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*
Division of Molecular Immunology, Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021;
The Immunology Program, Cornell University Weill Graduate School of Medical Sciences, New York, NY 10021; and
Immunex Corporation, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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S
, SµS
,
and SµS
class switch DNA recombination (CSR). This inhibition
is associated with decreased TNFR-associated factor-2 binding to CD40,
decreased NF-
B binding to the CD40-responsive element of the C3
promoter, decreased I3-C3 germline gene transcription, and
decreased expression of Ku70, Ku80, DNA protein kinase,
switch-associated protein-70, and Msh2 CSR-associated transcripts. In
addition, CD153 engagement inhibits IgG, IgA, and IgE production, and
this effect is associated with reduced levels of B lymphocyte
maturation protein-1 transcripts, and increased binding of B
cell-specific activation protein to the Ig 3' enhancer. These findings
suggest that CD30+ T cells modulate CSR as well as IgG,
IgA, and IgE production by inducing reverse signaling through B cell
CD153. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (10, 11),
a transcription factor that, together with IL-4-induced STAT-6, plays a
critical role in the germline transcription of downstream Ig
CH genes (12, 13). Within 2–3 days,
IH-CH germline
transcription of a targeted CH gene induces class
switch DNA recombination (CSR) to the same gene (12).
Although the inducers of CSR are largely known, the mechanisms deputed
to the negative modulation of this process remain elusive. Ag-stimulated CD4+ T cells initiate the Ab response by activating naive B cells in the T cell-rich extrafollicular areas of secondary lymphoid organs (4, 12). Upon recruitment to the lymphoid follicle, activated B cells undergo proliferation, germinal center (GC) phenotypic differentiation, Ig V(D)J gene somatic hypermutation, and Ig class switching (12, 14). Within the GC, Ag-activated CD95L (FasL)+ T cells modulate Ig production by deleting autoreactive and low affinity B cells through CD95 (Fas), a TNFR family member expressed by B cells upon CD40 engagement by CD154 (15, 16). Among CD95+ GC B cells, only high affinity (Ag-selected) B cells are rescued from CD95-mediated apoptosis and differentiate into Ig class-switched memory B cells or plasma cells (14, 15, 16, 17).
Recent studies suggest that additional T cell-dependent mechanisms modulate B cell selection in the GC. In the presence of CD154, IgD+ IgM+ B cells express CD30, a death domain-less TNFR family member (18). In these cells, CD30 engagement by CD153 (CD30L) on CD8+ T cells inhibits CD154 and IL-4-induced Ig class switching. Because B cell Ag receptor (BCR, surface Ig) engagement down-regulates CD30 expression (18), CD30 would represent a CD95-independent pathway limiting the CD40-mediated GC progression of low affinity B cells. In these cells, CD30 and CD95 might constitute complementary checkpoints at distinct B cell differentiation stages, as CD30 and CD95 are mainly expressed by perifollicular activated B cells and GC B cells, respectively (16, 17, 19).
The finding that CD95- and CD30-mediated signals are mainly effective in non-Ag-selected B cells suggests that additional mechanisms limit CSR and Ab production in B cells that have been positively selected by Ag. Few days after CD28 and IL-4R engagement, Ag-activated T cells express CD30 while undergoing clonal expansion as part of a specific immune response (20, 21). The expression of T cell CD30 is up-regulated in disorders associated with dysregulated IgG, IgA, and/or IgE production, including autoimmune and allergic diseases (22, 23, 24). This observation, together with the notion that human mature B cells express CD153 (CD30L) (25, 26, 27), prompted us to hypothesize that engagement of B cell CD153 by T cell CD30 modulates CSR.
CD153 is a member of the TNFL superfamily, a group of type II transmembrane glycoproteins that includes CD27L (CD70), FasL (CD95L), OX40L (CD134L), 4-1BB ligand (CD137L), and CD40L (CD154) (28). Several studies have shown that, while binding to their receptors, these molecules, including CD134L and CD154, elicit important biologic effects (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). The cytoplasmic domain of CD153 consists of 38 aa and is highly conserved across species (28, 42), suggesting that, like CD134L or CD154, whose cytoplasmic tails consists of 24 and 22 aa, respectively, CD153 can transmit intracellular signals. In agreement with this possibility, CD153 cross-linking by an immobilized agonistic mAb or a human CD30:Fc IgG1 chimeric protein has been shown to induce gene expression and metabolic activation in human T cells and neutrophils (43). The function of CD153 in human B cells remains unknown.
In this study, we show that, in the presence of CD154, IL-4, and BCR engagement, human IgD+ IgM+ B cells up-regulate surface CD153. In these cells, engagement of CD153 by T cell CD30 inhibits CSR as well as IgG, IgA, and IgE production. Our findings suggest that, by inducing reverse signaling through CD153, CD30+ T cells modulate the CD154-dependent entry of IgD+ and/or IgM+ B cells, including Ag-selected CD30- B cells, into the pool of B cells producing IgG, IgA, or IgE Abs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CL-01 B cell line has been described (2, 3, 13, 18, 44, 45). PB or tonsil B cells were isolated, as reported
(13, 44). IgD+,
IgD-, IgG+, and
IgG- B cell fractions were magnetically
segregated upon B cell incubation with FITC-conjugated mouse or goat
Abs to IgD or IgG (Southern Biotechnology Associates, Birmingham, AL)
and anti-FITC MicroBeads (Miltenyi Biotec, Auburn, CA). Tonsil
IgD+ CD38- and
IgD+ CD38+ B cells were
selected upon incubation of purified IgD+ B cells
with MultiSort Release Reagent (Miltenyi Biotec), FITC-conjugated mouse
mAb to CD38 (PharMingen, San Diego, CA), and anti-FITC MicroBeads.
Tonsil IgD- CD38+ and
IgD- CD38- B cells were
segregated in a similar fashion from purified
IgD- B cells. Plasmacytoid differentiation was
induced by culturing B cells with 1 µg/ml of htCD154 (Immunex,
Seattle, WA), 100 U/ml of IL-2 (Life Technologies, Grand Island, NY),
100 U/ml of IL-4, and 200 ng/ml of IL-10 (Schering-Plough, Kenilworth,
NJ). Under these conditions, activated B cells produced large amounts
of IL-6 (3). To obtain CD4+
CD30+ T cells, PBMCs were stimulated with 100
U/ml of IL-4 (Schering-Plough) and 1 µg/ml of anti-CD3 (from Dr.
K. Smith, Weill Medical College of Cornell University, New York, NY),
anti-CD28 (PharMingen), and anti-CD134 (anti-OX40; a gift
from Dr. Y. Tozawa, Kitasato University, Kanagawa, Japan) mAbs
immobilized on irradiated (7000 rad) mouse CD32 expressing L cells.
Anti-CD134 mAb was added 24 h after the onset of the culture. Four
days later, CD4+ T cells were magnetically sorted
by incubating monocyte-depleted PBMCs with FITC-conjugated mouse mAb to
CD4 (Southern Biotechnology Associates) and anti-FITC
mAb-conjugated MicroBeads. CD4+ CD30+ T cells
were purified by incubating sorted CD4+ T cells with
MultiSort Release Reagent, a biotinylated mAb to CD30 (PharMingen), and
SA MicroBeads. Upon expansion with 100 U/ml of IL-2, 1:100 (v/v) PHA
(Life Technologies), and 5 µM of TNF- protease inhibitor
(Immunex), CD30- and CD30+
T cells were fixed with 1% paraformaldehyde.
Cell cultures
Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Life Technologies), 2 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. IgD+ or IgD- B cells and CD4+ (total, CD30- or CD30+) T cells were seeded in 96-microwell plates at 0.5 x 105 and 2 x 105 cells/well, respectively. Mouse M81 or M80 mAbs to CD153 (Immunex), human CD30:Fc IgG1 (Immunex), human CTLA-4 (CD152):Fc IgG1 (R&D Systems, Minneapolis, MN), mouse control MOPC-21 mAb (Sigma, St. Louis, MO), mouse BU52 mAb to CD44 (The Binding Site, San Diego, CA), and mouse TAG34 mAb to CD134L (OX40L) (from Dr. T. Hori, Kyoto University, Kyoto, Japan) were immobilized on irradiated CD32 expressing L cells or on plastic plates at 5 µg/ml. In some experiments, anti-CD153 or control mAbs were added to naive B cells 24 h after htCD154 and cytokines. Blocking mouse mAbs to CD27 (M86; Immunex), CD30 (Ber-H2; Dako, Carpenteria, CA), and CD154 (24-31; Ancell, Bayport, MN) were used at 30 µg/ml. htCD154, IL-4, IL-10 (Schering-Plough), and IL-6 (Genzyme, Cambridge, MA), and Sepharose-conjugated Ab to Ig H and L chain (anti-BCR) (Irvine Scientific, Santa Ana, CA) were used at 1 µg/ml, 250 U/ml, 200 ng/ml, 100 U/ml, and 4 µg/ml, respectively.
Flow cytometry
FITC- or PE-conjugated mAbs to the following Ags were used: CD154 (Ancell), CD23, CD77 (Coulter, Miami, FL), CD30 (Dako), CD40, CD38 (PharMingen), CD3, CD4, CD8, CD19 (Sigma), CD10, CD19 (Becton Dickinson, San Jose, CA), CD38, IgD, IgM, IgA, IgG (Southern Biotechnology Associates), and CD138 (Serotec, Oxford, U.K.). To detect CD153 in tonsil B cell subsets, enriched B cells were labeled with unconjugated mouse mAb to CD153 (Immunex), washed, and further incubated with PE-conjugated Ab to mouse Igs (PharMingen). After extensive washes, these cells were incubated with FITC-conjugated anti-CD38 and biotin-conjugated anti-IgD (Southern Biotechnology Associates), washed, and finally labeled with peridinin chlorophyl protein-conjugated SA (Becton Dickinson). To analyze CD30 and CD154 on PB T cells, PBMCs were stained with FITC anti-CD3, biotin-conjugated anti-CD30 (labeled with peridinin chlorophyl protein-conjugated SA), and PE-conjugated anti-CD154, or anti-CD4 mAbs. Cell cycle analysis and apoptosis assays were performed with propidium iodide (Sigma) and annexin-V (R&D Systems). Cells (104) were acquired using a FACSCalibur analyzer (Becton Dickinson), and data processed using a MacIntosh CELL-Quest software program (Becton Dickinson).
Immunohistochemistry
Immunohistochemistry was performed by indirect immunoperoxidase staining of Formalin-fixed, paraffin-embedded tissue sections from different human lymphoid organs, including lymph nodes, tonsils, and spleen. A TechMate500TM BioTek automated immunostainer (Ventana Medical Systems, Tucson, AZ) was used with Abs recognizing IgD (Dako) and CD153 (Immunex). The pressure cooker and trypsinization Ag-retrieval methods were used for IgD and CDl53 staining, respectively.
ELISAs and cell proliferation assays
Culture supernatants were tested for IgG, IgA, IgE, and IL-6 concentration using specific ELISAs (Biosource International, Camarillo, CA). For proliferation assays, B cells (0.5 x 105) were seeded in 96-well plates and pulsed with 1 µCi of [3H]TdR at day 3 of culture. After 18 h, cells were harvested for the measurement of [3H]TdR uptake (18).
PCRs and Southern blots
cDNAs were reversed transcribed from equal amounts of total RNA
(3 µg) (18). PCR analysis was made semiquantitative by
varying the number of amplification cycles and performing dilutional
analysis so that there was a linear relationship between the amount of
cDNA used and the intensity of the PCR product. PCR conditions
consisted of 1-min denaturation at 94°C, 1-min annealing at 60°C,
and 1-min extension at 72°C. ß-actin, I3-C3,
VHDJH-C3, TdT, and RAG-2
were PCR amplified, as reported (18, 26, 46).
Extrachromosomal S-Sµ, S-Sµ, and S-Sµ were PCR amplified
from 500 ng of genomic DNA, blotted, and hybridized with probes
specific for the recombined switch regions
(2). The following sense and antisense primer pairs were
also used: Ku70, 5'-GTGATGTCCAATTCAAGATGAGTC-3' and
5'-GACCTCTTGGTATCGCTAGGCAG-3'; Ku80,
5'-GTATGGACGTGGGCTTTACCATGAG-3' and
5'-CCTAAGCGAAAGGGGCCATCTCCTC-3'; DNA-PK (cetelytic subunit)
5'-TACCAGAGAGCATTCCAGCACC-3' and 5'-TCTTGAATCACTCCTCCTTGATCC-3';
SWAP-70, 5'-GGAAGAAACTGGAGGAAGCAG-3' and
5'-CCATGATGGGCCACTTTGTCCT-3'; Msh2, 5'-CGTTCATGGCTGAAATGTTGG-3' and
5'-GCACTTCTTTGCGCTGGTTCC-3'; Blimp-1,
5'-AAGAGAAAAGCAACTGGATGCG-3' and
5'-GGGTGAAATGTTAGAACGGTAGAGG-3'. The human SWAP-70 cDNA sequence
was obtained from GenBank (accession no. AB014540) through comparative
analysis of mouse SWAP-70 cDNA (accession no. AF053974).
Immunoblotting and immunoprecipitation
Cytoplasmic proteins were extracted as described (13), fractionated on 10% SDS-PAGE gels, and transferred to nitrocellulose membranes. After blocking, membranes were incubated with a rabbit Ab to TRAF-2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500), washed, and further incubated with a HRP-conjugated Ab to rabbit Igs (Santa Cruz Biotechnology; 1:2000). Bound Abs were detected with an enhanced chemiluminescence detection system (Amersham, Little Chalfont, U.K.). After stripping, the same membranes were reprobed with a rabbit mAb to actin (Santa Cruz Biotechnology; 1:500). CD40-bound TRAF-2 was detected in a similar fashion after immunoprecipitation of total cell lysates with mouse BE1 (IgG1) mAb to CD40 (Ancell) and protein G plus Agarose (Santa Cruz Biotechnology) (7).
Luciferase reporter assays and EMSAs
A total of 2 x 106 CL-01 cells in
500 µl was mixed with 40 µl of plasmid DNA-TRIS-EDTA solution
containing 25 µg of ECS-I3 (-449/+265) pGL3 reporter construct
and 10 ng of pRL-CMV control vector. Electroporation was conducted at
525 V/cm and 950 µF using a Gene Pulser II apparatus (Bio-Rad
Laboratories, Hercules, CA). Transfected cells were cultured for
24 h, and luciferase activities were measured as reported
(13). NF-B and STAT-6 shift and supershift assays were
performed as described (13). A double-stranded probe
encompassing the BSAP site 1 of the mouse Ig H chain 3' enhancer was
prepared by annealing the 5'-CAGAATTGTGAAGCGTGACCATAG-3' primer to
the 5'-CTATGGTCACGCTTCACAATTCTG-3' primer. Inhibition assays were
performed using an Ab to BSAP/Pax5 (Santa Cruz Biotechnology).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tonsil B cells include IgD+
CD38- naive B cells, IgD+
CD38+ GC founder B cells,
IgD- CD38+ GC B cells
(further divided in CD77+ centroblasts and
CD77- centrocytes), and
IgD- CD38- memory B cells
(47, 48). These B cell subsets are localized in the
follicular mantle (FM), FM-GC junction, GC, and marginal zone of the
secondary lymphoid follicle, respectively (14, 17).
Although naive B cells, GC founder, and GC centroblasts mostly consist
of preswitched IgD+ IgM+
and IgD- IgM+ B cells,
respectively, centrocytes and memory B cells are mostly switching or
already class-switched IgD-
IgM- (IgG+ or
IgA+) B cells (14, 17). In this
study, CD153 as well as the transcription status of the Ig H chain
locus were analyzed in sorted tonsil B cell subsets. Naive B cells (B1)
expressed low levels of CD153 and lacked germline I3-C3 and
productive VHDJH-C3
transcripts (Fig. 1
A). Founder
GC B cells (B2) up-regulated CD153 and did not express I3-C3 or
VHDJH-C3 transcripts. GC
B cells (B3) coexpressed CD153 as well as I3-C3 or
VHDJH-C3 transcripts.
Among these cells, centroblasts expressed CD153 at higher density than
centrocytes (not shown). Finally, switched memory B cells (B4) lacked
CD153 and I3-C3, but expressed
VHDJH-C3.
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coreceptor, a
molecule expressed by all B cells), and CD153 on tonsil sections showed
that CD153 is expressed by IgD-
CD79a+ B cells within the GC of secondary
lymphoid follicles (Fig. 1
B). By contrast,
IgD+ CD79a+ B cells within
the FM surrounding the GC did not express significant CD153. This
latter was up-regulated in tonsil IgD+
IgM+ naive B cells 1 day after exposure to GC
differentiation and CSR-inducing stimuli, including htCD154, IL-4, and
anti-BCR Abs, and it was down-regulated 1 day later (Fig. 1C). These findings indicate that IgD+
IgM+ naive B cells up-regulate CD153 in the GC of
secondary lymphoid organs, and suggest that class-switched B cells,
including memory B cells, express CD153 at lower density than
preswitched B cells.
CD153 engagement inhibits CD154- and IL-4-induced I3-C3
germline transcription and SµS3 CSR
Previous studies have shown that mouse M81 anti-CD153 mAbs or
human CD30:Fc IgG1 fusion protein specifically bind B cell CD153
(25, 26, 27). These reagents were immobilized on CD32
(FcR)-expressing L cells, a condition that mimics
CD30:CD153-mediated cell:cell interaction and elicits significant
CD153-dependent signaling in T cells and neutrophils (43).
Upon exposure to htCD154 and IL-4 for 2 days, PB
IgD+ IgM+ B cells induced
I3-C3 germline transcripts (Fig. 2). In these cells, immobilized
anti-CD153 mAbs or human CD30:Fc IgG1 virtually abrogated the
expression of I3-C3 transcripts, whereas MOPC-21 mAb, Abs to B
cell CD44 or CD134L (17, 49), or human CD152 (CTLA-4, the
CD80 and CD86 receptor expressed by activated T cells):Fc IgG1 chimeric
protein did not. In addition to IgD+
IgM+ normal B cells, CD153 cross-linking
inhibited I-C germline transcription in Ramos and BL-2, two
CD153+ neoplastic B cell lines expressing an
IgD- IgM+
centroblastic-like phenotype (not shown). This inhibitory effect was
not associated with CD40 down-regulation, and was not dependent on the
secretion of endogenous cytokines, as a combination of neutralizing Abs
to IL-6, IL-10, TNF-, TGF-ß, and/or IFN- did not reverse the
inhibitory activity of anti-CD153 mAb (not shown).
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3-Sµ reciprocal DNA recombination products (Fig. 3B),
VHDJH-C3 productive
transcripts (Fig. 3C), and surface IgG (Fig. 3D)
4 days after exposure to htCD154 and IL-4. In these cells, switching to
C3 was associated with increased expression of transcripts encoding
proteins that are relevant to CSR (50, 51, 52, 53), including
Ku70, Ku80, DNA-PK, SWAP-70, and Msh2 (Fig. 3C). Similar
results were obtained when htCD154- and IL-4-stimulated B cells were
exposed to an anti-CD44 mAb (not shown). Compared with these cells,
B cells exposed to htCD154, IL-4, and anti-CD153 mAb strongly
inhibited SµS3 DNA recombination (Fig. 3B),
VHDJH-C3 transcription,
expression of CSR-associated transcripts (Fig. 3C), and
surface IgG expression (Fig. 3D). In similar cells,
anti-CD153 mAb did not down-regulate TdT, a gene involved in V(D)J
gene diversification (46), and, more importantly, did not
affect B cell survival or proliferation (Fig. 3A). These
findings indicate that CD153 cross-linking inhibits I3-C3
germline transcription and subsequent SµS3 CSR in CD154- and
IL-4-induced IgD+ and/or
IgM+ B cells.
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3 germline gene promoter
The role of CD153 engagement in C3 gene transcriptional
activation was assessed in human IgD+
IgM+ CL-01 cells. Two days after exposure to
htCD154 and IL-4, these B cells induced I3-C3 transcripts (Fig. 4A), and activated the C3
germline gene promoter (Fig. 4B). In similar B cells,
immobilized anti-CD153 but not MOPC-21 or anti-CD44 (not shown)
mAbs down-regulated I3-C3, but not RAG-2 transcripts
(46), and significantly hampered the transcriptional
activation of the C3 gene promoter (about 50% inhibition). This
promoter is induced upon NF-B and STAT-6 binding to specific CD40
and IL-4R DNA REs, respectively (13). In a 4-h culture,
anti-CD153 but not MOPC-21 or anti-CD44 (not shown) mAbs
significantly decreased the htCD154-induced binding of p50/p65,
p50/c-Rel, and p50/p50 NF-B/Rel complexes to the CD40 RE of the
C3 germline gene promoter, but did not affect the IL-4-induced
binding of STAT-6 to the IL-4R RE of the same promoter (Fig. 4C). Similar findings were obtained in
IgD- IgM+ Ramos and BL-2 B
cells (not shown), suggesting that CD153 engagement inhibits CD154- and
IL-4-induced transcriptional activation of the C3 germline gene
promoter by hampering NF-B binding to the CD40 RE of this
promoter.
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Because TRAF-2 has been suggested to play an important role in Ig
germline transcription (10), we reasoned that CD153
cross-linking could interfere with the CD154-induced assembly of the
CD40:TRAF-2 receptor complex. Cytoplasmic TRAF-2 was constitutively
expressed in unstimulated IgD+
IgM+ CL-01 B cells (Fig. 4D). Exposure
of these cells to htCD154 (with or without IL-4) but not IL-4
dramatically reduced the levels of cytoplasmic TRAF-2, which was
instead bound to CD40, suggesting that CD154:CD40 engagement depletes
cytosolic TRAF-2 at least in part by inducing TRAF-2 recruitment to the
CD40 cytoplasmic tail. Immobilized anti-CD153 but not MOPC-21 or
anti-CD44 (not shown) mAbs prevented htCD154-induced
down-regulation of cytoplasmic TRAF-2 as well as htCD154-induced
binding of TRAF-2 to CD40, indicating that CD153 cross-linking
interferes with the assembly of the CD40:TRAF-2 receptor complex.
CD153 engagement inhibits CD154- and cytokine-induced IgG secretion and plasmacytoid phenotypic differentiation
The role of CD153 engagement in plasmacytoid differentiation was
analyzed in purified tonsil IgD+
(IgM+ IgG-) and
IgG+ (IgD-
IgM-) B cells. Upon exposure to htCD154 and
cytokines for 8 days, IgD+ B cells secreted IgG
and differentiated to CD38++
CD138+ cells (Fig. 5A), all traits of
plasmacytoid differentiation (11). Compared with
IgD+ B cells, similarly induced
IgG+ B cells secreted higher amounts of IgG and
gave rise to a higher proportion of CD38++
CD138+ B cells (Fig. 5A). Upon
exposure to anti-CD153 but not MOPC-21 or anti-CD44 (not shown)
mAbs, stimulated IgD+ but not
IgG+ B cells secreted lower amounts of IgG and
gave rise to a significantly lower proportion of
CD38++ CD138+ B cells (Fig. 5A). These findings suggest that CD153 cross-linking
inhibits plasmacytoid differentiation in CD154- and cytokine-induced
IgD+ B cells.
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Upon exposure to htCD154 and cytokines for 4 days,
IgD+ IgM+ B cells secreted
IL-6, an inducer of plasmacytoid differentiation, and expressed
transcripts encoding Blimp-1 (Fig. 5B), a transcription
factor involved in the transition of mature B cells into plasma cells
(54, 55). Compared with B cells exposed to MOPC-21 mAb, B
cells exposed to anti-CD153 mAb increased secretion of IL-6
(56), but virtually lacked Blimp-1 transcripts (Fig. 5B). These CD153-stimulated B cells also increased the
binding of BSAP to the Ig 3' enhancer (Fig. 5B), a DNA
element that up-regulates the Ig H chain transcription in plasma cells
and is repressed by BSAP (57). Thus, in CD154- and
cytokine-induced IgD+ B cells, CD153 engagement
inhibits Blimp-1 transcription while up-regulating BSAP binding to
DNA.
T cells express functionally active CD30 and CD154 4 days after TCR, CD28, and CD134 engagement
Ag-activated CD4+ T cells express CD30 in
the presence of CD28 engagement and IL-4 (21), a cytokine
produced by T cells upon engagement of CD134 by CD134L on APCs
(36, 58). PBMCs were incubated with IL-4 and immobilized
agonistic mAbs to CD3, CD28, and CD134. After 8 h, 34 ± 8%
of CD4+ T cells expressed CD154, but not CD30
(Fig. 6A). Four days later,
12 ± 5% of these T cells still expressed CD154, whereas 62
± 14% of them expressed CD30 (Fig. 6B). These
4-day-stimulated CD4+ T cells were sorted, fixed
(to prevent cytokine secretion), washed, and cultured with
IgD+ IgM+ B cells in the
presence or absence of exogenous cytokines and anti-BCR Ab (the
latter was added to minimize CD30 expression by B cells). Under these
conditions, 4-day-stimulated CD4+ T cells induced
significant switching to IgG in IgD+ B cells, and
increased the IgG production in IgG+ B cells
(Fig. 6C). These effects were dependent on residual T cell
CD154, as they were abolished by preincubating 4-day-activated
CD4+ T cells with saturating amounts of a
blocking mAb to CD154. Preincubation of T cells with a blocking mAb to
CD30 (59), but not to CD27 (42), enhanced IgG
production in IgD+, but not
IgG+ B cells, suggesting that T cell CD30 limits
IgG synthesis in Ag-selected IgD+ B cells.
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, C, and C through B cell CD153
To formally verify the inhibitory ability of
CD4+ CD30+ T cells,
CD30- and CD30+ T cells
were sorted from 4-day-stimulated CD4+ T cells.
Sorted cells were fixed, washed, incubated with a mAb-blocking residual
surface CD154, washed, and then cultured with
IgD+ IgM+ B cells in the
presence or the absence of exogenous htCD154, cytokines, and
anti-BCR Abs. Purified CD30+ but not
CD30- T cells significantly inhibited htCD154-
and cytokine-induced S-Sµ, S-Sµ, and S-Sµ CSR (Fig. 7A) as well as IgG, IgA, and
IgE production (Fig. 7B). Preincubation of
CD30+ T cells with a saturating amount of
blocking mAb to T cell CD30 restored CSR as well as IgG, IgA, and IgE
secretion, while blocking mAb to T cell CD27 did not. These findings
indicate that CD4+ CD30+ T
cells negatively modulate CD154- and cytokine-induced CSR in
Ag-selected IgD+ B cells, and suggest that this
inhibitory activity is largely dependent upon CD30:CD153
interaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation, IH-CH germline
transcription, CSR as well as expression of transcripts encoding Ku70,
Ku80, DNA-PK, SWAP-70, and Msh2 CSR-related products. It also inhibits
IgG secretion and plasmacytoid differentiation, and this effect is
associated with increased BSAP binding to the Ig 3' enhancer and
down-regulation of Blimp-1 transcripts. Finally, purified
CD30+ T cells inhibit CSR as well as IgG, IgA,
and IgE production in CD154- and cytokine-stimulated
IgD+ IgM+ B cells. These
findings indicate that reverse signaling through CD153 inhibits Ig
class switching in B cells that are positively selected by Ag, and
suggest that CD30+ T cells play an important role
in the modulation of the Ab response. We have recently demonstrated that human IgD+ IgM+ B cells express CD30 upon CD40 engagement and in the absence of BCR coengagement (18). In these B cells, engagement of CD30 by CD153+ CD8+ T cells inhibits CD154- and IL-4-induced CSR as well as IgG, IgA, and IgE production. This CD30-dependent pathway would be critical to rapidly inhibit CSR in non-Ag-selected naive B cells, and would complement the CD95-dependent deletion of non-Ag-selected GC B cells (14, 16, 17). The observation that Ag-specific T cells up-regulate CD30 in disorders associated with dysregulated IgG, IgA, or IgE production (22, 23, 24) prompted us to postulate that engagement of B cell CD153 by T cell CD30 modulates CSR.
We show in this study that CD4+ CD30+ T cells inhibit CD154- and IL-4-induced CSR in IgD+ and/or IgM+ B cells, including Ag-selected CD30- B cells. The CSR-inhibitory activity of CD30+ T cells is dependent on B cell CD153, as emphasized by the finding that blockade of CD30:CD153 interaction restores CSR as well as IgG, IgA, and IgE secretion. Furthermore, CD153 cross-linking by immobilized CD30:Fc or specific agonistic anti-CD153 mAbs inhibits CD154- and cytokine-induced CSR. This CD153-mediated inhibitory activity seems to be CSR specific, as CD153 engagement does not affect CD40-mediated B cell proliferation or survival, nor does it affect CD23, CD54 (ICAM-1), CD80 (B7.1), or CD86 (B7.2) up-regulation. In addition to inhibiting CSR, CD153 engagement significantly enhances the production of endogenous IL-6 as well as DNA binding by BSAP, a transcription factor that critically modulates B cell proliferation and differentiation (57). Thus, CD153 transmits both positive and negative signals in CD40-activated B cells.
These findings are consistent with the reported signal-transducing
properties of other members of the TNFL superfamily, including CD70
(CD27L), CD95L (FasL), CD134L (OX40L), CD137L (4-1BBL), and CD154
(CD40L) (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41), and extend previous findings showing
that CD153 cross-linking by agonistic anti-CD153 mAbs or CD30:Fc
induces reverse signaling in T cells and neutrophils (43).
In CD154- and IL-4-activated IgD+
IgM+ B cells, CD153 engagement inhibits the
transcription of I3-C3 germline transcripts and turns off CSR to
C3. This inhibitory effect is associated with decreased levels of
transcripts encoding DNA-PK, Ku70, Ku80, SWAP-70, and Msh2. Because
DNA-PK, Ku, and Msh2 proteins play a role in CSR, and SWAP-70 has been
suggested to be an element of the switch recombinase machinery
(50, 51, 52, 53, 60), it is tempting to speculate that CD153
signaling modulates CSR not only by inhibiting the germline
transcription of downstream CH genes, but also by
modulating the expression of critical CSR-related molecules. This
inhibition appears to be specific, as CD153 engagement does not
significantly affect the level of transcripts encoding TdT and RAG-2,
two genes involved in Ig V(D)J gene diversification and recombination
(46).
Several studies have shown that Ig germline transcription and CSR are
critically dependent on the integrity of the CD40 signaling pathway
(12). Upon CD154-induced oligomerization, B cell CD40
recruits multiple TNFR adaptor proteins, including TRAF-1, TRAF-2,
TRAF-3, TRAF-5, and TRAF-6 (6, 7, 8, 9, 10, 11). By activating
downstream kinases, including NF-B-inducing kinase, IB kinase,
and c-Jun N-terminal protein kinase, TRAFs up-regulate the binding of
critical transcription factors, including NF-B and AP-1, to B cell
gene promoters (6, 7, 10, 11, 61, 62). In mouse B cells,
the recruitment of TRAF-2, TRAF-3, TRAF-5, and/or TRAF-6 to the CD40
cytoplasmic tail is critical for CD154-dependent induction of C gene
germline transcription and IgG production (8, 9). In human
IgD+ IgM+ B cells, CD153
cross-linking inhibits CD154-mediated TRAF-2 recruitment to CD40, and
concomitantly reduces the binding of NF-B to the CD40 RE of the
C3 germline gene promoter (Fig. 4). These findings, together with
our preliminary experiments showing the CD153 cross-linking decreases
the recruitment of TRAF-3 to CD40 (not shown), suggest that CD153
signaling may modulate the CD40-mediated transcriptional activation of
downstream CH genes by inducing an overall
desensitization of the CD40 signaling complex.
It has been recently shown that CD153+ B cells
coexpress high levels of a truncated form of CD30, referred to as CD30
variant (63). This CD30 variant encompasses the
cytoplasmic tail of transmembrane CD30; recruits TRAF-2, TRAF-3, and
TRAF-5; and induces NF-B activation when overexpressed in a myeloid
cell line (63, 64). Our preliminary experiments indicate
that, in CD40-activated B cells, engagement of CD153 up-regulates the
binding of TRAF-2 and TRAF-3 to CD30 variant (not shown). Thus,
CD30:CDl53 interaction would interfere with the CD40:NF-B-associated
signaling pathway by recruiting multiple TRAFs to CD30 variant, and
thereby hampering them from binding to the CD40 cytoplasmic tail. The
presence of the same signaling element (i.e., the CD30 cytoplasmic
tail) in both the CD153- and CD30-associated signaling pathways would
be consistent with our observation that engagement of CD30 or CD153
elicits partially overlapping functional effects in
CD154:CD40-activated IgD+
IgM+ B cells (18). It would also be
consistent with the possibility that bidirectional B cell CD30:B cell
CD153 signals synergistically inhibit the GC progression of
perifollicular non-Ag-selected CD30+
CD153+ B cells (18).
In addition to hampering CSR, CD153 engagement significantly inhibits IgG, IgA, and IgE as well as plasmacytoid phenotypic differentiation in CD154- and cytokine-induced IgD+ B cells. This inhibition is not associated with decreased production of B cell-derived IL-6 (56), but, rather, with decreased transcription of Blimp-1, a poorly defined molecule involved in the transition of mature B cells into plasma cells (54). It is also associated with up-regulation of BSAP, a transcription factor that, when overexpressed in GC-like B cells, represses the 3' Ig enhancer (a critical regulator of Ig H chain production) (57, 65). These findings suggest that, in CD154- and cytokine-induced IgD+ IgM+ B cells, CD153 signaling turns off the plasmacytoid differentiation program by modulating Blimp-l and BSAP transcription factors in a reciprocal fashion.
By defining a specific role for B cell CD153 in the regulation of Ig
CSR and plasmacytoid differentiation, our findings outline a novel
CD95-independent mechanism in the homeostasis of B cell
differentiation. They suggest that the process that initiates a
specific Ab response and the rapid expansion of
CD4+ CD154+ T cells with
CSR-inducing activity later on gives rise to CD4+
CD30+ T cells with CSR-inhibitory activity (Fig. 8). As early as 8 h after optimal
TCR, CD28, and CD134 activation by Ag and costimulatory molecules
(i.e., CD80, CD86, and CD134L) on APCs (5, 36, 58),
CD4+ T cells express CD154 that engages CD40 on
IgD+ IgM+ B cells. In the
presence of BCR engagement, these naive B cells undergo clonal
expansion, acquire GC phenotypic traits, and up-regulate CD153
expression. Within 4 days, a significant proportion of these GC B cells
completes CSR to IgG, IgA, or IgE; initiates plasmacytoid or memory
differentiation; and down-regulates CD153 expression. At this time and
in the presence of IL-4, activated CD4+ T cells
induce CD30 (20, 21) while still expressing functionally
significant levels of CD154 (66). These
CD4+ CD30+ T cells would
inhibit CSR and plasmacytoid differentiation in newly
CDl54:CD40-activated IgD+ and/or
IgM+ CD153+ B cells, while
sparing ongoing IgG, IgA, or IgE production in already class-switched
GC, plasmacytoid, or memory B cells. Thus, a few days after the
initiation of the Ab response, CD4+
CD30+ T cells would modulate the magnitude of
IgG, IgA, or IgE production by inhibiting the late CD154-dependent
entry of CD153+ B cells, including Ag-selected
CD30- B cells, into the pool of B cells
producing class-switched Abs.
|
By adding to previous reports indicating that CD30 signaling negatively regulates the cytotoxic activity of NK and CD8+ T cells (69, 70, 71), our experiments suggest that the CD30:CD153 interaction functions as a biological brake that physiologically limits the immune response by transmitting bidirectional negative signals. Given the CD30 ability to inhibit the in vivo clonal expansion of CD8+ cytotoxic T cells (71), human viruses, including HIV, measles, and hepatitis B and C viruses, might up-regulate CD30 expression on T cells to evade both Ab- and cell-mediated virus-specific immune responses (22, 23). In addition, expression of CD30 may confer immune privilege to a given tissue or organ. For instance, IL-4- and progesterone-induced CD30+ placental T cells as well as CD30+ decidual cells might contribute to inhibit the Ab response to fetal alloantigens by maternal CD153+ B cells (72). In conclusion, B cell CD153 could represent a novel target for the immunotherapy of disorders associated with dysregulated Ab production.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrea Cerutti, Division of Molecular Immunology, Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021.
3 Abbreviations used in this paper: L, ligand; BCR, B cell Ag receptor; Blimp-l, B lymphocyte maturation protein-1; BSAP, B cell-specific activation protein (Pax5); CSR, Ig class switch DNA recombination; DNA-PK, DNA protein kinase; FM, follicular mantle; GC, germinal center; htCD154, soluble human trimeric CD154; PB, peripheral blood; RAG, recombination-activating gene; RE, responsive element; SA, streptavidin; SWAP-70, switch-associated protein-70; TRAF, TNFR-associated factor.
Received for publication February 17, 2000. Accepted for publication April 24, 2000.
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References |
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) or IgG+ (
) B cells were cultured
with or without htCD154 and cytokines (IL-4 and IL-10), and in the
presence of MOPC-21 or anti-CD153 mAbs immobilized on CD32 L cells.
IgG concentration and CD38++ CD138+
plasmacytoid B cells were analyzed after 8 days (arrows indicate IgG
and plasma cell values <100 ng/ml and <0.1%, respectively).
B, Tonsil IgD+ B cells were cultured as
above. After 4 days, IL-6 was measured by ELISA; Blimp-1 (355-bp) and
ß-actin (593-bp) transcripts were PCR amplified (25 cycles) from
equal amounts of cDNA; and BSAP binding to the BSAP site 1 of the mouse
Ig 3' enhancer was determined by EMSA. These findings were derived from
one of three experiments yielding comparable results (error bars
indicate ±SD).
