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,
,
Departments of
*
Microbiology and
Internal Medicine,
Graduate Program in Immunology, and
Veterans Administration Medical Center, Iowa City, IA 52242; and
¶ Department of Pharmacology, 3M Pharmaceuticals, St. Paul, MN 55144
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B family
of transcription factors. Both R-848- and CD40-mediated B cell
differentiation were dependent upon NF-B activation, although the
relative importance of individual NF-B family members appeared to
differ between R-848- and CD40-mediated signals. Both signals were
partially dependent upon induction of TNF-
and IL-6, and the
cytoplasmic adaptor molecule TNF receptor-associated factor 2 is
involved in both R-848- and CD40-mediated
differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Understanding the mechanism of action of low molecular weight organic compounds such as R-848 in B lymphocyte activation is of considerable interest, as they are structurally quite different from the receptors whose signaling effects they mimic. Experiments in progress have revealed that R-848 does not exert its effects by engaging a cell surface receptor on B cells, but it is not yet clear to which intracellular structures or receptors it binds (M. A. Tomai, unpublished observations). We thus undertook this study to determine which signaling pathways are exploited by the drug to result in B cell activation. Information such as this can contribute to both increased understanding of B cell activation pathways as well as improved small molecule design for enhanced activation of immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse B cell lines CH12.LX and CHB3 have been previously
described (15, 16). Production and characterization of
CH12.LX cells expressing the Lac repressor (CH12.LAC) (17)
as well as those inducibly expressing a mutant form of IB
(18) or truncated, dominant negative, mutant
TNFR-associated factor 2
(TRAF2)3 and TRAF3
(19) were described previously. The human
EBV- B cell line Ramos was obtained from
American Type Culture Collection (Manassas, VA). Cell lines were grown
in RPMI 1640 containing 10% heat-inactivated FCS,
10-5 M 2-ME, and antibiotics (B cell medium
(BCM)). Resting splenic B cells from normal 7- to 10-wk-old female
(B6 x 129/J)F2 hybrid mice or
p50-/- knockout mice (provided by Arthur Krieg,
University of Iowa, Iowa City, IA) were purified on a discontinuous
Percoll density gradient as previously described (20).
Small dense B cells were recovered from the 70 to 75% interface for
use in experiments.
Drugs and reagents
The structures of R-848 (S-28463,
4-amino-2-ethoxymethyl-,-dimethyl-1H-imidazo-[4,5-c]quinoline-1-ethanol)
and its inactive analogue S-26424 have been previously described
(1). These compounds are proprietary molecules of 3M
Pharmaceuticals (St. Paul, MN) and are used as HCl salts. The drugs
were dissolved in BCM and stored as stock solutions at 4°C for up to
1 mo. Recombinant mouse IL-6 was purchased from PharMingen (San Diego,
CA), and recombinant mouse TNF- was purchased from Endogen (Woburn,
MA). Isopropylthio-
-D-galactoside (IPTG) was
purchased from Life Technologies (Gaithersburg, MD).
Antibodies
The 1C10 anti-mouse CD40 hybridoma was a gift from Dr.
Frances Lund (Trudeau Institute, Saranac Lake, NY). The EM95 hybridoma,
which served as an isotype control for 1C10 stimulation, was provided
by Dr. Thomas Waldschmidt (University of Iowa). The G28-5 hybridoma
producing anti-human CD40 mAb and hybridomas producing the
anti-mouse IL-6 mAbs 20F311 and 32C11 were obtained from American
Type Culture Collection. The above-mentioned mAbs were purified from
hybridoma supernatants using saturated ammonium sulfate precipitation.
The anti-TNF- mAbs MP6-XT22 and MP6-XT3 were gifts from Dr. John
Harty (University of Iowa). Abs specific for NF-B p50, p52, p65
(Rel-A), c-Rel, and Rel-B were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-rabbit IgG-HRP was purchased from Bio-Rad
(Hercules, CA).
c-Jun kinase assay
CH12.LX cells or resting splenic B cells (5 x 106 or 107/condition, respectively) were incubated with 1 µg/ml of R-848, its inactive analogue, the anti-CD40 mAb 1C10, or its isotype control EM95 for 5 min (CH12.LX) or 10 min (splenic B cells) at 37°C. Cells were then lysed, and c-Jun kinase activity was measured as described previously (18). Reactions were separated by SDS-PAGE. Gels were stained with Coomassie blue and dried. Phosphorylated c-Jun was visualized by autoradiography.
Assay for p38 kinase
CH12.LX cells or resting splenic B cells were stimulated with
R-848 or its inactive analogue for 5 min (CH12.LX) or 10 min (splenic B
cells) at 37°C. Cells were centrifuged at 500 x g
for 5 min at 4°C and washed once with ice-cold PBS. Cells were lysed,
and p38 kinase activity was assayed using a p38 mitogen-activated
protein kinase kit (New England Biolabs, Beverly, MA), according to the
manufacturer’s protocol. An in vitro kinase assay was performed in
supplied kinase buffer containing 20 µM ATP, 0.5 µCi
[
-32P]ATP, and GST-activating transcription
factor 2 as a substrate for 30 min at room temperature. The reaction
was terminated with an equal volume of 2x SDS buffer, and samples were
heated at 95°C for 5 min. Samples were resolved by 12% PAGE, and the
phosphorylated activating transcription factor 2 substrate band was
visualized using autoradiography.
Nuclear extraction and EMSA
CH12.LX, CHB3, Ramos, or splenic B cells
(107/sample) were stimulated at a concentration
of 106/ml with 1 µg/ml of R-848 or its inactive
analogue S-26424 for 5 min to 2 h at 37°C. Nuclear extracts were
then prepared from cells as previously described (21).
Extracts were recovered, quantitated against a BSA protein standard,
and stored at -70°C in the presence of 5 µg/ml antipain, 30
µg/ml leupeptin, 50 µg/ml aprotinin, and 400 µM sodium vanadate.
Double-stranded DNA probes were end labeled with
[-32P]ATP using T4 polynucleotide kinase.
The sequence of the probe for NF-B was described previously
(21). A total of 5 µg of nuclear extract was incubated
with 0.5 ng of probe for 30 min. Samples were separated on a 5% native
polyacrylamide gel at a constant current of 20 mA. X-ray film was
exposed to dried gels overnight at -70°C.
Ab secretion assays
To induce Ab secretion, CH12.LX cells (1500 cells/well in 150
µl) inducibly expressing IBAA or dominant negative TRAF2
(DNTRAF2) or DNTRAF3 were incubated for 24 h in 96-well
flat-bottom microtitration plates in the presence of 100 µM IPTG to
induce production of the IBAA or DNTRAF proteins. Various stimuli
were then added in a volume of 50 µl, and cells were incubated for an
additional 48 h. Stimuli included various combinations of the
following, all determined in preliminary experiments to be saturating
final concentrations: R-848 (200 ng/ml), the anti-CD40 mAb 1C10 (1
µg/ml), IL-6 (10 ng/ml), the anti-IL-6 mAbs 32C11 and 20F311 (3
µg/ml), TNF- (25 ng/ml), or the anti-TNF mAbs MP6-XT22 and
MP6-XT3 (5 µg/ml). CH12.LX cells inducibly produce IgM specific for
an Ag, phosphatidylcholine, which is present on the membranes of SRBC
(22). Differentiation to Ig-secreting cells can thus be
quantitated by counting the number of cells in each replicate culture
capable of forming lytic plaques on a lawn of SRBC at the end of the
culture period as previously described (23). Results are
presented as the mean number of plaque-forming cells per viable cells
recovered from replicate cultures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the earliest events induced by CD40 signaling in B cells is
the activation of the stress-activated protein kinases c-Jun kinase and
p38 (24, 25, 26). R-848 also rapidly induced both kinases in
the representative B cell line CH12.LX, as well as in purified splenic
B cells (Fig. 1
). Similar results were
found by cross-linking either mouse or transfected human CD40. The
inactive analogue of R-848 (S-26424), however, had no effect on the
activity of these kinases in either cell type.
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B activation in both mouse and human B
cells
CD40 signaling to B cells induces strong activation of the NF-B
family of transcription factors, and this event has been shown to be
necessary for CD40-mediated B cell differentiation and up-regulation of
the B7 costimulatory molecules (18). As it has been
recently shown that R-848, like CD40, induces B cell differentiation to
Ab secretion and can enhance CD40-mediated B7 up-regulation
(11), it was of interest to determine whether R-848 also
induces NF-B activation in B cells. Fig. 2A shows that within 5 min
R-848 (but not its inactive analogue) induced nuclear translocation of
NF-B in the mouse B cell lines CH12.LX and CHB3, both of which have
been previously shown to display an activated phenotype following R-848
treatment (G. A. Bishop and M. A. Tomai, manuscript in preparation).
Peak increases were seen between 15 and 60 min, with levels decreasing
in CHB3 cells by 4 h. In addition, R-848 induced NF-B
activation in human B cell lines, as shown in Fig. 2A in the
EBV- cell line Ramos. The kinetics of induction
were shifted to later time points in Ramos cells. Fig. 2B
shows that R-848 induced levels of NF-B translocation similar to
those stimulated via CD40, and the use of cold competitor probes
demonstrated that the activation was specific.
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B activation in normal resting mouse splenic B cells (Fig. 3), similar to the induction seen using
agonistic anti-CD40 mAbs. Of particular interest was the finding
that although a CD40 signal no longer induced NF-B nuclear
translocation in mice genetically deficient in the NF-B p50 subunit,
R-848 still induced NF-B activation in the B cells of
p50-/- mice. We have previously shown that
CD40-mediated NF-B activation in B cells involves nuclear
translocation of the family members p50, p65, and c-Rel
(21). The data shown in Fig. 3 raise the possibility that
R-848 activates the translocation of different NF-B subunits. In
Fig. 3, the shifted complex differs in size from that seen in wild-type
mice; however, this was not a consistent finding. To determine whether
different NF-B subunits are involved in CD40 vs R-848-mediated
NF-B activation, supershift analysis was performed. Fig. 4 presents data demonstrating that
R-848-induced NF-B nuclear translocation in B cells involves the
same subunits as those participating in CD40 signaling. It is likely
that different homo- or heterodimers of these subunits are
preferentially used by the two signals, and this may explain why
although R-848 and CD40 have remarkably similar effects on B cells, the
two signals can also act cooperatively in some signaling events (G. A.
Bishop and M. A. Tomai, manuscript in preparation).
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B activation
Previous studies showed that CD40-mediated differentiation of B
cells to Ab secretion is highly dependent upon CD40-induced NF-B
activation (18). We have found that R-848, like CD40,
induces Ab secretion and synergizes with signals delivered through the
B cell Ag receptor. It was thus of interest to determine whether the
R-848-mediated activation of NF-B is required for the ability of the
drug to induce Ab production. To do so, we used a subclone of CH12.LX
that stably and inducibly expresses a mutant form of the NF-B
inhibitory molecule, IB. In this mutant molecule (IBAA),
the two serine residues that are phosphorylated as a consequence of
activation signals, leading ultimately to the degradation of IB,
have been changed to alanines. Expression of the mutant protein is
normally repressed by constitutive expression of the bacterial Lac
operon repressor protein (LacR) in this cell line, but can be induced
by inclusion of IPTG in the culture medium. We have previously shown
that induced expression of IBAA in CH12.LX effectively blocks
NF-B activation (18). Fig. 5A shows that R-848-mediated
activation of B cells expressing only LacR (CH12.LAC) was not inhibited
by IPTG, but cells induced to express IBAA showed highly
diminished Ab secretion in response to the drug. This dependence of the
Ab response on NF-B activation was quite similar to that seen for
CD40 signaling, as shown in Fig. 5B, where the two stimuli
are compared in the same experiment. Thus, NF-B activation by R-848
is important for mediating the downstream effects of the drug on B
lymphocytes.
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We have recently found that R-848 treatment of B cells increases
both the gene expression and protein production of the cytokines
IL-6 and TNF (G. A. Bishop and M. A. Tomai, manuscript in
preparation), both of which have been shown to stimulate B cell
Ab production (27). To extend these findings, we wished to
determine whether either cytokine was involved in R-848-mediated B cell
differentiation. Fig. 6A shows
that treatment of CH12.LX cells with a saturating amount of exogenous
IL-6 (the maximal amount induced by R-848) induced Ab production,
although not to the same extent as R-848 itself. R-848-induced
differentiation could be completely blocked with a saturating
concentration of the IL-6-blocking mAb, 20F3–11 (second set of bars),
but was unaffected by a nonblocking IL-6-specific mAb (32C11). Fig. 6A also shows that R-848-induced Ab production was inhibited
50% by treatment with 20F3-11. This suggests that IL-6 is an
important component of the mechanism by which R-848 induces B cells to
secrete Ab, but cannot completely account for the effect. Fig. 6B shows that, similarly, a saturating amount of exogenously
added TNF- stimulated Ab secretion by CH12.LX, but was less
effective than R-848 itself. A mixture of two anti-TNF-blocking
mAbs also reduced R-848-induced Ab secretion, by 60%. Finally, the
results shown in Fig. 6C demonstrate that the presence of
blocking Abs to both IL-6 and TNF reduced CD40- and R-848-stimulated Ab
production to a greater extent than blocking either cytokine alone.
These results indicate that both IL-6 and TNF play important roles in
R-848-induced B cell differentiation.
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CD40 is a member of the TNF receptor (TNF-R) family of molecules,
and the cytoplasmic proteins known as TRAFs have been strongly
implicated as playing important roles in signaling through this family
of molecules (28). The TRAFs are thought to exert their
effects by direct binding to the cytoplasmic domains of the various
TNF-R family molecules in whose functions they are involved. However,
we have recently shown that TRAF2 can strongly influence CD40-mediated
B cell differentiation even when it cannot directly associate with CD40
(19). An indirect way in which TRAF2 could influence Ab
secretion is that CD40 signals are known to induce B cell TNF-
production, and TNF can induce Ab secretion (27, 29). In
addition, we have recently found that R-848 induces CH12.LX cells to
produce TNF- (G. A. Bishop and M. A. Tomai, manuscript in
preparation). TRAF2 binds to the cytoplasmic domains of both CD40 and
the TNF-R (28), so it could indirectly influence CD40-
and/or R-848-induced Ab secretion by affecting TNF-R signaling. We thus
tested whether induced expression of a truncated, DNTRAF2 molecule in B
cells affects R-848-induced differentiation, as Fig. 6
B
shows that this effect is partially dependent upon TNF production. Fig. 7A shows that induced
expression of DNTRAF2 inhibits both CD40- and R-848-mediated Ab
secretion to approximately the same extent as does treatment with
anti-TNF mAbs (see Fig. 6B). We have recently found that
DNTRAF2 also inhibits TNF-induced Ab production by CH12.LX cells
(G. A. Bishop and B. S. Hostager, manuscript in
preparation). However, induced expression of DNTRAF3, which binds
CD40 but not the TNF-R, inhibits only CD40-mediated differentiation and
does not affect R-848-mediated activation (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similar to the natural signal provided by CD40 ligation
(18), R-848 stimulates activation of stress-activated
protein kinases (Fig. 1). There is no evidence, however, that R-848
directly activates protein tyrosine kinases (M. A. Tomai,
unpublished observations). Similarly, although there is indirect
evidence that CD40 signaling may ultimately involve tyrosine kinases,
convincing in vivo evidence that CD40 associates with tyrosine kinases
has been difficult to obtain (reviewed in Ref. 32). R-848,
like CD40 (21) and another potential new adjuvant, CpG
oligonucleotides (33) also activates the NF-B family of
transcription factors (Figs. 2 and 3), and R-848-induced IgM secretion
depends upon NF-B activation (Fig. 4). NF-B activation by the
imidazoquinolines has also been demonstrated in human monocytes
(10). The activation of members of the NF-B family has
been shown to make important contributions to many immune activation
processes (34) and provides an important proviability
signal to cells (35, 36, 37, 38, 39). Of particular interest was the
finding that although CD40-induced NF-B activation was dependent
upon the p50 subunit, R-848-induced NF-B activation, although
inducing nuclear translocation of p50, was able to proceed in the
absence of this subunit (Fig. 3). This result suggests that although
the two types of signal are each able to induce nuclear translocation
of the same Rel family subunits, CD40 preferentially stimulates the
movement of hetero- and homodimers containing p50, while R-848 can also
stimulate translocation of dimers lacking p50.
Our previous studies showed that R-848 induces production of a variety
of cytokines in cells of the immune system, including B lymphocytes
(7, 40). The present study determined that R-848-induced B
cell production of both TNF- and IL-6 play important roles in
R-848-induced Ab secretion (Fig. 6). IL-6 has been shown to potently
preserve B cell viability (27, 41, 42) and stimulate B
cell differentiation (27, 43, 44, 45, 46). Additionally, IL-6
production has been demonstrated to play a significant role in
CD40-mediated B cell differentiation (47, 48, 49). Thus, R-848
again shows its ability to mimic signals delivered to B cells during
normal T-dependent Ab responses. Although TNF can induce apoptosis in a
variety of cells via the type I TNF-R (50), it has been
shown that TNF induces human B cells to secrete Ab (27, 51), a finding we have recently reproduced in mouse B cells
(B. S. Hostager and G. A. Bishop, manuscript in preparation).
Thus, TNF production induced by R-848 has a positive effect in
stimulating an Ab response. We also show that this effect requires
participation of the cytoplasmic adapter molecule TRAF2, which has been
shown to participate in signal transduction via the TNF-R
(52). The result that DNTRAF3, which binds CD40 but not
the TNF-R, does not affect R-848 signaling (Fig. 7) supports the
hypothesis that TRAF2 affects R-848 signaling indirectly via affects on
TNF-R signaling.
IL-6 and TNF are known mediators of inflammation in a variety of clinical situations (53, 54), and a number of therapeutic strategies have sought to block the actions of these cytokines. However, in designing more effective vaccines and adjuvants, it may clearly prove desirable to stimulate B lymphocyte production of IL-6 and TNF to obtain a more effective initial Ab response. The present study shows that R-848 can achieve this response, using molecular mechanisms remarkably similar to those used by CD40 signaling to B lymphocytes. The ability of R-848 to stimulate IL-12 production may also contribute to the potential of this compound as an adjuvant (7, 9). Mimicking the normal T cell-dependent pathways of B cell activation may prove a promising strategy to effective manipulation of the adaptive Ab response.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gail A. Bishop, Department of Microbiology, University of Iowa, 3-570 Bowen Science Building, Iowa City, IA 52242.
3 Abbreviations used in this paper: TRAF, TNFR-associated factor; BCM, B cell medium; TNF-R, TNF receptor; DNTRAF, dominant negative TRAF; IPTG, isopropylthio--D-galactoside; LacR, bacterial repressor of the Lac operon.
Received for publication May 5, 2000. Accepted for publication August 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation by a distinct subset of CD40-mediated effector functions in B lymphocytes. J. Immunol. 162:2804.B activation in lymphocytes. J. Immunol. 159:4898.[Abstract]
: autocrine and paracrine cytokines involved in B cell function. J. Immunol. 146:3462.[Abstract]
and lymphotoxin- via anti-CD40 in human B cells. Int. Arch. Allergy Immunol. 107:368.[Medline]
B and IB and sustained activation of NF-B. J. Immunol. 160:1240.B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
-induced apoptosis by NF-B. Science 274:787.B pathways. J. Exp. Med. 188:1381.B antiapoptosis: induction of TRAF1 and TRAF2 and cIAP1 and cIAP2 to suppress caspase-8 activation. Science 281:1680.B activation and reactive oxygen intermediates. J. Immunol. 162:1510.B-mediated up-regulation of BCL-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. USA 96:9136. is involved in the polyclonal B-cell activation induced by HIV-infected human T cells. Nature 363:464.[Medline]
in physiological and pathological conditions. Med. Sci. Res. 23:75.This article has been cited by other articles:
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  T. J. Vanden Bush, C. M. Buchta, J. Claudio, and G. A. Bishop Cutting Edge: Importance of IL-6 and Cooperation between Innate and Adaptive Immune Receptors in Cellular Vaccination with B Lymphocytes J. Immunol., October 15, 2009; 183(8): 4833 - 4837. [Abstract] [Full Text] [PDF]
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J. S. Poovassery, T. J. Vanden Bush, and G. A. Bishop Antigen Receptor Signals Rescue B Cells from TLR Tolerance J. Immunol., September 1, 2009; 183(5): 2974 - 2983. [Abstract] [Full Text] [PDF]
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 A. L. Peters, R. M. Plenge, R. R. Graham, D. M. Altshuler, K. L. Moser, P. M. Gaffney, and G. A. Bishop A novel polymorphism of the human CD40 receptor with enhanced function Blood, September 1, 2008; 112(5): 1863 - 1871. [Abstract] [Full Text] [PDF]
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 E. L. J. M. Smits, P. Ponsaerts, Z. N. Berneman, and V. F. I. Van Tendeloo The Use of TLR7 and TLR8 Ligands for the Enhancement of Cancer Immunotherapy Oncologist, August 1, 2008; 13(8): 859 - 875. [Abstract] [Full Text] [PDF]
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 H. J. Martin, J. M. Lee, D. Walls, and S. D. Hayward Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus J. Virol., September 15, 2007; 81(18): 9748 - 9758. [Abstract] [Full Text] [PDF]
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P. Xie, B. S. Hostager, M. E. Munroe, C. R. Moore, and G. A. Bishop Cooperation between TNF Receptor-Associated Factors 1 and 2 in CD40 Signaling J. Immunol., May 1, 2006; 176(9): 5388 - 5400. [Abstract] [Full Text] [PDF]
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S. Borsutzky, K. Kretschmer, P. D. Becker, P. F. Muhlradt, C. J. Kirschning, S. Weiss, and C. A. Guzman The Mucosal Adjuvant Macrophage-Activating Lipopeptide-2 Directly Stimulates B Lymphocytes via the TLR2 without the Need of Accessory Cells J. Immunol., May 15, 2005; 174(10): 6308 - 6313. [Abstract] [Full Text] [PDF]
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I. B. Bekeredjian-Ding, M. Wagner, V. Hornung, T. Giese, M. Schnurr, S. Endres, and G. Hartmann Plasmacytoid Dendritic Cells Control TLR7 Sensitivity of Naive B Cells via Type I IFN J. Immunol., April 1, 2005; 174(7): 4043 - 4050. [Abstract] [Full Text] [PDF]
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 C. L. Ahonen, C. L. Doxsee, S. M. McGurran, T. R. Riter, W. F. Wade, R. J. Barth, J. P. Vasilakos, R. J. Noelle, and R. M. Kedl Combined TLR and CD40 Triggering Induces Potent CD8+ T Cell Expansion with Variable Dependence on Type I IFN J. Exp. Med., March 15, 2004; 199(6): 775 - 784. [Abstract] [Full Text] [PDF]
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C. L. Doxsee, T. R. Riter, M. J. Reiter, S. J. Gibson, J. P. Vasilakos, and R. M. Kedl The Immune Response Modifier and Toll-Like Receptor 7 Agonist S-27609 Selectively Induces IL-12 and TNF-{alpha} Production in CD11c+CD11b+CD8- Dendritic Cells J. Immunol., August 1, 2003; 171(3): 1156 - 1163. [Abstract] [Full Text] [PDF]
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 D. M. Koelle and L. Corey Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research Clin. Microbiol. Rev., January 1, 2003; 16(1): 96 - 113. [Abstract] [Full Text] [PDF]
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 Y. Iwata, T. Wada, K. Furuichi, N. Sakai, K. Matsushima, H. Yokoyama, and K.-i. Kobayashi p38 Mitogen-Activated Protein Kinase Contributes to Autoimmune Renal Injury in MRL-Faslpr Mice J. Am. Soc. Nephrol., January 1, 2003; 14(1): 57 - 67. [Abstract] [Full Text] [PDF]
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B. S. Hostager and G. A. Bishop Role of TNF Receptor-Associated Factor 2 in the Activation of IgM Secretion by CD40 and CD120b J. Immunol., April 1, 2002; 168(7): 3318 - 3322. [Abstract] [Full Text] [PDF]
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