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of June 16, 2017.
Ligation of CD27 on Murine B Cells
Responding to T-Dependent and
T-Independent Stimuli Inhibits the
Generation of Plasma Cells
Vanitha S. Raman, Vineeta Bal, Satyajit Rath and Anna
George
J Immunol 2000; 165:6809-6815; ;
doi: 10.4049/jimmunol.165.12.6809
http://www.jimmunol.org/content/165/12/6809
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References
Ligation of CD27 on Murine B Cells Responding to
T-Dependent and T-Independent Stimuli Inhibits the
Generation of Plasma Cells1
Vanitha S. Raman, Vineeta Bal, Satyajit Rath, and Anna George2
W
hen naive B cells in the periphery encounter specific
Ag in the context of appropriate costimulatory molecules, they get activated and proliferate. Early clonal
expansion can be quite rapid, with doubling times estimated to be
as short as 6 – 8 h (1, 2). The expanded population has to be controlled to ensure that while enough numbers of activated cells are
generated to eliminate the Ag that initiated the response, a rapid
return to homeostasis is also possible. In addition, the population
has to be screened to ensure that any self-reactive cells that may be
generated during the process of somatic mutation do not survive
(3). Therefore, B cell activation is accompanied by extensive proliferation on the one hand and with cell death on the other, and
members of the TNFR family of proteins have emerged as important mediators of both events (4 –14).
Activated B cells differentiate into Ig-secreting effector cells and
to memory cells that are not immediately functionally active but
are capable of responding to a subsequent antigenic challenge with
rapid and enhanced proliferation and Ig secretion. Commitment to
effector preplasmablasts as well as to memory cells seems to occur
in germinal centers (15, 16), but the signals that lead cells down
one differentiation pathway rather than another have not yet been
elucidated. TNFRs are crucial for the formation of germinal centers and they also control the spatial arrangement of B and T cells
within lymphoid organs, and recent reports have shown that in
National Institute of Immunology, New Delhi, India
Received for publication December 20, 1999. Accepted for publication September
20, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants (to A.G. and V.B.) by the Department of Science
and Technology, Government of India. The National Institute of Immunology is supported by the Department of Biotechnology, Government of India.
2
Address correspondence and reprint requests to Dr. Anna George, National Institute
of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India. E-mail address:
[email protected]
Copyright © 2000 by The American Association of Immunologists
various TNFR knockout mice, in which optimal interactions between B cells, T cells, and follicular dendritic cells in germinal
centers do not occur, B cell responses are compromised (17–26).
CD27 is a member of the TNFR family, and there is evidence to
suggest, on the one hand, that it may be a potential marker for
human memory B and T cells (27–30) and, on the other, that engagement of CD27 by its ligand CD70 may promote the differentiation of human memory B cells to plasma cells (31, 32). Although CD27 is expressed on a substantial proportion of human
PBL, only a small proportion of murine B cells express CD27, and
they are found predominantly in the marginal zone of the spleen
and the germinal centers of tonsils and lymph nodes (33–35).
Thus, CD27 may identify a recently activated population of cells
in murine lymphoid tissues, and this raises the possibility that signaling through CD27 may influence the choice between terminal
differentiation and memory cell generation in activated B cells.
We have examined the effect of ligation of CD27 on events
following B cell activation using a T-dependent (TD)3 culture set
up in the presence or absence of anti-CD27. Because CD27 and its
ligand are present on activated B and T cells, we also looked for
B cell-specific effects of the Ab in T-independent (TI) culture. We
report that whereas CD27 engagement does not affect B cell activation, proliferation, or viability, it inhibits terminal differentiation
of B cells into Ig-secreting plasma cells.
Materials and Methods
Mice
Six- to 10-wk-old C57BL/6, BALB/c, and C3H/HeJ mice (The Jackson
Laboratory, Bar Harbor, ME), bred and maintained in the Small Animal
Facility of the National Institute of Immunology, were used for all experiments. Approval from the Institutional Animal Ethics Committee was obtained for all experimental procedures involving animals.
3
Abbreviations used in this paper: TD, T dependent; D10, D10.G4.1; DC, dendritic
cell; TI, T independent; NP, nitrophenyl; PNA, peanut lectin (agglutinin); PI, propidium iodide; NP-CGG, nitrophenyl-chicken gammaglobulin.
0022-1767/00/$02.00
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B cells can be stimulated either allogenically with the Th cell clone D10G4.1 and bone marrow-derived dendritic cells or polyclonally with LPS to proliferate and undergo terminal differentiation to Ig-secreting plasma cells in vitro. The addition of antiCD27 to such cultures inhibits Ig secretion, and inhibition is more marked in T-dependent cultures than in T-independent cultures.
Both IgM and secondary isotypes are affected, and addition of anti-CD27 even 4 days after culture initiation inhibits Ig secretion.
Anti-CD27 does not affect B cell proliferation or the acquisition of activation markers by B cells, and no marked loss of B cell
viability is detected in cells cultured in the presence of anti-CD27, suggesting that the inhibition of Ig secretion is not due to
inhibition of early activation events or to death of activated cells in vitro. However, the presence of anti-CD27 significantly inhibits
the induction of Blimp-1 and J chain transcripts, which are turned on in cells committed to plasma cell differentiation. Furthermore, mice immunized under cover of anti-CD27 make less Ag-specific IgM and IgG, but have equivalent T cell responses when
compared with control mice. These data suggest that ligation of CD27, a member of the TNFR family, on the B cell surface may
prevent terminal differentiation of activated B cells into Ig-secreting plasma cells. The Journal of Immunology, 2000, 165: 6809 –
6815.
6810
CD27 LIGATION PREVENTS PLASMA CELL GENERATION
FIGURE 1. Anti-CD27 inhibits Ig secretion in clonal TD culture. I-Ab
B cells were stimulated with ␥-irradiated (1000 rad) D10s and DCs in the
presence or absence of various Abs in Terasaki plates. One week later,
culture supernatants were diluted and tested for total IgM, IgG1, and IgA
by ELISA. The percentage of wells (of 60 plated for each set) that contained detectable Ig of each isotype is plotted, and one experiment, representative of three, is shown. ⴱⴱ, IgG1 was not estimated in wells that
contained CTLA-4-Ig because the CTLA-4-Ig cross-reacts with ELISA
reagents. No Ig was detected in wells containing only B cells and DCs (data
not shown).
Single-cell suspensions of splenocytes were obtained by mechanical disruption of the spleen, and RBCs were lysed by treatment of the cell pellet
with Gey’s solution. For some experiments, unfractionated splenocytes
were used as a source of B cells. For other experiments, B cells were
enriched either by depleting T cells with anti-Thy1.2 (Y-19, a kind gift of
Dr. C. A. Janeway, Jr., Yale Medical School, New Haven, CT) and complement (Cedarlane, Westbury, NY), followed by removal of plastic-adherent cells or by positive enrichment of total or IgM⫹ B cells on magnetic
columns (Miltenyi Biotec, Bergisch-Gladbach, Germany). For such separations, cells were labeled either with anti-CD19 beads (Miltenyi Biotec) or
with goat anti-mouse IgM-biotin (Southern Biotechnology Associates, Birmingham, AL) or anti-B220-biotin (PharMingen, San Diego, CA) followed
by streptavidin-coupled magnetic beads (Miltenyi Biotec). Purified cells
were used for experiments only if they were ⬎90% pure by flow cytometric analysis.
T cells
The conalbumin-specific I-Ak-restricted Th2 clone, D10.G4.1 (D10),
which also recognizes I-Ab molecules in alloreactive fashion, (36), was
maintained in Click’s medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS, 2 mM L-glutamine, 0.05 mM 2-ME (all from Life
Technologies, Grand Island, NY), and antibiotics (complete Click’s medium). The culture was maintained by weekly stimulation with conalbumin
and ␥-irradiated syngeneic splenocytes, and cells were used for assays
between 10 and 14 days after the last stimulation.
Dendritic cells (DCs)
DCs were grown from the bone marrow of C3H/HeJ (H-2k) mice in complete Click’s medium containing 20 ng/ml of GM-CSF (PeproTech, Rocky
Hill, NJ), and to get activated, mature DCs, 10 ng/ml of recombinant
TNF-␣ (PeproTech) was added 24 h before using them in culture (37).
B cell stimulation
For TI B cell activation, 105 B cells or 3 ⫻ 105 whole splenocytes were
stimulated with various doses of LPS (BBL-Difco, Becton Dickinson India, New Delhi, India) in 200-␮l cultures. For clonal TD experiments, 1–5
I-Ab B cells were allogenically stimulated with 3000 resting D10s and 400
DCs in a final volume of 15 ␮l in 60-well Terasaki plates (Falcon, Franklin
Lakes, NJ) as described earlier (16, 38). For bulk TD experiments, 100 –
300 B cells were stimulated with 3 ⫻ 104 resting D10s and 4000 DCs in
a final volume of 200 ␮l in 96-well flat-bottom plates (Falcon). Cultures
were set up in the presence or absence of azide-free anti-CD27 (PharMingen). The Ab is an Armenian hamster monoclonal IgG Ab (clone
LG.3A10) raised against an Armenian hamster fibroblast line transfected
with mouse CD27 DNA, and it has been reported to have potent costimulatory activity for T cells (39). No data on its effects on B cells have been
reported so far. Other Abs used (all from PharMingen) were anti-CD28,
anti-CD48, anti-CD54, anti-CD154 (all hamster IgG), anti-CD2 (rat
IgG2b), and anti-CD86 (rat IgG2a).
FIGURE 2. Anti-CD27 inhibits Ig secretion in bulk TD and TI cultures.
I-Ab B cells were stimulated in triplicate wells with ␥-irradiated (1000 rad)
D10s and DCs or with LPS in 96-well plates in the presence of titrating
amounts of anti-CD27. One week later, Ig in culture supernatants was
estimated. A shows IgM and IgG1 levels in TD culture. B shows the IgM
levels in TI cultures. The data shown are representative of four independent
experiments. C shows the specificity of inhibition of TI response by antiCD27 in comparison with various hamster anti-mouse IgG Abs with other
specificities. D and E show the kinetics of inhibition by anti-CD27 when
added to TI (D) and TD (E) cultures at various times after culture initiation,
and Ig was estimated on day 7. In E, the amount of each isotype is plotted
as a percentage of the amounts in controls that were stimulated in the
absence of anti-CD27. One experiment, representative of four, is shown.
Unstimulated cells produced negligible amounts of Ig.
B cell proliferation was assessed by stimulating cells in TD or TI culture
for 48 h, pulsing the wells with 0.5 ␮Ci/well of [3H]thymidine (NEN, Life
Science Products, Boston, MA) for 12–16 h and harvesting the cells onto
glass fiber filtermats for liquid scintillation counting (Betaplate; Wallac,
Turku, Finland). Data are expressed as mean ⫾ SE of triplicate cultures. Ig
secretion was estimated in replicate cultures stimulated for 6 – 8 days. Serial dilutions of culture supernatants were added to microtiter plates (Dynatech, Chantilly, VA) coated with 10 ␮g/ml of goat anti-mouse Ig (Southern Biotechnology Associates). Bound Ig was detected with biotinylated
goat anti-mouse IgM, IgG1, or IgA (Southern Biotechnology Associates)
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B cells
The Journal of Immunology
6811
CyChrome-avidin (PharMingen). Between 105 and 106 cells were incubated with staining reagents in buffer containing 0.1% sodium azide and
1% FBS. All incubations were for 45 min on ice. If stained cells could not
be analyzed immediately, they were fixed in 0.1% paraformaldehyde and
stored at 4°C. For determination of cell viability, 5 ␮g/ml of propidium
iodide (PI; Sigma) was added to samples before acquisition. Samples were
run on an Elite ESP flow cytometer (Coulter Electronics, Hialeh, FL) or on
a Bryte flow cytometer (Bio-Rad, Hemel-Hampstead, U.K), and data were
analyzed with WinMDI shareware or FlowJo software (Treestar, San
Carlos, CA).
RT-PCR
In vivo priming and recall responses
BALB/c mice were immunized s.c. with 100 ␮g of nitrophenyl-chicken
gammaglobulin (NP-CGG; Biosearch Technologies, Novato, CA), emulsified in CFA (Sigma), and were given either PBS or 30 ␮g of anti-CD27
i.p. on the day of immunization. To assess Ab responses, mice were bled
14 days after immunization, and anti-nitrophenyl (NP) Abs were estimated
by ELISA on NP-BSA-coated plates. To assess T cell priming, cells from
the draining lymph nodes were cultured in vitro with serial dilutions of
heat-inactivated chicken serum, and proliferation was assessed 72 h later
by scintillation spectroscopy as described earlier. The proliferative responses of cells to 1/10,000 dilution of chicken serum are shown.
FIGURE 3. Anti-CD27 does not inhibit the induction of activation
markers on B cells. I-Ab B cells stimulated with LPS (A–E) or with ␥-irradiated (1000 rad) D10s and DCs (F–J) in the presence or absence of
anti-CD27 were stained for two-color flow cytometry for expression of
CD24 (A and F), I-Ab (B and G), CD44 (C and H), PNA (D and I), or GL-7
(E and J) vs B220. The expression of the various activation markers on
gated B220⫹ cells is shown: shaded histogram, conjugate control; dotted
line, unstimulated cells; thin line, cells stimulated in the absence of antiCD27; thick line, cells stimulated in the presence of anti-CD27. One experiment, representative of three, is shown.
followed by streptavidin-HRP (Genzyme, Cambridge, MA). Hydrogen peroxide (Merck, Mumbai, India) was used as substrate, and o-phenylenediamine (Sigma, St. Louis, MO) as the chromogen, and color was read at 490
nm in a microplate reader (Sanofi, Redmond, WA). Ig amounts were calculated from a standard curve run in parallel with isotype-specific myeloma
standards (Sigma). The absorbance values shown in some experiments represent values for a 1/3000 dilution of supernatant in TI cultures and a 1/300
dilution in TD cultures.
Flow cytometry
Reagents used for single-step staining or for primary labeling were antiB220-biotin, anti-CD24, anti-GL-7, PE-anti-CD44 (all from PharMingen),
fluorescein-peanut lectin (agglutinin) (PNA;Vector Laboratories, Burlingame, CA), anti-I-Ab (culture supernatant from the hybridoma 212.A1 (a
gift of Dr. C. A. Janeway, Jr.), anti-Thy1.2 (Y-19 culture supernatant), and
anti-Mac-1 (culture supernatant from the hybridoma M1/70.16 (gift of Dr.
C. A. Janeway, Jr.). Secondary reagents used were PE-streptavidin
(PharMingen), fluorescein-mouse-anti-rat IgG (Jackson ImmunoResearch,
West Grove, PA), PE-donkey anti-rat IgG (Jackson ImmunoResearch), and
Results
Anti-CD27 inhibits Ig secretion in TD microcultures
Very small numbers (1–5) of naive I-Ab B cells can be stimulated
in microcultures containing the alloreactive Th2 clone D10 and
DCs isolated from the spleen or Peyer’s patches of mice to undergo proliferation, isotype switching to all secondary isotypes,
and terminal differentiation to plasma cells (16, 38). We find that
DCs that grow out from murine bone marrow cultured in the presence of GM-CSF can substitute for ex vivo DC, and we have used
the system to analyze the role of various cell surface molecules in
B cell activation and differentiation. Fig. 1 shows that the addition
of 5 ␮g/ml of anti-CD27 to such supportive cultures inhibits induction of secreted IgM, IgG1, and IgA, whereas the addition of
anti-CD2, anti-CD48, anti-CD54, and anti-CD86 have no effect.
CTLA-4-Ig (a kind gift of Dr. E. A. Clark, University of Washington, Seattle, WA) does inhibit IgA secretion substantially, but
has little effect on IgM secretion. Similar results were observed
with cultures containing DCs activated overnight with 10 ng/ml of
TNF-␣ (data not shown).
Anti-CD27 inhibits Ig secretion in TI and TD bulk cultures
The data in Fig. 1 represent the percentage of wells (of a total of
60) that are positive for a given isotype when very small numbers
of B cells are stimulated in TD microcultures in Terasaki plates
(Falcon). In the absence of anti-CD27, isotype switching does not
occur in each well, although B cells are driven to IgM production
in each well. Although this allows us to look at the two phenomena
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Total RNA was isolated from cells with RNeasy kits (Qiagen, Valencia,
CA) or with Trizol reagent (Life Technologies). RT-PCRs were set up with
100 ng of RNA using the Access RT-PCR System (Promega, Madison,
WI), and the reaction was set up according to the manufacturer’s recommendations. Briefly, cDNA was synthesized at 48°C for 45 min, the strands
were denatured at 94°C for 2 min, and the cDNA was amplified for 40
cycles (1 min at 95°C, 1 min at 55°C, and 1 min at 72°C), followed by a
final elongation step of 10 min at 72°C. Primers used for J chain were:
sense, 5⬘-ATGAAGACCCACCTGCTTCTC-3⬘; J chain antisense, 5⬘-GT
CAGGGTAGCAAGAATCGGG-3⬘, yielding a 430-bp product. Primers
used for Blimp-1 were: sense, 5⬘-TCCGGCTCCGTGAAGTTTCCA-3⬘;
antisense, 5⬘-GGTGGAACTCCTCTCTGGAAT-3⬘, yielding a 370-bp
product. Primers used for ␤-actin were: sense, 5⬘-CGTGGGCCGCCCTAG
GCACCA-3⬘; antisense, 5⬘-CGGTTGGCCTTAGGGTTCAGGGGGG-3⬘,
yielding a 245-bp band. All three sets of primers were added to a single
RT-PCR, and the PCR conditions were chosen to be within the optimal
range of amplification for all three products. Half the PCR products were
run on a 2% agarose gel, with a 100-bp ladder (Promega) for sizing the
bands.
6812
CD27 LIGATION PREVENTS PLASMA CELL GENERATION
FIGURE 4. Anti-CD27 does not inhibit B or T cell proliferation. A, I-Ab B cells were stimulated with 10 ␮g/ml of LPS in the presence of 5, 1, or 0.2 ␮g/ml
of anti-CD27. B, I-Ab B cells were stimulated with titrating numbers of ␥-irradiated (1000 rad) D10s and 4000 DCs in the presence or absence of 5 ␮g/ml
anti-CD27 (Ab). Background signals from B cells and DCs in the presence (‚) or absence (Œ) of anti-CD27 are also shown. C, D10s were stimulated with titrating
numbers of ␥-irradiated (1000 rad) I-Ab B cells in the presence or absence of 5 ␮g/ml of anti-CD27 (Ab). One experiment, representative of two each, is shown.
Anti-CD27 does not prevent acquisition of activation markers by
stimulated B cells
Signaling through TNFRs appears to be crucial for germinal center
formation and for the maintenance of the correct spatial relationships between B cells, T cells, and DCs in lymphoid organs that
are required for optimal humoral responses (17–26). Therefore, it
is possible that anti-CD27 in culture disrupts cellular interactions
and prevents optimal B cell activation. To examine this, we stained
cells for the up-regulation of various activation markers 60 –72 h
after TD or TI stimulation. As can be seen (Fig. 3), in both TI and
TD cultures, stimulated B cells up-regulate levels of CD24, MHC
class II, and CD44, and the enhancement is similar whether B cells
are cultured in the presence or absence of anti-CD27. Stimulated B
cells also bind high levels of PNA and up-regulate GL-7 (phenotypes characteristic of B cells in germinal centers) in the TD cultures, and the up-regulation is similar in B cells stimulated in the
presence or absence of anti-CD27 (Fig. 3). As expected, neither
PNA binding nor GL-7 expression is induced on B cells stimulated
with LPS (Fig. 3). Thus, B cell activation, including the acquisition
of germinal center markers, is unaffected by ligation of CD27.
Anti-CD27 does not inhibit B or T cell proliferation
B cell activation normally leads to clonal expansion, and we considered the possibility that anti-CD27 may inhibit the proliferation
of activated B cells. However, this appears not to be the case.
Proliferation of B cells stimulated with LPS (Fig. 4A) or with T
cells and DCs (Fig. 4B) is unaffected by the presence of anti-CD27.
Moreover, Fig. 4C shows that proliferation of D10s stimulated
with titrating numbers of irradiated I-Ab B cells is also not affected
by the presence of anti-CD27. These results indicate that ligation
of CD27 has no adverse effects on B or T cell proliferation.
Anti-CD27 does not affect B cell viability
Because CD27 is a member of the TNFR family of proteins that
also includes apoptosis-inducing receptors like CD95/Fas, and because CD27 has been shown to induce apoptosis by binding to the
proapoptotic protein Siva (10), it is possible that ligation of CD27
on activated cells may lead to cell death before terminal differentiation to Ig-secreting plasma cells occurs. We tested this possibility by adding anti-CD27 to LPS-stimulated cultures at the time
of culture initiation and counting the number of viable cells at
various times by trypan blue exclusion. As seen in Fig. 5A, cells
stimulated with LPS survive better than unstimulated cells, and
there is no difference in the number of viable cells recovered from
cultures stimulated in the presence or absence of anti-CD27. In
another set of experiments, anti-CD27 was added at various times
after culture initiation, and the number of viable cells was scored
on day 8. Again, the continued presence of anti-CD27 did not
affect the viability of the cultured B cells and their progeny (data
not shown). Because the TD cultures contained irradiated T cells
and DC, B cell viability was assessed by estimating the exclusion
of PI by gated Thy1.2-negative, Mac-1-negative cells harvested at
various times after culture initiation. As seen in Fig. 5, B and C, the
presence of anti-CD27 in culture does not affect the viability of
cells at either early (24 h) or late (7 days) times. The proportion of
PI-positive cells in the two groups was also similar at 48 h (data
not shown). Together, our results indicate that ligation of CD27
does not induce death of activated B cells.
Anti-CD27 prevents terminal differentiation of B cells to plasma
cells
So far, our data indicate that whereas B cells stimulated in the
presence of anti-CD27 undergo normal activation and proliferation, and whereas a significant proportion of activated cells are
viable at the end of 7– 8 days in culture, they fail to secrete Ig. The
results raise the possibility that CD27 may exert its effect by preventing terminal differentiation of activated B cells into Ig-secreting plasma cells. We tested this possibility by estimating levels of
mRNA for J chain and for the Blimp-1 transcription factor, both of
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separately, the assay does not quantitate the relative amounts of
IgM and IgG in a given well. Therefore, we tested the effect of
anti-CD27 in bulk TD cultures set up in 96-well plates with 100 –
300 B cells. As expected from the microculture data, anti-CD27
inhibits the secretion of primary and secondary isotypes in bulk
TD cultures, and the inhibition titrates with decreasing amounts of
anti-CD27 (Fig. 2A).
Because CD27 and its ligand CD70 are present both on activated B and T cells, we also set up TI cultures to look for B
cell-specific effects of anti-CD27. As seen in Fig. 2B, B cells stimulated with 10 ␮g/ml of LPS for 7 days in the presence of antiCD27 also make substantially less IgM than cells cultured in the
absence of Ab, although higher amounts of anti-CD27 are required
for reliable inhibition to be observed. Other hamster IgG Abs (antiCD28, anti-CD48, anti-CD54, and anti-CD154) did not inhibit Ig
secretion in such cultures (Fig. 2C), establishing the specificity of
inhibition. Furthermore, Ig secretion is inhibited even if anti-CD27
is added 2 or 4 days after culture initiation (Fig. 2D, TI; Fig. 2E,
TD), although secondary isotypes appear to be less affected if antiCD27 is added at later time points.
The Journal of Immunology
6813
FIGURE 5. Anti-CD27 does not induce death of activated B cells in TI or TD culture. A, B cells were
stimulated with LPS in the presence of 5 ␮g/ml or 0.5
␮g/ml of anti-CD27 (Ab), and viable cells were estimated at various times by trypan blue exclusion. B and
C, I-Ab B cells were stimulated with D10s and DCs,
and cells were harvested 24 h (B) or 1 wk (C) later and
stained for Thy1.2 and Mac-1 vs PI for two-color flow
cytometry. PI incorporation in the gated non-T, nonmyeloid cell populations is shown for cells stimulated
in the presence (dotted line) or absence (solid line) of
anti-CD27 (Ab). The shaded histograms represents negative control curves. The percentage of PI-positive cells
in each group is indicated. One experiment, representative of four, is shown.
Anti-CD27 inhibits Ag-specific B cell responses in vivo
Our experimental protocols so far rely on polyclonal TI or allogenic TD stimulation of B cells and do not directly address the role
of CD27 in physiologically relevant Ag-specific responses. To assess the physiological consequences of ligation of CD27 in vivo
directly, we immunized mice with a nominal Ag (NP-CGG emulsified in CFA) under cover of PBS or anti-CD27. As seen in Fig.
7, mice treated with 30 ␮g of anti-CD27 on the day of immunization make substantially less anti-NP IgM and IgG than PBStreated mice do. Significantly, T cells from the draining lymph
nodes of both groups of mice show equivalent proliferation to
NP-CGG in an in vitro recall response (Fig. 7). Thus, B cells
responding to specific Ags in vivo fail to differentiate efficiently
into plasma cells if CD27 is ligated.
Discussion
We have analyzed the effect of ligation of CD27, a member of the
TNFR family, on B cell responses. Earlier data with knockout
mice indicated that signaling through other TNFRs and their ligands affects TD responses (17, 20, 21, 22, 24, 25); therefore, we
initially looked for possible effects of CD27 ligation on splenic B
cells stimulated in vitro in TD culture. Ag-specific B cell responses
are difficult to analyze ex vivo because of the requirement for
enriching hapten-specific B cells. Very small numbers of Ag-specific B cells are recovered even from immunized mice; therefore,
the approach has to be limited to microcultures (40). Another
drawback with enrichment procedures is that B cells with low
levels of surface Ig do not bind well, so that germinal center cells
that have down-regulated surface Ig are excluded. Therefore, for
our TD cultures we have relied on the allogenic stimulation of I-Ab
B cells by the Th clone D10 in the presence of DCs. We found that
the addition of anti-CD27 to clonal or bulk TD cultures inhibited
Ig secretion by the B cells and that both primary and secondary
isotypes were affected (Figs. 1 and 2A). Because CD27 and its
ligand CD70 are present on activated B cells as well as on activated T cells, there is always the possibility that the Ab primarily
affects T cells and that the effects on Ig secretion are indirect.
However, a similar effect on Ig secretion was observed in TI cultures (Fig. 2B, C), suggesting that ligation of CD27 on B cells can
specifically affect B cell differentiation. Interestingly, TD responses appear to be more sensitive to inhibition by Ab. Although
1 ␮g/ml of anti-CD27 inhibits the secretion of all isotypes in TD
culture, 5 ␮g/ml is required for reproducible inhibition of IgM
secretion in TI culture (Fig. 2B and data not shown).
Our results appear to differ somewhat from earlier reports. It has
been shown, for instance, that engagement of CD27 with CD70
transfectants on human B cells stimulated with IL-4 and anti-CD40
induces IgE secretion by promoting the generation of plasma cells
(31). However, the reported enhancement of IgE secretion was
seen only if purified CD27⫹ (but not CD27⫺) cells were used in
culture, and such cells also proliferated better in the presence of the
CD70 transfectant. It has also been reported that greater differentiation to plasma cells occurs in human peripheral CD27⫹ (but not
CD27⫺) B cells in the presence of IL-10 and CD70 transfectant
(32). We have not estimated IgE in our culture supernatants, and it
is possible that IgE secretion may actually be enhanced. However,
CD27⫹ cells are undetectable in murine spleen (although they are
seen in the chronically activated Peyer’s patches, data not shown),
and therefore our data are compatible with the data on CD27⫺
human B cells. However, it is possible that anti-CD27 and the
CD70 transfectants used in these studies may have different effects.
Most B cells responding in vivo to TI stimuli differentiate into
plasma cells in the outer peri-arteriolar lymphoid sheath. However,
in the response to TD Ags, while some activated cells do differentiate into plasma cells at this site, others move into follicles,
where they initiate vigorous germinal center reactions. Ig secretion
in both cases is a relatively late event following B cell activation.
Because anti-CD27 modulates this late event significantly, we also
looked for anti-CD27-mediated inhibition of early events in B cell
activation. Our results indicate that at least two events that precede
Ig secretion, namely, the acquisition of activation markers and the
proliferation of stimulated B cells, are unaffected by anti-CD27. It
has been reported that anti-CD27 can enhance the proliferative
response of purified T cells to suboptimal mitogenic stimulation
(39), but no such enhancement of T cell proliferation was seen in
our system (Fig. 4). CD27 engagement also does not induce death
of activated cells because the viability of cells cultured in the presence or absence of anti-CD27 is similar (Fig. 5). Our results are
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which are induced in B cells committed to plasma cell differentiation, in B cells stimulated in the presence or absence of antiCD27. Fig. 6 shows that anti-CD27 inhibits the induction of both
transcripts. In the TI system, J chain induction is lower by 2-fold,
and Blimp-1 induction by 5-fold. In the TD system, J chain induction is lower by 5-fold, and Blimp-1 induction by 6-fold. However, the levels of ␤-actin mRNA are comparable. Addition of
anti-CD27 alone to cultures did not induce expression of either
gene product (data not shown).
CD27 LIGATION PREVENTS PLASMA CELL GENERATION
FIGURE 6. Anti-CD27 inhibits induction of transcripts for J chain and
Blimp-1. RNA was isolated from I-Ab B cells stimulated with LPS (B) (for
7 days) or with D10s and DCs (A) (for 24 h) in the presence or absence of
5 ␮g/ml of anti-CD27. Simultaneous RT-PCRs for J chain, Blimp-1, and
␤-actin were set up. The expected bands (430 bp for J chain, 370 bp for
Blimp-1, and 245 bp for ␤-actin) are indicated. The bands were scanned
and analyzed with NIH Image shareware, and the densitometric profiles are
shown for the lane with (thin line) or without (thick line) anti-CD27.
FIGURE 7. Anti-CD27 inhibits Ag-specific B cell responses in vivo.
Mice primed with NP-CGG in the presence or absence of anti-CD27 (Ab)
were bled on day 14, and levels of specific Ab were measured (IgM and
IgG). Preimmunization Ab levels were ⬍2 ␮g/ml. T cell priming was
assessed as proliferative response of draining lymph node cells to NP-CGG
(T cell). One experiment, representative of two, is shown.
consistent with earlier observations on the proliferation and survival of B cells from patients with chronic lymphocyte leukemia.
B cells from such patients do not proliferate well to B cell receptor
cross-linking or CD40 ligation, but they can proliferate almost as
well as normal B cells when stimulated with activated T cells, and
it has been shown that disrupting CD27-CD70 interactions during
such stimulation does not affect either proliferation or survival of
the B cells (41).
Because neither B cell activation nor survival were affected by
the presence of anti-CD27 in culture, we looked for possible effects of anti-CD27 on the generation of plasma cells. Our results
show that transcription of J chain and Blimp-1 are inhibited in cells
stimulated in the presence of Ab (Fig. 6). Because these gene products are specifically expressed in cells that have committed to Ig
secretion (42– 45), our results suggest that CD27 engagement prevents the differentiation of activated B cells into plasma cells. Signals that are responsible for commitment to the plasma cell lineage
have not been unambiguously identified, although some candidate
molecules have been reported. For instance, IL-10 has been shown
to inhibit the proliferation of CD20⫹ CD38⫺ B cells and to induce
their differentiation into plasma cells (46). In contrast, ligation of
CD40, another member of the TNFR family, has been shown to
inhibit terminal differentiation of murine B cells (47). Our demonstration that ligation of CD27 on B cells can inhibit the terminal
differentiation of B cells responding in vitro to TD and TI stimuli,
supports and extends this observation to another member of the
TNFR family. Importantly, we also found that mice immunized
under cover of anti-CD27 make less specific IgM and IgG than do
control mice, indicating that ligation of CD27 may have physiological consequences. The results raise two possibilities. One is
that the decreased generation of plasma cells may be accompanied
by a concomitant increase in memory cell generation, implying a
role for CD27 engagement in driving memory B cell commitment.
The second possibility is that engagement of CD27 may be the
mechanism by which activated B cells are prevented from differentiating completely into plasma cells in germinal centers. So far,
the ligand for CD70 has been identified on activated B and T cells,
and it is not known whether CD70 is expressed either on interdigitating DCs of the peri-arteriolar lymphoid sheath or on follicular
DCs of the B cell follicles. Differential expression of the ligand on
various DC subsets may explain our finding that TD responses are
much more sensitive to inhibition by CD27 engagement than are
TI responses.
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