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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-06-1855.
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Blood, 1 May 2003, Vol. 101, No. 9, pp. 3550-3559
IMMUNOBIOLOGY
Promoter IV of the class II transactivator gene is essential
for positive selection of CD4+ T cells
Jean-Marc Waldburger,
Simona Rossi,
Georg A. Hollander,
Hans-Reimer Rodewald,
Walter Reith, and
Hans Acha-Orbea
From the Institute of Biochemistry and Ludwig
Institute for Cancer Research, Lausanne Branch, University of Lausanne,
Epalinges, Switzerland; Department of Genetics and
Microbiology, University of Geneva, Medical School, Geneva,
Switzerland; Pediatric Immunology, Departments of Research
and Clinical-biological Sciences, and the Children's Hospital,
University of Basel, Switzerland; Department for
Immunology, University Clinics Ulm, Germany.
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Abstract |
Major histocompatibility complex class II (MHCII)
expression is regulated by the transcriptional coactivator CIITA.
Positive selection of CD4+ T cells is abrogated in mice
lacking one of the promoters (pIV) of the Mhc2ta gene. This
is entirely due to the absence of MHCII expression in thymic epithelia,
as demonstrated by bone marrow transfer experiments between wild-type
and pIV / mice. Medullary thymic epithelial cells
(mTECs) are also MHCII in pIV/ mice.
Bone marrow-derived, professional antigen-presenting cells (APCs)
retain normal MHCII expression in pIV/ mice, including
those believed to mediate negative selection in the thymic medulla.
Endogenous retroviruses thus retain their ability to sustain negative
selection of the residual CD4+ thymocytes in
pIV/ mice. Interestingly, the passive acquisition of
MHCII molecules by thymocytes is abrogated in pIV/
mice. This identifies thymic epithelial cells as the source of this
passive transfer. In peripheral lymphoid organs, the CD4+
T-cell population of pIV/ mice is quantitatively and
qualitatively comparable to that of MHCII-deficient mice. It comprises
a high proportion of CD1-restricted natural killer T cells,
which results in a bias of the V repertoire of the residual
CD4+ T-cell population. We have also addressed the identity
of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common  chain (CD132) or common chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.
(Blood. 2003;101:3550-3559)
© 2003 by The American Society of Hematology.
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Introduction |
Major histocompatibility complex class II
(MHCII) molecules are crucial for the development, survival, and
activation of CD4+ T cells. Recognition of
self-peptide-MHCII complexes on cortical thymic epithelial cells
(cTECs) determines whether or not immature CD4+ thymocytes
are positively selected.1 Negative selection, mediated by
MHCII+ thymic dendritic cells (DCs),2 results
in the deletion of autoreactive CD4+ T cells. In the
periphery, peptide-MHCII complexes expressed on antigen-presenting
cells (APCs) induce the activation, proliferation, and differentiation
of CD4+ T helper cells. The central importance of these
functions is illustrated by the phenotype of MHCII-deficient mouse
strains,3-5 by the clinical course of patients suffering
from bare lymphocyte syndrome (BLS),6-10 and by CIITA and
RFX5-deficient mice, 2 murine models of BLS.11-14 Patients
with BLS suffer from a severe primary immunodeficiency that is entirely
attributable to the nearly complete absence of MHCII
expression.6-10 Patients with BLS, as well as mice
deficient for MHCII, CIITA, and RFX5, exhibit impaired maturation of
CD4+ T cells and are unable to mount efficient
CD4+ T helper cell-dependent immune responses.
CIITA is one of the 4 genes affected in BLS.6,7 It now is
widely recognized to be the master regulator of MHCII expression. In
most instances, it is the expression of CIITA that dictates the tightly
controlled pattern of MHCII expression. Only MHCII+ APCs (B
cells, DCs, macrophages) express CIITA constitutively. The expression
of CIITA, and thus of MHCII genes, can be activated in
MHCII cells by stimulation with interferon (IFN).15-23 A large and complex regulatory region
containing several independent promoters controls transcription of the
Mhc2ta gene. Of the 4 promoters identified in the
human gene, 3 (pI, pIII, and pIV) are strongly conserved in the mouse.
Promoter I is highly specific for DCs. Promoter III is used primarily
in B cells but is also active in certain human DC
preparations.19,24,25 Promoter IV is largely responsible
for IFN-induced expression.
We have recently generated mice carrying a targeted deletion of
promoter IV (pIV) (Figure 1).
pIV/ mice were engineered to carry a deletion of
approximately 500 base pair (bp), encompassing exon IV and its
associated promoter. Transcription from the remaining promoters (pI and
pIII) is unaffected by the deletion of pIV.25 This ensures
normal levels of basal and activated MHCII expression on B cells,
macrophages, and DCs in the thymus and periphery of
pIV/ mice (Figure 1). IFN-induced MHCII expression
on extrahematopoietic cells is on the other hand completely abrogated
in pIV/ mice.
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| Figure 1.
Selective loss of MHCII expression in
pIV/ mice.
Mice with a targeted deletion of CIITA pIV lack IFN-induced MHCII
expression on nonhematopoietic cells and constitutive MHCII expression
on cortical thymic epithelial cells (cTECs) and mTECs. Professional
APCs retain IFN-induced CIITA expression via pI (microglia,
macrophages) and constitutive expression via pI (DCs) and pIII (B
cells, DCs).
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Surprisingly, pIV/ mice also lack MHCII
expression in the thymic cortex.25 This results in a
defect in cTEC-mediated positive selection and in drastically reduced
numbers of CD4+ T cells in the thymus and periphery. The
periphery of pIV/ mice contains ample numbers of
MHCII+ DCs, B cells, and macrophages. Interactions between
the T-cell receptor (TCR) of CD4+ T cells and MHCII
molecules on peripheral APCs are believed to be crucial for the
survival of CD4+ T cells.26,27
CD4+ T cells should thus encounter a favorable environment
for their survival in pIV/ mice. These mice therefore
represent an unprecedented model in which to study the fate of the
residual CD4+ T cells generated in the absence of the
"classical" positive selection pathway.
In this study we characterized the residual population of
CD4+ T cells in pIV/ mice. We further
defined MHCII expression in the thymus of pIV/ mice and
analyzed negative selection by endogenous retroviruses. To address the
question on the transcription factors required for constitutive MHCII
expression on cTECs, we analyzed positive selection of CD4+
T cells in mice lacking stromal expression of interleukin-7 receptor (IL-7R) or the common gamma chain.
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Materials and methods |
Mice and generation of bone marrow chimeras
Mice carrying a deletion of pIV of the Mhc2ta gene
were generated previously in our laboratory.25
I-A / mice were a generous gift from H. Bluethmann5 (Hoffman La Roche, Basel, Switzerland).
IL-7R-deficient mice were previously described.28 To
introduce a functional I-E allele in the original Sv129-C57Bl/6
background, pIV/ mice were backcrossed to B10BR
animals. F2 offspring were genotyped by polymerase chain reaction
(PCR) to distinguish between the wild-type (primers F1 + r1)
and deleted (primers F2 + r2) alleles of pIV: F1,
5'-CCTAGGAGCCACGGAGCTG-3'; r1, 5'-TCCAGAGTCAGAGGTGGTC-3'; f 2, 5'-CAGACTATCCTGAAA-TGCC-3'; r2, 5'-CGAGATCTAGATATCGATAAGCTTG-3'. pIV/ F2 progeny were tested for I-E expression by
fluorescence-activated cell-sorter scanner (FACS). All other
experiments were performed with CIITA pIV/ and
pIV+/ littermates on a mixed Sv129-C57Bl/6 background.
Mice 8 to 12 weeks of age were used to generate bone marrow chimeras. A
total of 107 cells depleted of mature CD4+
cells (by negative selection using monoclonal anti-CD4 antibodies and
magnetic beads [Dynal, Oslo, Norway] according to the manufacturer's instructions) were transferred per recipient (irradiated with 3 Gy).
Mixed bone marrow chimeras were reconstituted with an equal number of
bone marrow cells derived from CD45.2 pIV/ mice and
CD45.1 congenic C57BL/6 mice.29 All bone marrow transfer experiments involving pIV/ mice (Table 1; Figures 3 and
6) were performed using the congenic markers Ly5.1 and Ly5.2. Reconstitution of the chimeras was allowed for
2 months. The analysis of reconstituted IL-7R-deficient mice did not
require the use of congenic markers, because few endogenous CD4+ T cells are produced. The same applies to nude mice
reconstituted with c-kit, gamma-c-deficient thymuses (Figure
8). c-Kit, c-deficient thymuses30 were transplanted
under the kidney capsule of nude mice. The thymocytes were
analyzed 8 weeks later for CD4 and CD8 expression. Animals were housed
either under specific pathogen-free conditions at Research and
Consulting Company (RCC), Fullinsdorf, Switzerland, or under
standard conditions in a conventional mouse facility.
Cytofluorimetric analysis
Single-cell suspensions from peripheral lymph nodes, thymus, or
spleen were prepared by crushing the tissues between 2 frosted glass
slides. Peripheral blood lymphocytes (PBLs) were harvested by
tail blood sampling and isolated by centrifugation on a Ficoll gradient. Ice-cooled single-cell suspensions were pretreated with Fc-block (anti-CD16/CD32; PharMingen, San Diego, CA) and then incubated
with specific antibodies (PharMingen) directed against CD4 (RM4-5),
CD8 (53-6.7), I-Ab (AF6-120.1) I-Ed (14-4-4S), Ly5.1
(A20), Ly5.2 (104), NK-1.1 (PK136), CD62L (MEL-14), CD44H (TM-1), CD54
(3E2), TCR (H57-597), CD3 (17A2), V3 (KJ25), V4 (KT4), V5.1
and 5.2 (MR9-4), V6 (RR4-7), V7 (TR310), V8 (F23.1), V9
(MR10-2), V10b (B21.5), V11 (RR3-15), V12 (MR11-1), V13
(MR12-3), V14 (14-2). Then 104 (primary cultures) to
105 (suspensions from fresh organs) cells were analyzed
using a FACSCalibur (Becton Dickinson). -Galactosylceramide
(-GalCer)-loaded tetramers were a generous gift from M. Kronenberg,
San Diego, CA. Detection of CD1-restricted natural killer T
(NKT) cells with mCD1 tetramers loaded with -GalCer were performed
as described previously.31 Briefly, cells were pretreated
with Fc-block (anti-CD16/CD32; PharMingen) and neutravidin (Molecular
Probes, Eugene, OR) at 4°C and then stained for 20 minutes at 23°C
with anti-NK1.1 Ab, anti-CD4 Ab, anti-TCR Ab and -GalCer-loaded
mCD1 (tetramerized with neutravidin phycoerythrin).
Immunohistochemistry
Sections (8-µm) from frozen adult organs and whole
newborn mice were air dried, fixed in ice-cold acetone, blocked in
phosphate-buffered saline (PBS) containing 0.6%
H2O2, 5% goat serum and 0.1%
NaN3, and stained with digoxygenin-conjugated antibody
directed against MHCII (M5/114), F4/80 (Serotec no. MCAP497), CD11c
(HL3; Pharmingen), and keratin (antipankeratin serum provided by E. Reichmann, Zurich, Switzerland). Staining was revealed using
antidigoxygenin peroxidase and 3-amino-9-ethyl carbazole
(Sigma-Aldrich) to give a red precipitate. Sections were counterstained
with methylene blue.
Immunofluorescence
For detection of MHCII expression by medullary epithelial cells,
thymuses were isolated and embedded in cryoembedding media (Tissue-Tek;
Sakura Finetek Europe BV, Zoeterwoude, The Netherlands). Frozen samples
were cut into sections 6 mm thick, fixed with 4% paraformaldehyde/PBS,
and exposed to PBS with 1% fetal calf serum 0.1% Tween at pH
7.3 for 10 minutes before incubation with primary antibody for 1 hour
at room temperature. Washing was repeated before incubation with the
secondary antibody (20 minutes at room temperature). Isotype controls
were used in all experiments. Thymic sections were stained for MTS10
(Pharmingen) and costained against I-A (AF6-120.1;
Pharmingen).32 Medullary areas were microscopically localized and analyzed by 2-color immunofluorescence with a confocal microscope (Carl-Zeiss AG, Feldbach, Switzerland).
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Results |
Positive selection of CD4+ T cells is abrogated in
pIV/ mice
To our surprise, a severe depletion of CD4+ T cells
was observed when we examined thymocytes and mature peripheral T cells in pIV/ mice.25 The single-positive
CD4+ T-cell numbers and percentages in pIV/
thymuses did not exceed those found in MHCII-deficient mice (Figure 2A). In contrast, CD8+ and
double-positive CD4+ CD8+ thymocytes are
present in normal or slightly increased numbers. In the peripheral
lymphoid organs, the percentage of CD4+ T cells is reduced
to 1%-5% of total lymphocyte numbers (Figure 2B-C), while that of
CD8+ T cells is markedly increased. The extent of the
CD4+ T-cell deficiency in pIV/ mice was
surprising because MHCII expression is normal on B cells, macrophages,
and DCs in all organs, including the thymus.25 Since
peripheral MHC/peptide engagements have been shown to be necessary for
the survival and homeostatic expansion of CD4+ T
cells,26,27 the peripheral MHCII+ environment
of pIV/ mice should permit the survival and
accumulation of any positively selected CD4+ T cells. In
spite of this, CD4+ T-cell counts are as low as in mice
that have a complete lack of MHCII expression, such as CIITA, RFX5, and
MHCII-deficient mutants.
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| Figure 2.
The depletion of CD4+ T cells found in
pIV/ mice is comparable to that observed in
A/ mice.
CD4+ and CD8+ T-cell populations were analyzed
by FACS. Numbers indicate the percentage of CD4+,
CD8+, and CD4+CD8+ cells. (A)
Representative FACS analysis of thymocytes from pIV+/,
pIV/ and I-A/ mice.
(B) % of CD4+ and CD8+ T-cell
populations from the lymph nodes, spleen, and PBLs were analyzed for
pIV/ mice and pIV+/ control littermates.
(C) Representative FACS analysis showing the CD4+ and
CD8+ T-cell populations in lymph nodes from control
pIV+/, pIV/, and I-A/
mice.
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There are 2 explanations that could account for the loss of
CD4+ T cells. First, pIV could be essential for MHCII
expression in the thymic compartment that drives positive selection of
CD4+ T cells. The lack of MCHII expression in
pIV/ thymuses would have to be very tight because
MHCII+ DCs, B cells, and macrophages in the periphery of
pIV/ mice should ensure the survival of any positively
selected CD4+ T cells. The second possibility is that pIV
performs a function that is intrinsically important for T-cell
development. For instance, CIITA has been suggested to be involved in
the differentiation of peripheral CD4+ T cells into Th2
cells.33 To discriminate between these possibilities we
produced radiation bone marrow chimeras with various combinations of
recipient and donor phenotypes (Table 1). The development of T cells
was examined 2 months after engraftment. The origin of the lymphocytes
was assessed by using the congenic markers Ly 5.1 and Ly
5.2.29 Most CD3-negative lymphocytes were of donor origin.
In all chimeras, a small fraction of recipient cells persisted in the
CD3-positive subset.
When both the recipient and donor phenotypes are wild type, all
lymphocyte populations are reconstituted to normal levels (Table 1,
group A). This is also true when wild-type mice are reconstituted with
pIV/ donor cells (Table 1, group B). In contrast,
pIV/ mice reconstituted with either wild-type (Table 1,
group C) or pIV/ donor cells (Table 1, group F) had
very low CD4+ T-cell counts but normal levels of
CD8+ T cells. We also mixed wild-type with
pIV/ donor cells and reconstituted wild-type or
pIV/ mice (Table 1, groups D and E). Again,
CD4+ T cells were generated normally if the recipient was
wild type (Table 1, group D), whereas pIV/ recipients
were unable to produce normal numbers of CD4+ T cells
(Table 1, group E). Taken together these results exclude the
possibility that the lack of CD4+ T cells in
pIV/ mice could be due to an intrinsic defect in bone
marrow progenitors.
pIV/ mice specifically lack MHCII expression on
thymic epithelial cells
The thymus of pIV/ mice is characterized by an
abnormal pattern of MHCII expression (Figure
3A). In wild-type thymuses, the cortex
shows a fine reticular pattern of MHCII expression that is
characteristic of the epithelial cell matrix. In the medulla, the
staining is more diffuse and is attributable to DCs, B cells, macrophages, and medullary epithelial cells. In pIV/
mice, the cortical reticular staining is absent, indicating that the
cortical thymic epithelial cells (cTECs) lack MHCII expression. Only
patchy areas of MHCII expression remain in the cortex. In contrast, the
diffuse staining of the medulla is similar to wild-type controls. By
staining of adjacent sections, the patchy MHCII expression in the
cortex was found to colocalize with F4/80 positive macrophages. CD11c-positive cells, on the other hand, were restricted to the medulla
and corticomedullary junction, in accordance with previous studies.34 Finally, the fine reticular stain obtained with
an antikeratin antibody is consistent with a conserved architecture of
the thymic stroma. Medullary thymic epithelial cells (mTECs) were
analyzed by immunofluorescence for MHCII expression (Figure 3B). In
control pIV+/ thymic medulla, the majority of MTS10+
cells (major mTECs) express MHCII. Strikingly, in pIV/
mice MTS10+ mTECs do not express MHCII. The absence of MHCII expression
by mTECs has been confirmed by FACS experiments (data not
shown).
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| Figure 3.
pIV/ mice lack MHCII expression on
thymic epithelial cells, and passive transfer of MHCII molecules to T
cells is abrogated.
(A) Thymic sections were stained (brown) for MHCII, F4/80, CD11c, and
epithelial cells (keratin). The counterstain is methylene blue. The
strong reduction in MHCII expression in pIV/ thymuses
is restricted to the cortex (compare upper 2 panels). In
pIV/ thymuses, the residual patchy MHCII expression in
the cortex overlaps with F4/80+ thymic macrophages (compare middle 2 panels). CD11c-positive DCs are restricted to the medulla (bottom left
panel). The keratin stain indicates a normal architecture of the stroma
in both the cortex and the medulla (bottom right panel). m
indicates medulla; c, cortex. Original magnification, × 200 for all
images in panels A and B. (B) Immunohistology of thymic medullary
areas. MTS10+ and MHCII+ medullary cells appear blue and green,
respectively. Double-positive cells appear cyan in the thymic medulla
from the pIV+/ control. No costaining is observed in the
pIV/ medulla. (C-E) Passive transfer of MHCII
molecules to thymocytes does not occur in pIV/ mice.
(C) MHCII expression was analyzed by FACS on double-positive
CD4+CD8+ thymocytes from pIV/
(gray profile) and pIV+/ mice (open profile). (D)
Double-positive CD4+CD8+ thymocytes derived
from pIV/ bone marrow progenitors acquire MHCII
molecules by passive transfer if they develop in the thymus of an
irradiated wild-type recipient (open profile). On the other hand,
wild-type thymocytes developing in a pIV/ recipient do
not acquire MHCII molecules (gray profile). pIV/ donor
cells (open profile) were identified by gating on Ly5.2+ cells.
Wild-type donor cells (gray profile) were selected within the Ly5.1+
gate. (E) MHCII expression on double-positive
CD4+CD8+ thymocytes is compared between
pIV/ mice (gray profile) and I-A/
mice (open profile).
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Thymocytes acquire MHCII molecules by passive transfer
from TECs
Mouse T cells do not express significant amounts of CIITA and are
consequently devoid of MHCII molecules. However, experiments with
radiation chimeras35 have indicated that mouse thymocytes can acquire MHCII molecules in a cell contact-dependent fashion. Thymocytes acquire these MHCII molecules by passive transfer from an
unidentified thymic stromal cell.36 Strikingly, the
passive transfer of MHCII to CD4+CD8+
thymocytes is abrogated in pIV/ mice (Figure 3C). In
irradiated recipients reconstituted with pIV/ donor
cells, the passive MHCII transfer to thymocytes was corrected only if
the host thymus is wild type (Figure 3D and data not shown). In
contrast, wild-type thymocytes grafted into irradiated
pIV/ hosts remain MHCII. The lack of
MHCII transfer in pIV/ mice is complete, as shown by
the negative control (I-A/ mice) in Figure 3E. These
observations confirm that the presence of MHCII molecules at the
surface of thymocytes results from a passive transfer from cTECs
or mTECs.
Negative selection by endogenous mtv superantigens
is conserved in pIV/ mice
The data obtained from radiation chimeras and the histologic
findings in pIV/ thymuses strongly argue that the
CD4+ T-cell deficiency in pIV/ mice is
entirely attributable to the lack of MHCII expression by cortical
thymic epithelial cells (cTECs). In contrast to other MHCII-deficient
mutants, pIV/ mice retain normal MHCII expression on B
cells, DCs, and macrophages in both the thymus and the
periphery.25 This provided a unique opportunity to test
whether MHCII+ APCs can perform negative selection in the
absence of positive selection of CD4+ T cells. With this
aim, we examined clonal deletion of T cells in pIV/
mice. T cells expressing V5 and V11 TCRs are deleted in the thymus of mouse strains that express I-E and retroviral superantigens encoded by mtv-8 and -9.37 Because
pIV/ mice were generated in a mixed B6/129 background
(b haplotype), they lack a functional I-E gene. To obtain efficient,
superantigen-mediated deletion we therefore introduced a functional I-E
allele by crossing the pIV/ mice with B10.BR mice.
Figure 4A compares the frequency of
CD4+ T cells expressing V5, V11, and V8 in the
presence and absence of the I-E gene. In wild-type mice and in
pIV/ mice, introduction of the I-E gene has no
effect on the control V8 family. In wild-type mice, introduction of
the I-E gene leads to efficient deletion of the susceptible V5
and V11 families. A deletion of the V11 family and, to a lesser
extent, of the V5 subset was also observed in the residual
CD4+ T-cell population of pIV/ mice. The
fact that deletion in the CD4+ V5 subset is only partial
in pIV/ mice may be explained by the recently described
MHCI- and MHCII-independent population of CD4+ T
cells.38 This population displays a 6-fold increase in
V5 expression, which becomes apparent in the residual
CD4+ T-cell compartment of pIV/ mice. In
the CD8+ T-cell compartment, the I-E dependent,
superantigen-mediated deletion of the V5 and V11 families was, as
expected, similar in pIV/ and control littermates
(Figure 4B). There was again no change in the percentage of V8+
CD4+ T cells after introduction of the I-E gene. Taken
together, these results establish that superantigen-mediated negative
selection of CD4+ T cells occurs normally in
pIV/ mice. The residual CD4+ thymocytes in
pIV/ mice are negatively selected despite the defect in
their positive selection. This defect in positive selection is as
strong in the B10BR background as it is in the mixed Sv129-C57Bl/6
background (compare Figure 4C to Figure 2).
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| Figure 4.
Negative selection occurs normally in pIV/ mice.
V5, V11, and V8 subsets of CD4+ and
CD8+ T cells were analyzed by FACS of splenocytes extracted
from pIV/ mice and control pIV+/
littermates. The numbers indicated alongside the bar graphs represent
the percentages of the indicated V family relative to the total
CD4+ (A) or CD8+ (B) T-cell populations. White
and filled bars represent the mean value (2 to 4 mice) for a
nondeleting (I-E) and a deleting (I-E+)
background, respectively. (C) Representative FACS analysis
showing the CD4 and CD8 populations in B10BR thymuses and lymph nodes.
B10BR controls (I-E+ I-A+ pIV+/, on the left) are
compared with B10BR pIV/ mice (I-E+ I-A+
pIV/ , on the right).
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Thymocytes and peripheral CD4+ T cells exhibit the same
activated phenotype in pIV/ and MHCII-deficient
mice
CD4+ T cells in MHCII-deficient mice present typical
anomalies in the expression of several cell surface markers. Figure
5A shows that CD4+ thymocytes
of pIV/ mice present the same anomalies as their
counterparts isolated from animals fully deficient in MHCII molecules.
CD4+ thymocytes have reduced TCR levels in both
I-A/ and pIV/ mice. MHCII deficiency
also has been reported to have an impact upstream of the
CD4+ single-positive stage, leading to elevated CD4 and TCR
levels on double-positive CD4+CD8+
thymocytes.39 This phenotype is again reproduced in the
pIV/ animals.
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| Figure 5.
Thymocytes and CD4+ T cells from
pIV/ and I-A/ mice display a similar
phenotype.
(A) Double-positive CD4+CD8+ thymocytes from
pIV/ mice express higher levels of TCR and CD4 than
controls (top 2 histograms). CD8 levels are similar to controls (data
not shown). Single-positive CD4+ T cells display lower TCR
levels in pIV/ and I-A/ thymuses
compared to control littermates (middle 2 histograms). This is not
observed for CD8+ T cells (bottom 2 histograms). (B)
CD4+ splenocytes were analyzed by FACS for CD62L, CD54, and
TCR expression. CD62L and TCR levels are lower in
I-A/ and pIV/ CD4+ T
cells than in controls. CD54 is increased in both mutants. (C)
CD8+ T cells from the spleens of I-A/ , pIV/, and control littermates are similar with respect
to their expression levels of CD62L, CD54, and TCR.
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The similarity between pIV/ and MHCII-deficient mice
extends to the peripheral T-cell compartments. The residual
CD4+ T cells in the periphery of pIV/ mice
have the same activated phenotype as the CD4+ T cells
of MHCII-deficient mice (Figure 5B). They display a
down-regulation of CD62L with a concomitant increase in CD54. Moreover,
their TCR levels are markedly reduced. In contrast, CD8+ T
cells do not present these anomalies in either the pIV/
or MHCII-deficient mice (Figure 5C). We conclude that normal MHCII
expression in the periphery of pIV/ mice does not
prevent the residual CD4+ T cells from adopting an
activated phenotype.
There are 2 explanations that could account for the unusual activated
phenotype of the residual CD4+ T cells in the
pIV/ mice. First, the absence of pIV expression in the
CD4+ T cells could itself induce an abnormal phenotype.
Alternatively, the atypical phenotype could result not from an
intrinsic defect in CD4+ T cells but from the development
of these cells in the absence of MHCII expression on TEC. The analysis
of CD4+ T cells in the radiation chimeras described above
(Table 1) is consistent with the latter hypothesis. CD4+ T
cells that developed in wild-type hosts display wild-type levels of
cell-surface markers even if they are derived from pIV/
donor cells. On the other hand, wild-type CD4+ T cells that
developed in pIV/ recipients exhibit the same
characteristic alterations seen in the pIV/ and
MHCII-deficient mutants (Figure 6). These
results rule out the possibility that pIV expression in bone
marrow-derived cells is required to obtain a normal phenotype in
CD4+ T cells.
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| Figure 6.
The characteristic cell surface phenotype of thymocytes
and peripheral CD4+ T cells in pIV/ mice is
reproduced in bone marrow chimera experiments only if the irradiated
recipient is a pIV/ mouse.
Filled profiles represent T cells derived from pIV/
progenitors grafted into a wild-type host. Open profiles represent T
cells derived from wild-type progenitors grafted into a
pIV/ host. T cells were identified as donor-derived by
gating with the appropriate congenic marker (Ly5.1 or Ly5.2). Groups B,
C, D, and E refer to the groups of bone marrow chimeras listed in Table
1. The 6 histograms in the upper panel reveal that thymocytes derived
from wild-type bone marrow progenitors present alterations identical to
those described in pIV/ and I-A/
mice if they develop in a pIV/ host. The 4 histograms
in the lower panel represent a similar analysis of splenic
CD4+ T cells. Peripheral pIV/
CD4+ T cells display a normal surface phenotype when they
develop in wild-type hosts, whereas wild-type CD4+ T cells
that develop in pIV/ recipients up-regulate CD44 and
express lower levels of TCR.
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Taken together, our results show that the activated phenotype of
CD4+ T cells is a consequence of deficient MHCII expression
on cTECs and mTECs. This is consistent with the finding that the
residual CD4+ T cells in pIV/ and
MHCII/ mice share the same atypical phenotype.
Moreover, the fact that these 2 mutants exhibit the same phenotype
implies that it must arise independently of whether or not peripheral
APCs express MHCII molecules.
A large proportion of the residual CD4+ T cells in
pIV/ mice are CD1-restricted NKT cells
In addition to the activated phenotype, the residual
CD4+ T cells in pIV/ mice contain a higher
proportion of V8+ and V7+ cells (see Figure 7). These
features are typical of NKT cells.40,41 This led us to
suspect that the residual CD4+ T-cell population in
pIV/ mice contains a high proportion of NKT cells. We
therefore performed a 4-color FACS analysis with antibodies directed
against CD4, TCR-, and NK1.1, and with CD1 tetramers coupled to
GalCer (Figure 7A). The tetramers
allowed us to detect NKT cells that stain negative for the NK1.1
marker.42 CD4+ T cells from
pIV/, MHCII-deficient, and wild-type mice were found to
comprise similar absolute numbers of NK1.1+ and CD1-restricted cells.
In contrast, the NK1.1-CD1GalCer CD4+ T cells, which
comprise the classical MHCII-restricted CD4+ T cells, are
much less abundant in the MHCII-deficient and pIV/
animals. The consequence is a considerably higher proportion of NKT
cells in the pIV/ and MHCII-deficient animals (up to
half of the total number of CD4+ T cells).
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| Figure 7.
The proportion of CD1-restricted and NK1.1+ cells is
increased in pIV/ and I-A/ mice.
(A) The density plots show NK1.1 and CD1GalCer-tetramer staining of
CD4+, TCR+ T cells from pIV+/,
pIV/, and I-A/ mice. Numbers
indicate the percentage of CD4+ T cells in each quadrant
relative to the total number of cells. NK1.1, tetramer,
CD4+ T cells (lower left quadrant) are reduced 10- to
15-fold in pIV/ and I-A/ mice. In
contrast, similar percentages of NK1.1+ and tetramer+ cells are
detected in the control and mutant mice. (B) The higher proportion of
NKT cells in pIV/ and I-A/ mice
leads to an increase in the numbers of V8+ CD4+ T cells.
The bar graph shows the mean percentages of V8+ cells for the 3 mouse strains. Total CD4+ T cells comprise a higher
proportion of V8+ cells in pIV/ and
I-A/ mice as compared to control pIV+/
littermates (left). NK1.1+ CD4+ T cells exhibit a similar
V8 bias in all 3 mouse strains (right). (C) Representation of
various V families in CD4+ and CD8+ T cells
in pIV/ mice and control pIV+/
littermates. The upper bar graph indicates the mean percentage of the
CD4+ T cells belonging to the indicated V families (at
least 4 mice for each data point). The higher proportion of NKT cells
in the pIV/ mice leads to an increase in the percentage
of V8 and V7 CD4+ T cells. As a result, the
representation of most other families is reduced. The distribution of
V families in CD8+ T cells is identical between control
and pIV/ mice (lower bar graph).
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Since NKT cells display a strong V8 bias in their TCR
repertoire,41 V8+ cells are more frequent in the total
CD4+ T-cell population of the pIV/ and
MHCII-deficient mice (Figure 7B). Within the CD4+ NK1.1+
population, the same high proportion of V8+ cells is, of course,
evident in all strains.
To complete our characterization of the T-cell population in
pIV/ mice, we performed a detailed analysis of the V
repertoire of CD4+ and CD8+ splenocytes. For
each V subset, 4 to 6 pIV/ mice were compared to an
equivalent number of control littermates. No change in the CD8
repertoire was evident in the pIV/ mice (Figure 7C,
lower bar graph). On the other hand, the higher proportion of NKT cells
leads to an increase in the representation of V8 and V7 families
in the CD4+ T splenocytes of pIV/ mice
(Figure 7C, upper bar graph). The percentages of CD4+
V5+ T cells in pIV/ mice is also marginally
increased compared to control mice. This may be explained by the
recently described MHCI- and MHCII-independent population of
CD4+ T cells.38 This population displays a
6-fold increase in V5 expression and is proportionally increased in
pIV/ mice, given the lack of MHCII-restricted
CD4+ T cells. Most of the other V subsets in the
CD4+ T-cell compartment of pIV/ mice are
reduced to approximately 50% of the wild-type levels because of the
overrepresentation of V families of NKT cells.
No requirement for IL-7R signaling in MHCII expression by
cTECs
The Jak/signal transducers and activators of transription (STAT)
pathway is able to activate Mhc2ta transcription through promoter IV. We addressed the question whether the STAT-1,-3,-5 associated cytok |
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Abstract
Introduction