6 cDNA Encodes the CD21L.

After screening over 50 cell lines including Burkitt's lymphoma cells, B lymphoblastoid cells, T cells, myeloma cells, monocytic cells, and erythroblastoid cells, we found that Raji (Burkitt's lymphoma cell), UD123, UD261, IM9 (lymphoblastoid B cells), and K562 (erythroblastoid) expressed low, but significant, levels of 7D6 antigen (7–28% of cells were positive). Accordingly, we isolated a 4-kb cDNA clone from the IM9 cDNA library by FACS® sorting. Transfection of COS7 and L cells with p7D6 cDNA resulted in expression of the 7D6 antigen (not shown). The sequence of the p7D6 cDNA insert matched the sequence of long CR2/CD21 isolated from the Raji cell line (21), with several polymorphisms as described (22), and two additional ones: position 1979 [AGT (Ser) → A AT (Asn)] and position 2075 [CGT (Arg) → CAT (His)] (The sequence of the 7D6-reactive isoform of CD21 is available upon request). Consistent with this finding, all the anti-CR2/CD21 mAbs available from the Fifth International Leukocyte Typing Workshop that had been shown to stain both B cells and FDCs, stained p7D6 cDNA transfected Cos7 cells or L cells (not shown). However, mAb 7D6 is specific for FDC, and does not recognize CD21 expressed on other cells. Two CD21 isoforms have been described. A “long” form (CD21L) has an extracellular domain with 16 short consensus repeats (SCR) (Fig. 2), and is encoded by p7D6 and the clone described earlier (23). A short CD21 isoform was reported with an extracellular domain containing only 15 SCRs, the missing SCR (SCR10a) of 59 amino acids being encoded by 177 bp (Fig. 2) (23). Whether the mAb 7D6 epitope is encoded by SCR10a, or is a conformational determinant induced elsewhere in the molecule by the presence of SCR10a, is not known.

Human B Cells Selectively Express the CD21S; FDC Selectively Express CD21L.

The pattern of mAb 7D6 staining suggests that FDC specifically express CD21L, while B cells specifically express CD21S. To directly test this hypothesis, a PCR assay using a 5′ primer starting from basepair 1704 and a 3′ primer starting from basepair 2363 of the short CR2/CD21 sequence, was carried out on RNA from 104 highly purified FDC (Fig. 3) in parallel with follicular mantle B cells (FM) and GC B cells isolated by FACS® sorting. Fig. 4 shows that a single large PCR product was generated from FDC, and a single smaller PCR product was generated from both FM and from GC B cells of the same donor. Further, sequencing analysis of these two PCR products shows that the FDC-derived large PCR product is 836 bp containing the 177-bp insertion that encodes the SCR10a. The B cell–derived PCR product is 659 bp, which does not contain the 177-bp insert (Fig. 5).

mAbs DRC-1 and KiM4 have been widely used as human FDC-specific antibodies, but the target antigen(s) have not been characterized (16, 17). Interestingly, both DRC-1 and KiM4 strongly and specifically stain COS7 cells as well as L cells transfected with CD21L cDNA (not shown). This indicates that 7D6, DRC-1, and KiM4 specifically recognize the CR2/CD21L that is selectively expressed by FDCs. The weak staining of DRC-1 and KiM4 on tonsillar B cells may be explained by the cross-reaction of these two antibodies to CD21S expressed on B cells.

In conclusion, the present study demonstrates that FDC express the SCR10a-containing CD21L, and this long form of CD21 appears to be a specific cell surface marker for FDC. In contrast, B cells express the short form of CD21. The low frequency of isolation of CD21L cDNAs from a tonsillar cDNA library reported earlier was probably due to the fact that FDC are considerably less abundant in tonsil than B cells (23). Thus, FDC and B cells exhibit a cell type–specific splicing mechanism for CD21 expression.

CR2/CD21 has been shown to play key roles in B cell activation and humoral immune responses. Monoclonal anti-CD21 and a recombinant CD21-Ig fusion protein suppressed IgG responses to T cell–dependent antigens in mice (24, 25). CD21-deficient mice exhibit deficiencies in a B cell (B-1a) compartment, and in their ability to generate a T-dependent antibody response and GC reaction (26). Remarkably, antigen (hen egg lysozyme; HEL) attached to C3d (HEL-C3d) was 1,000–10,000-fold more immunogenic than HEL alone when these antigens were administered to mice (27). Several possible mechanisms have been proposed for the biological functions of CR2/ CD21: (a) long-term retaining and presenting native antigens in the form of immune complexes on FDC, (b) binding the B cell activation antigen CD23/FcεRII (28), and (c) serving as a co-receptor for B cell activation within the TAPA-1/CD19/CD21 complex (29).

The differential expression of CD21L and CD21S, respectively, on FDCs and B cells, may suggest their different functions. For example, understanding the functional significance of the additional SCR10a exon may provide a clue for explaining the mechanisms by which FDCs retain native antigen and costimulate GC B cells. The availability of anti-CD21L antibodies specific for FDCs will facilitate their purification after studies of their developmental pathway and their functional characterization.

Acknowledgments

We thank Dr. Wei-Feng Chen for contributions to the early phase of this project. We are grateful to S. Bonnet-Arnaud and M. Vatan for editorial assistance and to Dr. Chiller for support and discussions. Géraldine Grouard received a grant from Fondation Marcel Mérieux, Lyon, France.

References

1
Steinman
RM
,
Nussenzweig
MC
Dendritic cells: features and functions
Immunol Rev
1980
53
127
147
[PubMed]
2
Nossal
GJV
,
Abbot
A
,
Mitchell
J
,
Lummus
Z
Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles
J Exp Med
1968
127
277
290
[PubMed]
3
Tew
JG
,
Kosco
MH
,
Burton
GF
,
Szakal
AK
Follicular dendritic cells as accessory cells
Immunol Rev
1990
117
185
211
[PubMed]
4
Liu
YJ
,
Johnson
GD
,
Gordon
J
,
MacLennan
ICM
Germinal centers in T-cell–dependent antibody responses
Immunol Today
1992
13
17
21
[PubMed]
5
Berek
C
,
Berger
A
,
Apel
M
Maturation of the immune response in germinal centers
Cell
1991
67
1121
1129
[PubMed]
6
Jacob
J
,
Kelsoe
G
,
Rajewsky
K
,
Weiss
U
Intraclonal generation of antibody mutants in germinal centres
Nature (Lond)
1991
354
389
392
[PubMed]
7
Liu
YJ
,
Malisan
F
,
de Bouteiller
O
,
Guret
C
,
Lebecque
S
,
Banchereau
J
,
Mills
FC
,
Max
EE
,
Martinez-Valdez
H
Within germinal centers isotype switching of immunoglobulin genes occurs after onset of somatic mutation
Immunity
1996
4
241
250
[PubMed]
8
Liu
YJ
,
Joshua
DE
,
Williams
GT
,
Smith
CA
,
Gordon
J
,
MacLennan
ICM
Mechanisms of antigendriven selection in germinal centers
Nature (Lond)
1989
342
929
931
[PubMed]
9
Kosco
VMH
,
Burton
GF
,
Kapasi
ZF
,
Szakal
AK
,
Tew
JG
Antibody-forming cell induction during an early phase of germinal centre development and its delay with ageing
Immunology
1989
68
312
318
[PubMed]
10
Coico
RF
,
Siskind
GW
,
Thorbecke
GJ
Role of IgD and T cells in the regulation of the humoral immune response
Immunol Rev
1988
105
45
67
[PubMed]
11
Arpin
C
,
Dechanet
J
,
van Kooten
C
,
Merville
P
,
Grouard
G
,
Brière
F
,
Banchereau
J
,
Liu
Y-J
Generation of memory B cells and plasma cells in vitro
Science (Wash DC)
1995
268
720
722
[PubMed]
12
Burton
GF
,
Conrad
DH
,
Szakal
AK
,
Tew
JG
Follicular dendritic cells and B-cell costimulation
J Immunol
1993
150
31
38
[PubMed]
13
Grouard
G
,
de Bouteiller
O
,
Banchereau
J
,
Liu
Y-J
Human follicular dendritic cells enhance cytokine dependent growth and differentiation of CD40 activated B cells
J Immunol
1995
155
3345
3352
[PubMed]
14
Lindhout
E
,
Mevissen
ML
,
Kwekkeboom
J
,
Tager
JM
,
de-Groot
C
Direct evidence that human follicular dendritic cells (FDC) rescue germinal center cells from death by apoptosis
Clin Exp Immunol
1993
91
330
336
[PubMed]
15
Kosco-Vilbois
VMH
,
Gray
D
,
Scheidegger
D
,
Julius
M
Follicular dendritic cells help resting B cells to become effective antigen-presenting cells: induction of B7/ BB1 and upregulation of major histocompatibility complex class II molecules
J Exp Med
1993
178
2055
2066
[PubMed]
16
Naiem
M
,
Gerdes
J
,
Abdulaziz
Z
,
Stein
H
,
Mason
DY
Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue
J Clin Pathol (Lond)
1983
36
167
175
[PubMed]
17
Parwaresch
MR
,
Radzun
HJ
,
Feller
AC
,
Peters
KP
,
Hansmann
ML
Peroxidase-positive mononuclear leukocytes as possible precursors of human dendritic reticulum cells
J Immunol
1983
131
2719
2725
[PubMed]
18
Ho
AS-Y
,
Liu
Y
,
Khan
TA
,
Hsu
D-H
,
Bazan
JF
,
Moore
WK
A receptor for interleukin-10 is related to interferon receptors
Proc Natl Acad Sci USA
1993
90
11267
11271
[PubMed]
19
Elliott
JF
,
Albrecht
GR
,
Gilladoga
A
,
Handunetti
S
,
Neequaye
J
,
Lallinger
G
,
Minjas
JN
,
Howard
RJ
Genes for plasmodium falciparum surface antigens cloned by expression in COS cells
Proc Natl Acad Sci USA
1990
87
6363
6367
[PubMed]
20
MacNeil
I
,
Kennedy
J
,
Godfrey
DI
,
Jenkins
NA
,
Masciantonio
M
,
Mineo
C
,
Gilbert
DJ
,
Copeland
NG
,
Boyd
RI
,
Zlotnik
A
Isolation of a cDNA encoding thymic shared antigen-1. A new member of the Ly6 family with a possible role in T cell development
J Immunol
1993
151
6913
6923
[PubMed]
21
Moore
MD
,
Cooper
NR
,
Tack
BF
,
Nemerow
GR
Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes
Proc Natl Acad Sci USA
1987
84
9194
9198
[PubMed]
22
Fujisaku
A
,
Harley
JB
,
Frank
MB
,
Gruner
BA
,
Frazier
B
,
Holers
VM
Genomic organization and polymorphisms of the human C3d/Epstein-Barr virus receptor
J Biol Chem
1989
264
2118
2125
[PubMed]
23
Weis
JJ
,
Toothaker
LE
,
Smith
JA
,
Weis
JH
,
Fearon
DT
Structure of the human B lymphocyte receptor for C3d and the Epstein-Barr virus and relatedness to other members of the family of C3/C4 binding proteins [erratum published 168:1953–1954]
J Exp Med
1988
167
1047
1066
[PubMed]
24
Heyman
B
,
Wiersma
EJ
,
Kinoshita
T
In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody
J Exp Med
1991
172
665
668
[PubMed]
25
Hebell
T
,
Ahearn
JM
,
Fearon
DT
Suppression of the immune response by a soluble complement receptor of B lymphocytes
Science (Wash DC)
1991
254
102
105
[PubMed]
26
Ahearn
JM
,
Fisher
MB
,
Croix
D
,
Goerg
S
,
Ma
MH
,
Xia
JR
,
Zhou
XN
,
Howard
RG
,
Rothstein
TJ
,
Carroll
MC
Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen
Immunity
1996
4
251
262
[PubMed]
27
Dempsey
PW
,
Allison
ME
,
Akkaraju
S
,
Goodnow
CC
,
Fearon
DT
C3d of complement as a molecular adjuvant: bridging innate and acquired immunity
Science (Wash DC)
1996
271
348
350
[PubMed]
28
Aubry
JPS
,
Pochon
S
,
Graber
P
,
Jansen
KU
,
Bonnefoy
JY
CD21 is a ligand for CD23 and regulates IgE production
Nature (Lond)
1992
385
505
507
29
Fearon
DT
,
Carter
RH
The CD19/CR2/ TAPA-1 complex of B lymphocytes: linking natural to acquired immunity
Annu Rev Immunol
1995
13
127
149
[PubMed]
30
Ahearn
JM
,
Fearon
DT
Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21)
Adv Immunol
1989
46
183
219
[PubMed]

The present address of J. Xu is Corixa Corporation, Seattle, WA 88104.

Author notes

Address correspondence to Dr Yong-Jun Liu, Schering-Plough, Laboratory for Immunological Research, 69571 Dardilly Cedex, France.