By
§
From the * Howard Hughes Medical Institute and the Department of Medicine, National Jewish
Medical and Research Center, Denver, Colorado 80206; the § Department of Biochemistry, Biophysics,
and Genetics and the
Department of Immunology and Medicine, University of Colorado Health
Sciences Center, Denver, Colorado 80206; and ¶ Department of Molecular Microbiology and
Immunology, Oregon Health Sciences University, Portland, Oregon 97221
In normal mice, major histocompatibility complex (MHC) proteins are bound to many different peptides, derived from the proteins of their host. In the thymus, the diversity of this collection of MHC + peptide ligands allows thymocytes bearing many different T cell receptors (TCRs) to mature by low avidity reactions between the MHC + peptide ligands and the thymocyte TCRs. To investigate this problem, the selection of T cells specific for a well-studied combination of MHC + peptide, IEk + moth cytochrome c 88-103 (MCC), was investigated. Mice were created that expressed IEk bound to a single peptide, either a variant of MCC in which a critical TCR contact residue, 99K, was changed to A, or a variant of a mouse hemoglobin 64-76 (Hb) peptide, 72A. IEk bound to the MCC variant caused the clonal deletion of some T cells specific for the IEk + MCC ligand; nevertheless, it also positively selected many T cells that could react with this ligand. Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not. IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC. These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.
Many mysteries about the processes that drive thymocyte positive and negative selection have recently
been solved. It is now known that thymocytes are selected
to mature based on low avidity reactions between the Thymocytes bearing a particular TCR can be positively
selected by a particular MHC protein bound to a number of
different peptides (2, 8). Also, a particular MHC + peptide combination can positively select thymocytes bearing
many different TCRs (9). Perhaps these degeneracies
are due to the fact that many different TCR and MHC + peptide combinations can achieve the affinity and concentrations necessary to reach the avidity of reaction required for positive selection.
Positive selection is not a completely degenerate phenomenon, however. Some peptides bound to a certain MHC
protein cannot select thymocytes bearing a particular TCR,
even though another peptide bound to the same MHC can
manage this (2, 14, 15). Likewise, a single MHC + peptide combination probably does not select thymocytes bearing as diverse a collection of TCRs as does the same MHC
protein bound to the 2,000 or more peptides with which it
is engaged in normal thymuses (9). The first experiments done on this subject suggested that the selecting peptide might be related in structure to the peptide that would
later be able to drive a productive response by a T cell
bearing a particular TCR (2). Later experiments indicated that this view was too extreme and that the selecting
and activating peptides might not have to be obviously related (8, 15, 16). Some of these experiments were limited,
however, by the fact that they were done in vitro using
TCR transgenic thymocytes; thus, only a few different TCRs
could be tested. Also, most of the experiments involved selection on class I MHC and few involved selection on class II.
To address this problem, we created a collection of mice
in which almost all class II proteins of a particular type were
occupied by a single peptide (11). The current experiments
were done to find out whether the TCR repertoire selected by a single MHC + peptide combination would be
related to that selected by the same MHC bound to the
many mouse peptides with which it is engaged in normal
mice. We chose to study a T cell specificity which has been
very well examined in normal mice, that of T cells specific for the moth cytochrome c peptide 88-103 (MCC)1 bound
to IEk (17). We found that IEk bound to a variant of
MCC in which a central TCR contact residue (22), 99K,
had been exchanged for an A selected T cells which could
react with IEk + MCC and which bore TCRs which were
closely related in sequence to those selected in normal mice
by IEk bound to mouse peptides.
Construction of Transgenes.
We have previously described the
production of genes coding for soluble IE Mice.
B10.BR and B10.M mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). All other animals were
bred in the Animal Care Facility at the National Jewish Medical
and Research Center (Denver, CO).
/
TCRs they bear and combinations of MHC protein and
peptide in the thymus cortex (1). Reactions that are of
too high avidity cause thymocytes to die by clonal deletion (7). Likewise, reactions that are of too low avidity also
cause thymocytes to die, in this case of neglect.
k covalently linked via
a flexible linker at its NH2-terminal end to several peptides that
can bind to intact IEk protein (23). Two of these constructs, coding for IE
k bound to MCC with a substitution of A for K at position 99 (99A) or for mouse hemoglobin
d 64-76 (Hb) with a
substitution of A for N at position 72 (72A) were modified for injection into mice. Modifications included the removal of the thrombin sites from the flexible linkers that bound the peptide to the
IE
k protein and introduction of genetic material coding for
wild-type IE
k transmembrane and cytoplasmic domains (gift of
Dr. H. Fischer). These genes were cloned into a plasmid (DOI)
containing a promoter from IE
(gift of Drs. D. Mathis and C. Benoist, I.G.B.M.C. Strasbourg France; reference 24). cDNA
coding for IE
d (gift of Dr. R. Germain, NIAID, NIH) was also
introduced separately into DOI as was cDNA coding for the
wild-type IE
k protein without covalently linked peptides. The
chain construct was prepared for injection by digestion with
HhaI; the
chain constructs were cut with XbaI and NruI, and
the fragments were purified by electrophoresis and on glass beads
before injection.
gene
together with one of the IEk
genes into fertilized B10.M eggs.
Mice were screened for expression of the transgenes by the ability
of their peripheral blood cells to stimulate T cell hybridomas specific for IEk bound to the covalently linked peptides, KC99A-4.18
for the 99A transgene and KH-8.3 for the 72A transgene or, for
the wild type IEk transgene, by stimulation of KH-8.3 after addition of Hb peptide.
Antibodies and Flow Cytometry. Single cell suspensions from thymus or lymph nodes or spleen were filtered through nylon mesh (Falcon, Becton Dickinson, Franklin Lakes, NJ), washed in balanced salts solution (BSS) and stained with antibodies at 1-3 × 107 cells/ml in staining buffer (BSS, 0.1% sodium azide, 2% fetal bovine serum). Before analysis for class II MHC expression, thymus cells were enriched for large cells with a low speed spin, 500 g for 2 min, after which supernatant cells were discarded.
Cells were stained and analyzed for expression of TCR VProduction of Chimeric Mice.
8-12-wk-old mice were irradiated with 950 rads from a 137Cs source and immediately afterwards reconstituted with fetal liver cells from 7-8 pooled day 14 fetuses, the progeny of the cross of two IEk wild-type (wt) Ii+/
mice. Therefore, the majority of donors expressed Ii and the IEkwt transgene without covalently bound peptides. Mice were
maintained on acidified chlorinated water and immunized with
antigen 8 wk later.
Production and Analysis of Antigen-specific T Cell Hybridomas.
Chimeric and normal mice were primed with MCC in complete
Freund's adjuvant in the base of the tail. T cell hybridomas were prepared from these animals as previously described (28). In brief,
lymph node cells were harvested from the draining nodes of the
immunization 7 d later. The cells were incubated for 4 d with
MCC and then live cells were purified and cultured for 3 more d
with saturating amounts of IL-2. The live cells were then fused to
an variant of BW5147, BW
(28). Hybridomas were
assayed for their ability to react with IEk in the presence or absence of added MCC. Presenting cells were B10.BR spleen cells
which express IEk at high levels, or cells from IEwt transgenic
mice, which express IEk at low levels (Fig. 1). All hybridomas
used for further analysis expressed high levels of TCR and CD4.
In the past, T cell TCR repertoires for several peptides, including MCC and Hb, presented by IEk, have been very well studied (17, 29). The T cells bearing these TCRs were all selected in the thymus by reaction between their TCRs and IEk bound to one or more of the mouse peptides with which it is normally engaged in animals. We wished to compare these well-known TCR repertoires with those that might be selected by IEk bound to a single peptide. To this end we created transgenic mice expressing IEk bound covalently to single peptides and no other IE proteins. The peptides chosen were related to those used in the previous repertoire studies and were MCC with a substitution of A for K at position 99 or Hb with a substitution of A for N at position 72. These altered peptides will be called 99A and 72A, respectively, in this paper. Their sequences are shown in Table 1.
At the moment, mice that express IE transgenes and no
other class II proteins cannot be constructed. It is, however,
possible to breed mice that express transgenic IE proteins
and no other IE molecule. This is best done by introduction of IE and IE
transgenes into H2f or H2q animals
since these two haplotypes do not have functional IE
or
IE
genes of their own (30). Therefore, the IE transgenes described in this paper were injected into the fertilized eggs of H2f B10.M mice. As described in Materials and Methods, mice transgenic for IEk bound covalently to 99A, or for
IEk bound covalently to 72A, were identified by the ability
of their peripheral blood lymphocytes to stimulate T cell
hybridomas specific for the two IEk + peptide combinations. Mice transgenic for IEk with no covalently attached
peptide were identified by the ability of their peripheral
blood cells to stimulate a T cell hybridoma, KH-8.3, after
addition of its target peptide, Hb.
We have previously shown that the presence of Ii caused
the removal of the covalently bound peptide from class II
in cells (11). To prevent this, each of the transgenic lines
was crossed with invariant chain deficient mice (IiKO) and
the progeny were intercrossed to produce animals which
were H2f homozygous IiKO and lacked the 129/J-derived
mouse mammary tumor viruses that code for superantigens
which delete thymocytes bearing V3.
These crosses produced three lines of animals, all H2f
and IiKO, and expressing IEwt or IEk covalently bound to
99A or 72A. The mouse lines were called IEwtTg, 99ATg,
and 72ATg, respectively. H2f IiKO animals lacking any
transgenes were called Tg. A list of the mice used in this
paper, and their characteristics, is shown in Table 2.
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Thymus and spleen cells from these animals were stained
to determine expression of the transgenes. As shown in Fig. 1,
99ATg mice expressed the product of the transgenes at
high levels on the large cells in its thymus. They also expressed high levels of the product on their spleen cells, although not as many spleen cells in 99ATg mice were IEk+
as in control B10.BR animals. Levels approximated those
on the equivalent B10.BR H2k cells. 72ATg spleen cells
stained less well, and expression of this transgene was very
low in the thymus. Expression of the IEwt transgene was
very low on spleen cells, regardless of whether or not the
mice expressed Ii. The promoter used in these experiments, although derived from the IE gene itself, is known
to drive variable levels of expression of class II proteins in
transgenics (24). These results are probably manifestations
of this phenomenon.
To establish the distribution of IEk-bearing cells in the
thymuses of these mice, frozen sections of their thymuses
were stained with biotinylated anti-IEk and streptavidin-coupled horseradish peroxidase. Biotinylated anti-IAb or
anti-Kk served as controls. As expected, both the cortex
and medulla of B10.BR thymuses stained brightly with the
anti-IEk reagent. No staining above background was observed with the Tg or 72ATg thymuses, indicating that
expression of the transgene was very low on both cortical
and medullary epithelium in the latter mouse. Anti-IEk
stained the cortical epithelium of 99ATg animals almost as
brightly as it stained that of B10.BRs, with intense reticular
patterning. By contrast there was no staining above background in the medulla of the 99ATg animals. These results
demonstrated that the 99A transgene was well expressed
in the cells responsible for positive selection of class II-
restricted T cells, thymus cortical epithelium, but poorly, if
at all, expressed on medullary thymus epithelium. T cell
tolerance to the transgene in 99ATg mice must be established by contact with thymus cortical epithelium or bone
marrow-derived cells.
Staining experiments of this type did not reveal whether
or not the IE proteins in the 99ATg and 72ATg mice were
still bound to the covalent peptides with which they had
been engaged genetically. To find out whether displacement
with other peptides had occurred, cells from the transgenic
mice were tested for their ability to present exogenously
added peptides to T cells specific for IEk bound to the exogenous peptide. Thymus and spleen cells from the 99ATg
mice presented exogenous peptides very poorly (Fig. 2). By
contrast, thymus and spleen cells from the IEwtTg animals presented peptide fairly well, and thymus and spleen cells
from B10.BR animals had very good activity.
Spleen and thymus cells from 72ATg mice presented exogenous antigen even less well than those of 99ATg animals (data not shown), indicating that the 72A peptide, like
the 99A peptide, was not displaced from the IEk molecule
in Ii animals.
These experiments demonstrated that virtually all of the IEk molecules on the surfaces of 99ATg and 72ATg cells were occupied by the 99A and 72A peptides, respectively. Even though the IEwtTg cells bore only small amounts of IEk on their surfaces as detected by staining, they still had more IE molecules on their surfaces, which could present exogenous peptides, than 99ATg cells did.
Cells Bearing Either of Two TCRs Specific for IEk + MCC Are Deleted in 99ATgs.A number of mice have been produced that express TCR transgenes specific for IEk + MCC.
To find out whether the 99ATg or 72ATg mice could positively select thymocytes bearing these TCRs, mice expressing two of these TCRs, AD10 and AND (31), were
crossed with the 99ATg and 72ATg animals. Progeny (which
were still Ii+) were tested for expression of the TCR and
IE transgenes and for expression of H2k and H2f. They
were then tested for selection of the thymocytes bearing the
transgenic TCRs by estimation of the numbers of CD4+
mature thymocytes bearing high levels of the relevant V
and V
regions, V
11 and V
3.
The results in Fig. 3 show that CD4+ thymocytes bearing either of the two transgenes were selected with low efficiency in nontransgenic H2f homozygous mice. This was
not because H2f proteins caused deletion of thymocytes
bearing these TCRs since CD4+ thymocytes bearing either
the AND or AD10 TCRs appeared in large numbers in
H2fxk mice, positively selected by the wild-type IEk protein + some unknown mouse peptide.
Introduction of the 99ATg did cause deletion, however,
as witnessed by the fact that mature CD4+ T cells bearing
the transgenic TCRs were lower in number in H2fxk mice
that expressed the 99ATg than they were in animals of the same MHC type that did not express the transgene. T cells
bearing the AD10 TCR have previously been reported to
be antagonized and deleted by very high concentrations of
MCC99A bound to IEk (32), so this result was not surprising. Thymocytes bearing the AND TCR were more efficiently deleted in 99ATg mice than were thymocytes bearing the AD10 TCR. The AND and AD10 TCRs differ by
a single amino acid. The AND TCR has an alanine residue
in its V CDR3 region, four amino acids COOH terminal
to the invariant cysteine, whereas the AD10 TCR has a
threonine at this position (28). Apparently this change causes
the two TCRs to have slightly different affinities for IEk
bound to MCC 99A, resulting in different efficiencies of deletion of thymocytes bearing the two different TCRs.
These experiments were done with mice that still expressed Ii and hence the MCC99A peptide was probably displaced from some of the 99ATg class II proteins in the mice (11). Thus the fact that expression of the 99ATg in H2f homozygous mice increased the numbers of mature CD4+ thymocytes bearing the AD10 TCR could not be properly evaluated. It is possible that this indicated that the 99ATg could both positively and negatively select thymocytes bearing this TCR, i.e., the affinity of AD10 for IEk bound to 99A was in the marginal zone which has been noted before for other TCR, MHC + peptide combinations. Alternatively, positive selection in this case might have been driven by interactions between the AD10 TCR and 99ATg proteins from which the MCC99A peptide had been displaced. Future experiments will resolve this.
Expression of the 72ATg did not affect the fate of the thymocytes in any animal, suggesting either that this protein was not expressed at high enough levels to be effective and/or that the AND and AD10 TCRs had no productive affinity for this IEk-peptide combination.
These experiments showed that some TCRs specific for IEk + MCC have a high enough affinity for IEk-99A to cause thymocytes bearing them to be deleted in 99ATg mice. Hence, the extent of the TCR repertoire for IEk + MCC in 99ATg mice is limited, not only by failure to positively select, but also by clonal deletion of the relevant cells.
The 99ATg Selects T Cells that Can React with IEk + MCC.IiKO mice that express class II proteins covalently
bound to peptides that can occupy their grooves cannot be
primed with foreign proteins or peptides (data not shown).
Therefore, the ability of T cells from the class II-peptide
transgenic mice to react with foreign proteins or peptides
could not be evaluated in intact mice. To circumvent this
problem, the transgenic mice were irradiated and reconstituted with fetal liver from H2f animals transgenic for IEkwtTg genes and expressing Ii. In the chimeric animals that
were thus created, T cells were positively selected on host
thymus cortical epithelial cells, and could be primed with
foreign peptides presented on the IEkwt expressing fetal
liver derived cells. The T cells must also have been tolerant
to the low levels of IEk bound to mouse peptides expressed
on IEkwt Ii+ and Ii cells.
Three types of chimeric mice were produced in which
the hosts were 99ATg, 72ATg, or Tg. After T cells had
matured in the chimeras, they were primed with MCC peptide and the primed T cells were harvested and converted into T cell hybridomas as described in Materials and Methods. As additional controls, hybridomas were also produced
in the same fashion from MCC primed, nonchimeric H2f
mice with or without Ii expression, and from nonchimeric
IEwtTg mice with or without Ii expression.
Hybridomas were tested for their ability to respond to
B10.BR, IEwtTg Ii+, or 99ATg spleen cells. Examples of
the reactivities of some of the hybridomas from 99ATg
chimeric mice are shown in Fig. 4. None of the hybridomas responded to IEwtTg Ii+ or 99ATg cells. Thus the
hybridomas were tolerant to class II proteins at the levels
they were expressed in the chimeric mice. Several of the
hybridomas did, however, respond to B10.BR spleen cells.
Anti-IE addition to the stimulation cultures showed that this was due to reaction with IEk (data not shown). The
parent T cells for these hybridomas must have been tolerant
to IEk bound to the mouse peptides with which it is normally engaged at the very low levels at which it was expressed in the chimeric animals (Fig. 1), but were not tolerant to the same combination of IEk and peptides at the high
levels at which it is expressed on B10.BR cells. These hybridomas were probably another example of the fact that
selection on a particular MHC-peptide combination causes cells to mature, which are likely to react with that same
MHC protein bound to other peptides, particularly when
expressed at high levels (11).
The hybridomas were also tested for their ability to respond to different concentrations of MCC presented by
B10.BR cells (Fig. 5). Many of the hybridomas responded
well to this antigen and were as sensitive to low doses of
the peptide as a typical hybridoma prepared from primed
T cells from a normal mouse. Some of the hybridomas, for
example KMAC-92, which responded to B10.BR cells in
the absence of peptide responded better when increasing doses of MCC were added to the cultures.
The numbers of hybridomas obtained from two mice of
each type are shown in Table 3. When T cells cannot react
with the immunizing MHC + peptide combination, immunized lymph nodes give rise to few T cell blasts, and few
T cell hybridomas are produced (our unpublished observations). This was manifest in this experiment by the fact few
hybridomas were obtained from fusion of MCC-immunized T cells from some of the mice that did not express an
IEk protein and Ii in their thymus stromal cells. Analysis of
the numbers of hybridomas that could react with IEk + MCC supported this conclusion. No hybridomas with this
specificity were obtained from mice which did not express
IEk on their thymus stromal cells. This included the Tg
mice that contained fetal liver-derived cells expressing
IEwt and Ii. Hence, as previously reported, the MHC class
II proteins on the fetal liver-derived cells in these chimeras
could not participate in positive selection (33, 34).
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No hybridomas that could react with IEk + MCC were obtained from IEwtTg mice. Thus, either the peptides that occupied the grooves of IEk in IiKO animals did not allow selection of IEk + MCC-reactive T cells, or the levels of IEk in these animals were too low to allow selection of such cells (14). Also, no T cell hybridomas that could react with IEk + MCC were obtained from 72ATg chimeric mice. This might have been due either to an inability of IEk bound to the 72A peptide to select such cells, or it may have been due to the very low levels of expression of this combination in the thymuses of the transgenic animals. Some of the hybridomas that were produced in the fusion of T cells from 72ATg chimeric mice were specific for IAf bound to MCC (data not shown). It is interesting that T cells with this specificity were not produced from fusions of T cells from mice that lacked an IEk transgene. Future experiments will examine this phenomenon in more detail.
A number of hybridomas specific for IEk + MCC were
obtained from 99ATg chimeric mice. These hybridomas
could not have been positively selected on the fetal liver-
derived cells in the chimeras since similar hybridomas did
not appear in the other chimeras prepared with the same
fetal liver. The results with primed B10.M or Tg mice
demonstrate that these hybridomas could not have been selected on IAf. Finally, these hybridomas were not selected
on the small amounts of IEk on the 99ATg thymus epithelial cells from which the 99A peptide had been displaced
since there were fewer of these proteins in 99ATg thymuses than there were in IEwtTg thymuses (see data in Fig.
2) and mice of the latter type failed to produce T cells with
this specificity. These controls proved that the T cell parents of the IEk + MCC-reactive hybridomas obtained
from the chimeric 99ATg mice must have been positively
selected on the 99ATg.
The IEk + MCC-reactive T cell hybridomas
listed in Table 3 were stained with anti-V and anti-V
antibodies. Table 4 lists the combinations found on these cells.
We were surprised to find that only about a quarter of the
hybridomas from IEwt Ii+ animals bore the pairing of
V
11 V
3, which is usually associated with recognition of
IEk + MCC. Even though an overall analysis of this type
for responses to IEk + MCC 88-103 (in contrast to IEk + pigeon cytochrome C 88-104) is not available in the literature, the impression is that almost all T cells in normal mice specific for IEk + MCC bear V
11 and V
3. Perhaps the difference
between the results in the literature and those reported here
is due to the low levels of IEk on the selecting thymus epithelium in the IEwt Ii+ mice used in our experiments.
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Clearly, T cells selected on IEk bound to 99A could bear
the same V-V
combinations, including V
11 and V
3,
as those selected on IEk bound to many mouse peptides
(17).
The receptors from the 99ATg chimera derived T cells
bearing V11 and V
3 or V
8.3 were sequenced (Table 5
and 6). The TCR sequences from eight of the nine hybridomas bearing V
11 and V
3 were identical. The T cell
parents of these cells must have come from the same expanded clone. This result suggested that there were relatively few IEk + MCC-specific T cells in the 99ATg chimeric mice since hybrids derived from a single clone are not
usually found in fusions to antigen specific T cells from normal mice (P. Marrack, personal observation).
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Consensus sequences for the CDR3 regions of TCRs
specific for IEk + MCC and selected in normal IEk+ mice
were derived from the literature (17-21, 35; Tables 5 and 6).
Most of the chain CDR3s of these TCRs used V
11.1, had an E 3 amino acids COOH terminus to the conserved
C, and had CDR3s which were otherwise rich in compact
amino acids such as A and S. A notable exception to these
rules is the
chain of the well-known T cell hybridoma,
2B4, which uses V
11.2 and has a lengthier CDR3 (18, 20).
The literature suggests two different consensus sequences
for the CDRs of the TCR
chains. Both are shown in
Tables 5 and 6. Consensus sequence 1 was most commonly reported in the past (17), with consensus sequence 2 derived from that of 2B4 and a collection of sequences from
mice expressing IEk and heterozygous for Ii expression (15).
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The CDR3 sequences of the 99ATg T cells are also
shown in Tables 5 and 6. Like 2B4 and unlike the norm,
the two V3-bearing T cell hybridomas bore V
11.2.
V
11.1 in combination with V
3 may cause a higher reactivity for IEk + MCC99A than V
11.2 does since most of
the V
11.1-bearing, IEk + MCC reactive T cells from normal mice are activated or deleted by IEk + MCC99A, but
2B4 is not (21, 32, 35). Consequently, the V
11.1- and
V
3-bearing T cells may have been deleted in 99ATg mice,
leaving as IEk + MCC-reactive T cells only those which
bore, with V
3, V
11.2. The two V
11 family members
differ by only five amino acids (36). Only one of these
changes, position 72 in the CDR4 loop which is an R in
V
11.1 and S in V
11.2, maps to any region that might contact MHC and peptide (37, 38). Perhaps the shorter,
uncharged side chain of the S in V
11.2 and/or the presence of bulky amino acids in the TCR
and
chains reduced contact between the TCR and IEk + 99A, and thus
allowed IEk + MCC-reactive T cells bearing V
11.2 to be
positively selected and not deleted by the 99ATg.
Other than this, the chain CDR3 sequences of the
V
3- and V
8.3-bearing IEk + MCC-reactive TCRs obtained from 99ATg mice were similar in sequence and
length to the consensus with the exception that one lacked
the acidic amino acid usually found three residues COOH terminus to the conserved cysteine, and was thus similar in
sequence to the CDR3 of a TCR reported to cross-react
between MCC and MCC99E (39), and two contained a
bulky aromatic amino acid.
The CDR3 sequences of the two V3-bearing TCRs obtained from 99ATg mice were very similar in sequence to
that of consensus 2, the group which includes the 2B4 TCR
(Tables 5 and 6). As discussed above, this similarity may
have been driven by the requirement for a low reactivity
with IEk bound to MCC99A.
The results in Tables 5 and 6 did suggest one other idea:
most of the TCR and
chains which could contribute to
TCRs that could react with IEk + MCC might now be
known. Thus, several of the sequences from the 99ATg
mice were identical to sequences that had already been described. The
chain of KMAC-75 was identical to that of
a previously described hybridoma, 5C.C7 (20). The
chain
CDR3 sequence of KMAC-19 was identical to that previously reported for a TCR selected on wild-type IEk in Ii+/
mice (15). This previous publication did not mention
whether the TCR in question bore V
11.1 or V
11.2.2.
Likewise, the
chain sequence of KMAC-19 was identical
to that previously described for a T cell selected by IEk
bound to MCC (15).
The complete repertoire of T cells selected on the 99ATg and specific for IEk + MCC was not assessed in this paper since the T cells we isolated came from mice that contained fetal liver cells bearing IEk engaged by normal mouse peptides, albeit at low levels. The T cells in the chimeras were thus positively selected by IEk bound to a single peptide, and were tolerant to IEk bound to many normal mouse peptides at low levels, but not necessarily at high levels. This was manifested by the fact that none of the T cell hybridomas obtained from the chimeras reacted with IEk on IEwtTg Ii+ cells, although some of the hybridomas did react with IEk on B10.BR cells. Nevertheless, it is likely that expression of even the low levels of IEk found in the chimeras did cause deletion of some IEk + mouse peptide- reactive T cells, and thus reduce the total diversity of TCRs found in the chimeras. Moreover, all the mice contained an additional class II protein, IAf, and it is likely that at least some selected T cells were deleted by recognition of this protein, bound to its array of mouse peptides (11).
Assessment of the ability of IEk bound to 99A to select IEk + MCC-reactive T cells was also limited by the fact that some TCRs specific for this latter ligand also react with high affinity with the 99A selecting ligand (32). This was demonstrated in this paper by the deletion in 99ATg mice of T cells bearing the well known IEk + MCC reactive TCRs, AND or AD10 (20, 31, 32).
Overall, therefore, the repertoire for IEk + MCC in the transgenic chimeras was limited to some extent by negative selection on two types of ligands, low levels of wild-type IEk or IAf bound to mouse peptides, and IEk-99A.
In spite of these two limitations, it was clear that T cells reactive with IEk + MCC could be positively selected by IEk-99A, a closely related ligand. A previous publication showed that MCC with substitutions of E for K at position 99, or E for T at position 102, and indeed even MCC itself could positively select T cells reactive with IEk + MCC (15). Assuming that the affinity/avidity hypothesis of positive selection is correct, these results show that TCRs with high affinity for IEk + MCC can have an appropriate selecting affinity for IEk + any of these MCC variants or even for IEk + MCC itself at the concentrations that these ligands were expressed in the thymus.
Selection is peptide specific. T cells specific for IEk + MCC cannot be selected by IEk bound to any peptide. Such T cells were not positively selected in animals expressing IEk but not Ii (reference 14 and this paper). Apparently none of the peptides that were bound to IEk in IiKO animals could participate in selecting T cells that can react with IEk + MCC. Also, as shown in this paper, IEk + MCC-reactive T cells were not obtained from animals expressing IEk bound to 72A. Likewise, T cells with this specificity were not selected by IEk bound to a lambda repressor peptide (15).
Our result with the 72A peptide is quite surprising since it has previously been shown that unaltered Hb 67-76 could select MCC-reactive T cells (15). The 72A peptide differed from that previously used by the conservative change of A for N at the central, T cell contact residue of the peptide, and by a longer NH2-terminal tail protruding from the IEk-binding groove. MCC itself has a basic amino acid, K, at the equivalent position. Perhaps TCRs that will react with MCC are selected better by Hb with the hydrophilic amino acid N at this position than Hb with the shorter, more hydrophobic A. 99A, however, also has an A at the equivalent position and, as shown in this paper, 99A selected MCC-reactive TCRs well.
Alternatively or as well, perhaps the difference was due to the level of expression of the IEk-peptide conjugates. The IEk-99A transgenic protein was expressed at much higher levels per cell than the IEk-72A conjugate. This may have caused positive selection of thymocytes bearing TCRs with very low affinity for the 99A peptide, whereas low affinity positive selection may not have been possible in the 72ATg mice. Recent measurement of the affinity of a TCR on one of the IEk + MCC-specific T cells described in this paper showed that it did indeed have extremely low affinity for IEk-99A.
Finally, the difference may be due to the fact that the T cells used in this paper were tolerant to IEwt and, less likely, to IAf. Tolerance to IEk plus all the peptides to which it is bound in normal mice may have deleted all the T cells selected on IEk-72A that could react with IEk + MCC. We do not think this is the explanation, however, because preliminary experiments with 72ATg T cells that are not tolerant to IEwt suggest that these T cells are also unable to react with IEk + MCC.
Some of the TCRs specific for IEk + MCC that were
positively selected in the 99ATg mice were quite similar to
those found in normal animals. Again, this was quite surprising since many T cells with this specificity that bore
TCRs like those in wild-type animals must have been deleted in the 99ATg animals, as exemplified by the disappearance of T cells bearing the transgenic TCRs, AD10 and
AND, in these mice. It is worth noting, however, that the TCR repertoire of the 99ATg animals was not completely
included within that of normal animals since two different
TCRs which used V8.3 were obtained from the 99ATgs,
and IEk + MCC-reactive T cells bearing this V
have not
previously been reported from normal mice, although one
T cell with this specificity and this V
was reported from
mice heterozygous for Ii expression (15).
Finally, although IEk + MCC-reactive T cells bearing
identical TCRs have yet to be independently isolated, these
data support the notion that the complete TCR repertoire
for this MHC + peptide combination is nearing saturation.
Several of the chains of the TCRs isolated from our transgenic mice were identical in sequence to chains that have been
described previously. Presumably the numbers of TCR and
chains that can contribute to TCRs that can react
with IEk + MCC are limited, and this fact is reflected in
the fact that particular sequences for
or
are being reported with increased frequency.
Address correspondence to Dr. Chih-Pin Liu, Department of Medicine, Howard Hughes Medical Institute, National Jewish Medical and Research Center, Goodman Bldg., Fifth floor, 1400 Jackson St., Denver, CO 80206. Phone: 303-398-1322; FAX: 303-398-1396.
Received for publication 24 June 1997 and in revised form 28 August 1997.
1 Abbreviations used in this paper: Hb, mouse hemoglobin 64-76; Ii, invariant chain; MCC, moth cytochrome c peptide 88-103; wt, wild-type.The authors thank Dean Becker for his help with production and breeding of transgenic mice and Ella Kushnir, Patricia Mount, and Dr. Gail Ackermann for support in the Animal Care Facility at National Jewish Medical and Research Center, Denver, CO. They also thank Drs. Stephen Hedrick, Elizabeth Bikoff, Ron Germain, Laurie Glimcher, Diane Mathis, and Christophe Benoist for their generous gifts of transgenic and knockout mice, DNA constructs, and antibodies.
This work was supported by United States Public Health Service grants AI-17134, AI-18785, AI-22295, and AI-29544.
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