By
From the * Basel Institute for Immunology, CH-4005 Basel, Switzerland; and the Institut Necker,
Institut National de la Santé et de la Recherche Medicale 373, F-75730 Paris Cedex 15, France
Although individual T lymphocytes have the potential to generate two distinct T cell receptor
(TCR)- chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-
chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest
of further TCR-
rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-
rearrangements, we have studied the efficiency of allelic exclusion in mice lacking
the pre-TCR-
(pT
) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for V
6 or V
8 and a pool of antibodies
specific for most other V
elements, did not reveal any violation of allelic exclusion at the level
of cell surface expression. This was also true for pT
-deficient mice expressing a functionally
rearranged TCR-
transgene. Interestingly, although the transgenic TCR-
chain significantly
influenced thymocyte development even in the absence of pT
, it was not able to inhibit fully
endogeneous TCR-
rearrangements either in total thymocytes or in sorted CD25+ pre-T
cells of pT
/
mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.
Functional TCR genes are assembled by a program of
somatic gene rearrangements from variable (V In mature T cells, the rearrangement status of the TCR- While, in general, TCR- The role of the pre-TCR in allelic exclusion of the
TCR- Mice.
C57BL6 (B6) mice, which were used in most experiments as wild-type controls, were purchased from IFFA-Credo
(France). All other genetically modified mice (pT Antibodies and Flow Cytometry.
The following antibodies have
been used (all purchased from PharMingen, San Diego, CA, unless stated otherwise): FITC anti-mouse CD3
Depletion of CD4 and CD8 Expressing Thymocytes.
Single-cell
suspensions from thymi of three individual mice with the same
genotype were pooled, resuspended in 8 ml serum-free DMEM
medium, and incubated simultaneously with 1 ml of anti-CD8 (31M) and 1 ml of anti-CD4 (RL172.4) antibody supernatants on ice for 30 min. The cells were then washed in 10 ml DMEM, 2%
CS and resuspended in 9 ml of the same medium. After adding 1 ml of freshly dissolved rabbit complement (Low-Tox-M, Cedarlane, Hornby, Canada), the suspension was incubated at 37°C for
40 min. Surviving cells were purified by Ficoll density-gradient
centrifugation, washed and resuspended in PBS, 2% CS. The efficiency of CD4/CD8 depletion was usually more than 95% as determined by staining purified thymocytes with R-PE anti-mouse
CD4 (H129.19) and R613 anti-mouse CD8 Detection of Endogeneous TCR Rearrangements.
High molecular weight thymocyte DNA was prepared as follows: cells from
two thymi (pT Assuming sufficient time for
TCR- To alleviate this problem, we used a combination of antibodies directed against V Fig. 1 shows the result of a representative analysis using
the V To increase
further the sensitivity of our anti-V pT The observation that a TCR- Previous experiments in normal (pT
Introduction of a functionally rearranged TCR
The expression of productively rearranged TCR-
Because the proportion of various thymocyte subpopulations is markedly altered in pT
The data reported here demonstrate that expression of a
functional TCR- A priori, it would also be possible that neither the
TCR- The question arises whether the observed effects of the
transgenic TCR-)1 gene
segments, diversity (D
) genes, and joining (J
) elements at
the TCR-
loci and from V
and J
elements at the
TCR-
loci. At the TCR-
locus, D
J
rearrangements precede V
DJ
rearrangements. Although this
process of V(D)J recombination could theoretically give
rise to cells with two in-frame TCR rearrangements at corresponding alleles, and thus two functional
or
TCR
genes, virtually all T lymphocytes of the
lineage express
only one particular TCR-
chain, a phenomenon referred
to as allelic exclusion. Analysis of an increasing number of
T cell clones and hybridomas has revealed that allelic exclusion at the TCR-
locus is largely due to the fact that
T cells carry as a rule only one productive TCR-
rearrangement, whereas the rearrangement on the other allele
is either incomplete (DJ
) or out of frame (1). These findings are in line with the notion that a productive TCR-
rearrangement can somehow prevent further rearrangements at the TCR-
locus. Strong support for this hypothesis has been obtained in mice expressing productively rearranged TCR-
transgenes (2, 3), which enforce almost
complete inhibition of endogeneous V
DJ
rearrangements, whereas D
J
rearrangements were essentially
unimpaired. In contrast, no inhibition of endogeneous
TCR-
rearrangements could be observed in mice expressing a nonproductive TCR-
transgene (3).
locus differs from that of the TCR-
locus in that usually
both alleles carry V
J
rearrangements and cells with
two functional TCR-
alleles are easily detectable (1, 4, 5).
In fact, in TCR-
transgenic mice there is no or only very
inefficient inhibition of endogeneous V
J
rearrangements (6, 7, 8). Thus, it appears that rearrangements in the
TCR-
locus continue on both alleles until a receptor is
formed that can bind to thymic MHC molecules and induce positive selection, an event that leads to downregulation of RAG expression and complete termination of all
TCR gene rearrangements (6, 9, 10, 11).
rearrangements occur relatively late during thymocyte development, primarily at the
transition from the double-negative (DN) to the double-positive (DP) stage and during the DP stage itself (12, 13),
TCR-
rearrangements are initiated and completed much
earlier, namely at a CD4
8
(DN) stage defined by the expression of the IL-2 receptor
chain (CD25) (12, 14). Any
model postulating a negative feedback of functional TCR-
chains on rearrangement at the second
allele therefore
presumes a signaling function of TCR-
in the absence of
TCR-
. A similar situation is encountered in B cells where IgH chains are thought to inhibit further rearrangements at
the IgH locus, well before mature IgL chains become available. The discovery of the pre-B cell receptor (BCR) (15,
16) and pre-TCR (17, 18) provided likely candidates for
the signaling machinery mediating allelic exclusion at the
corresponding loci in the absence of mature light chains or
TCR
chains, respectively, because these receptors consist,
in the case of the pre-BCR, of a conventional IgH chain
paired with surrogate light chains
5 and VpreB (along with signal-transducing Ig
[mb-1] and Ig
[B29] proteins)
(19) and, in the case of the pre-TCR, of a conventional
TCR-
chain disulfide-linked to the invariant pre-TCR-
(pT
) chain (in association with components of the CD3
complex) (20). Surprisingly, however, analysis of the limited number of mature B cells that develop in
5-deficient
and therefore pre-BCR-defective mice did not provide any evidence for violation of allelic exclusion (21). On the other hand, more recent experiments seem to indicate that
allelic exclusion is not fully operating in the absence of
5
when precursor rather than mature B cells are studied (22).
locus has been investigated lately in mouse chimeras that were generated by injecting TCR-
-transgenic,
pT
/
embryonic stem (ES) cells into RAG
/
blastocysts (23). Analysis of ES cell-derived thymocytes in these
chimeric mice revealed equivalent inhibition of endogeneous V
DJ
rearrangements in the presence and absence of the pT
chain and provided no evidence for the
existence of T lymphocytes with more than one TCR-
chain (23). Here, we report on allelic exclusion in stable
lines of pT
/
and TCR-
-transgenic, pT
/
mice
rather than chimeric animals, which permitted a more rigorous analysis. Our results indicate an involvement of the
pre-TCR in allelic exclusion at the TCR-
locus, but they
also show that TCR-
signaling is not completely compromised in the absence of pT
, a finding that may explain
why it has been difficult so far to demonstrate an effect of the
pre-TCR, and by inference of the pre-BCR, on allelic exclusion when analysing pT
- (23) or
5-deficient mice (21).
/
, TCR-
-transgenic, RAG-2
/
, and the respective intercross offspring)
were bred and maintained in a specific pathogen-free facility of
the Basel Institute for Immunology (Switzerland). All animals
used were 6-12 wk of age. pT
-deficient mice were identified
by Southern blotting of PstI-digested tail DNA using as a probe a
genomic PstI-XhoI fragment (~610 bp) corresponding to positions 5,432 to 6,046 of the pT
gene (numbering according to
reference 24). TCR-
-transgenic mouse lines expressing functionally rearranged, V
8.2 transgenes have been described previously (2). In our experiments, we have used the line with ~20
copies. Transgenic mice were identified by PCR with 1 µg of genomic tail DNA as template and primers specific for V
8.2
(5
-GCATGGGCTGAGGCTGATCCATTA-3
) and a region
located immediately 3
of the J
2 cluster (5
-TGAGAGCTGTCTCCTACTATCGATT-3
). Cycling conditions were as
follows: 1 min at 94°C (denaturation); 30 s at 94°C; 1 min at
55°C; and 1 min 30 s at 72°C; 25 cycles. Amplification of a
~920-bp fragment indicated the presence of TCR-
transgenes.
Breeding stocks of RAG-2-deficient mice have been provided by
Dr. F.W. Alt (Boston, MA). RAG-2
/
animals were identified
based on the absence of B220+/surface Ig+ B lymphocytes as evidenced by double staining of peripheral blood cells with B220-
PE and sheep anti-mouse Ig-FITC antibodies.
(2C11); biotin
anti-mouse CD3
(500A2); FITC anti-mouse CD4 (H129.19);
R-PE anti-mouse CD4 (H129.19; GIBCO BRL, Gaithersburg, MD); FITC anti-mouse CD8
(53-6.7); R613 anti-mouse
CD8
(53-6.7; GIBCO BRL); FITC anti-mouse CD11b (Mac1
)
(M1/70); FITC anti-mouse CD19 (1D3); biotin anti-mouse
CD25 (7D4); R-PE anti-mouse CD44 (IM7); PE anti-mouse
TCR (GL3); FITC anti-mouse Ly-6G (GR1) (RB6-8C5); R-PE
anti-mouse CD45R/B220 (RA3-6B2); FITC anti-mouse V
6
(RR4-7); FITC anti-mouse V
8.1/8.2 (MR5-2); PE anti-
mouse V
2 (B20.6); PE anti-mouse V
3 (KJ25); PE anti-mouse
V
4 (KT4); PE anti-mouse V
5.1/5.2 (MR9-4); PE anti-mouse
V
6 (RR4-7); PE anti-mouse V
7; PE anti-mouse V
8.1/8.2
(MR5-2); PE anti-mouse V
10b (B21.5); PE anti-mouse V
11
(RR3-15); PE anti-mouse V
13 (MR12-3); FITC sheep anti-
mouse Ig F(ab
)2 fragment (Silenus, Australia). Streptavidin conjugates: Streptavidin-Tricolor (Caltag, CA) and streptavidin-APC
(Molecular Probes, OR).
reagents were used, it proved necessary to dialyze the antibody mix
containing all first-step antibodies immediately before the staining, to eliminate the cytotoxicity of the concentrated reagents.
Dialysis was performed at 4°C in ultra thimbles (Schleicher & Schuell UH 100/10; cutoff ~10,000 MM) against two changes of
PBS, 2% CS for 2 × 30 min. Phenotypes and proportions of thymocyte subsets were analyzed by three-color flow cytometry using a FACScan® (Beckton Dickinson, Mountain View, CA) and
the Lysis II program. The data depicted in Figs. 2 and 3 were analyzed with the program Cellquest (Becton Dickinson). Dead
cells were excluded from the analysis by forward- and side-scatter
gating, when analyzing numerically small subpopulations also by
addition of propidium iodine (PI) and gating on PI
cells. Four-color analyses and cell sorting were performed on a FACStar
Plus® (Beckton Dickinson) equipped with a pulse processor for
forward-scatter width (FSC-W) in order to gate out cell doublets.
Fig. 2.
V8.1/8.2 versus V
-pool staining of lymph node cells from
TCR-
-transgenic mice expressing (pT
+) or lacking (pT
/
) a functional pT
gene (four-color analysis). The transgene-encoded receptor
contains the V
8.2 element. Staining and gating was done as described in
Fig. 1.
[View Larger Version of this Image (38K GIF file)]
Fig. 3.
The effects of TCR- transgenes on various aspects of thymic T cell development in the absence of pT
(the right most panel in A,
B, and C). A TCR-
-transgenic mouse expressing functional pT
(TCR-
-transgenic, pT
+), a wild-type C57BL6 (WT [B6]), and a nontransgenic pT
-deficient (pT
/
) mouse are included as controls. (A)
CD4/CD8 profiles. The figures on top of each panel give the total number of thymocytes found in the particular mouse analyzed in this experiment. These values are very typical for mice with the respective genotype
as seen in many similar experiments. (B) Analysis of triple-negative
(CD3
CD4
CD8
) thymocytes for differential expression of the developmental markers CD25 and CD44. Thymocytes from three mice of the
same genotype were pooled. CD4- and CD8-expressing cells (single positive and DP thymocytes) from B6 and TCR-
-transgenic, pT
+ mice were
complement depleted (see Materials and Methods) before staining. For
cytofluorometric analysis, thymocytes were incubated with an antibody
mix containing CD4-FITC, CD8-FITC, CD3-FITC, GR1-FITC,
MAC1-FITC, CD19-FITC (the latter three antibodies are specific for
granulocytes, macrophages, and B cells, respectively), CD44-PE and
CD25-biotin, and in a second step with streptavidin-Tricolor. FITC-positive cells were excluded from the analysis by electronic gating (data
not shown). The cells shown in the four panels are thus highly enriched for
immature triple-negative thymocytes. (C) The generation of
-expressing
cells is suppressed in TCR-
-transgenic mice, even in the absence of
pT
. Thymocytes were stained with a PE-conjugated TCR-
-specific
antibody and CD3-biotin, in a second step with streptavidin-Tricolor.
Only thymocytes that are positive for both CD3 and TCR-
were considered as genuine
-expressing cells (as defined by the rectangular gate).
The data shown in A and C were obtained in the same experiment with
the same mice.
[View Larger Version of this Image (61K GIF file)]
Fig. 1.
V8.1/8.2 versus V
-pool staining of thymocytes and lymph
node cells from a wild-type C57BL6 (WT [B6]) and a pT
-deficient (pT
/
) mouse (four-color analysis). Thymocytes or lymph node cells were stained with an antibody mix containing CD3-biotin, V
8.1/8.2- FITC, and nine distinct PE-conjugated anti-V
antibodies (anti-V
2, 3, 4, 5.1/5.2, 6, 7, 9, 10b, 13). CD3+ cells were detected with a streptavidin-Tricolor reagent. Before acquisition, PI was added. (Top) Dot blots of
cells that have passed three gates: the FSC/SSC gate for live cells, the gate for PI
cells, and the pulse-width gate that eliminates cell doublets (data
not shown). (Bottom) The cells depicted have passed in addition the gate
for CD3high cells shown in the top.
[View Larger Version of this Image (43K GIF file)]
(53-6.7) antibodies, which bind epitopes distinct from those recognized by antibodies 31M and RL172.4.
+ mice) or three thymi (pT
/
mice) of the
same genotype were pooled and digested in proteinase K solution
(50 µg/ml proteinase K, 0.5% SDS) at 50°C for 24 h. The DNA
was phenol/chloroform extracted, precipitated with ethanol, recovered, and dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a concentration of 100 µg/ml. PCR-based analysis of TCR-
chain rearrangements was performed using an assay
originally described by van Meerwijk et al. (25) and modified by
Anderson et al. (26). V
4-J
1 rearrangements (nomenclature
according to reference 27) were detected in a similar fashion.
Primers specific for V
4 (5
-CCTGATATGCGAACAGTATCTAGGC-3
), V
6 (5
-GAAGGCTATGATGCGTATCG-3
), V
11 (5
-TGCTGGTGTCATCCAAACACCTAG-3
), V
12
(5
-AGTTACCCAGACACCCAGACATGA-3
), and a region
immediately downstream of the last J
segment in the J
2 gene
cluster (5
-TGAGAGCTTGTCTCCTACTATCGATT-3
) were
used to detect V
(D)J
rearrangements, and primers specific
for V
4 (5
-TGTCCTTGCAACCCCTACCC-3
) and J
1
(5
-CAGAGGGAATTACTATGAGC-3
) were used to detect
the respective rearrangement in the TCR-
locus. Template
DNA was used at three or more different concentrations in each
experiment to ensure linearity of the PCR signal. As a control for
the amount of template DNA, aliquots of a PCR mastermix were
amplified with oligonucleotides specific for the IgM constant region (5
-CACTAGCCACACCCTTAGCAC-3
and 5
-TGGCCATGGGCTGCCTAGCCCGGGACTT-3
). PCR amplifications were performed in 50 µl reaction buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin, 2.5 mM
MgCl2, 0.2 mM of each dNTP (Pharmacia, Piscataway, NJ), 1 pmol of each primer, 0.5U Perfect Match PCR enhancer (Stratagene, La Jolla, CA), 1 U AmpliTaq DNA polymerase (Hoffmann-La Roche, Basel, Switzerland) and the indicated amount of
template DNA. The amplification cycle consisted of 45 s at 94°C,
1 min 30 s at 62°C (V
[D]J
) or 50°C (V
4
J
4), and 2 min
at 72°C. The cycle was repeated 26 times. PCR products were
separated on 1.2% agarose gels, blotted with a vacuum blotter
(Bio-Rad; Richmond, CA) onto Hybond N+ nylon membrane
(Amersham, Arlington Heights, IL) and detected by hybridization
with end-labeled J
2.7-(5
-TATGAACAGTACTTCGGTCCC-3
), J
1-(5
-TGAATTCCTTCTGCAAATACCTTG-3
),
or Cµ-specific (5
-CCTGCCCAGCACCATTTCCTTC-3
) probes, as appropriate.
No Evidence for Cell Surface Expression of Two TCR-
Chains in pT
/
Mice.
rearrangements on both alleles, no feedback inhibition after a functional TCR-
chain has been generated
and no selective disadvantage of cells with two functional
TCR-
chains, one would expect that up to 1/5 (20%) of
all TCR-
+ cells express two types of
chains, indicating
the complete absence of allelic exclusion (see reference 4
for calculations). Any, even partial violation of these assumptions would reduce the percentage of double-expressing cells accordingly, albeit unpredictably. Given the low
frequency of cells that express a particular V
element, it is
clear that evidence for incomplete allelic exclusion is not
easy to obtain by simple cell surface staining with antibodies specific for just two particular V
elements.
6 or V
8.1/8.2 elements on
one hand and a pool of nine antibodies recognizing TCRs
that contain most of the remaining V
elements on the
other. V
6- or V
8.1/8.2-specific antibodies were chosen
as individual antibodies, because they detect the two most
frequent V
T cell populations on the B6 background, representing ~5% and 15% of all TCR-
+ cells, respectively. To increase further the sensitivity of our analysis, we
included as a third color a CD3
-specific antibody, which
allowed us to gate on CD3+ thymocytes/lymphocytes and
exclude all other cells that do not express a TCR or only at
low levels. Dead cells, which are known to absorb antibodies nonspecifically and which could therefore give rise to
apparently double-expressing cells, were excluded not only
by gating on live cells in a forward scatter/side scatter (FSC/SSC) analysis, but also by including PI staining as a
fourth color and subsequent gating on PI
cells. By using
the FSC pulse-width program on our flow cytometer, we
could also efficiently exclude cell doublets, which otherwise would have raised the background of false TCR double-expressing cells.
8.1/8.2-specific antibody as the individual antibody
and anti-V
2, 3, 4, 5.1/5.2, 6, 7, 9, 10b, and 13 as pooled
antibodies. In the B6 control mouse, the frequency of
CD3+ thymocytes and lymph node cells scoring positive
for V
8.1/8.2 and one of the V
s represented in the pool
is extremely low (~0.1-0.2%). This value may reflect the
low proportion of T cells that genuinely express two
chains, violating allelic exclusion, as suggested by studies of
human T lymphocytes (28, 29), or it could just indicate the
level of nonspecific background staining in our experiment. More important in this context, however, no significant increase in the percentage of potentially double-expressing
cells could be detected in the pT
/
mouse, despite this
very sensitive staining technique. These data clearly show
that T cells and thymocytes expressing intermediate to high
levels of the
TCR are allelically excluded even in pT
/
mice, at least at the level of cell surface expression. Equivalent results were obtained in a similar staining with V
6 as
the individual antibody versus a pool of antibodies including anti-V
8.1/8.2 (data not shown).
Chains in TCR-
-transgenic, pT
-deficient Mice.
staining, we included
TCR-
-transgenic mice in our analysis. As transgenic line,
we used mice expressing a functional V
8.2 TCR-
transgene, previously shown to prevent expression of endogeneous TCR-
chains (2). As a consequence, essentially all
mature T cells in these mice stain positive for V
8.2, while
no other V
elements can be detected on the cell surface.
To assess the efficiency of allelic exclusion in the absence of
pT
, we crossed mice of the TCR-
-transgenic line with
pT
-deficient animals and performed a four-color cytofluorometric analysis as described above, using V
8.1/8.2 to
detect the transgenic TCR-
chain and all other available
anti-V
reagents in a pool to detect expression of endogeneous TCR-
chains. Although the presence of the transgene allowed us to screen essentially all CD3+ cells for expression of two TCR-
chains rather than a fraction of
cells expressing a particular V
element (i.e., V
8.1/8.2 or V
6) as above, we still found no evidence for cell surface
expression of two distinct TCR-
chains, as endogeneous
TCR-
chains could not be detected either on thymocytes
(data not shown) or on mature T cells (Fig. 2) even in the
absence of pT
. These data, in conjunction with those
from nontransgenic pT
/
mice, strongly suggest that
functional TCR-
chains can regulate the expression of
other TCR-
genes even in the absence of an intact
pre-TCR.
Transgene Can Mediate Effects in Early T Cell
Development Despite the Absence of pT
.
-deficient mice
exhibit a severe defect in early T cell development, which
leads to a dramatic decrease in the relative proportion and
absolute number of DP thymocytes and a concomitant, more than 90% reduction in total thymic cellularity (30).
While analyzing V
surface expression in TCR-
-transgenic mice, we noticed that the presence of the TCR-
transgene on a pT
/
background had a mild, but reproducible, effect on thymic cellularity, causing an up to threefold increase in the total number of thymocytes as compared with nontransgenic pT
/
controls (Fig. 3; data not
shown). This was usually accompanied by a reversion in
the DP/DN thymocyte ratio (Fig. 3, compare the pT
/
and the TCR-
-transgenic, pT
/
panels), although the
relative increase in the proportion of DP versus DN thymocytes varied considerably between individual TCR-
-transgenic, pT
/
mice, possibly reflecting the weakness of the signal involved in this phenomenon.
transgene could influence thymic cellularity even in the absence of pT
, prompted
us to analyze the effect of the
transgene on early T cell
development in pT
-deficient mice. Early T cell development within the CD4
8
population can be subdivided
into four discrete stages that are defined by the differential
expression of CD44 and CD25 surface markers (31). In
normal mice, TCR-
rearrangements take place at or shortly
before the CD25+44
/low stage and only those thymocytes
that manage to express a functional TCR
chain can form
a complete pre-TCR, which is required to trigger expansion and progression to the next developmental stage, defined as CD25
CD44
/low. In pT
-deficient mice, which
cannot assemble an intact pre-TCR, this transition is severely impaired and relatively few CD25
CD44
/low cells
are generated (Fig. 3 B, compare the two left panels). On
the other hand, in pT
+ mice expressing a TCR
transgene, essentially all CD25+ thymocytes can form a pre-TCR, resulting in a strong increase in the proportion of
CD25
44
/low cells and a concomitant reduction in the
percentage of CD25+CD44
/low thymocytes (Fig. 3 B,
third panel from the left). Interestingly, analysis of TCR-
-transgenic mice lacking pT
revealed a marked down
regulation of CD25 in comparison with nontransgenic
pT
/
mice and the appearance of a significant population of CD25
44
/low cells (compare second and fourth
panel in Fig. 3 B). However, this effect of the TCR-
transgene was not as prominent as in the pT
+ background.
These data indicate that the TCR-
transgene can actually
influence the CD25+ compartment of CD4
8
thymocytes
in the absence of pT
in a similar way as in pT
+ mice, although to a much lesser extent.
+) mice have
shown that a TCR-
transgene inhibits TCR-
rearrangements and the generation of
expressing cells (32). Interestingly, the TCR-
transgene was able to suppress
rearrangements, as measured by PCR with primers specific for
V
4 and J
1, even in the absence of pT
(Fig. 4), although
somewhat less efficiently than in the presence of pT
. The
TCR-
transgene also mediated a strong reduction in the number of
cells in pT
-deficient mice (see Fig. 3 C).
This effect appears particularly striking, when taking into
account that the percentage and absolute number of
cells is strongly augmented in normal (nontransgenic) pT
/
mice in comparison to wild-type littermates (30) (Fig. 3 C, compare the two left panels). The inhibition of TCR-
rearrangements and the strong suppression of
T cell development illustrates once more that the TCR-
transgene is
not innocuous despite the absence of pT
.
Fig. 4.
Expression of TCR- transgenes inhibits V
-J
rearrangements. Thymocyte DNA from four types of mice (pT
+ or pT
/
mice
with or without TCR-
transgenes [
-TG]) was amplified with primers
specific for V
4 and J
1 (nomenclature according to reference 27). Three
different amounts of template DNA (300 ng, 200 ng, 100 ng) were tested
to ensure linearity of the signals. An aliquot of the PCR mastermix was
amplified with primers specific for the constant region of IgM to provide
an internal control for the amount of template used (Ig-C bands). Specific
bands were detected with appropriate end-labeled oligonucleotide probes.
N denotes a negative control (kidney DNA).
[View Larger Version of this Image (53K GIF file)]
transgene into mice lacking one of the recombination activating
genes (RAG-1 or RAG-2) leads to the induction of DP
cells and the generation of essentially normal numbers of
thymocytes (33), a result thought to reflect the physiological function of the pre-TCR. In this context, it was of interest to determine the potential effects of a TCR-
transgene on thymocyte development in RAG
/
mice lacking
pT
. To this end, we crossed RAG-2
/
mice carrying the
TCR-
transgene with mice deficient for both RAG-2
and pT
, and analyzed thymi from littermates of the F2 intercross. Fig. 5 shows the result of a representative staining
experiment. There was no difference between nontransgenic RAG-2
/
, pT
+ and RAG-2
/
, pT
/
mice
with respect to the total number of thymocytes (~1-4 × 106) and the CD4/CD8 profiles (virtually complete absence of CD4+8+ cells) (Fig. 5, top right; data not shown).
Introduction of a TCR-
transgene in RAG-2-deficient,
pT
+ mice resulted in a dramatic increase in total thymocyte numbers and efficient generation of DP thymocytes, as reported previously (33). Surprisingly, expression of the TCR-
transgene in RAG-2
/
mice lacking
pT
resulted in the generation of a significant number of
DP thymocytes as well, although the total thymic cellularity was raised only modestly (three- to fivefold over RAG-2
/
controls in four independent experiments), which was
also reflected by the limited reduction in the percentage of
DN thymocytes in these RAG
/
, pT
/
, TCR-
-transgenic mice (Fig. 5, bottom right). These findings are in full
agreement with our observations regarding TCR-
-transgenic, pT
/
mice with a wild-type RAG backgound,
reported above. Taken together, the data indicate that a
transgenic TCR-
chain can signal in the absence of pT
,
promoting the differentiation of DN thymocytes into DP
cells and inhibiting the generation of
-expressing cells, but that it cannot induce the massive cellular expansion,
which normally accompanies the selection of DN CD25+44
thymocytes expressing a productive TCR-
chain, unless
being associated with pT
.
Fig. 5.
Expression of TCR- transgenes in RAG-2-deficient mice
in the absence of pT
induce differentiation, but no substantial proliferation of immature thymocytes. CD4 and CD8 staining of thymocytes isolated from a TCR-
-transgenic, RAG-deficient mouse (pT
+) and from
a TCR-
-transgenic mouse that is RAG-deficient and in addition lacks
pT
(pT
/
). A wild-type C57BL6 (WT [B6]) and a RAG-deficient
mouse are included as controls. The figures on top of each panel refer to the total number of thymocytes found in the respective animal. Similar
results were obtained in four independent experiments. No differences
were found between nontransgenic RAG
/
× pT
/
and RAG
/
× pT
+ mice (data not shown).
[View Larger Version of this Image (56K GIF file)]
(D)J
Rearrangements Are Less Efficiently
Blocked in TCR-
-transgenic Mice Lacking pT
.
transgenes in thymocytes of normal (pT
+) mice inhibits V
(D)J
rearrangements at endogeneous TCR-
loci almost completely,
which is considered as a paradigm for allelic exclusion. To
determine the impact of a transgenic TCR-
chain on
TCR-
rearrangements in the absence of pT
, we compared the relative levels of endogeneous V
(D)J
rearrangements in thymocytes from TCR-
-transgenic and
nontransgenic pT
+ and pT
/
mice, using a sensitive
DNA-PCR assay (see Materials and Methods; Fig. 6 A, legend). As reported previously (2, 25, 26), TCR
-transgenic
thymocytes of pT
+ mice contained nearly undetectable
levels of endogeneous V
rearrangements (Fig. 6 A, lanes
2, 6, 10, 14, 18, 22, 26, 30, 34). Although the transgenic
TCR
chain was still able to inhibit endogeneous TCR-
rearrangements to a large degree even in the absence of
pT
, the inhibition was significantly mitigated, as bands
specific for endogeneous rearrangements involving all six
functional J
2 segments were apparent at very substantial
levels (Fig. 6 A, lanes 4, 8, 12, 16, 20, 24, 28, 32, 36).
Similar results were obtained with two additional V
primers (V
12, V
14; data not shown), clearly indicating an involvement of pT
in the inhibition process.
Fig. 6.
Inefficient inhibition of V (D)J
rearrangements in TCR-
-transgenic
mice lacking pT
. PCR-based
analysis of genomic DNA isolated from unfractionated thymocytes of nontransgenic and
TCR-
-transgenic pT
+ and
pT
/
mice, as indicated in the
panel below the autoradiographs
(
-TG,
-transgenic). Primers
complementary to one of several V
elements were used in
combination with a reverse
primer positioned immediately downstream of the J
2 gene
cluster, allowing amplification
of rearranged, but not germline
V
segments, as shown in the diagram and described previously (25, 26). Specific PCR products corresponding to rearrangements that involve each of
the six functional J
2 elements were identified by Southern blotting with a probe hybridizing to a region immediately upstream of the J
2 primer-annealing site. The numbers below the panels refers to the amount of template DNA used. Ig-C indicates bands corresponding to the IgM constant region, an internal control to estimate the actual amount of template used.
[View Larger Version of this Image (42K GIF file)]
-deficient mice, owing to
the severe block in
T cell development (30), we were
concerned that our results might have been influenced inappropriately by differences in the cellular composition of
thymi from TCR-
-transgenic pT
+ and pT
/
mice,
respectively, although it was difficult to envisage specifically how this could have mimicked enhanced endogeneous rearrangements in the absence of pT
. Nevertheless, to exclude such a possibility completely and to allow a comparison of equivalent thymic subpopulations, we verified our
PCR analysis of endogeneous V
(D)J
rearrangements with DNA from sorted CD25+CD44
/low DN thymocytes,
which represent the developmental stage at which TCR-
rearrangements predominantly occur (12, 14). Fig. 7 shows
that the result of this analysis was exactly the same as with
unfractionated thymocytes; although the transgenic TCR-
chain could inhibit endogeneous V
(D)J
rearrangements in the absence of pT
(Fig. 7, compare lanes 4, 8, 12 with lanes 3, 7, 11), the degree of inhibition was less pronounced than in CD25+ thymocytes expressing pT
.
Taken together, our data clearly indicate that the pT
chain contributes to the inhibition of endogeneous V
rearrangements by productive TCR-
transgenes.
Fig. 7.
Analysis of genomic DNA isolated from sorted CD25+ triple-negative (CD34
8
) thymocytes. (A) Gates used to isolate
CD25+CD44
/low triple-negative thymocytes. Thymocytes from three
mice of the same genotype were pooled and stained as described in Fig. 3
B. FITC-positive cells (macrophages, granulocytes, B cells, CD4-, and
CD8-expressing cells) were eliminated by electronic gating (data not
shown). The panels representing the B6 and the TCR-
-transgenic, pT
+ mice have a much higher density of dots, because CD4- and CD8-expressing thymocytes from these mice (but not from pT
/
animals)
had been depleted with complement before staining, so that triple-negative thymocytes were already strongly enriched. The lower panels show
the purity of the populations after sorting. (B) PCR-based analysis of genomic DNA from sorted CD25+ triple-negative thymocytes. For details,
see legend to Fig. 6. In this particular experiment, V
11 rearrangements
involving all J
2 elements, except J
2.6, were not appropriately amplified
at the highest template concentration (lane 4). This phenomenon is due
to excess template DNA and is not related to the mouse genotype, because a failure to amplify V
J
rearrangements that correspond to
larger PCR bands than V
J
2.6 rearrangements has also been observed
in a few other experiments at the highest DNA concentration when the
template DNA was derived from wild-type or nontransgenic pT
/
mice.
[View Larger Version of this Image (36K GIF file)]
chain efficiently prevents the expression
of a second functional chain on the cell surface of thymocytes and lymph node T cells in both nontransgenic and
TCR-
-transgenic mice even in the absence of pT
. At
first glance, this result seems to suggest that an intact pre-TCR is not required for mediating allelic exclusion. However, impaired cell surface expression of two functional
TCR-
chains in pT
/
mice does not automatically exclude a role for the pre-TCR in implementing allelic exclusion in normal mice. Recent data indicate that TCR
chains can, at least in part, substitute for pre-TCR-
chains, because they induce the generation of almost normal numbers of thymocytes in pT
-deficient mice, when
expressed at a relatively early developmental stage (34, 35).
Moreover, a recent analysis of pT
/
× TCR-
/
and
pT
/
× TCR-
/
mice has provided strong evidence
that a significant proportion of the
thymocytes that develop in pT
/
mice are actually derived from precursors
that have been selected for further development based on
the presence of a functional TCR-
chain and early expression of a conventional TCR-
chain (35). The underlying assumption that some V
J
rearrangements and
TCR-
expression can occur already in the CD25+ DN
subpopulation is supported by a PCR-based analysis of
TCR-
rearrangements in sorted CD25+ thymocytes from
normal mice (36). If early expression of TCR
chains in a
few CD25+ pre-T cells was responsible for the generation
of most mature
T cells in pT
/
mice, the early formation of an
TCR in these cells might as well be accountable for the allelic exclusion of TCR-
chains that is
observed despite the absence of pT
.
nor the pT
chain are involved in mediating allelic exclusion and that TCR-
chains can signal independently, even in the absence of these two partner chains.
Our analysis of TCR-
-transgenic, pT
/
mice clearly
demonstrates that TCR-
chains can signal without being
associated with a pT
chain, resulting in marked effects on
early T cell development; for instance, a small increase in the number of DP thymocytes, partial downregulation of
CD25 in the pre-T cell population, significant inhibition of
V
J
and V
(D)J
rearrangements, suppression of the
generation of
-expressing cells and, most striking, induction of DP thymocyte formation in RAG
/
× pT
/
mice. These effects of a transgenic TCR-
chain in the absence of pT
could be due to the association of TCR-
chains with some other proteins, like TCR-
, TCR-
(37), or the hypothetical VpreT subunit (20), giving rise to
a signaling complex that can assume part but not all of pre-TCR function. The idea that the transgenic TCR-
chain
mediates its effects in the absence of pT
via a signal-transducing complex including CD3 components is consistent
with the observation that a functionally rearranged TCR-
transgene can no longer inhibit V
(D)J
rearrangements in CD3-deficient mice (B. Malissen, personal communication).
chain in the absence of pT
have some
physiological relevance or whether they are solely due to a
peculiarity of the transgenic system; for instance, expression
of the TCR-
transgene at inappropriately high levels. In
this context, it should be mentioned that the TCR-
chains in our TCR-
-transgenic mice can be expressed on
thymocytes in gpi-linked form, a feature not found in nontransgenic animals (38). If the significant inhibition of endogeneous V
(D)J
rearrangements in pT
-deficient
mice was due to a transgenic artifact, one might anticipate a
variation in the degree of inhibition when analyzing different TCR-
-transgenic lines. This may explain, why Xu et
al. (23) have seen an equally strong inhibition of endogeneous TCR-
rearrangements in both pT
+ and pT
/
thymocytes, while in our experiments the potentially artifactual, transgene-specific effects of the TCR-
chain may
be fortuitously less pronounced, allowing us to detect significant differences in the absence and presence of pT
.
Moreover, the use of a stable line of transgenic mice allowed us to compare endogeneous rearrangements within
the same transgenic background, which was not possible in
the chimeric mice of Xu et al. (23), because they were generated from pT
+ and pT
/
ES cell clones that had been
transfected individually with the respective TCR-
transgene, almost certainly giving rise to pT
+ and pT
/
animals with distinct transgene copy numbers and/or insertion sites. Whatever the reason for the discrepant results
may be, our observation that the inhibition of endogeneous
rearrangements at the TCR-
locus is less profound in the
absence of pT
, despite the capacity of the transgenic
chain to signal in part independently of pT
, clearly indicates a role of pT
and the pre-TCR in the regulation of
V
(D)J
rearrangements. The importance of an intact pre-TCR for allelic exclusion at the level of TCR gene rearrangements therefore may be even more obvious in normal (nontransgenic) mice, in which a newly formed, functional TCR-
chain is by definition expressed at physiological
levels and therefore possibly less prone to pT
-independent signal transduction. This will be assessed by comparing
the rearrangement status at the TCR-
locus in a statistically significant number of sorted DN CD25+ thymocytes
from pT
-deficient mice and wild-type littermates at the
single cell level.
Address correspondence to H.J. Fehling, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland. Phone: 41-61-605-1245; FAX: 41-61-605-1364; E-mail: fehling{at}bii.ch
Received for publication 28 April 1997.
1 Abbreviations used in this paper: BCR, B cell receptor; CS, calf serum; D, diversity; DN, double-negative; DP, double-positive; ES, embryonic stem; FSC, forward scatter; J, joining; PI, propidium iodine; pTWe thank C. Laplace for skillful and efficient technical support; M. Dessing and A. Pickert for expert assistance with cell sorting and four-color cytofluorometric analysis; W. Metzger, E. Wagner, and B. Aschwanden for their care in maintaining the mouse colonies; J. Hatton and R. Schulze for oligonucleotide synthesis; B. Pfeiffer and H. Spalinger for photography, and T. Rolink and S. Gilfillan for critical reading of the manuscript. A. Krotkova is affiliated with the Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow. H. von Boehmer is supported by the Human Frontier Science Foundation. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Basel.
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