| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Allelic Exclusion During T Cell Differentiation1
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain is functional only in mature thymocytes. A significant
proportion of immature thymocytes (TCRlow) express
more than one cell surface -chain, but mature thymocytes
(TCRhigh) show phenotypic allelic exclusion and express
only a single -chain. We have analyzed thymocytes for both surface
and intracellular -chain expression and find that the majority of
mature thymocytes express a second -chain intracellularly. This
result is predicted by a model in which the developmentally regulated
allelic exclusion of the TCR -chain is caused by competition between
-chains for the ß-chain rather than by models in which one
-chain is down-regulated or in which selection favors cells with
only a single -chain species. Changes in the relative amounts of
- and ß-chains available for pairing may therefore allow
competition between the two -chains for the ß-chain. Peripheral T
cells also frequently express second -chains in the cytoplasm
(18–27%), despite a rather low frequency of dual -chain expression
on the cell surface (2–4%). The frequency of nonsurface expressed
-chains is reduced somewhat compared with thymocytes, indicating
that an additional level of control of allelic exclusion operates
during the maturation of peripheral T cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- and ß-chains is strikingly different. In-frame
rearrangement of a ß-chain gene effectively blocks further
rearrangement at this locus (reviewed in 1 . When the ß-chain
protein associates with the pre-TCR , it starts a phase in
development during which the cells proliferate and start to express CD4
and CD8. The rearrangement of -chain genes starts in these
TCR-, "double positive"
(DP)3 cells, resulting in a
TCRlow DP population (2). Positive selection is first
detectable in these cells (3). However, -chain rearrangement is not
turned off until the cell has successfully undergone positive selection
(3, 4, 5). Thus most mature T cells have -chain rearrangements on both
chromosomes and many have two in-frame, expressible -chain genes
(reviewed in 1 . Sequential rearrangements on the same chromosome
also occur, so that a single cell can test the selectability of several
TCRs (4, 6, 7). A significant proportion of immature TCRlow
thymocytes (TCRlow/DP/CD69low) express more
than one cell surface TCR -chain (8). However, following positive
selection, mature TCRhigh thymocytes
(TCRhigh/mostly single positive (SP)/CD69high)
display only a single -chain. Hemizygous TCRAo/+ mice do
not have the dual V-expressing cells, showing that these cells are
due to expression of the rearranged genes on both chromosomes, rather
than to "left-over" protein from an earlier rearrangement on the
same chromosome. Thus, there is functional or "phenotypic" allelic
exclusion in the mature thymocytes that is developmentally regulated in
concert with positive selection (8).
In view of the relaxed nature of allelic exclusion of the TCR
-chain, it is perhaps not surprising to find that both alleles
rearrange, giving rise to two different -chain proteins on
TCRlow cells. But what causes a mature thymocyte to lose
the surface expression of one of its -chains? There can be several
possible explanations: 1) single -chain-expressing thymocytes are
selected in preference to dual -chain expressors; 2) the expression
of the second -chain is down-regulated following thymic selection
and TCR up-regulation; and 3) competition between two -chains for a
single ß-chain becomes evident only when thymocytes undergo positive
selection, up-regulate their TCR level, and become TCRhigh
cells ("-chain competition model"). The first two possibilities
have not previously been experimentally tested, but analysis of T cell
clones has shown that these frequently have in-frame -chain
rearrangements on both chromosomes, and that both proteins can be
expressed (1, 9, 10, 11, 12). Allelic exclusion operates phenotypically
however, so that only one of the -chains is generally present on the
cell surface. Phenotypic allelic exclusion appears to be maintained by
competition between the -chains for the ß-chain (1, 4, 9, 12).
We have previously argued that phenotypic allelic exclusion in the
TCRhigh thymocytes operates via -chain competition, and
that it does not function in the TCRlow cells because of
differences in the expression of the - and ß-chains at different
stages of thymocyte development (8). This argument was based on the
finding that -chain proteins are extremely unstable in immature
thymocytes. After their production, they are rapidly degraded so that
ß-pairing is limiting (13). After signaling through the TCR,
-chain expression is up-regulated so that a larger quantity of
-chains are available for pairing (14). The stability of -chains
is higher in mature thymocytes (13). The higher level of -chain
protein found in mature thymocytes (15) results in the 
10-fold
higher level of cell surface TCR in mature thymocytes than in immature
thymocytes (16, 17). We proposed that in the immature thymocytes,
ß-chain is in excess due to the instability of the -chains; thus,
both -chain proteins can pair with ß-chains and are expressed on
the cell-surface. Since -chain expression is higher in the mature
thymocytes, the ß-chains are limiting. Competition between the
-chains for the ß-chain occurs, resulting in phenotypic allelic
exclusion (8). The outcome of all this is that allelic exclusion of
-chain occurs at the level of protein and will be functional only in
TCRhigh mature thymocytes. Thus mature thymocytes, although
still capable of producing more than one -chain, will usually
express a single -chain on the cell surface: the one that has the
higher affinity for pairing to the ß-chain. If the difference in
affinities between the two -chains is not too great, the second
chain may be expressed on the surface. If phenotypic allelic exclusion
operates by competition, then it should be possible to detect more than
one intracellular -chain in mature thymocytes. This would not be the
case if there were selection against dual -chain expressors or if
the expression of the second -chain were down-regulated during
thymic selection.
Expression of dual -chains on normal peripheral T cells has been
reported in both humans and mice at frequencies estimated at 30%
and 10 to 20%, respectively (18, 19, 20). Accurate measurements are
difficult because of the lack of allelic serologic markers for the TCR
-chain. As mentioned above, evidence from T cell clones has
suggested that phenotypic allelic exclusion operates
posttranslationally, and it is therefore of interest to determine and
compare the expression of intracellular and cell surface dual
-chains in peripheral T cells.
We performed flow cytometric analysis of thymocytes and peripheral T
cells for both surface and intracellular -chain expression. We found
that, as predicted by the chain competition model but not the other two
models, both mature thymocytes and peripheral T cells can express more
than one -chain intracellularly. In most cases, however, only a
single -chain is present on the surface. We also found that the
proportion of mature T cells with a second intracellular -chain
species was lower than in the mature thymocytes, approximating the
proportion of mature T cells reported to have two in-frame -chain
rearrangements. Thus, there is a difference in the mechanism of
phenotypic allelic exclusion between peripheral T cells and mature
thymocytes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 (B6) mice were bred and maintained at the rodent
breeding facility of The Scripps Research Institute. The -chain
knockout (C57BL/6J-Tcratm1Mom (21))
(TCRAo/o) mice were purchased from The Jackson Laboratory
(Bar Harbor, ME), and hemizygous TCRA+/o mice were
generated from a B6 x TCRAo/o cross.
Ab staining and flow cytometry
Both the preparation of cell suspensions from thymuses and the
staining protocol for FACS analysis were essentially the same as
described previously (8). Flow cytometry data were acquired on a Becton
Dickinson FACSort instrument (Mountain View, CA), and the Abs used
were all purchased from PharMingen (San Diego, CA) except for
streptavidin-red 613 (Life Technologies, Grand Island, NY). The
anti-V Abs used in this study (aV2 (22), aV3.2 (23),
aV8 (24), and aV11 (25)) were either biotinylated or directly
conjugated to phycoerythrin (PE) or FITC. Anti-Cß (H57-597 (26, 27))
was used similarly.
For cytoplasmic staining, a single-cell suspension of thymocytes from
6- to 8-wk-old mice was prepared. Due to the relatively large number of
V2+ cells compared with other V regions present
in the thymus, the cells were initially stained for surface V2
expression and then permeabilized for intracellular staining as
described previously (28). Briefly, this involved resuspension of cells
in a mixture of RPMI 1640 and FCS (1:1). Next, ethanol was added
dropwise while vortexing the cell suspension gently to a final
concentration of 50%. After these cells were incubated on ice for 3 to
5 min, the permeabilized cells were gently spun down on a relatively
slower speed (200 g). After two additional washes in
a bed of FCS, the permeabilized cells were stained with
anti-V3.2, V8, and V11 Abs. Data acquisition and analysis
were performed as described previously (8).
The cell sorting of V2+ thymocytes and
Thy-1.2+/V2+ splenic T cells was conducted
as described in Reference 8, except that sorting was performed on a
Becton Dickinson FACS Vantage instrument.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
expression on immature but not on mature thymocytes
During differentiation in the thymus, when DP
TCRlow cells develop to become SP TCRhigh
cells, the surface expression of TCR -chain is regulated
differentially in TCRlow and TCRhigh
thymocytes. This is evident from the observation that a significant
number of TCRlow cells express more than one -chain,
while TCRhigh cells carry on their surface a single
-chain (Fig. 1
) (8). Usually, 3 to
5% of V2low cells express V3.2, V8, or V11 as
a second -chain (8). This has been consistently observed on
TCRlow cells and, as reported earlier, the dual
V-expressing cells on V2high cells are not easily
detectable (8). The dual expressors within the TCRhigh
population represent rare events, and we estimated that these cells, if
present, must be <0.5% of the tested V2+ cells. Thus,
there is a clear difference between immature and mature thymocytes in
terms of surface expression and phenotypic allelic exclusion of TCR
-chain.
|
-chain proteins by
TCRhigh thymocytes
To test whether the production of the second -chain is
down-regulated following thymic selection and TCR up-regulation, we
analyzed surface and intracellular staining of thymocytes for different
V regions. Figure 2 shows an
experiment in which V2+ surface-stained cells have
also been stained for both surface and intracellular expression of
V8 and V11. As shown in Figure 2, A–D, there is
frequently surface expression of a second -chain within the
V2low population, but this is rare on
V2high cells. This is not the case for intracellular
staining (Fig. 2, E–I). Figure 2I shows
that all thymocytes positive for surface V2 (both TCRlow
and TCRhigh) also stain intracellularly with anti-V2
and provide a positive control for the specificity of the intracellular
staining. Interestingly, this same plot also shows the presence of
thymocytes staining intracellularly for V2 but negative for surface
V2. This strongly indicates that thymocytes are capable of producing
-chains other than those expressed on their surface. This phenomenon
is more clearly demonstrated in Figure 2, F–H. These plots
show that a significant number of V2high thymocytes,
like the V2low cells, express a second -chain protein
intracellularly. However, unlike V2low cells, mature
thymocytes rarely express more than one -chain on the surface (Fig. 2, B–D). The number of dual -chain-expressing
cells within the TCRlow population is similar for both
surface and intracellular expression.
|
2high cells positive for
intracellular staining with anti-V8 and -V11 is slightly
higher than that calculated for V2low thymocytes. This
could be an overestimate due to the relatively low number of
TCRhigh cells present in the thymus. However, this is
unlikely, as the numbers obtained from a highly enriched
V2high population were very similar to those shown in
Figure 2
(see below). A more feasible explanation is that, due to
increased production of -chains in mature thymocytes, the
steady-state level of -chains is higher in TCRhigh
thymocytes and, thus, allows increased efficiency of detection. In
immature thymocytes, the -chains are either rapidly degraded or, if
able to form stable ß complexes, are exported to the surface (13, 14). Together, these events lower the available pool of intracellular
-chains and are likely to contribute toward lower efficiency of
detection. This is evident in Figure 2I, in which the
intracellular V2 staining is less bright in the cells expressing
cell surface V2low than in those expressing
V2high.
To determine the resolution of this cell surface/intracellular staining
protocol, we compared staining of normal B6 thymocytes with TCR
-chain knockout (TCRAo/o) and hemizygous mice
(TCRAo/+) mice. Figure 3
shows the number of V2high cells expressing a second
-chain in the cytoplasm of normal B6 mice as compared with the two
groups of control mice. Since the TCRAo/+ mice carry a
single -chain allele and can therefore express only a single
-chain, the numbers obtained for intracellular staining represent
the nonspecific background staining. This is significantly lower than
the number of dual-labeled cells in the B6 thymus, while the
TCRAo/o thymus shows little or no background staining
(<0.5%). The intracellular staining for a second -chain is lower
in the TCRAo/o mice than in the TCRAo/+ mice,
probably due to the fact that there is no staining with the
anti-V2 reagent for cell surface -chain in these mice. This
most likely results in a lower overall background staining.
|
Abs shows that the numbers are
additive in the B6 thymus and allows an assessment of the signal to
noise ratio from the TCRAo/+ and TCRAo/o
thymus. It is notable that the signal to noise (B6 to
TCRAo/+) is lower for the TCRhigh thymocytes,
probably for reasons similar to those noted in the previous paragraph.
This experiment rules out the possibility that the intracellular
staining is the result of translation of mRNA expressed from
transcriptionally active unrearranged V genes. If such expression
contributed to the intracellular staining, it should be equally evident
in the TCRAo/o mice, since the targeted mutation in these
mice affects only the C region of the TCRA locus (21).
Dual -chain expression in peripheral T cells
In human peripheral blood, a substantial proportion (30%) of T
cells were estimated to express two cell surface -chains (18).
Murine peripheral T cell populations were estimated to have a slightly
lower number of dual -chain-expressing cells (7–21% (19) or
10% (20)). Although it is possible to generate mouse T cell clones
that carry dual -chains on their surface from these peripheral
cells, Ag-stimulated T cells seemed to predominantly express only a
single -chain (19). To analyze dual -chain expression and
phenotypic allelic exclusion in peripheral T cells, we stained cells
from normal B6, TCRAo/+, and TCRAo/o
spleens for both surface and intracellular -chain expression. A
representative experiment is shown in Figure 4. As compared with the two groups of
control mice, the B6 splenic T cells have a significant population of
cells that express a second -chain in the cytoplasm. Thus, the
capacity to produce a second -chain is not lost when thymocytes
leave the thymus to become mature peripheral T cells. However, it
should be noted that the number of V2+ T cells that
stain intracellularly for V8 and V11 (Fig. 4) is roughly twofold
lower than we find in V2high thymocytes (Fig. 2).
|
-expressing T cells and to confirm the difference with
TCRhigh thymocytes, we decided next to enrich surface
V2+ T cells by cell sorting and then restain them for
cell surface and intracellular V proteins. The sorting of the
V2+ cells allows us to analyze a much higher number of
these cells than in experiments using gating (as in Fig. 4 and Refs. 19
and 20), making the data statistically more valid. Figure 5 shows that the sorted
V2+ T cells include cells that produce other -chain
proteins intracellularly; it also confirms the results shown in Figure 4. The numbers derived from sorted V2+ T cells (Fig. 5)
are very close to what was calculated from the gated analysis of
V2+ cells (Fig. 4). There is clearly a lower proportion
of such cells in the peripheral T cells than in the mature thymocytes.
The double V surface expressors are also distinguishable in this
experiment, representing 0.12% and 0.24% of the V2+
cells for V8 and V11, respectively. These percentages are 19x
and 13x less than the percentages of these V regions expressed
intracellularly as second -chains (see below).
|
-chains in thymocytes and peripheral T cells
Table I presents a summary of data
obtained from two separate experiments on the expression of dual
-chains both on the cell surface and intracellularly. These data
were obtained by sorting V2+/Thy-1+
splenocytes or thymocytes and then restaining for the other aV
reagents either with or without permeabilization for intracellular
staining. Gates for TCRhigh and TCRlow were set
as before. The number of cells expressing particular V elements in
the total TCRhigh or TCRlow population was
determined by the level of staining with an anti-Cß reagent,
while those for peripheral T cells were determined by gating on
Thy-1.2+ cells. The background observed in
TCRAo/+ mice with each of the anti-V reagents was
noticeably higher for intracellular staining than that observed with
the control Ab (see Figs. 3 and 4). The use of TCRAo/+ mice
allows a better estimate of the nonspecific intracellular staining for
aV mAbs. In assessing the frequency of the dual -chains in the
periphery and thymus (Table I), we therefore subtracted the background
obtained from the thymocyte and splenocyte populations from the surface
V2+/TCRAo/+ mice. In the TCRlow
thymocytes, a large proportion of cells (50–60%) express two
-chains on the cell surface, which is very similar to the number
that express the protein as determined by intracellular staining. The
TCRhigh cells show more discrimination. Like the
TCRlow cells, a similar proportion of these mature cells
express dual intracellular -chains, but only 10% of the dual
V+ cells (4.5–6.8% of total) express both chains on
the surface. This regulation is maintained and strengthened in the
periphery, as only 2 to 4% of T cells express two different
-chains. Interestingly, there is roughly a twofold reduction in both
the number of T cells that can express two -chains intracellularly
(18–27%) when compared with the frequency in the TCRhigh
populations (43–65%) and the percentage that express two -chains
on the surface.
|
-chain protein
following transition of immature thymocytes to the mature stage. Thus,
there is no apparent disadvantage during positive selection for
thymocytes that are capable of producing two different -chain
proteins. Thymocytes that are capable of productive rearrangement at
both alleles continue to produce both -chain proteins at the
TCRhigh stage. The analysis of peripheral T cells provides
good evidence that mature thymocytes producing two different -chains
intracellularly can make their way out into the periphery, although
they are somewhat less frequent than in the thymus. As with the
thymocytes, these mature T cells mostly express a single -chain on
their surface. These results are compatible with the -chain
competition model of phenotypic allelic exclusion in thymocytes and
suggest that there is some selective disadvantage to dual -chain
expressors postthymically. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We previously demonstrated that immature,
TCRlow/CD69low/DP thymocytes commonly
express two TCR -chains on the cell surface. As the cells undergo
positive selection and become
TCRhigh/CD69high/SP cells, the surface
expression of the second -chain is lost (8). Here, we show that the
loss of surface expression of the second -chain is not due to
down-regulation of expression of the protein, since it is readily
detected by cytoplasmic staining. This finding rules out one of the
possible mechanisms for this regulation of phenotypic allelic
exclusion: that transcription or translation of the second -chain is
turned off in the mature thymocytes. Another possibility is that
positive selection preferentially chooses cells that can make only one
-chain (8). The demonstration that the mature thymocytes express two
-chain proteins would seem to disprove this idea. However, we cannot
rule out that these "second" -chains are not able to pair with
the ß-chain and thus were never expressed on the surface at the
earlier stage.
A more compelling interpretation is that the phenotypic allelic
exclusion is performed by competition between the -chains for the
ß-chain. Earlier TCR transfection experiments have provided good
experimental data supporting this hypothesis (1, 9, 12). However, this
"chain competition" model requires that the competition occur in
the mature TCRhigh cells but not in the immature
TCRlow cells. We have suggested that this requirement
related to the finding that -chains are quickly degraded in immature
thymocytes (8, 13). This results in limited formation of ß pairs,
which are stable and exported to the cell surface, and leaves an excess
of ß-chain compared with the level of -chain (13). After positive
selection, the net amount of cell surface TCR increases 10-fold (16, 17), as a result of the increased quantity of -chain now available
(15) because of its markedly increased stability (14). We postulate
that this results in competition between the -chains for binding to
the more limited supply of ß-chains (8), which are synthesized at a
lower level in SP thymocytes (15). However, there is evidence for a
pool of unassociated ß-chains even in mature T cells (13), so the
limiting factor could in fact be the CD3 
-chain (D. L. Wiest,
personal communication). This chain is required for stabilizing the
TCR/CD3 complex and allowing it to be transported to the cell surface
(29). Immature DP cells have excess relative to the intermediate
TCR/CD3 complexes to which they bind (13). Expression of is reduced
after signaling through the TCR (14), but is much more stable in SP
than in DP thymocytes (15). This is most likely because it is
stabilized as part of complete TCR/CD3 complexes, as in mature T cells
(13). Thus might be limiting for the formation of complete TCR/CD3
complexes in the mature thymocytes. This model still relies on
differences in stability of different ß pairs, however.
As we have previously discussed (8), if -chain competition is the
mechanism for phenotypic allelic exclusion, then a proportion of the
cells that receive a positive selection signal will lose cell surface
expression of the selectable -chain, leaving the nonselectable
-chain because it binds better to the ß-chain. The maintenance of
the nonselectable -chain occurs because positive selection acts
first on TCRlow cells (3). The requirement for continued
TCR ligation after TCR up-regulation would be expected to ensure that
such cells do not complete positive selection and die (8).
The finding that dual expression of intracellular -chains is
frequent in mature thymocytes (and peripheral T cells; see below)
indicates a posttranslational mechanism for phenotypic allelic
exclusion in these thymocytes. The frequency of surface dual
-chain-expressing cells drops 10-fold (from roughly 5% staining
with the available reagents to <0.5%) following the differentiation
of TCRlow immature thymocytes to TCRhigh mature
thymocytes. In contrast, intracellular expression of both -chains is
maintained in both of these populations. The staining profile of V2
for both surface and cytoplasmic staining (Fig. 2I)
clearly demonstrates that thymocytes can express -chain proteins
other than those expressed on the surface. These dual-expressing
thymocytes make up a significant number of the total V2+
cells in the thymus; the V2low population represents
53% of the total, while the V2high cells represent
13%. Approximately 34% of V2+ cells express V2 only
intracellularly. These findings are predicted by the -chain
competition model but not by the other models suggested.
We have estimated the general number of cells that express two
-chains by comparison of the number of cells expressing V8 or
V11 within the V2+ population with the number in the
total population (Table I). As we have shown previously, the number of
cells expressing two different -chains is greatly reduced in the
TCRhigh cells compared with the TCRlow cells
(8). We calculate that 50 to 60% of the TCRlow cells
have two cell surface -chains; at this stage, apparently most or all
cells that express two -chains intracellularly are also capable of
expressing them on the surface. But as these immature thymocytes
progress to the TCRhigh stage, the number of dual
expressors is reduced 10-fold. However, for cytoplasmic expression, the
frequency of dual -chain-expressing cells within the
TCRhigh population is maintained at 50 to 60%. This
proportion is 2-fold higher than the predicted potential number
(30%) of thymocytes that can undergo productive rearrangement at
both alleles (30). We previously noted a similar discrepancy between
the experimental and theoretical numbers for the cell surface
-chains in TCRlow thymocytes (8). This discrepancy has
yet to be resolved.
Allelic exclusion and dual TCR expression in peripheral T cells
Peripheral T cells with two V elements expressed on the cell
surface have been found in both humans and mice (18, 19). They have
been suggested as potentially autoreactive cells, in that only one of
the ß combinations may have been subjected to thymic selection.
However, only one of the combinations is likely to be restricted by
self-MHC, and if this combination is restricted by self-MHC then it
will have been subject to positive and negative selection (31). In one
TCR transgenic system, however, potential autoimmune cells remained in
the presence of autoantigen. These cells used a second, nontransgenic
-chain (32). Genetic experiments indicate that the ability to
produce double -chain cells does not contribute to susceptibility to
autoimmunity (diabetes, experimental autoimmune encephalomyelitis,
lupus) in disease-prone strains (20, 33). However, expression of dual
TCR can give selective advantage to some autoreactive thymocytes, thus
allowing their escape from negative selection (34).
The lack of allelic markers of the TCR -chain, with the exception of
infrequently expressed V-region markers, has made enumeration of the
dual -chain cells difficult. We have attempted to estimate this
number by first sorting peripheral T cells on the basis of expression
of one V region and then staining and reanalyzing the cells for
expression of other V regions. By this method, a greater number of
events can be analyzed, and we calculate the percentage of double cell
surface -chain expressors to be 2 to 4% (see Table I). In contrast,
estimates of 7 to 21% and 10% dual expressors were made for mouse
lymph node T cells (19, 20) and an estimate of 30% was made for
human peripheral blood T cells (18).
For the cytoplasmic expression of a second V region, we get
remarkably different results. The expression of a second -chain in
the cytoplasm occurs in 18 to 27% of peripheral T cells. This is
5- to 10-fold higher than the level of cell surface dual -chain
expressors calculated above. Yet this expression is 2-fold lower
than for the TCRhigh thymocytes, so there could be
some other allelic exclusion mechanism, such as down-regulation of one
chain or selection of single expressors. It is notable that the
percentage of cells with the ability to produce two -chain proteins
is close to the percentage (30%) of T cell clones found to have two
in-frame rearrangements (1, 9, 10, 11, 12), suggesting that there may be
selection against dual expressors that reduces the number of peripheral
cells with two in-frame rearrangements.
Our data indicate that both mature TCRhigh thymocytes and
peripheral T cells are capable of producing more than one -chain
protein intracellularly, even though very few of them express the
second -chain on the cell surface. The proportion of cells
expressing a second -chain protein is similar in both immature
TCRlow thymocytes, which frequently express the second
-chain on the cell surface, and mature TCRhigh cells,
which do not. This demonstrates that the phenotypic allelic exclusion
found in the mature thymocytes is regulated posttranslationally, most
likely by some form of -chain competition. There is a difference in
the frequency of expression of the second, cytoplasmic, -chain
between the thymus and the periphery, which could reflect an additional
level of control that is manifested during extrathymic maturation of
the peripheral cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Nicholas R. J. Gascoigne, Dept. of Immunology, IMM1, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: DP, double positive; SP, single positive; PE, phycoerythrin.
Received for publication October 15, 1997. Accepted for publication December 17, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß gene allelic exclusion during T-cell development. Immunol. Today 13:315.[Medline]
ß T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
and ß T cell receptor alleles. Cell 69:529.[Medline]
to J genes replaces the preexisting V-J complexes in a T cell receptor ,ß positive T cell line. Cell 55:291.[Medline]
chain genes maximize the production of useful thymocytes. J. Exp. Med. 178:615. chains occurs at the time of thymocyte TCR up-regulation. Immunity 3:449.[Medline]
genes but uses one ß heterodimer for allorecognition and self MHC-restricted antigen recognition. Cell 55:49.[Medline]
chain in a T cell clone. Int. Immunol. 3:75.-gene rearrangements found in both the A10 and BM 3.3 T cell clones give rise to an chain which can contribute to the constitution of a surface-expressed ß dimer. Int. Immunol. 3:719.ß T cell antigen receptor expression results from differential stability of nascent TCR proteins within the endoplasmic reticulum of immature and mature T cells. EMBO J. 13:4504.[Medline]
protein is an early response to TCR signaling that compensates for TCR- instability, improves TCR assembly, and parallels other indicators of positive selection. J. Exp. Med. 181:193. chains in immature CD4+CD8+ thymocytes. Proc. Natl. Acad. Sci. USA 89:9494. chains: dual receptor T cells. Science 262:422. chains on the surface of normal murine T cells. Eur. J. Immunol. 25:1617.[Medline]
chain T cells in autoimmunity. J. Exp. Med. 182:953. and ß block thymocyte development at different stages. Nature 360:225.[Medline]
2+CD8+ T cells in mouse strains expressing both H-2k and T cell receptor Va haplotypes: determination with a V2-specific monoclonal antibody. Eur. J. Immunol. 22:399.[Medline]
-chain variable region (V3) of the mouse T cell antigen receptor. J. Immunol. 143:2602.[Abstract]
8-, Vß2-, or Vß10-bearing T cells. Eur. J. Immunol. 21:3035.[Medline]
11 gene family: analysis of allelic sequence polymorphism and demonstration of J region-dependent recognition by allele-specific antibodies. J. Immunol. 147:3185.[Abstract]
ß T cell receptors. J. Immunol. 142:2736.[Abstract]
chains in TCRs. Int. Immunol. 6:881. locus are susceptible to diabetes and sialitis. Eur. J. Immunol. 26:953.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. P. Morris and P. M. Allen Cutting Edge: Highly Alloreactive Dual TCR T Cells Play a Dominant Role in Graft-versus-Host Disease J. Immunol., June 1, 2009; 182(11): 6639 - 6643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. V. Serreze, C. M. Choisy-Rossi, A. E. Grier, T. M. Holl, H. D. Chapman, J. R. Gahagan, M. A. Osborne, W. Zhang, B. L. King, A. Brown, et al. Through Regulation of TCR Expression Levels, an Idd7 Region Gene(s) Interactively Contributes to the Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in Nonobese Diabetic Mice J. Immunol., March 1, 2008; 180(5): 3250 - 3259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Warmflash and A. R. Dinner A Model for TCR Gene Segment Use J. Immunol., September 15, 2006; 177(6): 3857 - 3864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Warmflash, M. Weigert, and A. R. Dinner Control of Genotypic Allelic Inclusion through TCR Surface Expression J. Immunol., November 15, 2005; 175(10): 6412 - 6419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Niederberger, L. K. Buehler, J. Ampudia, and N. R. J. Gascoigne Thymocyte stimulation by anti-TCR-{beta}, but not by anti-TCR-{alpha}, leads to induction of developmental transcription program J. Leukoc. Biol., May 1, 2005; 77(5): 830 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. D. Lacorazza and J. Nikolich-Zugich Exclusion and Inclusion of TCR{alpha} Proteins during T Cell Development in TCR-Transgenic and Normal Mice J. Immunol., November 1, 2004; 173(9): 5591 - 5600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Niederberger, K. Holmberg, S. M. Alam, W. Sakati, M. Naramura, H. Gu, and N. R. J. Gascoigne Allelic Exclusion of the TCR {alpha}-Chain Is an Active Process Requiring TCR-Mediated Signaling and c-Cbl J. Immunol., May 1, 2003; 170(9): 4557 - 4563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mantovani, B. Palermo, S. Garbelli, R. Campanelli, G. Robustelli della Cuna, R. Gennari, F. Benvenuto, E. Lantelme, and C. Giachino Dominant TCR-{alpha} Requirements for a Self Antigen Recognition in Humans J. Immunol., December 1, 2002; 169(11): 6253 - 6260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. R. Santori, I. Arsov, M. Lili, and S. Vukmanovic Editing Autoreactive TCR Enables Efficient Positive Selection J. Immunol., August 15, 2002; 169(4): 1729 - 1734. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. B. Sant'Angelo, P. Cresswell, C. A. Janeway Jr., and L. K. Denzin Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers PNAS, May 24, 2001; (2001) 121179998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Fujio, Y. Misaki, K. Setoguchi, S. Morita, K. Kawahata, I. Kato, T. Nosaka, K. Yamamoto, and T. Kitamura Functional Reconstitution of Class II MHC-Restricted T Cell Immunity Mediated by Retroviral Transfer of the {alpha}{beta} TCR Complex J. Immunol., July 1, 2000; 165(1): 528 - 532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Fossati, A. Cooke, R. Q. Papafio, K. Haskins, and B. Stockinger Triggering a Second T Cell Receptor on Diabetogenic T Cells Can Prevent Induction of Diabetes J. Exp. Med., August 16, 1999; 190(4): 577 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Sant'Angelo, P. Cresswell, C. A. Janeway Jr., and L. K. Denzin Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers PNAS, June 5, 2001; 98(12): 6824 - 6829. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
