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Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have been studying the B cell response to phosphocholine (PC),3 an Ag expressed on the cell wall of Streptococcus pneumoniae. PC-specific Abs have been shown to be highly protective against infection with S. pneumoniae, (13, 14, 15, 16, 17). Concordantly, the absence of anti-PC Ab-producing B cells in X-linked immune-deficient (Xid) mice leaves this host highly susceptible to infection with S. pneumoniae (16, 17). Analysis of B cell development in Xid mice indicated that PC-specific B cells behave like autoreactive B cells, in that they are clonally deleted (18, 19). Paradoxically, in normal mice, these PC-specific B cells are positively selected into peripheral lymphoid tissues. These data suggest that it is advantageous to the host to retain these potentially autoreactive PC-specific B cells rather than delete them.
During normal B cell development, B lymphocytes that recognize self Ags in the bone marrow (BM) are arrested at the transition from pre-B to immature (sIgM+) cells and are subsequently clonally deleted (20, 21, 22, 23, 24, 25). The interaction between the B cell receptor and membrane-displayed self-Ags or polymeric self-Ags (i.e., DNA) signals the reactivation and up-regulation of the recombinase-activating genes (Rag) and results in efficient receptor editing of IgL chain genes (26, 27, 28, 29, 30). This editing process replaces the original L chain with one that can pair with the IgH chain to form a receptor that binds an Ag other than a self Ag (24, 28). However, in Rag knockout (KO) mice, B cells expressing rearranged H and L chain transgenes encoding autoreactive Ig receptors cannot edit their transgene-encoded Ig receptors because they cannot rearrange their endogenous Ig genes. Therefore, virtually all the B cells expressing autoreactive Ag receptors on a Rag KO background are developmentally arrested at the pre-B/immature B cell transition stage and undergo apoptosis (31).
Additionally, B lymphocytes that recognize self-Ags can undergo tolerance induction. Different states of anergy can result depending on the nature of the autoantigen. In the soluble HEL/anti-HEL system developed by Goodnow and colleagues (32, 33), the anergic B cells down-regulate their IgM receptors and are unable to proliferate in vitro following stimulation with anti-IgD or anti-IgD-dextran (Dex). On the other hand, Xu et al. (34) found that anergic ssDNA-binding B cells did not produce Ab in vivo, but proliferated in vitro in response to anti-µ plus IL-4, and Andersson et al. (31) found that anergic DNA-binding B cells could be induced to differentiate into plasma cells in response to a cross-reactive thymus-independent (TI)-type 2 Ag, trinitrophenyl-Ficoll.
We present evidence that B cells expressing transgenes encoding PC-specific Abs are indeed autoreactive; however, rather than being developmentally arrested and clonally deleted, these autoreactive PC-specific B cells persist by expressing more than one Ab specificity, thereby compromising allelic exclusion and the paradigm of one Ag-specific Ig receptor per lymphocyte.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice carrying the MOPC-167 (M167) myeloma-derived µ
plus 
transgenes (line 207-4, designation Tg[Igh + Igk]Bri12)
(35) were obtained from Dr. U. Storb (University of
Chicago, Chicago, IL) through Dr. R. L. Brinster (School of
Veterinary Medicine, University of Pennsylvania, Philadelphia, PA).
1 transgenic mice were obtained from K. Rajewsky, University of
Cologne (Cologne, Germany), through F. Alt, Children’s Hospital
(Boston, MA) and were backcrossed to Rag-2 KO mice in our laboratory.
Rag-2 KO mice were obtained from Taconic Farms (San Francisco, CA) and
were crossed to Ig transgenic mice in our animal facility. KO mice
were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6
µMT/µMT mice (36, 37) were obtained from Dr. K.
Rajewsky through Dr. C. Sidman (University of Cincinnati,
Cincinnati, OH).
Flow cytometric analysis
Spleen, BM, and peritoneal (PerC) cells were prepared and stained as previously described (38, 39). PE conjugation of anti-VH1, anti-M167-id (clone 28-6-20) (38), and anti-CD23 (40) was conducted by Molecular Probes (Eugene, OR). FITC-conjugated PC-Dex was a gift from Dr. H. Dintzis (The Johns Hopkins University, Baltimore, MD) and was used at a final concentration of 2.5 µg/106 cells. The synthesis of PC-Dex was previously described (19). Stained cells were analyzed using a FACScan, and 5% probability contour profiles were generated using CellQuest Graphics Software (Becton Dickinson, San Jose, CA). Images presented in the figures are representative of three or more experiments per background. In each experiment five or more mice per genotype were analyzed.
ELISA analysis of serum
Blood was collected by retro-orbital puncture, and serum was
titrated on PC-BSA-coated plates as previously described
(41). The plates were washed three times with
Tris-buffered saline and Tween 20 and were tested for the presence of
the transgene-encoded µa allotype, the
endogenous µb allotype, and total Ig using
biotinylated anti-µa (clone DS-1),
anti-µb (clone AF6), and anti-
(clone HB58), respectively. The plates were washed and developed using
streptavidin alkaline phosphatase (Calbiochem, La Jolla, CA) for 1
h at room temperature. The plates were washed, and substrate
(p-nitrophenyl phosphate at 1 mg/ml in 1 M
diethanolamine; 104–100, Sigma, St. Louis, MO) was added for 30 min at
room temperature. Substrate cleavage was read on a Bio-Tek ELx800 ELISA
reader (Bio-Tek, Burlington, VT) at 405 nm, and Ab values were
determined from internal IgM anti-PC standards included on each
plate.
Proliferation assays
Spleen cells were stimulated in vitro with the TI type 2 Ags, PC-Sepharose, PC-Dex, and anti-id conjugated to Sepharose as previously described (19, 42).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used the Rag-2 KO mouse to determine whether PC-binding Ag
receptors were autoreactive in vivo. This was previously predicted from
the finding that these PC-specific B cells appear to clonally delete in
Xid mice (18, 19). Mice carrying the M167 V1 H chain
variant and the -24 L chain as transgenes (M167H:24L)
(35) encoding an anti-PC Ab were crossed onto the
Rag-2-/- (43) background, and B
lymphocyte development was analyzed by characterizing the surface
immunophenotype of cells isolated from the spleens of these mice. Fig. 1
(B and D)
illustrates that there were virtually no mature B cells present in the
spleens of the M167H:24L:Rag-2-/- mice. What
few B cells were observed expressed very low levels of sIgM (Fig. 1B), and no detectable mature
CD23+/sIgM+ B cells were
present (Fig. 1D). Similar analyses of
Rag-2-/- KO mice expressing µ anti-hen
egg lysozyme (anti-HEL) transgenes (32) are presented
as a control for development of B cells expressing non-self Ig
receptors. In contrast to the
M167H:24L:Rag-2-/- mice, large numbers of
mature, sIgMhighB220bright
B cells (Fig. 1A) and
CD23+/sIgM+ B cells (Fig. 1C) were observed in the
anti-HEL:Rag-2-/- mice. These data
demonstrate that B cells expressing the M167H:24L transgene receptor
are arrested at the pre-B/immature B cell transition point typical of B
cells expressing autoreactive Abs. In the phenotypically normal
M167H:24L:Rag-2-/+ littermates, large numbers
of sIgM+:B220+ cells (Fig. 1E) were observed. All the splenic B cells were
VH1+:M167-id+
(Fig. 1F) and bound PC-Dex (Fig. 1H); therefore,
they express the autoreactive B cell receptor. The 28-6-20 Ab used to
identify the M167-Id recognizes the 24L chain when paired with a
heavy chain and therefore identifies 24+
cells.
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24L PC-specific B cells develop in the BM of
TG+ Rag-2-/- mice
The developmental profile of B cells in the BM of
M167H:24L:Rag-2-/- and their phenotypically
normal M167H:24L:Rag-2-/+ littermates is
presented in Fig. 2. B cell development
was clearly arrested at the
B220+/S7+, pro-B cell stage
in the Rag-2-/- mice (Fig. 2H), and
there were no detectable
B220+/sIgM+ or
M167id+/sIgM+ B cells (Fig. 2, G and I, respectively). Expression of the
M167H:24L transgenes in Rag-2-/- mice allows
the B cells to make the pro-B to pre-B transition
(B220+/S7-; Fig. 2B). Similar percentages of
B220+/sIgM+ and
M167id+/sIgM+ B cells were
observed in the M167H:24L:Rag-2-/- as well
as the TG+Rag-2-/+
littermates (Fig. 2, A/C and
D/F, respectively). However, a distinct immature
B cell population, sIgMbright, was clearly
observed in the M167H:24L Rag-2-/+ mice (Fig. 2D), but was missing in the
M167H:24L:Rag-2-/- (Fig. 2A).
Additionally, both the M167H:24L:Rag-2-/-
mice and their TG+ Rag-2-/+
littermates expressed the transgene-encoded 24L chain, as shown by
the expression of M167-id (Fig. 2, C and F);
however, a small population of more mature
sIgMbright B cells was observed in the
phenotypically normal TG+
Rag-2-/+ mice (Fig. 2F), which was
clearly absent in the TG+
Rag-2-/- mice (Fig. 2C).
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In addition to receptor editing, homing to anatomically privileged
sites such as the peritoneal cavity has been shown to be another way
that autoreactive B cells escape clonal deletion (44, 45).
In the transgenic system devised by the Honjo laboratory, transgenic B
cells expressing anti-autoerythrocyte Abs were present in the
peritoneal cavity, but were clonally deleted in the BM. These
peritoneal autoreactive erythrocyte-specific B cells developed into
self-renewing B1 (CD5+) B cells. In our
transgenic system of anti-PC Abs, cells isolated from the
peritoneal cavity of Rag-2-/- KO mice
expressing the M167H:24L transgenes exhibited large numbers of
VH1+ B cells (Fig. 3, A–C). Approximately 42%
(20% of the total events) of these
VH1+ peritoneal B cells
expressed CD5 on their surface and 100% expressed Mac-1 (CD11b; Fig. 3, A and B, respectively), while 20% (9.1% of
the total events) of the
VH1+ B cells present in the
peritoneal cavity of Rag-2+/- anti-PC
transgenic mice expressed CD5, and 
50% (24% of the total events)
expressed Mac-1 (Fig. 3, D and E, respectively). Forty
percent (17 and 14% of the total events) of peritoneal
VH1+ B cells in
M167H:24L:Rag-2-/+ mice and
IgM+ C57BL/6 control mice expressed CD23 (Fig. 3, F and I, respectively), whereas the
TG+ Rag-2-/- mice lacked
these CD23+ B2 B cells (Fig. 3C). All
the peritoneal B cells were both M167-id+ and
bound PC-Dex (data not shown). Thus, like autoreactive
erythrocyte-specific B cells (44, 45), PC-specific B cells
can escape deletion by homing into the peritoneal cavity and
differentiating into B1 B cells.
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In several model systems in which potentially autoreactive B cells
are generated, overexpression of bcl-2 does not alter their
susceptibility to induction of central tolerance in the BM, but can
prevent induction of peripheral clonal deletion (20, 46, 47, 48). We crossed Rag-2-/-
M167H:24L, anti-PC transgenics to
Rag-2-/- bcl-2 transgenic mice to
determine the effect overexpression of bcl-2 would have on B
cell development in these mice. As we previously observed in Xid mice
(19), overexpression of bcl-2 prevented the
clonal deletion of PC-specific cells in the
Rag-2-/- M167H:24L/bcl-2 double-transgenic
mice (Fig. 4, D–F compared
with A–C). The absolute number of
M167-id+/sIgM+-staining B
cells increased 10-fold in the spleen of many of these mice,
corresponding to an 10-fold increase in the percentage of
sIgM+ cells (compare Fig. 4, A and
B to D and E). All these B cells were
PC specific when stained with FITC-conjugated PC-Dex (Fig. 4F). However, the level of sIgM was lower on B cells from
the Rag-2-/- bcl-2+
M167H:24L B cells compared with B cells from the
Rag-2+/+ M167H:24L mice (compare Fig. 4, D and E to G and H).
Although introduction of the bcl-2 transgene resulted in
increased numbers of sIgM+ B cells compared with
those in bcl-2-/TG+
Rag-2-/- mice (Fig. 4, A and
B vs D and E), these B cells were
still anergic, as reflected by the absence of anti-PC Abs in their
serum (Table I).
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While the specific autoantigen recognized by PC-binding Abs is
still undefined, it is clear from previous data in Xid mice and the
above data in Rag-2-/- KO mice that B cells
expressing M167H:24L, PC-binding Abs are characteristically
autoreactive, i.e., they undergo developmental arrest in the BM and
home to privileged sites in the periphery. However, normal mice
carrying these transgenes generated near normal numbers of
B220+/sIgM+ cells (Fig. 1
E). These B cells expressed the V1 H chain gene product and
were positive for the M167-id, which indicates that they are expressing
the 24 L chain in association with the M167 H chain variant (Fig. 1F). Additionally, all these
VH1+ B cells also bound
PC-Dex (Fig. 1H). The first insight into a possible
explanation for the persistence of these B cells, which were obviously
expressing the autoreactive anti-PC Ig receptor, was gained by
examining Fig. 1G. This figure illustrates that 10% of
the VH1+ B cells (4.1% of
the total events) also expressed the L chain. All the
VH1+ cells also expressed
24 (identified by anti-M167-id (28-6-20), which recognizes
24L when associated with an IgH chain) and bound PC-Dex. These data
suggested that these PC-specific B cells coexpress more than one Ig
receptor.
The above data suggest that coexpression of multiple Ig receptors,
which compromises the central tenet of allelic exclusion, may provide a
mechanism by which autoreactive B cells can elude negative selection.
To directly confirm that coexpression of multiple Ig receptors would
rescue autoreactive PC-specific B cells from developmental arrest,
M167H:24L Rag-2-/- mice were crossed to 1
L chain Rag-2-/- mice. The offspring of this
cross have one heavy chain and two L chains from which to make an Ab.
One H:L combination is autoreactive, and one is not. In these double L
chain transgenic mice, substantial numbers of
B220+/sIgM+ B cells
developed (Fig. 5A). These
VH1+ B cells expressed the
autoreactive M167-id (Fig. 5B), bound PC-Dex (Fig. 5E), and expressed the L chain (Fig. 5C).
Detection of M167-id and binding to PC-Dex both demonstrate the
expression and association of the 24L chain with the M167H
chain. Finally, coexpression of both 24L chain (as shown by the
presence of the M167-Id) and L chain in association with the
VH1 H chain is demonstrated in Fig. 5D.
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24L transgenes,
20% of the B cells coexpress an endogenous
µb-allotype H chain along with the
transgene-encoded µa -allotype H chain
(41). Many H chain transgenic mice have been described as
leaky or poor at allelic exclusion (5, 49, 50, 51), in that
they coexpress endogenous µb-allotype H chain
along with the transgene-encoded µa H chain. To
ascertain what contribution coexpression of endogenous H chains makes
to facilitating the escape of autoreactive B cells from clonal
deletion, we backcrossed M167H:24L, anti-PC transgenic mice with
µMT H chain KO mice (36, 37). Because the transmembrane
exons of the µ H chain gene are eliminated in these mice, the B cells
cannot insert endogenous H chains into the cell membrane; thus, the
loss of endogenous H chain in this rescue process in µMT mice would
force the B cell to increase coexpression of endogenous L chains to
rescue the autoreactive PC-specific B cells. As shown in Fig. 5F, normal numbers of
B220+/sIgM+ B cells were
observed in the spleens of M167H:24L µMT mice. All the
autoreactive sIgM+ B cells expressed the M167-id
(Fig. 5G) and bound PC-Dex (Fig. 5J). In this
genetic background where the endogenous H chains cannot be used to
modify the autoreactive receptor, 40–50% of the autoreactive,
PC-binding, M167H:24L-expressing B cells coexpressed L chains
(Fig. 5, F–J). Additionally, Fig. 5I clearly
demonstrates that 40–50% of the L chain-expressing B cells also
express sIg receptors containing the L chains. Presumably, the
remainder of the autoreactive PC-binding, M167H:24L-expressing B
cells have rearranged and coexpressed additional endogenous L
chains that do not form an autoreactive Ab when paired with the M167 H
chain.
Coexpression of endogenous L chains is difficult to analyze,
inasmuch as available reagents cannot discriminate between the 24
transgene product and endogenous L chains at the protein level. To
determine whether coexpression of endogenous L chains plays a major
role in modulating the developmental fate of autoreactive,
anti-PC-expressing B cells, the M167H:24L anti-PC transgenes
were backcrossed onto the -/- KO
(52) mouse background. Fig. 5K reveals that in
the absence of endogenous L chain expression, 100% of the
autoreactive, M167-id+
(24L+) B cells coexpressed endogenous L
chains while still binding low levels of PC-Dex (Fig. 5L).
These data are consistent with the observations presented in the other
genetic backgrounds described above and contrast dramatically with the
10% of
M167-id+/+-coexpressing
B cells (4.0 and 4.1% of the total events, respectively) observed in
either +/- M167H:24L (Fig. 5M)
or +/+ M167H:24L (Fig. 1G) mice.
All these data indicate that the developmental fate of these
autoreactive, PC-specific, M167H:24L-expressing B cells is being
altered by coexpression of additional H and/or L chains.
Receptor coexpression reverses anergy and permits serum Ig production
M167-id+ B cells that developed in both M167
µ Rag-/- and
bcl-2+ M167 µ
Rag-/- mice were anergic. This is illustrated
by the data in Table I, which show that no anti-PC Abs were
detected in the serum of these mice. When the tolerant M167 µ
Rag-/- B cells were rescued by coexpression of
a L chain, low levels of TG+ anti-PC Abs
were produced (Table I). High levels of anti-PC Abs were detected
in all strains of M167 µ mice with functional Rag genes, including
M167 µ nude mice. This suggests that not only does receptor
coexpression reverse tolerance, but the in vivo production of
anti-PC Abs is driven by endogenous or environmental TI type 2 Ags.
H chain coexpression is once again demonstrated by the presence of
mixed µa/µb anti-PC
molecules in all M167 µ mice with functional Rag genes, except the
µMT KO mice, which cannot insert the endogenous
µb H chain into the B cell membrane (Table I).
We previously demonstrated mixed
µa/µb anti-PC
molecules in normal M167 µ transgenic mice (41).
M167 µ anergic B cells proliferate in vitro
The data in Fig. 6 show that the
PC-specific B cells in M167 µ Rag-/- and
bcl-2+ M167 µ
Rag-/- mice appear to be
developmentally arrested at a stage similar to anergic ssDNA-binding B
cells (34). When whole spleen cells were stimulated in
vitro with PC-conjugated to Dex or Sepharose or with
anti-M167id-conjugated Sepharose, these anergic B cells
proliferated. When one normalizes for the number of B cells present
in the spleens, the magnitude of proliferation seen in the
B cells from M167 µ Rag-/- and
bcl-2+ M167 µ
Rag-/- mice would be equivalent to that in the
M167 µ Rag+/+ controls. Spleen cells from
Rag-2-/- mice did not proliferate in response
to any of these TI-type 2 Ags (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B cells expressing anti-PC Abs exhibit characteristics of
autoreactive B cells when these cells develop in a Rag-2 deficient
genetic background. Thus, the BM and splenic B cells express low levels
of the transgene-encoded H and L chains and no mature B cells are
present. These M167 µ B cells are clearly anergic, as judged by
the lack of serum anti-PC Abs in either the M167 µ
Rag-/- mice or the
bcl-2+ M167 µ
Rag-/- mice (Table I). However, these low
affinity PC-specific B cells are not as completely tolerized as those
found in sHEL/anti-HEL double-transgenic mice, since they
proliferate in vitro when stimulated with PC-conjugated TI type 2 Ags
(Fig. 6). In this respect these B cells are similar to those found in
transgenic mice with Ig receptors specific for ssDNA
(34).
We have previously shown that B cells expressing anti-PC transgene
Ig receptors in the Xid background are clonally deleted in the absence
of a functional btk signaling pathway. Btk appears to play a critical
role in the positive selection of long-lived B cells following low
affinity receptor engagement by autoantigens that behave like TI type 2
Ags (53, 54). Here we have demonstrated that on the
Rag-2-/- KO background
these same anti-PC transgene-expressing B cells are developmentally
arrested at the pre-B to immature B cell stage. This is similar to B
cells expressing other known autoreactive Ig receptors, such as
anti-DNA, anti-MHC, or anti-erythrocyte receptors. We have
also shown that these autoreactive anti-PC-Ig-expressing B cells
home to the peritoneal cavity, an immunologically privileged site,
where they develop into B1 B cells. When the PC-specific receptors are
modulated by coexpression of endogenous H or L chains, the subsequent
signaling intensity is presumably lowered so that the B cells are not
clonally deleted, and they no longer become anergic. Furthermore, the B
cells rescued by receptor coexpression develop into
CD23+ B2 B cells as well as B1 B cells (Fig. 3).
Clarke and Arnold (55) demonstrated a similar shift from
B1 to B2 cell development in the phosphate dylcholine-binding B cells
that develop in VH12 4 transgenic mice in the
absence of a functional btk signaling pathway. It would therefore
appear that the signal intensity from the Ig receptor regulates which B
cell subset will develop. B1 B cells dominate when receptor engagement
and signaling are high, while lower signaling promotes B2 subset
development.
We have also demonstrated that autoreactive M167H:24L
anti-PC-expressing B cells can be rescued from clonal deletion by
overexpression of bcl-2 in both the Xid and
Rag-/- KO backgrounds.
However, the B cells that develop in bcl-2+
M167 µ Rag-/- mice remain anergic, appear
to down-regulate their sIgM, and do not develop into mature
CD23+ B cells, whereas the B cells rescued in Xid
M167 µ mice are predominantly CD23+ and are
not anergic (19). Once again, the intensity of the signal
and the signaling pathway activated by the sIg receptor alter both the
subset and the functional state of the B cell. Although the B cells
from bcl-2+ M167 µ
Rag-/- mice were anergic in vivo, they
proliferated in vitro in response to TI type 2 Ags (Fig. 6).
Introduction of the 1 L chain into M167H:24L
Rag-2-/- mice was able to rescue B cells
expressing the autoreactive Ig receptor. However, the total number of B
cells in the spleen was still significantly below normal. A similar
decrease in the total number of B cells was observed in the experiments
with -/- KO mice. The decrease in splenic
cell numbers in these two models persists even though near normal
numbers of B220+/sIgM+
cells were observed in the BM. Additionally, normal numbers of splenic
B cells were observed in the µMT experiments. These data could be
explained if the capacity to populate and proliferate within the spleen
varied between B cells expressing different sIg receptors. For B cells
at the same developmental stage, the capacity to populate and
proliferate within the spleen could reflect the level of cell surface
expression of the Ig receptor, the affinity and avidity of the sIg
receptors for some positive-selecting Ag, or the quantity and quality
of the signal induced by sIg receptor engagement.
Surprisingly, in a M167H:24L transgenic Rag+
mouse, the number of anti-PC-expressing B cells observed was
similar to the number of normal B cells observed in transgenic or
normal mice that express nonautoreactive Ig receptors.
Membrane-displayed PC in the BM should lead to the clonal deletion of
immature PC-specific B cells during their ontogeny (22).
However, PC-specific B cells not only escape clonal deletion in the BM,
but appear to be positively selected via an Ag-mediated,
receptor-driven process (19, 38). In
TG- mice, T15-id+ B cells
get selected into the self-renewing, autoreactive,
CD5+ subset of B cells (56). This
apparent incongruity was explained by demonstrating that these
autoreactive anti-PC-expressing B cells escaped clonal deletion by
coexpressing more than one functional Ig receptor.
In other models of B cell development that have examined the fate of B cells that express autoreactive Ig receptors, coexpression of multiple Ig receptors has been described (26, 28, 31, 57). The coexpression of multiple Ig receptors has been interpreted within the context of the autoreactive B cells undergoing the receptor-editing process, suggesting that they are transitional. It is evident from our data that 1) B cells expressing anti-PC receptors are autoreactive; 2) coexpression of multiple Ag receptors by the introduction of multiple transgenes can rescue the autoreactive B cells; 3) in a permissive system (i.e., Rag-2+/+) these autoreactive B cells express more than one functional Ag receptor on their cells; and 4) this population of autoreactive B cells, which expresses more than one functional Ag receptor, is a stable population of B cells and can constitute a significant portion of the B cell population. The data presented here are certainly concordant with the model of receptor editing; however, it would relax the stringency of application of the principle of allelic exclusion.
It should be noted that the immunological consequence of rescuing autoreactive B cells by coexpressing multiple sIg receptors is entirely different from that achieved by receptor editing. Following receptor editing, the initial Ag specificity of the rescued B cell is lost. However, by coexpressing multiple sIg receptors, one autoreactive and one not autoreactive, B cells retain the autoreactive, yet potentially beneficial, sIg receptor and still escape negative selection by clonal deletion. One possible mechanism by which coexpression of multiple receptors may effect this escape from negative selection is by lowering the number of autoreactive receptors. This could functionally raise the threshold for tolerance induction and allow the B cell to be positively selected into the peripheral B cell repertoire. Experiments in normal mice are currently underway to test this hypothesis.
Coexpression of multiple Ig receptors is not limited to transgenic
mouse systems. Gollahon et al. (58) have shown that 20%
of + B cells coexpress L chains. This
observation could not be adequately explained by either a stochastic
model of V gene rearrangement or a feedback model for controlling V
gene rearrangement. It is possible that these endogenous double
L chain B cells represent cells in which the original µ receptor
was autoreactive, and the coexpression of allowed the B cell to
escape tolerance. If our hypothesis is correct, these double L
chain-positive B cells will exhibit a high degree of autoreactivity.
Additionally, B cells that express two H or L chains generally lose one
of these chains after hybridoma formation and in vitro passage
(50, 58). This may account for the fact that few
anti-PC hybridomas or myelomas express more than one L chain.
However, there is at least one T15-id myeloma, the HOPC-8 anti-PC,
in which two different functional L chains are expressed
(59).
Generally, the escape of autoreactive B cells from clonal deletion or anergy is considered to be detrimental to the host. However, in our system compromising the tenet of allelic exclusion and rescuing the autoreactive anti-PC binding B cells confers a clear immunological advantage to the host. The germline-encoded T15-id+, anti-PC Abs dominate the immune response in most strains of mice following vaccination with avirulent S. pneumoniae or PC-conjugated protein Ags (60, 61, 62, 63, 64). Anti-PC Abs are highly protective against pathogens such as S. pneumoniae and filariae, and the inability to produce anti-PC Abs results in a dramatic increase in susceptibility to infection by S. pneumoniae (13, 14, 15, 16, 17). It is likely that this mechanism of escape from clonal deletion and tolerance will account for the frequent observation of potentially beneficial, low affinity autoantibodies in normal individuals. This mechanism is not likely to be used in B cells with high affinity for self Ags; thus, it should not occur when the receptor affinity is >109, as it is in anti-HEL transgenic mice. High affinity autoreactive B cells would be forced to use receptor editing to escape clonal deletion.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James J. Kenny, Room 4C19, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224.
3 Abbreviations used in this paper: PC, phosphocholine; BM, bone marrow; Dex, dextran; HEL, hen egg lysozyme; KO, knockout; Rag, recombinase-activating gene; sIg, secretory Ig; TG, transgene; TI, thymus-independent; Xid, X-linked immune-deficient.
Received for publication August 19, 1999. Accepted for publication February 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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