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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 705-710
NEOPLASIA
Altered lymphoid development in mice deficient for the mAF4
proto-oncogene
Patricia Isnard,
Nathalie Coré,
Philippe Naquet, and
Malek Djabali
From the Centre d'immunologie INSERM-CNRS de Marseille Luminy,
Marseille Cedex 9, France.
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Abstract |
Some chromosomal translocations in acute leukemias involve the
fusion of the trithorax-related protein Mll (also called HRX, All1,
Htrx,) with a variety of heterologous proteins. In acute lymphoblastic
leukemia associated with the t(4;11)(q21;q23) translocation, the
4q21 gene that fuses with Mll is AF4. To gain insight
into the potential role of AF4 in leukemogenesis and
development, this gene was inactivated by homologous recombination in
mice. As expected from the tissue distribution of the AF4 transcript,
development of both B and T cells is affected in AF4 mutant mice. A
severe reduction of the thymic double positive CD4/CD8
(CD4+/CD8+) population was observed; in
addition most double- and single-positive cells expressed lower levels
of CD4 and CD8 coreceptors. Most importantly, the reconstitution of the
double-positive compartment by expansion of the double-negative cell
compartment was severely impaired in these mutant mice. In the bone
marrow pre-B and mature B-cell numbers are reduced. These results
demonstrate that the function of the mAF4 gene is critical for
normal lymphocyte development. This raises the possibility that
the disruption of the normal AF4 gene or its association
with Mll function by translocation may orient the oncogenic process
toward the lymphoid lineage. This represents the first functional
study using a knock-out strategy on one of the Mll partner genes
in translocation-associated leukemias.
(Blood. 2000;96:705-710)
© 2000 by The American Society of Hematology.
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Introduction |
The most common chromosome abnormality among infants
with acute lymphoblastic leukemia is a t(4;11)(q2l;q23) and patients with this 4;11 translocation have a very poor prognosis.1,2 This genetic rearrangement fuses the Mll/ALL-1/HRX-Htrx gene at 11q23 with the AF4/FEL gene at 4q21.5-7 The
Mll gene is involved in different acute leukemias through a
series of chromosome translocations5,8,9 and fusion to a
variety of genes, most frequently to AF4 and
AF9.10 The critical feature of these chromosomal
rearrangements is the generation of an in-frame chimeric fusion
transcript consisting of 5'-Mll and 3' sequences of the
gene on the partner chromosome.11 The resulting chimeric
messenger RNAs (mRNAs) presumably encode chimeric proteins that
contribute to the leukemogenic state.12,13 However, it has
been shown that hematopoietic precursors are greatly reduced in Mll
mutant mice, suggesting that Mll functions as a regulator of the growth
of hematopoietic precursors.14
Distinguishing features of Mll alterations include the striking
heterogeneity of its recombinations, with more than 30 chromosome partners described in Mll rearrangements among which 17 Mll partner genes have been characterized (for a review see reference 2). Furthermore, each partner is preferentially associated to a different subset of myeloid or lymphoid leukemias.2 Because these
genes do not all share the same structural or functional features,
their putative roles in Mll activation in a specific hematopoietic
lineage still need to be clarified.
The function of the AF4 gene remains poorly understood. The AF4
protein contains a domain with transcriptional activation activity that
is consistently retained by Mll-AF4 fusion proteins created by t(4;11)
translocations in leukemia15 and shows a nuclear
localization, thus supporting a potential role in regulating transcription.16,17 In previous studies, it has been shown that the AF4 transcripts are present in a variety of hematopoietic and
nonhematopoietic human cells.18,19 In mice, we and others have previously shown that the mAF4 gene is expressed
predominantly in developing thymocytes3 and is strongly
expressed in developing and adult lymphoid organs.4 These
characteristics support the hypothesis that the AF4 gene might
be important for the leukemic phenotype.
To investigate the effects of the mAF4 gene on lymphocyte
development, we have inactivated this gene by homologous recombination in the mouse. We show that in the hematopoietic system only the lymphoid compartment is severely affected in mAF4 mutant mice. Our data
show that the mAF4 gene affects early events in lymphopoiesis such as precursor proliferation or recruitment, but it is not required
for the terminal stages of lymphocyte differentiation.
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Materials and methods |
Construction of the targeting vector
A XbaI-SalI fragment containing the pGK-neo gene flanked
with 2 loxP sites was subcloned in the EcoRV site of pBluescript (pBS)
SK vector (Stratagene, San Diego, CA).20 Two
DNA fragments from the mAF4 locus were isolated from a  phage
screened from a mouse 129/Ola genomic library: the 5-kb BamHI-HindIII
fragment contains part of the 3' intron 11 region of the
mAF4 gene; the 3-kb HindIII fragment, from the 5' region,
contains intron 10 and part of exon 11. After filling the ends of
restriction sites, the 2 mAF4 homologous fragments were cloned,
respectively, in the SmaI and HindIII sites of the modified pBS-neo
vector, on each side of the loxP-neo-loxP cassette. This targeting
vector pBS-neo-mAF4 was designed to allow the interruption of the exon 11 and the excision of a part of this exon up to the BamHI site in the
following intron after homologous recombination in embryonic stem (ES) cells.
Transfection of ES cells and generation of chimeric mice
The E14 (129/Ola) strain ES cells (2×107) were
electroporated with 20 µg pBS-neo-MAF4 targeting construct DNA.
Twenty-four hours later, cells were positively selected with 400 µg/mL G418. Isolated G418-resistant colonies were picked after 7 days
of selection. Homologous recombinants were tested by hybridization of
EcoRI-digested genomic DNA using a HindIII fragment (5' probe) as
an external probe. A unique integration event was checked with a
neomycin probe. For Northern analysis, RNA was extracted from kidney
and expression was checked with a 1.4-kb complementary DNA (cDNA) fragment as a probe.4
Manipulations of the embryos were performed as described.21
Two male chimeras (distinguished by the coat color), originating from 2 independent ES clones, gave a germline transmission of the targeted ES
cells when mated to BALB/c females. Tail DNA from the F1 offspring were
analyzed by Southern blotting for germline transmission of the mAF4
targeted allele. Homozygous mutant mice were obtained by mating the
heterozygous mutant mice.
Flow cytometric analysis
Single cell suspensions from thymus were incubated at
1 × 107 cells/mL in 100 µL of staining solution
(phosphate-buffered saline [PBS] complemented with 0.2% fetal calf
serum [FCS] and 0.2% mouse serum) for 30 minutes on ice with
fluorescein isothiocyanate (FITC)-conjugated anti-CD3, -CD69, and
H-2Kb, phycoerythrin (PE)-conjugated anti-CD8 (53-6.7)
and biotin-conjugated anti-CD4 (H129.19) monoclonal
antibodies. After 2 washes in PBS/0.2% FCS solution, the cells were
incubated with 50 µL of streptavidin-cy-chrome reagent in staining
solution for 30 minutes on ice. The stained cells were washed 3 times
and then fixed in 150 µL of 1% paraformaldehyde in PBS. Analysis was performed on a Becton Dickinson FACScan. The monoclonal antibodies and
the streptavidin-cy-chrome reagent were purchased from Pharmingen (San
Diego, CA).
Glucocorticoid treatments
Two injections were performed at day  2 and day 1
before the time-course experiment. Mice were injected intraperitoneally with 2 mg/20 g hydrocortisone (Roussel, Paris, France).
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Results |
Generation of mutant mice at the AF4 locus
To investigate the effects of the mAF4 gene on lymphocyte
development we have inactivated this gene by homologous recombination in the mouse by inserting a neomycin-resistance (neo) gene in reverse orientation to the mAF4 transcription (Figure
1A). The targeting vector was
electroporated into E14 ES cells and selected by G418. Two independent,
homologous recombinant E14 ES cell clones generated several chimeric
males, which transmitted the disrupted allele to their offspring
(Figure 1B). Male and female mAF4 heterozygous mice appeared normal and
fertile and were interbred to generate homozygous mice. From these
crosses 25% of the mice observed at birth were of the
mAF4/ genotype indicating that the mutants survive
embryogenesis. Genomic Southern blot analysis as well as Northern blot
analysis and reverse transcriptase-polymerase chain reaction (RT-PCR,
data not shown) confirmed the disruption of the mAF4 gene and
the absence of the mAF4 transcript in the homozygous
mAF4/ mice (Figure 1C).
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| Fig 1.
Targeted disruption of the mAF4 gene.
Homologous recombination at the mAF4 locus in ES cells. (A) Structure
of the targeting vector and partial restriction map of the mAF4 locus
before and after targeted integration. The targeting vector contains
the neomycin-resistance (neo) gene flanked by genomic mAF4
sequences. After the recombination event, the neo gene replaces
a BamHI-HindIII fragment interrupting exon 11 of the mAF4 gene.
E indicates EcoRI; H, HindIII. (B) Southern blot analysis of a
representative litter showing alleles from a wild-type (+/+), a
heterozygous (+/), and a homozygous (/) animal.
Hybridization of genomic DNA with an external probe (5' probe)
reveals a 20-kb EcoRI fragment for the mAF4 wild-type allele and a 8-kb
EcoRI fragment corresponding to the inactivated allele. (C) Northern
blot analysis of a representative litter showing the mAF4 transcript of
RNA (10 µg) extracted from the kidney of a heterozygous mouse
(+/) and from 2 homozygous mice (/).
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Among the mAF4/ mice on a mixed 129/BALB/c background,
20% were significantly smaller than the control littermates
(P < .01) and had a life span not exceeding 10 days when
the reduced size was exaggeratedly marked; otherwise the size
retardation was no longer visible after 6 weeks of age. Analysis of
lymphoid organs in those mutant mice revealed a significant reduction
in thymic cellularity (average 108 cells in mutant mice as
compared to an average of 2.3 × 108 in wild-type
mice; Table 1), whereas bone marrow and
spleen were less affected (Figure 2A; Table
1). However, the mutant mice with no growth retardation had a normal
lymphocyte compartment.
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| Fig 2.
Phenotypic analysis of mAF4/ mice.
(A) Body weight (g), lymphoid cell content of spleen, bone marrow, and
thymus (×106) of 3-week-old mAF4 mutant ( ) and
control ( ) mice. (B) Absolute number of thymocyte subpopulations
from 3-week-old mAF4/ and wild-type mice (6 mice per
group) stained to reveal CD4 and CD8 distribution.
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Analysis of the thymocyte populations in mAF4/ mutant mice
To evaluate which thymic subpopulations were modified, in the
affected mutant mice, a flow cytometric analysis using conventional markers was performed. The most striking observation was a reduction in
the number of cortical CD4+CD8+ thymocytes
(Figure 2B; Table 1). In contrast, the number and the different subsets
of CD4CD8 precursor thymocytes are normal, indicating
that the seeding and the early cytokine-dependent expansion phase of
T-cell antigen receptor (TCR)-prothymocytes were not significantly
affected by the mAF4 mutation.22 Similarly, the presence of
mature CD4+ or CD8+ thymocytes indicates that
there is no block in thymocyte differentiation despite a 3-fold
reduction of CD4+ cells (Figures 2B and 3A; Table 1). This
point is further substantiated by the fact that these cells express
normal levels of TCR/CD3 molecules (Figure
3B) and based on the increased expression
of the H-2 class I major histocompatibility complex (MHC) marker (Figure 3C, gate R3) complete their maturation
program.23,24 These mature thymocytes colonize efficiently
peripheral lymphoid organs in appropriate proportions (data not shown).
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| Fig 3.
Flow cytometric analysis of thymocytes in 3-week-old mAF4
mutant mice.
(A) Distribution of the CD4 and CD8 markers in mutant and control mice.
(B) CD3+ cells (gate R1) on CD4 and CD8 thymocytes in
mutant versus control mice. Histograms are plotted as overlays of the
control thymocytes (black line) and AF4 mutant thymocytes (gray line).
(C) H-2Kb distribution on CD4 and CD8 cell populations. Gates are H-2Kb
low (R1), H-2Kb intermediate (R2), and H-2Kb high (R3). (D) CD69
distribution on CD4 and CD8 thymocytes. Data are displayed as dot plots
with log scale. Data are representative of 6 control mice and 6 mAF4/ mice.
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An important difference was a lowered expression (50%) of the CD4 and
CD8 coreceptor molecules on a fraction of double and single positive
thymocytes (Figure 3A), and this might suggest modifications in the
efficiency of T-cell selection or increased susceptibility to
apoptosis.25,26 This latter possibility seems unlikely
because cultured mAF4/ thymocytes show a normal rate of
spontaneous apoptotic cell death in vitro (data not shown). Then the
expression of the CD69 molecule was evaluated as a marker of positive
selection.27 As shown in Figure 3D, CD69 is normally expressed by DP thymocytes at the transition toward the SP stage because the preferential expression of 1 coreceptor is detectable on
most cells. Interestingly, the lowered CD4 (×5) and CD8
(×1.5) expression is observed on thymocytes undergoing positive
selection (Figure 3D, gate R2) and on terminally differentiated
thymocytes (Figure 3C, gate R3). In total, the effect of the AF4
mutation mainly concerns the immature CD4+ CD8+
H-2 low thymocyte population, which contains the cells undergoing positive selection (Figure 3C, gate R1). Thus, these results indicate that both expansion and selection might be affected in mutant animals.
Glucocorticoid treatments
To evaluate if the mutant mice had a normal rate of thymocyte
expansion, the potential of the DN cell population to reconstitute the
DP compartment was assessed.23 Nonaffected mutant and
control wild-type mice were treated with glucocorticoid, known to
eliminate the majority of DP but not DN thymocytes.28,29 In
a time-course experiment, the kinetics of reconstitution is clearly
retarded in mutant versus control mice. Over a 2-week period, first the number of thymocytes recovered per thymus increased up to
1.2 × 108 in wild-type and heterozygote samples
compared with an increase to an average of
0.6 × 108 in mAF4/ mice (Figure
4A). This reduction in cell number is accompanied by a marked retardation in maturation, although
representatives of all stages of thymocyte differentiation were
observed as seen with CD4 and CD8 markers. Indeed, FACS profiles at day
12 in mutant mice are similar to those observed at day 9 in
wild-type animals (Figure 4B). These results show that in mutant mice,
precursor cells can transit from the immature DN to the most mature SP
thymocytes, but generate far fewer T cells.
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| Fig 4.
AF4 is required for normal thymocyte expansion.
Thymus from mAF4/ glucocorticoid-treated mice show a
decrease in the reconstitution of the number of DP cells. (A) Kinetic
analysis of thymocyte reconstitution. T-cell counts after
glucocorticoids in mutant ( ) and control () mice over a 2-week
period. Data are representative of 6 control mice and 6 mutant mice
except when indicated. Two-tailed Student P value is shown,
where significant. **P < .05; ***P < .001. (B)
FACS analysis was performed on thymocytes of control and mutant mice at
day 0 (d0), day 9, and day 12 after the glucocorticoid injections as
indicated in "Materials and methods." T-cell maturation was
assayed using the CD4 and CD8 markers. Results shown are representative
of 6 separate experiments.
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Analysis of bone marrow hemopoiesis in AF4/ mutant
mice
A similar study was performed on the B-lymphocyte lineage in the
bone marrow. As shown in Figure 5A and D
the size of the B-cell compartment is reduced by 3-fold and this
diminution concerns the B220 intermediate population that contains
mainly the immature B cells.30 To refine which subset
of the B-cell progenitors is specifically affected, the pro-B and pre-B
cell compartments were analyzed using the CD43 maturation
marker.30 As shown in Figure 5B, the CD43+
B220+ pro-B cells are not affected in mutant mice. In
contrast B220+ CD43 or B220+
IgMlow pre-B cells and mature B220+
IgMhigh B cells are reduced without evidence of a block in
B-cell differentiation (Figure 5C-D; Table1). These results
suggest that the pre-B cell receptor-dependent expansion phase
(pre-BCR) is affected in mAF4 mutant mice. On the contrary, the
numbers of monocytes (CD11b+) and granulocytes
(Gr1+) were unaltered in the mutant bone marrow.
Moreover, all the other hematopoietic compartments were unaltered in
mAF4 mutant mice (data not shown).
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| Fig 5.
Analysis of bone marrow hemopoiesis.
(A) Representative flow cytometry analysis of bone marrow B cell.
Absolute number of B220+ cells in the bone marrow of mAF4
mutant mice and controls. Cell counts are given
(×106). (B) and (C) Representative flow cytometry
analysis of bone marrow B-cell-stained populations from 3-week-old
mAF4/ and wild-type mice; cell counts
(×106) are indicated in the corresponding quadrant.
Mature, immature, and transitional B cells are boxed. Staining was
performed with B220, IgM, and CD43. (D) Total B cell numbers in the
bone marrow of mAF4 mutant and wild-type mice. The data are the mean of
6 mAF4 null mice and of an equal number of wild-type littermates. The
relative SD is shown. For statistical analysis, see Table 1.
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Altogether our results demonstrate that the absence of the mAF4
gene specifically affects lymphoid development despite its large domain
of expression in the hematopoietic system.
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Discussion |
We generated mAF4-deficient mice and found that the
mAF4 gene is not essential for embryonic development. From
mAF4 heterozygous mice crosses, 25% of the mice observed at
birth were of the mAF4/ genotype indicating that the
mutants survive embryogenesis. In a 129/BALB/c mixed background 20% of
the mice showed a pronounced effect on growth and on lymphoid
development. This incomplete penetrance could be due to a partial
complementation by a related gene. Recently, a novel human gene,
LAF4, was isolated from a subtracted cDNA library that showed
strong sequence similarity to AF4. AF4 and LAF4
are homologous throughout their coding regions. Human
LAF4 hybridized with genes in mouse and chicken, showing that
this gene family has been highly conserved during vertebrate evolution.31 In mouse tissues, although the LAF4
and mAF4 genes are differentially expressed, the highest levels
for both of them are observed in lymphoid tissues. It is thus possible
that LAF4 could partially substitute for the mAF4 gene
function during lymphoid development.
During early T- and B-lymphoid maturation the expansion of immature
lymphocytes depends on the expression of a functional pre-T or pre-B
cell receptor. In mutant mice the prolymphoid cell population, which
precedes this stage, is not affected. In contrast all the
lymphoid-committed populations beyond that critical first checkpoint
are reduced in number, inferring that the mAF4 gene controls
this expansion phase. This analysis was refined in the case of T-cell
development. Two genetic checkpoints regulate thymocyte differentiation
and cell proliferation.32 The second checkpoint controls
the progression from CD4+CD8+ immature to
mature CD4+ or CD8+ thymocytes associated with
positive selection.33 In the absence of positive selection,
thymocytes undergo apoptosis. Our results indicate that the mAF4
mutation affects the size of the immature double-positive thymocyte
subset. We found no evidence that mAF4/ thymocytes had a
higher rate of cell death in vitro (data not shown); in addition, their
sensitivity to cell death-inducing factors such as
glucocorticoids28,34 is not modified in
hydrocortisone-treated mutant mice (Figure 4, day 0). Thus, the most
likely possibility would be that the mAF4 gene has a role in
the expansion or differentiation of early DP thymocytes. Our
experiments using glucocorticoid treatment on mutant mice reveal that
the DN population is affected in its potential to generate a normal
number of DP and SP thymocytes. Indeed, mAF4/ thymocytes
are depleted of CD4+CD8+
TCR/low thymocytes expressing low levels of H-2
molecules prior to any TCR-dependent selection step
(11 × 106 in mutant mice versus
50 × 106 in wild-type mice). Furthermore, we
observed a lowered expression of coreceptor molecules. The fact that DP
thymocytes express normal levels of the CD69 molecule and become mature
CD3high/ H-2high SP thymocytes suggests that
positive selection is operational in mAF4 mutant mice. However, the
threshold of selection, which is dependent on the expression of high
levels of CD4, CD8, and TCR molecules, might be
affected35,36; indeed, in mutant mice both CD4 and CD8
coreceptor molecules are down-regulated in CD69+ DP
thymocytes and in the fully selected SP thymocytes. Thus the absence of
the mAF4 gene might render thymocytes more susceptible to
TCR-dependent signal transduction. However, it has been shown using
Gal4-AF4 constructs that AF4 is a potent transactivator15; it is then also possible that mAF4 product controls directly the transcriptional rate of different target genes involved in
signal transduction including the coreceptor molecules in
T-lymphocyte development and similar molecules involved in
B-lymphocyte maturation.
The AF4/FEL5,37 gene was first identified in the
human by its involvement with Mll in the translocation t(4;11)(q21;q23) in acute mixed-lineage leukemia.5,38 The most frequent
reciprocal translocation found in infant leukemias is the
t(4;11)(q21;q23); it has a poor prognosis. The leukemic cells express
the stem cell marker CD34, HLA-DR, and a pro-B cell marker CD19 as well
as the myelomonocytic marker CD15.39,40 These
characteristics of the blast cells suggest that the t(4;11) occurs and
transforms a multipotential progenitor cell. The mechanism
by which the fusion protein is able to induce neoplasia is not
fully understood and 2 models are proposed. The first model
implies a gain of function of the MLL/AF4 chimeric protein
in which the DNA binding domain is fused to a transactivation
domain of AF4 altering the regulation of target genes.13
Some proposed that the truncation of Mll is sufficient to induce
neoplasia.41
A previous in vitro study demonstrated that an HRX-ENL fusion cDNA
transduced into a cell population enriched in hematopoietic stem cells
confers to the infected cells an enhanced potential to generate myeloid
colonies with primitive morphology, demonstrating a direct role for
HRX-ENL in the immortalization and leukemic transformation of a myeloid
progenitor and supporting a gain-of-function mechanism for
HRX-ENL-mediated leukemogenesis.13 Moreover, in other
experiments AF9 sequences were fused by homologous
recombination12 into the mouse Mll gene so that
expression of the Mll-AF9 fusion gene occurred from endogenous
Mll promoter, as in t(9;11) found in human leukemia. Chimeric mice
carrying the fusion gene developed tumors, which were also restricted
to acute myeloid leukemias. Those results demonstrate that the Mll-AF9
fusion protein is oncogenic predominantly in myeloid or
myeloid-committed cells despite the large domain of activity of the Mll
promoter and its demonstrated activity in B and T cells. Chimeric
mice expressing an Mll-LacZ fusion gene42
develop acute leukemia similar in phenotype to the Mll-AF9 fusion
mice.43 This could indicate that Mll-mediated oncogenesis is on the myeloid pathway by default.44 Then
the lymphoid leukemic phenotype may require an additional
specific fusion partner. Our results show that the mAF4 gene is
important for lymphoid cell development because in mAF4/
mice only the lymphoid compartment is affected although this gene is
expressed in the other hematopoietic cell populations. This emphasizes
that the partner gene in translocation-associated leukemias could be an
important factor for the lineage of the leukemic phenotype. However
t(4;11) positive acute lymphocytic leukemia with a T phenotype is
extremely rare. Alternatively, the timing of the Mll translocation during cell commitment could alone influence the lineage of the associated leukemia. Further studies on the function of AF9 or ENL and
creating an Mll-mAF4 fusion product in mice should clarify this point.
| |
Acknowledgments |
We thank C. Schiff for critical reading of the manuscript. The authors
are grateful to G. Warcollier and M. Pontier for helping with the
animal facility and to A. Boned for technical assistance.
| |
Footnotes |
Submitted December 14, 1999; accepted March 9, 2000.
Supported by grants from CNRS, ARC, and from the Ligue contre
le cancer. P.I. is supported by an ARC and an FRM fellowship.
Reprints: Malek Djabali, Centre d'immunologie INSERM-CNRS de
Marseille Luminy, Case 906, 13288 Marseille Cedex 9, France; e-mail:
djabali{at}ciml.univ-mrs.fr.
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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Abstract
Introduction