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Presidential Address |
,
,¶,
,¶
*
Howard Hughes Medical Institute,
Department of Immunology, National Jewish Medical and Research Center and University of Colorado Health Sciences Center, and Departments of
Pharmacology,
Biochemistry and Molecular Genetics, and
¶ Medicine, University of Colorado Health Sciences Center, Denver, CO 80206
Early
in the 20th century, scientists realized that grafts and transplanted
tumors that differed from their hosts at the MHC were rapidly rejected.
Later work showed that lymphocytes were unstoppably interested in MHC
differences, so much so that Jerne, in a visionary article in the first
issue of the European Journal of Immunology
(1), proposed that lymphocyte receptors were selected
evolutionarily to react with MHC proteins. His article continued with
the proposal that lymphocytes would mutate their receptors during
development in the thymus, such that, once mature, they could no longer
react with the MHC of their hosts, but would retain the ability to
react with other MHC alleles of their species. Jerne’s ideas have been
modified as our understanding of thymocyte development has increased.
For example, it is now known that T cells rarely, if ever, mutate their
and 
TCR genes (2, 3). Also, it is now
recognized that thymocytes are selected in two ways for the reactivity
of their TCRs, by positive selection for TCRs that react with self-MHC
plus self-peptides with low but appreciable affinity
(4, 5, 6, 7, 8, 9), and by deletion if their TCRs react too well with
self-MHC plus self-peptides (10, 11, 12).
Despite the modifications to Jerne’s hypothesis, which were forced by
these findings, immunologists clung to the idea that MHC proteins and
the - and -chains of TCRs must coevolve to have some affinity for
each other. There is quite a lot of evidence for the notion.
TCR+ T cells do react with appreciable
frequency in the absence of priming with foreign MHC proteins
(13, 14, 15, 16). The frequency of allo-MHC-reactive T cells in
the unprimed population is much higher than for any other Ag except the
superantigens. Such a high alloreactivity could be because of an
evolutionarily conserved fit between TCRs and MHC. On the other hand,
the germline repertoire of TCRs may actually be completely
random and the high frequency with which TCRs react with MHC may be
because of the fact that allogeneic MHC proteins, with the many
host-derived peptides to which they are bound, actually comprise
thousands of Ags, not one (17), an idea that is supported
by the fact that many alloreactive T cells recognize both the
allogeneic MHC protein and the peptide bound to it (18).
Alternatively, high frequency reaction with foreign MHC may simply be
because of positive selection in the thymus for low reaction with
self-MHC (plus peptides) and heteroclitic cross reaction between self
and foreign
MHC.
To deal with the complication that positive selection almost certainly
biases the repertoire of TCRs toward MHC reaction, several
groups have tested the allo-MHC reactivity of TCRs from thymocytes that
have not been positively and negatively selected or that have been
formed by random combinations of TCR - and -chains (19, 20). The experiments suggested that the unselected TCR
repertoire had some intrinsic affinity for MHC. However, neither
experiment dealt with the problem of the many ligands offered by MHC
plus host peptides. Others tackled this question by studying whether
particular TCR - or -chains were particularly likely to be used
in reactions with particular MHC proteins, class I vs class II
(21, 22, 23, 24) or MHC alleles (24, 25, 26). Again, in
all cases evidence of preferential use was observed, suggesting some
overall reactivity of particular TCR V regions for MHC. However, others
have shown that receptors differing by only a single amino acid residue
can be expressed on CD4+ or
CD8+ T cells (27).
Therefore, with all of these results in hand, it was very disappointing
when the x-ray crystallographically solved structures of TCRs
bound to MHC proteins began to appear with no sign from one structure
to another of conserved interactions (28, 29, 30, 31, 32, 33, 34). Admittedly
the structures show a generally conserved alignment, with the TCR
-chain CDR1 and CDR2 loops over the C-terminal end of the helix
of the class I MHC 2 domain/class II 1 domain and the TCR
-chain CDR1 and CDR2 loops over the C-terminal end of the helix
of the class I or class II I domains (Fig. 1
). However, the angle at which the TCR lies on MHC can vary by as much
as 30° and the tilt of the TCR on MHC also varies, such that
sometimes almost all of the TCR -chain loops are lifted completely
off the MHC. This variability in the geometry of TCR/MHC engagement
precludes interactions that are conserved in all structures (34, 35). The people who worked out these structures have had to
reluctantly conclude that TCR and MHC may not have some conserved fit
with each other. The loose conservation of alignment may be because of
ridges on the MHC protein at the C-terminal ends of the helices of
the class I MHC 2 domain/class II 1 domain and the class I or
class II I domains and an alignment forced by CD4 or CD8 as they
join the TCR/MHC complex on the surfaces of the interacting cells
(34, 35).
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TCRs and MHC.
The result in question comes from examination of the reactivities of T
cells from mice in which all detectable class II MHC proteins are bound
to a single peptide. To make such animals, a transgene coding for
IAb bound at its N-terminal end via a
flexible peptide linker to a peptide that can bind to
IAb, is bred in to animals that are knocked out
for the wild type IAb chain, and for invariant
chain (C2-Ii-). In the
transgene-positive
C2-Ii- mice, all
detectable class II proteins are bound to the introduced peptide
(36).
We have now prepared two mice of this type. The peptides bound to
IAb in these mice, Ep and 2W1S, are shown in
Table I. In one of the strains
(C2-Ii-Ep), all
detectable IAb is bound to a peptide from the
IE chain (36). In the second strain
(C2-Ii-2WIS), all
detectable IAb is bound to a peptide (2W1S) that
differs from Ep in three amino acids. Previous work from our laboratory
has shown that this peptide binds more tightly to
IAb than Ep does (37).
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To circumvent this priming problem we used dendritic cells from
C2-Ii- animals, infected
with a retrovirus expressing IAb bound to a
single peptide, the 3K peptide shown in Table I
. The two single peptide
mice and C2-Ii- mice were
primed with these cells. Seven days later, T cells were harvested from
the immunized animals, expanded by culture with
IAb/3K-presenting cells and then IL-2, and fused
to BW--. Hybrids
were screened for reactivity with tetramers bearing
IAb bound to the 3K peptide. Positive hybrids
were tested for their ability to react with spleen cells from mice of
different MHC types, and for their ability to react with
IAb-3K adhered to plastic dishes. The results are
shown in Table II.
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In 1996, when we first reported that CD4+ T cells from single peptide mice were very likely to react with allogeneic MHC we thought the result was an unremarkable illustration of the ability of TCRs to react with configurations of MHC that are conserved between different MHC alleles. However, the fact that crystallographic solutions of TCRs bound to MHCs have failed to demonstrate any conserved interactions led us to reconsider these data.
To reconcile the various results we first looked carefully at the
structure of MHC proteins. Fig. 1 shows the surface of class II as it
appears to the TCR. Residues in white are those that are conserved in
sequence between all known mouse class II IA proteins. About two-thirds
of this surface of class II is completely conserved with a few other
amino acids expressed in most class II proteins. Therefore, TCRs are
exposed to many IA conserved amino acids as they interact with MHC and
peptide.
Comparison of the actual configurations of these amino acids between
structures of the same MHC protein (IEk) bound to
different peptides shows that the bound peptide changes the exposure
and configuration of these conserved amino acids (Fig. 2). Comparison of different IAs bound to different
peptides (Fig. 3) demonstrates this same phenomenon. For example, the arginine that is
present at position 70 on the -chain of almost all IA proteins
varies tremendously in configuration depending on the peptide
bound.
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The idea that the bound peptide affects the conformation of MHC bears on the observation that, even from wild-type mice, alloreactive T cells are often peptide specific. This result is often interpreted as an indication that the receptors on alloreactive T cells must engage amino acid residues of both the allogeneic MHC and the bound peptide, a conclusion that is supported by the x-ray-solved structures of a few such combinations. However, peptide specificity does not prove peptide engagement. In some cases alloreactive TCRs may actually bind only to MHC residues pushed in to a certain configuration by a particular peptide or set of peptides.
CD4+ T cells from single peptide mice will have been exposed to only one of the possible configurations of IAb controlled by the single peptide engaged in these mice. Consequently they will not be tolerant to any of the other configurations of IAb allowed by engagement of IAb by other peptides. This lack of deletion, combined with positive selection of thymocytes that can react with low avidity with the configuration of MHC that is present, plus the expressed peptide, which allows the very high frequency with which these T cells react with IAb from wild type mice. The same lack of deletion allows the appearance of T cells bearing receptors that can react with other conserved configurations of MHC, that is, allogeneic IA proteins.
These ideas bear on the failure of the x-ray-solved structures to illustrate conserved interactions between TCRs and MHC. In normal mice and human beings all configurations of the conserved MHC residues will be present, expressed by the proteins bound to some of the many peptides that are engaged in such animals. Therefore, T cells that can react with appreciable affinity with these configurations will be deleted. Thus powerful illustrations of TCRs reacting with conserved MHC residues are automatically absent from mature T cell repertoires and will not be apparent in structures derived from T cells from normal animals interacting with MHC plus Ag peptides. However, such T cells will be available in the single peptide mice because only the T cells that react with the IA configuration present in these mice will have been deleted.
Finally, if TCRs that react well with conserved MHC configurations are absent from the mature T cell repertoire, what are the evolutionary pressures that lead to coevolution of TCR/MHC binding? We suggest that the mature TCR repertoire, on which natural selection can act, does contain remnants of the conserved interactions, expressed as a few TCR amino acids that react with a few of the conserved MHC residues. However, the amino acids that manage this vary from one TCR/MHC pair to another. So far, too few structures have been solved to allow such interactions to become apparent.
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We thank Dean Becker for production of the transgenic mice, Frances Crawford for her help in tetramer production, and Janice White for sequencing the TCRs on some of the hybridomas.
Footnotes
1 This work was partially supported by U.S. Public Health Service Grants AI-17134, AI-18785, and AI-22295. B.C.S. is a Leukemia and Lymphoma Society Special Fellow. 
2 Address correspondence and reprint requests to Dr. Philippa Marrack, Department of Immunology, National Jewish Medical and Research Center, Howard Hughes Medical Institute, 1400 Jackson Street, Denver, CO 80206. E-mail address: marrackp{at}njc.org
3 Abbreviation used in this paper: HEL, hen egg lysosome.
Received for publication June 1, 2001. Accepted for publication June 1, 2001.
References
gene segments. Nature 316:517.[Medline]
2+CD8+ T cells in mouse strains expressing both H-2k and T cell receptor V a haplotypes: determination with a V2-specific monoclonal antibody. Eur. J. Immunol. 22:399.[Medline]
CDR1 and CDR2. Science 273:963.[Abstract]
gene usage by alloreactive T cells responding to defined HLA-DR alleles. Transplantation 62:1332.[Medline]
T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 274:209. T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization. Proc. Natl. Acad. Sci. USA 94:13838. T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. 19:5611.[Medline]
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