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Defective T Cell Development and Function in the Absence of
Abelson Kinases1
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
24, 2007.
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: email@example.com
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
Page 2
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.
Apoptosis assays
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
Page 3
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.
Statistics analysis
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
are indicated.
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
Page 4
Thymocyte development is abnormal in Abl/Arg-conditional
knockout mice
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
Page 5
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
Page 6
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
Page 7
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
Page 8
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
knockout mice.
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
Ag) experiments.
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
Page 9
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
Page 10
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
and advice.
The authors have no financial conflict of interest.
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33. Ravichandran, K. S., K. K. Lee, Z. Songyang, L. C. Cantley, P. Burn, and
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34. Walk, S. F., M. E. March, and K. S. Ravichandran. 1998. Roles of Lck, Syk and
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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,
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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,
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receptor-mediated proliferation and cytoskeletal remodeling. Curr. Biol. 16:
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You've never seen comfort food like this. (There's one that's layered with creamed spinach!)
8 Unusual Potato Chip Flavors
This quick and hassle-free recipe makes a satisfying snack that's healthy, too.
Recipe: Fruit-and-Nut Trail Mix
Although this snack may be blood-red, it is not meant for bloodsuckers, but for nut-lovers to enjoy!
Recipe: Vampire Bites Nut Mix


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From the Publisher"Every renter in California should know about [California Tenants' Rights]..." San Francisco Chronicle
"Want to break a lease? The landlord won't make needed repairs? Want to get your deposit back when you move out? The answer to these and many other questions about the magic of renting are answered in detail in California Tenant's Rights." Oakland Tribune
"Are you shopping for an apartment, or already renting one and wondering about repairs, your rights as a tenant or collecting a security or cleaning deposit when you move? Your legal rights and how to pursue them are spelled out in California Tenant's Rights." San Diego Union-Tribune


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^ It is moments like these that truely test us as fans....
^ It is moments like these that truely test us as fans....
i wonder if its because of Air Gear : /
So like...remember last time when you guys said Nagi tore off that girl's head, how come it's still attached to her body in this chapter?! harharhar =D
Street Fighter > All
yeah i was wondering that too. i thought she got decapitated unless Nagi made him envision the whole thing
And we are talking of Souhaku, don't expect compassion from him.
Anyway, great chapter.
Waiting for the HQ scan and translaton to enjoy it even more.
Last edited by Gawl; 09-21-2008 at 06:20 PM.
Street Fighter > All


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H. A. Fertig
Herbert Fertig is a Professor of Physics at Indiana University in Bloomington. He received his undergraduate degree from Princeton University and his Ph.D. from Harvard University under the direction of Bert Halperin in 1988. After a postdoctoral fellowship with Sankar Das Sarma at the University of Maryland, he joined the faculty of the University of Kentucky in 1991, moving to Indiana in 2004. Professor Fertig was elected a Fellow of the American Physical Society in 2001, was chosen as a Lady Davis Fellow for 2006–7, and in 2008, he was recognized as an Outstanding Referee for the Physical Review journals. His research focuses mostly on low-dimensional electron systems, with particular interests in quantum Hall effects, and recently on graphene.
Semiconductor Physics | Mesoscopics | Nanophysics
Measurements of the heat transport at the edges of two-dimensional electron systems appear to provide explanations about the quantum Hall state that have not been forthcoming via charge transport experiments.


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I consider myself a hard-working, honest man with passion for living that I take into everything I do. It's very important to me to maintain a positive attitude about life, because I believe our lives are what we make of them. It's also very important to me to be a trusted, trusting, loyal friend to the people in my life. They always know I'm there for them if they ever need my help. I have the strength and confidence in myself to handle whatever life throws my way with poise determination and the belief that there is a lesson to be learned in every experience. I take my happiness into my own hands, stir up my own fun and adventure in whatever I do, and I go through life with a genuine smile.
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Making friends, helping the less privileged ones and making people happy.
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by Tess WilloughbyWhen Jack was just a baby, Nate had rejected Jack's personality, and had set out to beat and berate it out of him. When Jack reached middle school age, or perhaps a little bit before, the seeds that his father had planted suddenly sprouted. Jack began to come unraveled. "Something is wrong with me," he said. "I'm messed up."Jack began to abuse his little sisters. His grades slipped. He had been my comedian, making jokes about everything all day. Now, he moped. He refused to play sports. He would not bathe.If anyone touched Jack, even to restrain him or remove dangerous objects from his hands, Nate took Jack to magistrates to swear out warrants that Jack was being … [Read more...]
Justice is No Lady: Chapter 11: Jack, the One I Lost
by Bruce GerencserNine years have passed since I preached my last sermon, since my wife was called the "pastor's wife." Almost four years have passed since my wife and I determined to stop attending church. We are now among the "unchurched", casualties of a life spent in Evangelical Christianity. Worse yet, at least according to our critics, we are now enemies of Christ and his church.You see, we not only left the Church, we left Jesus. Regardless of how some Christians parse our lives through their peculiar theological system, we are indeed two people who once were devoted, committed followers of Jesus, who are now described as an atheist husband and his agnostic wife. … [Read more...]
Raised Quiverfull: Introductory Questions
by Libby AnneWelcome to Raised Quiverfull! Nine young adults who grew up in the Christian Patriarchy and Quiverfull movements have come together to answer a series of questions about their experiences. All of these young adults have since questioned and left these ideologies and have chosen their own life paths. The goal of the Raised Quiverfull project is to inform. Click here for the Raised Quiverfull introduction.This section, Introductory Questions, showcases the participants' responses to three introductory questions. If you would like to read the questions and answers to these three questions in one document, click here. To go to the other sections and read the par … [Read more...]
We Are Spiritual Abuse Survivors!
by Paradise Recovered(I wrote this piece to honor those who have been hurt by high-demand churches and cults. I also wrote it for their loved ones, clergy, and therapists that they might understand how to handle us with care. Our experiences are unique, but much of the aftermath we experience can be similar. I hope this is helpful to someone. Peace, Andie Redwine)We are spiritual abuse survivors.To paraphrase the late Jan Groenveld, one of the pioneers in the spiritual abuse recovery movement, we believed a dangerous lie that closely resembled the truth. And we have paid dearly for that belief with the sacrifice of our very souls.Life can really be hard at times for human … [Read more...]
Millipede: Part 6
by MillipedeI hated the the underlying spirit which had crept into our marriage; it was a peevish, disapproving air that my husband had taken on. In our "heathen" days my husband had a happy go lucky air. This was endearing and made up for the fact that he could be irresponsible.Under Christian patriarchy this changed. Not only was he now "head of the household", but he was responsible for making sure that we lived a "Godly life". This permeated every bit of our life as we didn't want to be "Sunday Only" Christians; God was not someone that we turned our back on for the other six days of the week.This affected how I dressed, what we ate, what entertainment we watched or listened … [Read more...]
Introducing, Raised Quiverfull!
by Libby AnneNine young adults who were raised in families involved in the Christian Patriarchy and Quiverfull movements have come together here to answer questions about their upbringing, their questioning, and their transition to lives in the normal world. Ranging in age from their early twenties to their early thirties, all have questioned and left the Christian Patriarchy and Quiverfull movements and all but one blog against what they see as the destructive results of those movements.These nine young adults come from an array of backgrounds (no two families in the Christian Patriarchy and Quiverfull movements are identical) and have today arrived at a variety of perspectives … [Read more...]
Millipede: Part 5
by Millipede Peevish men, wives who watched every word and worse they really embraced the submission beliefs. The wives would gripe very rarely about their husbands and the work and if they did they were sure to say that they weren't in disagreement with the system, ,merely letting off a little steam.The women were just as much true believers. This isn't as I first thought/hoped in that Patriarchy can be a humane system for women. Instead I realized that there will always be those who for varying reasons, become ardent followers of movements which harm them. There are women who will accept or even come to love degradation if it comes approval and "love". Look at people in … [Read more...]
Update: Lori Wick, Christian Author Involved in Child Abuse Probe
There has been some resolution of last year's allegations of child abuse of children as young as two months old at the Black Earth, Wisconsin Aleithiea Baptist Church. Philip Caminiti, the former pastor of Aleitheia Bible Church of Black Earth Wisconsin was found guilty in March of conspiracy to commit child abuse. Yesterday he was sentenced to 2 years in prison and 6 years if probation. His probation stipulates that that during his probation term, Caminiti is not to have contact with the members of his church, though he is allowed contact with his wife, children, and grandchildren.Lori Wick and the others have yet to stand trial.Wisconsin State Journal: Black Earth pastor gets 2 … [Read more...]
Cult of Personality – Adventures in Recovery
by CaluluA few weeks ago I took my daughter out for an celebratory lunch at her favorite Greek restaurant. She's gotten acceptance letters from all of the colleges she's applied to plus we really needed to touch base, take a time out together from the busy of our lives. Over sovlaki and hummus she started talking about what she would say to our former pastor Patrick if she ran into him again. She had run into him an few months ago and had been so surprised she'd just hurriedly muttered out pleasantries before leaving him as rapidly as a man with his pants on fire would run for the lawn sprinklers.I had to ask her what she would say to Patrick if they were face to face. She blurted … [Read more...]
Karma Will Run Over Your Dogma & Squash It
by Calulu(Editorial note: What follows below is my own personal thoughts on this, no one elses, I'm not speaking for the group, just me. I cannot stay silent to this. If you're offended or triggered I'm so sorry. Warning if you are triggered by scriptures or the like.)This morning several of us at NLQ were directed to look at "Women's Prayer Group Praying That the Women at MRFF All Get Incurable Breast Cancer" posting on Free Thought Blogs.Apparently there is a group of ladies calling themselves Christians who've decided to make praying for the death of anyone connected with the Military Religious Freedom Foundation their number one priority. And why is that? Perhaps because t … [Read more...]


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A Superfly Shambles: Weekend with a Superman
Sometimes we come across government films that are so well-produced and visually appealing that it stands to reason that they must also have an effective message. Sometimes that's just not the case. Curious Alice is one such film. Weekend with a Superman is another. Produced in 1975, "Weekend with a Superman" was part of the Army's "Our […]
Posted by Jim Konicek on August 5, 2014, under Military, Motion Pictures.
1930s 1950s 1960s 1970s Academy Awards AEF American Expeditionary Forces animation Army Air Forces art aviation Civil Rights D-Day declassified Defense Visual Information Center digitization Fashion Film First Motion Picture Unit Guest blogger horses Inventions Lyndon Baines Johnson Motion Picture Preservation Motion Pictures NARA National Archives newsreels New York New York City Preservation restoration Still Pictures Training films UFOs United States Air Force (USAF) United States Army United States Information Agency Universal News Universal Newsreel US Army US Navy Washington DC World War I World War II


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Hilton Houston - Post Oak
This Houston hotel is a block from the Galleria retail and office complex. Williams Tower and Memorial Park are within two miles.
Hotel Features.
Hilton Houston - Post Oak has an outdoor pool, a sauna, and a fitness facility. Business amenities include a business center and wireless Internet access. Hilton Houston - Post Oak has a restaurant and a coffee shop/café. 24-hour room service is available. The hotel also offers a complimentary shuttle to destinations within 3 miles of the hotel.
Amenities featured in guestrooms include balconies, coffee/tea makers, and minibars. Refrigerators and microwaves are available upon request. Guestrooms have televisions with premium TV channels and pay movies. In-room safes accommodate laptops. Bathrooms feature designer toiletries and hair dryers. All guestrooms at Hilton Houston - Post Oak are non-smoking.
Friendly staff, comfortable bed, and up to date rooms. I would definitely stay here again…
Hotel was very nice, clean, staff was friendly. Didn't like that you could hear the doors…
Enter dates to check prices
Hotel Info: 866-539-8117from $109
- Standard 2 Queens
- Two Double Beds
- Standard King
- Executive, Two Double Beds
- One King Acc W Ri Shwr
- One Bedroom Suite, One King Bed
- Executive, One King Bed
- One King Bed, Non-Smoking, Accessible
- One Bedroom Suite, Two Double Beds
- Executive, Two Queen Beds
Recommended hotels
At a glance
Key facts
Hotel size
- This hotel has 448 rooms
- This hotel is arranged over 14 floors
- Check-in time starts at 3 PM
- Check-out time is noon
- Express check-in/out
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 21
Travelling with others
- Pets allowed *
- Up to 75 lb
Extra people
- Roll-away beds available, charges may apply*
- WiFi and wired Internet access in public areas *
- WiFi in rooms *
- Valet parking *
- Accessible parking
- Free area shuttle within 3 mi
- Free shopping center shuttle
In the hotel
- In-room childcare (surcharge)
- Breakfast (surcharge)
- Restaurant
- Bar/lounge
- Coffee shop/café
- 24-hour room service
- Outdoor pool
- 24-hour fitness facilities
- Golfing nearby
- Hiking/biking trails nearby
- 24-hour business center
- Number of meeting rooms - 23
- Conference space
- Meeting rooms
- Conference space size (feet) - 30000
- Conference space size (meters) - 2787
- 24-hour front desk
- Dry cleaning/laundry service
- Laundry facilities
- Hair salon
- Luggage storage
- Wedding services
- Elevator/lift
- ATM/banking
- Safe-deposit box at front desk
- Disabled parking
- Accessible path of travel
- Braille or raised signage
- Accessibility equipment for the deaf
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- Climate control
- Air conditioning
- Coffee/tea maker
- Iron/ironing board
- Hypo-allergenic bedding available
- In-room massage available
- Balcony
- Shower/tub combination
- Designer toiletries
- Hair dryer
- Pay movies
- Premium TV channels
- Video-game console
- Free newspaper
- Phone
- Refrigerator (on request)
- Microwave (on request)
- Daily housekeeping
- In-room safe (laptop compatible)
Hilton Houston - Post Oak's small print
Also known as
- Hilton Houston
- Houston Hilton
- Hotel Hilton Houston Post Oak
- Houston Hilton Hotel
- Hilton Hotel Houston
- Hilton Hotel Houston Post Oak
- Hilton Houston Post Oak
- Hilton Houston - Post Oak
- Hilton Houston - Post Oak Houston
Optional extras
Self parking costs USD 16 per night with in/out privileges
Valet parking costs USD 27 per night with in/out privileges
Pets are allowed for an extra charge of USD 75 per pet, per stay
WiFi is available in guestrooms for USD 10.95 for 24 hours (rates may vary)
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.


text: Back to Top ▲ Upgrade Your Home Cinema 0 Shares Share Tweet LG 50_A6500 50-Inch 1080p 600Hz Plasma HDTV 1/10 Upgrade Your Home Cinema List Price: $799.99Discount Price: $629.99 + Free ShippingMassive and beautiful HDTVs have never been cheaper. Do yourself a favor and bring the crystal-clear picture of the LG 50_A6500 50-Inch 1080p 600Hz Plasma HDTV to your living room. Your eyes won't know what hit them. Take off 21% until May 1st at Dell. Next Slide Comments Show Comments More Like This Drinks Toast Of The Week: Raise A Glass To These Outstanding Women Read More Wine & Dine Pot Edibles Are About To Go Fine Cuisine... Are You Ready? Read More Wine & Dine Five Mouth-Destroying Hot Sauces Only The Truly Brave Would Dare Try Read More Wine & Dine Toast Of The Week: March Madness Read More Wine & Dine This Is The Fastest And Easiest Comfort Food You've Ever Had Read More Wine & Dine This New London Gin Trend Is About To Affect What Guys Are Drinking Worldwide Read More Promoted Around the Web Special Features Special Feature From Dating To Fitness, Over 75 Ways Men Are Winning 2015 Read More AskMen Exclusive Want To Win $1,000 To Stock Your Home Bar? Read More Special Feature 50 Ways To Look Better Than You Do Now. Smooth Up, Man. Read More


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Silent Tears of A Military Mom
in Blogs
Recently my cousin messaged me, I would die if my son will be in the military. How can you live with the fear that any moment he could be sent to a war? I couldn't do it .. I would have done all I could to stop him from going in the military, and for that I admire you.
My answer to her was…I will never forget how my heart dropped when my son came to tell me he wanted to join the military. I asked him if he was sure and if that's what he wanted then.. I will support his decision 100%. He explained his reasons and I noticed he had really thought it over and was sure of his decision. I knew if he saw that his decision was breaking my heart he wouldn't go through with it. The next few months were emotionally torturing and that's when my silent tears began. The day came when we had to drive him so he could begin his journey. On my way to drop him off, I prayed for strength. The time to say our goodbyes came, he said bye to my daughter, my boyfriend and then me. That hug almost made me break down, as I hugged him in my head I scream to God for strength and to please help me hold my tears. As we pull away I saw his tears, I took a deep breath and said "Hey, don't cry you see me I'm strong and time will fly, we will see each other soon." He cracked a smirked and said "true," took a deep breath and walked away. I held my tears in till I got in my car, and I just let it out. I was not seeing my son leaving but one of my best friends.
I'm very proud of him and his achievements so far in his military journey, but I couldn't be selfish. A mother or any parent must let their child free and let them spread their wings. My faith gives me strength and yes I'm hurting but I have faith that the Lord and my grandmother are watching over him and keeping him safe. Only I know how many silent tears I've shed.
Tears To Triumph Inaugural Show
in Women
From the authors of the national best selling series Souls of My Sisters Dawn Marie Daniels, Candace Sandy and co-author Dr. Jarralynne Agee discuss their newest installment Tears To Triumph: Women Learn To Live Love and Thrive (Soulsof My Sisters/Kensington Publishing Corp.). Listen in as the women talk about why they decided to write Tears To Triumph, behind the scenes experiences and their personal thoughts. If you are facing your toughest challenge tune into Tears To Triumph on Blogtalk Radio and you will be uplifted and inspired.
Tears of Hope with Author Aimmee Kodachian
in Self Help
Aimmee is the author of "Tears of Hope," her riveting true life story of a young Middle Eastern Christian girl, who lived 13 years through the Lebanese Civil War where she escaped death 3 times and lost many close family members. Aimmee was faced with enormous obstacles and many challenges to overcome. Due to the war and her severe dyslexia, she was forced to leave school at the 4th grade, and lost her own Dream and Vision of becoming a teacher, and was left with no opportunities or Hope.
In 1988, Aimmee took the opportunity to come to the United States and start a new life. Soon after her arrival, she was told to go back to her country because, "You will never make it here." After years of persistence and determination, Aimmee found a way to rise above her obstacles and turn her Dream and Vision into Reality. Aimmee did not become a teacher; instead, she became a powerful, sought after Inspirational and Transformational Speaker, helping people see light through their darkness, and guiding them to live their True Purpose!
To learn more about how Aimmee can help you, Please visit: www.ReachingYourLight.com
Happy New Years, Tears, Hugs, Memories, The Highs and Good byes.....
As we are about to celebrate another New Year, I want to thank everyone for joining in and being a part of the show. The Americas Voice Show was started to give everyone people a say in everyday events.
Now as we close out yet another year, let us take time to look back at our past, look forward to our furture and remember and pay tribute to all of those that have helped us to get to the point in our life that we are at. Make a promise to carry them in your heart and as you think of a loved one and memories bring tears, let your mind bring back all of the good and happy times, and let this be a celebration of their lives. Hug those near you, hold them close and tell those you love how much...Remember that tomorrow is guaranteed to no one!
As I close lets all take a moment of silence for all of those that put their lives in jeopardy everyday to protect us, for those far away from their homes and in foreign lands. Say a Special prayer that they are protected and will return to their loves ones.
May God Bless America ! Everyone Have a Happy and Safe New Year !!!!!!
Tears of Hope
in Motivation
Hundreds of thousands of ordinary innocent children and women are suffering in Syria every single day. They have no HOPE and voice to TELL their story. Aimmee Kodachian, author of Tears of Hope, understands the horror and the FEAR that they go through when they hear a bomb. Get an inside feel for the war in Syria from the life of a Lebanese War Victim. Don't miss our "Artist Spotlight" featuring Jake Hughes.
John Carver Show - Bear Grylls - Adversity (Mud Sweat & Tears)
in Self Help
On today's Show I am sharing a small part of Bear Grylls book "Mud, Sweat and Tears."
Here are some quotes from this amazing book:
"Never depend on those luck moments – they are gifts – but instead always build your own back-up plan."
"You can't become a decent horseman until you fall off and get up again, a good number of times.
There's life in a nutshell."
"Time and experience have taught me that fame and money very rarely go to the worthy, by the way - hence we shouldn't ever be too impressed by either of those impostors. Value folk for who they are, how they live and what they give - that's a much better benchmark."
"Many people find it hard to understand what it is about a mountain that draws men and women to risk their lives on her freezing, icy faces - all for a chance at that single, solitary moment on the top. It can be hard to explain. But I also relate to the quote that says, Iif you have to ask, you will never understand."
"To me, my Christian faith is all about being held, comforted, forgiven, strengthened, and loved--yet somehow that message gets lost on most of us, and we tend only to remember the religious nutters or the God of endless school assemblies. This is no one's fault, it is just life. Our job is to stay open and gentle, so we can hear the knocking on the door of our heart when it comes. The irony is that I never meet anyone who doesn't want to be loved or held or forgiven. Yet I meet a lot of folk who hate religion. And I so sympathize. But so did Jesus. In fact, He didn't just sympathize, He went much further. It seems more like this Jesus came to destroy religion and to bring life."
"If you can find a path with no obstacles, it probably doesn't lead anywhere."
Tears of Silence with Lakisha Louissaint
in Religion
Living Out Loud Radio Broadcast I www.larussiacainministries.com I Host La Russia Cain
Author Lakisha Louissaint will be our Co- Host, and we will be discussing her book "Tears Of Silence."
Silence is deadly. Just because you don't acknowledge the pain, does not mean that it is not there. It simply means that it is waiting for something or someonme to trigger it to resurface. Lakisha will share her story of how she overcame depression, rejection, being molested, high school drop out, losing a mother and much more.
To connect with Lakisha Louissaint:
Facebook- www.facebook.com/lakishalouissaint
Website: www.IamLakisha.com
Years of Tears
In observance of Domestic Violence Awareness Month we will be talking Maxine Browne, author of "Years of Tears". We will talk to about what influenced her to write this book, what is domestic violence and how can it be prevented. Don't miss the "Artist Spotlight" featuring C.J. Luckey.
Open Treasures: "Tattoo Tears Of A Child"
in Christianity
We're dealing with a generation of children who are coming from broken homes, dysfunctional families, abuse, and neglect that has created what we call "Tattoo Tears". You may wonder why they act out the way they do and why they appear to carry so much anger that you can't seem to understand. You'll be surprised to know that many of them don't even understand what's going on with themselves. Join us as we attempt to dissect the war going on within and what is truly behing these tears of anger, bitterness, and resentment. This is a show you truly don't want to miss.
Oh that my head were waters, and mine eyes a fountain of tears
in Religion
"Oh that my head were waters, and mine eyes a fountain of tears that I might weep day and night for the slain of the daughter of my people! Oh that I had in the wilderness a lodging place of wayfaring men; that I might leave my people, and go from them! for they be all adulterers, an assembly of treacherous men. And they bend their tongues like bow for lies: but they are not valiant for the truth upon the earth;......"
LETTING THE TEARS ROLL......are you able to do that?
in Self Help
Are you able to do that? I believe it is very healthy, and GOOD for us to CRY!
It releases stress, negativity, tension, and all unhealthy blocks in our bodies. Think about it, Children cry and just because our bodies have grown up we still have within the child and sometimes the child to be able to LET THE TEARS ROLL.....are you able to do that?
Join me Tuesday February 3rd @ 4pmPAC/7pmEST to discover WHY it is healthy to CRY! I will talk about the importance of tear ducts to our body, why God created tears to begin with and the benefits you receive from it!
We will have a great meditation, powerful affirmations,and more. So, join me Tuesday Feb.3rd right here @www.blogtalkradio.com/buzzardskorner-of-love for a powerful converastion, and the importance of. LETTING THE TEARS ROLL......are you able to do that?
Raising Your Daughter Through the Joys, Tears and Hormones! By C. Lynn Williams
in Parents
The "What Books Sound Like" Series will launch on Chicago's Black Business RADIO Network (CBBRN) on Tuesday, February 10, 2015.
This is where authors from around the world bring their books to life for YOU the reader and lover of words. CBBRN will feature book excerpts from authors from around the world.
Join us for this treasured reading from author C. Lynn Williams on "Raising Your Daughter Through the Joys, Tears and Hormones!"
C. Lynn Williams, also known as Ms. Parent Guru, is an:
Keynote speaker
Workshop presenter
Generational Development Strategist
Wife and Mother
More information and other books at http://www.clynnwilliams.com
Are you a published author? You will find more information on how you can join us at YOUR BOOK ON FIRE!
Ms. Sonja Cassandra Perdue, Executive Producer - Chicago's Black Business Radio Network
Author of "Black America: Asking Ourselves The Tough Questions." Now available on Amazon.com
Click HERE to order your autographed copy of "The Tough Questions."
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text:
Rotterdam-based architects MVRDV recently won the Gwanggyo City Centre Competition with their design of this incredible new city just south of Seoul, South Korea. Envisioned as a verdant acropolis of organic 'hill' structures, the proposed complex is a fully self-sufficient city for up to 77,000 inhabitants. Similar nodes, common in South Korea, concentrate residences, work and play all in one interactive center, reducing dependency on auto or train travel and building a strong sense of community.
MVRDV's masterplan provides space for housing, offices, shops, and educational facilities. Designed as a cluster of structures rising up in concentric rings, each floor in the city is lined with lush box hedges that improve ventilation while reducing energy and water usage. An internal irrigation system stores extra water from the buildings and uses it to sustain these green facades. MVRDV states: "This diverse program has different needs for phasing, positioning and size. To facilitate this all elements are designed as rings. By pushing these rings outwards, every part of the program receives a terrace for outdoor life."
The concept is currently being reviewed for development and feasibility at Gyeonggi provincial authority's Urban Innovation Corporation. Development is to be completed in 2011 and construction will start shortly after.
6 Comments
Real life influences | ... March 3, 2015 at 9:57 am
tomako3a March 9, 2013 at 1:31 am
"Development is to be completed in 2011 and construction will start shortly after"
It's 2013, has any real progress been made on this? I can't find anything on the web, did this project just die?
Bogdi January 2, 2013 at 10:31 am
And here are some other similar projects from early 2000:
baoan January 2, 2013 at 10:18 am
This project amazingly reminds me the Zaha Hadid's projects Galaxy Soho:
or Wangjin SOHO: http://www.archdaily.com/313492/zaha-hadid-seeing-double-in-china/05northwestaerialnight/
elale33 June 30, 2009 at 10:37 pm
Wow Super!!!!!!
MrSustainable December 23, 2008 at 4:02 pm
As Buckminster Fuller has stated. The most inefficient part of any city is that the pencil shaped sky scraper. It is the most inefficient for heating and transportation. This is a good concept on the wrong shape. If all the tall building were in a circle with the wide short building in the middle all connected by rings and spokes lets say it would be more functional.
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