*Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada;
and
Program in Human Immunogenetics, Fred Hutchinson Cancer Research Center, Seattle, Washington
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Abstract |
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Introduction |
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Despite this growing body of evidence, it is still sometimes assumed that -TCRs function similarly to
ß-TCRs (Bartl, Baltimore, and Weissman 1994
; Flajnik 1998
), recognizing antigens only when they are presented by some sort of specialized antigen-presenting molecule analogous to an MHC molecule (we refer to this as "indirect antigen binding"). However, recent studies (Schild et al. 1994
; Rock et al. 1994
; Chien and Jores 1995
) indicate that the process of antigen recognition by
T cells may be fundamentally different from that in
ß T cells. In fact,
-TCRs appear to recognize antigens in a manner similar to the antigen recognition processes of immunoglobulins, the receptors of B cells. Immunoglobulins bind directly to antigens and do not require specialized antigen processing and presentation as do
ß T cells.
TCRs and immunoglobulins (Igs) are the most closely related members of a large protein family called the immunoglobulin superfamily (IGSF), many of whose members are involved in immune recognition or cellular adhesion (Hunkapiller and Hood 1989
). TCR and Ig molecules are clearly more similar to each other than to other IGSF molecules (Marchalonis, Schluter, and Edmundson 1997
), and the genes that encode them display highly similar patterns of organization and rearrangement during transcription and translation (Hunkapiller and Hood 1989
) that are not observed in any other genes. In phylogenetic terms,
ß- and
-TCR molecules and genes are often thought of as a monophyletic group whose sister group is the Igs, and this assumption is one reason that
- and
ß-TCRs have been assumed to function similarly. As illustrated in figure 1A,
since
ß- TCRs exhibit indirect antigen binding, and since they are closely related to the
-TCRs, it has been assumed that this mode of antigen binding evolved before the split between the two. However, it would be equally parsimonious to assume that indirect antigen binding is a derived characteristic of
ß T cells (fig. 1B
). Moreover, it is possible that the phylogenetic relationships in figure 1
are incorrect, specifically, that the TCR genes are not monophyletic. This would lead to different conclusions about the evolution of immune cells and antigen recognition and binding by
ß-TCRs,
-TCRs, and also Igs.
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Materials and Methods |
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Sequences were aligned using the program PILEUP (Feng and Doolittle 1987
) in the GCG package, version 8.1-UNIX (Devereaux, Haeberli, and Smithies 1984
). The program CLUSTAL W (Higgins and Sharp 1988
) was also used and gave similar results, but results presented here are based primarily on the PILEUP alignments. Various gap penalties were tried. A lower gap penalty results in the insertion of more gaps in the alignments and greater similarity among sequences. An extremely low gap penalty tends to eliminate any phylogenetic signal, since aligned sequences are either highly similar or full of missing characters. An excessively high gap penalty is likely to lead to misalignment in cases in which insertions and deletions have occurred. A gap penalty of 2.0 (using PILEUP) guaranteed perfect alignment of two highly conserved motifs common to all TCR and Ig sequences, so this gap penalty was adopted for all alignments. The two motifs are the highly conserved WYRK/Q and YFCA motifs in the second and third framework regions of the V region genes. Overall, then, the alignments were conservative. The V region alignment, including the complementarity determining regions (CDRs), was truncated at a consensus length of 134 amino acids, ending at the highly conserved G-G hinge region between the V and D/J regions in the fourth framework region. Excluding the CDRs resulted in a consensus length of only 64. The C region alignment was truncated at a consensus length of 150 amino acids, the approximate length of the IgL C regions.
Two different tree-building methods were used. The parsimony criterion was used to find the set of trees that minimizes the number of changes in character states across the whole tree, ignoring invariant or uninformative characters. Using the program PAUP, version 3.1 (Swofford 1993
), parsimony trees were found by heuristic search with the branch-and-bound search option and 1,000 iterations. Each iteration starts the search with a different tree (i.e., set of branching patterns) and finds the shortest set of trees based on that starting point. Through dozens of trials and hundreds of hours of tree building with this method, we found that the set of shortest trees was almost always found in the first 10 iterations. Successive approximations character weighting (SACW; Carpenter 1988
) was used to further improve tree resolution. Each character was reweighted by its goodness of fit to the most parsimonious trees, and the trees were recalculated. The process was repeated until a constant tree topology was obtained. Thus, each parsimony consensus tree should represent the best fit between tree topology and the current data set.
The second tree-building method was based on the use of genetic distance matrices and the neighbor-joining method of Saitou and Nei (1987)
. These trees were calculated using the PHYLIP (version 3.57c) programs PROTDIST and NEIGHBOR (Felsenstein 1993
). One thousand neighbor-joining trees were calculated based on a bootstrap of 1,000 separate genetic distance matrices; the final trees are consensuses of these 1,000 trees.
Trees based on outgroup comparison can be used to infer both patterns of relationship among taxa and branching order over time. Several members of the IGSF have been proposed to be representative of a primordial IGSF molecule which probably had only one Ig-like region that would have been ancestral to both the C and the V regions of TCRs and Igs (Hunkapiller and Hood 1989
). One of these is the TCR-associated cell surface molecule CD3. CD3 exists in three forms, two of which, CD3-
and CD3-
, were used as outgroup sequences. Several other molecules were also tested as outgroups, including the cell surface molecules CD3-
, Thy-1, CD4, and CD8 (Williams 1987
), but this did not result in improved resolution of TCR and Ig topologies.
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Results |
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Using the parsimony criterion, an initial set of 119 trees of length 894 steps was found. These were reduced to 2 trees of length 896 using SACW (fig. 4
). The IgH sequences, including IgW (Cpu40560), comprise the basal branch, giving rise to the TCRs as a whole, which in turn eventually give rise to the IgL group. IgL and TCR sequences are more closely related to each other than either is to the IgH sequences. None of the TCR groups is monophyletic. The position of axolotl TCR-ß (Amttcrb) is problematic, rendering the IgL sequences paraphyletic. We note that if a second amphibian sequence, Xenopus TCR-ß (U60436; Chretien et al. 1997
), is added to the analysis, the axolotl sequence remains associated with IgL, although the Xenopus sequence groups with all other TCR-ß sequences (data not shown). If Amttcrb is removed, TCR-ß and IgL become monophyletic (data not shown). However, the tetrapod IgL sequences (those not including the chondrichthyan group I, II, and III sequences; table 1
) do comprise a monophyletic group. The position of IgNARC (Gcu51450) is unclear: while it is probably associated with the IgH group, it appears as a separate branch intermediate between the IgH and TCR sequences. However, the shark NAR is clearly associated with TCR-
and embedded in the TCR+IgL group.
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![]() |
Discussion |
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If, as indicated by the C region trees, TCR- and -
/
sequences represent the earliest immune receptor sequences, and since the TCR-
/
sequences are highly convergent (Guglielmi at al. 1988
), this suggests that
-TCRs represent the oldest of the extant immune receptors, whereas
ß-TCRs and Igs are more recent. The V region trees, which may reflect functional relationships among antigen-binding sequences, indicate that the IgHs are the earliest, implying that the earliest immune receptors had Ig-like antigen-binding properties. Despite these important distinctions in V and C region tree topologies, both data sets indicate that direct antigen-recognition is the primitive condition for T and B cells, while indirect antigen recognition is clearly a derived characteristic of
ß T cells. Indeed, it would be startling if this were not the case, since direct ligand recognition and binding is typical of other cell surface adhesion molecules in the IGSF (Hunkapiller and Hood 1989
).
Even before the antigen receptors of T cells were identified and long before it was realized that two classes of T cells existed, it was suggested that the ancestral immune cells were probably T cells (Marchalonis 1977
). Since then, several authors (Marchalonis and Schluter 1990
; Stewart 1992
; Thompson 1995
; Flajnik 1998
) have suggested that primordial immune cells were
-like. Many characteristics of
T cells fit the role of a first line of immune defense (Brenner, Strominger, and Krangel 1988
; Raulet 1989
; Allison and Havran 1991
). The primordial immune cell would have to have been capable of self- versus nonself-recognition, as well as of protective effector functions such as cytolysis, and its receptor would likely have been membrane-bound rather than secreted. Both of these capabilities are present in
T cells. It might also be expected that the primordial immune cells would have been capable of recognizing a wide array of antigens, many of which could be ingested through the gut. Despite their limited V-gene repertoire in comparison with
ß V genes,
TCRs display far greater N region (a region of random nucleotide addition between the V and D regions) variability and are potentially capable of recognizing far more antigens than are
ß T cells.
T cells can respond to antigen challenge very quickly, without a requirement for professional APCs, and have been suggested as a first line of immune defense in mammalian immune systems. On the other hand,
ß T cells require professional APC and antigen presentation by MHC molecules and exhibit highly specialized interactions with B cells in the maturation of antibody responses. A characteristic of most receptors in the IGSF is that they are membrane-bound, an ancestral characteristic that is retained in
and
ß T cells. However, B cells can secrete immunoglobulins, a trait that is likely derived (Flajnik 1998
). In fact, while many characteristics of
T cells are suggestive of an ancient and primary role in immune defense, the roles of
ß T cells and B cells imply highly specialized, and probably derived, roles in the immune system, supporting the conclusion that
-like T cells gave rise to
ß T cells and B cells.
Several lines of structural evidence also support the suggestion that T cells exhibit ancestral antigen-binding characteristics.
-TCRs recognize a wider variety of antigens than do
ß-TCRs, including both peptides and phospholigands, and they are especially reactive to conserved molecules such as mycobacterial and heat shock proteins (Constant et al. 1994
; Burk, Mori, and DeLibero 1995
). An analysis of the length distributions of V region chains, those that actually interact with antigen, in T and B cells indicates greater similarity of
-TCRs to Igs than to
ß-TCRs (Rock et al. 1994
), and this also strongly suggests that
-TCRs bind directly to antigens, as do immunoglobulins.
ß T cells display two cell surface markers, CD4 and CD8, that are required for binding of the
ß-TCR to MHC molecules, but most
T cells carry neither of these; thus, they are not MHC-restricted, and no alternative presentation molecules have been discovered. The differences in antigen binding are probably reflected in the three-dimensional structures of the two types of TCR. Although the structure of
-TCR molecules has not been elucidated, the structure of an
ß-TCR bound to a peptide+MHC complex has been studied (Garcia et al. 1996
). The antigen-binding surface of the
ß-TCR is fairly flat and similar to the undulating surfaces where Igs bind to proteins, but it is also much smaller than the Ig-binding areas, perhaps bestowing greater complementarity of fit with antigen (Garcia et al. 1996
). It may therefore be predicted that the binding surface of the
-TCR is also relatively flat but more like an Ig than an
ß-TCR in size, as would be expected for recognition of a wider variety of antigens.
Origin of VJ-VDJ Heterodimeric Immune Receptors
All modern TCRs and Igs are heterodimers composed of one VJC molecule and one VDJC molecule. The only exceptions appear to occur in animals such as camelids that have functioning and abundant IgG molecules composed of heavy-chain dimers without light chains (Hamers-Casterman et al. 1993
), and possibly in NAR-bearing cells (Marchalonis et al. 1998
). However there is no evidence in our study or in previous studies (e.g., Greenberg et al. 1995
; Schluter, Bernstein, and Marchalonis 1997
) that the VJC (TCR-
, TCR-
, and IgL) and the VDJC (TCR-ß, TCR-
, and IgH) sequences constitute monophyletic groups, as might be predicted if they evolved separately from proto VJ and VDJ molecules (Thompson 1995
; Rast et al. 1997
). In fact, our analyses suggest that the TCR-
and TCR-
sequences are equally ancient and could even represent the two most ancient TCR-V sequences (fig. 5
). The branching order in figure 5
could be incorrect if the true relationship between TCR-
and TCR-
sequences is obscured by convergence: mammalian TCR-
and TCR-
variable genes are so highly convergent that they even display overlapping usage by
ß- and
-TCRs (Guglielmi at al. 1988
; Takihara et al. 1989
). Alternatively, if the branching order is correct, then the oldest V region genes are IgH, which gave rise to TCR-
+
sequences, from which TCR-
and TCR-ß subsequently arose through duplication (Rast et al. 1997
), and from which the IgL sequences also evolved. Thus, the original dimeric molecule could have been a VDJC/VDJC molecule, implying that as TCR and Ig molecules evolved, the D segment has been either retained or lost in different lineages. Thus the origin of the TCR sequences could have coincided with the origin of the VJC/VDJC heterodimeric immune receptor, perhaps replacing some earlier, homodimeric receptor composed of two IgH-like chains. Rast and Litman (1998)
have also noted the evolutionary lability of D regions, as is evident in vertebrate IgH genes where receptor molecules may be VJ, VDJ, VDDJ, or even VDDDJ (Marchalonis, Schluter, and Edmundson 1997
). Although presence or absence of a D segment does not appear to be a phylogenetically useful characteristic, it must be of functional significance at the level of the entire receptor molecule.
Close Relationship of TCR and IgL
Previous studies utilizing fewer TCR sequences have found that the TCR and IgL variable region genes formed one cluster and the IgH genes formed a second cluster (Greenberg et al. 1995
; Bernstein et al. 1996
; Schluter, Bernstein, and Marchalonis 1997
). We also found that the IgL and TCR variable sequences were more closely related to each other than to the IgH sequences, with the IgH variable genes giving rise to the TCR and IgL genes. Interestingly, when the conformations of the binding surfaces (encoded by V region genes) of
ß-TCR, IgL, and IgH molecules were compared, there seemed to be a closer match of the TCR with IgL than with IgH (Garcia et al. 1996
; Marchalonis, Schluter, and Edmundson 1997
), as might be expected if the similarity in molecular conformation reflects an orthologous relationship between TCRs and IgLs.
IgL genes make up two basic groups. The first group comprises the chondrichthyan IgL genes (groups I, II, and III; see table 1 ), and the second group comprises the and
light-chain genes. In our analyses, the IgL constant genes as a whole do not form a monophyletic group, because the group I, II, and III genes form separate branches that actually appear to be younger than the remaining, "modern," light-chain sequences (figs. 2 and 3 ). However, the variable region sequences of all light chains appear to be monophyletic (if Amttcrb is removed; figs. 4 and 5 ), with the group I, II, and III sequences being the most ancient, as found by Rast et al. (1994)
.
Relationships of NAR to IgW and TCR
Strongly supported in our analyses of the C region genes (figs. 2 and 3 ) is a close relationship between the recently discovered shark NAR (Gcu18701) and the IgHs, including IgW (Cpu40560) and IgNARC (Gcu51450). When NAR was first discovered (Greenberg et al. 1995
), it was speculated that a NAR-bearing cell might bind to antigen in a manner resembling that of immunoglobulins (Greenberg et al. 1995
), and this seems very likely, since comparisons of IgW, NAR, and IgNARC C region genes confirm that they are all genuine immunoglobulins (Schluter, Bernstein, and Marchalonis 1997
). Although IgM was long thought to represent the primordial immunoglobulin type (Fellah et al. 1992
), the fact that the IgW class is found only in sharks and their allies may indicate that IgW is primordial (Bernstein et al. 1996
; Shen et al. 1996
; Schluter, Bernstein, and Marchalonis 1997
). Our analyses of the C region genes offer little support for this, since they indicate that the IgH sequences are in fact younger than the IgL sequences. On the other hand, the V region sequences do support the hypothesis that IgW sequences evolved earlier than IgM (figs. 4 and 5
). There is little support in our analyses for the hypothesis that among the non-IgW heavy-chain sequences, the chondrichthyan IgM sequences are archaic (Shen et al. 1996
), but we may not have included sufficient diversity of sequences to address this issue.
Although the C region trees unequivocally group IgW, IgNARC, and NAR molecules with other immunoglobulin heavy chains, the same cannot be said of the V region trees, in which NAR is more closely allied with TCR than with Ig sequences (figs. 4 and 5 ). This divergent evidence from C and V region trees with respect to the relationship between NAR and other receptor sequences has also been noted in several previous studies (Greenberg et al. 1995, 1996
; Schluter, Bernstein, and Marchalonis 1997
) and suggests that NAR is not simply a rather odd member of the IgW class, but a molecule with its own distinct role. Klein (1998)
has interpreted the "chimeric" nature of the NAR molecule in two ways. First, the NAR V region gene sequence is either highly convergent with or actually orthologous to a fully evolved (sic) TCR V region sequence. This is plausible if NAR represents an early branch in TCR phylogeny, as is indicated in figure 5 (and in fig. 4
to a lesser extent). Kleins (1998)
second suggestion (and the one he prefers) is that the similarity between TCR and NAR V regions is an artifact of phylogenetic analysis, because over the last 400 Myr, any phylogenetic signal would have been obliterated by mutations in the NAR protein sequence. We do not accept this argument, because there is no reason to believe that this should be more true for NAR than for any of the other TCR or Ig sequences which we and others have analyzed, including the IgH V region sequences, which appear to be the most ancient. In general, we believe that our own and other analyses support Rast and Litmans (1998)
suggestion that NAR is a divergent IgH gene type. NAR might be divergent not in effector function (since it is a bona fide immunoglobulin), but in the way it binds antigens or in the type of antigens bound.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Keywords: T-cell receptor,
immunoglobulin,
immune system evolution.
2 Address for correspondence and reprints: M. H. Richards, Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada. E-mail: mrichard{at}spartan.ac.brocku.ca
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