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Transgenic Expression of a Human
Polyreactive Ig Expressed in Chronic
Lymphocytic Leukemia Generates
Memory-Type B Cells That Respond to
Nonspecific Immune Activation
George F. Widhopf II, Diana C. Brinson, Thomas J. Kipps
and Helen Tighe
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 172:2092-2099; ;
doi: 10.4049/jimmunol.172.4.2092
http://www.jimmunol.org/content/172/4/2092
The Journal of Immunology
Transgenic Expression of a Human Polyreactive Ig Expressed
in Chronic Lymphocytic Leukemia Generates Memory-Type B
Cells That Respond to Nonspecific Immune Activation1
George F. Widhopf II,* Diana C. Brinson,† Thomas J. Kipps,* and Helen Tighe2†
T
he elimination of B cells expressing Ig with self-reactivity
is critical for establishing and maintaining immunological
self-tolerance. Most autoreactive B cells are deleted or
rendered anergic at the immature stages in the marrow, as has been
clearly demonstrated in studies of Ig transgenic mice (1–5). Autoreactive, nonanergic B cells that escape the marrow generally are
eliminated upon encounter with autoantigen in the periphery
(6 – 8).
Although anergy is the primary mechanism of tolerance to soluble self-Ags in the periphery, a notable exception is tolerance to
self-IgG. High-affinity rheumatoid factor (RF)3-expressing B cells
can be deleted by activation-induced apoptosis upon encounter
with soluble human (h)IgG (9, 10). In contrast, B cells expressing
Ig with low affinity for self-IgG are apparently unaffected by the
presence of autoantigen (11). Many such Abs are also polyreactive, because they bind with low affinity to two or more distinct
self-Ags. Unlike the pathologic high-affinity RF found in patients
Divisions of *Hematology/Oncology, and †Rheumatology, Allergy, and Immunology,
Department of Medicine, University of California, San Diego, La Jolla, CA 92093
Received for publication September 4, 2003. Accepted for publication December
4, 2003.
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 in part by an Arthritis Foundation Biomedical Sciences
award, a Multiple Myeloma Foundation grant, and Leukemia and Lymphoma Society
translational awards to H.T., G.F.W., and T.J.K., and this work was also funded in
part by R37 49870.
2
Address correspondence and reprint requests to Dr. Helen Tighe, University of
California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 920930663. E-mail address: [email protected]
3
Abbreviations used in this paper: RF, rheumatoid factor; GVHD, graft-vs-host disease; CLL, chronic lymphocytic leukemia; BCR, B cell receptor; HSA, heat-stable
Ag; h, human; m, mouse; s, surface; MZ, marginal zone; PALS, periarteriolar sheath;
HEL, hen egg lysozyme.
Copyright © 2004 by The American Association of Immunologists, Inc.
with rheumatoid arthritis, these natural autoantibodies are present
in the circulation of many normal healthy individuals where they
have no apparent pathological significance. However, similar Abs
are also frequently associated with graft-vs-host disease (GVHD),
mixed cryoglobulinemia, or Waldenströms macroglobulinemia,
and are expressed by the malignant B cells of patients with various
lymphoproliferative diseases, such as chronic lymphocytic leukemia (CLL) (12). The link between Ab specificity and these disease
processes is unclear.
Polyreactive autoantibodies are naturally occurring Ig, primarily
of the IgM isotype, that bind with low affinity to two or more
distinct self- or nonself-Ags, such as hIgG, ssDNA, dsDNA, histones, cardiolipin, actin, and/or cytoskeletal components (13, 14).
The autoreactive B cells that produce these Ig are not rendered
anergic, because the autoantibodies they produce constitute a large
fraction of total serum Ig. Such Abs additionally account for a
large proportion of the early human B cell repertoire and are speculated to contribute to homeostasis and/or competence of the primary humoral immune response. Polyreactive autoantibodies generally are encoded by nonmutated germline V region genes (V
genes). In both humans and mice, the cells that frequently produce
these autoantibodies are B-1 B cells that coexpress B cell surface
Ags and the CD5 molecule. In mice, these cells are commonly
found in the peritoneum (15, 16).
Mounting evidence exists from several recent studies that engagement of the B cell receptor (BCR) with self- or autoantigen
can influence the fate and differentiation of B cells in lymphoid
tissues (17–22). Antigenic selection also has been suggested for
the development and maintenance of natural autoantibodies (13,
14, 20, 23). This is supported by studies showing that such polyreactive Ig are encoded by a limited and conserved set of Ig VH
genes (24). Also, structure-function analyses of Ig H chains have
demonstrated that the somatically generated third complementarity
determining region contributes significantly to the polyreactive
0022-1767/04/$02.00
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We generated transgenic mice, designated SMI, expressing unmutated H and L chain Ig genes encoding a low-affinity, polyreactive
human (h)IgM/␬ rheumatoid factor. These animals were compared with control AB29 transgenic mice expressing a hIgM/␬
rheumatoid factor specific for human IgG, with no detectable reactivity with mouse proteins. SMI B cells expressed significantly
lower levels of surface hIgM/␬ than did the B cells of AB29 mice, but still could be induced to proliferate by surface Ig cross-linking
in vitro and could be deleted with anti-Id mAb in vivo. Transgene-expressing B cells of AB29 mice had a B-2 phenotype and were
located in the primary follicle. In contrast, a relatively high proportion of hIgM-expressing B cells of SMI mice had the phenotype
of B-1 B cells in the peritoneum or marginal zone B cells in the spleen, where they were located in the periarteriolar sheath,
marginal zone, and interfollicular areas that typically are populated by memory-type B cells. Although the relative proportions
of transgene-expressing B cells in both types of transgenic mice declined with aging, SMI mice experienced progressive increases
in the serum levels of IgM transgene protein over time. Finally, SMI transgene-expressing B cells, but not AB29 transgeneexpressing B cells, were induced to secrete Ab when cultured with alloreactive T cells. These results indicate that expression of
polyreactive autoantibodies can allow for development of B cells that are neither deleted nor rendered anergic, but instead have
a phenotype of memory-type or Ag-experienced B cells that respond to nonspecific immune activation. The Journal of Immunology, 2004, 172: 2092–2099.
The Journal of Immunology
Materials and Methods
Transgenic mice
Transgenic mice were generated that expressed the genes encoding a polyreactive hIgM/␬ RF isolated from the leukemia B cells of a patient with
CLL. The V regions of the H and L chains of SMI, respectively, are encoded by an unmutated allele of the VH1-69 gene, designated 51p1, and an
unmutated V␬III gene, designated A27 (23). The vectors containing human
Ig constant regions are those previously described for the generation of the
AB29 mice that express the rearranged Ig H and L chains encoding the
LES hIgM/␬ RF, and include the murine H chain enhancer for B cellspecific expression (29). The S12 and S6 transgenic lines were generated
from individual founder mice expressing identical H and L chain genes
encoding the SMI polyreactive IgM Ab. Data from experiments using S12
mice are shown, although numerous experiments have been repeated using
both strains. All transgenic lines were maintained by backcrossing heterozygous males with C57BL/6 females (The Jackson Laboratory, Bar
Harbor, ME). Positive progeny are identified at 4 –5 wk of age by testing
sera for the presence of hIgM and human ␬ L chains, as previously described (29). All mice were housed in the animal facility of the University
of California, San Diego.
Flow cytometry analyses
Harvested mouse spleens were teased into single-cell suspensions using
RP-10 medium (RPMI 1640 medium supplemented with penicillin, streptomycin, 2 mM L-glutamine, 5 ⫻ 10⫺5 M 2-ME, and 10% heat-inactivated
FBS). Peritoneal cells were harvested before splenectomy by injecting the
peritoneum of each mouse with 3 ml of RP-10 medium followed by withdrawal of the peritoneal exudate. Single-cell suspensions were purged of
RBCs by hypotonic lysis with ACK lysis solution (BioWhittaker, Walkersville, MD). Washed spleen cells were incubated for 10 min at 4°C with
anti-CD16/32 (2.4G2) to block FcR-mediated cytophilic binding (BD
PharMingen, San Diego, CA). Cells were stained on ice for 20 min with
optimized amounts of the appropriate mAbs conjugated to FITC, PE,
PerCP, allophycocyanin, or biotin; washed; and, when necessary, stained
for an additional 20 min with streptavidin-allophycocyanin (BD PharMingen). Cells were examined by four-color, multiparameter flow cytometry
using a dual-laser FACSCalibur (BD Biosciences, San Jose, CA). Data
were analyzed using FlowJo analysis software (Tree Star, San Carlos, CA).
Viable lymphocytes were defined by exclusion of propidium iodide and
light scatter characteristics.
mAbs used included B220 (RA3-6B2), CD23 (B3B4), CD43 (S7),
CD21 (7G6), CD5 (53-7.3), heat-stable Ag (HSA; M1/69), CD3 (17A2),
hIgM (G20-127), h␬ (G20-193), and mouse (m)IgM (R6-60.2). These
mAbs were purchased from BD PharMingen. The G6 anti-idiotypic mAb
was as described (30).
Immunohistochemistry
Spleens were snap frozen in optimal temperature medium (OCT; Miles
Laboratories, Naperville, IL). Five-micrometer sections were prepared
from tissue blocks. After air-drying and acetone fixation, endogenous peroxidase activity was blocked using 0.03% hydrogen peroxide. Slides were
blocked with 10% goat serum/1% BSA in PBS (pH 7.4) and then stained
first for mouse T cells using biotinylated anti-CD3⑀ (BD PharMingen),
followed by HRP-streptavidin (Jackson ImmunoResearch, West Grove,
PA), and diaminobenzidine substrate (DAKO, Carpinteria CA). Human
IgM-expressing cells were detected by sequential addition of alkaline
phosphatase-conjugated anti-hIgM (Southern Biotechnology Associates, Birmingham, AL), followed by Vector Blue (Vector Laboratories,
Burlingame, CA).
B cell proliferation assays
Freshly harvested splenocytes were washed, counted, and then suspended
at 3.5 ⫻ 106 cells/ml in RP-10 medium. We added 100 ␮l of this cell
suspension to each well of a 96-well round-bottom plate. To each well, we
added another 100 ␮l of RP-10 medium containing either F(ab⬘)2 goat
anti-hIgM (Jackson ImmunoResearch), aggregated hIgG, or LPS (Salmonella minnesota; Sigma-Aldrich, St. Louis, MO), as indicated in the text.
Aggregated hIgG was prepared by incubating a solution of IgG at 10 –20
mg/ml in PBS (pH 7.4) at 63°C for 1 h followed by cooling on ice for 2 h.
All tests were performed in triplicate. Proliferation was measured on day 2
or 3 of culture by adding 1 ␮Ci of [3H]thymidine to each well. The assay
was terminated 18 h later by harvesting plates using an automated cell
harvester.
Injection of deaggregated hIgG or G6 anti-Id mAb
Human IgG (Miles Laboratories) was purified using a protein A-Sepharose
column, as described (29). The G6 mAb reactive with Ig encoded by the
51p1 VH gene was prepared from the ascites fluid of SCID mice (The
Jackson Laboratory) injected with the G6 hybridoma. G6 mAb was partially purified by a 50% ammonium sulfate precipitation, followed by protein A purification and repeated dialyzes into PBS. All preparations of
hIgG and G6 mAb were tested by the Limulus amebocyte lysate assay
(Associates of Cape Cod, Falmouth, MA) and found to contain ⬍0.2 ng of
endotoxin for each milligram of protein. Aggregated hIgG or G6 were
removed from the preparations immediately before injection by ultracentrifugation at 40,000 rpm for 150 min at 4°C using an SW60 rotor. The top
25% of the preparation was removed gently and then diluted in cold PBS
to achieve a final Ig protein concentration of 3– 4 mg/ml. Immediately
thereafter, 2 mg of the deaggregated hIgG or G6 was injected into the
peritoneum of each recipient mouse. All mice were sacrificed 7 days later,
and the spleen cells were removed for immune phenotypic analyses.
Induction of hIgM secretion in vitro
Spleen cells were added to 96-well round-bottom plates at 3.5 ⫻ 105 cells/
well in 100-␮l volume. We then added either medium or hIgG or aggregated hIgG to a final concentration of 10 or 0.5 ␮g/ml, respectively. To
determine whether T cell help alone was sufficient to stimulate secretion of
hIgM, C57BL/6bm12 allogenic spleen cells were used as a source of T cells,
as described previously (9). Briefly, 5 ⫻ 105 SMI, AB29, or nontransgenic
littermate spleen cells were added to 96-well flat-bottom plates followed by
5 ⫻ 105 C57BL/6bm12 allogenic spleen cells or 5 ⫻ 105 C57BL/6 control
filler spleen cells per well in a total volume of 200 ␮l. All cultures were
performed in duplicate wells and using duplicate plates, and incubated at
37°C in 5% CO2. One set of plates was used for measuring hIgM-secreting
cells by ELISPOT after 4 days of culture. Supernatants were harvested
from the other plate after 7 days of culture to determine levels of hIgM in
the culture supernatant by ELISA (29).
Measurement of hIgM-secreting cells by ELISPOT and ELISA
The numbers of hIgM-secreting cells were measured by ELISPOT. Briefly,
96-well flat-bottom microtiter plates were incubated overnight at 4°C with
goat anti-hIgM at 10 ␮g/ml in PBS. After washing, the plates were washed
with PBS containing 1% BSA to block nonspecific protein-binding sites.
Cultured spleen cells were washed to remove secreted hIgM, suspended in
fresh RP-10, and then added at various concentrations to the ELISPOT
plates. After incubation at 37°C for 4 h, Ab-producing cells were detected
using sequential addition of goat anti-hIgM-biotin (Accurate Chemical,
Westbury, NY) and streptavidin-alkaline phosphatase (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) diluted in PBS followed by 5-bromo-4chloro-3-indolyl phosphate substrate (Sigma-Aldrich). After developing
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binding activity of natural autoantibodies (25–27). Several of the
latter studies were conducted with polyreactive Ig isolated from
leukemic B cells of patients with CLL, a disease in which there is
frequent neoplastic B cell expression of polyreactive IgM autoantibodies, such as those that bind IgG, cardiolipin, ssDNA, actin,
and/or thyroglobulin (12, 28). How cells expressing polyreactive
or natural autoantibodies are regulated or are able to evade immune tolerance is not known. Neither is it clear why such Abs are
disproportionately represented in CLL, mixed cryoglobulinemia,
Waldenström’s macroglobulinemia, or during GVHD following
allogenic stem cell transplantation.
To study this, we generated transgenic mice with B cells that
express a hIgM/␬ Ab with low-affinity RF activity (23). This Ab
was derived from a patient with CLL (designated SMI), whose H
and L chain genes were encoded by 51p1 and A27, respectively.
This Ab also binds myoglobin, thyroglobulin, actin, and ssDNA.
The SMI mice were compared with transgenic mice, designated
AB29, that have B cells that express a hIgM/␬ RF with high affinity for hIgG, but without reactivity with other known Ags, and
in this case, represent a negative control (29). We demonstrated
that SMI hIgM/␬-expressing B cells were neither deleted nor rendered anergic, but populated distinct lymphoid compartments,
were responsive to BCR ligation or to nonspecific T cell help, and
displayed the phenotype of Ag-experienced, or memory-type B
cells.
2093
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POLYREACTIVE HUMAN RF TRANSGENIC MICE
the plates, the wells containing 5–100 spots were counted. The mean numbers of spots per 106 hIgM⫹ cells were calculated from the percentage of
hIgM⫹ cells at the initiation of the spleen cell cultures.
Results
Generation and characterization of SMI transgenic mice
SMI B cells are responsive to ligation of their transgenic BCR
To examine whether SMI hIgM/␬ B cells were anergic, we examined whether we could induce proliferation of splenic hIgM/␬expressing B cells through ligation of the BCR complex. For this,
we cultured splenocytes of SMI or AB29 transgenic mice with
either F(ab⬘)2 anti-hIgM or aggregated hIgG. F(ab⬘)2 anti-hIgM
induced significant proliferation of the hIgM⫹ splenocytes from
either SMI (mean ⫾ SD, 90 ⫾ 12 ⫻ 103 cpm; n ⫽ 4) or AB29
(136 ⫾ 45 ⫻ 103 cpm) (Fig. 2). However, 10 ␮g/ml aggregated
hIgG did not induce significant proliferation of SMI splenocytes
(1.4 ⫾ 0.5 ⫻ 103 cpm), as compared with medium alone (1.2 ⫾
0.2 ⫻ 103 cpm). In contrast, the same amount of aggregated hIgG
induced proliferation of AB29 splenocytes (26 ⫾ 6 ⫻ 103 cpm).
LPS induced proliferation of splenocytes from SMI, AB29, or nontransgenic mice (data not shown). We conclude that SMI B cells
are neither deleted nor rendered anergic in vivo.
FIGURE 1. Phenotypic characterization of SMI hIgM/␬ splenic B cells.
A, Spleen cells of 8-wk-old SMI, AB29, and age-matched nontransgenic
littermates were analyzed by flow cytometry for B cell-restricted expression (upper panel) and allelic exclusion (lower panel) of the human IgM
and human ␬ transgenes. Contour plots depict staining of live lymphocytes,
as determined by propidium iodide exclusion and light scatter characteristics, with fluorochrome-conjugated mAbs specific for the surface Ags
indicated on the axes. B, Human Ig-expressing splenic B cells were examined for expression of surface Ags that distinguish various defined B
cell subsets, as indicated. The upper panel denotes the expression of hIg␬
and CD23 on live lymphocytes. The two lower panels depict staining of
gated human Ig␬⫹ lymphocytes only. The differential expression of CD23
and CD21 allows for the identification of T1-transitional (T1) and MZ B
cells (middle panel). The differential expression of HSA and CD23 allows
for further identification of the T2-transitional (T2) and mature (MAT) B
cells (bottom panel). Data are representative of groups of mice of similar
age and of multiple experiments.
SMI splenic B cells are enriched in T1 and marginal zone (MZ)
B cell subsets
Human Ig-expressing splenic B cells from SMI mice were examined by flow cytometry for expression of surface Ags that distinguish various defined B cell subsets. We found that the proportion
of the hIgM⫹ B cells from 8- to 10-wk-old SMI mice that expressed CD23 (78%) (Fig. 1B, Table III) was significantly lower
than that of hIgM⫹ B cells of age-matched AB29 control mice
(97%; n ⫽ 4; p ⬍ 0.0001, Student’s t test). The subset of B cells
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We generated transgenic mice (designated SMI) that expressed a
hIgM/␬ polyreactive, low-affinity RF derived from the leukemic B
cells of a patient with CLL (23). These animals were compared
with control transgenic mice, designated AB29, that expressed a
hIgM/␬ high-affinity RF that reacts with hIgG, but not with other
known Ags, including mouse IgG (data not shown) (9, 23, 29, 31).
We found that expression of a low-affinity polyreactive Ab does
not lead to cell deletion. Approximately 12% (mean ⫾ SD, 12.0 ⫾
1.9%; n ⫽ 4) of the splenic mononuclear cells of SMI transgenic
mice expressed surface (s)hIgM/␬ at 8 –10 wk of age (Fig. 1A).
The H and L chain Ig transgenes were coordinately expressed on
the surface of 32% (31.7 ⫾ 6.0%; n ⫽ 4) of the splenic B cells.
Furthermore, hIgM/␬ expression was restricted to B cells (Table I).
Allelic exclusion of the H chain was nearly complete, because cells
expressing mIgM did not coexpress hIgM. In some experiments, a
small proportion (⬍5%) of B cells expressed hIgM in the absence
of human ␬, suggesting that L chain allelic exclusion was not
absolute. Lastly, we found that SMI splenic B cells expressed levels of sIgM (mean fluorescence intensity ratio, 41.1 ⫾ 3.4) that
were significantly lower than that found on AB29 splenic B cells
(mean fluorescence intensity ratio, 67.0 ⫾ 3.2; n ⫽ 4; p ⬍ 0.0001,
Student’s t test), consistent with the phenotype of anergic B cells (2).
To examine whether the hIgM/k⫹ B cells of SMI were resistant
to deletion, we injected a high-affinity ligand (G6) into the peritoneum of SMI mice. G6, a mouse mAb specific for the crossreactive Id present on the SMI hIgM H chain, does not compete
with anti-hIgM mAb for binding to SMI-expressing B cells. Injection of G6 reduced the mean proportion of hIgM/␬-expressing
SMI splenic B cells from 48 to ⬍3% (Table II). The reduction of
shIgM/␬-expressing cells was not due to BCR down-modulation,
because SMI mice that received deaggregated G6 had a concomitant and corresponding loss of splenic B220⫹ cells (Table II). This
depletion of hIgM/k⫹ B cells was similar to that noted for AB29
mice injected with deaggregated hIgG. However, SMI animals injected with deaggregated hIgG had negligible loss of transgeneexpressing splenocytes, as did either mouse strain after injection
with saline alone (Table II), demonstrating the significance of the
affinity of the Ag-Ab interaction in B cell clearance.
The Journal of Immunology
2095
Table I. Phenotype of splenic lymphocyte populations of SMI and
AB29 Ig-transgenic micea
SMI
B220
hIgM/h␬
mIgM
CD3
AB29
Of total
Of B cells
Of total
Of B cells
39.2 ⫾ 2.0
12.0 ⫾ 1.9
15.1 ⫾ 1.8
50.4 ⫾ 4.0
100
31.7 ⫾ 6.0
42.0 ⫾ 2.0
47.0 ⫾ 1.6
32.3 ⫾ 6.3
6.8 ⫾ 0.4
43.2 ⫾ 6.4
100
68.4 ⫾ 11.2
16.8 ⫾ 1.8
a
Values shown represent the mean percentage ⫾ SD of viable splenic mononuclear cells (of total) or splenic B220⫹ cells (of B cells) that express each surface
Ag(s). Values are the average of four 8- to 10-wk-old mice per group and are representative of multiple experiments.
SMI peritoneal B cells are enriched in both B-1a and B-1b
subsets
We also examined the peritoneal lymphocytes of SMI mice for
coexpression of the hIgM transgenes and surface Ags that distinguish various defined B cell subsets. As noted for other mouse
strains, the peritoneal B cells of SMI and control AB29 transgenic
mice had higher proportions of B-1-type B cells than did the B
cells in the spleen (data not shown) (33). B cells that express
hIgM/␬ constituted 7.2 ⫾ 1.9% or 20 ⫾ 2.0% of the peritoneal
lymphocytes from 8- to 10-wk-old SMI or AB29 mice, respectively ( p ⬍ 0.0001; n ⫽ 4; Student’s t test). However, SMI mice
exhibited significantly higher proportions of hIgM/k B-1 B cells in
their peritoneum, because 21 ⫾ 4.4% (n ⫽ 4) of the peritoneal
transgene-expressing B cells from SMI mice were B-1a B cells
(CD5⫹CD23⫺CD11b⫹) (Fig. 3) and 4.4 ⫾ 1.2% (n ⫽ 4) were
B-1b cells (CD5⫺CD23⫺CD11b⫹). In contrast, in AB29 mice,
FIGURE 2. SMI splenic B cells are responsive to ligation of their
hIgM/␬ BCR. Splenocytes from SMI, AB29, or nontransgenic littermates
were stimulated in vitro with 10 ␮g/ml F(ab⬘)2 anti-hIgM or cultured in
medium alone. Proliferation was measured on day 2 or 3 of culture by
addition 1 ␮Ci of [3H]thymidine per well. Cells were harvested 18 h later
using an automated cell harvester, and the incorporated [3H]thymidine was
assessed by scintillation counting. A, Graphs depict data expressed as the
mean counts per minute incorporated ⫾ SD per 3.5 ⫻ 105 input spleen
cells. Data represent groups of four mice, with all tests performed in triplicate. B, Graphs depict the same data normalized for equal input of 3.5 ⫻
105 hIgM⫹ B cells, calculated based on the percentage of spleen cells
expressing hIgM at the start of the culture. Background proliferation in the
presence of medium alone has been subtracted from all values.
only 2.8 ⫾ 1.9% (n ⫽ 4) or ⬍1% (0.7 ⫾ 0.4%; n ⫽ 4) of transgene-expressing peritoneal B cells were B-1a- or B-1b-type B
cells, respectively. The remaining human transgene-expressing
peritoneal B cells were B-2 B cells, as indicated by their coexpression of the CD23 (Fig. 3).
SMI B cells localize within the periarteriolar sheath (PALS),
MZ, and interfollicular space, as well as in the primary follicle
Fresh-frozen sections of spleens from SMI or AB29 control mice
were examined to study the tissue distribution of hIgM/␬-expressing B cells. We found that both strains of mice had transgeneexpressing B cells scattered throughout the primary B cell follicles
Table II. Deletion of SMI hIgM/␬ B cells upon introduction of high-affinity ligand in vivoa
SMI
SMI
SMI
AB29
AB29
Treatment
% hIgM of Total
None
hIgG
G6 mAb
None
hIgG
19.3 ⫾ 7.7
18.8 ⫾ 4.3
0.8 ⫾ 0.4
32.8 ⫾ 6.2
5.0 ⫾ 0.3
% hIgM of B Cells
47.7 ⫾ 20.2
46.1 ⫾ 5.5
2.9 ⫾ 2.1
67.9 ⫾ 5.9
20.8 ⫾ 3.5
% B Cells of Total
41.2 ⫾ 3.1
40.5 ⫾ 4.6
30.5 ⫾ 5.7
48.2 ⫾ 8.6
24.6 ⫾ 5.8
a
SMI and AB29 mice were injected in the peritoneum with 2 mg of G6 mAb, 2 mg of deaggregated hIgG (hIgG), or PBS
alone (none), and sacrificed 7 days later for immune phenotypic analysis. Splenic lymphoid cells were stained for expression of
hIgM or B220, and analyzed by flow cytometry. Data are expressed as the mean percentage of positive cells ⫾ SD of either total
viable lymphocytes (of total) or B220⫹ cells (of B cells). The data represent the average of four mice per treatment group and
are representative of duplicate experiments.
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that express CD23 is comprised of both mature B-2 cells
(CD23⫹CD21lowHSAlow) and T2 transitional B cells
(CD23⫹CD21⫹HSAhigh) (32). In SMI mice, 57 ⫾ 3.9% of the
splenic hIgM⫹ B cells were B-2 type cells and 19 ⫾ 2.1% were T2
B cells. In contrast, 82 ⫾ 4.4% of the splenic hIgM⫹ B cells of
AB29 mice were B-2-type B cells and 14 ⫾ 4.4% were T2-type B
cells. As such, the proportion of the hIgM transgene-expressing B
cells of SMI mice that were mature B-2 type B cells was significantly lower than that found in AB29 mice ( p ⬍ 0.0001; n ⫽ 4;
Student’s t test).
The population of splenic B cells that do not express CD23 is
comprised of both immature T1 (CD23⫺CD21⫺HSAhigh) and MZ
B cells (CD23⫺CD21highHSAint) (Table III). Eight- to 10-wk old
SMI mice had significantly higher proportions of transgene-expressing splenic T1 B cells (15.3 ⫾ 2.2%) and MZ B cells (6.8 ⫾
1.8%) than did age-matched AB29 mice, which have only 1.5 ⫾
0.2% ( p ⬍ 0.0001; n ⫽ 4; Student’s t test) and 1.3 ⫾ 0.5% ( p ⬍
0.002; n ⫽ 4; Student’s t test) T1-type and MZ-type transgeneexpressing splenic B cells, respectively.
2096
POLYREACTIVE HUMAN RF TRANSGENIC MICE
Table III. Phenotype of splenic hIgM/␬ B cell subsets of SMI transgenic micea
% of hIgM/␬ B Cells
CD23⫹
CD23⫺
MZ CD23⫺CD21highHSAint
T1 CD23⫺CD21⫺HSAhigh
Mature CD23⫹CD21lowHSAlow
T2 CD23⫹CD21⫹HSAhigh
No. of hIgM/␬ B Cells
SMI
AB29
SMI
AB29
78.2 ⫾ 2.6
21.8 ⫾ 2.6
6.5 ⫾ 1.8
15.3 ⫾ 2.2
57.2 ⫾ 3.9
18.7 ⫾ 2.1
97.3 ⫾ 0.7
2.7 ⫾ 0.7
1.3 ⫾ 0.5
1.5 ⫾ 0.2
81.7 ⫾ 4.4
14.3 ⫾ 4.4
9.2 ⫾ 2.3
2.5 ⫾ 0.3
0.7 ⫾ 0.1
1.8 ⫾ 0.3
6.7 ⫾ 1.5
2.2 ⫾ 0.5
26.8 ⫾ 6.2
0.7 ⫾ 0.1
0.3 ⫾ 0.1
0.4 ⫾ 0.1
22.7 ⫾ 5.4
4.0 ⫾ 1.4
a
Values shown represent the mean percentage ⫾ SD or total number of cells (⫻106) ⫾ SD of viable splenic hIgM/␬ B cells
that express each surface Ag(s). Values are the average of four 8- to 10-wk-old mice per group and are representative of multiple
experiments. int, intermediate.
Age-related changes in transgene expression
We examined for age-related changes in the proportion of transgene-expressing B cells and serum hIgM/␬ over time. We found
the relative proportions of B cells that expressed hIgM/␬ declined
as the mice aged (Table IV). In addition, the total number of hIgM/
␬-expressing splenic B cells decreased upon aging from 9 to 45
wk, from 10.8 ⫻ 106 to 5.4 ⫻ 106 in SMI mice, and from 14.2 ⫻
106 to 9.7 ⫻ 106 in AB29 mice. In contrast, SMI mice had significantly higher levels of serum hIgM/␬ than did AB29 mice at
any age ( p ⬍ 0.001; Student’s t test). For example, at 4 – 6 wk of
age, the mean serum concentration of hIgM/␬ was 33 ␮g/ml
(32.5 ⫾ 9 ␮g/ml; n ⫽ 7) in SMI mice, but only 12 ␮g/ml (12.4 ⫾
3.1 ␮g/ml; n ⫽ 5) in AB29 mice. The differences in the serum
concentrations of the transgene IgM/␬ between the two types of
transgenic mice increased as the animals got older. Whereas SMI
mice at 30 –32 wk of age had an elevated mean serum concentration of hIgM/␬ of 55 ␮g/ml (54.7 ⫾ 21.3 ␮g/ml; n ⫽ 7), comparably aged AB29 mice showed a decline in mean serum concentration to only 4 ␮g/ml (4.4 ⫾ 1.4 ␮g/ml; n ⫽ 5).
SMI B cells secrete Ab in response to T cell help alone
Given the indications that a proportion of the SMI B cells had an
Ag-experienced phenotype and the well-documented association
FIGURE 3. Phenotypic characterization of SMI
hIgM/␬ peritoneal B cells. Peritoneal B cells of
8-wk-old SMI and AB29 mice were analyzed by
flow cytometry for expression of human Ig transgenes and other cell surface markers that define B
cell subsets. Contour plots depict staining of live
lymphocytes, as determined by propidium iodide exclusion and light scatter characteristics, with fluorochrome-conjugated mAbs specific for hIgM and either murine CD5 or CD23 (upper panel). The
bottom panels depict the logarithmic fluorescence
intensity (x-axis) of CD5 (lower left panel) or CD23
(lower right panel) of gated hIgM⫹ SMI (shaded
histogram) or AB29 (open histograms) peritoneal B
cells.
of polyreactive autoantibodies with GVHD, we attempted to determine whether T cell help alone is sufficient to stimulate secretion of SMI hIgM, by culturing splenocytes from SMI mice with
C57BL/6bm12-alloreactive T cells. Addition of C57BL/6bm12 alloreactive T cells to SMI splenocytes induced differentiation of IgM/
␬-secreting cells, as assessed by ELISPOT assay on day 4 of culture (2.7 ⫾ 0.7 ⫻ 103 spots per million B cells; n ⫽ 4)) (Fig. 5).
We also noted increased hIgM in the culture supernatants of wells
containing the C57BL/6bm12-alloreactive T cells by ELISA (data
not shown). In contrast, addition of C57BL/6bm12-alloreactive T
cells to AB29 splenocytes generated significantly fewer IgM/␬secreting cells per million B cells (173 ⫾ 50; n ⫽ 4; p ⬍ 0.001,
Student’s t test). All animals had negligible responses to either
medium alone or to control autologous C57BL/6 splenocytes (Fig.
5). AB29 splenocytes, but not SMI splenocytes, also responded to
aggregated hIgG (4.8 ⫾ 0.3 ⫻ 103 vs 48 ⫾ 55; n ⫽ 4).
Discussion
In this study, we found that expression of human low-affinity,
polyreactive Ig, encoded by unmutated Ig V genes that are used
frequently by CLL B cells, could induce mouse B cells to differentiate into nonnaive, memory-type B cells that are hyperresponsive to nonspecific T cell help. The human Ig-expressing B cells
from SMI mice were neither deleted nor rendered anergic, because
they were found in the peripheral lymphoid compartments and
responded well to stimulation via their transgenic Ag receptors,
respectively. Importantly, we found SMI mice, but not control
AB29 mice, developed some hIgM/␬⫹ B cells in memory-type B
cell compartments in the absence of human Ags. Furthermore,
SMI mice had significantly higher proportions of hIgM⫹ B cells
that were T1-type splenic B cells (CD23⫺CD21⫺HSAhigh),
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of the white pulp and mantle zones of secondary follicles, areas
where resting mature B cells typically are located (Fig. 4). However, the SMI mice were distinctive in that many transgene-expressing cells were found to reside within the MZ. In addition,
SMI hIgM/␬-expressing cells also were found in the interfollicular
space and PALS, areas typically inhabited by B cells that have
encountered Ag. In contrast, AB29 mice had few transgene-expressing B cells in these subanatomic sites.
The Journal of Immunology
2097
splenic MZ B cells (CD23⫺CD21highHSAint), and B-1-type peritoneal B cells (CD5⫹CD11b⫹) than did control AB29 mice, which
had mostly CD23⫹ T2-transitional and primary follicular hIgM/␬⫹
splenic B cells. Moreover, a significant proportion of the splenic
hIgM⫹ B cells of SMI mice were located within the PALS, MZ,
and the interfollicular areas that typically are populated by memory or Ag-experienced B cells. Finally, some SMI B cells were
able to secrete hIgM/␬ when provided with nonspecific T cell help,
further demonstrating that these cells are Ag experienced, rather
than naive B cells.
The influence that expression of hIgM/␬ has on B cell differentiation is not solely a consequence of the expression of a rearranged human Ig transgene, as noted by the differences in splenic
B cell populations of SMI vs control AB29 mice. Whereas the SMI
Ig molecule is derived from the B cells of a patient with CD5⫹
CLL and is a low-affinity RF that also binds to a variety of self-Ags
(23), the mutated high-affinity hIgM/␬ RF of AB29 reacts only
with hIgG. Unlike AB29 mice, SMI mice do not experience deletion of transgene-expressing B cells upon treatment with
deaggregated hIgG. Nevertheless, SMI hIgM/␬ B cells can be de-
leted by injection of deaggregated high-affinity anti-idiotypic G6
mAb, indicating that SMI transgenic B cells are not protected from
clearance by high-affinity Ag.
A number of other Ig transgenic mice have been used to examine the development and differentiation of B cells, particularly
those that express Ag receptors that are reactive against self-Ags.
They have demonstrated that differentiation of these B cells is
influenced by a combination of mechanisms operating in both the
central and peripheral lymphoid compartments (34 –36). Although
BCR expression is critical for B cell differentiation (37, 38), engagement of the BCR with self-Ag can profoundly influence the
fate of B cell development. The ultimate fate of an autoreactive B
cell depends upon several factors, such as the form and location of
Ag, Ag concentration, structure, the affinity of the expressed BCR,
and the availability of T cell helper activity (34, 36). Mice that are
made transgenic for both Ig with high Ag-binding affinity and specific Ag, experience either anergy or deletion of B cells that expressed the Ig transgenes, unless they undergo Ig receptor editing
to express BCR that no longer react with the transgenic Ag (39).
Unlike transgenics expressing higher affinity receptors for soluble
IgG (29) or reacting to membrane-bound hen egg lysozyme (HEL)
(3) or H-2 Ags (4), SMI B cells are not deleted and are found in
peripheral lymphoid compartments. Additionally, they are not rendered anergic as a consequence of maturational arrest or exclusion
Table IV. Age-related changes in the phenotype of splenic lymphocyte populations of SMI transgenic micea
SMI
6
Total cells (⫻10 )
% B220 of live cells
Total B cells (⫻ 106)
% hIgM/␬ of B cells
Total hIgM/␬ B cells (⫻ 106)
hIgM/␬ in sera (␮g/ml)
AB29
Nontransgenic
Young
Old
Young
Old
Young
Old
78.0 ⫾ 13.9
35.2 ⫾ 5.2
27.2 ⫾ 4.9
39.5 ⫾ 2.9
10.8 ⫾ 2.4
32.5 ⫾ 9.0
92.0 ⫾ 11.8
41.2 ⫾ 4.8
37.6 ⫾ 3.3
14.3 ⫾ 4.0
5.4 ⫾ 1.5
54.7 ⫾ 21.3
46.8 ⫾ 11.2
39.8 ⫾ 3.6
18.6 ⫾ 5.0
76.7 ⫾ 0.9
14.2 ⫾ 3.8
12.4 ⫾ 3.1
67.5 ⫾ 16.6
42.3 ⫾ 5.4
28.1 ⫾ 5.5
33.8 ⫾ 5.8
9.7 ⫾ 3.2
4.4 ⫾ 1.4
89.1 ⫾ 14
57.8 ⫾ 4.0
51.4 ⫾ 7.9
93.5 ⫾ 13.9
59.0 ⫾ 1.5
55.3 ⫾ 9.6
a
Values shown represent the mean percentage ⫾ SD or total number of cells (⫻ 106) ⫾ SD of viable cells, as indicated. Young animals are 8 –10 wk of age, and old animals
are 42– 45 wk old. Values represent the average of four to six mice per group and are representative of multiple experiments.
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FIGURE 4. SMI hIgM B cells are located in splenic areas typically
populated by Ag-experienced or memory B cells. Immunohistochemical
analysis of sections prepared from the spleens of SMI (A), AB29 (B), and
nontransgenic (C) mice. Five-micrometer spleen sections were stained for
human IgM (blue) and murine CD3 (brown) to examine the tissue distribution of hIgM/␬-expressing B cells. Each panel is divided to show both
low-power (⫻100, upper panels) and high-power (⫻200, lower panels)
views of the same section. Although the AB29 hIgM⫹ B cells were restricted to the primary follicles (B), SMI hIgM/␬⫹ B cells were also present
in the PALS (T) and interfollicular areas (IF).
FIGURE 5. Alloreactive T cell help alone promotes differentiation and
Ab secretion by SMI hIgM/␬ B cells. SMI and AB29 spleen cells or spleen
cells from nontransgenic littermates were stimulated in vitro with alloreactive C57BL/6bm12 spleen cells to provide T cell help (T cell help), control
filler C57BL/6 spleen cells (no T cell help), 10 ␮g/ml hIgG, 0.5 ␮g/ml
heat-aggregated hIgG (agg hIgG), or medium as a control. The numbers of
cells secreting hIgM were measured at day 4 by ELISPOT. Data are expressed as the mean number of hIgM-secreting cells per 106 hIgM-positive
cells, calculated based on the percentage of spleen cells expressing hIgM
at the start of the culture. Data are expressed as the mean ⫾ SD for groups
of four mice. All tests were performed in duplicate.
2098
encoded Ig expressed by CLL B cells encode a third complementarity determining region with conserved amino acid motifs (69).
More recently, a study using microarray analysis revealed that the
gene expression pattern of CLL B is distinct from that of other B
cell lymphomas or of adult or cord blood B cells (70). Moreover,
this CLL gene expression profile is similar to that of nonnaive, or
memory-type, B cells that frequently reside in the MZ of secondary lymphoid tissue. In addition, CLL B cells that have unmutated
Ig genes express several genes that are up-regulated during BCR
ligation. As such, these leukemia cells in particular appear to have
genetic features in common with B cells that have encountered Ag.
Although expression of such Ig by normal B cells does not by itself
induce the development of CLL, low-level chronic stimulation
may result in prolonging the life of an autoreactive B cell and thus
provides an expanded window of time in which the requisite
events for leukemogenesis can occur. Additionally, because B
cells expressing SMI IgM/␬ are susceptible to nonspecific immune
activation by the provision of T cell help alone, such encounter
could render them more susceptible than naive or primary B cells
to incurring somatic mutations and cytogenetic changes that result
in CLL. Conceivably, these cells could be predisposed to leukemogenesis by virtue of their expressed Ig. As such, the Ig expressed by such B cells might be relevant to the etiopathogenesis
of CLL, independent of specific human or environmental Ags.
Acknowledgments
We thank Patty Charos, Josh Kohrumel, and Esther Avery for their excellent technical assistance, and Drs. Dennis Carson and David Engel for their
numerous helpful discussions.
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from the B cell follicles of the spleen, as are those of other mice
such as those specific for dsDNA (40, 41) or sHEL (2, 6, 42).
The SMI mice share some, but not all, characteristics with other
transgenic mice that express Ig with low affinity for self-Ag, such
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B cells of SMI mice, most of these other transgenic mice have loss
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