| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Max-Planck-Institut für Immunbiologie, Freiburg, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/
and/or p56lck. While the
numbers of DP cells are strongly reduced in the single-deficient mice,
a further drastic reduction in the generation of DP thymocytes is seen
in the double-deficient mice. The poor generation of DP cells in the
mutant mice is primarily due to an impaired ability of
CD25+ DN thymocytes to proliferate following expression of
a TCRß-chain. Nevertheless, the residual DP cells in all mutant mice
are strictly selected for expression of TCRß polypeptide chains. DN
thymocytes of mutant mice expressed TCRß and CD3
at the cell
surface and contained mRNA for pre-T
, but not for clonotypic
TCR-chains, together suggesting that TCRß selection is mediated by
pre-TCR signaling in all cases. The data suggest differential
requirements of pre-TCR signaling for cell survival on the one hand,
and for the proliferative burst associated with TCRß selection on the
other. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T lineage cells pass through two main
phases of selection: in the first, termed TCRß selection,
CD4-8- double-negative
(DN)3 thymocytes are screened
for productive rearrangement of a TCRß VDJ gene. Successful cells
express the pre-TCR on the cell surface, consisting of a TCRß-chain,
a surrogate TCR-chain termed pre-T, and components of the CD3
complex. Expression of the pre-TCR is a prerequisite for survival and
is followed by maturation to the CD4+8+
double-positive (DP) stage, associated with pronounced proliferation
(reviewed in Refs. 1 and 2). Following rearrangement of the TCR
locus, DP thymocytes express the complete ßTCR on the cell surface
and are screened for recognition of MHC/peptide complexes in the thymic
microenvironment. This second phase of selection generates the
MHC-restricted, self-tolerant repertoire of peripheral T
lymphocytes (reviewed in 3 .
A host of evidence suggests that the pre-TCR drives the generation of
DP cells by a process involving signal transduction through the CD3
complex. Most known pre-TCR-dependent differentiation events can be
induced in TCRß-deficient mice by treatment with anti-CD3 mAb
4, 5, 6, 7 . Deletions of components of the CD3 complex, such as CD3 8
or CD3/ 9, 10, 11 , are associated with defective pre-TCR-dependent
differentiation. Similar defects are seen in mice in which the
src family protein tyrosine kinases
p56lck (Lck) and p59fyn
(Fyn), known to be involved in CD3 complex signaling 12 , have been
genetically manipulated: mice deleted for Lck show a partial defect
13 , whereas double deletion for Lck and Fyn is associated with a more
pronounced block 14, 15 . Moreover, whereas mice bestowed with a
dominant-negative Lck transgene are compromised in the generation of DP
thymocytes 16 , a constitutively active Lck transgene promotes
differentiation of DP cells in RAG-deficient mice 17 . Double deletion
for ZAP-70 and Syk, protein tyrosine kinases with partially redundant
functions in lymphocyte receptor signal transduction 12 , is also
associated with compromised pre-TCR-dependent maturation 18 .
A hallmark of TCRß selection is that the vast majority of DP
thymocytes expresses intracellular TCRß-chains 19 . Nevertheless, a
small proportion of DP cells lacks intracellular TCRß-chains 19 ,
and a small number of DP cells is generated in TCRß-deficient mice
20 and in pre-T-deficient mice 21 . These results suggested
pre-TCR-independent ways for thymocytes to mature to the DP stage 22 ,
and it was subsequently shown that a prematurely expressed TCR-ß
as well as the presence of 
thymocytes can promote the generation
of DP cells 23 . The former mechanism generates TCRß-selected DP
cells; the latter does not 2, 23 . In wild-type (wt) mice, DP cells
generated by these alternative pathways do not account for more than a
few percent of the total DP population.
The relationships between survival, differentiation, and proliferation
following TCRß selection, and the pre-TCR signaling requirements for
each of these aspects of the DN to DP transition, have to date not been
unequivocally delineated. We therefore studied TCRß selection in mice
with graded defects in CD3 complex signaling. In mice deficient for Lck
or for CD3/, the generation of up to 15% of the wt number of DP
thymocytes suggests significant residual pre-TCR signaling activities.
In mice double deficient for CD3/ and Lck, we observe a drastical
further reduction in DP cells, suggesting a more pronounced impairment
in pre-TCR function. We demonstrate that the poor generation of DP
cells in mice with compromised CD3 signaling is due to the inability of
CD25+ DN thymocytes to enter the cell cycle following
expression of a TCRß-chain. Nevertheless, the residual DP cells
generated in the single-deficient and in the double-deficient mice are
strongly enriched for cells expressing intracellular TCRß-chains. The
results suggest that even in the presence of severely crippled CD3
signaling, TCRß selection is involved in the generation of DP cells.
TCRß selection may thus be independent of or may require minimal CD3
signaling, in contrast to the more stringent signal requirements of the
expansion of the TCRß-selected population.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice deficient of p56lck 13 , mice
deficient of CD3/ 11 , and mice deficient of TCRß 20 were
bred in the specific pathogen-free animal facilities of the
Max-Planck-Institute (Freiburg, Germany). Crosses of the
p56lck and CD3/ mutations were
monitored by PCR analysis of DNA isolated from tissue obtained by
earpunching, using primers described in the original papers. Mice were
analyzed at different ages as indicated.
mAb and flow cytometry
Flow cytometry utilized the following mAb, which were labeled with either FITC, phycoerythrin, allophycocyanin, or biotin, purchased from PharMingen (San Diego, CA): anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-Vß8 (F23.1), anti-TCRß (H57-597), and anti-CD44 (IM7). Anti-CD25 (5A2) was purified and labeled with FITC in our own laboratory. Red-670 coupled to streptavidin was used for biotin-labeled Abs. Thymocytes were preincubated with supernatant of anti-FcR mAb 2.4G2 before analysis. Two- and three-color FCM analysis employed a FACScan (Becton Dickinson, Mountain View, CA). Intracellular staining was done on cells fixed with paraformaldehyde and permeabilized with saponin (Sigma, Heidelberg, Germany), as previously described 4, 19 . Controls for cell surface stainings employed isotype-matched mAb labeled with the same fluorochrome; controls for intracellular stainings employed blocking with an excess of the same unlabeled mAb.
Cell cycle analysis
Cell cycle analyses in DN thymocyte subpopulations were done by four-color FCM using a FACSCalibur (Becton Dickinson, Mountain View, CA): first color (allophycocyanin), surface CD4 + CD8 + CD44; second color (phycoerythrin), surface CD25; third color (FITC), intracellular TCRß; and fourth color, DNA staining with 7-amino-actinomycin D (7AAD; Molecular Probes, Eugene, OR). FCM analysis was done as previously described 24, 25 .
Semiquantitative RT-PCR for pre-T and clonotypic TCR mRNA
Total RNA was isolated using TRISOLV (Biotex, Houston, TX), as
directed by the manufacturer. To eliminate remaining genomic DNA, RNA
preparations were subjected to DNase I (Boehringer Mannheim, Mannheim,
Germany) digestion for 20 min at 37°C. Oligo(dT)-primed cDNA was
prepared from total RNA using RNaseH-Reverse Transcriptase (Life
Technologies, Eggenstein, Germany), according to the recommendations of
the manufacturer. cDNAs were adjusted to equal concentrations by
competitive PCR between hypoxanthine phosphoribosyltransferase (249 bp)
and an internal control fragment (200 bp) using the primers previously
described 26 . To semiquantify mRNA for pre-T and for clonotypic
V2 and V4 TCR-chains, equal amounts of cDNA were amplified in
5-fold serial dilution steps, using oligonucleotide primer combinations
shown in Table I
.
|
, a regimen of 94°C for 30 s, 66°C for 40 s, and
72°C for 40 s for 5 cycles, followed by 25 cycles with an
annealing temperature of 64°C. To amplify V2 and V4 family
members, the annealing temperature was 58°C in the first 5 cycles and
55°C in the last 25 cycles. Ten microliters of the resulting
amplified material were subjected to gel electrophoresis in
Tris-borate-EDTA buffer with 1% agarose. |
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/ and Lck
Thymocyte development was compared in mice double deficient (dd)
of CD3/ and Lck (/Lck-dd), in the two types of
single-deficient (sd) mice (-sd and Lck-sd), and in mice bearing wt
alleles for both genes. Fig. 1 shows FCM
analyses of thymocytes from 2–3-wk-old mice for CD4, CD8, and cell
surface TCRß, as well as absolute cell numbers for total thymocytes
and the subpopulations defined by these markers. Results are shown for
representative individual mice. While -sd or Lck-sd mice are able to
generate approximately 3 and 12%, respectively, of the normal number
of DP cells, hardly any of the thymocytes from /Lck-dd mice proceed
to the DP stage. Occasional older /Lck-dd mice generate a small
population of DP cells (see below, Fig. 4). Surface expression of
ßTCR shows the previously described reduced level on DP cells of
-sd mice 27 and the slightly increased level on DP cells of Lck-sd
mice 13 . Thymocytes of /Lck-dd mice fail to express detectable
levels of ßTCR on their surface.
|
|
shows FCM analyses by CD44 and
CD25 of the DN thymocytes of the four types of mice. By these marker
Ags, the DN stage of thymocyte development can be divided into four
consecutive subsets: CD44+CD25-,
CD44+CD25+,
CD44-CD25+, and
CD44-CD25- 28 . TCRß locus VDJ
rearrangement begins during the CD44+CD25+
stage and peaks at the CD44-CD25+ stage 29, 30 . TCRß polypeptide chains are first detected in
CD44-CD25+ DN cells 19, 31 . TCRß-positive
cells proceed to the CD44-CD25- stage and
subsequently become DP cells 19, 31, 32, 33 . The data show a proportion
of about 16% CD44-CD25- DN cells in wt mice,
with a progressive loss of this subset in sd and dd mice. In addition,
the CD44+CD25- subset appears somewhat
diminished in sd and dd mice. Together, the results in Figs. 1
and 2
suggest that the block in pre-TCR-dependent thymic development is more
pronounced in /Lck-dd mice than in mice single deficient for
CD3/ or Lck.
|
/ and/or Lck
Fig. 3 shows flow-cytometric
analyses of intracellular (IC) TCRß expression among gated DN and DP
cells of the four types of mice under study. In wt mice, an average of
about 50% of DN thymocytes are TCRßIC+, with a rather
wide individual variation between about 35 and 70%. DN cells of sd
mice have a reduced proportion of about 20% of TCRßIC+
cells. The DN thymocytes of /Lck-dd mice show an even lower
proportion of TCRßIC+ cells, ranging from 4–12% in
individual mice. As we have shown previously, sd and particularly
/Lck-dd mice are deficient in the expression of rearranged TCRß
VDJ genes at the mRNA and protein level 25 . In the present context,
we focus on TCRß expression in the DP populations. Almost all DP
cells in wt mice display intracellular TCRß, suggesting that TCRß
selection takes place upon differentiation of DN to DP cells 19, 23 .
Similarly, the DP cells generated in both -sd and Lck-sd mice are
equally enriched for TCRßIC+ cells. The results suggest
that DP cells of both kinds of sd mice, even though reduced in absolute
number, are generated by TCRß selection.
|
/Lck-dd mice, we took
advantage of our observation that some of these mice generate a small
proportion of DP cells with increasing age. Two representative examples
are shown in Fig. 4. The data reveal that
the few DP cells generated in /Lck-dd mice are enriched
significantly for expression of TCRß-chains, although enrichment
appears to be somewhat less pronounced than in wt and sd mice. The
specificity of the intracellular TCRß staining is documented in this
experiment by comparison with the DN and DP cells of a
TCRß-deficient mouse. These data suggest that the generation of
the small population of DP thymocytes in occasional older /Lck-dd
mice involves TCRß selection.
CD25+ DN thymocytes of mice single deficient and double
deficient for CD3/ and/or Lck are defective in entering cell
cycle following expression of a TCRß polypeptide chain
What is the basis for the paucity of DP thymocytes in mice with
deficient CD3 complex signaling? As previously shown, fetal
CD25+ DN thymocytes enter the cell cycle following
expression of a TCRß polypeptide chain and subsequently go through
nine cell divisions before they return to a resting state as DP cells
31 . As shown by the experiment in Fig. 5, CD25+ DN thymocytes of
adult wt mice that do not express a TCRß-chain contain a small
proportion of cells in S/G2/M phase of cell cycle, whereas
CD25+ DN cells that express TCRß-chains contain 4- to
5-fold more cells in S/G2/M. These results suggest that,
similar to fetal thymus, adult CD25+ DN thymocytes enter
the cell cycle upon expression of a TCRß polypeptide chain. In the sd
mice, a less pronounced increment in the proportions of cells in
S/G2/M from TCRß- to
TCRß+CD25+ DN cells is seen, whereas
CD25+ DN thymocytes of /Lck-dd mice contain minimal
proportions of cells in S/G2/M, before and after expression
of a TCRß-chain. These results suggest that in mice with compromised
CD3 signaling, CD25+ DN thymocytes do not efficiently enter
the cell cycle following expression of a TCRß-chain. The results in
Fig. 5 also confirm our previous results that mice with CD3 complex
malfunctions poorly express TCRß VDJ genes in CD25+ DN
thymocytes 25 . The paucity of DP thymocytes in these mice therefore
has two causes: the reduced generation and the reduced proliferation of
TCRß+CD25+ DN thymocytes.
|
/ and Lck possess
functional TCRß and CD3 chains at the cell surface
Is TCRß selection of DP thymocytes in sd and dd mice mediated by
a pre-TCR? Expression of the pre-TCR at the surface of DN thymocytes is
very low, and physical detection is ambiguous. However, CD3 chains
have previously been demonstrated at the surface of DN cells of wt and
TCRß-deficient mice by the induction of DP cells upon cross-linking
with anti-CD3 mAb 4, 5, 6, 7 . As shown in Fig. 6, injection of anti-CD3 into
/Lck-dd mice generates a significant proportion of DP cells as well
as induces a three- to 4-fold increase in thymic cellularity. Injection
of anti-Vß8 mAb into /Lck-dd mice results in but a small
induction of DP cells without a significant increase in thymic
cellularity. Such DP cells, however, are highly enriched for cells that
express intracellular Vß8, in contrast to DP cells induced in
/Lck-dd mice by anti-CD3 or to DP cells of wt mice, either of
which express Vß8 at the expected proportion of just below 20%. The
selective induction of Vß8 DP cells suggests a direct recognition of
cell surface Vß8 by the injected mAb. Similar results have been
obtained also with the sd mice (data not shown). Together, these data
strongly suggest that TCRß as well as CD3 are expressed at the
surface of DN thymocytes of /Lck-dd mice, connected to a signaling
machinery that can be activated by Ab cross-linking.
|
, but not for clonotypic
TCR-chains in DN thymocytes of mice single and double deficient for
CD3/ and Lck
The demonstration of TCRß and CD3 at the cell surface of DN
thymocytes does not rule out the possibility that TCRß selection in
sd and /Lck-dd mice is mediated by prematurely expressed ßTCR,
as previously suggested for a small subset of wt DP thymocytes 2, 23 .
If this were the case, we should detect clonotypic TCR mRNA in the
DN cells of these mice at levels greater than in that of wt mice.
Semiquantitative RT-PCRs, performed to estimate the levels of mRNA for
clonotypic V2 and V4 and for pre-T, are shown in Fig. 7. V2 and V4 transcripts were
readily detected in DP cells of wt and sd mice. No V2 and only trace
amounts of V4 transcripts were detected in DN cells of wt and -sd
mice. Most importantly, no V2 or V4 transcripts were seen in DN
cells of Lck-sd and /Lck-dd mice, and the levels of V4
transcripts in DN cells of -sd mice were even lower than that in DN
cells of wt mice. As expected, pre-T mRNA was found strongly
expressed in DN cells of all mice, in wt and sd mice, decreasing upon
transition to DP cells. Unexpectedly, in the mutant mice, expression of
pre-T was generally enhanced, proportionally in DN and in DP cells.
These latter findings warrant further investigation. Together, these
data do not support the possibility that TCRß selection in mice
deficient in CD3 signaling is atypically mediated by prematurely
expressed ßTCR.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/Lck-dd mice extend this information by
demonstrating that, in the absence of Lck, efficient use of the salvage
pathway through Fyn depends on the / module of the CD3 complex.
The unimpaired pre-TCR function in Fyn-sd mice 34, 35 as well as the
dependence of constitutive -phosphorylation on Lck 36, 37 suggests
that the CD3/ module can make use of Lck as well as Fyn.
Conversely, the present work suggests that the CD3 module is mainly
dependent on Lck. Together, the data suggest at least two synergistic
signaling pathways for the pre-TCR, one dependent on CD3/ and
either Fyn or Lck, the other on CD3 and Lck only. It is possible
that this is based on preferences in the cooperation between CD3
modules and src-PTKs 12 . Alternatively, the poor
cooperation between CD3 and Fyn in /Lck-dd mice may be a
consequence of reduced surface expression of the CD3 complex in the
absence of CD3 38 , which may also apply to the pre-TCR.
In spite of the reduced CD3 signaling capacity in both kinds of sd mice
and of the aggravated defect in the /Lck-dd mice, residual DP
thymocytes are generated. It is known that the maturation of DN to DP
cells is not absolutely dependent on signaling through a pre-TCR. The
mere presence of CD3-positive cells 22 , including T cells
23 , can drive a low level of maturation to the DP stage 39 . DP
thymocytes generated in this way, however, can be distiguished from DP
thymocytes produced as the result of TCRß selection by the fact that
they are not enriched for TCRß+ cells 23 . Our results
therefore clearly show that the DP cells seen in CD3
signaling-deficient mice are not generated by this indirect pathway. In
addition, the results presented in this work may shed some light on
this to date unknown indirect mechanism for generating DP cells. In wt
mice, about 3% of DP cells are negative for TCRß and are thus likely
to be generated without TCRß selection. If the generation of these
cells was independent of CD3 complex signaling, the mutant mice
described in this study would be expected to possess wt numbers of
these DP cells, which would then account for a strong proportion of
their DP cells. This is obviously not the case, suggesting that the
TCRß-independent generation of DP cells also requires CD3 complexes
competent of signal transduction. Possibly, as previously suggested 1, 39 , activated CD3-positive cells contribute to a thymic
microenvironment that allows for a low level of differentiation through
trans-activation.
The present results clearly show that the majority of DP cells in both
kinds of sd mice as well as in the /Lck-dd mice are generated by
TCRß selection. TCRß-selected DP cells may arise either by
stimulation through a pre-TCR or a prematurely expressed ßTCR
23 . Our data cannot unequivocally rule out the latter possibility.
However, our failure to detect clonotypic TCR mRNA in DN cells of
/Lck-dd mice argues against this possibility.
The presence of TCRß and of CD3 components at the surface of
/Lck-dd DN thymocytes was demonstrated by Ab-induced proliferation
and differentiation to DP cells. In addition, these experiments show
that forceful cross-linking of these components can generate signals
that translate into a developmental response. Therefore, together with
the spontaneous generation of a few DP thymocytes with increasing age,
these data are consistent with a minimal residual signaling competence
of the pre-TCR in /Lck-dd mice. The defects in CD3 signaling studied
in this work primarily result in reduced proliferation of the
TCRß+ population, whereas the survival of TCRß-positive
cells and their differentiation to the DP stage appear to be
maintained, at least to some extent. Particularly the data on
/Lck-dd mice suggest that a severely crippled pre-TCR/CD3 complex
may suffice to induce the selective survival resulting in TCRß
selection, without being able to induce proliferation. Alternatively,
TCRß selection may be dependent on functions of the pre-TCR other
than signal transduction through the CD3 complex.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Klaus Eichmann, Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany. E-mail address:
3 Abbreviations used in this paper: DN, double negative; 7AAD, 7-amino-actinomycin D; dd, double deficient; DP, double positive; FCM, flow cytometry; IC, intracellular; sd, single deficient; wt, wild-type.
Received for publication August 24, 1998. Accepted for publication November 30, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
determines clonal deletion or induction of developmental program. J. Exp. Med. 177:707.. Proc. Natl. Acad. Sci. USA 90:11401.-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCRß chain expression. Int. Immunol. 6:995. gene. EMBO J. 14:4641.[Medline]
-/- mutant mice and identification of a novel T cell population in the intestine. EMBO J. 12:4863.[Medline]
chain of the T cell antigen receptor complex. Science 261:918./ gene. EMBO J. 12:4347.[Medline]
ß T cell development is abolished in mice lacking both lck and fyn protein tyrosine kinases. Immunity 5:429.[Medline]
and maturation to the CD4+CD8+ stage are highly correlated in individual thymocytes. J. Exp. Med. 178:1867. and ß genes block thymocyte development at different stages. Nature 360:225.[Medline]
gene in the development of ß but not of cells. Nature 375:795.[Medline]
in murine thymocytes and lymph node T cells. Immunity 1:675.[Medline]
chain in T cell development and selection. Science 266:1047.This article has been cited by other articles:
|
|
 |
|
  X. Zheng, J.-X. Gao, X. Chang, Y. Wang, Y. Liu, J. Wen, H. Zhang, J. Zhang, Y. Liu, and P. Zheng B7-CD28 Interaction Promotes Proliferation and Survival but Suppresses Differentiation of CD4-CD8- T Cells in the Thymus J. Immunol., August 15, 2004; 173(4): 2253 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Blank, I. Brown, R. Marks, H. Nishimura, T. Honjo, and T. F. Gajewski Absence of Programmed Death Receptor 1 Alters Thymic Development and Enhances Generation of CD4/CD8 Double-Negative TCR-Transgenic T Cells J. Immunol., November 1, 2003; 171(9): 4574 - 4581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Baur, G. Nerz, A. Nil, and K. Eichmann Expression and selection of productively rearranged TCR{beta} VDJ genes are sequentially regulated by CD3 signaling in the development of NK1.1+ {alpha}{beta} T cells Int. Immunol., August 1, 2001; 13(8): 1031 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Baird, J. A. Lucas, and L. J. Berg A Profound Deficiency in Thymic Progenitor Cells in Mice Lacking Jak3 J. Immunol., October 1, 2000; 165(7): 3680 - 3688. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wack, M. Coles, T. Norton, A. Hostert, and D. Kioussis Early Onset of CD8 Transgene Expression Inhibits the Transition from DN3 to DP Thymocytes J. Immunol., August 1, 2000; 165(3): 1236 - 1242. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
