Article
The Rockefeller University Press $30.00
J. Exp. Med. 2017 Vol. 214 No. 3 793–814
https://doi.org/10.1084/jem.20160049
The Journal of Experimental Medicine
793
INT ROD UCT ION
Acute lymphoblastic leukemia (ALL) is the most common
childhood malignancy and the leading cause of childhood
cancer–related mortality (Smith et al., 2010; Hunger and
Mullighan, 2015). The majority of childhood ALL cases are of
the precursor B cell subtype of acute leukemia (pre–B ALL),
which arise upon transformation of developing progenitor
B cells in the BM. pre–B ALL is characterized by genomic
lesions, including the Philadelphia chromosome, a transloca-
tion involving the breakpoint cluster region (
BCR
) and the
ABL1
gene (Ph
+
or BCR-ABL1
+
; Wong and Witte, 2004). In
addition, loss-of-function mutations in hematopoietic tran-
scription factors occur in many pre–B ALL cases (Somasun-
daram et al., 2015). More than 80% of BCR-ABL1
+
pre–B
ALL harbor deletions or mutations in the
IKZF1
gene, en-
coding the zinc nger (ZnF) transcription factor Ikaros (Mul-
lighan et al., 2008). The majority of Ikaros lesions in pre–B
ALL involve an aberrant Rag-mediated deletion of the exons
encoding the DNA-binding ZnFs, resulting in expression of
a dominant-negative (DN) isoform called IK6 (Mullighan et
al., 2008). Furthermore, deletion or mutation of the
IKZF1
gene correlate with poor prognosis in other subgroups of
pre–B ALL, providing evidence that Ikaros is an important
tumor suppressor in pre–B ALL (Mullighan et al., 2009; van
der Veer et al., 2013). Despite an established tumor suppressor
role for Ikaros, it remains unclear how Ikaros functions as
a tumor suppressor, and insight into the mechanism of ac-
tion and its downstream target genes might aid in the de-
velopment of targeted therapies for treatment of aggressive
IKZF1
-mutated pre–B ALL.
Ikaros is an important regulator of hematopoiesis and
is required for B cell development, as demonstrated by the
lack of B cells in
Ikzf1
null
mice (Wang et al., 1996). Ikaros
tumor suppressor activity in the lymphoid lineage has been
demonstrated in mouse models, as
Ikzf1
mutant mice develop
spontaneous thymic lymphoma caused by activating Notch1
mutations in the developing precursor T cells (Winandy et
Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (
IKZF1
) is a hallmark of BCR-ABL1
+
precursor B cell acute lymphoblastic leukemia (pre–B ALL). However, the mechanisms by which Ikaros functions as a tumor
suppressor in pre–B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1
+
pre–B ALL together with
a new model of inducible expression of wild-type Ikaros in
IKZF1
mutant human BCR-ABL1
+
pre–B ALL. We performed inte-
grated genome-wide chromatin and expression analyses and identied Ikaros target genes in mouse and human BCR-ABL1
+
pre–B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic
depletion of different Ikaros targets, including
CTN ND1
and the early hematopoietic cell surface marker CD34, resulted in
reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper develop-
mental stage–specic expression of multiple genes through chromatin compaction at its target genes.
Genetic analysis of Ikaros target genes and tumor
suppressor function in BCR-ABL1
+
pre–B ALL
HildeSchjerven,
1
EtapongF.Ayongaba,
1,2
AliAghajanirefah,
1
JamiMcLaughlin,
3
DonghuiCheng,
4
HuiminGeng,
1
JosephR.Boyd,
6
LinnM.Eggesbø,
1,2
IdaLindeman,
1,2
JessicaL.Heath,
7,8
EugenePark,
1
OwenN.Witte,
3,4,5
StephenT.Smale,
3,4,5
SethFrietze,
9
* and MarkusMüschen
10
*
1
Department of Laboratory Medicine, University of California, San Francisco, CA 94143
2
Department of Biosciences, University of Oslo, 0316 Oslo, Norway
3
Department of Microbiology, Immunology, and Molecular Genetics,
4
Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and
5
Molecular
Biology Institute, University of California, Los Angeles, CA 90095
6
Department of Biochemistry and University of Vermont Cancer Center,
7
Department of Pediatrics,
8
Department of Biochemistry, and
9
Department of Medical
Laboratory and Radiation Science, University of Vermont, Burlington, VT 05405
10
Department of Systems Biology, Beckman Research Institute and City of Hope Comprehensive Cancer Center, Pasadena, CA 91016
© 2017 Schjerven et al. This article is distributed under the terms of an Attribution–Noncommercial–Share
Alike–No Mirror Sites license for the rst six months after the publication date (see http ://www .rupress .org
/terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial–
Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /).
*S. Frietze and M Müschen contributed equally to this paper.
Correspondence to Hilde Schjerven: Hilde.Schjerven@ucsf.edu; or Seth Frietze: Seth.
Frietze@med.uvm.edu
Present address for Linn M. Eggesbø and Ida Lindeman is Department of Immunol-
ogy, University of Oslo, 0424 Oslo, Norway
Present address for Eugene Park is Dept. of Haematology, University of Cambridge,
UK
Abbreviations used: ALL, acute lymphoblastic leukemia; COG, Children’s Oncology
Group; DN, dominant-negative; EMT, epithelial to mesenchymal transition; GMP,
granulocyte-macrophage progenitor; GSEA, gene set enrichment analysis; HSC,
hematopoietic stem cell; IPA, Ingenuity Pathway Analysis; MRD, minimal residual
disease; OS, overall survival; Ph, Philadelphia chromosome; pre-B ALL, progenitor
B-cell ALL; RFS, relapse-free survival; TKI, tyrosine kinase inhibitor; TSS, transcription
start sites; ZnF, zinc nger.
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Ikaros tumor suppressor function in Ph
+
pre–B ALL | Schjerven et al.794
al., 1995; Papathanasiou et al., 2003; Dumortier et al., 2006).
Although the complete lack of B lymphoid lineage cells in
the original germline
Ikzf1
null
and
Ikzf1
DN
mice precluded
the use of these homozygote mutants as models for studies of
B cell malignancies, a heterozygote hypomorphic
Ikzf1
L
mu-
tant was shown to collaborate with a BCR-ABL1-Tg mouse
model, indicating that the loss of Ikaros in human BCR-
ABL1
+
pre–B ALL can be modeled in mice (Georgopoulos
et al., 1994; Wang et al., 1996; Virely et al., 2010).
Recently, we reported the generation of two
Ikzf1
mu-
tant mice with targeted deletions of the exons encoding the
DNA-binding ZnF1 or ZnF4 (
Ikzf1
Δ
F1/
Δ
F1
and
Ikzf1
Δ
F4/
Δ
F4
,
respectively), and found that whereas the exon encoding
ZnF1 was expendable, the exon encoding ZnF4 was required
for Ikaros tumor suppressor function (Schjerven et al., 2013).
Furthermore, both mutant strains of mice retained B cell
lymphopoiesis, but displayed dierent partial defects in B cell
development associated with deregulation of distinct subsets
of genes, suggesting that they can be useful in elucidating
Ikaros function and target genes. Upon retroviral transduc-
tion of BCR-ABL1 in BM cells from these mice,
Ikzf1
Δ
F4/
Δ
F4
mutant cells gave rise to a more aggressive growth pheno-
type than either WT or
Ikzf1
Δ
F1/
Δ
F1
BCR-ABL1–transformed
pre–B ALL cells. This demonstrated selective ZnF4-dependent
loss of Ikaros tumor suppressor function and presents a new
mouse model of BCR-ABL1
+
pre–B ALL.
Herein, we use the
Ikzf1
Δ
F4/
Δ
F4
mutant mouse strain to
gain insight into the functional consequence of loss of Ikaros
tumor suppression in BCR-ABL1
+
pre–B ALL. Further-
more, we present a new model of inducible expression of
WT Ikaros in
IKZF1
mutant human patient–derived BCR-
ABL1
+
pre–B ALL. We have dened the Ikaros target genes
in human BCR-ABL1
+
pre–B ALL and compared this to
the
Ikzf1
Δ
F4/
Δ
F4
mouse model of BCR-ABL1
+
pre–B ALL to
uncover conserved functions of Ikaros. Our analysis reveals
new target genes and pathways not previously associated with
Ikaros function. Specically, we found that repression of key
developmentally restricted cell surface receptors, as well as
the intracellular protein p120-catenin, are conserved func-
tions of Ikaros that restrict leukemic growth. Overall, these
results further our understanding of how Ikaros functions as
a tumor suppressor and dene downstream targets of Ikaros
that promote leukemic growth.
RES ULTS
Targeted deletion of the fourth Ikaros DNA-binding ZnF
domain in a mouse model of BCR-ABL1
+
pre–B ALL results
in enhanced cell proliferation
BCR-ABL1–transduced pre–B cells from
Ikzf1
Δ
F4/
Δ
F4
mice
lacking the exon encoding the fourth Ikaros ZnF domain
(ZnF4) exhibited an increased growth rate relative to trans-
duced pre–B cells from WT or
Ikzf1
Δ
F1/
Δ
F1
mice lacking the
exon encoding the rst ZnF domain (ZnF1; Schjerven et
al., 2013). To investigate the cellular process underlying the
increased growth rate, we examined cell cycle status and
apoptosis by ow cytometry analysis and found that BCR-
ABL1–transduced
Ikzf1
Δ
F4/
Δ
F4
pre–B ALL cultures had a con-
sistently increased fraction of cells in S/G2-M as compared
with WT and
Ikzf1
Δ
F1/
Δ
F1
cultures (Fig.1 A). However, we
did not see any evidence of increased growth caused by re-
duced apoptosis in
Ikzf1
Δ
F4/
Δ
F4
cultures. The increased frac-
tion of cells engaged in active cell cycle corresponded to an
increase in the protein and mRNA levels of the cell cycle
regulators Cyclin D1 and Cdk6, and reduced levels of the
negative cell cycle regulator p21 (Fig.1, B and C). In contrast,
the negative cell cycle regulator p16 was selectively induced
in
Ikzf1
Δ
F1/
Δ
F1
cultures (Fig.1, B and C), corresponding to
the observed reduced rate of growth and senescence of the
Ikzf1
Δ
F1/
Δ
F1
cultures.
Ikzf1
Δ
F4/
Δ
F4
BCR-ABL1
+
cells give more
aggressive leukemia in vivo
To examine the leukemic potential of the
Ikzf1
Δ
F4/
Δ
F4
-
mutated model of BCR-ABL1
+
pre–B ALL, we performed
i.v. transplantation of BCR-ABL1
+
pre–B ALL cells into non-
irradiated immunocompetent WT-recipient mice (Williams
et al., 2006). Whole BM from WT or
Ikzf1
Δ
F4/
Δ
F4
mice were
transduced with BCR-ABL1, and cultured in vitro for 7 d to
allow for expansion of BCR-ABL1–transformed pre–B ALL
cells before i.v. injection (Fig. 1 D). Transplantation of 10
5
BCR-ABL1–transformed cells was sucient to give rise to
leukemia with both WT and
Ikzf1
Δ
F4/
Δ
F4
donor cells (Fig.1E,
left), but mice that received
Ikzf1
Δ
F4/
Δ
F4
BCR-ABL1
+
cells
developed a more aggressive leukemia and had a shorter lifes-
pan than those that received WT BCR-ABL1
+
cells. Further-
more, whereas 10
4
WT BCR-ABL1
+
cells were not sucient
to induce leukemia, 10
4
Ikzf1
Δ
F4/
Δ
F4
BCR-ABL1
+
cells were
sucient to induce aggressive leukemia at 100% penetrance
(Fig.1E, right). This indicates that loss of Ikaros tumor sup-
pression in the
Ikzf1
Δ
F4/
Δ
F4
mutant leads to a higher frequency
of cells that can initiate leukemia upon transplantation to a
nonirradiated immunocompetent host.
Loss of Ikaros tumor suppressor function corresponds
to a less mature cell surface phenotype, but does not block
successful IgH V(D)J recombination
Previous phenotypic analysis had revealed an aberrant ex-
pression of c-Kit and lack of CD25 expression on BCR-
ABL1–transformed
Ikzf1
Δ
F4/
Δ
F4
cells (Schjerven et al., 2013).
We found that this was also accompanied by a higher ex-
pression level of CD43 than on WT and
Ikzf1
Δ
F1/
Δ
F1
cells
(Fig. 2 A). Together, this suggests that the BCR-ABL1–
transformed
Ikzf1
Δ
F4/
Δ
F4
cells are at a less mature stage of
B cell development than WT and
Ikzf1
Δ
F1/
Δ
F1
(Hardy et al.,
1991; Rolink et al., 1994). Because Ikaros has been reported
to be required for
Rag1
and
Rag2
expression as well as IgH
recombination (Reynaud et al., 2008), we tested the ex-
pression of
Rag1
and
Rag2
by RT-qPCR and found that
Ikzf1
Δ
F1/
Δ
F1
and
Ikzf1
Δ
F4/
Δ
F4
cells expressed
Rag1
and
Rag2
mRNA at higher levels than WT cells (Fig. 2B). We also
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795JEM Vol. 214, No. 3
found that BCR-ABL1–transformed
Ikzf1
Δ
F4/
Δ
F4
cells were
able to undergo IgH V(D)J recombination to the same ex-
tent as BCR-ABL1–transformed WT and
Ikzf1
Δ
F1/
Δ
F1
cells
(Fig.2C), thus demonstrating that the cells are not defective
in Rag-mediated recombination of IgH. Furthermore, we
analyzed the levels of intracellular μ chain by ow cytom-
etry analysis, and found that WT, as well as both mutants,
were able to express cytoplasmic μ (Fig.2, D and E). These
results indicate that
Ikzf1
Δ
F4/
Δ
F4
pre–B ALL cells can develop
to the stage of successful IgH recombination and expression
of cytoplasmic μ, but fail to down-regulate c-Kit and in-
duce expression of CD25.
Figure 1. Loss of Ikaros tumor suppression in
Ikzf1
∆F4/∆F4
mutant mice results in an increase
in active cell cycle and aggressive leukemia
in a nonirradiated in vivo model. (A–C) BM
from WT,
Ikzf1
Δ
F1/
Δ
F1
, and
Ikzf1
Δ
F4/
Δ
F4
mutant mice
were transduced with BCR-ABL1-p185-IRES-YFP
and grown in vitro on BM stroma–derived feeder
layers. (A) Cell cycle ow cytometry analysis was
performed by Hoechst incorporation, and one rep-
resentative experiment (at day 14 of cell culture) is
shown. (B) Cells were harvested on different days
of in vitro cell culture and protein was extracted
for Western blot analysis of Cyclin D1, Cdk6, p16,
and p21, with ERK as loading control. Space in-
dicates that intervening lanes have been spliced
out. (C) RNA was isolated from the in vitro cul-
tures, cDNA was prepared, and mRNA for
Ccnd1
,
Cdk6
,
p16
, and
p21
was analyzed by quantitative
real-time PCR.
Ubc
was used as a reference gene.
(D) Schematic of the experimental set up for the in
vivo transplantation assay. BM cells from WT and
Ikzf1
Δ
F4/
Δ
F4
mutant mice were transduced and cul-
tured in vitro as in A–C for 7 d before i.v. injection
into nonirradiated immunocompetent WT c57BL/6
recipient mice. Animals were monitored and eu-
thanized upon signs of leukemia development.
(E) Kaplan-Meyer curve of cohorts receiving 10
5
(left) or 10
4
(right) BCR-ABL1–transformed cells.
(A–C) are representative of three independent ex-
periments and (E) represents one experiment with
ve recipient mice for each of the four cohorts.
The result in right panel was repeated in a separate
independent experiment displayed in Fig.6F.
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Ikaros tumor suppressor function in Ph
+
pre–B ALL | Schjerven et al.
796
Figure 2.
Ikzf1
∆F4/∆F4
mutant BCR-ABL1
+
cells display immature cell surface phenotype but undergo successful IgH V(D)J recombination, and
increased cell cycle correlates with CD43 expression. (A) Flow cytometry analysis of CD43 on in vitro grown BCR-ABL1–transformed WT,
Ikzf1
Δ
F1/
Δ
F1
, and
Ikzf1
Δ
F4/
Δ
F4
pre–B ALL cells. (B) Quantitative RT-PCR analysis of
Rag1
and
Rag2
mRNA expression on in vitro grown BCR-ABL1–transformed WT,
Ikzf1
Δ
F1/
Δ
F1
,
and
Ikzf1
Δ
F4/
Δ
F4
pre–B ALL cells.
UBC
was used as a reference gene. (C) IgH V(D)J recombination was analyzed by PCR (Schlissel et al., 1991) with genomic DNA
from BCR-ABL1–transformed WT,
Ikzf1
Δ
F1/
Δ
F1
, and
Ikzf1
Δ
F4/
Δ
F4
cells, and genomic DNA from WT tail and spleen was used as negative and positive controls,
respectively. (D and E) Expression of cytoplasmic μ chain was analyzed by ow cytometry. Whole mouse BM was used as positive control. One representative
is shown in (D) and results from multiple samples analyzed in independent experiments are summarized in E as fraction of cells expressing cytoplasmic
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797JEM Vol. 214, No. 3
Correlation between cell cycle prole and the
developmentally regulated cell surface marker CD43
Given the less mature cell surface phenotype of the
Ikzf1
Δ
F4/
Δ
F4
pre–B ALL cells, we hypothesized that BCR-ABL1–trans-
formed
Ikzf1
Δ
F4/
Δ
F4
cells are maintained at a highly prolifer-
ative stage of B cell development (C' or transitional pre–BI/
large pre–BII; Hardy et al., 1991; Rolink et al., 1994; Mårtens-
son et al., 2010), with the failure of repressing key genes from
the earlier progenitor stages (such as c-Kit). In contrast, WT
and
Ikzf1
Δ
F1/
Δ
F1
cells are able (at a certain rate) to mature be-
yond this proliferative stage, express CD25, and exit cell cycle,
thus limiting the aggressiveness of growth. This model is in
agreement with reports that Ikaros is required for the nor-
mal progenitor B cell dierentiation at this point and pre–
BCR-mediated cell cycle exit, as well as earlier reports that
Ikaros is required to turn o the gene expression programs
for self-renewal and multipotency (Ng et al., 2009; Trageser et
al., 2009; Ferreirós-Vidal et al., 2013; Heizmann et al., 2013).
To test if proliferation is linked to developmental cell
surface markers, we analyzed the cell cycle prole of vari-
ous sub-populations in the BCR-ABL1–transformed in vitro
cultures. We found that CD43 expression correlated well
with cell cycle in all three genetic backgrounds (Fig.2F).
CD43
+High
cells had a higher percentage of cells in S/G2-M
phase, whereas cells with CD43
+low
expression had the lowest
percentage of cells in S/G2-M phase (Fig. 2 F). Although
CD43 could merely be a marker for these more highly pro-
liferative cells, this observation is also consistent with the re-
ported growth-promoting function of CD43 (Balikova et al.,
2012). Collectively, the loss of tumor suppressor function in
Ikzf1
ΔF4/ΔF4
leukemia cells correlated with a failure to repress
the expression of progenitor cell surface markers, resulting in
a less mature cell surface phenotype as compared with WT
and Ikzf1
ΔF1/ΔF1
leukemia cells.
Expression proling of
Ikzf1
Δ
F4/
Δ
F4
BCR-ABL1–transformed
cells reveals Ikaros target genes relevant
for tumor suppressor function
To investigate the changes in gene expression associated with
the increased proliferative growth and loss of tumor sup-
pression, we compared global expression proles of replicate
BCR-ABL1–transduced WT,
Ikzf1
Δ
F1/
Δ
F1
and
Ikzf1
Δ
F4/
Δ
F4
mutant cell cultures from independent experiments at 14 d
after transduction. Clustering of dierentially expressed genes
demonstrated that the
Ikzf1
Δ
F4/
Δ
F4
mutant pre–B ALL expres-
sion patterns were distinct from the WT and
Ikzf1
Δ
F1/
Δ
F1
pro-
les, and also revealed a cluster of genes that were selectively
up-regulated in
Ikzf1
Δ
F1/
Δ
F1
cells compared with either WT or
Ikzf1
Δ
F4/
Δ
F4
mutant cells (Fig.3A; cluster 1). As the
Ikzf1
Δ
F1/
Δ
F1
cells retain tumor suppressor function, the cluster of dif-
ferentially expressed genes specic for
Ikzf1
Δ
F1/
Δ
F1
mutant
cells are not expected to be tumor suppressor target genes,
whereas clusters of genes deregulated in
Ikzf1
Δ
F4/
Δ
F4
mutant
cells represent candidate genes regulated by Ikaros tumor sup-
pressor function. This selective ZnF-dependent gene regula-
tion underscores the utility of ZnF mutant mice to dene
the subset of Ikaros-regulated genes associated with Ikaros
tumor suppressor function.
To investigate the tumor suppressor function of Ikaros,
we examined genes that were dierentially expressed in
Ikzf1
ΔF4/ΔF4
mutant cells as compared with WT cells, and
found that 365 and 414 genes were up- and down-regulated,
respectively (P < 0.005; fold change ≥ 2; Fig.3, B and C).
To identify potential direct regulatory target genes of a tran-
scription factor, a common analysis is to evaluate the binding
sites within a given distance to gene transcription start sites
(TSS; Wang et al., 2013). We found that 68 and 62% of the
Ikzf1
Δ
F4/
Δ
F4
down- and up-regulated genes, respectively, were
bound by Ikaros in mouse BM progenitor B cells within 100
kb of the gene TSS (Bossen et al., 2015; Fig.3C). We noted
that Ikaros was not enriched at deregulated genes compared
with all expressed genes, and this was the case whether ana-
lyzing the aforementioned dataset in isolation, or when using
a high-condence dataset of Ikaros peaks obtained by analy-
sis of datasets from three dierent laboratories (Fig.3D and
Fig. S1; Ferreirós-Vidal et al., 2013; Schwickert et al., 2014;
Bossen et al., 2015). However, further analysis by integrat-
ing dierential gene expression between the
Ikzf1
Δ
F4/
Δ
F4
and
WT pre–B ALL cells with Ikaros ChIP-Seq data using BETA
(Wang et al., 2013), predicted a signicant repressive function
for WT Ikaros for genes that were up-regulated in
Ikzf1
Δ
F4/
Δ
F4
BCR-ABL1
+
cells compared with WT BCR-ABL1
+
cells (P
= 0.0003; Fig.3E). This supports the transcriptional repressor
function of Ikaros, and although it still remains dicult to
distinguish direct from indirect Ikaros targets, we nevertheless
note that several of the Ikaros-dependent genes dened in
this study contain Ikaros binding sites in proximity of the
gene loci, including at gene promoters (Fig.3F).
Deregulated signaling pathways in
Ikzf1
Δ
F4/
Δ
F4
leukemic cells
Consistent with recent ndings from both progenitor T cells
(thymocytes) and progenitor B cells in
Ikzf1
mutant mouse
models (Schjerven et al., 2013; Joshi et al., 2014; Schwickert
et al., 2014; Churchman et al., 2015), adhesion was one of the
most signicantly enriched pathways in
Ikzf1
Δ
F4/
Δ
F4
compared
with WT pre–B ALL cells (Fig.3G). In agreement with our
phenotypic analysis of
Ikzf1
Δ
F4/
Δ
F4
mutant cells demonstrating
the failure to down-regulate the cell surface markers c-Kit
and CD43, the deregulated genes in
Ikzf1
Δ
F4/
Δ
F4
mutant cells
were enriched for genetic pathways involving cell surface re-
μ (% of live; E). (F) Flow cytometry analysis of CD43 and cell cycle analysis on in vitro cultures of BCR-ABL1–transformed WT,
Ikzf1
Δ
F1/
Δ
F1
, and
Ikzf1
Δ
F4/
Δ
F4
cells (gated on YFP
+
), displaying cell cycle activity of cells gated on low, medium, or high expression of CD43, respectively. All results are reproduced in two
or more independent experiments.
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