REVIEW
Peripheral B lymphocyte tolerance
Amanda Gavin, Djemel Aı̈t-Azzouzene, Annica Mårtensson, Bao Duong, Laurent Verkoczy, José Luis Skog,
Patrick Skog and David Nemazee
Department of Immunology, and Macromolecular Structure and Chemistry Program, The Scripps ResearchInstitute, La Jolla, CA, USA(Received for publication on February 9, 2004)

Abstract. This lecture discusses two interrelated topics, B cell tolerance in the peripheral immune
system and BAFF. Using the 3-83 antibody transgenic mouse bred to mice carrying cognate antigen
in the liver, we previously found that clonal elimination drastically reduced the precursor frequency
of autoreactive cells. The consensus model to explain this tolerance is the 2-signal hypothesis, which
proposes that in the absence of T cell help BCR stimulation is a negative signal for B cells. However,this model fails to explain how these same B cells can respond to T-independent type II (TI-2) antigens, raising the question of how they distinguish TI-2 antigens from multimeric self determinants. We
propose that B cells use NK-like missing self recognition to provide the needed speciﬁcity, as foreign
antigens are unlikely to carry self markers. The model has implications for the evolution of the immune
system, B lymphocyte signaling, tissue speciﬁcity of autoimmunity, and microbial subversion of theimmune system. Overexpression of the critical B cell survival cytokine BAFF/BLyS has been associated with autoimmunity. We have discovered a novel splice isoform that regulates BAFF activity andmay play a role in limiting B cell activity. The novel form, called DBAFF, is able to heteromultimerizewith normal BAFF and can suppress receptor binding and proteolytic release from the cell surface.
Preliminary studies from transgenic mice overexpressing wild type or DBAFF are consistent with apossible regulatory role for DBAFF, raising the possibility that the relative expression levels of BAFF
and DBAFF regulates tolerance. (Keio J Med 53 (3): 151–158, September 2004)
Key words: antibody, BAFF, TNF13B, inhibitory signaling, tolerance, B lymphocyte

Introduction
A new theory of self/non-self discrimination in mature
B cells
A coherent theory to explain mature B cell responsiveness and tolerance does not exist. A number of lines
of evidence indicate that mature B cells are tolerizable.
The consensus model to explain this tolerance is the 2signal hypothesis,1 which proposes that in the absenceof T cell help BCR stimulation is a negative signal for B
cells, leading to death or functional inactivation. However, these same B cells can respond to T-independent
type II (TI-2) antigens, which have the properties ofhigh multivalency and an absence of T cell determinants. (TI-2 antigens by deﬁnition lack intrinsic mitogenicity and therefore likely do not activate toll-likereceptors.) Indeed, responses to TI-2 antigens can occur
normally in T cell deﬁcient mice. These considerations
raise the question of how B cells can distinguish TI-2
antigens from multimeric self determinants.
A competing alternative to the 2-signal model,
initially suggested by Lederberg, is that tolerance sensitivity of B cells is dependent on the age of the individual; cells from immature individuals are tolerance
sensitive, whereas those of adults are activatable only.
Work of several laboratories has indeed indicated thatrecently formed splenic B cells or neonatal B cells may
be especially susceptible to tolerance induction.2 Unlike newly formed bone marrow B cells,3–5 these cells
do not appear to undergo efﬁcient receptor editing, but
are tolerized through an apoptotic process.6 In addition, the ability of B cells to respond to TI-2 antigens is
acquired relatively slowly after birth.7 While transi-

Presented at the 1338th Meeting of the Keio Medical Society in Tokyo, December 12, 2003.
Reprint requests to: Dr. David Nemazee, The Scripps Research Institute, Mail drop IMM-29, 10550 N. Torrey Pines Rd., La Jolla, CA 92037,
USA, email: nemazee@scripps.edu
151

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Gavin A, et al: Missing self recognition in B lymphocytes

Fig. 1 TI-2 Ags (A) may be distinguished from self (B) by a second marker on self cells. Receptors for the putative second signal could be
expressed by soluble factors in the plasma, by putative non-T-cell leukocyte ‘‘helpers,’’ or, as indicated here, by the B-cell itself.

tional B cells are more easily tolerized in some experimental situations, mature B cells are clearly tolerizable
in vivo, at least in the case of some experimental ligands
such as anti-IgD, lysozyme or MHC class I.8–13 Given
that these tolerizable cells are able to respond to TI-2
antigens, despite the high multivalency of TI-2 antigens
and their ability to evoke antibody responses even in
the total absence of T cells,14–15 it is not clear how TI-2
antigens avoid inducing tolerance and promote activation. In an attempt to resolve this paradox, we haverecently proposed the hypothesis that B cells use NKlike missing self recognition to provide the self/non-self
discrimination needed to distinguish foreign TI-2 antigens from multivalent self.16‘‘Missing self’’ recognition is believed to be an important aspect of natural killer (NK) cell signaling.17 In
NK cells, several receptors are expressed that can stimulate activation of killer function.18 These activatory
receptors are counteracted by the signaling of more
numerous inhibitory receptors. Both activating and inhibitory receptors often recognize MHC class I molecules or related structures. Because of the balance of
signals mediated by these receptors, NK cells can be
activated under physiological conditions by target cells
that lose the expression of a subset of self MHC molecules. This is the basis for such phenomena as hybrid
resistance, which is the ability of an F1 hybrid of two
inbred strains to reject parental bone marrow grafts
through NK-mediated killing. NK recognition is thought

to be important for killing of infected or defective hostcells, which may lose MHC class I expression as part ofa microbial strategy or tumor selection to hide from theCD8 cytotoxic T cells of the adaptive immune system.
As illustrated in Fig. 1, we propose that unlike
foreign antigens, self antigens, even those that are displayed in a multimeric array, are associated with one
or more widely expressed ‘‘self markers’’ that arecapable of stimulating a B cell’s inhibitory receptors.
Putative self markers would include particular carbohydrate moieties, such as Siaa2-6Galb1-4GlcNAc, thewidely expressed host ligand for the B cell-restricted
inhibitory receptor CD22. Another established class ofself markers is the MHC class I antigens; B cells carry atleast one cell surface inhibitory receptor with apparent
reactivity to MHC class I molecules, PirB, but its functional signiﬁcance is unknown. Interestingly, both PirB
and CD22 genes have strong homology to NK inhibitory receptors (KIRs) and their genes are located in the
genome adjacent to the major KIR homology cluster inboth human (chr 19q13) and mouse (chr 7), within theleukocyte receptor cluster (LRC).19 Upon activation,
PirB and CD22 proteins are phosphorylated at immunoreceptor tyrosine inhibitory motifs (ITIMs; consensus[L/I/V/S]xYxx[L/V]) and recruit the tyrosine phosphatase SHP-1,20–23 a mode of action essentially identicalto the authentic NK inhibitory receptors.24 Other B cell
receptors with inhibitory function also map to the LRC
region, including LAIR and CD66a.19 Additional in-Keio J Med 2004; 53 (3): 151–158

hibitory receptors on B cells that contain ITIM motifs
and recruit SHP-1, SHP-2 or SHIP-1 phosphataseactivities include PD-1, FcgRIIb, CD72, CD31 and additional members of the FcR superfamily.25–30 Therefore, B cells express a diversity of inhibitory receptors
similar to those of NK cells that may participate in‘‘missing self’’ recognition.
In contrast to their expression of a multiplicity ofNK-like inhibitory receptors, B cells do not appear
to express any NK-like activatory receptor except
the BCR itself. Activatory receptors on NK cells are
similar to BCR and TCR in their modes of intracellularsignaling. Like the antigen receptors, activatory NK
receptors do not have intrinsic signaling function; they
are transported to the plasma membrane with signaltransducer proteins.18 The transducers themselves are
small dimeric transmembrane proteins with conserved
cytoplasmic immunoreceptor associated tyrosine activatory motifs (ITAMS); consensus: [D/E]xxYxx[L/I]xxxxxxxYxx[L/I].31 These ITAM motifs are sites oftyrosine phosphorylation and recruitment of ZAP70
and or syk protein tyrosine kinases. Signal transducers
for T cells, NK cells, and myeloid cells include CD3z; g; d; e, DAP12 and FceRIg. B cells lack these transducers, but express the related heterodimer Iga/b.
Interestingly, like the NK cell ITAM-containing transducer DAP12, Iga is encoded near the KIR locus at
19q13.2. Iga and Igb are required both for B cell antigen receptor transport to the plasma membrane andcoupling of antigen recognition to the signaling machinery of the cell through cytoplasmic ITAM motifs.31
A common feature of activatory receptors is thepresence in the transmembrane region of polar or
charged amino acid residues (usually K or R) that interact with a residue of opposite charge (D or E) in achain of one of the associated transducers.18–31 A consequence of this design is that expression of functional
activatory receptors is restricted by transducer expression and speciﬁcity of association. Because Iga and
Igb do not appear to directly associate with activatory
receptors other than sIg (nor to dimerize with othertransducers), the lack of expression in B cells of transducers besides Iga/b insures that B cells cannot use nonimmunoglobulin activatory receptors, even those thatare expressed inside the cells, such as PirA.32 In contrast, NK and myeloid cell types express multiple
transducers that convey signals from a broader arrayof receptors. For example, NK cells express CD3z andFceRIg in addition to DAP12. Some activatory receptors, such as NKp46, interact with all of these transducers, while others associate solely with particularhomodimers of DAP12 (human KIR2DS) or FceRIg(mouse NKR-PI)18; NKp46 can also signal throughCD3z-FceRIg heterodimers. In any case, the suppression by B cells of alternative signal transducers along

153

Fig. 2 Hypothetical evolutionary progression differentiating lymphocyte function from its primordial NK activity, to NK and adaptive
lymphocyte subsets. Shapes in gray represent inhibitory receptors,
those in white, activatory receptors. The model suggests that with
the invention of V(D)J recombination at a midpoint in evolution, a
subset of lymphocytes discontinued expression of most or all nonrearranging activatory receptors, increasingly focussing on the rearranging activatory receptors BCR or TCR, while retaining multiple
inhibitory receptors.

with the speciﬁcity of Iga/b for association with sIg
appears to insure that a single type of activatory receptor, the BCR, is functional in B cells.
We therefore propose that in the evolution of theadaptive immune system, B and T lymphocytes suppressed expression of innate activatory receptors after
the invention of V(D)J recombination, but retained
the recombining activatory receptor, along with a signiﬁcant collection of inhibitory receptors (Fig. 2). One
vestige of the proposed NK origin of lymphocytes is thefocus of NK and CD8 T cells on similar MHC class I
antigens. A second vestige is the continued expression
on a subset of T cells of the NK activatory receptor
NK1.1, which in part deﬁnes the NKT cell.33 (Upon
activation, CD8 cells express a different type of NK
receptor associated with the PI3K-recruiting adapter
DAP10).34 This hypothesis may also help explain odd
NK-like features sometimes seen in lymphocytes that
promote monoallelic distribution of receptors, such as
parental imprinting of the PirB gene.35
The notion that B cells express inhibitory receptors that recognize widely expressed self ligands has
numerous implications for immune tolerance and autoimmunity. It is well known that tissues protect themselves from the toxic effects of antibody-mediated complement activation by expressing various regulators of
the complement system, such as CD46, CD55 and154

Gavin A, et al: Missing self recognition in B lymphocytes

CD35; such inhibition works at the effector phase ofantibody responses.36 We suggest that the role of selfmarker expression by tissues is in part to suppress theinitiation phase of such antibody responses through engagement of inhibitory receptors on B cells.
However, tissues may differ in their expression levelsof self markers because there are likely to be multiple
self markers and cognate receptors involved in the tolerance process. This heterogeneity, coupled with allelicvariation in self markers such as MHC class I molecules, may lead to a situation in which in certain individuals particular tissues are more vulnerable thanothers to a breakdown in tolerance. This target tissue
component of immune tolerance may explain how evengeneral defects in lymphocytes can lead to tissue speciﬁc autoantibody disease.37 A second hypotheticalcontributor to autoantibody disease could be aberrant
expression of non-BCR activatory receptors on B cells.
For example, aberrant expression of FceRIg and
FcgRIII in mature B cells is predicted to result in B
cell hyperreactivity. Interestingly, these receptors are
expressed by early progenitors of T and B cells, but arenormally turned off with maturation.38
Pathogenic microbes have evolved means to suppress antibody responses. One way they can do so is to
produce ligands for inhibitory receptors. The humancytomegalovirus is known to produce a pseudo MHC
class I ligand UL18 that engages not only NK inhibitory
receptors, but also the B cell inhibitory receptor ILT2,
the human homologue of PirB.39–40 B cells and phagocytes recognize sialic acid moieties on self cells using a
number of inhibitory receptors of the so called Siglec
family.39 B cells may express as many as 5 different
Siglecs, including CD22.39 Because the ability to produce sialic acids is a relatively recent evolutionary invention, lacking in most non-deuterostome organisms,
it is signiﬁcant that several pathogenic bacteria have
independently evolved or captured the enzymatic machinery to do so from their hosts.39 This bacterial
adaptation should suppress antibody responses early
in infection, prior to the development of robust T cell
help. Another potential way that microbes could perturb the B cell system is to introduce activatory signaling that is independent of the BCR. This is known tooccur in the case of two viruses, Epstein-Barr virusof humans and Bovine leukemia virus, which encode
ITAM-containing proteins that can promote cell activation, growth and survival of infected cells.41 Suchdysregulation probably promotes B cell neoplasia.

Table 1 Genes Implicated in B Cell Tolerance or Autoimmunity

The regulation of B cell survival by BAFF
In the preceding section, we have proposed that B
cell recognition of self by inhibitory receptors, a cell
intrinsic mechanism, facilitates the self/non-self dis-B cell intrinsic
Fas
Cr2 (CD21/CD35)
CD40
FcgRII
Lyn
SHP-1
CD22
Bcl-2
Bim
TLR9
TACI
B cell extrinsic
FasL
Complement C1q, C4
CD40L
IL-4
BAFF
Tlr4 (LPS)

References(59)(60, 61)(62, 63)(64)(65–67)(68, 69)(70, 71)(72)(73, 74)(75)(76)(77–79)(80, 81)(63, 82–84)(62, 85, 86)(50, 87, 88)(89)crimination. The outcome of BCR signaling leading to
B cell anergy, activation, survival or death will reﬂect
thresholds of positive and negative signals, along withextrinsic signals that shift these thresholds. Table 1 lists
genes expressed in B cells or non-B cells whose overexpression or deﬁciency is implicated in promoting
autoimmunity and breakdown in B cell self-tolerance.
In transgenic models where autoreactive B cells havebeen studied, the fate of autoreactive cells is dependent
on the presence of competing, non-autoreactive B cells,
indicating that B cells compete for a limiting survival
factor.42–43 As we discuss below, a key B cell survival
factor whose overexpression is implicated in humoral
autoimmunity is BAFF.
Most B cells require at least two signals to survive
in the peripheral immune system: intrinsic expressionof BCR (i.e., activatory receptor) on the cell surface44
and extrinsic availability of the TNF family cytokine
BAFF.45 Furthermore, BCR and BAFF signaling arealso important in the TI-2 antibody response.46,47 BAFF
overexpression in vivo promotes autoimmune lupuslike disease and potentiates antibody responses.47–50
On the other hand, mice deﬁcient in BAFF or the
BAFF receptor (BAFF-R) lack long-lived follicular B
cells and are hyporesponsive to immunization and germinal center maturation.48,51–53 BAFF also interacts
with two other receptors, TACI and BCMA, which arealso able to bind to APRIL, BAFF’s closest homologue.
Importantly, treatment of lupus-prone mice or a mousemodel of collagen-induced arthritis with soluble Fc
fusion proteins of TACI, which binds to BAFF, canreduce disease incidence and severity.51 On a normal,
non-autoimmune background, short-term treatment

Keio J Med 2004; 53 (3): 151–158

155

Fig. 3 BAFF transgenic (Tg) mice, but not DBAFF transgenics, have preferential augmentation of serum IgA levels. Independent malefounder mice transgenic for BAFF, DBAFF, or non-Tg controls were tested for serum levels of IgM, IgA, IgG1, and IgG2a/b.

with BCMA-Fc fusion protein reduced B cell numbersby two-fold.54 Because overexpression or underexpression of BAFF has pathogenic consequences, it is likelythat natural antagonists of BAFF action exist.
We recently identiﬁed a novel splice isoform of
BAFF that exists in both mouse and human.55 This
form, which is called DBAFF because it lacks a 57-bp
exon, appears to be abundant in quiescent macrophages, while the full-length form predominates in
activated macrophages, in which total BAFF levels are
greatly induced. Furthermore, DBAFF can assemble
disulﬁde-linked complexes both with itself and withwild type BAFF. Interestingly, unlike full length BAFF,
DBAFF fails to be efﬁciently cleaved from the cell
membrane. These features, along with the ability of
DBAFF coexpression to limit BAFF bioactivity in amouse model system, suggest that DBAFF may play arole in limiting antibody, and autoantibody, responses.
DBAFF may modulate BAFF activity by heterotrimerizing with newly made BAFF to affect receptor
speciﬁcity or potency, or by preventing efﬁcient releaseof BAFF from the plasma membrane. Alternatively, orin addition, DBAFF may heterotrimerize with other
TNF family molecules, such as APRIL, which may
suppress B cell response or survival through TACI or
BCMA signaling. We have begun to probe these possibilities using a transgenic overexpression strategy.
MethodTransgenic constructs were generated by introducing
BAFF or DBAFF cDNA sequences downstream of
a 2.9 kb human CD68 promoter.56 The human CD68
promoter construct has been used in transgenic miceto generate myeloid speciﬁc expression.56 Vector
sequences were removed and isolated insert DNA was
microinjected into [(DBA/2XC57Bl6/J)F1 X (C3H/

HeJXC57Bl6/J)F1] zygotes. Young adult mice wereanalyzed for plasma immunoglobulins by solid phase
ELISA assay. Flow cytometry analysis was used toquantify the numbers of B lymphocyte subsets in thelymphoid organs of young adult offspring of foundermice bred to C57Bl6/J. A detailed description of thetransgene constructs and transgenic mouse characterization will be presented elsewhere (Gavin, et al. in
preparation).
Results
Disparate in vivo biological effects of BAFF and DBAFF
We have compared the in vivo functions of BAFF
and DBAFF in a series of transgenic mice expressing
BAFF or DBAFF under the control of the human CD68
promoter, which drives expression in myeloid and dendritic cells, where BAFF is normally expressed.56 As
shown in Fig. 3, several independent founder lines carrying BAFF transgenes have roughly 100-fold increased
levels of IgA, but only @10 fold higher levels of IgM
and marginal increases in IgG. In contrast, DBAFF
transgenics lack elevated (or reduced) IgA levels relative to wild type. While preliminary, this ﬁnding suggests that BAFF may have an especially signiﬁcant,selective adjuvant effect on humoral mucosal immune
responses. In addition, DBAFF transgenics manifested
a reduced level of plasma IgG2a/b and reductions inoverall B cell numbers, particularly in the spleen where
marginal zone B cell numbers were much lower (Table
2).
Discussion
While our analyses of BAFF and DBAFF transgenic mice are still quite preliminary, our results are156
Table 2

Gavin A, et al: Missing self recognition in B lymphocytes
Quantitation of Splenic Marginal Zone B Cell Numbers

MZ cells*

Mean (Std Dev)n¼
P value**

Littermate
controls

Delta BAFF
Tg

BAFF Tg

3.8 (1.2)  10 6
15N/A

2.4 (0.7)  10 6
13
>0.001

13.2 (4.6)  10 6
11>0.0000001* Phenotype deﬁned by ﬂow cytometry staining (B220þ CD21hi
CD23lo). ** P value from two tailed, two sample equal variance Student’s T Test comparing transgenic to control.

consistent with other studies indicating that BAFF
overexpression can increase B cell numbers, and preferentially augment IgA and, to a lesser extent, IgM serum levels.57 Of greater novelty were the results using
the DBAFF expressing transgene. We had guessedthat mice expressing this construct should suppress theeffects of wild type BAFF, resulting in a phenotype
resembling BAFF-deﬁcient mice.48 Our results seem to
conﬁrm the prediction that DBAFF expression partly
neutralizes BAFF activity, because the DBAFF overexpressing mice had the predicted reductions in B cellnumbers. However, little reduction in serum IgM or
IgA levels was seen relative to wild type mice. In contrast, IgG2a/b levels were reduced in DBAFF transgenic mice. These results may suggest a role for DBAFF
in regulating isotype switch, antibody forming cell survival, or the expression of other cytokines that regulatethese processes. Gorelik et al demonstrated that a major source of BAFF expression is radio resistant stromalcells.58 The CD68 expression of these is unknown andmight explain why our DBAFF transgenic mice do not
more resemble a BAFF-deﬁcient phenotype. So far, themost dramatic phenotype in DBAFF transgenic micehas been the reduced numbers of marginal zone phenotype cells, perhaps reﬂecting the myeloid cell expression pattern of the transgenic promoter.
However, the phenotype of DBAFF transgenics
might be based not only on the ability of DBAFF to act
as a dominant negative of BAFF in CD68 positive cells;
DBAFF subunits might associate with other TNF family
proteins, such as APRIL, or form homotrimers withunique biological functions. DBAFF homomultimers
can form and be transported to the plasma membraneof transfected 293 cells or S17 stromal cells.55
Acknowledgements: The authors thank Professor Masayuki Amagaifor the lecture invitation, David Greaves for providing the CD68
promoter construct, and the National Institutes of Health for ﬁnancial
support (RO1GM44809 and P01AG01743).

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