By
From Abgenix, Inc., Fremont, California 94555
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Abstract |
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The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered
with yeast artificial chromosomes (YACs) containing different sized fragments of the human
heavy (H) chain and light (L) chain loci. Each of the two H and the two
chain fragments
encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique VH (5 versus 66) or V
genes (3 versus 32). Although each of
these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V
gene complexity restored efficient development throughout all the different recombination and
expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation
with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by VH and V
gene complexity: (a) production of functional µ chains at the transition from the pre B-I to the pre B-II stage; (b) productive V
J
recombination at the small pre B-II stage; (c) formation of
surface Ig molecules through pairing of µ chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell
lineage.
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Introduction |
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Generation of Igs by B cells is an ordered and highly
controlled process of gene recombination and expression that plays a key role in the regulation of B cell development. This process is initiated in the bone marrow by a
series of steps in which the genes that encode the different
Ig variable segments (V, D, and J) are joined by sequential
rearrangements, followed by expression of functional Ig
molecules on the surface of B cells. Distinct developmental
stages were identified based on the rearrangement and expression of H and L chain genes and the expression of characteristic sets of cell surface antigens (for a review, see references 1 and 2). The immature pro B/pre B-I cells are
B220loCD43+HSA+c-kit+Ig and in the process of rearranging H chain genes. The more mature pre B-II cells are
B220loCD43
HSA+BP-1+c-kit
CD25+, cytoplasmic µ+
and are rearranging the L chain genes. Finally, there are
two surface (s)Ig1-expressing B cell populations
the immature B220loµhi
and the mature B220hiµlo
+ cells.
The regulation of B cell differentiation by proper rearrangement and expression of the H and L chain genes is
well documented, primarily as a result of studies of mutant
mice in which mediators or cis control elements of antibody production were inactivated by gene targeting technology. Elucidation of structure-function relationships of
the Ig loci focused primarily on cis-acting sequences 3' of
the mouse V region. These studies demonstrated the importance of coding (e.g., J, Cµ, C) and noncoding (e.g.,
Eµ, E
, 3'E, 3'
) sequences in regulation of antibody diversification, assembly, and selection (for a review, see reference 3). However, the role of the V genes, the largest
and most diverse gene family in the Ig loci, in controlling
Ig production and B cell differentiation is still not fully understood. Introduction of rearranged Ig transgenes with defined single specificities into wild-type or Ig-deficient mice
demonstrated the importance of antibody surface expression and its specificity both for development and for positive and negative selection of B cells (4). However, these
model systems did not permit study of the effects of the V
gene pool on either successful rearrangement, expression,
and assembly of functional Ig molecules, or selection and
expansion of sIg-expressing B cells. The availability of
mouse strains containing varying portions of the VH or V
germline repertoire could clarify the extent to which the
number and complexity of the V gene repertoire influences development of B lineage cells.
We have engineered strains of mice, collectively designated XenoMouse, that contain both inactivated mouse Ig
genes and different portions of the human H and L chain
loci cloned on yeast artificial chromosomes (YACs; references 9 and 10). The two human H chain YACs used, yH1
and yH2, encompass in germline configuration the same
core variable and constant sequences (D, JH, Cµ, C
), but
contain different numbers of VH genes, either 5 or 66, respectively. The two human
chain YACs used, yK1 and
yK2, contain in germline configuration the same J
and C
regions, but different numbers of V
genes, either 3 or 32, respectively. Each of the combinations, yH1 and yK1 or
yH2 and yK2, restored in Ig-inactivated mice a humoral
immune system and produced fully human antibodies, indicating the compatibility of these human Ig transgenes with
the mouse machinery for antibody recombination and expression (9, 10). Evaluation of antigen-specific human antibodies produced by mice engineered with a limited number
of human V genes on small YACs or minigenes (9, 11, 12)
in relation to those generated by mice engineered with the
large antibody gene repertoire suggested the importance of V
gene complexity in supporting production of high-affinity
antibodies to multiple human antigens (10). These XenoMouse strains, equipped with different portions of the human V gene repertoire, also provided a unique model system
to determine the impact of the structure and content of V
gene complexity on shaping differentiation and proliferation
of B lineage cell populations. Our results reveal the critical
role of V gene complexity in supporting efficient B cell differentiation, and demonstrate the different developmental
checkpoints controlled by VH and V
gene repertoire.
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Materials and Methods |
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Mice.
All mice were born, bred, and kept in a barrier facility until 1 or 2 d before killing. TG;mJHFlow Cytometry Analysis.
Bone marrow, peripheral blood, and spleen were obtained from 8-10-wk-old mice, and the lymphocytes were separated from erythrocytes on Lympholyte M (ACL-5035; Accurate Chemical & Scientific Corp., Westbury, NY). Approximately 106 cells for each sample were treated with purified anti-mouse CD32/CD16 Fc receptor (01241D; PharMingen, San Diego, CA) to block nonspecific binding to Fc receptors, stained with antibodies, and analyzed on a FACStarPLUS using CellQuest software (Becton Dickinson, San Jose, CA). Antibodies used except where indicated were from PharMingen: FITC anti-mouse IgM (02084D); FITC anti-human IgM (08184D); FITC goat F(ab')2 anti-human IgD (2032-02; Southern Biotechnology Associates, Inc., Birmingham, AL); FITC anti-mouse IgDa (05064D); FITC anti-mouse IgDb (05074D); FITC anti- mouseIn Vitro Proliferation Assays.
Spleens from three to eight mice of each genotype were isolated and ground with a frosted glass microscope slide. The cell suspension in DME was then spun over a Lympholyte M step gradient. Lymphocytes at the gradient interface were collected and washed twice in DME, then resuspended in a solution of PBS, 5 mM EDTA, and 0.5% FCS. T cells and macrophages were depleted from the spleen cell suspensions by magnetic cell sorting using anti-CD5 and anti-CD11b magnetic beads (493-01 and 496-01; Miltenyi Biotec Inc., Auburn, CA) and a Type B column according to the manufacturer's instructions. Enrichment for live B cells was assayed by staining the cells for B220 and µ and with propidium iodide before and after depletion, followed by FACS® analysis. This treatment resulted in a 70-90% B220+µ+ cell population. For each assay, 1.5 × 105 live B220+µ+ cells were grown in 96-well plates in DME supplemented with 10% FCS, 2 mM glutamine, and penicillin-streptomycin plus one of the following: LPS at a final concentration of 20 µg/ml, goat anti-mouse IgM F(ab')2 (115-006-075; Jackson ImmunoResearch Labs, West Grove, PA) at a final concentration of 150 µg/ml, goat anti-human IgM F(ab')2 (109-006-043; Jackson ImmunoResearch Labs) at a final concentration of 150 µg/ml, or medium alone as a negative control. After 2 d incubation at 37°C, 1 µCi [3H]thymidine (1 mCi/ml; Amersham Corp., Arlington Heights, IL) was added to each well, and after an additional 1 d of incubation, the samples were counted for incorporation of label into DNA. Each sample was assayed in triplicate. ![]() |
Results |
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The effect of increasing V gene repertoire on antibody
production and B cell development was evaluated in Ig-inactivated mice engineered with either of two pairs of human
H and chain Ig YACs
yH1 and yK1, or yH2 and yK2
(Fig. 1). The yH1 H chain YAC had a 220-kb insert of the
human IgH locus, containing in germline configuration the
µ and
constant regions, the intronic enhancer, all six functional JH regions, the entire D complex, and the five most
proximal variable genes from four VH families (Fig. 1; references 9 and 13). The yH2 H chain YAC contained the
entire yH1 YAC as the core and 61 additional upstream VH
genes (34 functional VH in total), all in germline configuration, plus the human
2 constant region (C
2) and the
murine 3' enhancer (m3'E) appended downstream of C
(10). The
L chain YAC, yK1, had a 170-kb insert containing in germline configuration the
deleting element, the intronic and 3' enhancers, the C
region, all five functional J
regions, and the three most proximal V
regions
in the B cluster, two of which are functional (9, 13). The
larger
chain YAC, yK2, had an 800-kb insert, with yK1
at the core and 29 additional V
genes from the proximal
V
cluster, with 18 functional V
genes in total (10).
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Mice homozygous for both the inactivated mouse H and
the inactivated chain alleles (DI) were generated as described previously (9). Into this DI genetic background, we
introduced either the yH1 and yK1, or the yH2 and yK2
transgenes to yield XenoMouse I or XenoMouse II strains,
respectively. Using these strains, we evaluated the ability of
these human H and
L chain loci to restore B cell development and to produce fully human antibodies. To demonstrate the ability of individual H or
chain transgenes to
replace their corresponding mouse counterparts, intermediate mouse strains were generated. One set of mouse strains
was generated with either yH1 or yH2 on a mouse H
chain-inactivated background (yH;mJH
/
) (14). Another
set of mouse strains was generated with yK1 or yK2 on a
mouse
chain-inactivated background (yK;mC
/
).
B cell populations in the bone marrow and peripheral
lymphoid tissues were analyzed by multiparameter flow cytometry, staining for cell surface markers specific to different stages of development. Precursor B cell populations
were separated as described by Hardy and Hayakawa using
the differential expression of B220, CD43, HSA, and BP-1
(1), or by Rolink and Melchers using the expression of
B220, µ, c-kit, CD25, and cell size (2). These two systems complemented each other and allowed the identification of
the developmental stages controlled by the V gene repertoire on both the human H and chain loci.
The ability of the H chain
YACs, yH1 and yH2, to restore B cell development was
first evaluated in mice homozygous for the JH deletion
(mJH/
) and engineered with one of these YACs. JH-inactivated mice were devoid of mature B cells (B220+µ+) and
deficient in antibody production (Fig. 2, A and E; references 9 and 14). The bone marrow from mJH
/
mice lacked
B220+µ+ cells due to terminal arrest of B cell development
at the pro B/pre B-I, B220+µ
c-kit+CD25
stage (Fig. 2,
A and B). By Hardy's convention, developing pro B/pre
B-I cells at Fraction B (B220+CD43+HSA+BP-1
) accumulated to levels three- to fourfold over wild-type, and terminally arrested at Fraction C (B220+CD43+ HSAmed
BP-1+) (Fig. 2 D). The inability to develop beyond the pro
B/pre B-I stage was consistent with a block in H chain rearrangement at the stage of DH-JH recombination (1).
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The presence of one allele of the yH1 transgene in
mJH/
mice partially alleviated the arrest at the pro B/pre
B-I stage, resulting in incomplete reconstitution of B cell
compartments with B220+hµ+ cells (Fig. 2 A) and formation of human µ chain-containing antibodies (9). Pro B/
pre B-I cells in yH1;mJH
/
bone marrow successfully developed into large pre B-II (B220+µ
c-kit
CD25+) or
B220+CD43+HSAhiBP-1+ (Fraction C') cells, although at
levels ~30% of wild-type mice (Fig. 2, B-D). The partial
differentiation was consistent with the continued accumulation of pro B/pre B-I cells at the B220+µ
c-kit+CD25
stage or at Fraction B, similar to that observed in mJH
/
mice. Cell accumulation was also detected in the Fraction
C compartment, which was three- to fourfold larger than in
both wild-type and mJH
/
mice. A reconstitution level of
~50% was detected at the small pre B-II stage (Fig. 2 C)
and was sustained in the newly emerging B220loµ+ B cell
population (Fig. 2 A). However, the levels of mature, recirculating B220hiµ+ cells dropped to only 10% of wild-type. Consistent with the mature B cell population in bone
marrow, spleens of yH1;mJH
/
mice exhibited ~10% B cell
reconstitution (Fig. 2 E). The percentage of B220+µ+ cells
that were
+HSAlo in the spleen was similar to that of wild-type mice, demonstrating normal differentiation to mature
B cells in yH1;mJH
/
(reference 15, and data not shown).
The proper pairing between human µ protein and mouse
and
L chains was supported by the wild-type-like L chain
distribution on the mouse B cells (
/
= 95:5; not shown).
These results indicate that yH1 can induce complete but inefficient B cell differentiation in H chain-inactivated mice, leading to a partial reconstitution of mature B cell populations in the different lymphoid organs. The stage at which the differentiation block is observed (Fractions B and C) suggests that a large fraction of pro B/pre B-I cells are unable to complete a productive VDJ rearrangement and/or to express a µ protein capable of pairing with surrogate L chains (SLC) and forming a pre B cell receptor (pre-BCR), a prerequisite for differentiation to the pre B-II cell stage (3).
The effect of a second yH1 allele on B cell development
was evaluated in mice homozygous for the YAC (yH1/
yH1;mJH/
). This strain exhibited a twofold increase in B
cell reconstitution over the hemizygous strain, starting at
stages correlated with completion of a productive VDJ rearrangement and µ chain expression. Accumulation of cells
at the pro B/pre B-I stage in yH1/yH1;mJH
/
mice was
still observed (Fig. 2 B). However, the large pre B-II population was 50% of wild-type, and the small pre B-II population (or Fraction D) reached wild-type levels (Fig. 2, C and
D). The newly emerging B220loµ+ cells were ~80% of
wild-type, but the mature, recirculating B220hiµ+ cells
demonstrated a 20% reconstitution level, similar to that observed in the spleen and double the levels detected in yH1; mJH
/
mice (Fig. 2, A and E). A proportional increase in
the percentage of mature B220+HSAloµ+
+ cells in the
spleen of yH1/yH1;mJH
/
mice relative to yH1;mJH
/
mice was detected as well (not shown). Human µ in yH1/
yH1;mJH
/
serum, averaging 60 µg/ml, represented 10%
of the mouse µ serum levels in wild-type mice housed in
the same pathogen-free facility. Thus, the second yH1 allele in mJH
/
mice doubled the population of B cells that
completed productive VDJ recombination, expressed surface µ protein, and progressed to mature B cells.
To test the effect of increased VH repertoire on B cell
development, the yH2 transgene was bred onto the mJH/
background. yH2 was able to improve significantly all
stages of B cell development and reconstitution in bone
marrow and in the peripheral lymphoid compartments.
yH2 fully relieved the accumulation of developing B cells
at the pro B/pre B-I stage observed in yH1-bearing mJH
/
strains and restored fully the large and small pre B-II populations (Fig. 2, B-D). Reconstitution was also complete in
the newly emerging B cell population, but it decreased to
~70% of wild-type in the mature, recirculating B cell population in both the bone marrow and spleen (Fig. 2, A and
E). However, this decrease in reconstitution level upon
maturation from B220loµ+ to B220hiµ+ was significantly
lower than that observed in yH1-bearing mJH
/
strains.
Consistent with the improved B cell development, the levels of circulating human µ and
2 chains in sera from yH2; mJH
/
mice averaged 200 µg/ml for both, only twofold
lower than the mouse µ levels in normal mice kept under
pathogen-free conditions.
yH2 differed from yH1 not only by its increased VH
gene content but also by the presence of the human C2
and the m3'E sequences. To study the contribution of
these downstream sequences to the improved restoration of
B cell development by the yH2 YAC, mJH
/
mice bearing a yH2µ YAC transgene, containing all of yH2 except for the human C
2 and the m3'E sequences, were analyzed. Similar to the yH2 hemizygous strain, yH2µ;mJH
/
mice exhibited complete reconstitution of all stages of B
cell development, up to and including the newly emerging
B cell compartment, and a mature B cell population ~70%
of wild-type (not shown). These results indicate that the
human C
2 and m3'E sequences are dispensable for the
improved reconstitution of B cell development by yH2,
and that the enhanced B cell development was the result of
the increased number and complexity of the VH genes.
We compared reconstitution of B cell development in
mJH/
mice by the H chain YACs to that obtained by a
rearranged human µ transgene (TG). TG had previously
been shown to be expressed at significant levels on mouse
B cells resulting in a complete allelic exclusion of the murine H chains in wild-type mice (4), and to support B cell
development in recombination activating gene (RAG)-
deficient mice (7). Consistent with previous observations (7), TG;mJH
/
mice had greatly reduced populations at
the pro B/pre B-I, large and small pre B-II, and the newly
emerging B cell stages, probably due to the acceleration of
B cell development by early expression of the rearranged µ transgene (Fig. 2). Reconstitution of mature B220+µ+
populations in the bone marrow and spleen was ~50 and
70% of wild-type mice, respectively, similar to that observed in yH2;mJH
/
mice (Fig. 2 A). Thus, a partially reconstituted mature B cell population was also obtained
with a rearranged human transgene, which is properly expressed and selected in wild-type mouse B cells. This observation may indicate that other components, in addition
to a large VH repertoire, are required for complete maturation and/or expansion and survival of the recirculating
B220+µ+ cells (see Discussion).
The ability of yK1 and
yK2 YACs to substitute for the inactivated mouse chain
locus was first evaluated in mice homozygous for the deletion of mouse C
(mC
/
). These mice displayed a complete absence of
+ B cells, and all B cell populations expressed the mouse
L chain exclusively (Fig. 3). Analysis
of the different B cell subpopulations in the bone marrow
demonstrated a wild-type-like distribution in the developmental stages that precede L chain expression, the pro B/
pre B-I and the large pre B-II populations (not shown). However, a twofold accumulation at the small pre B-II
stage was detected (Fig. 3 B). As a result of the partial differentiation arrest, the newly emerging B cell compartment
and the mature recirculating population reached only 50-
60% of wild-type levels (Fig. 3 A), consistent with previous
reports (16). In the serum of mC
/
mice, levels of circulating mouse
chains averaged 580 compared with 70 µg/
ml in wild-type mice. These results indicate that the mouse
locus can substitute for the inactivated
locus only partially, resulting in a lower efficiency of differentiation to
sIg-expressing B cells.
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The first human transgene tested, yK1, with its three
V
genes, two of which are functional, could partially replace the inactivated mouse
locus and compete with the
mouse
chain genes. mC
/
mice with either one or
two yK1 alleles exhibited an accumulation at the small pre
B-II stage (Fig. 3, A and B). The percentage of newly
emerging B220loµ+ B cells in yK1 hemizygous or homozygous mice, relative to the mC
/
mice, increased to ~70
and 100% of wild-type mice, respectively, whereas their
mature bone marrow B220hiµ+ populations did not change
significantly (Fig. 3 A). The B cell populations in the peripheral blood and lymph nodes of the yK1-bearing mC
/
strains increased by 20-25% over mC
/
mice, indicating
improved B cell development by the yK1 YAC (Fig. 3, C
and D).
Another manifestation of the ability of the yK1 YAC to
compete effectively with the mouse chain and restore
chain expression in mC
/
mice was the appearance of a
significant human
+ B cell population, in particular in the
presence of two yK1 alleles. In peripheral blood (Fig. 3 E),
lymph nodes, and spleen (data not shown) of yK1;mC
/
mice, there were equivalent numbers of h
+ and m
+ B
cells, whereas in yK1/yK1;mC
/
mice, h
+ B cells outnumbered m
+ B cells by a 2:1 ratio. Similar results were
observed in the newly emerging B220loµ+ population in
the bone marrow, indicating that the preferential usage of
human
over mouse
occurs at the stage of L chain rearrangement, as shown previously for wild-type mice (17).
The circulating human Ig
levels in yK1 homozygotes
were higher than those detected in the hemizygotes, averaging 720 and 250 µg/ml, respectively. The levels of circulating mouse
chain were reduced in both strains to ~300
µg/ml.
The second human Ig transgene, yK2, with its increased V
repertoire, was able to substitute fully for the
mouse
chain locus and to dominate L chain use. Both
hemizygous and homozygous yK2;mC
/
mice exhibited
full restoration of B cell development in the bone marrow,
with a complete relief of the cell accumulation at the small
pre B-II stage detected in both mC
/
and yK1;mC
/
mice and the appearance of wild-type-like newly emerging
and mature B cell populations (Fig. 3, A and B). Complete
reconstitution of B cell compartments was also detected in
the peripheral blood, lymph nodes (Fig. 3, C and D), and
spleen (not shown) of yK2;mC
/
mice.
The apparent ability of yK2 to substitute for the mouse
Ig locus was also observed at the level of h
+-expressing
B cells (Fig. 3 E). In hemizygous yK2;mC
/
mice, the
majority of the peripheral blood lymphocytes (~75%) expressed human
chain exclusively, whereas only a minority (15%) expressed mouse
chain. A similar
to
chain
distribution was detected in mice with only one functional
mouse Ig
locus (16). In yK2/yK2;mC
/
mice, the human
+ B cell population increased to >90%, and the
mouse
+ B cell population decreased to 5%, reaching a
wild-type-like
to
distribution ratio. Similar results
were obtained in the spleen and lymph nodes, and in the
newly emerging B220loµ+ and mature B220hiµ+ cells in
the bone marrow (not shown). The average levels of circulating h
and m
in yK2;mC
/
mice were 1,400 and
100 µg/ml, respectively, equivalent to the levels of mouse
and
in wild-type mice kept under similar conditions. As the only known difference between the yK1 and yK2
YACs resides in the number and diversity of their V genes,
these results provide evidence for the role of V
gene repertoire in supporting normal
chain recombination and
expression.
Equivalency of the human chain locus on yK2 with
the mouse Ig
locus was further demonstrated in mice with
one functional mIg
allele and either one or two alleles of
yK2 (Table 1). In the peripheral blood of mice with functional H chain loci, one functional mIg
allele, and one
yK2 allele (yK2;mC
+/
), the percentages of h
+ and m
+
B cells were equivalent (Table 1). Similarly, equivalent usage of yK2 and mIg
in a yH2;mJH
/
background (yH2;
yK2;mJH
/
;mC
+/
mice) was demonstrated. In mice
with two yK2 alleles and one functional mIg
allele (yH2;
yK2/yK2;mJH
/
;mC
+/
), the ratio of h
+ to m
+ B
cells in the spleen increased to 2:1 (Table 1). Thus, yK2 competes effectively with the mouse
chain locus. These
data also demonstrated the lack of an apparent preference of
the H chain for a
chain of the same species.
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The combined effects of yH1 and yK1 or yH2 and yK2 in replacing their inactivated mouse counterpart loci, thereby restoring B cell
development and inducing human antibody production in
DI (mJH/
;mC
/
) mice, was examined in the XenoMouse I (yH1;yK1;DI) and II (yH2;yK2;DI) strains. In the
bone marrow of DI mice, the pattern of arrested B cell
development was similar to that observed in mJH
/
mice.
Developing B cells accumulated at the pro B/pre B-I stage (Fraction B) and were terminally arrested at Fraction C,
or the B220+µ
c-kit+CD25
stage (Fig. 4; references 9
and 10).
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As demonstrated above (Figs. 2 and 3), a second yH1 allele affected B cell development in yH1;mJH/
mice significantly, whereas only a small effect was observed with a
second yK1 allele in yK1;mC
/
mice. Moreover, no obvious differences in reconstitution of B220+µ+ compartments in XenoMouse strains with one versus two alleles of
either yK1 or yK2 were observed (not shown). Therefore,
we concentrated on analyzing B cell development in XenoMouse I and II strains homozygous for the yK transgenes,
in conjunction with either one or two alleles of the yH
transgene.
yH1 in conjunction with yK1 partially restored B cell
development and B220+hµ+h+ compartments in XenoMouse I. Similar to yH1;mJH
/
strains, XenoMouse I
strains with one or two yH1 alleles still exhibited a threefold accumulation of developing B cells at the pro B/pre
B-I stage (B220+µ
c-kit+CD25
or Fraction B; Fig. 4, B
and D). XenoMouse with one yH1 allele had ~30% of
wild-type levels at Fraction C' (or the large pre B-II stage),
whereas the homozygous strain exhibited ~60% reconstitution level. The sizes of B cell populations in Fractions D
and E in XenoMouse I were substantially lower than the
respective ones in the yH1;mJH
/
or yK1;mC
/
mouse
strains (Figs. 2-5). Hemizygous and homozygous XenoMouse I strains represented reconstitution levels of 30 and
70% for the small pre B-II cells, and 10 and 30% for the
newly emerging and mature B220+µ+
+ populations.
These results illustrated a consistent twofold improvement in B cell development by the second yH1 allele, which was
also manifested in the spleen (Fig. 6 A, and data not
shown). In the B220+µ+ compartment of XenoMouse I,
there was an overrepresentation of B220+HSAhi cells and
an underrepresentation of the mature B220+HSAlo cells
compared with wild-type (Fig. 6 B). The B220+ population had a twofold lower level (50% of wild-type) of
hµ+h
+ cells. The B220+HSAlo cells were CD5
CD40+
(not shown). These results indicate that yH1 and yK1 are
capable of restoring the progression of precursor B cells
through development and maturation, but with a limited
efficiency that is likely to reflect the combined deficiencies
associated with the small VH and V
gene repertoires (see
Discussion).
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We then evaluated the potency of yH2 and yK2, with
their greatly increased V gene repertoires, for improving B
cell development in XenoMouse II strains compared with
the respective intermediate strains. Complete reconstitution of the pro B/pre B-I and the large and small pre B-II
populations in the bone marrow of XenoMouse II was observed (Fig. 7), consistent with results from both yH2; mJH/
and yK2;mC
/
strains. The newly emerging
B220+µ+
and the mature B220hiµ+
+ populations both
exhibited reconstitution of 50-70%. These reconstitution levels were similar to those detected in yH2;mJH
/
mice,
whereas complete restoration of the B220hiµ+ population
was demonstrated in yK2;mC
/
mice (Figs. 2, 3, and 5).
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In the spleen of XenoMouse II, the B220+µ+ population exhibited ~70% reconstitution level (Fig. 6 A).
Within this compartment, the ratio of HSAhi (immature) to
HSAlo (mature) B cells was reminiscent of that detected in
wild-type mice, with both immature and mature populations ~70% of wild-type (Fig. 6 B). Furthermore, within
the XenoMouse II B220+ population, the ratio of µ++ to
µ+
cells was identical to wild-type mice (Fig. 6 C). The
majority (80%) of the B220+HSAlo B cells in XenoMouse
II spleen were hµ+h
+ and CD5
CD40+, identical to the
mµ+m
+ cells in the B220+HSAlo population in wild-type
mice (not shown). Thus, the yH2 and yK2 YACs supported proper maturation of XenoMouse II B cells to
HSAloµ+
+. The percentages of h
+- and m
+-expressing
B cells were identical to the L chain distribution in wild-type mice (Fig. 6 D). The restoration of normal B cell development in XenoMouse II was also manifested in circulating levels of human µ and
chains (300 and 530 µg/ml,
respectively) that were higher than those detected in XenoMouse I (150 and 120 µg/ml, respectively). The average
levels of h
2 in XenoMouse II were 100-200 µg/ml.
Mouse
levels were lower in XenoMouse II compared
with XenoMouse I
10 versus 40 µg/ml.
A similar reconstitution pattern of the B cell lineage by
yH2 and yK2 was observed in XenoMouse II.3 (Fig. 6), a
mouse strain in which the two YACs were cointegrated on
the same chromosome by fusing embryonic stem cells with
a yeast strain containing both the yH2 and yK2 YACs. In
transgenic mice, the two transgenes cosegregated through
>1,000 progeny, strongly suggesting close genetic linkage.
In the XenoMouse II.3 spleen, B cell reconstitution was
70% of wild-type, and the majority of the B cells were
B220+HSAloµ++, as observed in the XenoMouse II strain
(Fig. 6). However, a higher percentage of mouse
+-expressing B cells was observed in the periphery of XenoMouse II.3 compared with XenoMouse II strains (Fig. 6 D). Serum Ig levels in the XenoMouse II.3 strain were higher
than in XenoMouse II, with average levels of 420 µg/ml
for hµ, 700 µg/ml for h
2, and 600 µg/ml for h
. Thus,
human H and
chain loci, when arrayed in cis-configuration on the same chromosome, can support essentially proper recombination and expression of both chains and
induce efficient B cell development.
Formation of a functional BCR complex, through
assembly of the µ chain with the other components such as
Ig and Ig
, is a prerequisite for transduction of proliferation signals (18). The ability of mouse B cells expressing
yH-encoded human µ chains to respond properly to proliferation signals such as LPS and anti-µ antibodies was evaluated. Proliferation of B cells from spleens of yH1;mJH
/
,
yH2;mJH
/
, or yH2µ;mJH
/
(not shown) mice was
equivalent to that of wild-type mouse B cells after in vitro
stimulation with either LPS (Fig. 8 A) or anti-µ F(ab')2 antibodies (Fig. 8 B). Induction of expression of B7-1 and
B7-2, surface markers for B cell activation, was also observed (not shown). These results indicate that the human
H chains encoded by both yH1 and yH2 YACs can functionally substitute for their murine counterparts in the
BCR complex and can support B cell proliferation in vitro.
The functionality of XenoMouse-derived B cells in vivo was
confirmed by the ability of these mice to mount a strong human antibody response to multiple antigens and to produce
high-affinity fully human mAbs against different antigens, including human IL-8, human TNF-
, and the human epidermal growth factor receptor (10).
|
![]() |
Discussion |
---|
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---|
This report elucidates the role of the size and complexity
of the native human antibody repertoire in shaping B cell
differentiation and proliferation pathways by directly comparing B cell development in XenoMouse strains transgenic
for segments of the human H and chain loci differing
only in the spectrum of their V gene repertoire. As the V
gene arrays on the integrated Ig YACs are in germline configuration and in a single copy, our study evaluated the human H and
variable gene repertoires in their native organization and structure. Furthermore, the availability of
XenoMouse and their precursor strains bearing only a human H or
chain YAC allowed us to study both the discrete and the combined contribution of each of the VH or
V
gene repertoires to B cell development, and the pairing
compatibility of the human and mouse Ig chains.
Our studies identified four developmental stages affected
by V gene complexity (Fig. 5): (a) production of a functional µ chain at the transition from pre B-I to pre B-II
stage; (b) productive recombination of VJ
genes; (c) formation of functional Ig molecules by pairing of specific µ and conventional L chains; and (d) maturation to recirculating B cells. All of these checkpoints are critical to successful
B cell development. Therefore, any decline in the efficiency of cell progression at these stages can severely impair the entire humoral immune system, as demonstrated in this
report.
The first developmental checkpoint regulated by VH
gene complexity is the production of a functional µ protein. yH1, the human H chain YAC with core variable and
constant sequences and only five VH genes, was sufficient to
relieve the block at H chain gene recombination in JH/
mice and to induce B cell differentiation all the way to mature B cells, but only in a small fraction of the precursor B cells (Fig. 2). Accumulation of the majority of the yH1;mJH
/
B
cell population in Fractions B and C is indicative of a reduced efficiency in completing productive VH-DJH recombination and/or an impaired ability of the generated µ protein specificities to pair successfully with the SLC, the
proposed function of which is to select for µ chains capable
of pairing with conventional L chains (19, 20). Inability to
form a functional µ chain-SLC complex triggers attempts
to generate a compatible µ protein by a recombination at
the second H chain allele or by H chain replacement with
an upstream VH segment via cryptic recombination signals (21, 22). Indeed, the presence of a second yH1 allele in
mJH
/
mice doubled the number of pre B-II and mature
B cells.
The ability of the yH2 and yH2µ YACs, with their additional 61 VH genes, to restore normal B cell development
indicated that the limited VH gene repertoire on the yH1
YAC is the cause for the inefficient B cell development in
yH1;mJH/
mice. The large VH gene complexity, even
when presented on only one yH2 allele, restored a wild-type-like probability of productive H chain recombination
events and ensured a wide range of V gene specificities that
could form functional pre-BCR complexes.
The importance of VH gene specificity for formation of a
functional pre-BCR complex, and thus for the development of the B cells expressing it, was suggested by previous
studies investigating the expression pattern of Ig transgenes
or specific mouse endogenous V genes. For example, adult
mouse pre B cells expressing VH81X or Q52 genes were
unable to progress efficiently to the CD43 stage due to
the failure of these VH genes to form functional pre-BCR
complexes that deliver signals required for further differentiation (23).
The limited VH diversity on the yH1 YAC reduced significantly the number of B cells that progressed from the
immature B220loµ+ stage to the recirculating B220hiµ+
stage (Fig. 5). The reduced mature B cell population could
reflect elimination of B cells expressing autoreactive specificities, lack of specificities that trigger positive selection
and expansion, or impaired cell survival in the periphery.
This block at B cell maturation was relieved, although not
fully, by the yH2 YAC, suggesting a necessity for diverse
VH specificities to support efficient B cell maturation and
expansion. The reduced mature B cell population in yH2
mice compared with wild-type mice may reveal possible
deficiencies, such as a need for specificities of the human chain genes that comprise 40% of the repertoire presented
on human B cells (28). The existing mature B cell population in yH1- and yH2-bearing mJH
/
mice indicates that
human µ can be stably produced by the mouse B cells and
can be assembled with the mouse Ig
and Ig
to form a
functional BCR capable of receiving and transmitting extracellular differentiation and proliferation signals (Fig. 8).
Studies with the two human chain YACs demonstrated the critical role of V
gene repertoire in efficient L
chain recombination, in production of functional IgM
protein, and thus in proper B cell differentiation (Fig. 5).
yK1;mC
/
mouse strains, containing similar numbers of
functional V
and V
genes, provided a unique tool to
study the mechanisms underlying the regulation of L chain
isotype use. One allele of yK1, with only two functional
V
genes, competed effectively with the two
chain alleles, each containing three functional V genes, and two
yK1 alleles dominated L chain use. The dominance of yK1
can be attributed not to a larger repertoire but rather to intrinsic structural differences between the human
and
mouse
chain loci, such as the inferior
chain recombination signal sequences (29), thus favoring a stochastic model
for
/
usage (30). In yK2;mC
/
strains, the
to
chain usage ratio equaled that of wild-type mice (16), indicating that the structural elements controlling L chain use
are similar in humans and mice. The ability of yK2, with its
increased number of V
genes, to restore a normal mouse
to
ratio indicated the importance of V
gene repertoire
for wild-type-like L chain recombination.
yK1 supported modest improvements in B cell reconstitution in mC/
mice (Figs. 3 and 5). In contrast, yK2,
with its 18 functional V
genes, restored normal B cell development. Therefore, the limited number of V
genes on
yK1 is likely to be the reason for the low efficiency of productive V
J
recombination events. Fewer V
genes could
also potentially reduce the efficiency of
chain editing, a
valuable mechanism for rescuing incompatible
chain
specificities (31). Finally, the lower efficiency of recombination by inversion associated with the functional V
genes on yK1 (35) could also contribute to the reduced
number of recombination products.
The importance of V gene specificity for B cell development was suggested by previous studies with Ig transgenes. For example, a
transgene paired with the TG
transgene in a RAG-deficient background failed to generate a mature B cell compartment (7), in contrast to the substantial reconstitution we detected when the TG transgene
could pair with the normal mouse L chain repertoire (Fig.
2). Spanopoulou et al. also demonstrated that a µ
transgene derived from a native antibody was able to support
full reconstitution in RAG-deficient mice (7). In addition,
differential abilities of two V
J
constructs, in combination
with endogenous mouse H chains, to generate a mature B
cell compartment were proposed to originate from formation of incompatible specificities (36). The contribution of
gene number or complexity to the ability of V
repertoire
to support normal B cell development can be evaluated from comparison of our yK2;mC
/
strains, with 18 different V
genes, to mC
/
mice homozygous for a YAC
containing 20 copies of the same 5 V
genes, i.e., 100 functional V
genes but with limited complexity. This
transgene restored B cell development to a degree similar to that observed in yK1 homozygous strains, and clearly
less efficiently than the yK2 YAC, as judged by the h
+ B
cell population and the human
serum levels (37). Therefore, the diversity of the V
gene repertoire seems critical
for proper L chain regulation.
The XenoMouse I (yH1;yK1;DI) strain exhibited the
same developmental blocks at the pro B/pre B-I and small
pre B-II stages, and twofold improvement in B cell development by the second yH1 allele, as observed in yH1;mJH/
mice (Figs. 5 and 7). However, the impairment of B cell
development at Fractions D-F, stages associated with generation of sIg+ B cells, was more severe in XenoMouse I
than in either yH1;mJH
/
or yK1;mC
/
mice (Fig. 5),
likely due to the combined limitations associated with the
limited V gene repertoire on both the H and the
chain YACs. These limitations may have further reduced both
the frequency of B cells with productive and compatible µ and
chains (38), and the ability of the B cells to progress
to mature HSAlo
+ cells (Fig. 6). Consistent with our findings, mice engineered with multicopy human Ig minigenes
containing four VH and four V
genes exhibited low levels
of B cell reconstitution compared with wild-type mice (11).
In contrast to XenoMouse I, the XenoMouse II strains exhibited wild-type-like B cell development similar to that
observed in yH2 or yK2 intermediate strains, demonstrating proper regulation of the H and
chain gene recombination and expression (Fig. 5).
Significant but not fully restored mature B cell populations were detected in the bone marrow and in the periphery of XenoMouse II, exhibiting markers associated with B
cell maturation (HSAlo+). As stated previously, the lack of
complete reconstitution may stem from specific features of
the human antibody repertoire. For example, some of the
generated human antibody specificities may be recognized
as autoreactive by the mouse and trigger B cell elimination
in the bone marrow (39), or may have a decreased life-span
due to lack of appropriate antigen stimulation (for a review,
see references 20 and 40). Positive selection and expansion of
the mature B cell population could also be impaired by the
absence of specificities, such as those associated with the human V
genes. Nevertheless, the existing mature B cells respond properly to antigen stimulation in vivo, as demonstrated by efficient class switching, somatic hypermutation, and generation of high-affinity antigen-specific human mAbs (10).
Our previous report demonstrated the critical role of a
large V gene repertoire in providing the diverse specificities
required for production of high-affinity human antibodies
against a broad spectrum of antigens (10). This report
proved that V gene complexity is also essential to support
efficient B cell development, and thus to reconstitute a
normal humoral immune system in Ig-inactivated mice.
The findings reported here suggest the utility of XenoMouse strains as a tool to elucidate the molecular mechanisms underlying the shaping of the human antibody gene
repertoire during developmental and disease states that are
not accessible for analysis in humans, including differential
expression of V, D, and J genes, human H and chain editing, and identification of autoreactive specificities.
![]() |
Footnotes |
---|
Address correspondence to Aya Jakobovits, Abgenix, Inc., 7601 Dumbarton Circle, Fremont, CA 94555. Phone: 510-608-6599; Fax: 510-608-6511; E-mail: jakobovits_a{at}abgenix.com
Received for publication 6 April 1998 and in revised form 5 June 1998.
We are grateful to Donna Louie, Cathy Roth, and Kate Maynard for help with tissue harvesting and animal husbandry, Xiao-Chi Jia and Shulah Iflah for performing ELISAs, Michael Scott for technical assistance with flow cytometry, Sia Kruschke for sample preparation, and Kevin Moscrip for figure preparation. We thank Drs. Michael Gallo, Michel Nussenzweig, Anthony DeFranco, and Klaus Rajewsky for critical discussions, and Drs. Shoshana Levy, Geoff Davis, and Xiao-dong Yang for comments on the manuscript.
Abbreviations used in this paper BCR, B cell receptor; h, human; m, mouse; RAG, recombination activating gene; sIg, surface immunoglobulin; SLC, surrogate light chain(s); TG, rearranged µ chain transgene; YAC, yeast artificial chromosome.
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