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B Activation
*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Lausanne, Switzerland; and
Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B inhibitor IB
(mut-IB)
and to study the maturation defects occurring when NF-B activation
is inhibited during fetal development. Fetal thymocytes infected with
adenovirus containing mut-IB were found to develop normally until
the CD44-CD25+,
CD4-CD8- double-negative stage, while
production of more mature double-positive and single-positive
populations was strongly decreased. Proliferation, as measured by the
percentage of cells in cycle appeared normal, as did rearrangement and
expression of the TCR ß-chain. However, apoptosis was much higher in
FTOC infected with adenovirus containing mut-IB than in FTOC
infected with a control virus. Taken together, these results suggest
that NF-B plays a crucial role in ensuring the differentiation and
survival of thymocytes in the early stages of their
development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As stem cells expressing low levels of CD4 migrate into the cortex of
the thymus, they down-regulate CD4 and become
CD4-CD8-
(DN)3 prothymocytes, which
express CD44 but not CD25 3 . Then CD25 is up-regulated, closely
followed by down-regulation of CD44. As DN thymocytes reach their most
mature state, expression of CD25 is gradually lost 4 . During the DN
stage, the TCRß gene is rearranged 5, 6 , and upon expression of the
pT gene product, as part of the pre-TCR complex, further
differentiation and expansion occur 7 . After transition through an
immature single-positive (SP) stage, during which CD8 is expressed but
not the mature TCR, cells become double positive (DP), expressing both
CD4 and CD8. At this stage TCR is rearranged, and a mature TCRß
is expressed at intermediate levels on the cell surface. Next, the
cells move to the medulla through the cortico-medullary junction where
they are positively selected for self-MHC recognition and negatively
selected for recognition of self peptides. Finally, either the CD4 or
the CD8 coreceptor is down-regulated, and TCRß is up-regulated to
form mature, SP cells that ultimately migrate to the periphery 8, 9 .
The differentiation steps described above result from a sequence of
signaling events. Among candidate mediators of maturation signals are
members of the transcription factor family NF-B/Rel. NF-B is a
ubiquitous, constitutively expressed factor that is normally kept in an
inactive form in the cytoplasm by IB. Upon receiving an activation
signal, IB is rapidly phosphorylated and degraded. NF-B then
moves to the nucleus, where it induces the transcription of a variety
of genes 10, 11, 12 . A role for NF-B in the regulation of thymocyte
development is suggested by its high expression in the thymus and the
fact that NF-B family members are differentially expressed in the
thymic compartments; RelA is expressed in the cortex, while RelB and
c-Rel are preferentially expressed in the medulla 13, 14, 15 . Several
groups have tried to elucidate the role of NF-B in thymocyte
development by studying mice deficient in the genes coding for
individual family members or by overexpressing factors that inhibit
NF-B activation. Mice deficient for individual NF-B/Rel subunits
exhibit no intrinsic defects in T cell development. However, gene
targeting approaches are complicated by the functional redundancy that
exists in the family; for example, complexes containing either c-Rel or
RelA have the potential to stimulate transcription from the same
promoter 16, 17 . To circumvent this problem two groups have recently
made transgenic mice expressing the NF-B inhibitor IB under a
lymphocyte-specific promoter. This inhibitor functions as a
constitutive repressor of multiple NF-B/Rel proteins. Although it
was shown that the total number of thymocytes was decreased in these
mice and that there was a defect in their development 18, 19 , this
system does not allow us to pinpoint the exact developmental stage at
which NF-B is involved. We used a novel technique combining
adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC)
to determine at which stage of fetal thymic development NF-B is
critical. We infected FTOC with an adenovirus containing an IB cDNA
(mut-IB) from which the N-terminal phosphorylation sites have been
removed, resulting in an IB with a prolonged half-life 20 . Using
this technique, we show that inhibition of NF-B activation results
in impaired transition from the CD44-CD25+ to
the CD44-CD25- developmental stage and an
increased sensitivity to apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The AdCMVß-gal recombinant virus was constructed as previosuly described 21 . Briefly, a 3.5-kb HindIII/BamHI fragment containing the Escherichia coli lacZ gene was ligated into the viral plasmid pACCMV95 to produce pACß-gal. pACCMV95 contains the 5' 6242 bp of Ad-5 from which the region 454-3328 bp has been deleted (the deletion incorporates all of the E1A and part of the E1B region) and includes a CMV intermediate-early promoter upstream of the cloning site and an SV40 polyadenylation signal downstream. The pACß-gal was cotransfected together with JM17 (which contains an Ad-5 viral backbone) into 293 cells. Homologous recombination between the two plasmids resulted in the production of infectious recombinant adenovirus expressing the bacterial ß-galactosidase (ß-gal) protein.
A similar strategy was used to create AdCMVmut-IB 22 . To make the
mut-IB construct, the Xho-BamHI fragment of
MAD3pBS (containing a human IB cDNA lacking phosphorylation
sites) 20 was ligated to the nuclear localization sequence of the
proto-oncogene c-myc. The blunt/XbaI fragment
containing mut-IB without a promoter was then ligated into the viral
plasmid pACCMV95 and cotransfected with JM17 into 293 cells.
Transfected 293 cells were harvested, and individual plaques were isolated and purified as described previously 23 . Large stocks were produced and concentrated before being titrated by plaque and limiting dilution assays. Concentrated stocks typically had titers in the range of 108–1010 pfu/ml.
Mice
Pregnant C57BL/6 mice were purchased from Harlan CPB (Zeist, The
Netherlands) or were bred under standard conditions in the animal
facilities of the Swiss Institute for Experimental Cancer Research
(Epalinges, Switzerland). IB transgenic mice 18 were
generated and bred under standard conditions.
Fetal thymus organ culture
Fetal thymi were isolated from C57BL/6 mice or IB
transgenic mice on day 14 of gestation and transferred to membranes of
a transwell system (Costar, Cambridge, MA). Eight to ten lobes were
cultured per well. The lower compartment was filled with complete
DMEM-10 medium 24 containing 2-ME and 107 pfu of either
AdCMVmut-IB or AdCMVß-gal. To obtain the data shown in Fig. 2
and
Table I, either 107 or
109 pfu of virus was used. Both medium and virus were
changed every 3 days. After 6 days or as indicated in the figure
legends, thymic lobes were harvested and mechanically disrupted.
Approximately 80 lobes/experiment were analyzed, which were normally
divided into pools of eight lobes. Single cells were stained and
analyzed by FACS.
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Single-cell suspensions of thymocytes were incubated on ice with
saturating concentrations of the following Abs: anti-CD4-CyChrome,
anti-CD4-PE, anti-CD8-CyChrome, anti-CD3-PE,
anti-TCRß-PE, anti-TCRß-CyChrome, and
anti-CD44-CyChrome (PharMingen, San Diego, CA); hamster IgG-PE,
anti-TCR
tri-color and anti-CD25-PE (Caltag, San
Francisco, CA); anti-CD4-R613 and anti-CD8-R613 (Life
Technologies, Gaithersburg, MD); FITC-labeled anti-CD44 (Pgp-1)
25 ; and FITC-labeled anti-CD8 H35 26 . Intracellular staining
was performed as previously described 27 . Three- and four-color
analyses were performed on FACScan and FACStar Plus cytofluorometers,
respectively, using LYSIS II software (Becton Dickinson, Mountain View,
CA).
Fluorescent ß-gal assay
Detection of ß-gal by FACS was performed as previously described 28 . Cells previously stained with surface Abs were incubated at a density of 106–107 cells/ml in 100 µl of PBS/FCS during 5 min at 37°C. Then they were hypotonically loaded with the ß-gal substrate, fluorescein di-ß-D-galactopyranoside (Sigma, Buchs, Switzerland), for 1 min. Loading was stopped by the addition of PBS containing 300 µM chloroquine. After 1 h of incubation on ice, cells were analyzed by FACS.
Electrophoretic mobility shift assay
Fetal thymocytes were put into culture and were infected with
either AdCMVß-gal or AdCMVmut-IB. After 2 days, thymocytes
were harvested, and electrophoretic mobility shift assays were
performed on crude nuclear extracts as described previously 29 .
Briefly, extracts were incubated with a 32P-labeled
double-stranded oligonucleotide derived from the B enhancer of the
mouse light chain locus before separation in an 8% acrylamide gel
in 0.25x TBE. The amount of probe trapped in a DNA/protein complex was
quantitated by exposing the dried gel to an imaging plate of an
autoradiography system (BAS, Fuji, Tokyo, Japan). Protein
concentrations in extracts were determined using the bicinchoninic acid
protein assay (Pierce, Rockford, IL) to ensure equal loading.
Bromodeoxyuridine (BrdU) assay
To detect cell proliferation, BrdU (1 µg/ml) was added to the cultures 10 h before harvest. Thymic lobes were mechanically disrupted, and single cells were stained with Abs detecting cell surface Ags. Cells were then resuspended in 25 µl of 0.15 M NaCl, after which 70 µl of ice-cold 95% ethanol was added. After 30 min incubation on ice, cells were washed and incubated in 100 µl of PBS containing 1% paraformaldehyde and 0.01% Tween-20. Fixed cells were washed and stained with anti-BrdU-FITC (Becton Dickinson) in the presence of 0.5% Tween after blocking nonspecific binding with rabbit anti-mouse IgG for 1 h.
Cell cycle analysis
Different populations within the DN compartment were sorted by FACS. Cell suspensions were then fixed with 70% ethanol at 4°C for 30 min, washed in PBS, and stained with 50 µg/ml propidium iodide. DNA content was analyzed using doublet discrimination on FACS.
Quantitation of apoptosis by annexin V-FITC
To quantify apoptosis, thymic lobes were mechanically disrupted and incubated at 37°C for 1 h. After incubation, 105–106 cells were washed in 100 µl of binding buffer. Then they were incubated with a 1/500 dilution of annexin V-FITC (NeXins Research, Leiden, The Netherlands) for 20 min on ice before apoptosis was quantified by FACS.
Quantitation of apoptosis by TUNEL
Thymic lobes were mechanically disrupted and incubated at 37°C for 3 h. Cells undergoing DNA fragmentation were detected with the TUNEL method (terminal deoxyribonucleotidyl transferase labeling of DNA strand breaks with BrdU; Boehringer Mannheim, Indianapolis, IN), according to the manufacturer’s instructions. Stained cells were analyzed by FACS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of NF-B in fetal thymocyte development, we
developed a system for adenovirus-mediated gene expression in FTOC. To
monitor whether fetal thymocytes can be infected with adenovirus, fetal
thymi were placed in culture on days 14–15 of gestation in the
presence of 107 pfu AdCMVß-gal. This virus directs
expression of ß-gal, allowing easy monitoring of infection based on
enzymatic activity. Fetal thymi cultured under the same conditions in
the presence of 107 pfu AdCMVmut-IB were used as a
control for the ß-gal assay. At different times after infection cells
were harvested and treated with a ß-gal substrate (fluorescein
di-ß-D-galactopyranoside) that fluoresces after cleavage,
and fluorescence was measured by FACS. Virtually all fetal thymocytes
cultured with AdCMVß-gal (Fig. 1B,
hatched histograms) showed fluorescent staining regardless of their
stage of maturation, while control thymocytes infected with
107 pfu AdCMVmut-IB remained negative (empty
histograms). Similar infection levels were found at all time points
analyzed (after 2, 4, 6, or 7 days of culture), and development
kinetics of AdCMVß-gal-infected thymocytes were comparable to those
of uninfected thymocytes (data not shown).
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B in fetal thymocyte development
To investigate the influence of NF-B on fetal thymic
development, fetal thymi were taken on days 14–15 of gestation,
transferred to Transwell plates (Costar, Cambridge, MA), and
infected with AdCMVmut-IB. This virus expresses a mutant IB
with a prolonged half-life, which functions as a constitutive repressor
of NF-B 20 . Electrophoretic mobility shift assays using nuclear
extracts from fetal thymocytes cultured for 2 days in the presence of
either viral vector revealed a fourfold or higher reduction in the more
slowly migrating, active form of NF-B in the AdCMVmut-IB-infected
thymocytes (Fig. 1
A). Earlier studies had shown that this
band consists of a mixture of RelA, c-Rel, and presumably RelB
complexed to NF-B1/p50 14, 15, 30 .
Thymocyte development was analyzed after 6 days of culture in the
presence of the virus and compared with the development of thymocytes
that were infected with the control virus AdCMVß-gal. Fig. 2A shows that in the
thymocytes infected with 107 pfu AdCMVmut-IB there was a
twofold relative increase in the CD4-CD8- DN
population compared with that in thymocytes infected with the same
amount of AdCMVß-gal, while there was a twofold relative decrease in
the DP compartment. Although less pronounced than in the DP population,
a decrease could also be observed among the CD4+ SP cells.
The total CD8+ SP compartment was not significantly
decreased, but if only mature CD8+ SP cells are considered
(as defined by high levels of TCRß expression), a significant
reduction in the proportion of these cells was observed following
AdCMVmut-IB infection. As the total number of thymocytes in
AdCMVmut-IB-infected thymi was twofold less than that in those
infected with AdCMVß-gal (Table I), there was no significant
difference in absolute numbers in the DN population, while the
reduction in the DP and mature SP populations was highly significant
(p < 0.0001; Fig. 2C).
Interestingly, FTOC from IB transgenic mice 18 showed a similar
impairment of thymocyte maturation to the DP stage as FTOC infected
with 107 pfu AdCMVmut-IB, while FTOCs infected with
107 or 109 pfu AdCMVß-gal developed in a
similar way as uninfected FTOC from C57BL/6 mice (Fig. 3). When thymocytes were infected with
109 pfu of AdCMVmut-IB, DP and SP populations were
virtually absent (Fig. 2, B and C).
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, we observed a twofold decrease in the
most mature, CD44-CD25- DN population, in
AdCMVmut-IB-infected thymic lobes relative to those infected with
AdCMVß-gal. This decrease is paralleled by a proportional increase in
the CD44-CD25+ population that directly
precedes it in the thymic development pathway, suggesting a
developmental block at the stage where the expression of CD25 is
down-regulated.
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Since the developmental block we observed after AdCMVmut-IB
infection occurs at the same stage as those found in
recombinase-activating gene (RAG)-/- and
TCRß-/- mice, which are both unable to express the
TCRß gene 31, 32, 33, 34 , we checked whether TCRß was normally expressed
in the CD25+CD44- DN population. Intracellular
staining for TCRß revealed that its expression was not decreased in
AdCMVmut-IB-infected CD44-CD25+ DN
thymocytes; 68 ± 3% stained positive for intracellular TCRß
compared with 52 ± 3% of those infected with AdCMVß-gal
(Fig. 5).
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shows that there were more cells of
the AdCMVmut-IB-infected FTOC proliferating in the
CD44-CD25+ population, while there was no
significant difference in proliferation rate in the
CD44-CD25- population. These data were
confirmed by cell cycle analysis using propidium iodide (Fig. 7).
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Because NF-B was found to play an important role in ensuring
the survival of cells 35, 36 , we also investigated whether
AdCMVmut-IB-infected thymocytes were more susceptible to
apoptotic death than AdCMVß-gal-infected thymocytes. After 6 days of
culture thymocytes were stained with annexin V, a ligand of the
phosphatidylserine that is expressed on the surface of apoptotic cells,
together with Abs that detect surface markers. Fig. 8A shows that there was a
significant increase in apoptosis, as measured by the proportion of
annexin+ cells, in the CD44-CD25-
DN population of AdCMVmut-IB-infected cells compared with that in
the population of AdCMVß-gal-infected cells. Apoptosis in the
CD44-CD25+ DN population was also increased,
although less so than in the CD44-CD25- DN
population. Similar results were obtained using the TUNEL method 37
(Fig. 8B). The higher level of apoptosis-positive cells seen
in the TUNEL assay is caused by a longer incubation at 37°C. In
populations containing more mature thymocytes (DP and SP), no
significant differences in levels of apoptosis were found (data not
shown).
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B-infected
sorted cells remained alive as judged by uptake of trypan blue, while
12% of the AdCMVß-gal infected cells survived. As
CD44-CD25+ cells are known to down-regulate
CD25 within this time frame, these data support the results obtained
with the annexin and TUNEL assays. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B expression targeted to lymphoid
lineage cells showed maturational defects very similar to those of
wild-type thymocytes infected with AdCMVmut-IB. This strongly
suggests that the observed effects are intrinsic to the thymocytes and
are not due to an effect in trans of the stromal cells. Our system
makes it possible to pinpoint the exact stage in fetal development at
which NF-B is involved, since such critical parameters as the time
in development at which NF-B is inhibited can be rigorously
controlled, and thymocyte development can be studied in isolation of
other organs.
As a result of NF-B inhibition, we observed a partial block in the
maturation of CD4-CD8- DN thymocytes between
the CD44-CD25+ and
CD44-CD25- stages. A complete block at this
stage was also observed in RAG and TCRß knockout mice 31, 32, 33, 34 . In
both, the TCR ß-chain cannot be rearranged, and cells therefore do
not enter the cell cycle due to the inability to form a pre-TCR
complex. In either of these mouse strains, cross-linking of CD3
results in proliferation and transition to the DP stage 42, 43 . In
thymocytes infected with AdCMVmut-IB we have rescued maturation at
the CD44-CD25+ stage in a similar manner by
cross-linking CD3 (data not shown). In our system, however, TCRß
expression appeared to occur normally. Indeed, a slightly higher
percentage of cells at the CD44-CD25+ DN stage
expressed intracellular TCRß protein in the AdCMVmut-IB-infected
thymocytes compared with the AdCMVß-gal-infected thymocytes, implying
that NF-B is not necessary for expression of TCRß. Furthermore,
the percentage of cycling cells in the
CD44-CD25+ DN subset was similar in
AdCMVmut-IB-infected thymocytes to that observed in
AdCMVß-gal-infected thymocytes, suggesting that the pre-TCR complex
is functional.
Since NF-B family members are differentially expressed in the
cortical and medullary compartments, it is likely that NF-B has more
than one function in thymocyte development. Our results could be
interpreted as the consequence of two concurrent effects: first, a
delayed down-regulation of CD25, and second, a massive apoptosis in
CD44-CD25- cells. It is also likely that the
function of NF-B in thymocyte development is not identical in adults
or fetuses, as maturation pathways in these two life stages are known
to differ 3 . Multiple functions for NF-B could explain the
differences between the maturation defects observed in adult IB
transgenic mice, on the one hand, and in FTOC from the same transgenic
mice or from normal mice infected with AdCMVmut-IB, on the other
hand. In the first case, cells develop normally until the DP stage
18 , whereas in the other two cases perturbation of maturation starts
at earlier stages.
Earlier studies indicate that NF-B plays an important role in
regulating apoptosis in different cell types. In RelA knockout embryos,
hepatocytes undergo massive apoptosis on day 15 of gestation, and
RelA-deficient fibroblasts and macrophages are more sensitive to
killing by TNF 35, 36 . It has also been suggested that Rel-related
proteins protect B lymphocytes from apoptosis 44 . Boothby et al.
found that primary T cells expressing a mutant IB are more
susceptible to apoptosis induced by stimulation through the TCR, which
suggests a protective role for NF-B 19 . Here we show that
CD44-CD25- DN thymocytes infected with
AdCMVmut-IB are more susceptible to apoptosis than those
infected with AdCMVß-gal. This suggests that NF-B plays a role in
protecting thymocytes from undergoing apoptosis in early stages of
development. It is not yet clear whether the antiapoptotic functions of
NF-B also involve extracellular pathways. Transferred into normal
mice, fetal liver cells deficient for NF-B1/p50 and RelA were did
not undergo any T cell development. However, when doubly deficient
fetal liver cells were mixed with normal fetal liver cells, both the
normal liver cells and the deficient liver cells developed normally
45 . This suggests that NF-B can influence thymocyte development by
means of an extracellular factor. Furthermore, many cytokines are known
to be regulated by NF-B 46 , while other studies show that defined
cytokines are required for proliferation and development at different
stages of thymocyte development 47, 48 . It thus seems reasonable to
speculate that NF-B-regulated cytokines may play a role in ensuring
the survival of early thymocytes. Besides cytokines, it is also
possible that other intra- or extracellular factors are involved.
Bcl-2, for example, is known to be highly expressed in
CD44-CD25+ thymocytes and has been shown to
protect different cell types from undergoing apoptosis 49 . Taken
together, our data indicate that if NF-B is kept inactive during
early fetal thymocyte development, cells develop normally until the
CD44-CD25+ DN stage. At this stage, activation
of NF-B is required to convey a signal without which the cells will
fail to develop further and will eventually die by apoptosis. Whether
this signal induces the production of intrinsic or extrinsic factors
remains to be resolved.
In summary, we have established a novel infection system to express
relevant genes in FTOC, allowing a detailed dissection of the complex
molecular and cellular interactions implicated in thymic development.
We were able to precisely describe thymic maturation defects that occur
when NF-B is inactivated from day 14 of gestation. We are currently
in the process of generating new adenoviral constructs to study the
role of other genes of potential importance in lymphocyte development.
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Acknowledgments |
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B transgenic mice, and Anne Wilson, Myriam Capone, and
H. Robson MacDonald for discussion and critical comments on the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. C. V. Jongeneel, Ludwig Institute for Cancer Research, chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address:
3 Abbreviations used in this paper: DN, double negative; SP, single positive; DP, double positive; FTOC, fetal thymic organ culture; pfu, plaque-forming units; PE, phycoerythrin; BrdU, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling; ß-gal, ß-galactosidase; RAG, recombinase-activating gene.
Received for publication August 10, 1998. Accepted for publication December 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-like activity. Cell 65:1281.[Medline]
B is necessary for activation of transcription factor NF-B. Nature 365:182.[Medline]
B transcription factor involves dissociation of an apparently cytoplasmic NF-B/inhibitor complex. Cold Spring Harb. Symp. Quant. Biol. 53:789.
B activities in mouse thymus and spleen. Oncogene 9:3289.[Medline]
B-related proteins in thymocytes. J. Immunol. 154:3213.[Abstract]
B site-dependent activation of the interleukin-2 receptor -chain gene promoter by human c-Rel. Mol. Cell. Biol. 12:4067.B/Rel proteins and interferon regulatory factors. J. Virol. 68:4707.B. J. Immunol. 158:5075.[Abstract]
B. J. Exp. Med. 185:1897.B proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485.B inhibitor sensitises melanoma to tumour necrosis factor-mediated apoptosis. Int. J. Cancer 80:320.[Medline]
B by TNF. FEBS Lett. 351:45.[Medline]
B/Rel protein complexes. J. Immunol. 155:4653.[Abstract]
and ß block thymocyte development at different stages. Nature 360:225.[Medline]
-induced apoptosis by NF-B. Science 274:787.B in preventing TNF-induced cell death. Science 274:782.ß T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
B/Rel induces apoptosis of murine B cells. EMBO J. 15:4682.[Medline]
B-deficient fetal liver cells. Immunity 6:765.[Medline]
B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
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