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IMMUNOBIOLOGY
Quantitative trait loci regulating relative lymphocyte proportions in
mouse peripheral blood
Jichun Chen and David E. Harrison
Relative proportions of peripheral blood
(PB) B lymphocytes (B220%) as well as
CD4 (CD4%) and CD8 (CD8%) T lymphocytes differ significantly among inbred
mouse strains: B220% is high in C57BL/6J
(B6) and C57BR/cdJ, intermediate in
BALB/cByJ (BALB) and DBA/2J (D2), and
low in NOD/LtJ (NOD) and SJL/J (SJL)
mice, whereas CD4% and CD8% are high
in NOD and SJL mice and low in the other
4 strains. By following segregating genetic markers linked to these traits in
(B6 ⴛ D2) recombinant inbred (BXD RI)
mice, the study defined 2 quantitative
trait loci (QTLs) for the B220% phenotype: Pbbcp1 (peripheral blood B cell
percentage 1, logarithm of odds [LOD]
4.1, P < .000 01) and Pbbcp2 (LOD 3.7,
P < .000 04) on chromosome 1 (Chr 1) at
about 63 cM and 48 cM; one suggestive
locus for the CD4% phenotype (LOD 2.6,
P < .000 57) on Chr 8 at about 73 cM; and
one QTL for the CD8% phenotype: Pbctlp1
(peripheral blood cytotoxic T lymphocyte
percentage 1, LOD 3.8, P < .000 02) on
Chr 19 at about 12 cM. The study further
segregated PB lymphocyte proportions
in B6SJLF2 mice by using DNA markers
adjacent to these mapped QTLs and found
that the Pbbcp1 locus (LOD 5.6,
P < .000 01) was also important in this
mouse population. In both BXD RI and
B6SJLF2 mice, QTLs regulating B-cell
proportions showed no significant effect
on T-cell proportions and vice versa. Thus,
PB B- and T-lymphocyte proportions are
regulated separately by different genetic
elements. (Blood. 2002;99:561-566)
© 2002 by The American Society of Hematology
Introduction
Mature leukocytes in mouse and human peripheral blood (PB)
express specific cell surface molecules (markers) that can be
detected by fluorescence-activated cell staining (FACS) using
specific antibodies.1,2 The major categories of PB leukocytes
include B lymphocytes that express B220, T-helper lymphocytes
that express CD4, cytotoxic T lymphocytes that express CD8,
and granulocytes that express Gr1. Under physiologic conditions, proportions of PB leukocyte subsets are relatively constant in mice from a particular genotype, showing a precise
regulation of hematopoietic lineage commitment, differentiation, maturation, recruitment, and elimination.3 Proportions of
PB leukocyte subsets can be dramatically altered by spontaneous and induced mutations; for example, a mutation in the
DNA-dependent protein kinase gene in severe combined immunedeficient mice results in the absence of mature B and T
lymphocytes in blood circulation.4-6 In addition, stromal cells,
cytokines, receptors, ligands, signaling molecules, and transcription factors have been shown to affect levels of B and T
lymphocytes.7-10
Previous studies identified molecular factors that play important
roles in the regulation of B-and T-cell commitment and balance
such as cytokines interleukin-3 (IL-3) and IL-7, signaling receptor
and ligand Notch and Jagged, paired-box gene Pax5, and the
transcriptional factors E2A and EBF.3,11-13 Mutations that change
the relative levels of other leukocyte subsets may also indirectly
affect B- and T-cell proportions.14 Earlier studies found that the
CD4/CD8 ratio is under genetic control in the mouse as well as in
humans.15-17 A study using inbred mouse strains identified a
quantitative trait locus (QTL) that regulates peripheral B220% on
mouse chromosome 15 (Chr 15).18 Differences in the percentage
of pre-B cells (BP-1⫹B220⫹) in the bone marrow of SL/Kh
and NFS/N mice helped to map another regulatory QTL on mouse
Chr 3.19 These studies illustrated that there are specific genetic
loci that regulate CD4/CD8 ratio, B-cell apoptosis, and pre–Bcell expansion.
We and others have found significant strain differences in
primitive immunohematopoietic progenitor cell functions between
B6, BALB, and D2 mice and have defined QTLs that regulate
hematopoietic stem cell development, proliferation, and senescence.20-23 It is important to also define strain differences in mature
blood cell proportions. The current study is focused on the genetic
elements that regulate strain differences in blood cell proportions
and tests whether the same QTL affects both B- and T-cell
proportions.
We examined PB B220%, CD4%, and CD8% in healthy young
mice of 6 inbred strains from 3 separate original stocks and found
significant strain differences. We then segregated these strain
differences in 35 strains of (C57BL/6 ⫻ DBA/2) recombinant
inbred (BXD RI) mice and mapped 2 QTLs for the B220%
phenotype, one QTL for the CD8% phenotype, and one suggestive
locus for the CD4% phenotype. We further analyzed 98 intercross
F2 mice derived from C57BL/6J (B6) and SJL/J (SJL) inbred
strains (B6SJLF2) and found that one of the QTLs for the B220%
phenotype was also mapped to the same location in the B6SJLF2
mice. In both studies, PB B- and T-lymphocyte proportions are
regulated separately by different QTLs.
From The Jackson Laboratory, Bar Harbor, ME.
e-mail: [email protected].
Submitted April 16, 2001; accepted August 6, 2001.
Supported by R01 grant HL58820 and a core grant CA34196 from the National
Institutes of Health.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Jichun Chen, The Jackson Laboratory, Bar Harbor, ME 04609-1500;
© 2002 by The American Society of Hematology
BLOOD, 15 JANUARY 2002 䡠 VOLUME 99, NUMBER 2
561
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562
BLOOD, 15 JANUARY 2002 䡠 VOLUME 99, NUMBER 2
CHEN and HARRISON
Table 1. Strain effects on peripheral blood lymphocyte composition
Strain
C57BL/6J
WBCs
(106/mL)
B220%
CD4%
CD8%
Gr1%
11.2 ⫾ 0.5
67.0 ⫾ 1.1
13.3 ⫾ 0.6
7.7 ⫾ 0.3
11.1 ⫾ 0.3
C57BR/cdJ
14.4 ⫾ 0.3
59.6 ⫾ 1.3
23.2 ⫾ 1.2
10.4 ⫾ 1.5
13.2 ⫾ 0.6
BALB/cByJ
10.3 ⫾ 0.6
45.8 ⫾ 2.7
29.3 ⫾ 1.7
10.8 ⫾ 0.6
13.3 ⫾ 1.2
DBA/2J
8.6 ⫾ 1.1
46.1 ⫾ 4.6
18.3 ⫾ 0.1
5.9 ⫾ 0.2
15.5 ⫾ 2.9
NOD/LtJ
3.2 ⫾ 0.6
17.6 ⫾ 0.9
49.5 ⫾ 1.5
17.1 ⫾ 0.8
12.9 ⫾ 2.8
SJL/J
5.7 ⫾ 1.0
16.1 ⫾ 1.3
57.5 ⫾ 1.1
20.1 ⫾ 1.6
8.3 ⫾ 1.2
B6D2F1
9.6 ⫾ 0.2
63.7 ⫾ 1.0
14.9 ⫾ 0.3
8.9 ⫾ 0.4
12.1 ⫾ 0.6
CByB6F1
8.6 ⫾ 1.3
59.3 ⫾ 2.0
12.8 ⫾ 0.7
6.2 ⫾ 1.4
11.9 ⫾ 1.6
Strain effect
P ⬍ .01
P ⬍ .01
P ⬍ .01
P ⬍ .01
NS
Data shown as mean ⫾ SEM of 3 to 4 males measured for each strain at 2 to 3
months of age.
WBC indicates white blood cell.
Materials and methods
Mice
Mice of the B6, C57BR/cdJ, BALB/cByJ (BALB), DBA/2J (D2), NOD/LtJ
(NOD), and SJL inbred strains, of 35 BXD RI strains, and of the B6SJLF2
intercross were all produced and raised at The Jackson Laboratory (Bar
Harbor, ME) using standard animal care and nutrition.24 Mice of both
genders were used as specified in each experiment.
Sample collection and FACS analysis
Procedures for FACS analysis were adapted from previous studies.25 In
brief, 2 micro-hematocrit tubes (75 ␮L) of blood were taken from the
orbital sinus of each mouse and mixed with 1350 ␮L Hanks balanced salt
solution in the presence of 5 mM ethylenediaminetetraacetic acid. Diluted
blood samples were incubated in Gey solution twice for 10 minutes each
time to lyse erythrocytes. Leukocytes were stained with specific antibody
cocktails in FACS buffer for 30 minutes. When a biotinylated antibody was
used, samples were stained with streptavidin red 670 for an additional 30
minutes. Cells were kept on ice or at 4°C during incubation, staining, and
centrifugation procedures. Monoclonal antibodies specific for mouse B220
(clone RA3-6B2), CD4 (clone GK1.5), CD8 (clone 53-6.72), Gr1 (clone
RB6-8C5), and the streptavidin-conjugated dye red 670 were purchased
from Pharmingen (San Diego, CA). Stained cells were analyzed by either
FACScan II or FACScalibur flow cytometry (Becton Dickinson Immunocytometry Systems, Mansfield, MA). We collected 10 000 to 20 000 leukocytes for each sample.
Laboratory (http://www.bioinformatics.jax.org). We used Map Manager
QTXb11 software, also available online at the same Web site, for QTL
mapping analyses.26 Significance of linkage was judged by using the LOD
(logarithm of odds) score in both the RI lines and the B6SJLF2 intercross
mice.26-28 The B220%, CD4%, and CD8% of B6SJLF2 mice were also
tested by variance analysis, using the JMP statistical discovery software
(SAS Institute, Cary, NC) on the Fit Model platform to define allelic
difference.29
Results
Strain differences in PB lymphocyte proportions
We analyzed PB leukocyte proportions in healthy young (2-3
months) B6, C57BR/cdJ, BALB, D2, NOD, and SJL mice. Data
presented in Table 1 show that concentrations of circulating white
blood cells (WBCs) were high in B6 and C57BR/cdJ mice, slightly
lower in BALB and D2 mice, and significantly lower (P ⬍ .01) in
NOD and SJL mice. Percentages of B cells (B220%) were
significantly different among the 6 inbred strains (P ⬍ .01): high in
B6 (67%) and C57BR/cdJ (60%) mice, intermediate in BALB
(46%) and D2 (46%) mice, and low in NOD (18%) and SJL (16%)
mice. Percentages of CD4 (CD4%) and CD8 (CD8%) T lymphocytes were significantly higher (P ⬍ .01, P ⬍ .01) in NOD (50%,
17%) and SJL (58%, 20%) mice than in the other 4 strains (CD4,
13%-30%; CD8, 6%-11%), whereas percentages of granulocytes
(Gr1%) were similar in the 6 strains (Table 1, Figure 1). Thus, there
are significant strain differences in mouse PB B220%, CD4%,
and CD8%, suggesting that PB lymphocyte proportions are
genetically regulated.
Polymerase chain reaction
Genomic DNA samples were prepared from mouse tail tips, and DNA
markers were analyzed by polymerase chain reaction (PCR) for each of the
98 B6SJLF2 mice. Primers for the Gli2 locus, Gli2-pA: TTCAGGCAGACCAAAGATAGAACATT and Gli2-pB: CACTGACATATGTACCATTTTCAT and primers for the Bcl2 locus, 24.MMBCL2A: CATTATCAATGATGTAC CATG and 24.MMBCL2B: GCAGTAAATAGCTGATTCGAC were
purchased from One Trick Pony Oligos (Ramona, CA). Primers for regular
Mit DNA microsatellite markers were purchased from Research Genetics
(Huntsville, AL). PCRs were carried out in a GeneAmp PCR system 9600
(Perkin Elmer) using Taq DNA polymerase. We used a program with a 97°C
touchdown for 30 seconds followed by 40 cycles of 94°C for 30 seconds,
55°C for 30 seconds, and 72°C for 30 seconds ⫹ 1 second. A 10-minute
enlongation at 72°C was added at the end of the reaction. Each sample was
amplified in 10 ␮L volume, electrophoresed in a 3% agarose gel, and
stained with ethidium bromide.
QTL mapping and statistical analysis
Genotype data for the 35 BXD RI mouse strains were retrieved online from
the Mouse Genome Database, which is maintained at The Jackson
Figure 1. Peripheral blood leukocyte composition. Peripheral blood from 2- to
3-month-old mice were stained with B220-FITC ⫹ CD4-Cy3 ⫹ CD8-PE ⫹Gr1-biotin
and streptavidin-red 670 after erythrocytes were lysed with Gey solution. Data shown
are dot plots of representatives from 3 to 4 male mice measured for each strain.
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BLOOD, 15 JANUARY 2002 䡠 VOLUME 99, NUMBER 2
QTL FOR BLOOD LYMPHOCYTE PROPORTIONS
563
To define the pattern of inheritance, we measured B220%,
CD4%, CD8%, Gr1%, and total WBCs in B6D2F1 and CByB6F1
hybrid mice. Interestingly, both hybrid F1 stocks had leukocyte
proportions similar to those of B6 mice (Table 1), indicating that
B6 genetic elements are dominant to those of the BALB or D2
genetic elements in the regulation of PB lymphocyte proportions.
Mapping QTLs for PB lymphocyte proportions in BXD RI mice
To segregate the genetic factors that regulate lymphocyte proportions, we measured B220%, CD4%, and CD8% in PB of healthy
young mice from 35 BXD RI strains. Individuals from the same RI
strain share the same genotype and are homozygous at almost all
(⬎ 99.99%) loci, with about half of the loci carrying alleles from
each parental strain.30,31 Many polymorphic loci and DNA markers
have been analyzed in the BXD RI strains, and the allele
information is available online through the Mouse Genome Database. The mean B220%, CD4%, and CD8% values for the 35 BXD
RI strains shown in Table 2 were used in mapping analyses, which
relied heavily on data from BXD 1-32 (Table 2) because many
marker loci have not been defined in BXD 33-42.
Table 2. Concurrence of B220%, CD4%, and CD8% with marker loci
in BXD RI mice
BXD
Pbbcp2
B%
Pbbcp1
CD4%
D8Mit156
CD8%
Pbctlp1
1
B
60
B
20
D
7.7
B
2
B
64
B
9
B
6.1
B
5
D
49
D
25
D
11.5
B
6
D
38
D
18
D
9.0
D
8
B
58
B
14
D
6.4
D
9
B
61
B
14
D
11.7
B
11
D
44
D
18
B
7.1
D
12
B
61
B
16
D
7.1
B
13
D
55
D
12
D
5.6
D
14
B
67
B
6
B
2.5
B
15
D
53
D
16
D
7.5
B
16
D
34
D
19
D
14.6
D
18
D
44
D
11
B
7.1
B
19
B
60
B
10
B
6.4
B
20
D
9
D
25
D
25.4
D
21
B
48
B
13
D
6.9
B
22
B
58
B
19
D
10.0
B
23
D
39
D
15
B
9.0
D
24
D
44
D
20
D
8.0
B
25
B
69
B
11
D
4.5
B
27
—
53
B
11
D
13.5
D
28
—
56
—
10
B
6.9
B
29
D
52
D
15
D
11.7
B
30
D
49
D
16
B
11.7
D
31
B
61
B
7
B
6.7
B
32
D
46
D
12
D
5.4
B
33
—
58
—
12
B
8.4
D
34
—
46
—
20
D
7.3
B
35
—
42
—
18
D
15.7
D
36
—
54
—
16
B
9.6
D
37
—
44
—
21
D
14.9
D
38
—
43
—
18
D
15.7
D
39
—
49
—
19
D
9.0
D
40
—
63
—
11
B
7.3
B
42
—
50
—
12
D
7.6
B
Two males 3 to 5 months of age were measured for each BXD RI line.
Pbbcp2 indicates peripheral blood B cell percentage 2; Pbbcp1, peripheral blood
B cell percentage 1; Pbctlp1, peripheral blood cytotoxic T lymphocyte percentage 1;
B, B6 allele; D, D2 allele; and —, genotype data not available.
Figure 2. QTLs for the B220%, CD4%, and CD8% phenotypes in BXD RI mice.
Peripheral blood B220%, CD4%, and CD8% were measured in 35 BXD RI strains for
QTL mapping by using predetermined genotype data and the Map Manager QTXb11
software, both available from The Jackson Laboratory (http://www.bioinformatics.jax.org) Web site.56 Two QTLs were defined for the B220% phenotype (A), designated
peripheral blood B cell percentage 1 (Pbbcp1, LOD 4.1, P ⬍ .00001) and Pbbcp2
(LOD 3.7, P ⬍ .00004), on chromosome 1 (Chr 1) at 63.1 cM and 48.4 cM,
respectively. A suggestive locus was found for the CD4% phenotype (LOD 2.6,
P ⬍ .000 57) at 73 cM on Chr 8 (B). A QTL for the CD8% phenotype, peripheral blood
cytotoxic T lymphocyte percentage 1 (Pbctlp1, LOD 3.8, P ⬍ .000 02), was mapped
to the 9- to 24-cM region of Chr 19 (C).
For the B220% phenotype, we mapped 2 QTLs. One of these
QTLs showed linkage to the 62- to 64-cM segment of Chr 1 with
the strongest linkage to the D1Mit188 marker at 63.1 cM (LOD 4.1,
P ⬍ .000 01). We have provisionally designated this locus peripheral blood B cell percentage 1, or Pbbcp1 (Figure 2A). The other
locus also mapped to Chr 1 with the strongest linkage to the
D1Ncvs45 marker at 48.4 cM (LOD 3.7, P ⬍ .000 04). We
designated this locus Pbbcp2 (Figure 2A). Both P values passed the
P ⬍ .0001 threshold and are considered significant in genomewide mapping analyses.27,32 The Bcl2 locus is located in between
the 2 QTLs at 59.8 cM.
For the CD4% phenotype, we found a putative linkage to a
DNA marker D8Mit156 (LOD 2.6, P ⬍ .000 57) at 73 cM on Chr 8
(Figure 2B). This P value is considered suggestive and reportable
but not fully significant.27,32 For the CD8% phenotype, we mapped
a QTL to the 9- to 24-cM region of Chr 19 with the strongest
linkage to the D19Mit28 marker at 12 cM (LOD 3.8, P ⬍ .000 02).
We have designated this locus peripheral blood cytotoxic T
lymphocyte percentage 1, or Pbctlp1 (Figure 2C).
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BLOOD, 15 JANUARY 2002 䡠 VOLUME 99, NUMBER 2
CHEN and HARRISON
From the mapping analyses, we noted that the QTL for the
B220% phenotype showed no linkage to either the CD4% or the
CD8% phenotype. However, the Pbbctlp1 locus had no linkage to
the B220% phenotype either. Thus, B- and T-cell proportions are
regulated by different QTLs in the BXD RI mice.
Mapping of the Pbbcp1 locus to the same location in
B6SJLF2 mice
The existence of RI lines with preanalyzed genotype data provides
a good resource for QTL mapping. However, other genetic methods
should be used to test QTLs defined by RI lines. Toward this end,
we produced a panel of B6SJLF2 mice and measured PB B220%,
CD4%, and CD8% at 2 to 3 months of age. We then tested DNA
markers on each mouse near each putative locus defined earlier by
using the BXD RI lines. When a marker of interest was not
polymorphic between B6 and SJL mice, a nearby polymorphic
marker was analyzed.
We first tested the hypothesis that Pbbcp1 and Pbbcp2 are
important loci in the regulation of B220% difference between B6
and SJL mice. This test was done by analyzing 12 polymorphic
markers in the 41- to 70-cM region of Chr 1 on 98 B6SJLF2 mice,
followed by a mapping analysis. Three markers adjacent to the
Pbbcp1 locus, D1Mit387 (62 cM), Gli2 (63 cM), and D1Mit419
(63.1 cM), all showed strong linkage to the B220% phenotype
(LOD 5.4, 5.6, and 5.6, all at P ⬍ .000 01), whereas the linkages to
the B220% were lower but still significant at Bcl2 (59.8 cM, LOD
4.9). D1Mit139 (65 cM, LOD 5.4), D1Mit286 (67 cM, LOD 5.2),
and D1Mit446 (70 cM, LOD 4.9) loci (Figure 3). Note that the Gli2
and D1Mit419 loci overlap on Figure 3. At the Gli2 locus where
Pbbcp1 showed the strongest linkage (LOD 5.6), B220% was
significantly higher (P ⬍ .01) in the carriers of the B6 (36.6% ⫾
1.8%) and F1 (32.6% ⫾ 1.0%) alleles than in the carriers of the SJL
(6.3% ⫾ 1.3%) allele. Thus, we mapped the Pbbcp1 locus to the
same 60- to 70-cM Chr 1 location in the B6SJLF2 intercross mouse
population. It is of particular interest to note that the Pbbcp1 locus
had no effect on either the CD4% or the CD8% phenotype in
B6SJLF2 mice, further indicating that PB B- and T-lymphocyte
proportions are regulated by different genetic elements.
The other 5 Chr 1 markers we have tested on B6SJLF2 mice that
cover the Pbbcp2 locus region showed decreasing significance
levels in the linkage analysis: D1Mit135 (59.7 cM, LOD 4.5,
P ⬍ .000 01), D1Mit48 (54.0 cM, LOD 3.5, P ⬍ .000 05),
Figure 3. Mapping the Pbbcp1 locus in B6SJLF2 mice. A total of 98 B6SJLF2 mice
(51 males, 47 females) were produced; measured for PB B220%, CD4%, and CD8%
phenotypes at 2 to 3 months of age; tested for selected polymorphic markers
adjacent to the Pbbcp1, Pbbcp2, and Pbctlp1 QTL regions; and proceeded for linkage
analyses with the use of the Map Manager QTXb11 software. Only the Pbbcp1 locus
was mapped to the same Chr 1 region with statistic significance (LOD 5.6,
P ⬍ .000 01).
Table 3. Quantitative trait loci for peripheral blood lymphocyte proportions
LOD
P
Name
Phenotype
Chromosome
Location
Pbbcp1
B220%
1
63 cM
5.6 ⬍ .00001
In B6SJLF2
Yes
Pbbcp2
B220%
1
48 cM
3.7 ⬍ .00004
No
Pbctlp1
CD8%
19
12 cM
3.8 ⬍ .00002
No
—
CD4%
8
73 cM
2.6 ⬍ .00057
No
LOD indicates logarithm of odds.
D1Mit216 (49.7 cM, LOD 2.5, P ⬍ .000 87), D1Mit46 (43.1 cM,
LOD 2.3, P ⬍ .001 18), and D1Mit24 (41.0 cM, LOD 2.0,
P ⬍ .002 30). Thus, on the basis of the markers we have tested,
Pbbcp2 was not an independent locus for the B220% phenotype in
the B6SJLF2 mouse population.
For the suggestive CD4% locus, we tested 12 standard Mit
markers in the 69- to 73-cM region of Chr 8: D8Mit140 and
D8Mit324 at 69 cM; D8Mit122 at 70 cM; D8Mit42, D8Mit52,
D8Mit92, D8Mit279, and D8Mit325 at 71 cM; D8Mit93, D8Mit245,
and D8Mit326 at 72 cM; and D8Mit156 at 73 cM. None of these
markers showed detectable polymorphism between B6 and SJL
mice by using our assay.
For the Pbctlp1 locus, we tested 5 polymorphic markers in the
8- to 41-cM region of mouse Chr 19 on B6SJLF2 mice: D19Mit69
(8 cM), D19Mit61 (9 cM), D19Mit106 (22 cM), D19Mit40 (25
cM), D19Mit11 (41 cM), and we performed a mapping analysis.
None of the 5 tested markers was linked to the CD8% phenotype in
the B6SJLF2 intercross mouse population. It is possible that the
SJL strain may not differ from the B6 strain at the Pbctlp1 locus.
Overall, we mapped 2 QTLs for the B220% phenotype, one
QTL for the CD8% phenotype and one suggestive locus for the
CD4% phenotype by using the BXD RI mice (Table 3). The
Pbbcp1 locus was also mapped to the same Chr 1 region in the
B6SJLF2 intercross mouse population.
Discussion
Genetic regulation of PB lymphocyte proportions
Mouse PB B220%, CD4%, and CD8% are genetically regulated, as
shown by the consistent differences among inbred mouse strains
derived from different original stocks (Table 1). The B6 and
C57BR/cdJ inbred strains both originated from the mating of
female 57 with male 52 from Miss Abbie Lathrop’s stock,24 and
both have high levels of B cells and low levels of CD4 and CD8 T
cells. The BALB and D2 inbred strains both originated from Castle
mice received from Lathrop,24 and both have intermediate levels of
B cells and low levels of CD4 and CD8 cells. The NOD and SJL
inbred strains both originated from Swiss mice at the Pasteur
Institute. NOD was derived from outbred ICR/Jcl mice in the 1970s,
and SJL was derived from noninbred Swiss Webster mice in the
1960s.33 Both of these Swiss-derived strains have low levels of B
cells and high levels of CD4 and CD8 cells (Table 1). With low
concentrations of circulating WBCs (Table 1), NOD and SJL mice
have significantly lower levels of circulating B cells than the other
4 strains.
Importance in the regulation of PB lymphocyte proportions
Regulation of PB lymphocyte proportions may have biomedical
significance. B6 and C57BR/cdJ mice have high B220%, low
CD4% and CD8%, and low tumor incidences.34-39 BALB and D2
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BLOOD, 15 JANUARY 2002 䡠 VOLUME 99, NUMBER 2
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Table 4. Potential candidate genes and human homologies for the Pbbcp1 locus
Gene name
Mouse gene
Mouse position
Human gene
Human position
B-cell leukemia/lymphoma 2
Bcl2
59.8 cM
BCL2
18q21.33
Troponin I, skeletal, slow 1
Tnni1
60.0 cM
TNNI1
1q32-q32
Troponin T2, cardiac
Tnnt2
60.0 cM
TNNT2
1q32-q32
Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2
Serpinb2
61.1 cM
SERPINB2
18q22.1
GLI-Kruppel family member Gli2
Gli2
63.0 cM
GLI2
2pter-qter
5-Hydroxytryptamine (serotonin) receptor 5B
Htr5b
63.0 cM
HTR5B
2q11-q13
Engrailed 1
Enzyme
64.1 cM
Enzyme
2q13-q21
Inhibin beta-B
Inhbb
64.1 cM
INHBB
2cen-q13
Gastrulation brain homeobox 2
Gbx2
65.0 cM
GBX2
2q37-q37
Chemokine (C-X-C) receptor 4
Cmkar4
67.4 cM
CXCR4
2pter-qter
Pbbcp1 indicates peripheral blood B cell percentage 1.
mice have intermediate B220%, CD4%, and CD8% and intermediate tumor incidences.34,35,39,40 SJL mice have low B220%, high
CD4% and CD8%, and high tumor incidences, especially of the
type B–reticulum cell sarcoma.41-43 NOD mice also have low
B220% and high CD4% and CD8%. They develop many types of
tumors when fed a specific diet to prevent diabetes.44 Thus, mouse
PB B220%, CD4%, and CD8% measured early in life may be
associated with tumor incidences later in life.
Regulation of PB lymphocyte proportion may also be related to
average life span. In 22 strains of inbred mice and 5 F1 hybrids, we
found that mean life span is positively correlated with B220%
(r ⫽ 0.67, P ⬍ .0001), negatively correlated with CD4%
(r ⫽ ⫺0.54, P ⬍ .0040) and CD8% (r ⫽ ⫺0.23, P ⫽ .26).24 These
correlations may be related to tumor incidences because strains
with high tumor incidences tend to die relatively early. In the BXD
RI mice, however, average life spans showed no significant
correlation with B220% (r ⫽ ⫺0.16, P ⫽ .47), or CD4% (r ⫽ 0.13,
P ⫽ .55), or CD8% (r ⫽ 0.29, P ⫽ .20).45 Thus, future studies are
needed to clarify the relationship between B220%, CD4%, CD8%,
and life expectancy.
QTL mapping
Our mapping analyses on the BXD RI mice used 3 phenotypes;
thus, the threshold P value for significant linkage on genome-wide
scan should be adjusted to .000 03 for each phenotype (.0001 ⫼ 3).
Judging by this adjusted P value, the Pbbcp1 and Pbctlp1 loci are
still significant, whereas the Pbbcp2 locus is marginally significant.
Because the Pbbcp1 locus was also mapped to the same chromosomal region by using the B6SJLF2 mice, this locus regulates
B220% in both the B6-D2 and B6-SJL genetic combinations.
The Pbbcp2 locus had the strongest linkage to the D1Ncvs44
marker at 48.4 cM on Chr 1 in the BXD RI mice.46 Interestingly,
another locus, Ncl, also located at 48.4 cM on Chr 1,47,48 showed no
linkage to the B220% phenotype in the same BXD RI strains (data
not shown) because 8 of the 26 BXD RI strains showed crossover
between the D1Ncvs44 locus and the Ncl locus. Whether or not the
D1Ncvs44 locus is polymorphic between B6 and SJL mice is yet to
be defined. Thus, further analyses are needed to clarify whether
Pbbcp2 is a real locus, and whether the Pbbcp2 locus is really
located at 48.4 cM on mouse Chr 1.
The fact that neither the Pbctlp1 nor the suggestive CD4% locus
were found in the B6SJLF2 intercross mice indicates that the B6
and SJL mice are probably not polymorphic at these loci. It is
possible that the suggestive CD4% locus may not be a real locus. It
is also possible, although less likely, that the Pbctlp1 locus is a
false-positive QTL for regulating the CD8% phenotype.
Importantly, in both the BXD RI and the B6SJLF2 mice,
B220%, CD4%, and CD8% are each regulated by different genetic
elements. The Pbbcp1 and Pbbcp2 loci had no significant effect on
the CD4% and CD8% phenotypes, whereas the Pbctlp1 locus had
no significant effect on the B220% and CD4% phenotypes.
Potential gene candidates for the Pbbcp1 locus
There are many genes in the 60- to 70-cM Chr 1 region that are
potential candidates for the Pbbcp1 locus (Table 4); however, we
emphasize 2 possibilities: Gli2 at 63 cM and En1 at 64.1 cM, both
of which are important genes in the regulation of embryonic
development. The Gli2 locus is polymorphic between B6 and D2
and between B6 and SJL mice. It encodes the vertebrate GLI2 zinc
finger protein that is a putative transcription factor responding to
sonic hedgehog signaling.49,50 Mutant mice lacking Gli2 function
exhibit defects in embryonic development.49,51,52 To date, there has
been no report showing that Gli2 is involved in lymphohematopoiesis. However, the Gli2 polymorphism marked the B220% phenotype in both BXD RI (LOD 3.7, P ⬍ .000 04, Figure 2A) and
B6SJLF2 mice (LOD 5.6, P ⬍ .000 01, Figure 3), suggesting that
Gli2 may be a candidate gene for the Pbbcp1 locus. The En1 locus
encodes a homeobox-containing transcriptional factor that controls
pattern formation during development of the central nervous
system.53-55 En1 showed significant linkage to the Pbbcp1 locus in
the BXD RI lines (LOD 3.6, P ⬍ .000 05). Other potential
candidate genes for the Pbbcp1 locus and their locations and
human homologies are listed in Table 4. However, all candidates
are very tentative at the current level of genetic resolution.
Overall, the mapping of the Pbbcp1 locus in 2 independent
studies suggests that this locus is important in the regulation of PB
B220% in both the B6-D2 and B6-SJL genetic combinations. In
both studies, B- and T-cell proportions are regulated independently
by different QTLs. Such genetically based regulation of PB
lymphocyte proportion may be associated with health and longevity. Because the Pbbcp1 locus maps to a well-conserved chromosomal region that harbors genes regulating embryonic development, it is possible that the gene or genes encoded by this locus may
regulate cell fate both during embryonic development and during
adult lymphohematopoiesis.
Acknowledgments
We thank Drs Edward H. Leiter and Brian Soper for critical review
comments, Dr Stephen Sampson for technical editing, Mr Ted
Duffy for technical assistance in FACS analyses, and Ms Elaine
Shown for technical assistance in PCR analyses.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
566
BLOOD, 15 JANUARY 2002 䡠 VOLUME 99, NUMBER 2
CHEN and HARRISON
References
1. Spangrude GJ, Klein J, Heimfeld S, Aihara Y,
Weissman IL. Two monoclonal antibodies identify
thymic-repopulating cells in mouse bone marrow.
J Immunol. 1989;142:425-430.
2. Wu L, Scollay R, Egerton M, et al. CD4 expressed on earliest T-lineage precursor cells in
the adult murine thymus. Nature. 1991;349:7174.
3. Busslinger M, Nutt SL, Rolink AG. Lineage commitment in lymphopoiesis. Curr Opin Immunol.
2000;12:151-158.
4. Kirchgessner CU, Patil CK, Evans JW, et al.
DNA-dependent kinase (p350) as a candidate
gene for the murine SCID defect. Science. 1995;
267:1178-1183.
5. Blunt T, Gell D, Fox M, et al. Identification of a
nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic
subunit in the SCID mouse. Proc Natl Acad Sci
U S A. 1996;93:10285-10290.
6. Greiner DL, Hesselton RA, Shultz LD. SCID
mouse models of human stem cell engraftment.
Stem Cells. 1998;16:166-177.
7. Obinata M, Okuyama R, Matsuda KI, Koguma M,
Yanai N. Regulation of myeloid and lymphoid development of hematopoietic stem cells by bone
marrow stromal cells. Leuk Lymphoma. 1998;29:
61-69.
8. Borge OJ, Adolfsson J, Jacobsen AM. Lymphoidrestricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands. Blood.
1999;94:3781-3790.
9. Metcalf D. Lineage commitment in the progeny of
murine hematopoietic preprogenitor cells: influence of thrombopoietin and interleukin 5. Proc
Natl Acad Sci U S A. 1998;95:6408-6412.
10. Colucci F, Di Santo JP. The receptor tyrosine kinase c-kit provides a critical signal for survival,
expansion, and maturation of mouse natural killer
cells. Blood. 2000;95:984-991.
11. Thompson A, Brouns GS, Schuurman RK, Borst
J, Timmers E. A pro-B-cell stage characterized by
germline Ig transcription without surrogate light
chain expression. Immunogenetics. 1998;48:305311.
12. Nutt SL, Heavey B, Rolink AG, Busslinger M.
Commitment to the B-lymphoid lineage depends
on the transcription factor Pax5. Nature. 1999;
401:556-562.
13. Enver T. B-cell commitment: Pax5 is the deciding
factor. Curr Biol. 1999;9:R933–935.
14. Dave VP, Allman D, Keefe R, Hardy RR, Kappes
DJ. HD mice: a novel mouse mutant with a specific defect in the generation of CD4⫹ T cells.
Proc Natl Acad Sci U S A. 1998;95:8187-8192.
15. Clementi M, Forabosco P, Amadori A, et al. CD4
and CD8 T lymphocyte inheritance. Evidence for
major autosomal recessive genes. Hum Genet.
1999;105:337-342.
16. Kraal G, Weissman IL, Butcher EC. Genetic control of T-cell subset representation in inbred mice.
Immunogenetics. 1983;18:585-592.
17. Amadori A, Zamarchi R, De Silvestro G, et al. Genetic control of the CD4/CD8 T-cell ratio in humans. Nat Med. 1995;1:1279-1283.
18. Hoag KA, Clise-Dwyer K, Lim YH, et al. A quantitative-trait locus controlling peripheral B-cell deficiency maps to mouse Chromosome 15. Immunogenetics. 2000;51:924-929.
19. Lu LM, Shimada R, Higashi S, Zeng Z, Hiai H.
Bone marrow ptr-B-1 (Bomb1): a quantitative trait
locus inducing bone marrow pre-B-cell expansion
in lymphoma-prone SL/Kh mice. Cancer Research. 1999;59:2593-2595.
20. Chen J, Astle CM, Harrison DE. Development
and aging of primitive hematopoietic stem cells in
BALB/cBy mice. Exp Hematol. 1999;27:928-935.
21. Chen J, Astle CM, Harrison DE. Genetic regulation of primitive hematopoietic stem cell senescence. Exp Hematol. 2000;28:442-450.
22. de Haan G, Van Zant G. Intrinsic and extrinsic
control of hemopoietic stem cell numbers: mapping of a stem cell gene. J Exp Med. 1997;186:
529-536.
23. de Haan G, Nijhof W, Van Zant G. Mouse straindependent changes in frequency and proliferation
of hematopoietic stem cells during aging: correlation between lifespan and cycling activity. Blood.
1997;89:1543-1550.
24. Staff of The Jackson Laboratory. Handbook on
Genetically Standardized JAX Mice. Bar Harbor,
ME: The Jackson Laboratory; 1997.
25. Chen J, Astle CM, Harrison DE. Delayed immune
aging in diet-restricted B6CBAT6F1 mice is associated with preservation of naive T cells. J Gerontol Biol Sci. 1998;53A:B330–337.
26. Manly KF, Olson JM. Overview of QTL mapping
software and introduction to Map Manager QT.
Mamm Genome. 1999;10:327-334.
27. Lander ES, Schork NJ. Genetic dissection of
complex traits. Science. 1994;265:2037-2048.
28. Kruglyak L, Lander ES. A nonparametric approach for mapping quantitative trait loci. Genetics. 1995;139:1421-1428.
29. SAS Institute Inc. JMP Statistics and Graphics
Guide, Version 3. Cary, NC: SAS Institute, 1998.
30. Taylor BA. Recombinant inbred strains. In: Lyon
MF, Rastan S, Brown SDM, eds. Genetic Variants
and Strains of the Laboratory Mouse. Vol. 2. New
York, NY: Oxford University Press; 1996:15971633.
31. Bailey DW. Recombinant inbred strains and bilineal congenic strains. In: Foster HL, Small JD, Fox
JG, eds. The Mouse in Biomedical Research. Vol.
I. New York, NY: Academic Press; 1981:223-239.
32. Belknap JK, Mitchell SR, O’Toole LA, Helms ML,
Crabbe JC. Type I and type II error rates for
quantitative trait loci (QTL) mapping studies using
recombinant inbred mouse strains. Behav Genet.
1996;26:149-160.
33. Committee on Immunologically Compromised
Rodents, Institute of Laboratory Animal Resources, Commission on Life Science, and National Research Council. Immunodeficient Rodents: A Guide to Their Immunobiology,
Husbandry, and Use. Washington, DC: National
Academy Press; 1989.
34. Hoag WG. Spontaneous cancer in mice. Ann N Y
Acad Sci. 1963;108:805-831.
35. Myers DD, Meier H, Huebner RJ. Prevalence of
murine C-type RNA virus group specific antigen
in inbred strains of mice. Life Sci. 1970;9:10711080.
36. Murphy ED. Characteristic tumors. In: Green EL,
ed. Biology of the Laboratory Mouse, 2nd ed.
New York, NY: McGraw-Hill; 1966:521-562.
37. Festing MFW, Blackmore DK. Life span of specified-pathogen-free (MRc category 4) mice and
rats. Lab Anim. 1971;5:179-192.
38. Staats J. Standardized nomenclature for inbred
strains of mice: sixth listing. Cancer Res. 1976;
36:4333-4377.
39. Storer JB. Longevity and gross pathology at
death in 22 inbred strains of Mice. J Gerontol.
1966;21:404-409.
40. Heston WE. Genetic aspects of experimental animals in cancer research. Jpn Cancer Assoc Gann
Monogr. 1968;5:3-15.
41. Crispens CG. Some characteristics of strain SJL/
JDg mice. Lab Animal Sci. 1973;23:408-413.
42. Murphy ED. SJL/J, a new inbred strain of mouse
with a high, early incidence of reticulum-cell neoplasms. Proc Am Assoc Cancer Res. 1963;4:46.
43. Fujinaga S, Poel WE, Williams WC, Dmochowski
L. Biological and morphological studies of SJL/J
strain reticulum cell neoplasms induced and
transmitted serially in low leukemia-strain mice.
Cancer Res. 1970;30:729-742.
44. Leiter EH. The NOD mouse meets the “Nerup
hypothesis”: Is diabetogenesis the result of a collection of common alleles presented in unfavorable combinations? In Shafrir E, ed. Frontiers in
Diabetes Research. Lessons from Animal Diabetes III. London: Smith-Gordon; 1990:54-58.
45. Gelman R, Watson A, Bronson R, Yunis E. Murine chromosomal regions correlated with longevity. Genetics. 1988;118:693-704.
46. Hughes DC, Allen J, Morley G, et al. Cloning and
sequencing of the mouse Gli2 gene: localization
to the Dominant hemimelia critical region.
Genomics. 1997;39:205-215.
47. Aitman TJ, Hearne CM, McAleer MA, Todd JA.
Mononucleotide repeats are an abundant source
of length variants in mouse genomic DNA. Mamm
Genome. 1991;1:206-210.
48. Huang H, Acuff CG, Steinhelper ME. Isolation,
mapping, and regulated expression of the gene
encoding mouse C-type natriuretic peptide. Am J
Physiol. 1996;271:H1565–H1575.
49. Ding Q, Motoyama J, Gasca S, et al. Diminished
Sonic hedgehog signaling and lack of floor plate
differentiation in Gli2 mutant mice. Development.
1998;125:2533-2543.
50. Borycki AG, Mendham L, Emerson CP Jr. Control
of somite patterning by Sonic hedgehog and its
downstream signal response genes. Development. 1998;125:777-790.
51. Park HL, Bai C, Platt KA, et al. Mouse Gli1 mutants are viable but have defects in SHH signaling
in combination with a Gli2 mutation. Development. 2000;127:1593-1605.
52. Mo R, Freer AM, Zinyk DL, et al. Specific and redundant functions of Gli2 and Gli3 zinc finger
genes in skeletal patterning and development.
Development. 1997;124:113-123.
53. Danielian PS, McMahon AP. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate
midbrain development. Nature. 1996;383:332334.
54. Joyner AL. Engrailed, Wnt and Pax genes regulate midbrain—hindbrain development. Trends
Genet. 1996;12:15-20.
55. Wurst W, Auerbach AB, Joyner AL. Multiple developmental defects in Engrailed-1 mutant mice:
an early mid-hindbrain deletion and patterning
defects in forelimbs and sternum. Development.
1994;120:2065-2075.
56. Mouse Genome Database (MGD). Mouse genome informatics Web site, The Jackson Laboratory, Bar Harbor, ME. Available at: http://www.
informatics.jax.org. Accessed April 10, 2001.
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2002 99: 561-566
doi:10.1182/blood.V99.2.561
Quantitative trait loci regulating relative lymphocyte proportions in mouse
peripheral blood
Jichun Chen and David E. Harrison
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