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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 216-224
By
From the Departments of Pathology, Microbiology, and Immunology,
University of Arkansas for Medical Sciences, Little Rock, AR.
Follicular dendritic cells (FDCs) reside within germinal centers of
secondary lymphoid tissue where they play a critical role in
antigen-driven immune responses. FDCs express numerous adhesion molecules that facilitate cellular interactions with B and T cells within the germinal center microenvironment. Although human FDCs have
been shown to influence B-cell development, very little is known about
the ability of FDCs to regulate T-cell responses. To investigate this
functional aspect of FDCs, highly enriched preparations were isolated
by magnetic cell separation using the FDC-restricted monoclonal
antibody HJ2. We found that isolated human FDCs inhibited proliferation
of both autologous and allogeneic T cells, and were dependent on the
number of FDCs present. Inhibition by FDCs was observed using two
serologically distinct superantigens at multiple concentrations
(Staphylococcus enterotoxin A and B). In contrast, B cells
failed to inhibit, and often augmented superantigen-induced T-cell
proliferation. Antibody-blocking studies showed that CD54 and CD106
were involved in the ability of FDC to inhibit T-cell proliferative
responses. When FDCs and T cells were separated by a semipermeable
membrane, the inhibitory effect was partially abrogated, demonstrating
that in addition to cell-cell interactions, a soluble factor(s) was
also involved in the process. The addition of indomethicin to cultures
improved the proliferative response in the presence of FDCs, indicating
that inhibition was mediated, in part, by prostaglandins. These results
indicate that FDCs regulate T-cell proliferation by two molecular
mechanisms and that FDC:T-cell interactions may play a pivotal role in
germinal center development.
FOLLICULAR DENDRITIC CELLS
(FDCs) are distinct antigen-presenting cells that reside within
germinal centers (GCs) of secondary lymphoid tissue.1
Although FDCs are a minor component of GCs, comprising only 1% to 2%
of all cells, they exhibit numerous cytoplasmic extensions that form an
extensive network throughout the GC.1 This enables FDCs to
be in intimate contact with neighboring B cells (90% of GC cells) and
T cells (5% to 10% of GC cells).1-3 FDCs express large
amounts of the adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) that
facilitate cellular interactions.3,4 Indeed, in vitro
binding studies have shown that adhesion between either B cells or T
cells with FDCs is mediated by adhesion pathways involving interactions
between CD54 with CD11a/CD18 (LFA-1 Because FDCs represent a minor population of cells within lymphoid
tissue, functional studies have been hard to perform because of
difficulties in obtaining adequate numbers of highly purified human
FDCs. Several studies have been performed using FDC clusters containing
approximately 1 FDC to 20 contaminating lymphocytes. FDC clusters have
been shown to prolong survival,16,17 augment proliferation,16-18 and promote differentiation of B
cells.18,19 Other groups have studied FDC function by
generating FDC-like cell lines. These lines were produced by culturing
FDC-enriched tonsil cell preparations with or without exogenous
cytokines,20-22 by Epstein-Barr virus
transformation,23 or by fusion and immortalization with a
mouse myeloma cell line.24 Studies have shown that FDC-like cells promote B-cell survival,22,23
differentiation,25 and either augment or inhibit B-cell
proliferation.20-23 However, the origin of these FDC-like
cell lines is questionable because of the impurity of the starting
population and the observation that surface antigens associated with
freshly isolated FDCs are often lost upon culture.
Monoclonal antibodies (MoAbs) that are relatively specific for human
FDCs have been developed, such as HJ2,26 7D6,4
DRC-1,27 and Ki-M4.28 These antibodies can be
used to identify FDCs in situ and for isolating FDCs in single-cell
suspension. 7D6, DRC-1, and Ki-M4 have recently been shown to recognize
an isoform of CD21 (long form) that is expressed by human FDCs and not
B cells.29 It is important to note that these
FDC-associated antibodies do not react with other types of dendritic
cells such as GC dendritic cells30 and interdigitating
dendritic cells found in T-cell areas of lymphoid
tissue.30,31 A limited number of studies have been
performed using highly purified human FDCs. Interestingly, isolated
FDCs were shown to either inhibit or augment B-cell proliferation, depending on the stimulating agent that was used.32,33 For instance, purified FDCs inhibited B-cell proliferation when
Staphylococcus aureus strain Cowan I was used as the activating
agent,32 whereas B-cell proliferation was augmented using
antibodies against CD40 or surface immunoglobulin.33
Although further studies are needed to identify specific molecules
produced by isolated FDCs that control B-cell responses, these data
show that human FDCs regulate B-cell responses and, as a result, play a
pivotal role in B-cell development within GCs.
Although FDC:B-cell interactions have been studied, the ability of
human FDCs to regulate T-cell responses has not been examined. This is
important because T cells are critical for GC formation34 and provide B-cell help through cellular interactions and the production of cytokines.35 It is well known that FDCs do
not process antigens.36 However, FDCs can acquire processed
antigens consisting of major histocompatibility complex (MHC) class
II-peptide complexes from neighboring B cells for presentation to T
cells.37 Indeed, murine studies have shown that FDCs
express processed antigens in a form capable of inducing
antigen-specific T-cell proliferation.37 With the long-term
goal of characterizing molecular mechanisms by which FDCs control
T-cell responses, we examined the ability of highly purified human FDCs
to regulate T-cell proliferative responses. We discovered that purified
human FDCs inhibit superantigen (sAg)-induced T-cell proliferation by
two distinct mechanisms: one is mediated by cellular interactions
involving CD54 and CD106, and the other is mediated by prostaglandins (PGs).
Antibodies and reagents.
Tissue-culture supernatants from hybridomas (purchased from American
Type Culture Collection, Rockville, MD) producing the following mouse
MoAbs were used: OKT3 (anti-CD3, IgG2a); Lym-1 (anti-DR, IgG2a); HB180
(anti-class II, IgG2a); HB45 (anti-kappa light chain, IgG1); CRL1763
(anti-lambda light chain, IgG2a); and OKM1 (anti-CD11b, IgG2b). HJ2, an
IgM mouse MoAb that recognizes human FDCs has been previously
described.26 Ascites containing Ki-M4 (IgG3) was kindly
provided by Dr M. Parwaresch (University of Kiel, Kiel,
Germany). R-phycoerythrin (PE)-conjugated mouse MoAbs
against CD19, DR, CD3, CD4, CD13, and CD22 were obtained from Becton
Dickinson (Mountain View, CA). Purified MoAbs against CD106 (VCAM-1,
IgG1) and CD86 (B7-2, IgG1) were obtained from PharMingen (San Diego,
CA), and MoAbs against CD54 (ICAM-1, IgG1) and CD80 (B7-1, IgG1) were
obtained from Immunotech (Westbrook, ME). Indomethicin, and the
bacterial sAgs, Staphylococcus enterotoxin A (SEA) and B (SEB),
were obtained from Sigma Chemical Co (St Louis, MO).
FDC isolation.
Tonsils were obtained from Arkansas Children's Hospital after routine
tonsillectomies. Tonsil tissue was cut into small fragments and
digested in 20 mL of RPMI 1640 containing 2 mg/mL collagenase (type IV;
Worthington Biochemical Corp, Freehold, NJ), 0.1 mg/mL DNase (type I;
Sigma Chemical Co), 10% fetal bovine serum (FBS; Atlanta Biologicals,
Inc, Norcross, GA), and 5 mmol/L EDTA for 1 hour at 4°C, as
described.3 Residual red blood cells and dead cells were
eliminated by centrifugation over a Ficoll-Hypaque density gradient
(Sigma Chemical Co). The resulting cells were resuspended at
107/mL and centrifuged at 250g for 4 minutes. The
low-density cells in the supernatant contained the majority of FDCs
(3% to 10% FDCs) and was used to obtain highly purified FDCs.
Low-density cells were stained with HJ2 (25 µL of tissue-culture
supernatant per 5 × 105 cells), followed by rat
anti-mouse IgM-coated microbeads (15 µL beads per 107
cells; Miltenyi Biotec Inc, Auburn, CA) and fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse IgM antibodies (µ-chain specific with no cross-reactivity with IgG antibody; Jackson ImmunoResearch Laboratories, West Grove, PA). HJ2+ cells (FDCs) were
isolated by positive selection using the magnetic cell separation
system from Miltenyi. Labeled cells were applied to a magnetic
separation column (type WHM) equilibrated with column buffer according
to the manufacturer's instructions. Flow rates through the column were
10 to 15 drops per minute. The column was removed from the magnet and
adherent cells were obtained (HJ2+ cells) by forcing the
cells through the column using 1 mL of column buffer and the rubber
plunger supplied by the manufacturer. Adherent cells were subsequently
reapplied to a second column and collected as above. We typically
obtained between 6 and 12 × 105 cells after
application to two columns when starting with a cell suspension from an
individual tonsil donor. The purity of the FDC preparations used in all
the described experiments was always greater than 80%, based on
HJ2+ cells.
T- and B-cell isolation.
T cells and B cells were isolated from the FDC-depleted cell fraction
(cells in the pellet after low-speed centrifugation) by negative
selection using the magnetic cell separation system described above.
Single-cell suspensions were stained with MoAbs against class II
molecules (Lym-1 and HB180), kappa light chain (HB45), lambda light
chain (CRL1763), and monocytes (HB180), or antibodies against T cells
(CD3) and monocytes (OKM1) to isolate T cells and B cells,
respectively. After incubation with goat anti-mouse IgG-coated
microbeads, cells were applied to a magnetic separation column
(Miltenyi Biotec Inc) and cells passing through the column when
attached to the magnet (nonadherent) were collected. The purity of T
cells and B cells isolated by this method was always greater than 90%,
based on reactivity with CD3 (T-cell purity) or CD19 (B-cell purity).
For experiments requiring more highly purified T cells, the nonadherent
cells were applied to a second magnetic separation column and the
nonadherent cells collected. This resulted in a tonsil T-cell
preparation (>97% CD3+) that failed to proliferate to
sAg without adding APCs.
Flow cytometry.
Single-cell suspensions (0.5 to 1 × 105 cells) of
HJ2+ cells (labeled with HJ2 and FITC-conjugated goat
anti-mouse IgM antibodies) were stained with PE-conjugated MoAbs
against CD19, DR, CD3, CD4, CD13, and CD22. Cells were incubated on ice
with each antibody for 20 minutes followed by two washes with
phosphate-buffered saline. HJ2+ cells were also stained
with Ki-M4 followed by PE-conjugated goat anti-mouse IgG antibodies
(Tago, Burlingame, CA). The anti-mouse IgG reagent was IgG-specific and
exhibited no cross-reactivity with mouse IgM antibodies. Flow cytometry
was performed on a FACScan (Becton Dickinson) using WinMDI software.
The instrument was calibrated with CaliBRITE beads (Becton Dickinson)
using AutoCOMP software. Dead cells were excluded from analysis and
10,000 cells were collected either ungated (HJ2 v Ki-M4), or
gated based on HJ2 positivity.
Proliferation assays.
Varying numbers of FDCs or B cells (2 × 104 to 1.25 × 103; Boyden chamber assays.
To physically separate T cells from FDCs or B cells, some proliferation
assays were performed using polyethylene terephtalate cell culture
inserts with 3-µm pores (Becton Dickinson) that were placed into
wells of a 24-well cluster plate. In preliminary experiments, we
determined that optimal T-cell proliferation was obtained by culturing
4 × 105 T cells and SEB in cell culture inserts in a
500-µL final volume. FDCs or B cells ( Statistics.
Means from individual experiments were used and background cpm (T cells
alone) were subtracted from experimental values for all statistical
analyses. T-cell cultures stimulated with sAg in the presence of either
FDC or B cells were compared to cultures containing T cells and sAg
using a paired t-test. A one-way analysis of variance (ANOVA)
test and Tukey's post-hoc analysis were used for comparing multiple
experimental groups. Experiments involving antibody treatment (blocking
studies) or indomethicin were compared using an ANOVA and Tukey's
post-hoc analysis. A P value <.05 was considered
statistically significant. Statistical analysis was performed using
SigmaStat for Windows 2.0 software (Jandel Corp, San Rafael, CA).
FDCs downregulate autologous T-cell proliferation in response to sAgs.
FDCs were isolated from human tonsil tissue using HJ2, a mouse MoAb
that binds human FDCs both in tissue sections and in single-cell suspension.26 Low-density tonsil cells (enriched for FDCs)
were stained with HJ2 followed by anti-mouse IgM microbeads, and
HJ2+ cells were isolated by magnetic cell separation. Cells
obtained by this method were always greater than 80% HJ2+,
and virtually all of the HJ2+ cells were also
Ki-M4+ (Fig 1A), confirming
their identity as FDCs and distinguishing these cells from the
interdigitating dendritic cells found in T-cell regions of tonsils that
are Ki-M4
FDCs downregulate allogeneic T-cell proliferation.
We next examined the ability of human FDCs to regulate sAg-induced
proliferation using allogeneic tonsil T cells, because sAg presentation
by APC is not MHC class II-restricted.38 As shown in Fig
2C, FDCs significantly inhibited SEB-induced allogeneic T-cell proliferation in an FDC dose-dependent manner by 54% and 21%
at FDC to T-cell ratios of 1:5 and 1:20, respectively (n = 5, P < .05). Similar results were obtained using SEA to induce allogeneic
T-cell proliferative responses (Fig 2D). FDCs also significantly downregulated tetanus toxoid-induced allogeneic T-cell
proliferation (52% to 62% reduction at a 1:5 ratio at 2 tetanus
toxoid concentrations), indicating that the inhibitory nature of FDCs
is a general finding not restricted to sAg-induced T-cell proliferation
(data not shown). Thus, inhibition of T-cell proliferation is a unique
functional property associated with FDCs that is not dependent on the
type or dose of stimulating agent used, and is active in both
autologous and allogeneic culture systems.
FDCs can present sAgs to T cells.
The observation that FDCs inhibit rather than stimulate T-cell
proliferation prompted us to examine whether FDCs can present sAg and
stimulate T-cell proliferative responses in the absence of other APCs.
For these experiments, highly purified tonsil T cells were isolated
that were free from contamination with residual APCs (cells were passed
over a second magnetic separation column). As shown in Fig 3 (n = 3),
highly purified T cells were unable to proliferate to sAg, based on a
proliferative response that was only 3% of the response observed when
B cells were added at a 1:5 ratio (maximum proliferative response). The
addition of FDCs to cultures containing T cells and SEB resulted in a
modest increase in T-cell proliferation that was only 18% of the
response observed using B cells at the same ratio. Interestingly, when smaller numbers of FDCs were used, T-cell proliferation dramatically increased (Fig 3). For
instance, the addition of FDCs at a 1:20 and 1:80 ratio resulted in
augmentation of the T-cell proliferative response by 41% and 73%
(P < .05), respectively, compared with the response using
FDCs at 1:5. T-cell proliferation using FDCs at a 1:80 ratio was
comparable to the response obtained using B cells at a much higher
ratio of 1:5. Thus, FDCs can present sAg and provide costimulatory
signals that induce vigorous T-cell proliferation when FDCs are used in
limiting numbers.
Inhibition of T-cell proliferation by FDCs is mediated by CD54 and
CD106.
FDCs may alter T-cell proliferative responses by delivering inhibitory
signals during cellular interactions and/or through the production of
biologically active molecules. To determine the role of cellular
contact in the ability of FDCs to downregulate T-cell proliferation,
antibodies against molecules expressed on the surface of FDCs were used
to block specific FDC:T-cell interactions. Because human FDCs express
large quantities of the adhesion molecules CD54 and
CD106,4,5 FDCs were treated with purified antibodies against these molecules before addition to T-cell cultures. For these
studies, FDCs were used at an FDC to T-cell ratio of 1:5. Residual
unbound antibody was removed by extensive washing. As shown in
Fig 4A (n = 5), pretreatment of FDCs with
either anti-CD54 or anti-CD106 antibodies resulted in an increase in
T-cell proliferation from 47% in untreated cultures to 82% and 86%,
respectively. Pretreatment of FDCs with an isotype-matched irrelevant
control antibody failed to reverse the inhibitory effect of FDCs on
T-cell proliferation (data not shown). Further increases in T-cell
proliferation in the presence of FDCs were not observed using higher
antibody concentrations (data not shown). In addition, pretreatment of
T cells with antibodies against CD54 or CD106 failed to alter
SEB-induced T-cell proliferation (data not shown), indicating that FDC
function and not T-cell function was blocked by the antibody treatment.
We also examined the regulatory role of the costimulatory molecules
CD80 and CD86 and found that pretreatment of FDCs with antibodies
against CD80 and CD86 failed to reverse the ability of FDC to inhibit
T-cell proliferation in response to SEB (Fig 4A). In contrast, the
ability of B cells to augment T-cell proliferation at a 1:5 ratio was abrogated by pretreating B cells with either anti-CD80 or anti-CD86 antibodies at a similar concentration (Fig 4B), showing that these antibodies were capable of blocking cellular interactions in our culture system.
Inhibition of T-cell proliferation by FDCs can be partially overcome
by preventing cellular contact.
To further evaluate the role of cellular interactions on the ability of
FDCs to inhibit T-cell proliferative responses, FDCs and T cells were
cultured in chambers separated by a membrane that prevents cellular
interactions while allowing free exchange of soluble molecules between
chambers. As expected, addition of FDCs to the chamber containing T
cells (insert) resulted in a significant reduction in SEB-induced
T-cell proliferation of 63%, compared to T cells cultured only with
SEB (Table 1, n = 3). When FDCs were
separated from T cells by a membrane (FDC in the well and T cells in
the insert), the T-cell proliferative response increased by 19%
(P < .05). Although T-cell proliferation was not completely
restored when FDCs were separated from T cells, these data indicate
that FDCs produce a soluble factor(s) that has anti-T-cell
proliferative activity. In contrast to FDCs, the ability of B cells to
augment SEB-induced T-cell proliferation was compromised when B cells
were placed in a separate chamber, indicating that B cells augment
SEB-induced T-cell proliferation through cellular interactions (Table
1).
Inhibition of T-cell proliferation by FDCs is partially reversed by
indomethicin.
It has previously been reported that FDCs within lymphocyte clusters
are capable of producing prostaglandin E2
(PGE2) .41 Because PGE2
has been shown to inhibit peripheral blood T-cell proliferation,42 one possible mechanism by which FDCs
inhibit sAg-induced T-cell proliferation is through the production of PGs. To examine the role of PGs in this process, indomethicin was used
to block PG production by FDCs. Indomethicin acts on the cyclooxygenase
pathway and blocks production of several PGs, including
PGE2.43 The addition of indomethicin to
cultures containing FDCs and T cells at a 1:5 ratio resulted in a
significant increase in T-cell proliferation of 50%, compared to
cultures containing FDC without indomethicin
(Fig 5, n = 3). In the absence of FDCs, SEB-induced T-cell proliferation was not altered by indomethicin, indicating that the inhibitory effect of FDCs is mediated through the
production of PGs by FDCs and not a direct effect of indomethicin on T
cells (Fig 5).
Results from this study show that isolated human FDCs have the ability
to downregulate sAg-induced T-cell proliferative responses. The ability
of FDC to inhibit T-cell proliferation was observed when FDCs were
isolated using either HJ2 or antibodies against CD14 (data not shown),
suggesting that the inhibitory effect is not mediated through
cross-linking the HJ2 antigen because HJ2 and anti-CD14 antibodies
recognize different surface molecules (unpublished data).
To our knowledge, this is the first report examining regulation of
T-cell proliferative responses by human FDCs. Interestingly, T-cell
proliferation was significantly inhibited at FDC concentrations that
mimic the ratio of FDCs to T cells normally found within GCs (1:5
ratio), based on immunohistological studies of tonsil tissue. Although
the majority of studies were performed with SEB, similar results were
obtained with SEA, a serologically distinct sAg, with limited sequence
homology to SEB.40 Because SEA activates T cells expressing
TCR V We thank Dr Moon H. Nahm for providing HJ2 antibody and for his
continued encouragement, and Dan Ayers for statistical consultation.
Submitted October 8, 1998; accepted February 18, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Anthony W. Butch, PhD,
Department of Pathology, Slot 502, University of Arkansas for Medical
Sciences, Little Rock, AR 72212; e-mail: twbutch{at}bloodbank.uams.edu.
1.
Tew JG, Thorbecke GJ, Steinman RM:
Dendritic cells in the immune response.
J Reticulo Society
31:371, 1982
2.
Nieuwenhuis P, Opstelten D:
Functional anatomy of germinal centers.
Am J Anat
170:421, 1984[Medline]
[Order article via Infotrieve]
3.
Schriever F, Freedman AS, Freeman G, Messner E, Lee G, Daley J, Nadler LM:
Isolated follicular dendritic cells display a unique antigenic profile.
J Exp Med
169:2043, 1989
4.
Liu YJ, Grouard G, de Bouteiller O, Banchereau J:
Follicular dendritic cells and germinal centers.
Int Rev Cyto
166:139, 1996
5.
Koopman G, Parmentier HK, Schuurman HJ, Newman W, Meijer CJLM, Pals ST:
Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways.
J Exp Med
173:1297, 1991
6.
Schriever F, Korinth D, Salahi A, Lefterova P, Schmidt-Wolf IGH, Behr SI:
Human T lymphocytes bind to germinal centers of human tonsils via integrin
7.
Tew JG, Phipps RP, Mandel TE:
The maintenance and regulation of the humoral immune response: Persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells.
Immunol Rev
53:175, 1980[Medline]
[Order article via Infotrieve]
8.
Reynes M, Aubert JP, Cohen JHM, Audouin J, Tricottet V, Diebold J, Kazatchkine D:
Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens.
J Immunol
135:2687, 1985[Abstract]
9.
Szakal AK, Holmes KL, Tew JG:
Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic cell morphology.
J Immunol
131:1714, 1983[Abstract]
10.
Szakal AK, Hanna MG:
The ultrastructure of antigen localization and virus-like particles in mouse spleen germinal centers.
Exp Mol Pathol
8:75, 1988
11.
Kosco MH, Szakal AK, Tew JG:
In vivo obtained antigen presented by germinal center B cells to T cells in vitro.
J Immunol
140:354, 1988[Abstract]
12.
Kraal G, Weissman IL, Butcher EC:
Germinal center cells: Antigen specificity, heavy chain class expression and evidence of memory.
Adv Exp Med Biol
186:145, 1985[Medline]
[Order article via Infotrieve]
13.
Coico RF, Bhogal BS, Thorbecke GJ:
Relationship of germinal centers in lymphoid tissue to immunologic memory. VI. Transfer of B cell memory with lymph node cells fractionated according to their receptors for peanut agglutinin.
J Immunol
131:2254, 1983[Abstract]
14.
Jacob J, Kelsoe G, Rajewsky K, Weiss U:
Intraclonal generation of antibody mutants in germinal centres.
Nature
354:389, 1991[Medline]
[Order article via Infotrieve]
15.
Berek C, Berger A, Apel M:
Maturation of the immune response in germinal centers.
Cell
67:1121, 1991[Medline]
[Order article via Infotrieve]
16.
Cormann N, Heinen E, Kinet-Denoel C, Tsunoda R, Simar LJ:
Isolation of follicular dendritic cells from human tonsils and adenoids.
Adv Exp Med Biol
237:171, 1988[Medline]
[Order article via Infotrieve]
17.
Tsunoda R, Cormann N, Heinen E, Lesage F, Kinet-Denoel C, Simar LJ:
Immunohistochemical study on cultured FDC-C enriched lymphoid cell populations.
Adv Exp Med Biol
237:177, 1988[Medline]
[Order article via Infotrieve]
18.
Grouard G, de Bouteiller O, Banchereau J, Liu YJ:
Human follicular dendritic cells enhance cytokine-dependent growth and differentiation of CD40-activated B cells.
J Immunol
155:3345, 1995[Abstract]
19.
Cormann N, Lesage F, Heinen E, Schaaf-Lofontaine N, Kinet-Denoel C, Simar LS:
Isolation of follicular dendritic cells from human tonsils and adenoids. V. Effect on lymphocyte proliferation and differentiation.
Immunol Lett
14:29, 1986[Medline]
[Order article via Infotrieve]
20.
Tsunoda R, Bosseloir A, Onozaki K, Heinen E, Miyake K, Okamura HO, Suzuki K, Fujita T, Simar LJ, Sugai N:
Human follicular dendritic cells in vitro and follicular dendritic-cell-like cells.
Cell Tissue Res
288:381, 1997[Medline]
[Order article via Infotrieve]
21.
Clark EA, Grabstein KH, Shu GL:
Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.
J Immunol
148:3327, 1992[Abstract]
22.
Kim HS, Zhang X, Klyushnenkova E, Choi YS:
Stimulation of germinal center B lymphocyte proliferation by an FDC-like cell line, HK.
J Immunol
155:1101, 1995[Abstract]
23.
Lindhout E, Lakeman A, Mevissen MLCM, de Groot C:
Functionally active Epstein-Barr virus-transformed follicular dendritic cell-like cell lines.
J Exp Med
179:1173, 1995
24.
Orscheschek K, Merz H, Schlegelberger B, Feller AC:
An immortalized cell line with features of human follicular dendritic cells. Antigen and cytokine expression analysis.
Eur J Immunol
24:2682, 1994[Medline]
[Order article via Infotrieve]
25.
Clark EA, Grabstein KH, Gown AM, Skelly M, Kaisho T, Hirano T, Shu GL:
Activation of B lymphocyte maturation by a human follicular dendritic cell line, FDC-1.
J Immunol
155:545, 1995[Abstract]
26.
Butch AW, Hug BA, Nahm MH:
Properties of human follicular dendritic cells purified with HJ2, a new monoclonal antibody.
Cell Immunol
155:27, 1994[Medline]
[Order article via Infotrieve]
27.
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
36:167, 1983
28.
Parwaresch MR, Radzun HJ, Hansmann ML, Peters KP:
Monoclonal antibody Ki-M4 specifically recognizes human dendritic reticulum cells (follicular dendritic cells) and their possible precursor in blood.
Blood
62:585, 1983
29.
Liu YJ, Xu J, de Bouteiller O, Parham CL, Grouard G, Bjossou O, DeSaint-Vis B, Lebecque S, Banchereau J, Moore KW:
Follicular dendritic cells specifically express the long CR2/CD21 isoform.
J Exp Med
185:165, 1997 |
TOP
ABSTRACT
INTRODUCTION
/
) and CD106 with CD49d
(VLA-4).
-irradiated 3,000 rads) were
cultured with 105 T cells in proliferation media (RPMI-1640
containing 10% FBS, L-glutamine, and penicillin-streptomycin) for 3 days (autologous assays) or 5 days (allogeneic assays) at 37°C in
5% CO2. Each stimulation condition was performed in
triplicate wells of a 96-well microtiter plate in a final volume of 200 µL. Indomethicin (100 µg/mL) and sAg (SEA or SEB at 100 ng/mL) were
added at the initiation of each culture. Where indicated, FDCs and B
cells (2 × 104 cells) were pretreated with 1 µg/mL
of MoAb against various surface molecules for 30 minutes at 37°C.
Cells were extensively washed to remove unbound antibody before their
addition to T-cell cultures. One microcurie of tritiated
thymidine (New England Nuclear, Boston, MA) was added during the last
24 hours of culture and the contents of each well was harvested onto
glass fiber filters using a Skatron cell harvester (Sterling, VA), and
counted for radioactivity after being placed in a aqueous scintillation
cocktail. To normalize for the variability among individual
experiments, background cpm (T cells alone) were subtracted from all
experimental values. The means of triplicates from individual
experiments were then pooled. Data points were expressed as the mean
cpm + SEM or as a percentage of the maximum response, as indicated.
and
CD19
chains differing from those recognized by
SEB,