Defective T cell development and function in the absence of Abelson kinases.
ABSTRACT Thymocyte proliferation, survival, and differentiation are tightly controlled by signaling from the pre-TCR. In this study, we show for the first time that the Abelson (Abl) kinases regulate proximal signaling downstream of the pre-TCR. Conditional deletion of Abl kinases in thymocytes reveals a cell-autonomous role for these proteins in T cell development. The conditional knockout mice have reduced numbers of thymocytes, exhibit an increase in the percentage of the CD4(-)CD8(-) double-negative population, and are partially blocked in the transition to the CD4(+)CD8(+) double-positive stage. Moreover, the total number of T cells is greatly reduced in the Abl mutant mice, and the null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine production. Notably, Abl mutant mice are compromised in their ability to produce IFN-positive CD8 T cells and exhibit impaired CD8(+) T cell expansion in vivo upon Listeria monocytogenes infection. Furthermore, Ab production in response to T cell-dependent Ag is severely impaired in the Abl mutant mice. Together these findings reveal cell-autonomous roles for the Abl family kinases in both T cell development and mature T cell function, and show that loss of these kinases specifically in T cells results in compromised immunity.
- SourceAvailable from: Thomas Harding[Show abstract] [Hide abstract]
ABSTRACT: Chronic myeloid leukemia (CML) can be responsive to T-cell-mediated immunity. K562/granulocyte macrophage-colony stimulating factor (GM-CSF) is a GM-CSF producing vaccine derived from a CML cell line that expresses several CML-associated antigens. A pilot study was developed to determine if K562/GM-CSF immunotherapy could improve clinical responses to imatinib mesylate (IM) in patients with chronic myeloid leukemia. Patients with chronic phase CML who achieved at least a major cytogeneic response but remained with persistent, measurable disease despite one or more years on imatinib mesylate were eligible. Each was given a series of four vaccines administered in three-week intervals, with or without topical imiquimod, while remaining on a stable dose of imatinib mesylate. CML disease burden was measured serially before and after vaccination. Nineteen patients were vaccinated, with a median duration of previous imatinib mesylate therapy of 37 (13-53) months. Mean PCR measurements of BCR-ABL for the group declined significantly following the vaccines (P = 0.03). Thirteen patients had a progressive decline in disease burden, 8 of whom had increasing disease burden before vaccination. Twelve patients achieved their lowest tumor burden measurements to date following vaccine, including seven subjects who became PCR-undetectable. K562/GM-CSF vaccine appears to improve molecular responses in patients on imatinib mesylate, including achieving complete molecular remissions, despite long durations of previous imatinib mesylate therapy.Clinical Cancer Research 01/2010; 16(1):338-47. · 7.84 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design.Journal of Medicinal Chemistry 03/2011; 54(7):2359-67. · 5.61 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In the previous paper of ours we compared, prior to start any treatment, a number of immunological parameters in 24 chronic myeloid leukemia patients with the same number of healthy subjects matched by age and sex. We found significant differences in the levels of immunoglobulins, the C4 component of complement, the C-reactive protein, interleukin 6, the composition of lymphocyte population and the production of some cytokines by stimulated CD3+ cells. Eleven of these patients were followed longitudinally. After treatment with hydroxyurea, interferon alpha, imatinib mesylate and dasatinib, or various combinations thereof, hematological remission was achieved in all patients and complete cytogenetic remission in nine of them. There was a nearly general tendency towards normalization of the abnormalities observed in the patients at their enrollment.Clinical and Developmental Immunology 01/2010; 2010:137320. · 3.06 Impact Factor
Defective T Cell Development and Function in the Absence of
Jing Jin Gu,* Nu Zhang,†You-Wen He,†Anthony J. Koleske,‡and Ann Marie Pendergast2*
Thymocyte proliferation, survival, and differentiation are tightly controlled by signaling from the pre-TCR. In this study, we show
for the first time that the Abelson (Abl) kinases regulate proximal signaling downstream of the pre-TCR. Conditional deletion of
Abl kinases in thymocytes reveals a cell-autonomous role for these proteins in T cell development. The conditional knockout mice
have reduced numbers of thymocytes, exhibit an increase in the percentage of the CD4?CD8?double-negative population, and
are partially blocked in the transition to the CD4?CD8?double-positive stage. Moreover, the total number of T cells is greatly
reduced in the Abl mutant mice, and the null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine pro-
duction. Notably, Abl mutant mice are compromised in their ability to produce IFN-positive CD8 T cells and exhibit impaired
CD8?T cell expansion in vivo upon Listeria monocytogenes infection. Furthermore, Ab production in response to T cell-dependent
Ag is severely impaired in the Abl mutant mice. Together these findings reveal cell-autonomous roles for the Abl family kinases
in both T cell development and mature T cell function, and show that loss of these kinases specifically in T cells results in
compromised immunity. The Journal of Immunology, 2007, 179: 7334–7343.
mental program that is regulated by signals downstream of the
pre-TCR, and subsequently the TCR (1, 2). Immature thymocytes
lack expression of the CD4 and CD8 cell surface receptors, and
proceed from the CD4?CD8?double-negative (DN)3stage
through a CD4?CD8?double-positive (DP) stage to become mature
CD4?or CD8?single-positive (SP) T cells. The transition from
DN into DP cells is regulated by signaling through the pre-TCR,
whereas differentiation from DP cells to mature T cells requires a
functional TCR?? (2, 3). A subset of protein tyrosine kinases has
been shown to play pivotal roles as proximal signaling molecules
downstream of both the pre-TCR and the TCR??. Mice deficient
for Lck, a member of the Src family of tyrosine kinases, show a
partial block in thymocyte development at the DN stage and lack
mature T cells (4). Mice deficient for both the Syk and ZAP70
tyrosine kinases fail to initiate clonal expansion of DN cells,
which fail to differentiate into DP cells (5). In addition to these
tyrosine kinases, adaptor proteins such as linker for activation
of T cells (LAT), SH2-domain-containing leukocyte protein of
uccessful T cell development in the thymus is critical for
both cellular and humoral immunity. Bone marrow-de-
rived prothymocytes undergo a tightly controlled develop-
76 kDa, and Shc have been shown to contribute to pre-TCR
signaling and regulate T cell development (6–9).
The Abelson (Abl) kinases, Abl (Abl1) and Arg (Abl2), are
highly regulated nonreceptor tyrosine kinases that transduce sig-
nals downstream of receptor tyrosine kinases and other receptors
(10, 11). Abl kinases have been shown to regulate cell prolifera-
tion, migration, and survival (12), and altered forms of the Abl
kinases produced from chromosomal translocation events, such as
Bcr-Abl and Tel-Arg, are implicated in the development of some
human leukemias (13). We have previously demonstrated that en-
dogenous Abl tyrosine kinase activity is elevated following acti-
vation of the TCR, and that inhibition of Abl kinase activity or
partial loss of Abl and Arg proteins results in down-regulation of
mature TCR-induced signaling in vitro (14). Whether the Abl fam-
ily kinases regulate pre-TCR and TCR signaling in vivo, thereby
affecting T cell development and function, remained to be
Both Abl and Arg are expressed throughout embryonic devel-
opment and in the adult (15). Expression of Abl is higher in the
thymus, spleen, and testes compared with other tissues in postnatal
mice (16), and Arg expression levels are highest in the brain, fol-
lowed by thymus and spleen (17). Single deletion of Abl (Abl1) in
mice results in severe immune system dysfunction, including
splenic and thymic atrophy, lymphopenia, and increased suscepti-
bility to infection (18, 19). However, the cellular and molecular
basis for the immune phenotypes observed in the Abl1 single-
knockout mice have remained elusive, and have been compounded
by the pleiotropic roles of the Abl kinases in the regulation of
multiple cell types, including B cells and stromal cells (11, 12, 18,
19). In contrast to the Abl1 knockout mice, Arg-deficient mice do
not display obvious abnormalities in the thymus and spleen (17).
Notably, whereas mice lacking either abl1 or abl2 can survive to
adulthood, mice knockout for both abl1 and abl2 genes die during
embryogenesis, thereby suggesting that these kinases share over-
lapping functions during embryonic development (17).
To study whether Abl kinases are involved in T cell develop-
ment and dissect the role of the Abl kinases in T cell function in
vivo, we have conditionally inactivated the Abl kinases in T cells
*Department of Pharmacology and Cancer Biology, and†Department of Immunology,
Duke University Medical Center, Durham, NC 27710; and‡Department of Molecular
Biophysics and Biochemistry, and Department of Neurobiology, Yale University, New
Haven, CT 06520
Received for publication May 25, 2007. Accepted for publication September
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI056266 (to
A.M.P.) and CA92123 (to Y.-W.H.), and National Institute of Neurological Disorders
and Stroke Grant NS39475 (to A.J.K.).
2Address correspondence and reprint requests to Dr. Ann Marie Pendergast, Depart-
ment of Pharmacology and Cancer Biology, Duke University Medical Center, Box
3813, Durham, NC 27710. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: DN, double negative; Abl, Abelson; DP, double
positive; LAT, linker for activation of T cells; NP, nitrophenylacetyl; PI, propidium
iodide; PLC, phospholipase C; SP, single positive.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by crossing the Lck-Cre transgenic mice with mice carrying loxP-
flanked abl1 sequences in the abl2?/?background. In this study,
we report that Abl/Arg-conditional knockout mice have impaired
thymocyte development and show for the first time that Abl kinase
activity is required for pre-TCR signaling. We have found that
specific deletion of Abl family kinases in mature T cells results in
impaired proliferation in response to TCR stimulation, as well as
reduced IL-2 and IFN-? production. Significantly, Abl/Arg-con-
ditional knockout mice are compromised to mount a humoral im-
mune response to T cell-dependent Ag in vivo, and are severely
impaired in the production of CTL during Listeria monocytogenes
infection. Together these findings reveal an important role for Abl
family kinases in T cell development and T cell function in vitro
and in vivo.
Materials and Methods
Generation of Abl/Arg T cell-conditional knockout mice
The Ablfloxmice were generated, as previously described (20), and were
crossed into the arg?/?background to generate abl flox/flox arg?/?mice.
These mice were subsequently crossed with the Lck-Cre transgenic mice
(Taconic Farms) to generate conditional loss of Abl kinases in the T cell
lineage. Genotypes were confirmed by PCR. All mice were in the C57BL/6
genetic background. Mice were housed under specific pathogen-free con-
ditions in the Duke University Cancer Center Isolation Facility. All studies
using mice have followed the protocols reviewed and approved by Duke
Institutional Animal Care & Use Committee.
Cell culture and Abs
Primary T cells purified from mouse spleen were cultured in RPMI 1640
medium supplemented with 10% heat-inactivated FBS, 55 ?M 2-ME, and
100 ?g/ml each penicillin and streptomycin. The pre-TCR SL-12?12 cell
line (gift from D. Wiest, Fox Chase Cancer Center, Philadelphia, PA) was
maintained in DMEM supplemented with 10% heat-inactivated FBS, 10
mM HEPES, 1 mM sodium pyruvate, 55 ?M 2-ME, 0.1% gentamicin, and
0.5 mg/ml geneticin. Anti-Abl Ab specific for the Abl kinase domain
(clone 8E9) was obtained from BD Pharmingen, and anti-Abl Ab specific
for the Abl C-terminal region (Ab-3) was obtained from Calbiochem.
Flow cytometric analysis and cell staining
Thymocytes or splenocytes in single-cell suspension were lysed of RBC
and washed in FACS buffer (PBS containing 2% heat-inactivated FBS,
0.05% sodium azide). For surface staining, ?1 ? 106cells were first in-
cubated 5 min with anti-CD16/CD32 mouse Fc block (BD Pharmingen)
and followed by staining with FITC-, PE-, PE-CY5-labeled anti-CD4,
CD8, B220, TCR?, CD44, and CD25 (BD Pharmingen; eBioscience) on
ice for 30 min. Cells were washed twice, collected on a FACScan flow
cytometer, and analyzed using CellQuest software (BD Biosciences). For
BrdU staining, 50 ?M BrdU was added at the end of T cell activation for
30 min. After fixation in EtOH, cells were then treated with 0.08% pepsin
in 0.1 N HCl to isolate nuclei, followed by denaturing DNA with 2 N HCl
and neutralizing with 0.1 M sodium borate (pH 8.5). After wash, cells were
stained with FITC anti-BrdU Ab (BD Pharmingen) and propidium iodide
(PI; Sigma-Aldrich) (10 ?g/ml) in the presence of RNase A (250 ?g/ml) in
staining buffer (10 mM HEPES (pH 7.5), 150 mM NaCl, 4% FBS, 0.5%
Tween 20, and 0.1% sodium azide).
T cell proliferation and cytokine ELISA
T cells were purified from total splenocytes using Pan T Cell Isolation Kit
with LS columns (Miltenyi Biotec). Approximately 2 ? 105T cells in 100
?l were plated in triplicates into 96-well plate precoated with anti-CD3
(clone145-2C11; BD Pharmingen) or anti-CD3 plus anti-CD28 Abs (clone
37.51; BD Pharmingen), incubated in a 37°C 5% CO2incubator for 48 h.
[3H]Thymidine (PerkinElmer Life & Analytical Sciences) (1 ?Ci/well)
was added during the last 16–20 h, and cells were harvested using a scin-
tillation counter (PerkinElmer Life & Analytical Sciences). For MLR,
splenocytes from DBA mice (H-2d) were first treated with 50 ?g/ml mit-
omycin C (Sigma-Aldrich) and mixed with splenocytes isolated from either
wild-type or Abl/Arg-conditional null mice at E:T ? 1:2.5 for 72 h;
[3H]thymidine was added during the last 16 h. For cytokine production, T
cells were stimulated as for the proliferation assay, and culture supernatant
was collected at 24 h. Levels of IL-2, IL-4, and IFN-? were measured using
DuoSet ELISA kits for mouse IL-2, IL-4, and IFN-? (R&D Systems).
In vitro Cre-mediated Abl deletion
Splenocytes isolated from either Ablflox/flox/Arg?/?or Ablflox/flox/Arg?/?,
lck-Cre-negative mice were stimulated with anti-CD3 Ab (5 ?g/ml) for
24 h. Cells were then retrovirally transduced with either vector-encoding
GFP alone or Cre-GFP-containing vector in the presence of mouse rIL-2
(10 ng/ml; R&D Systems) and 8 ?g/ml polybrene (Sigma-Aldrich). GFP-
positive cells were sorted 3 days after infection using FACS and were used
for experiments. For proliferation, 5 ? 104cells were cultured in the pres-
ence of mouse rIL-2 for 24 h, and [3H]thymidine (1 ?Ci/well) was added
during the last 12 h. For IFN-? secretion, cells were restimulated with
anti-CD3, and culture supernatant was collected between 3 and 8 h.
For analyzing TCR-induced apoptosis, purified T cells were stimulated
with plate-bound anti-CD3 for 48 h, and stained with annexin V and 7-ami-
noactinomycin D using Annexin V-PE Apoptosis Detection Kit (BD
Pharmingen). For caspase 3 activation, purified T cells were plated onto
96-well plate precoated with anti-CD3 (5 ?g/ml) Ab for the indicated
times. Cell lysates were then analyzed using both anti-caspase 3 and anti-
cleaved caspase 3 Abs (Cell Signaling Technology).
TCR activation and Western blot analysis
For pre-TCR activation, SL-12?12 cells in DMEM plus 0.1% BSA were
incubated with or without imatinib (5 ?M) for 1 h at 37°C. Cells were then
incubated with biotinylated anti-TCR? Ab (10 ?g/ml; BD Pharmingen) on
ice for 30 min. After wash, streptavidin (25 ?g/ml; Sigma-Aldrich) was
added to each sample, incubated at 37°C for the indicated time, and
stopped by adding 1 ml of cold PBS. Cells were lysed in lysis buffer
containing 50 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 150 mM NaCl,
1 mM EDTA, 1 mM sodium orthovanadate (Na3VO4), and 1? proteinase
and phosphatase inhibitors (Sigma-Aldrich). Purified splenic T cells were
plated onto 24-well plate precoated with anti-CD3 (5 ?g/ml) Ab at 37°C
for the indicated time and stopped by adding 5? radioimmunoprecipitation
assay buffer (1? buffer: 50 mM HEPES (pH 7.0), 150 mM NaCl, 2 mM
EGTA, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM Na3VO4,
and proteinase and phosphatase inhibitors). For analyzing I?B? protein, T
cells were pretreated with proteosome inhibitor MG132 (5 ?M) at 37°C for
1 h and then stimulated with either anti-CD3 (5 ?g/ml) or anti-CD3 (2.5
?g/ml) plus anti-CD28 (5 ?g/ml) for 20 min. Protein was quantitated by
Bio-Rad Protein Assay (Bio-Rad). Equal amount of protein was separated
on SDS-PAGE. Western blots were performed per manufacturer’s instruc-
tion with the following phospho-Abs: anti-ZAP70 (Tyr319/Syk352), anti-
phospholipase C (PLC) ?1 (Tyr783), anti-Shc (Tyr239/240), anti-ERK
(Thr202/Tyr204), anti-SAPK/JNK (Thr183/Tyr185), anti-p38 (Thr183/Tyr185),
and anti-I?B? (Ser32) from Cell Signaling Technology, and anti-LAT
(Tyr132) from BioSource International. Phospho-specific Ab against the
autophosphorylation site of Lck (Y394) was a gift from A. Shaw (Wash-
ington University School of Medicine, St. Louis, MO). Membranes were
washed with stripping buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 0.7%
A, Total thymocytes were isolated from the four different mouse genotypes,
as indicated. Lysates were analyzed using anti-Abl (8E9, specific for Abl
kinase domain) and anti-c-Abl (Ab-3, C-terminal-specific) Abs. The trun-
cated Abl protein band is indicated by the arrow. B, Splenic T cells and
non-T splenocytes were isolated from wild-type (lanes 1 and 3) and Abl/
Arg-null (lanes 2 and 4) mice. Western blot analysis was performed using
anti-Abl (8E9) Ab. Splenic T cell purity ranged from 91 to 97% using the
Pan-T Cell Isolation kit.
Abl protein expression in T cell-conditional knockout mice.
7335The Journal of Immunology
2-??) and reprobed with the following Abs for total protein: anti-ZAP70,
anti-LAT, and anti-p38 (Cell Signaling Technology); anti-PLC?, anti-
ERK1, anti-JNK1, and anti-I?B? from Santa Cruz Biotechnology; anti-Shc
from BD Transduction Laboratories; and anti-p56lckfrom BioSource
Immunization and serum Ig measurements
Wild-type and Abl/Arg-conditional null mice were immunized i.p. at day
0 with 50 ?g of alum-precipitated nitrophenylacetyl (NP)-chicken ?-glob-
ulin (T cell-dependent Ag) or NP-LPS (T cell-independent Ag) (Biosearch
Technologies). For T cell-dependent Ag response, mice were challenged
with same dose at day 21. Serum was collected from tail vein at indicated
times, and NP-specific IgM and IgG titers were determined by ELISA on
96-well plates coated with NP-BSA (Biosearch Technologies), followed by
alkaline phosphatase-conjugated goat anti-mouse IgM or IgG, and devel-
oped in pNPP substrate (Southern Biotechnology Associates). NP-specific
mouse IgM and IgG (gifts from W. Zhang, Duke University Medical Cen-
ter, Durham, NC) were used as standards.
Generation of Ag-specific T cells after
L. monocytogenes infection
A recombinant L. monocytogenes strain engineered to secrete chicken
OVA and pMHC/peptide tetramers was originally provided by M.J. Bevan
(University of Washington, Seattle, WA). Wild-type and Abl/Arg-condi-
tional null mice were infected i.v. with 5 ? 103CFU of bacteria. Spleno-
cytes were prepared at day 7 after infection and were then challenged with
or without 0.2 ?M of either OVA (257–264) peptide for CD8 or listerio-
lysin O (190–201) peptide for CD4 in the presence of monensin (3 ?M) at
37°C for 5.5 h. Cells were surface stained with PE-conjugated anti-CD8 or
anti-CD4 Abs, fixed in 2% paraformaldehyde, and permeabilized with
0.1% saponin, followed by staining with FITC anti-IFN-? (BD Pharmin-
gen) Ab. Activation of Ag-specific CD4 cells was also analyzed by staining
for both CD4 and CD154. For H-2Kb-OVA-binding experiment, DimerX
I (BD Biosciences) was loaded with OVA peptides (257–264) overnight at
37°C and preincubated with PE-conjugated anti-mouse IgG1 for 4 h before
incubation with splenocytes harvested 7 days after L. monocytogenes in-
fection. The number of cells binding to the H-2Kbpeptide was analyzed by
gating on the CD8?cells. A total of 200,000 cells was collected on
FACScan and analyzed by CellQuest.
All statistics were performed using Student’s unpaired, two-tailed t test.
Generation of Abl/Arg T cell-conditional knockout mice
The Ablfloxmice were generated, as previously described (20), and
were crossed into the arg?/?background to generate abl flox/flox
arg?/?mice. These mice were subsequently crossed with the Lck-
Cre transgenic mice to generate conditional loss of Abl kinases
in the T cell lineage. Protein analysis of Abl/Arg-double-null
thymocytes and purified splenic T cells demonstrated the ab-
sence of full-length Abl proteins by immunoblotting with an Ab
specific for the kinase domain of Abl (Fig. 1, A, upper panel,
and B, lane 2). In contrast, Abl protein was present in cells other
than T cells, indicating specific inactivation of the abl1 gene in
T cells (Fig. 1B). Consistent with previous findings (20), a small
amount of a truncated Abl protein was detected in Abl/Arg-null
thymocytes by blotting with Ab specific for the Abl C-terminal
region (Fig. 1A, bottom panel). This truncated version of the
Abl protein was previously demonstrated to completely lack
Abl kinase activity (20).
development in Abl/Arg-conditional
total thymocytes (wild type (WT), n ?
7, 166.7 ? 10.4; Abl/Arg null, n ? 12,
73.5 ? 9.5, mean ? SEM). B, Thymo-
cytes were stained with anti-CD4 and
anti-CD8 Abs, followed by flow cy-
tometry analysis. This result is repre-
sentative of at least five independent
experiments. C, Absolute numbers of
the DP cells (WT, 137.1 ? 10.3; Abl/
Arg null, 58.7 ? 8.3, mean ? SEM),
DN cells (WT, 4.3 ? 0.5; Abl/Arg null,
3.8 ? 0.5, mean ? SEM), CD4-SP
cells (WT, 18.8 ? 1.6; Abl/Arg null,
8.4 ? 1.1, mean ? SEM), and CD8-SP
cells (WT, 6.2 ? 0.3; Abl/Arg null,
2.7 ? 0.4, mean ? SEM). Horizontal
bars indicate mean values. Values of p
stage in the absence of Abl and Arg kinases. A, Thymocytes were stained
with anti-CD4, anti-CD8, anti-CD44, and anti-CD25, and flow analysis
was gated on the CD4?CD8?DN population and analyzed for CD25/
CD44 expression. The data are representative of at least five independent
experiments. B, Mice were injected i.p. with 20 ?g of anti-CD3? Ab, and
thymocytes were stained and analyzed as in A. The data are representative
of three independent experiments.
Development of thymocytes is partially blocked at the DN3
7336Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION
Thymocyte development is abnormal in Abl/Arg-conditional
We observed that Abl/Arg-conditional knockout mice consistently
exhibited greater than 50% reduction in the total number of thy-
mocytes compared with wild-type controls (Fig. 2A). Thymocytes
isolated from the Abl/Arg-conditional knockout mice and their
wild-type littermate controls (4–6 wk old) were surface stained
with anti-CD4 and anti-CD8 Abs and analyzed by flow cytometry.
Although the percentage of CD4?CD8?DP and CD4?or CD8?
SP cells was not significantly different between wild-type and mu-
tant mice, the percentage of the CD4?CD8?DN population was
consistently increased by 2- to 4-fold in the Abl/Arg-null mice
(Fig. 2B). Comparison of the absolute cell numbers for each sub-
population revealed that the reduction in the total number of thy-
mocytes in the Abl/Arg mutant mice compared with wild-type
mice was primarily due to a ?50% decrease in DP cells as well as
TCR signaling (A–E). SL-12?12 cells were pretreated
with or without the Abl kinase inhibitor imatinib (5
?M) for 1 h. Cells were then activated with biotinylated
anti-TCR? plus streptavidin for the indicated times at
37°C. Equal amount of protein lysates was loaded onto
SDS-PAGE and analyzed by Western blotting with Abs
specific for phosphorylated (p) ZAP70/Syk (A), LAT
(B), PLC?1 (C), Shc (D), and ERK (E). Membranes
were stripped and reprobed with Abs to each of the cor-
responding total proteins.
Abl kinase activity is required for pre-
duced in the Abl/Arg-conditional
knockout mice. A, Splenocytes iso-
lated from 5- to 9-wk-old mice were
stained with anti-TCR?, CD4, CD8,
and B220 Abs, followed by flow cy-
tometry analysis. Percentage of posi-
tive cells in the gated region is indi-
cated. B, Absolute numbers of total
splenocytes (n ? 10, WT, 102.6 ?
10.8; Abl/Arg null, 93.9 ? 9.8,
mean ? SEM), total T cells (n ? 8,
WT, 43.3 ? 4.4; Abl/Arg null,
28.5 ? 3.5, mean ? SEM), CD4?
cells (n ? 8, WT, 26.5 ? 2.3; Abl/
Arg null, 17 ? 1.6, mean ? SEM),
and CD8?cells (n ? 10, WT, 11.2 ?
1.1; Abl/Arg null, 6.6 ? 0.8, mean ?
SEM). Horizontal bars indicate mean
values. Values of p are indicated.
Splenic T cells are re-
7337 The Journal of Immunology
similar decreases in CD4-SP and CD8-SP cells, with no significant
difference in the total number of DN cells (Fig. 2C). These results
suggested potential developmental defects during the DN to DP
transition in the absence of Abl and Arg kinases.
The DN thymocytes can be subdivided into four developmental
stages (DN1, DN2, DN3, and DN4) based on the expression of the
CD25 and CD44 cell surface molecules (2, 3). These stages
progress in thefollowing order:
CD25?CD44?(DN2), CD25?CD44?(DN3), and CD25?CD44?
(DN4). TCR? gene rearrangements are initiated at the DN2 stage
and continue during the DN3 stage. The rearranged TCR? chain
heterodimerizes with the pre-T? chain to form the pre-TCR at the
cell surface. Signals derived from the pre-TCR at the DN3 stage
are important for maturation to the DN4 stage and are implicated
in proliferative expansion, survival, allelic exclusion of the TCR?
locus, and induction of TCR? rearrangement (2). To define the
defect in thymocyte development in the Abl/Arg-conditional
knockout mice, we analyzed the expression of CD25 and CD44
among the DN populations. Thymocytes from Abl/Arg-condi-
tional knockout mice consistently presented a higher percentage of
CD25?CD44?(DN3) cells with a corresponding decrease in DN4
stage cells compared with wild-type mice (Fig. 3A), which sug-
gests that loss of Abl kinases may affect the DN3 to DN4 transition
during thymocyte development. Successful TCR? gene rearrange-
ment and expression of the pre-TCR occur at DN3 stage, and func-
tional signaling through pre-TCR is critical for thymocyte devel-
opment (2). Therefore, we analyzed the expression of TCR? on
subsets of thymocytes by FACS analysis. There was no significant
difference in surface expression of TCR? in all four thymocyte
subpopulations between wild-type and Abl/Arg mutant mice (data
not shown). Thus, Abl kinases are not involved in TCR? chain
rearrangement and surface expression. Similarly, analysis of pos-
itive selection by staining thymocytes for DP expression of TCR?
and CD69 (21) demonstrated that there was no significant differ-
ence between Abl/Arg-null thymocytes and their littermate con-
trols (data not shown).
To address whether the defect in the transition from DN3 to
DN4 elicited by the absence of Abl kinases could result from im-
paired signaling downstream of the pre-TCR, we performed in
vivo injections with anti-CD3? Ab, which activates the pre-TCR
wild-type, c-Abl-null, Arg-null, and Abl/Arg-double-null mice were stimulated with either anti-CD3 alone (top panel) or anti-CD3 plus anti-CD28 (bottom
panel) Abs at the indicated concentrations for 48 h, and [3H]thymidine was added during the last 16–20 h of culture. Data are mean ? SD of triplicate wells.
with allogeneic stimulators (MLR) for 72 h. Proliferation was measured by [3H]thymidine incorporation in triplicate wells (mean ? SD). This experiment
is representative of at least five independent experiments. C, Wild-type or Abl/Arg-double-null T cells were stimulated with either anti-CD3 (5 ?g/ml)
alone, or anti-CD3 (1 ?g/ml) plus anti-CD28 (5 ?g/ml) Abs, as indicated. Culture supernatant was collected after 24 h, and the levels of IL-2 (top panel),
IL-4 (middle panel), and IFN-? (bottom panel) were measured by ELISA in triplicates. This result is representative of four independent experiments. The
percentage of decrease in cytokine levels in mutant T cells compared with wild-type T cells stimulated with anti-CD3 or anti-CD3 plus CD28 was calculated
as follows. For IL-2: 72.8% ? 10.7 (CD3); 72.9% ? 5.6 (CD3 ? CD28). For IL-4: 40.1% ? 32.3 (CD3); 31.6% ? 28.5 (CD3 ? CD28). For IFN-?: 63.7%
? 21.6 (CD3); 74.3% ? 17.3 (CD3 ? CD28). D, Abl protein was absent in Cre-GFP cells after in vitro retroviral-Cre infection (left panel). Vector-GFP
(wild-type) and Cre-GFP (Abl/Arg-null) T cells were stimulated with anti-CD3 Ab before retroviral infection and analyzed for proliferation in the presence
of IL-2 for 24 h (middle panel). Supernatant was collected after restimulation of indicated cells with anti-CD3 for 6 h, and IFN-? levels were measured
(right panel). The data are representative of three independent experiments and retroviral infections.
Proliferation and IL-2 secretion are impaired in mice lacking Abl and Arg in the T cell compartment. A, Splenic T cells isolated from
7338Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION
and accelerates thymocyte maturation (22, 23). Mice were injected
i.p. with anti-CD3? Ab and thymocytes were analyzed 4 days later.
As expected, wild-type mice exhibited accelerated maturation
through the DN3 stage with few cells remaining at the DN2 and
DN3 stages (Fig. 3B). In contrast, a high number of DN3 cells
accumulated in the Abl/Arg-double-null thymocytes (Fig. 3B). To-
gether our data support a role for Abl kinases in thymocyte devel-
opment and pre-TCR signaling in vivo.
Inhibition of Abl kinases impaired pre-TCR signaling
To directly examine the role of Abl kinases in pre-TCR signaling,
we used the pre-T cell line SL-12?12, derived from a spontaneous
SCID mouse thymoma that stably expresses functionally rear-
ranged TCR? with endogenous pre-TCR? chains on the cell sur-
face (24). To assess whether loss of Abl kinase activity affected
pre-TCR signaling, SL-12?12 cells were pretreated with the Abl
kinase inhibitor imatinib mesylate (5 ?M for 1 h), followed by
stimulation with biotinylated anti-TCR? Ab and cross-linking with
streptavidin for the indicated times (Fig. 4). We observed en-
hanced phosphorylation of ZAP70/Syk, LAT, and PLC?1, as well
as the adaptor protein Shc following pre-TCR activation in control
cells. In contrast, the tyrosine phosphorylation of these molecules
was greatly diminished in cells treated with the Abl kinase inhib-
itor (Fig. 4, A–D). Phosphorylation of ERK was also strongly en-
hanced after pre-TCR cross-linking in control cells, and it was only
slightly reduced at the earliest time point after pre-TCR stimula-
tion in the imatinib-treated cells (Fig. 4E). Together these results
suggest that Abl kinases are involved in pre-TCR signaling.
Decreased proliferation and cytokine production of
Abl/Arg-double-null mature T cells
Splenocytes were isolated from Abl/Arg-conditional knockout
mice and their littermate controls (5–9 wk), and stained with cell
surface markers for T and B cells, including TCR?, CD4, CD8,
and B220. We observed a ?40–50% decrease in the percentage of
total T cells, as well as CD4?and CD8?cell populations, and a
relative small increase in the percentage of B cells in the Abl/Arg-
conditional null compared with wild-type mice (Fig. 5A). B cell
proliferation was not affected in these mice because there was no
significant difference in proliferation following stimulation of total
splenocytes with the B cell mitogens anti-IgM or LPS (data not
shown). Although the absolute number of total splenocytes was
not significantly different between mutant and wild-type mice, the
total T cell numbers were significantly reduced in the absence of
Abl family kinases, with significant reductions in both CD4?and
CD8?subsets in the Abl/Arg-conditional knockout mice (Fig. 5B).
The reduction in splenic T cells in the mutant mice suggested that
Abl kinases might be involved in T cell proliferation or survival.
Splenic T cells from wild-type, Abl-null, Arg-null, and Abl/Arg-
double-null mice were stimulated with either anti-CD3 alone or
anti-CD3 plus anti-CD28 for 48 h, and proliferation was measured
by [3H]thymidine incorporation (Fig. 6A). Resting T cells did not
show significant [3H]thymidine incorporation in both wild-type
and mutant cells (data not shown). Abl- and Arg-single-null T cells
showed a significant reduction in proliferation upon TCR stimu-
lation with low amounts (1 ?g/ml) of anti-CD3 (Fig. 6A). At
higher doses of anti-CD3 (5 ?g/ml) alone or anti-CD3 plus higher
dose of anti-CD28 (5 ?g/ml), Arg-null T cells proliferated similar
to wild-type controls. Notably, loss of both Abl and Arg resulted
in a more profound defect in T cell proliferation compared with the
loss of either kinase alone at both low and high doses of anti-CD3
in the absence or presence of CD28 costimulation (Fig. 6A). Addition
of IL-2 failed to reverse the proliferation defect in the Abl/Arg-dou-
ble-null T cells (data not show). We did not observe significant dif-
ferences in proliferation between wild-type and Abl/Arg-double-null
T cells in response to PMA and ionomycin stimulation (data not
shown). We also observed significantly less proliferation in lympho-
cytes from Abl/Arg-conditional null mice compared with wild-type
mice in response to allogeneic stimulation in a MLR (Fig. 6B). Thus,
loss of Abl/Arg kinases does not result in a general defect in T cell
proliferation, but specifically inhibits TCR-induced cell proliferation,
and suggests that Abl kinases function to transduce proximal signals
downstream of the TCR.
To study whether T cell function is impaired in the absence of
Abl kinases, wild-type or Abl/Arg-double-null T cells were stim-
ulated with anti-CD3 in the absence or presence of anti-CD28 Abs
for 24 h, and the levels of IL- 2, IL-4, and IFN-? secreted into
culture supernatant were measured by ELISA (Fig. 6C). Both IL-2
and IFN-? levels were markedly reduced by ?70% in Abl/Arg-
double-null T cells compared with wild type (Fig. 6C, top and
bottom panels), whereas a smaller reduction of IL-4 (?30%) was
observed in the Abl/Arg mutant mice (Fig. 6C, middle panel).
Thus, Abl kinases regulate Th function, and specifically TCR-me-
diated IL-2 and IFN-? secretion.
To determine whether the defect in mature T lymphocyte pro-
liferation and cytokine secretion in the absence of Abl kinases is
due to defective T cell development or to an intrinsic cell-auto-
nomous defect, we have deleted the abl gene in vitro following
transduction with a retroviral Cre vector. T cells were stimulated
with anti-CD3 before retroviral infection, and deletion of the abl
Wild-type or Abl/Arg-double-null T cells were stimulated with anti-CD3
for 48 h, and BrdU was added during the last 30 min of culture. For
treatment with the Abl kinase inhibitor, imatinib (5 ?M) was added to
wild-type T cells. Cells were stained with anti-BrdU Ab and PI, and ana-
lyzed by flow cytometry. Percentage of S-phase cells is indicated. B, Wild-
type or Abl/Arg-null T cells were stimulated with plate-bound anti-CD3 (5
?g/ml) for 48 h. Cells were stained with annexin V and 7-aminoactino-
mycin D, followed by flow analysis. C, Wild-type or Abl/Arg-null T cells
were stimulated with plate-bound anti-CD3 (5 ?g/ml) for the indicated
times. Cell lysates were separated on SDS-PAGE and analyzed using either
anti-caspase 3 or anti-cleaved caspase 3 Abs, as indicated.
Decreased cell cycle entry in the absence of Abl kinases. A,
7339The Journal of Immunology
gene was confirmed by the absence of Abl protein (Fig. 6D, left).
Proliferation of the CD3-stimulated T cells was measured in the
presence of IL-2 for 24 h, and IFN-? secretion was measured 3–8
h after restimulation with anti-CD3 Ab. We observed a significant
reduction in both proliferation and IFN-? secretion in the Abl/Arg-
double-null T cells compared with wild-type T cells (Fig. 6D, mid-
dle and right panels). Thus, Abl kinases have a T cell-autonomous
role and are required for maximal proliferation and cytokine se-
cretion in mature T cells. Prolonged restimulation of these acti-
vated T cells with anti-CD3 Ab resulted in apoptosis of both wild-
type and Abl/Arg-null cells (data not shown).
To further investigate the possible mechanism(s) responsible for
the reduced T cell proliferation induced by loss of the Abl kinases,
cells were pulse labeled for a short time with BrdU at the end of
anti-CD3 stimulation, and the cell cycle was analyzed by the DNA
profile. Abl/Arg-double-null T cells or wild-type T cells treated
with the Abl kinase inhibitor imatinib showed a consistently lower
percentage of cells entering into S phase compared with wild-type
T cells (Fig. 7A). There was no significant difference in the per-
centage of sub-G1cells, suggesting no increased apoptosis in Abl/
Arg-double-null T cells. Moreover, analysis of T cell survival
upon TCR stimulation using annexin V and PI staining (Fig. 7B),
as well as Western blot analysis of caspase 3 activation (Fig. 7C)
did not show differences in apoptosis between wild-type and Abl/
Arg-double-null T cells. These results indicate that the reduced T
cell proliferation in the absence of Abl kinases is primarily due to
decreased cell cycle entry.
Abl kinases are involved in multiple TCR signaling pathways
To define the possible mechanism(s) responsible for impaired T
cell proliferation, we examined expression of early T cell surface
markers following TCR activation. Wild-type and Abl/Arg-dou-
ble-null T cells were stimulated with anti-CD3 Ab for 18 h and
stained with either anti-CD25? (IL-2R? chain) or the CD69 early
T cell activation marker. There was no significant difference in the
induction of either CD25? or CD69 expression between wild-type
and mutant T cells (data not shown), indicating that the prolifer-
ation defect in the Abl/Arg-double-null T cells is not due to de-
creased IL-2R on the cell surface. These findings suggested that
inhibition of cell proliferation in T cells lacking Abl and Arg ki-
nases may result from impaired TCR-mediated signaling. Indeed,
we found that TCR-induced phosphorylation of ZAP70, PLC?1,
and LAT was greatly reduced in Abl/Arg-double-null T cells com-
pared with wild-type cells (Fig. 8, A–C). These results are consis-
tent with our previous findings of reduced phosphorylation of these
signaling proteins in imatinib-treated primary T cells and splenic T
cells derived from abl1?/?abl2?/?mice (14). Notably, TCR-in-
duced Lck activation measured by tyrosine 394 phosphorylation
was unchanged in T cells lacking Abl family kinases compared
with wild-type controls (data not shown). We also analyzed the
phosphorylation of the Shc adaptor protein following TCR stim-
ulation of purified T cells derived from control and Abl/Arg-con-
ditional knockout mice, and show for the first time that Abl/Arg-
null T cells exhibit a dramatic decrease in Shc tyrosine
phosphorylation following TCR stimulation (Fig. 8D). Activation
in Abl/Arg-double-null T cells, with loss of the Abl kinases leading to
activation (Fig. 8, E and F). In contrast, we found that phosphoryla-
tion of the p38 MAPK was unchanged in Abl/Arg-double-null T cells
compared with wild-type T cells (Fig. 8G).
We have shown in this study that IL-2 production was markedly
reduced in Abl/Arg-null T cells, and have previously observed that
inhibition of Abl kinase activity reduced activation of the
indicated. Equal amounts of lysates were analyzed by Western blotting with Abs specific for phosphorylated (p) ZAP70 (A), LAT (C), PLC?1 (B), Shc (D),
JNK (E), ERK (F), p38 (G), and I?B? (H). Membranes were stripped and reprobed with Abs to measure total levels of the corresponding proteins.
TCR signaling is impaired in Abl/Arg-double-null mature T cells (A–H). Wild-type and Abl/Arg-double-null T cells were stimulated, as
7340 Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION
CD28RE/AP response element of the IL-2 promoter following
TCR activation in Jurkat T cells (14). The NF-?B transcription
factor is known to bind to the CD28RE region and activate IL-2
gene transcription (25), and is also required for chromatin remod-
eling of the IL-2 promoter upon TCR stimulation (26). Thus, we
examined whether NF-?B activation was impaired in T cells lack-
ing the Abl kinases. Inactive NF-?B is sequestered by binding to
I?B in the cytoplasm of resting T cells. Upon TCR activation,
I?B? is phosphorylated, which triggers the ubiquitination and deg-
radation of I?B?, thereby releasing NF-?B and promoting its
translocation to nucleus to regulate transcription (27, 28). As
shown in Fig. 8H, phosphorylation of I?B? was markedly de-
creased in Abl/Arg-double-null T cells compared with wild-type
controls following stimulation with either anti-CD3 alone or anti-
CD3 plus anti-CD28. Thus, decreased IL-2 production in the ab-
sence of Abl kinases may be mediated in part by decreased NF-?B
Abl kinases are required for T cell function in vivo
To determine whether Abl kinases play a role in T cell function in
vivo, mice were injected with T cell-dependent or T cell-indepen-
dent Ags, and Ab production was analyzed in Abl/Arg-conditional
knockout mice and control littermates. As shown in Fig. 9A, wild-
type mice produced high levels of IgM in response to initial in-
jection of T cell-dependent Ag, and high levels of IgG upon sec-
ondary boost with same Ag. In contrast, Abl/Arg-conditional
knockout mice were markedly impaired in both early IgM secre-
tion and secondary isotype switch to IgG secretion, suggesting that
Th function is severely impaired in these mice. As expected,
wild-type and mutant mice secreted equal amounts of IgM in
response to a T cell-independent Ag (Fig. 9B), indicating that B
cell function is not affected in the Abl/Arg T cell-conditional
conditional null mice. Wild-type and Abl/Arg-conditional null mice
were injected i.p. with either T cell-dependent Ag (NP-CGG) (A) or T
cell-independent Ag (NP-LPS) (B). Serum was collected from the tail
vein at the indicated time points. Levels of IgM and IgG were measured
by ELISA; data are mean ? SD of triplicate wells. This result is rep-
resentative of four (T cell-independent Ag) and five (T cell-dependent
T cell-dependent Ab secretion is reduced in Abl/Arg-
with L. monocytogenes through tail vein injection. After 7 days, splenocytes were cultured with or without specific OVA (A) or listeriolysin O (D)
peptides for 5.5 h. Cells were then surface stained with anti-CD8 (A) or anti-CD4 (D), respectively, and intracellular stained with anti-IFN-? Abs,
followed by flow cytometry analysis. B and E, Quantitation of absolute numbers of CD8?IFN-??(B) or CD4?IFN-??(E) DP cells from wild-type
vs Abl/Arg-conditional null mice. For IFN-??CD8?, WT, n ? 9, 1.2 ? 0.5; Abl/Arg null, n ? 8, 0.2 ? 0.1. For IFN-??CD4?, WT, n ? 4, 4.9 ?
1.6; Abl/Arg null, n ? 4, 2.2 ? 1.2. (mean ? SD). Horizontal bars indicate mean values. C, Total number of CD8?T cells binding to H-2Kbpeptide.
This is a representative experiment of four independent infections with L. monocytogenes (WT, 2.6 ? 0.8; Abl/Arg null, 0.7 ? 0.5, mean ? SD).
F, Total number of CD4?/CD154?DP cells. This is a representative experiment of four independent Listeria infections (WT, 1.9 ? 0.6; Abl/Arg
null, 0.8 ? 0.3, mean ? SD). Values of p are indicated.
Defective production of CTL in Abl/Arg-conditional null mice. A and D, Wild-type and Abl/Arg-conditional null mice were infected
7341 The Journal of Immunology
To investigate whether deficiency in the expression of Abl fam-
ily kinases results in altered susceptibility to infection, Abl/Arg-
conditional knockout mice and littermate controls were infected
with a low dose of L. monocytogenes, and the mice were analyzed
for the development of Ag-specific CD8?and CD4?T cells. We
found that Abl/Arg mutant mice developed very few Ag-induced
IFN-?-producing CD8?effector T cells compared with wild-type
mice (Fig. 10A). Notably, the total number of IFN-?-positive
CD8?T cells in the Abl/Arg-conditional knockout mice was re-
duced by greater than 80%, and it was only 18% compared with
the wild-type controls (Fig. 10B). The number of specific CD8?T
cells binding to the H-2Kbpeptide was also significantly reduced
in the Abl/Arg-null mice (73% reduction compared with the wild
type; Fig. 10C). Thus, expansion of specific CD8?T cells upon
Listeria infection was impaired in Abl/Arg-null mice. In contrast,
the percentage of IFN-?-positive CD4?T cells was similar be-
tween wild-type and mutant mice (Fig. 10D). However, the total
number of IFN-?-positive CD4?T cells was ?50% of the wild
type (Fig. 10E), which most likely reflects the reduced number of
splenocytes in the mutant mice after infection (52.6% of the wild-
type mice). The total number of CD4?/CD154?DP cells in the
mutant mice was also ?50% of the wild-type mice (Fig. 10F).
Together, these results reveal that Abl kinases are principally re-
quired for the development of effector CD8?T cells in vivo in
response to Listeria infection.
Our work has uncovered cell-autonomous roles for Abl family
kinases in the regulation of both thymocyte development and ma-
ture T cell function, and showed that Abl kinases signal down-
stream of both the pre-TCR and TCR. The generation of T cell-
specific Abl/Arg-null mice permitted us to bypass the embryonic
lethality associated with complete loss of Abl and Arg, and re-
vealed a previously unappreciated role for these kinases in thymo-
cyte development. We found that Abl/Arg-double-null thymocytes
were partially blocked at the DN3 stage, a developmental stage
that requires signaling through the pre-TCR to progress to the DN4
stage (2). Abl/Arg-double-null thymocytes expressed wild-type
levels of TCR? on the DN populations, which suggests that the
Abl/Arg-double-null thymocytes have normal amounts of pre-
TCR surface expression. Rather, we found that signaling through
the pre-TCR was impaired in the absence of Abl kinases. In this
regard, Abl/Arg-double-null thymocytes failed to progress through
the DN3 stage induced by in vivo injection of anti-CD3? Ab,
which results in complete mobilization of wild-type thymocytes to
the DN4 stage with few remaining DN3 stage cells. Moreover, loss
of Abl kinase activity produced a marked decrease in the activation
of several signaling molecules in pre-TCR cells.
Knockout mouse studies have identified a critical role for the
Lck and ZAP70/Syk tyrosine kinases in thymocyte development
(4, 5). Loss of Lck or ZAP70 and Syk results in arrested thymocyte
development with a partial or complete block at the DN3 stage.
Similarly, genetic inactivation of adaptor proteins regulated down-
stream of these tyrosine kinases, such as LAT (9) and Shc (8),
results in arrested thymocyte development. Our finding that Abl
kinase activity is required for maximal activation of ZAP70/Syk,
LAT, PLC?1, and Shc following pre-TCR activation defines a new
role for Abl family kinases downstream of the pre-TCR in
As expected from our finding that CD4?and CD8?SP thymo-
cytes were decreased in the Abl/Arg-conditional null mice, mature
splenic T cells were also significantly reduced by ?40–50% in the
mutant mice. Although Abl- and Arg-single-null T cells showed a
limited defect in proliferation, Abl/Arg-double-null T cells exhib-
ited a more profound proliferative defect upon anti-CD3 and/or
anti-CD28 stimulation. Thus, both Abl and Arg kinases are re-
quired for maximal immune responses. The basis for the prolifer-
ative defect in T cells lacking Abl kinases is most likely due to
delayed cell cycle entry into S phase. A role for Abl kinases in cell
cycle progression is consistent with previous findings that Abl is
required for growth factor-induced DNA synthesis in fibroblasts
(29–31). Furthermore, inhibition of TCR-induced proliferation in
cells lacking Abl and Arg is also consistent with a role for Abl
kinases in the regulation of mature TCR signaling. Our current
findings and previous data (14) suggest that Abl kinases may link
TCR engagement and Lck activation to the activation of ZAP70/
Syk, LAT, and PLC?1. Alternatively, Abl kinases may not directly
activate ZAP70 and specific signaling molecules, but rather Abl
kinases may regulate the assembly of signaling complexes and/or
cytoskeletal scaffolds required for proper activation of ZAP70 and
downstream signaling targets.
We have also shown that Abl kinases are involved in maximal
activation of Shc, ERK, and JNK in mature T cells. Following
TCR engagement, the adaptor protein Shc is recruited to the TCR
through interactions with ZAP70 and CD3? (32, 33). Shc proteins
have been reported to be phosphorylated by Lck and Syk/ZAP70
tyrosine kinases (34), and subsequently bind to Grb2 and son of
sevenless, leading to activation of the Ras-MAPK pathway (35).
We found that phosphorylation of Shc at tyrosines 238/240 fol-
lowing TCR stimulation was greatly reduced in Abl/Arg-double-
null T cells. Thus, Abl kinases may regulate Shc activity indirectly
through the regulation of ZAP70/Syk phosphorylation, or may di-
rectly phosphorylate Shc at Y238/Y240. Tyrosine phosphorylation
of Shc is important for both T cell proliferation and IL-2 produc-
tion (35, 36), and Abl-mediated phosphorylation of Shc may play
a role in the regulation of these processes. Furthermore, decreased
IL-2 production in Abl/Arg-null T cells is most likely linked to
decreased transcriptional activity of factors required for IL-2 gene
expression, such as NF-?B. In this study, we showed that TCR-
induced NF-?B activity, as measured by the phosphorylation of
I?B?, is decreased in the absence of the Abl kinases. Additionally,
JNK activity is reduced in Abl/Arg-double-null T cells, which may
contribute to the impaired production of cytokines, including
IFN-?, observed in Abl/Arg T cell-conditional knockout mice in
response to TCR stimulation and bacterial infection. In this regard,
JNK activity was shown to be required for production of IFN-? in
T cells (37).
A most striking finding derived from the analysis of the Abl/Arg
T cell-conditional null mice was the discovery that these mice are
impaired in the development of Ag-specific CTL and display a
marked reduction of IFN-?-positive CD8?T cells compared with
wild-type mice in response to L. monocytogenes infection. More-
over, the Abl/Arg-double-null mice fail to mount a response to T
cell-dependent Ags. Thus, Abl kinases are required for both hu-
moral and cellular immunity. The findings presented in this study
with the Abl/Arg-conditional knockout mice support the notion
that loss of Abl family kinases specifically in T cells underlies the
increased susceptibility to infection observed in Abl1 knockout
mice (18, 19), as well as the reported immunosuppressive effects of
imatinib treatment (38, 39).
L. monocytogenes is a potent inducer of CD8 T cell responses,
and once primed to proliferate by the pathogen, CD8 T cells un-
dergo proliferation as well as differentiation into effector and mem-
ory T cells (40). Recent work has suggested that asymmetric cell
division of CD8 T cells proliferating in response to L. monocyto-
genes infection results in the segregation of immune receptors and
polarity proteins following formation of the immunological syn-
apse, thereby producing effector CTL derived from the daughter
7342 Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION
cell near the synapse, and long-lived memory T cells derived from
the daughter cell distal to the synapse (41). We have previously
shown that Abl kinases regulate synapse formation at the neuro-
muscular junction (42). Moreover, we showed that the Abl targets
Abi and Wave are critical for immunological synapse formation
and the accumulation of actin of the T cell-B cell interface (43,
44). Thus, loss of Abl kinases may impair adhesive interactions
between T cells and APCs, thereby affecting immunological syn-
apse formation and maintenance. The availability of the T cell-
conditional Abl/Arg-double-knockout mice described in this work
will allow us to examine whether Abl tyrosine kinases have a role
in the regulation of the immunological synapse in primary T cells,
as well as asymmetric T cell division in response to pathogens.
We thank Weiguo Zhang for advice and reagents, Emily Riggs for tech-
nical assistance, and Mike Cook and Lynn Martinek for FACS assistance
The authors have no financial conflict of interest.
1. Hayday, A. C., and D. J. Pennington. 2007. Key factors in the organized chaos
of early T cell development. Nat. Immunol. 8: 137–144.
2. Von Boehmer, H. 2005. Unique features of the pre-T-cell receptor ?-chain: not
just a surrogate. Nat. Rev. Immunol. 5: 571–577.
3. Cantrell, D. A. 2002. Transgenic analysis of thymocyte signal transduction. Nat.
Rev. Immunol. 2: 20–27.
4. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran,
E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, and A. Veillette. 1992.
Profound block in thymocyte development in mice lacking p56lck. Nature 357:
5. Cheng, A. M., I. Negishi, S. J. Anderson, A. C. Chan, J. Bolen, D. Y. Loh, and
T. Pawson. 1997. The Syk and ZAP-70 SH2-containing tyrosine kinases are
implicated in pre-T cell receptor signaling. Proc. Natl. Acad. Sci. USA 94:
6. Clements, J. L., B. Yang, S. E. Ross-Barta, S. L. Eliason, R. F. Hrstka,
R. A. Williamson, and G. A. Koretzky. 1998. Requirement for the leukocyte-
specific adapter protein SLP-76 for normal T cell development. Science 281:
7. Pivniouk, V., E. Tsitsikov, P. Swinton, G. Rathbun, F. W. Alt, and R. S. Geha.
1998. Impaired viability and profound block in thymocyte development in mice
lacking the adaptor protein SLP-76. Cell 94: 229–238.
8. Zhang, L., V. Camerini, T. P. Bender, and K. S. Ravichandran. 2002. A nonre-
dundant role for the adapter protein Shc in thymic T cell development. Nat.
Immunol. 3: 749–755.
9. Zhang, W., C. L. Sommers, D. N. Burshtyn, C. C. Stebbins, J. B. DeJarnette,
R. P. Trible, A. Grinberg, H. C. Tsay, H. M. Jacobs, C. M. Kessler, et al. 1999.
Essential role of LAT in T cell development. Immunity 10: 323–332.
10. Hernandez, S. E., M. Krishnaswami, A. L. Miller, and A. J. Koleske. 2004. How
do Abl family kinases regulate cell shape and movement? Trends Cell Biol. 14:
11. Pendergast, A. M. 2002. The Abl family kinases: mechanisms of regulation and
signaling. Adv. Cancer Res. 85: 51–100.
12. Zipfel, P. A., M. Grove, K. Blackburn, M. Fujimoto, T. F. Tedder, and
A. M. Pendergast. 2000. The c-Abl tyrosine kinase is regulated downstream of
the B cell antigen receptor and interacts with CD19. J. Immunol. 165:
13. Advani, A. S., and A. M. Pendergast. 2002. Bcr-Abl variants: biological and
clinical aspects. Leuk. Res. 26: 713–720.
14. Zipfel, P. A., W. Zhang, M. Quiroz, and A. M. Pendergast. 2004. Requirement for
Abl kinases in T cell receptor signaling. Curr. Biol. 14: 1222–1231.
15. O’Neill, A. J., T. G. Cotter, J. M. Russell, and E. F. Gaffney. 1997. Abl expres-
sion in human fetal and adult tissues, tumors, and tumor microvessels. J. Pathol.
16. Muller, R., D. J. Slamon, J. M. Tremblay, M. J. Cline, and I. M. Verma. 1982.
Differential expression of cellular oncogenes during pre- and postnatal develop-
ment of the mouse. Nature 299: 640–644.
17. Koleske, A. J., A. M. Gifford, M. L. Scott, M. Nee, R. T. Bronson, K. A. Miczek,
and D. Baltimore. 1998. Essential roles for the Abl and Arg tyrosine kinases in
neurulation. Neuron 21: 1259–1272.
18. Schwartzberg, P. L., A. M. Stall, J. D. Hardin, K. S. Bowdish, T. Humaran,
S. Boast, M. L. Harbison, E. J. Robertson, and S. P. Goff. 1991. Mice homozy-
gous for the ablm1 mutation show poor viability and depletion of selected B and
T cell populations. Cell 65: 1165–1175.
19. Tybulewicz, V. L., C. E. Crawford, P. K. Jackson, R. T. Bronson, and
R. C. Mulligan. 1991. Neonatal lethality and lymphopenia in mice with a ho-
mozygous disruption of the c-abl proto-oncogene. Cell 65: 1153–1163.
20. Moresco, E. M., S. Donaldson, A. Williamson, and A. J. Koleske. 2005. Integrin-
mediated dendrite branch maintenance requires Abelson (Abl) family kinases.
J. Neurosci. 25: 6105–6118.
21. Fischer, A. M., C. D. Katayama, G. Pages, J. Pouyssegur, and S. M. Hedrick.
2005. The role of erk1 and erk2 in multiple stages of T cell development. Im-
munity 23: 431–443.
22. Levelt, C. N., A. Ehrfeld, and K. Eichmann. 1993. Regulation of thymocyte
development through CD3. I. Timepoint of ligation of CD3? determines clonal
deletion or induction of developmental program. J. Exp. Med. 177: 707–716.
23. Shinkai, Y., and F. W. Alt. 1994. CD3?-mediated signals rescue the development
of CD4?CD8?thymocytes in RAG-2?/?mice in the absence of TCR? chain
expression. Int. Immunol. 6: 995–1001.
24. Berger, M. A., V. Dave, M. R. Rhodes, G. C. Bosma, M. J. Bosma, D. J. Kappes,
and D. L. Wiest. 1997. Subunit composition of pre-T cell receptor complexes
expressed by primary thymocytes: CD3? is physically associated but not func-
tionally required. J. Exp. Med. 186: 1461–1467.
25. Lai, J. H., G. Horvath, J. Subleski, J. Bruder, P. Ghosh, and T. H. Tan. 1995.
RelA is a potent transcriptional activator of the CD28 response element within the
interleukin 2 promoter. Mol. Cell. Biol. 15: 4260–4271.
26. Rao, S., S. Gerondakis, D. Woltring, and M. F. Shannon. 2003. c-Rel is required
for chromatin remodeling across the IL-2 gene promoter. J. Immunol. 170:
27. DiDonato, J., F. Mercurio, C. Rosette, J. Wu-Li, H. Suyang, S. Ghosh, and
M. Karin. 1996. Mapping of the inducible I?B phosphorylation sites that signal
its ubiquitination and degradation. Mol. Cell. Biol. 16: 1295–1304.
28. Henkel, T., T. Machleidt, I. Alkalay, M. Kronke, Y. Ben-Neriah, and
P. A. Baeuerle. 1993. Rapid proteolysis of I?B-? is necessary for activation of
transcription factor NF-?B. Nature 365: 182–185.
29. Plattner, R., and A. M. Pendergast. 2003. Activation and signaling of the Abl
tyrosine kinase: bidirectional link with phosphoinositide signaling. Cell Cycle 2:
30. Boureux, A., O. Furstoss, V. Simon, and S. Roche. 2005. Abl tyrosine kinase
regulates a Rac/JNK and a Rac/Nox pathway for DNA synthesis and Myc ex-
pression induced by growth factors. J. Cell Sci. 118: 3717–3726.
31. Furstoss, O., K. Dorey, V. Simon, D. Barila, G. Superti-Furga, and S. Roche.
2002. c-Abl is an effector of Src for growth factor-induced c-myc expression and
DNA synthesis. EMBO J. 21: 514–524.
32. Milia, E., M. M. Di Somma, F. Baldoni, R. Chiari, L. Lanfrancone, P. G. Pelicci,
J. L. Telford, and C. T. Baldari. 1996. The aminoterminal phosphotyrosine bind-
ing domain of Shc associates with ZAP-70 and mediates TCR dependent gene
activation. Oncogene 13: 767–775.
33. Ravichandran, K. S., K. K. Lee, Z. Songyang, L. C. Cantley, P. Burn, and
S. J. Burakoff. 1993. Interaction of Shc with the ? chain of the T cell receptor
upon T cell activation. Science 262: 902–905.
34. Walk, S. F., M. E. March, and K. S. Ravichandran. 1998. Roles of Lck, Syk and
ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein
Shc. Eur. J. Immunol. 28: 2265–2275.
35. Ravichandran, K. S. 2001. Signaling via Shc family adapter proteins. Oncogene
36. Pratt, J. C., M. R. van den Brink, V. E. Igras, S. F. Walk, K. S. Ravichandran, and
S. J. Burakoff. 1999. Requirement for Shc in TCR-mediated activation of a T cell
hybridoma. J. Immunol. 163: 2586–2591.
37. Yang, D. D., D. Conze, A. J. Whitmarsh, T. Barrett, R. J. Davis, M. Rincon, and
R. A. Flavell. 1998. Differentiation of CD4?T cells to Th1 cells requires MAP
kinase JNK2. Immunity 9: 575–585.
38. Dietz, A. B., L. Souan, G. J. Knutson, P. A. Bulur, M. R. Litzow, and
S. Vuk-Pavlovic. 2004. Imatinib mesylate inhibits T-cell proliferation in vitro and
delayed-type hypersensitivity in vivo. Blood 104: 1094–1099.
39. Mattiuzzi, G. N., J. E. Cortes, M. Talpaz, J. Reuben, M. B. Rios, J. Shan,
D. Kontoyiannis, F. J. Giles, I. Raad, S. Verstovsek, et al. 2003. Development of
Varicella-Zoster virus infection in patients with chronic myelogenous leukemia
treated with imatinib mesylate. Clin. Cancer Res. 9: 976–980.
40. Lara-Tejero, M., and E. G. Pamer. 2004. T cell responses to Listeria monocyto-
genes. Curr. Opin. Microbiol. 7: 45–50.
41. Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer,
A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, et al. 2007.
Asymmetric T lymphocyte division in the initiation of adaptive immune re-
sponses. Science 315: 1687–1691.
42. Finn, A. J., G. Feng, and A. M. Pendergast. 2003. Postsynaptic requirement for
Abl kinases in assembly of the neuromuscular junction. Nat. Neurosci. 6:
43. Nolz, J. C., T. S. Gomez, P. Zhu, S. Li, R. B. Medeiros, Y. Shimizu,
J. K. Burkhardt, B. D. Freedman, and D. D. Billadeau. 2006. The WAVE2 com-
plex regulates actin cytoskeletal reorganization and CRAC-mediated calcium en-
try during T cell activation. Curr. Biol. 16: 24–34.
44. Zipfel, P. A., S. C. Bunnell, D. S. Witherow, J. J. Gu, E. M. Chislock, C. Ring,
and A. M. Pendergast. 2006. Role for the Abi/wave protein complex in T cell
receptor-mediated proliferation and cytoskeletal remodeling. Curr. Biol. 16:
7343The Journal of Immunology