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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2312-2320
IMMUNOBIOLOGY
Phenotypic and functional characteristics of hematopoietic cell
lineages in CD69-deficient mice
Pilar Lauzurica,
David Sancho,
Miguel Torres,
Beatriz Albella,
Mónica Marazuela,
Teresa Merino,
Juan A. Bueren,
Carlos Martínez-A, and
Francisco Sánchez-Madrid
From the Departamento de Fisiología, Universidad de
Barcelona, Barcelona, Spain; Servicio de Inmunología, Hospital
de la Princesa, Universidad Autónoma de Madrid, Spain; Department
of Immunology and Oncology, Centro Nacional de Biotecnología,
CSIC-UAM, and Departamento de Biología Molecular y Celular.
CIEMAT. Madrid.
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Abstract |
AIM/CD69 is the earliest leukocyte activation antigen and is
expressed mainly by activated T, B, and natural killer (NK) cells. It
is also constitutively expressed by platelets, by bone marrow myeloid
precursors, and by small subsets of resident lymphocytes in the
secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including
Ca++ flux, cytokine synthesis, and cell proliferation.
To investigate the physiological relevance of CD69, we generated mice
deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells.
CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell
receptor transgenic model demonstrated that these processes were not
altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T
lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The
B220hiIgMneg bone marrow pre-B cell compartment
was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led
to a slight increase in immunoglobulin (Ig) G2a and IgM responses to
immunization with T-dependent and T-independent antigens. Nevertheless,
CD69-deficient lymphocytes had a normal proliferative response to
different T-cell and B-cell stimuli. Together, these observations
indicate that CD69 plays a role in B-cell development and suggest that
the putative stimulatory activity of this molecule on bone
marrow-derived cells may be replaced in vivo by other signal
transducing receptors.
(Blood. 2000;95:2312-2320)
© 2000 by The American Society of Hematology.
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Introduction |
The development of the immune response leads to
lymphocyte activation by antigens or mitogens, which readily express
genes known as early genes (immediate-early genes); these include
proto-oncogenes, growth factors, and cytokine receptors.1
CD69, also termed "Activation Inducer Molecule"
(AIM),2 is expressed on leukocytes during the activation
process (reviewed in Sánchez-Madrid3 and Testi et
al4). This molecule is a disulfide-linked homodimer (24 kd)5 that belongs to the type 2 C-type lectin family of surface receptors, characterized by a carbohydrate recognition domain
in the C-terminal region.6,7 The CD69 gene is located in
the long arm of mouse chromosome 6, syntenic of chromosome 12 in
humans.6,7 It is found within the region designated "NK
complex," which comprises several genes from the family of C-type
lectins specific for natural killer (NK) cells.4 The genomic organization, promoter regions, and transcriptional activity of
the human CD69 gene have been reported.6,8,9 The genetic and biochemical characteristics of mouse CD69 are very similar to its
human homologue.4,7
Lymphocyte expression of CD69 can be induced in vitro by a wide variety
of agents, such as anti-CD3/T-cell receptor (TCR) and anti-CD2
monoclonal antibody (mAb), activators of protein kinase C
(PKC), and phytohemagglutinin (PHA). Soon after
stimulation of T lymphocytes through the TCR, CD69 messenger RNA levels
are transiently increased.6 CD69 expression is absent in
vivo in peripheral blood lymphocytes, but it is expressed by small T- and B-cell subsets in secondary lymphoid tissues, as well as by platelets and bone marrow (BM) myeloid precursors.10-12 The
expression of CD69 by thymocytes undergoing positive
selection13 is associated with the activation process that
occurs during thymic development.14 In this regard, CD69 is
a useful marker for defining the molecular map of T-cell
development,15 and a putative role for CD69 in T-cell
selection has been postulated by its restricted expression during the
late stages of thymocyte development.13 Because the CD7+
thymic precursors do not express CD69, it appears that its expression
is regulated intrathymically. Furthermore, CD69 expression on
thymocytes has been associated with apoptosis of these
cells.16 However, T lymphocytes in the inflammatory cell
infiltrates of various chronic inflammatory diseases express
CD69.17,18 The expression of CD69 associated with
inflammatory processes seems to be induced, at least in part, by
proinflammatory cytokines. Up-regulation of CD69 expression is observed
in vitro after incubation of lymphocytes with exogenous
cytokines.19,20 Likewise, it has been shown that tumor
necrosis factor  is able to promote the transcriptional activity of
the 5' gene region of CD69 gene, thereby inducing CD69-cell
surfaceexpression.8 Although the putative ligands
for CD69 are so far unknown, it has been demonstrated that CD69
functions as a signal transducer molecule.2-4 The
engagement of CD69 induces an increase in intracellular
Ca++, synthesis of cytokines and their receptors, increase
in the expression of the proto-oncogenes c-myc and c-fos, as well as cellular proliferation.2,4,21-23 CD69 thus seems to play an important role in the activation and proliferation of human
lymphocytes, although its precise role in leukocyte physiology remains
undetermined. Here we have studied this issue by generating
CD69-deficient mice. The phenotypic and functional characterization of
different hematopoietic cell lineages in the absence of CD69 is the
subject of this work.
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Materials and methods |
Generation of CD69-deficient mice
A genomic DNA clone, containing the complete coding
sequence of CD69 and including more than 1.5 kb upstream of translation start site and 9 kb 3' to exon 5 end, was isolated from a murine 129-Sv phage genomic library (Stratagene, La Jolla, CA). The targeting construct was developed, using the 4.5-kb SalI-EcoRI fragment (long
arm) 5' containing exon 1 (intracytoplasmic domain) and the
3-kb XbaI fragment (short arm) containing the exon 5 untranslated region and 3' region of genomic 129 v-derived DNA
and subcloned in a double-selection vector kindly provided by Drs. D. Milstone and R. Mortensen (Brigham and Women's Hospital, Boston, MA).
This vector contains the neomycin-resistance gene (neo) for positive selection of the transfected embryonic stem (ES) cells and a copy of
the thymidine kinase gene for the negative selection of randomly integrated constructs. ES cells24 were grown,
transfected, and selected as described.25 Southern blot was
used to identify clones with the expected recombination event, which
were analyzed and confirmed using several restriction enzyme digestions
(XbaI, BamHI, and EcoRI/EcoRV) and verified using two different probes by Southern blot analysis. The probes used were 5' flanking, a 2-kb HindIII-EcoRI fragment from the genomic Xba-EcoRI fragment mapping
5'of the targeting construct; 3'Exon-1, a 2-kb HindIII fragment isolated from the genomic fragment SalI-EcoRI carrying the
exon 1. Three successfully targeted ES cell clones were aggregated with
CD1 morula cells and reimplanted in the uteri of 2.5-day pseudopregnant females. Germline transmission was obtained, and mice
were bred to homozygosity on a C57BL/6 genetic background. Consecutive
litters were analyzed by polymerase chain reaction using peripheral
blood samples.
Mice
Mice were bred at the Centro Nacional de Biotecnologia
(Madrid, Spain) under specific pathogen-free conditions. Mice used for
experiments were 8 to 16 weeks of age, and all experiments used either
littermate controls or age-matched litters whose parents were
littermates. In some cases, mice bearing the TCR transgene F526 were intercrossed with CD69 (-/-) mice to obtain
F5 transgenic TCR, CD69 (-/+) heterozygous mice that, when
intercrossed, produced F5 TCR, CD69 (-/-) and F5 TCR, CD69 (+/+) littermates.
Peptide treatment of F5 TCR transgenic mice
Mice were injected intraperitoneally with the peptide 9-mer
Ala-Asn-Glu-Asn-Met-Asp-Ala-Met NP-366-374 from the
influenza virus nucleoprotein A/NT/60/68 in phosphate-buffered saline
(PBS) at the indicated concentration.
Cell culture
All cell cultures were maintained in RPMI 1640 medium
supplemented with 5% heat-inactivated fetal calf serum, 10 mmol/L
HEPES, 10-5 mol/L 2-mercaptoethanol, 100 U/mL
penicillin/streptomycin, 2 mmol/L L-glutamine, and 1 mmol/L sodium
pyruvate at 37°C, 5% CO2.
Determination of hematological parameters
To measure blood cell parameters, whole blood was collected in tubes
containing K-EDTA and analyzed on an automatic hematology counter
(Technicon H*1E, Bayer, Tarrytown, NY).
Flow cytometry analysis
BM, thymus, spleen, lymph node, peripheral blood, and peritoneal
exudate cell suspensions were obtained from CD69 (-/-) or control
C57BL/6 littermates. Cells were stained with FITC-, PE-, TC-, or
SPRD-conjugated antibody reagents or indirectly with biotinylated antibodies followed by streptavidin-FITC, -PE, -TC, or -SPRD. The
antibodies used for staining were anti-CD69 (H1.2F3), anti-CD2 (RM2-5),
anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD5 (53-7.3), anti-CD8
(53.6.7), anti-CD25 (PC61), anti-CD44 (IM7), anti-NK (D × 5),
anti-TCR (H57-597), anti-V3 (53), anti-V8 (F2.3.1), anti-V11 (RR3-15), anti-V2 (B20.1), anti-TCR  (GL3),
anti-B220/CD45R (RA3-6B2), anti-CD43 (S7), anti-BP1 (6C3),
anti-immunoglobulin (Ig) M (AF6-78), anti-IgD (AMS15.1.5), and anti-HSA
(M1/69) (PharMingen, San Diego, CA). Propidium iodide was used at 5 µg/mL to detect dead cells. Cells were analyzed with a FACScan flow
cytometer (Becton Dickinson, Mountain View, CA). Cell staining and flow cytometry were performed according to standard methods, and, for 4-color analysis, a total of 50 000 events were acquired.
Analysis of hematopoietic progenitors
Progenitors of the granulocyte/macrophage lineage were assayed by
culturing 105 BM cells in Bactoagar medium (Difco, Detroit,
MI), as previously described.27 Briefly, cells were
resuspended in Iscove's modified Dulbecco's medium (Gibco-life
Sciences, Gaithersburg, MD) supplemented with 25% horse serum (Gibco)
and 10% WEHI-3b conditioned medium as an interleukin-3 (IL-3) source,
and then mixed with Bactoagar (0.3% final concentration) and seeded
into 35-mm plastic tissue culture dishes (Nunc, Roskilde, Denmark). The
megakaryocyte colony-forming units were assayed in serum-free cultures.
BM cells (3 × 105 cells per dish) were cultured on
Iscove's modified Dulbecco's medium supplemented with 10 mg/mL bovine
serum albumin, 3 µg/mL transferrin, 25 µg/mL soy bean lipids (all
from Boehringer Mannheim, Mannheim, Germany), 45 µg/mL linoleic acid
(Sigma, St Louis, MO), 7.8 µg/mL cholesterol (Sigma), 110 µg/mL
sodium pyruvate (Sigma), 10-4 mol/L L-thioglycerol (Fluka,
Buchs, Switzerland), 2 × 10-2 mmol/L glutamine
(Gibco), and 20% WEHI-3b conditioned medium. The pre-B colony-forming
units were assayed by culturing 105 BM cells in
methylcellulose culture medium supplemented with recombinant human IL-7
(Methocult M3630; StemCell Technologies, Vancouver, Canada) as
described.28 In all instances, triplicates of each sample
were seeded and incubated for 7 days at 37°C in 95% humidified
atmosphere with 5% CO2 in air. Colonies were scored under
a dissecting microscope, and, in the case of the megakaryocyte cultures, colonies were stained with acetylcholinesterase prior to
scoring.29 The exogenous spleen colony forming-units were assayed as described previously.30 Briefly, groups of 10 C57Bl/6 mice were irradiated with a split dose of 9 Gy (2 doses of 4.5 Gy spaced 4 hours apart), using Philips MG 324 x-ray equipment (Philips, Hamburg, Germany) at 300 kV, 10 mA, and a dose rate of 1.03 Gy/min. Each recipient was injected with 5 × 104
cells through the lateral vein tail. Twelve days after transplantation, recipients were killed and their spleens removed and fixed in Telleyeniczky's solution. Spleen colonies were then determined using a
dissecting microscope.
Cell proliferation assays
Cell suspensions were prepared from spleen, lymph node, or thymus
and cultured in triplicate in the presence of plate-bound anti-CD3
antibody, staphylococcus enterotoxin B, CD40L, anti-IgM, or
lipopolysaccharide (LPS) at various concentrations in culture medium
for 3 days. Cells were pulsed with [3H]dT (1 µCi/well)
12 hours before harvesting onto glass fiber filters for determination
of [3H]dT. For measurement of lymphocyte proliferation in
vivo, mice were intraperitoneally injected with 0.6 mg
bromodeoxyuridine (BrdU) in 100 µL PBS 18 hours before flow cytometry
analysis of BrdU incorporation.31
51Cr release assay
Specific NK cell and cytotoxic T lymphocyte (CTL) activity was
determined using a standard 51Cr release assay. Splenocyte
suspensions were prepared and erythrocytes were lysed by
NH4Cl treatment. Cr-labeled (100 µCi sodium chromate) YAC-1, NK-resistant (RMA), or NK-sensitive (RMA-S) target cells were
plated with the appropriate effector cells at different ratios. NK
cytotoxic activity was measured at 2.5 hours. In CTL experiments, splenocytes stimulated in vivo for 3-4 days by intraperitoneal injection of F5 peptide (18 nmol) in PBS were used as effector cells.
RMA and RMA-S unsensitized and F5 peptide-sensitized by preincubation
with 100 nmol NP 366-374 in 1 mL medium overnight at 26°C were used
as targets cells. Cytotoxicity was measured after an incubation period
of 4 hours. The percentage of specific lysis was calculated as (sample
cpm  spontaneous cpm)/(maximal cpm spontaneous
cpm)] × 100.
Immunizations and enzyme-linked immunosorbant assay (ELISA)
Age- and sex-matched mice were immunized intraperitoneally with 10 µg of 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (DNP-KLH) in
complete Freund's adjuvant (CFA) or in alum, or intravenously with
DNP-KLH in PBS at day 0. Each mouse was boosted 7 days later using the
same protocol. Blood was collected at days 6 and 14 after the first
immunization. Other mice were immunized intraperitoneally with 10 µg
LPS-TNP (Sigma) in PBS at day 0, and bled at day 0 and 14. NP-specific
antibodies were measured by ELISA using DNP-ovalbumin-coated plates at
3 µg/mL. Previously tested horseradish peroxidase-labeled isotype-specific anti-mouse immunoglobulin antibodies (Southern Biotechnology Associates, Birmingham, AL) were used in these assays. Sera were diluted 1:200 and 1:2000 and immunoglobulin levels were analyzed.
Immunohistochemistry
Frozen spleen, lymph node, and Peyer's patch sections from DNP-KLH
immunized CD69-deficient and wild-type mice (day 10 after immunization)
were stained with the indicated biotinylated antibodies, followed by
streptavidin-peroxidase. Sections were developed with diamino-benzidine
and counterstained with Mayer's hematoxylin.
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Results |
Generation and characterization of lymphoid populations in CD69
(-/-) mice
The CD69-mouse genomic DNA consists of 5 exons and 4 introns and is
approximately 9 kb in length.7 To disrupt CD69-gene expression by homologous recombination in ES cells, a targeting vector
was constructed by replacing with the neomycin-resistance gene, a
fragment of genomic DNA of 4 kb containing exons 2, 3, 4 and translated
region of the exon 5 (Figure 1A). Two
chimeric mice were obtained by morula aggregation, one of which
successfully contributed to the germ line. Homozygous mice were
obtained by interbreeding of heterozygous mice (Figure 1B). CD69 (-/-)
mice appeared normal, with no apparent developmental defects. Mice were
fertile and no obvious defects were appreciated in any organ analyzed
after autopsy.
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| Fig 1.
CD69 gene disruption by homologous recombination.
(A) Schematic representation of the partial restriction maps of the
mouse genomic CD69 locus, the targeting construct, and the targeted
allele. The exons of the wild-type CD69 gene are shown as solid bars,
boundaries of homology between the wild-type gene and the targeting
construct are denoted by thin lines. (B) Southern blot analysis of
BamHI-digested tail DNA from wild-type (+/+) and heterozygous (+/)
or homozygous (-/-) CD69 mice were analyzed with a 2-kb HindIII-EcoRI
fragment from the genomic Xba-EcoRI fragment mapping 5'of the
targeting construct. The unmutated CD69 gene produced a 12-kb fragment,
whereas the mutated allele results in a 9-kb fragment. (C) CD69
expression in wild-type, CD69 (+/), and in CD69 (-/-) mice. Flow
cytometry analysis of CD69 expression in thymus from wild type (+/+),
heterozygous(+/), or homozygous (-/-) CD69 mutant mice. (D) CD69
expression in spleen, lymph node, and peripheral blood lymphocytes from
wild type (+/+) or CD69 (-/-) mutant mice. Cells were activated with
PMA at 10 ng/mL for 15 hours. Cells were double-stained with anti-CD69
and anti-CD2 labeled monoclonal antibodies and analyzed by flow
cytometry.
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To demonstrate the lack of CD69 expression in CD69 (-/-) mice,
thymocytes were stained with anti-CD69 mAb and CD2 as control and
analyzed by flow cytometry. As expected, CD69 expression was absent in
CD69 (-/-), while in wild-type CD69 (+/+) mice, approximately 29% of
CD2+ thymocytes coexpressed CD69 (Figure 1C). Intermediate
CD69 expression was found in heterozygous CD69 (+/) mice (Figure
1C). Moreover, spleen, lymph node, and peripheral blood T cells from
CD69 (-/-) mice activated with PMA did not express CD69
(Figure 1D). All these results confirmed the inactivation of the CD69
gene and the inheritance of this trait at the expected Mendelian ratio.
Comparison of CD69 (-/-) and CD69 (+/+) mice from the same litter
showed no significant differences in the CD3+ lymphoid
populations in thymus, spleen, and lymph nodes (Figure 2A, and data not shown). Moreover, no
differences were appreciated in the major T-cell subsets, as assessed
by CD4 and CD8 expression (Figure 2B). Similar results were obtained
using other cell markers such as CD2, CD5, CD25, and CD44 (data not
shown). Mature and immature thymic subsets in CD69 (-/-) mice showed a
normal pattern of development (Figure 2B). Proportions of mature
TCR and TCR were similar in CD69 (-/-) and CD69 (+/+) mice
(data not shown). Likewise, normal cell numbers and a normal T:B
lymphocyte ratio were observed in the peripheral lymphoid organs of
CD69 (-/-) deficient mice (Figure 2B). Lymphoid organs, including
thymus, spleen, lymph nodes, and Peyer's patches from CD69 (-/-) mice, had a normal appearance by histological analysis (Figure 2C, and data
not shown). No significant differences in other cell subsets from
peripheral lymphoid organs were found, as determined with a panel of
mAb specific for B cells (B220, IgM, IgG, CD43), granulocytes (Gr), and
monocytes (CD11b, CD71, I-Ab, ICAM-I). Peritoneal exudate cells were
also present in normal proportions in CD69 (-/-) mice (data not shown).
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| Fig 2.
Lymphocyte subpopulation analysis in CD69 (-/-) and
wild-type mice.
Cells from lymphoid organs were obtained from CD69 (-/-) mice and
wild-type littermate controls, then immunofluorescently stained and
analyzed using flow cytometry. (A, B) CD3 expression on thymocytes and
CD4 and CD8 markers on thymocytes, splenocytes, and lymph node cells.
Numbers indicate the percentage of lymphoid populations. Data from a
single mouse are shown, they are representative of more than five mice
per group and from more than one litter. (C) Immunochemical analysis of
lymphoid organs from CD69 (-/-) mice. Frozen sections of lymph nodes
and spleens from unimmunized mice were immunostained for immunoglobulin
M. Peyer's patches obtained from mice 10 days after challenge by
intraperitoneal injection of 10 µg of alum absorbed
2,4-dinitrophenyl-keyhole limpet hemocyanin mice were stained with
anti-CD5.
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Hematopoiesis in CD69 (-/-) mice: altered pre-B immature cells
Analysis of CD69 expression in normal BM revealed its presence in
the B220+ pre-B cells (Figure
3A). Interestingly, CD69 was expressed with high intensity on 20%-30% of B220+ pre-B cells; it was,
therefore, of interest to analyze B cells at different maturation
stages within CD69 (-/-) mouse BM. Increased percentages of pre-B
(B220+int, IgMneg) and immature B
(B220+int, IgM+) cells were consistently
detected in CD69 (-/-) mice compared with wild-type mice. The pre-B
cell compartment, defined as B220+int IgMneg,
was the most affected, whereas B220+int IgM+
pre-B and immature B cells were only moderately augmented (Figure 3B).
The overall increase in the B220+int IgMneg
lymphoid cell subset was statistically significant. The relative change
mean for this subset was 1.523 (P < .01; Student t
test, sample size = 20). The detection of another pre-B cell marker (the Heat Stable Antigen, HSA) confirmed the difference in this B-cell
subset between wild-type and CD69-deficient mice (Figure 3C). These
differences correlate with a slight lymphocytosis in CD69 (-/-) mice
(1.35 relative change mean, P < .01). In contrast, the
mature B-cell compartment (B220bright IgM+) was
not significantly altered. Analysis of pro-B cells defined by CD43,
BP-1, and HSA markers revealed no alteration in CD69 (-/-) mice (data
not shown). Our results, therefore, show that CD69 is constitutively
expressed on B-cell precursors and appears to play a role in cell
maturation. To further analyze the effect of the disruption of CD69
gene in B-cell development, clonogenic assays with BM cells from CD69
(-/-) and wild-type mice were performed, but no significant differences
were found (Table 1).
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| Fig 3.
Phenotypic analysis of B-cell precursors in CD69 (-/-)
and wild-type bone marrow.
(A-C) Two-color immunofluorescence analysis with anti-B220 and
anti-CD69 monoclonal antibody shows that
B220+CD69+ cells are enriched in the
B220int lymphocyte fraction. In CD69 (-/-) mice, the
increase in IgM B220+ and
HSA+B220+ cell subsets is also shown.
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Table 1.
Comparative analysis of the hematopoietic progenitors
present in femoral bone marrow of CD69 (/) and wild-type
mice
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The expression of CD69 has been described on BM myeloid
precursors.10 In CD69 (-/-) mice, erythroid and myeloid
precursors were normal, according to both their number and specific
surface markers (Table 1, and data not shown). Clonogenic assays with BM cells of myeloid progenitors showed no significant differences in
the values of granulocyte/macrophage and megakaryocyte colony-forming units obtained between CD69 (-/-) and wild-type mice. Similarly, no
differences were found in analyses of a more primitive and multipotential precursor by exogenous spleen colony-forming unit assays
(Table 1). Hematologic analyses demonstrated that the cellular
components of peripheral blood were normal in number and distribution
(data not shown). In addition, although CD69 is expressed in a
constitutive manner in normal platelets, platelet numbers were similar
in the CD69 (-/-) and wild-type mice. Finally, analyses of different
serum biochemical parameters in CD69 (-/-) mice showed no abnormality
in the protein, lipid, and enzyme content studied (data not shown).
Positive and negative selection of thymocytes
The definition of thymocyte subsets is currently based on the
detection of different molecules (TCR, CD4, CD8), including CD69. CD69
is absent in double-positive TCRneg/lo cells and is not
expressed in TCR-deficient mice and in major histocompatibility
complex Class I and Class II double-deficient mice.32 CD4loCD8lo and
CD4loCD8+ TCRint cells express CD69. Therefore,
CD69 expression is induced just after the initiation of TCR
positive selection in thymocytes, when these cells are signaled via
their TCR.13,33 To study the role of CD69 in thymocyte
selection, CD69 (-/-) TCR F5 transgenic mice were generated. The F5
receptor is specific for influenza virus A/NT/60/68 nucleoprotein NP
366-379 and utilizes the V4 and V11 TCR gene
segments. CD8+T cells expressing F5 are positively selected in TCR
transgenic H-2Db wild-type mice. The process of positive
selection into the CD8 compartment was not altered in F5 TCR CD69 (-/-)
mice (Figure 4A). Both F5 TCR CD69 (-/-)
and F5 TCR wild-type mice thus have a high CD8:CD4 cell ratio.
Accordingly, the thymocyte expression of transgenic V11 chain by T
cells was high in both mice (not shown).
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| Fig 4.
Analysis of thymic selection and in vivo proliferation of
mature T cells in CD69 (-/-) F5 T-cell receptor (TCR) transgenic mice.
(A) Two-color flow cytometry analysis was performed in thymocytes of F5
TCR transgenic mice untreated or after the administration of
NP366-374 as described in Materials and methods section.
(B) The proliferative capability of mature T cells in CD69 (-/-) mice
was analyzed in vivo after bromodeoxyuridine (BrdU) treatment as in the
Materials and methods section. Numbers indicate the percentage of
positive, BrdU-incorporating lymph node cells. Data shown are
representative of results obtained in three mice per group and from
more than one litter.
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Negative selection was induced in F5-TCR transgenic mice by
intraperitoneal injection of the antigenic peptide of the influenza nucleoprotein (NP) 366-374.26 This negative selection was
reflected in a threefold reduction of thymus cellularity, a decrease in double-positive thymocytes, a depletion of V11-bearing T cells, and
an apparent increase in CD4-CD8- thymocytes
detected at 2 or 4 days after peptide administration. A similar pattern
of expression of CD4, CD8, V11, and CD25 was observed in both F5 TCR
CD69 (-/-) and F5 TCR CD69 (+/+) mice (Figure 4A, and data not shown).
Positive and negative selection of thymocytes were, therefore,
unaltered by CD69 deficiency.
Lymphocyte proliferative responses
To explore the possible involvement of CD69 in B- and T-cell
functions, we first determined the proliferative response of thymus,
spleen, and lymph node cells from CD69 (-/-) mice to different stimuli.
Cells from CD69-deficient mice displayed proliferative responses to
T-cell-specific stimuli, such as anti-CD3, Con-A, and staphylococcus
enterotoxin B, similar to those of cells from wild-type mice (Table
2). In addition, lymph node and spleen cells from CD69 (-/-) mice showed a normal response to B-cell stimuli,
such as LPS and CD40L+ IL-4 (Table 2 and data not shown). These results
indicated that CD69 is not necessary for the proliferation of
lymphocytes induced by different stimuli.
To assess the in vivo role of CD69 in T-cell proliferation, lymph node
and spleen transgenic CD8+ T-cell expansion was studied in TCR F5
transgenic CD69 (-/-) mice by BrdU incorporation. It has been described
that exposure of mice transgenic for a F5 TCR to NP366-374
peptide results in expansion and activation of peripheral
V11-bearing CD8 T cells.26 Both F5 TCR CD69 (-/-) and F5
TCR wild-type mice showed a comparable high rate of peripheral CD8
T-cell proliferation (Figure 4B, and data not shown). These results
showed that T cells are able to proliferate in vivo in response to a
specific antigen in the absence of CD69. T-cell cytokine production
was analyzed by intracytoplasmic staining of PMA-activated
Th1-derived spleen cells. A comparable cytokine synthesis profile was
observed in cells from CD69 (-/-) and wild-type mice (data not shown).
Cytolytic activity in CD69 (-/-) mice
Previous studies33 suggested that CD69 may be involved
in the regulation of cytolytic activity in NK and T lymphoid
cells. We, therefore, analyzed the cytolytic activity of CD69 (-/-)
lymphocytes against NK-sensitive and NK-resistant target cells. The
number and phenotype of splenic NK and CD8+ cytotoxic
cells were found to be similar in both CD69 (-/-) and wild-type mice
(data not shown). We assayed two different effector cells, spleen cells
cultured in the presence of phytohemagglutinin plus IL-2, and
unstimulated spleen cells. CD69 (+/+) and CD69 (-/-) splenocytes
displayed similar cytolytic activity against RMA-S or RMA syngeneic
target cells (Figure 5A).
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| Fig 5.
Cytotoxic T lymphocyte activity in CD69 (-/-) mice.
(A) Natural killer (NK) cytotoxity against NK-resistant and
NK-sensitive cells using unfractionated spleen cells. Cytotoxic
activity was assayed in the presence or absence of phytohemagglutinin
(PHA) plus IL-2. Specific lysis is shown for three mice per group,
representative of five experiments. (B) Nucleoprotein (NP)
peptide-specific cytotoxic response of spleen cells from F5 CD69 (+/+)
and F5 CD69 (-/-) mice, treated with influenza virus nucleoprotein
peptide as in Materials and methods section. Cells were assayed in the
presence or absence of the NP366-374. Results shown are the
average of three mice per group, representative of three experiments.
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Because a major CD8 T-cell subset differentiates in F5 TCR transgenic
lymphocytes on the C57BL/6 (H-2b haplotype) genetic
background, we compared the lytic activity of in vivo activated CTL
from CD69 (-/-) and CD69 (+/+) F5 TCR mice against RMA-S and RMA cells
loaded with NP peptide. We found no differences in peptide-specific
lysis mediated by splenocytes from either type of mice when the lytic
assay was performed using an optimal concentration peptide loading (100 µmol/L) (Figure 5B).
Antibody responses
The possible role of CD69 in adaptive B-cell responses was analyzed.
Different immunization protocols were used to evaluate the humoral
immune response of CD69-deficient mice against thymus-dependent (TD)
and -independent antigens. For TD B-cell responses, age- and
sex-matched CD69 (+/+) and CD69 (-/-) mice of the same progeny were
divided into groups receiving 10 µg of DNP-KLH in PBS intravenously, or adsorbed to alum or mixed with CFA intraperitoneally. Basal immunoglobulin concentrations were in the same range in all mice tested
prior to immunization (Figure 6).
CD69-deficient mice were able to mount DNP-specific primary and
secondary responses of all immunoglobulin isotypes. The magnitude of
IgG1, IgG2b, and IgG3 antibody responses was similar in the wild-type
and CD69 (-/-) mice. A slight increase in the IgG2a primary response
was observed in CD69 (-/-) mice, regardless of the immunization
protocol used (Figure 6A). IgG2a anti-DNP antibodies were also slightly increased in secondary responses when immunization was intravenous. Moreover, a slightly enhanced IgM response was observed in immunized CD69 (-/-) mice, but the differences in antibody titers were not statistically significant between CD69 (-/-) and CD69 (+/+) mice. Nevertheless, CD69-deficient mice produced specific antibodies to the
DNP-LPS thymus-independent antigen at a similar level to that of
wild-type mice (Figure 6B). As was found for TD antigens, immunized
CD69 (-/-) mice produced slightly more anti-DNP-LPS IgM and IgG2a than
wild-type mice.
View larger version (21K):
[in this window]
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| Fig 6.
Specific antibody responses in CD69 (-/-) mice.
Several immunization protocols were used to assess the immunoglobulin
responses in CD69-deficient mice. The responses of individual mutant
(filled circles) and wild-type (open triangles) mice are shown. (A)
T-cell-dependent B-cell response. Results shown are from different
groups: (IV), immunized with 2,4-dinitrophenyl (DNP)-keyhole limpet
hemocyanin (DNP-KLH) in phosphate-buffered saline (PBS), (ALUM)
immunized intraperitoneally with DNP-KLH adsorbed to alum, and complete
Freund's adjuvant (CFA) immunized intraperitoneally with DNP-KLH mixed
with CFA. CD69 (-/-) mice immunized with DNP-KLH mixed with CFA and
DNP-KLH in phosphate-buffered saline produce augmented amounts of
immunoglobulin (Ig) G2a (P  0.05, two-way analysis of
variance). (B) T-cell-independent B-cell response. DNP-LPS CD69 (-/-)
mice produce increased amounts of IgM (top panel) and IgG2a (bottom
panel) antibodies after immunization. IgM and IgG2a anti-DNP titers
differed significantly (P < .05) between the two groups
(+/+ and -/-) by two-way analysis of variance. Other DNP-specific
antibody isotypes were at similar levels in CD69 (-/-) and wild-type
mice (not shown).
|
|
It has been postulated that CD69 plays a role in T-B cell
collaboration, as it is highly expressed by germinal center CD4 T
cells12; we, therefore, performed immunohistochemical
stainings of spleen and lymph node sections after DNP-KLH immunization. We found that mAb specific for IgM, B220, CD3, and CD5 exhibited a
similar staining pattern in lymph node sections from CD69 (-/-) and
CD69 (+/+) mice. T- and B-cell areas and germinal centers were clearly
appreciated in immunized lymph nodes from both types of mice (Figure
2C, and data not shown). In addition, no differences were observed in T
cells located in the periphery of lymphoid follicles and B cells within
the follicles. Likewise, no significant differences were detected in
spleen sections, Peyer's patches, and intraepithelial lymphocytes from
CD69 (-/-) and CD69 (+/+) immunized mice (Figure 2C, and data not shown).
| |
Discussion |
The pattern of expression of CD69 as well as its putative role as a
signal transducing receptor on leukocytes points to an important role
for CD69 in the biology of these cells. In addition, CD69 expression
during thymocyte and B-cell development, as well as during activation
of mature lymphocytes, strongly suggests its involvement in the
differentiation and proliferation of these cells.3,4 We
explored the physiological role of CD69 in vivo and studied the
phenotypic and functional characteristics of lymphocyte development in
CD69 (-/-) mice. T-lymphocyte ontogeny was apparently normal, whereas a
subtle alteration in B-cell development was detected. CD69 is
constitutively expressed by a significant percentage of B-cell
precursors in normal BM, and the pre-B-cell subset was significantly
augmented in CD69-deficient mice. Furthermore, CD69 (-/-) mice
exhibited a slight but significant increase in the humoral immune
response against a TD antigen. No additional alterations were found in
any other cell compartments or functions studied, including NK and T
cells, macrophages, granulocytes, and platelets.
It has been described that CD69 is detected on activated B
cells.12 The CD69 expression by BM B-cell precursors at the
B220+ pre-B-cell stage found by us is thus probably a
consequence of the cell activation process that occurs during
B-lymphocyte development. CD69 could be involved in the positive
selection of B cells in BM, paralleling what it is thought to occur
during T-cell development.32,34 It has been described that
surface immunoglobulin crosslinking on mature B cells induces CD69
expression. It is feasible that in pre-B cells, which do not bear
surface immunoglobulins, different activatory stimuli such as cytokines
are able to induce CD69 expression. In addition, the possibility of
CD69 expression induction on pre-B cells by the interaction of a
pre-B-cell receptor with its putative ligand cannot be ruled out.
Interestingly, CD69 (-/-) mice showed a significant increase in the
number of pre-B and immature B cells compared with CD69 (+/+) wild-type
mice. These data support a regulatory role for CD69 in B-cell
differentiation. It is feasible that CD69 acts as a signal transducing
molecule in pre-B cells, with a role in the induction of
differentiation of these cells. Under such circumstances, the absence
of CD69 may be responsible for slow pre-B-lymphocyte differentiation,
with accumulation (and increase in the number) of cells at this
differentiation stage. An alternative, but not exclusive, possibility
is that CD69 is involved in the induction of apoptosis associated with
pre-B-cell activation (activation-induced cell death). In this case,
the absence of CD69 would be responsible for the augmented number of
pre-B cells in CD69 (-/-) mice due to defective deletion of these
cells. This possibility is supported by a report on the association
between CD69 expression and programmed cell death in
thymocytes.16 Another possibility is that CD69 might act in
pre-B cells as an inhibitory membrane receptor, generating negative
regulatory signals that would constrain cell proliferation. Although
all available information on CD69 strongly suggests that this molecule
acts as a costimulatory receptor with an important role in lymphocyte
activation and proliferation (reviewed in
Sánchez-Madrid3 and Testi et al4), we
think it is possible that CD69 may exert an inhibitory effect in some
cells. In this regard, the CD94-NKG2 lectin-like receptors, which
exhibit some degree of homology with CD69, are inhibitory/triggering
molecules in NK cells.35 CD94-NKG2A is thus an inhibitory
receptor coupled to SHP tyrosine phosphatases, whereas CD94-NKG2C forms
a triggering complex in these cells. Nevertheless, other membrane
receptors, such as those for IL-10 and transforming growth factor ,
exert stimulatory and inhibitory effects on different leukocyte
subsets.36-39
Other inhibitory/stimulatory coreceptors have been described on
lymphoid cells, such as the killer inhibitory receptors that are
expressed, aside from NK cells, by some T lymphocytes, in which they
regulate TCR-dependent functions.40 Finally, the CD81
tetraspanin regulates lymphocyte functions, acting as positive or
negative mediator of lymphocyte proliferation depending on the type of
stimulus and modulating the immunoglobulin isotype balance.41 It is thus feasible that CD69 exerts an
inhibitory effect on certain cell types at specific differentiation
stages. In addition, the absence of CD69 could lower the signal
threshold necessary for the effect of receptors involved in negative
signaling on B-cell activation/proliferation such as CD22 and
FcRII.42,43 It is, therefore, conceivable that CD69 acts
in pre-B cells by modulating the signaling function of other membrane
receptors involved in cell activation. In this regard, CD22 is
described as both a positive and negative modulator of B-cell antigen
receptor complex signal transduction in mature and immature B
cells.42
The lack of effect of CD69 deficiency on T-cell function is of
interest. CD69 has been also termed "Activation Inducer Molecule" and is expressed on leukocytes during and after their
activation.2-4 CD69 expression can be induced in vitro on
lymphocytes by several stimuli, including anti-CD3 and anti-CD2 mAb,
PMA, and phytohemagglutinin. The functional characteristics and
molecular structure of CD69, a type 2 C-type lectin with a carbohydrate
recognition domain in the C-terminal region,6,7 as well as
its expression pattern, suggest its involvement in cell activation.
Different data support the role of this molecule as a signal
transducing receptor in leukocytes. Although the putative ligands for
CD69 have not been characterized yet, it has thus been found that the
engagement of CD69 with mAbs induces an increase in intracellular
Ca++, as well as cytokine synthesis and expression of
proto-oncogenes. When the effect of CD69 engagement is combined with a
PKC activator, lymphocyte activation proceeds to DNA synthesis and cell
proliferation is observed.2,22 We, therefore, expected to
detect abnormalities in T-cell activation or development in
CD69-deficient mice. Despite CD69 expressed during thymocyte
differentiation in normal mice, no significant changes were observed in
T-lymphocyte development in CD69 (-/-) mice. The number and phenotypic
characteristics of T cells in different lymphoid tissues were normal,
and the positive and negative selection of thymocytes was unaffected in the absence of CD69. T cells from CD69 (-/-) mice also showed normal
proliferative capability and were able to provide apparently adequate
help in antibody-specific responses. Furthermore, we have detected no
alterations in the activation-induced cell death of T lymphocytes
triggered by anti-CD3 mAb in CD69 (-/-) mice (Lauzurica et al,
unpublished observations). These findings suggest that the function of
CD69 in T cells is not essential or that it may be replaced by other
molecules. It is also possible, however, that subtle defects in T-cell
function may occur in CD69-deficient mice and that the assays employed
in this study were not able to detect them.
There are several possibilities to explain the slight but significant
increase in the humoral immune response against TD antigens observed in
CD69 (-/-) mice. Immunoglobulin synthesis by B cells is under the
control of different mechanisms, including those exerted by regulatory
T cells. It is feasible that the absence of CD69 has subtle
consequences on the immunoregulatory activities of T lymphocytes and
that the increased synthesis of certain isotypes observed in
CD69-deficient mice is related to defective modulation of B-cell
function. Another possibility is that, as stated above, CD69 may exert
a direct modulatory/inhibitory activity on certain cell subsets,
including mature antibody-producing B lymphocytes. In this case, the
absence of CD69 expression by antibody-producing cells may have as
consequence a disregulation in isotype-specific immunoglobulin
synthesis. Since only one ES cell line was used to generate the CD69
(-/-) mice, it is feasible, but highly unlikely, that the phenotype
differences might be due to a defect in the ES cells at a locus linked
to but different from CD69. Further studies are necessary to elucidate
the precise mechanism of enhanced antibody production in CD69-deficient mice.
Although CD69 is expressed by platelets, myeloid precursors, activated
neutrophils, and eosinophils, we found no abnormalities in these cells
in CD69 (-/-) mice. Different mechanisms may account for these results,
including (i) the activities previously reported for CD69 in these
cells in vitro may not occur in vivo; (ii) because most CD69 functions
have been defined in human cells, it is possible that mouse CD69 may
not exert the same functions in different cells; and (iii) as stated
above, CD69 function may be replaced by other molecules in CD69 (-/-)
mice. The cloning has recently been reported of AICL, a gene with a
highly similar sequence to that of CD69, although its cellular and
tissue distribution at the protein level has not been described
yet.44 In addition, other yet uncharacterized molecules
from the C-lectin NK cell complex may also participate, as well as
different costimulatory molecules that have been extensively studied
such as CD28.
In conclusion, CD69 is expressed by both B-cell precursors and
activated B cells and seems to exert a subtle modulatory effect on
B-cell development and antibody synthesis. This study also indicates
that CD69 is not essential for TD T-lymphocyte development and cell
proliferation. Nonetheless, subtle defects in some regulatory function
of T cells cannot be ruled out. Functional redundancy of costimulatory
molecules may account for the lack of gross abnormalities in the
differentiation and activation of T cells in the absence of CD69.
Because our CD69-deficient mouse line has been maintained in
pathogen-free conditions, it is not known whether or not these mice
exhibit increased susceptibility to some infectious agents; experiments
to address this question are in progress. Other mouse lines deficient
in immune response-related molecules (eg, IL-10, CCR-1, MIP-1) are
also apparently normal, but important abnormalities emerge when they
are challenged with different pathological agents. Finally, the
possible role of CD69 deficiency in autoimmune diseases deserves
further research.
| |
Acknowledgments |
We thank Dr Kioussis for the gift of the F5 transgenic mice, and C. Mark for editorial assistance. We are very grateful to Dr Roberto
González-Amaro for the excellent critical review. Lucio
Gómez and Juan Caballero were very helpful in their technical assistance.
| |
Footnotes |
Submitted August 16, 1999; accepted November 30, 1999.
Supported by grant No. SAF 99/0034-C01-C02 from the Ministerio
de Educacion y Cultura, and grant 08/001/1997 (to FS-M) from the
Comunidad Autónoma de Madrid. The Department of Immunology and
Oncology was founded and is supported by the Spanish Research Council
(CSIC), Pharmacia, and Upjohn.
Reprints: Francisco Sánchez-Madrid, Servicio de
Immunología, Hospital de la Princesa, C/ Diego de León
62. Madrid 28006, Spain; e-mail: smadrid/princesa{at}hup.es.
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.
| |
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H. S. Kang, M. J. Lee, H. Song, S. H. Han, Y. M. Kim, J. Y. Im, and I. Choi
Molecular Identification of IgE-Dependent Histamine-Releasing Factor as a B Cell Growth Factor
J. Immunol.,
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A. S. Llera, F. Viedma, F. Sanchez-Madrid, and J. Tormo
Crystal Structure of the C-type Lectin-like Domain from the Human Hematopoietic Cell Receptor CD69
J. Biol. Chem.,
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H. Zhou, V. Kartsogiannis, Y. S. Hu, J. Elliott, J. M. W. Quinn, W. J. McKinstry, M. T. Gillespie, and K. W. Ng
A Novel Osteoblast-derived C-type Lectin That Inhibits Osteoclast Formation
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[Abstract]
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Abstract
Introduction