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- and ß-Chains Is Highly Dependent on the Level of Selecting Ligand1
Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß loci. We found that this transgenic TCR is also
very inefficient at mediating ß selection, thereby showing a direct
linkage between ß selection and allelic exclusion of TCR ß. We have
also examined these mice on MHC backgrounds that have reduced levels of
I-Ek and found that positive selection of cells with high
levels of the transgenic TCR depends greatly on the ligand density.
Decreasing the selecting ligand density is a means of reducing the
number of available selecting niches, and the data reveal that the 3.L2
TCR is used sparingly for positive selection under conditions where the
number of niches becomes limiting. The results, therefore, show a way
that T cells may get to the periphery with two self-restricted TCRs:
one that efficiently mediates positive selection, and another that is
inefficient at positive selection with the available niches. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß lineage is the
successful rearrangement of a TCR ß-chain gene (1, 2, 3, 4). This first
major checkpoint in the thymic developmental process has been called
ß selection (5, 6). The initial ß rearrangement takes place in
CD4-CD8- thymocytes, and successful
rearrangements produce functional TCR ß-chains that pair with the
pre-T-chain (7). The expression of the ß/pre-T complex on the
cell surface leads to three main consequences: 1) proliferation of
these thymocytes, 2) up-regulation of the CD4 and CD8 coreceptors, and
3) an end to rearrangement at the ß locus (2). The result is an
expanded CD4+CD8+ population of thymocytes that
all have at least one ß-chain that is competent for cell surface
expression. Rearrangement is stopped quickly at the ß locus once one
functional ß-chain has been produced, and it is observed that almost
all T cells have only a single functionally rearranged ß (8). This
mechanism is therefore termed allelic exclusion, but does not exclude
the possibility that both ß loci can functionally rearrange. Indeed,
studies on both human and mouse lymphocytes have shown that 1% of
peripheral T cells can have two functional TCR ß-chains expressed on
the cell surface (9, 10, 11), but it is not known whether this level of
allelic exclusion is determined solely by the efficiency of the ß
selection step or if other mechanisms might be involved.
Once thymocytes become CD4+CD8+, they can then
start rearrangements at the TCR locus. Double-positive thymocytes
will keep trying to rearrange until they can produce a functional
TCR complex on the cell surface that is competent for positive
selection (12). Therefore, at this stage many functional -chains are
produced that are not competent for positive selection, but are
nevertheless expressed along with the second, selectable TCR. It has
been estimated that as many as 30% of peripheral ß T cells
express two -chains (13).
Others have considered the effect that multiple - and ß-chains
might have on an immune response and have suggested that they may be
involved in autoimmunity (14). A T cell that has a single TCR against
self may not ever be activated and find the site of its self antigen,
or it may not have sufficient avidity to react, but if it also has a
second TCR that is activated by a pathogen, the T cell could then
become competent to attack self via its other TCR. The argument that
has been raised against this hypothesis is that T cells with two TCRs
are likely to have only one of the TCRs that is specific for self MHC
plus peptide (15). This is a natural consequence of the fact that
CD4+CD8+ thymocytes stop trying to rearrange at
the locus as soon as an -chain is produced that is competent for
positive selection (12).
This latter counterargument would certainly be true if a TCR always immediately started the process of positive selection when the proper ligand was also present in the thymus, but expression of a selectable TCR is not sufficient for positive selection, since this process is limited by the number of available selecting niches (16). This concept stems from the fact that in TCR transgenic mice in which all the CD4+CD8+ thymocytes express a receptor competent for positive selection, only 20% of these CD4+CD8+ cells are positively selected (17). There are a limited number of niches available to select these TCRs. Although the number of niches can be reduced experimentally by lowering the number of cells that promote selection, a major factor that determines the number of available thymic niches in the mouse is the expression level of selecting molecules. For at least two cytochrome c-specific TCRs, it has been reported that positive selection can depend greatly on the expression level of the selecting ligand (18, 19). Thus, a TCR that is specific for self-MHC may not mediate positive selection due to a lack of a thymic niche, but this TCR may still be able to make it to the periphery if it is expressed on a cell that has a second, more efficiently selected TCR.
We have recently produced TCR transgenic mice that use a TCR specific
for the d allele of murine hemoglobin
(Hbbd)4 presented
by I-Ek. These mice show extensive usage of endogenous TCR
ß- and -chains and also show a dramatic dependence on
I-Ek levels for positive selection. These mice, therefore,
gave us the ability to look at mechanisms other than ß selection that
might act to keep only one functional TCR ß-chain per cell. These
mice also gave us an opportunity to examine the effect of selecting
ligand density on positive selection and endogenous receptor usage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Minigenes of TCR 3.L2 - and ß-chains were constructed by
inserting V-J and V-D-J ß exon cassettes into TCR and ß
shuttle vectors (20). 3.L2 V18 V-J and 3.L2 Vß8.3 V-D-J segments
were amplified by PCR using cDNA from the 3.L2 T cell clone (21). The
gene fragments were cloned into pBluescript (Stratagene, La Jolla, CA),
sequenced using ABI Dye Terminator Cycle Sequencing (Perkin-Elmer,
Foster City, CA), and cloned into the TCR shuttle vectors. To verify
that the V18 and Vß8.3 chains were involved in the recognition of
Hb64–76, the minigene constructs were transfected
into the TCR-negative 58 -ß-hybridoma line, and identical
specificity to the original clone was observed (data not shown). The
minigene constructs were then coinjected into C57BL/6 pronuclei in the
Department of Pathology’s Transgenic Core Facility as previously
described (22). Mice were screened by Southern blotting of genomic DNA
using probes to the C and Cß regions. The founder mice were bred
to the B6 congenic strain B6.AKR-H-2k/FlaEg or to the
RAG-1-deficient strain C57BL/6J-Rag1tm1 Mom (The
Jackson Laboratory, Bar Harbor, ME). Transgenic F2
generation mice were identified that were H-2k homozygous
by FACS analysis of the peripheral blood using
anti-H-2Kk and anti-H-2Kb Abs. After
the establishment of the line, transgenic mice were identified by PCR
amplification of 3.L2 transgene from tail DNA. The PCR conditions
for the 3.L2 -chain were 35 cycles of 94°C for 30 s, 60°C
for 30 s, and 72°C for 45 s. The forward primer was
5'-AAACTCGAGACCTGTGTGGATAAAAACCTCTCTGATTCTGGTTTGCTTTTCTGTTTCCAAGCAGGGGCAAAGAGCCAATGGGGAGGA-3',
and the reverse primer was
5'-AAAATTGCGGCCGCTTGGGCCCAAGAAACTGTCATCAAAACGTACATGGATGAACCACGAGGCTGGTC-3'.
All mice were housed in a specific pathogen-free barrier facility at
Washington University.
Ab production
Clonotypic Ab (CAb).
The 3.L2 CAb was made following a rapid immunization protocol (23).
C57BR/cdJ mice (The Jackson Laboratory) were immunized s.c. with 2
x 107 3.L2 clone T cells emulsified in CFA and
boosted 13 days later with 2 x 107 T cells in IFA.
Draining lymph node cells were fused 3 days later with the B cell
fusion partner P3X63Ag8.653 (24). Supernatants were screened by FACS
for positive staining of the 3.L2 hybridoma. The supernatant from one
well strongly stained the 3.L2 T cell hybridoma and was shown to be
clonotype specific by the absence of staining to a panel of T cell
hybridomas expressing Vß8.3 receptors and those expressing V18
family receptors. Specificity for the 3.L2 TCR was further shown by
specific staining of the
58-ß--transfected cells described above.
The CAb line was subcloned twice and was determined to be an IgG2a 
isotype. The CAb Ab was purified from tissue culture supernatants using
protein A-Sepharose (Sigma, St. Louis, MO) and biotinylated.
1B3.3. The anti-Vß8.3 Ab (1B3.3) was made by immunizing Armenian hamsters i.p. with 1 x 107 3.L2 clones in saline three times, with each injection 14 days apart. Hamsters were rested for 2 mo and then given 5 x 106 T cells i.v. Spleens were fused 3 days later with P3X63Ag8.653 (24). Supernatants were screened by activation of the 3.L2 hybridoma by plate-bound Ab. 1B3.3 clone specificity was shown by the ability to activate or stain by FACS Vß8.3 cells and not Vß8.1 or 8.2 hybridomas. The Ab was subcloned four times, and supernatants were purified on protein A-Sepharose and biotinylated.
Peptides
The peptides used in this study were synthesized by standard F-moc chemistry using either a Synergy Peptide Synthesizer (model 432A, Applied Biosystems, Foster City, CA) or a multiple peptide synthesizer (Symphony/Multiplex, Protein Technologies, Tuscon, AZ). The peptides were purified by HPLC, and their purity and accuracy were confirmed by mass spectrometry (Washington University Mass Spectrometry Resource). The concentration of the peptides was determined on an amino acid analyzer (model 6300, Beckman, Fullerton, CA). Altered peptide ligands have been defined previously and are referred to using the one-letter amino acid code of the substituted amino acid residue and its position (25). The amino acid sequences for the peptides used in this study are: Hb64–76, GKKVITAFNEGLK; T72, GKKVITAFTEGLK; I72, GKKVITAFIEGLK; A72, GKKVITAFAEGLK; and Q72, GKKVITAFQEGLK.
Flow cytometry
Single cell suspensions were prepared from thymus and spleen.
One million cells per sample were stained at 4°C for 30 min with the
Abs diluted in PBS with 0.5% BSA and 0.02% NaN3, and
then washed. When necessary, cells were also stained in an identical
manner with a second step reagent. The Abs used in this study were
53.6.7-FITC (rat anti-mouse CD8; PharMingen, San Diego, CA),
H129.19-phycoerythrin (rat anti-mouse CD4; PharMingen),
H57-597-biotin (hamster anti-mouse TCRß; PharMingen),
17-3-3-biotin (mouse anti-mouse I-Ek; PharMingen),
AF6-88.5-biotin (mouse anti-mouse H-2Kb; PharMingen),
36-7-5-biotin (mouse anti-mouse H-2Kk; PharMingen),
G155-178-biotin (mouse anti-TNP (control IgG2a; PharMingen),
KT4-biotin (rat anti-mouse Vß4; PharMingen), 14-2-biotin (rat
anti-mouse Vß14; PharMingen), TR310-FITC (rat anti-mouse
Vß7; PharMingen), streptavidin-tricolor (Caltag, South San Francisco,
CA), streptavidin-FITC (Caltag), CT-CD8a-tricolor (rat anti-mouse
CD8; Caltag), CAb-biotin (rat anti-mouse 3.L2 clonotype), and
1B3.3-biotin (hamster anti-mouse Vß8.3). Cells were analyzed on a
FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using
CellQuest analysis software.
T cell proliferation assay
Proliferation of primary 3.L2tg T cells was performed as described previously (21). Briefly, 5 x 105 splenocytes were cultured in RPMI 1640 supplemented with 10% FCS, 5 x 10-5 M 2-ME, 1 mM Glutamax (Life Technologies, Gaithersburg, MD), and 50 µg/ml gentamicin in a 200-µl total volume in a 96-well tissue culture plate with the indicated concentration of peptide. Culture wells were pulsed at 48 h with 0.4 µCi [3H]thymidine and harvested 18 to 24 h later.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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18 and Vß8.3 genes of the Th1 clone
3.L2 (21, 26). This founder was generated by coinjection of the
constructs into C57BL/6J (H-2b)-fertilized eggs, and
the resulting line was named 3.L2tg. Because the 3.L2 clone is specific
for residues 64 to 76 of murine hemoglobin (Hbbd) complexed
to I-Ek, the founder was bred to the congenic strain B6.AKR
(H-2k) to introduce the I-Ek selecting ligand
and also to maintain homozygosity of the nonstimulatory
Hbbs allele.
To follow expression of the 3.L2 TCR in these mice, we have generated
two mAbs: one is against the 3.L2 clonotype, which we call CAb, and the
other is specific for Vß8.3. Initial analysis of these mice was
performed on thymocytes of 3.L2tg mice on the H-2k/k
background. As shown in Figure 1
A, the percentages of CD4 and
CD8 single-positive cells are similar to those observed in
nontransgenic B6.AKR mice. The thymocytes express the transgenic
ß-chain (Fig. 1B) and the combination of the
transgenic and ß as shown by CAb staining (Fig. 1C). The CAb+ cells can be divided into
three groups: low, intermediate, and high. The intermediate cells are
mostly the immature double-positive cells, and the high CAb cells are
those we consider to be undergoing selection. When we gated on these
high CAb cells, we observed that they were preferentially selected into
the CD4 lineage (Fig. 1D). The histograms in Figure 1, E and F, demonstrate the specificity of these
Abs. These panels show staining performed on the CD4+
thymocytes from nontransgenic B6.AKR mice. There are essentially no
CAb+ cells, whereas there are about 7%
Vß8.3+ cells among CD4+ thymocytes in
these mice.
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, the
Hb64–76 peptide induced strong proliferation in 3.L2tg
splenocytes at a concentration as low as 10 nM. Furthermore, peptide
analogues of Hb64–76 that are known to stimulate the 3.L2
clone also stimulated the 3.L2tg splenocytes with a relative activity
similar to that found with the original T cell clone (26). Therefore,
the 3.L2tg mice on an H-2k/k background have cells that
express the same TCR as the 3.L2 clone. These cells are selected into
the CD4 lineage and recapitulate the reactivity observed in the 3.L2
clone.
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B shows Vß expression on
CD4+ splenocytes in 3.L2tg mice. Although 85% of these
CD4+ splenocytes express the transgenic Vß8.3 chain, they
also express significant levels of the three endogenous ß-chains that
we examined (Vß4, Vß14, and Vß7). There are many other
Vß-chains that we did not examine, but we would presume that all the
endogenous chains are being used. The extent of endogenous ß usage in
these mice suggests that most of these T cells are expressing more than
one ß-chain on the cell surface. Similar results were obtained with
CD4+ thymocytes. As a control, we also examined Vß usage
on CD4+ splenocytes in a class II-restricted TCR transgenic
mouse that was generated using the same expression constructs as the
3.L2tg mouse, but with the 3A9 TCR (20). This TCR is comprised of the
Vß8.2 and V3 chains, and in these mice (as expected) there is very
little usage of endogenous, nontransgenic TCR ß-chains (Fig. 3A). We also found that the transgenic Vß8.2 chain
is expressed on >97% of CD4+ splenocytes in 3A9
transgenic mice (data not shown).
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indicate that most T cells in the 3.L2tg
mouse are expressing more than one ß-chain on the cell surface. We
wanted to show this directly by dual staining of CD4+
splenocytes for expression of the transgenic Vß8.3 and an endogenous
TCR ß-chain. Figure 4 shows staining of
CD4+ splenocytes for both Vß8.3 and Vß14. As expected,
the normal B6.AKR mouse expresses these two TCR chains on distinct
populations of T cells (Fig. 4A). In contrast, in the
3.L2tg mouse, Vß14 is expressed almost exclusively on cells that also
have the transgenic Vß8.3 chain (Fig. 4B). In
addition, dual staining of CD4+ splenocytes in 3.L2tg mice
for the two endogenous chains Vß14 and Vß4 found that these two
receptors are expressed on distinct cell populations (data not shown).
The data indicate that almost all the CD4+ T cells in this
mouse have both the transgenic Vß8.3 and one additional, endogenous
Vß on the surface.
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, both
the transgenic and ß are expressed on most thymocytes in
H-2k/k mice. In these mice, although most cells are
expressing more than one TCR, the majority of these cells express the
transgenic TCR at high levels, and it is this TCR that is likely to be
used for positive selection. This fits with models that suggest that in
thymocytes that have two TCRs, only one will be able to mediate
positive selection (15). We next wanted to test this idea by asking
whether the transgenic TCR would continue to be used on a nonselecting
MHC background and also how the transgenic TCR would be used on a
background that limited the number of available selecting niches by
reducing the levels of I-Ek.
We were able to investigate these questions by breeding the 3.L2tg,
H-2k/k mice to the H-2b strain C57BL/6 and the
H-2h4 strain B10.A(4R). By such breeding, we were able to
obtain 3.L2tg mice that expressed varying levels of I-Ek.
H-2k/k mice have the highest levels of I-Ek,
whereas H-2h4 mice do not express I-Ek due to a
defect in the E promoter (28). H-2k/h4 mice have levels
of I-Ek that are reduced compared with those of
H-2k/k because only one chromosome has a functional E
gene. H-2k/b mice have levels of I-Ek that are
even lower than those of H-2k/h4. This is because the
H-2b chromosome is also unable to contribute a functional
E gene, and the functional E-chain from the H-2k
chromosome must be shared between Eßk and
Eßb (see Table I). The
result is that I-Ek levels vary in these MHC backgrounds:
k/k > k/h4 > k/b, and h4/h4 is I-Ek negative.
We have confirmed that these mice really do have these relative amounts
of I-Ek, both by staining with an Ab against
Eßk and by measuring the ability of APCs from these
strains to stimulate a hybridoma derived from the 3.L2 clone
(Table I).
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).
What is striking about the CD4+ T cells that do develop is
that their usage of the 3.L2 transgenic - and ß-chains is highly
dependent on the level of the I-Ek-selecting ligand. Figure 5A shows CAb staining
performed in a single experiment on CD4+ thymocytes in
3.L2tg mice bred onto the four indicated MHC backgrounds, and Table II
shows cumulative data from numerous mice and experiments. The data show
that as the level of I-Ek is reduced, the percentage of
thymocytes maturing with high levels of the 3.L2 TCR is also reduced.
Correspondingly, Figure 5B shows that the percentage of
CD4+ thymocytes using the transgenic ß-chain is also
reduced as I-Ek levels are lowered. Surprisingly, in the
absence of I-Ek, usage of Vß8.3 in CD4+ cells
is reduced to levels seen in nontransgenic mice, even though 85% of
CD4+ T cells use this chain on a 3.L2tg;H-2k/k
background (Fig. 3B). The down-regulation of
transgenic ß must mean that these cells are using an endogenous
ß-chain, since levels of total TCR ß remain uniformly high (data
not shown). These data demonstrate that this TCR is highly dependent on
the density of the selecting ligand for positive selection.
Furthermore, CD4+CD8+ thymocytes that express
more than one TCR specificity will favor expression of the TCR used for
positive selection (29). However, in conditions where the number of
available selecting niches is limiting (e.g.,
3.L2tg;H-2k/b) these mice use mostly nontransgenic TCRs for
positive selection even though the transgenic TCR has the requisite
specificity for positive selection.
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displays dual staining for Vß8.3 and
Vß14 on CD4+ splenocytes from 3.L2tg mice with four
different levels of I-Ek. Indeed, as I-Ek
levels were reduced we did observe an increase in the percentages of
cells using endogenous Vß14. Similar increases were seen with Vß4
and Vß7 (data not shown). Overall, on the H-2k/h4 and
H-2k/b backgrounds, similar levels of endogenous TCR
ß-chains were used, and this was at a level similar to that found in
nontransgenic mice. Therefore, our conclusion is that under conditions
where the number of thymic niches for the transgenic TCR is limiting,
the endogenous TCR repertoire is expressed at near normal levels. In
3.L2tg;H-2h4 mice, the percentage of cells using high
levels of Vß8.3 is reduced to the levels found in normal mice,
although a fairly high percentage of CD4+ cells maintains
Vß8.3 expression at a low level.
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or ß
loci and will therefore only be able to express the transgenic TCR.
3.L2tg;RAG-/- mice with an H-2k/k MHC have
thymi about 10 times smaller than those in 3.L2tg;RAG+/+
mice. These small thymi are made up of about 85%
CD4-CD8- cells that are also CD44 low and
CD25+ (Fig. 7 and data not
shown). CAb staining of the different subsets in these mice shows that
the CD4-CD8- cells are negative for surface
TCR, but cells at the CD4 single-positive stage express uniformly high
levels of the 3.L2 TCR (Fig. 7B). Surprisingly, we
also see cells that are CD8 single positive and expressing high levels
of 3.L2 TCR. We are currently investigating the selection and function
of these cells. Overall, these data suggest that the process of
positive selection works well in these mice, but the
3.L2tg;RAG-/- mice are very inefficient at the transition
from CD4-CD8- to
CD4+CD8+. Because we see this phenotype when
endogenous TCR chains are unable to rearrange, the endogenous ß must
be required in 3.L2tg;RAG+/+ mice to mediate efficient ß
selection.
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and 3.L2 Vß8.3. A precedent for the latter
explanation has been seen in the inability of certain Ig heavy chains
to mediate maturation of B cell precursors in adult bone marrow due to
poor pairing with the surrogate light chain (31).
These data show that in the 3.L2tg mice, ß selection is not working
efficiently to prevent multiple ß-chain expression on individual
thymocytes. This has given us an opportunity to determine whether any
other mechanisms might be at work to limit multiple ß expression. We
have seen one possibility in the examination of transgenic TCR levels
on CD4+CD8+ thymocytes. Figure 8 shows the levels of CAb staining on
double-positive thymocytes in 3.L2tg mice bred onto the four MHC
backgrounds that differ in their levels of I-Ek. The
3.L2tg;H-2k/k mouse that is shown is representative of most
of the 3.L2tg;H-2k/k mice examined; the levels of CAb
staining on CD4+CD8+ thymocytes is mostly in a
peak that we call TCR intermediate. On the MHC backgrounds that have
reduced levels of I-Ek, we have observed that the
double-positive thymocytes use lower levels of the transgenic TCR, even
though the level of total TCR ß is the same in all these mice (data
not shown). This is surprising given the fact that interactions between
the TCR and MHC ligands are only thought to influence the few
CD4+CD8+ cells that are starting the process of
positive selection (32). Therefore, these data suggest that in normal
mice, the few CD4+CD8+ thymocytes that have two
functional ß-chains due to the occasional imperfection of the ß
selection process may have a tendency to down-regulate any ß-chain
that does not result in efficient positive selection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß-chains. Surprisingly, these mice use many endogenous ß-chains
despite the fact that the transgenic - and ß-chains are expressed
well on both CD4+CD8+ and CD4 single-positive
thymocytes. Although the transgenic chains are only expressed at low
levels on some backgrounds, this does not indicate a down-regulation of
TCR in general, because T cells in these mice always have normal,
uniform levels of TCR ß, as measured by staining with an
anti-TCR ß Ab. Therefore, in these mice, the level of ligand
expression is critical in determining TCR usage.
This observation has important implications for the mechanism of
determining the naive T cell repertoire. The data suggest that many
cells at the immature CD4+CD8+ stage have
multiple -chain rearrangements and, in the case of our 3.L2tg mice,
- and ß-chain rearrangements. However, during the process of
positive selection, a specific TCR is up-regulated as cells progress
from the double-positive to the single-positive stage. It is striking
that in our 3.L2tg mice under conditions where the 3.L2 TCR is poorly
selected, most positively selected cells fail to up-regulate the 3.L2
TCR. Instead, this TCR seems to remain on many cells, but only at a low
level. Thus, up-regulation of TCR during positive selection seems to be
specific for the TCR that can efficiently mediate this process. This
type of phenomenon has also been observed in a careful analysis of
multiple -chain usage on immature vs mature thymocytes
(29).
At this time, we do not know the mechanism by which the developing
thymocyte determines that a particular TCR ß pair is useful for
selection and therefore up-regulates only that particular TCR. T cells
seem to have a type of feedback mechanism that instructs a cell that a
particular TCR has bound ligand. In mature cells this results in
specific down-regulation of the ligated TCR (33). We would argue that a
similar mechanism acts in immature thymocytes such that cells whose TCR
binds a ligand will up-regulate that TCR specifically. In addition, we
cannot rule out that competition for pairing among - and ß-chains
may play some role, particularly in the 3.L2tg mice that can
potentially have two ß- and three -chains functionally rearranged
in double-positive thymocytes.
Previous studies have reported that mice transgenic for the 2B4 and 5C.C7 TCR are also both dependent on ligand density, whereas the AND TCR transgenic mice show little dependence on ligand levels (18, 19). This phenomenon may also explain observations regarding selection of 3A9 TCR transgenic mice. Thymocytes bearing this TCR are selected well on an H-2k background, but poorly on an H-2k/b background (34). Although it is unknown exactly what makes some TCRs highly dependent on ligand density, it seems that those TCRs that are not selected efficiently will be more dependent on the level of selecting ligand. Thymocytes with these TCRs need more time to find their selecting niche, and if the number of niches is effectively reduced by lowering the level of ligand expression, they may not find the niche before death. One way that these cells might be saved is for them to express a second TCR specificity that might be more efficiently selected. We would conclude that the efficiency of selection of the 3.L2tg TCR is low, and when the number of niches is lowered, thymocytes have a better chance for selection using an endogenous TCR specificity.
This interpretation has implications for the involvement of second TCRs in autoimmune recognition. There is evidence to suggest that a T cell that has a receptor against self may be ignorant of self until that cell is activated through a second TCR against some foreign Ag (14). Once activated, this cell may then find the self Ag and become competent to mediate some effector function in response to the self Ag. This scenario depends on the cell having two receptors that are both restricted to self MHC. It has been argued that this is an unlikely event, because once a CD4+CD8+ thymocyte rearranges a TCR that is competent for positive selection, it should begin that process and stop rearrangements (12, 15). That would be true if TCRs were all uniformly efficient, like the AND TCR. The data presented here, however, would argue that under conditions where selecting niches are limiting, a thymocyte may express one self-restricted TCR, but get selected on a second self-restricted TCR because rearrangements continue until an efficient TCR is produced. The second TCR may not be up-regulated during positive selection, but could be maintained at a low level until activation of the T cell makes its specificity relevant. Due to the large number of T cells produced, it seems inevitable that T cells with two self-restricted TCR specificities will exist in the mature T cell repertoire.
These data also have implications for the mechanism of allelic
exclusion at the TCR ß locus. A variety of experiments have pointed
to a direct link between the ß selection step and allelic exclusion
at the ß locus (3, 12). It is clear that when a functional ß-chain
makes it to the cell surface of a CD4-CD8-
thymocyte, a very efficient shut-off of rearrangement at the ß locus
ensues. What we have observed in the 3.L2tg mouse also points to such a
linkage. Breeding of the 3.L2tg mouse to the RAG-/-
background shows that the 3.L2 transgenes are not capable of inducing
efficient ß selection on their own. These mice are not able to
produce a greatly expanded CD4+CD8+ population,
although some cells using the 3.L2 TCR do make it through development.
These transgenes are not efficient at ß selection, and they are also
not efficient at excluding endogenous TCR ß rearrangements. We would
conclude that the same signal that goes through ß/pre-T to induce
ß selection also induces allelic exclusion at the ß locus. This has
been suggested by studies involving lck transgenes and
studies on the role of pre-T in allelic exclusion of TCR ß
(35, 36).
We know that this mechanism of allelic exclusion is not 100% accurate. Studies in both humans and mice have found that about 1% of peripheral T cells have two TCR ß-chains (9, 10, 11). It was therefore of interest to determine whether there might be any other mechanisms to help cells guard against expressing two functional ß-chains. We did observe one possible mechanism in the 3.L2tg mice. Only on the H-2k/k background did we observe good expression of the 3.L2 TCR in the double-positive population. In cases where the ligand density was limiting or the selecting ligand was absent, we saw a much lower level of transgenic TCR on the CD4+CD8+ cells. This phenomenon seems to act on the whole double-positive population and may not be related to the process of TCR up-regulation on cells undergoing positive selection. Further evidence for this interpretation comes from our observation that 3.L2tg;H-2k/k mice bred to express the stimulatory Hbbd allele have a level of 3.L2 TCR on the CD4+CD8+ cells even higher than that in 3.L2tg;H-2k/k;Hbbs mice (data not shown). This occurs despite the fact that this ligand is sufficient to mediate negative selection. We can therefore offer two possible interpretations of this data: 1) CD4+CD8+ thymocytes may up-regulate a useful TCR even before the process of positive selection starts; or 2) positive selection starts at an early double-positive stage and is noticeably working before negative selection is evident.
From our studies on 3.L2tg mice we conclude that a TCR used during positive selection is the predominant TCR expressed on the surface of the mature T cell. The efficiency with which a TCR mediates positive selection will determine whether it is used for that step. Thus, a self-restricted TCR may get to the periphery on the surface of a T cell even if it was not the receptor used for positive selection. This finding can have important implications for T cell cross-reactivity and autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Current address: Rheumatology Division, University of Pennsylvania, 545 Maloney Building, 3400 Spruce St., Philadelphia, PA 19104.
3 Address correspondence and reprint requests to Dr. Paul Allen, Department of Pathology, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8118, St. Louis, MO 63110.
4 Abbreviations used in this paper: Hbb, hemoglobin; CAb, clonotypic antibody.
Received for publication December 18, 1997. Accepted for publication March 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß T cell receptor alleles. Cell 69:529.[Medline]
chains: dual receptor T cells. Science 262:422. immune response gene. Cell 32:745.[Medline]
chains occurs at the time of thymocyte TCR up-regulation. Immunity 3:449.[Medline]
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