By
From the * Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York 10029; the Howard Hughes Medical Institute, The Rockefeller University, New York 10021; the § Howard
Hughes Medical Institute, Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York
10029; and the
Immunology Program, Graduate School of Medical Sciences, and the Department of
Medicine, Cornell University Medical College, New York 10021
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Abstract |
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Superantigens are defined as proteins that activate a large number of T cells through interaction
with the V region of the T cell antigen receptor (TCR). Here we demonstrate that the superantigen produced by Mycoplasma arthritidis (MAM), unlike six bacterial superantigens tested, interacts not only with the V
region but also with the CDR3 (third complementarity-determining region) of TCR-
. Although MAM shares typical features with other superantigens, direct interaction with CDR3-
is a feature of nominal peptide antigens situated in the antigen
groove of major histocompatibility complex (MHC) molecules rather than superantigens. During peptide recognition, V
and V
domains of the TCR form contacts with MHC and the
complex is stabilized by CDR3-peptide interactions. Similarly, recognition of MAM is V
-dependent and is apparently stabilized by direct contacts with the CDR3-
region. Thus,
MAM represents a new type of ligand for TCR, distinct from both conventional peptide antigens and other known superantigens.
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Introduction |
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In the first step of a specific immune response, T lymphocytes are activated by interaction between the TCR and
peptide antigen bound to the MHC. Specific peptide recognition is achieved by the unique structure of TCRs,
formed by somatic rearrangement of their composite gene
segments (V, J
, V
, D
, and J
). Most contacts with
peptide are formed by very diverse CDR3 (third complementarity-determining region) regions that are encoded by
the V-D-J junctional region (1, 2). During recombination,
a second level of diversity is provided by the trimming and
addition of nucleotides (N-additions)1 at the junctions (3).
As a result of the huge diversity of TCRs created, one peptide antigen activates only a small number of T cells bearing
specific receptors. However, several groups of microorganisms have developed a way to activate large numbers of T
cells. They produce superantigens, potent stimulators of T
cells, that form contacts with lateral surfaces of both the MHC and TCR. In superantigen recognition, the complexity of the TCR is ignored and interaction occurs between the V
region and the superantigen. In this way, a
single superantigen can activate >10% of all T cells (4).
Superantigens can be produced by bacteria, viruses, and
even plants (7). Their biological role remains unclear,
except for several mouse mammary tumor virus (MMTV)
superantigens that are essential during the viral life cycle.
By activating T and B cells in neonatal mice, MMTV superantigens form a pool of dividing cells that amplify the
viral load and allow viral persistance until maturation of the
main target site, the mammary tissue. The endogenous
form of MMTV superantigen, acquired by integration of
the viral gene into the mouse genome, has the opposite
role. Mice expressing endogenous superantigens are protected from viral infection because of deletion of the superantigen-reactive T cell V subsets in early development
(10, 11). Other superantigens also cause proliferation and
expansion of T cells with a responding V
phenotype, often followed by deletion of the targeted subset. It has been
hypothesized that nonspecific stimulation of large numbers
of T cells by superantigens could include self-reactive T
cells and lead to autoimmunity (5). A recent report suggests involvement of a human endogenous retrovirus (HERV-K)-
encoded superantigen in induction of autoimmune diabetes
(12). Unlike MMTV-encoded superantigens that are products of the 3
LTR, this superantigen seems to be encoded
by the env gene. It activates the V
7+ subset of T cells,
which was previously found to be enriched in pancreatic
infiltrates of diabetic patients (13).
The superantigen produced by Mycoplasma arthritides
(MAM) does not have significant homology to the primary
sequence of other known bacterial or viral superantigens
(14). Nevertheless, MAM shares several features common
to many superantigens, including requisite presentation by
MHC class II to responding CD4 and CD8 T cells, lack of
restriction of the presenting MHC class II alleles, lack of an
antigen processing requirement, and the ability to stimulate
T cells expressing specific V genes (for review see reference 15). However, functional differences between MAM
and other superantigens have been noted. MAM is a relatively weak mitogen for polyclonal human T cells, but it is
an efficient stimulator of B cells and induces IgM production better than do staphylococcal superantigens (16). It has
been suggested that the poor mitogenic activity of MAM
reflects a lower frequency of MAM-reactive T cells among
peripheral lymphocytes compared with other superantigens (16, 17).
Here we demonstrate that the MAM represents a new
type of ligand for the TCR, intermediate between superantigens and conventional peptide antigens, because it
forms contacts both within the V region and the CDR3-
region of the TCR.
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Materials and Methods |
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Human T Cell Clones.
CD4+ clones were obtained after stimulation of fresh PBL with MAM by limiting dilution in 96-well plates. Every 2 wk clones were stimulated by sodium periodate-treated non-T PBL as previously described (18) and maintained in RPMI 1640 with 10% FCS and 5% IL-2 (Pharmacia Biotech, Piscataway, NJ).Proliferative Response.
2 × 104 T cells and 1.5 × 104 irradiated (15,000 rads) lymphoblastoid 8866P cells were incubated with 10-fold dilutions of superantigens (from 1 to 10TCR Sequencing.
cDNAs obtained from human T cell clones were amplified with primers specific for human VTransfection of TCR Chains.
IL-2 Production Assay.
Activation of the TCR transfectants was tested according to reference 27. 105 cells in a 100 µl/well were mixed with an equal number of 8866P lymphoblastoid cells. 10-fold dilutions of each superantigen were tested in triplicates. After 24 h, supernatants were frozen at ![]() |
Results |
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To characterize MAM-reactive TCRs, we tested a panel of
16 human T cell clones for proliferative responses to 7 different superantigens (Table 1, Fig. 1). Clonal T cells were
incubated with APCs in the presence of gradual 10-fold dilutions of each superantigen, then pulsed with [3H]-thymidine and harvested. Among T cell clones using the same V genes, similar patterns of reactivity were observed with
most superantigens, whereas reactivity to MAM often differed (Fig. 1, shaded field). T cell clones expressing either
V
5.1, V
8, V
12, or V
17 could respond to MAM, but
within each of these V
subsets there were both reactive
(MAM+) and nonreactive (MAM
) clones. Lack of reactivity to MAM was not dose dependent, as we were unable
to find concentrations of MAM that would activate MAM
clones (Table 1). The MAM
cells were competent to respond to TCR-mediated signals from other superantigens
(Fig. 1). In addition, the sequences of TCR V
regions from
MAM+ and MAM
T cell clones were identical, ruling
out the possibility that V
allelic variants resulted in differences in MAM reactivity.
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Strikingly, all T cell clones that did not respond to MAM
used the TCR J2.1 gene segment, whereas MAM-reactive clones used other J
2 segments (Fig. 2). Alignment of
the CDR3 regions showed that a tyrosine (Y), present
within the conserved motif Q(Y/F)FG, correlated with responsiveness to MAM. Nonreactive clones had phenylalanine (F) at this position. The same correlation was observed in polyclonal cell lines obtained after repeated stimulation
of fresh T cells with MAM. Dominant clones within these
cultures (Fig. 2; SEL) represent T cells expanded in vitro
after stimulation with MAM. All V
17 MAM+ and SEL
clones have isoleucine at position 94 in addition to Y
within the Q(Y/F)FG motif in their CDR3-
region.
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Staining data indicated that in the polyclonal cell lines
only the V17+ subset was expanded in response to MAM,
to ~50% of all T cells, in agreement with previous data
(21). The minor MAM-responsive phenotypes (V
5, V
8,
and V
12) were not expanded in these polyclonal cell
lines. However, within these minor V
subsets, dominant
clones were also found after three to five stimulations with
MAM. We hypothesize that these clones represented relatively infrequent cells within the V
5, V
8, or V
12 subsets, which may depend on the other TCR elements for
MAM reactivity. Consistent with this view, V
2 and V
8
were frequently observed in clones of minor V
phenotypes (Fig. 2). Thus, reactivity may depend on V
usage with these minor V
phenotypes, as previously described
for low affinity TCR V
-superantigen interactions (29).
However, the major MAM-responsive V17 phenotype
is not
chain-dependent. To prove this point, we transfected
chains derived from clones N17 and J17 into two
mouse T hybridoma cell lines, DS 23.27 and YL
-, that
are
chain-deficient (Table 2). Surface expression of the
endogenous mouse TCR-
chain, either V
2 or V
3.1,
respectively, is rescued by TCR complex formation with the transfected
chain.
chains N17 and J17 retained
their differential MAM reactivity regardless of which
chain they were paired with (Table 2).
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Presentation of superantigens to T cell clones can be sensitive to minor differences among MHC class II alleles (32)
and the nature of the peptide in the MHC class II groove
(33). Therefore, we tested the reactivity of MAM+ and
MAM clones with three superantigens presented by lymphoblastoid cell lines expressing different MHC class II alleles (Fig. 3). In each of four groups (V
17, V
8, V
12,
and V
5), MAM+ and MAM
clones retained their specific reactivity regardless of the MHC class II alleles expressed on APCs. The magnitude of the proliferative responses differed from one APC type to another, probably
due to different levels of expression of MHC class II or accessory molecules, but the pattern of reactivity remained
the same. MAM+ clones were always MAM-reactive and
MAM
clones did not respond to MAM presented by
other APCs.
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Sequence alignment of
TCR- chains implicated a single residue (Y/F) of the
CDR3 region in determining MAM reactivity (Fig. 2). To
test whether Y103 of clone N17 (MAM+) and the equivalent residue, F106, of clone J17 (MAM
) determine MAM
reactivity, we attempted to reconstitute reactivity of J17 by
mutagenesis in the CDR3-
region (Fig. 4). Wild-type and mutant TCRs were expressed in DS23.27 cells and
tested for IL-2 production in response to several superantigens. A single amino acid substitution (JY, F106Y) at position 106 was insufficient to restore reactivity to MAM. A
second candidate amino acid was isoleucine in position 94. This position is encoded by the V
-Db junctional region
and in all V
17+ clones reactive with MAM, it was represented by isoleucine (Fig. 2). Therefore, we introduced a
second substitution at position D94 of J17. Strikingly, the
combination of D94I and F106Y (transfectant JIY) resulted
in full reconstitution of MAM reactivity (Fig. 4) while the
single change, D94I, was insufficient (JIF).
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To demonstrate that other regions of CDR3 were not
involved, we tested three deletion mutants including either
one or two residues between I94 and Y106 (JIY-SY, JIY-NE, JIY-E). These mutants would be expected to differ in
their peptide/MHC reactivity, because recognition of conventional peptide antigen is extremely sensitive to changes
in CDR3 (34, 35). All three deletion mutants retained MAM reactivity (Fig. 4). Together with the data on MAM
presentation by different MHC class II alleles (Fig. 3) these
experiments exclude a role for MHC II-bound peptides in
these MAM-TCR interactions. A direct contact between
TCR CDR3- and MAM is the most likely explanation
for dependence on residues I94 and Y106. Based on recent
crystallographic data, these two amino acids are located at
the base of the CDR3 loop lying in close proximity to one
another (36). The side chain of Y106 extends towards the
exposed surface of the TCR (Y107 in reference 2). It appears that residues I94 and Y106 are accessible for direct interaction with MAM. These interactions are specific for
MAM since none of the tested mutations affected reactivity
with staphylococcal superantigens (Fig. 4).
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Discussion |
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In this study we show
that the CDR3 region of TCR- chain mediates specific
recognition of the MAM superantigen. Complementarity-determining regions (CDR loops) (2, 37) represent hypervariable loops of the TCR that face the MHC during antigen recognition. On the
chain, both CDR1 and CDR2
as well as the HVR4 loops can be involved in interactions
with superantigens. TCR-
point mutations that affect
specific interaction with a particular superantigen are shown
in Fig. 5. The cocrystals of TCR
chain with staphylococcal enterotoxins SEC2 and SEC3 revealed that these staphylococcal superantigens interact with CDR1, CDR2, FR3,
and HVR4 but not with the CDR3 loop (38). Most contacts (47%) are formed between the superantigen and the
CDR2 loop. Since Y106 of CDR3 (2) and exposed CDR2
residues are located at opposing faces of the TCR
chain
(1), our data suggest that recognition of MAM and staphylococcal superantigens is substantially different. That CDR2
regions are not involved in MAM recognition is supported by the observation that the primary sequence of the homologous human V
17 and mouse V
6 chains, both
MAM-reactive, differs in the CDR2 region.
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Mutational and structural studies have shown that the
CDR3 loops of the and
chains are central in peptide
recognition (1, 2, 34, 35, 39, 40). CDR3 regions are the
most diverse parts of the TCR produced by extensive processing of end joints during antigen receptor rearrangement
(3). Junctional diversity is increased by N-additions contributed by the lymphoid-specific enzyme terminal deoxytransferase (TdT; references 41). Although an N-region-
depleted TCR repertoire from TdT-knockout mice was shown to be as efficient as normal TCR repertoire (44), the N-region-depleted TCRs were more promiscuous in peptide recognition. These TCRs recognized a significantly
larger number of different peptides than did the wild-type
TCRs when tested for reactivity against a library of random
peptides (45).
The structural basis for the role of N-encoded TCR residues was revealed in a recently solved structure of the
TCR-MHC interface (1). In this crystal, three interactions
are formed between residue p5 of the peptide and the
TCR. Two of these are contacts of p5 with N-encoded
residues of the V-D
and V
-D
junctional regions.
The specific interaction of MAM with the TCR appears
to imitate the natural process of peptide/MHC recognition. During peptide recognition, CDR3 regions of and
chains form contacts with MHC. The complex is stabilized by CDR3-peptide interactions (46, 47). Recognition
of MAM is clearly V
-dependent, as is the case with other
superantigens, but it appears to be stabilized by additional contacts with the CDR3-
region.
In all MAM-reactive V17+ clones that we have sequenced, position 94 was represented by isoleucine. I94 is
adjacent to the conserved end of V
region CASS and corresponds to position 2 of the CDR3 region (48, 49). In
random CDR3 sequences, position 2 is rarely represented
by isoleucine (49) but in V
17+ sequences I94 is more
common, occurring in ~20% of the sequences (50). This
residue is often assumed to be part of the N-region, but in
the case of isoleucine it may be encoded by the 3
end of
the germline V
17 sequence, codon ATA of the genomic
sequence (51). However, in ~80% of the cases this codon
is removed during V
-D
recombination and replaced by
N-additions.
The second CDR3- position critical for interaction
with MAM is tyrosine within the Q(Y/F)FG motif of J
2
(T cell clones using J
1 are rare and they were not analyzed
in this study). At this position Y is encoded by J
2.3,
J
2.4, J
2.5, and J
2.7. F, which does not allow MAM reactivity (Fig. 4), is encoded by J
2.1 and J
2.2. The J
2.1
segment, which was used by the MAM
T cell clones studied here (Fig. 2), is used by ~40% of all T cells from peripheral blood (52).
The major subset of T cells targeted by MAM is the
V17 subset (21). Yet not all V
17+ T cells respond to
MAM, and the response is limited to cells expressing two
appropriate residues at the base of the CDR3-
loop. This
suggests that T cell activation by MAM is dependent on
TCR junctional diversity, thus limiting the number of potential MAM-reactive T cell clones. The frequency of T
cells responding to this superantigen could be significantly
lower than to other superantigens (for example SEB,
SEC1, SEC2, and SEC3). Among the minor MAM-responsive TCR phenotypes (V
5, V
8, and V
12), the frequency is even lower than within the V
17 subset,
probably because V
usage is an additional requirement as
suggested by the data in Fig. 2. As previously shown,
MAM-responsive T cells expressing V
5, V
8, and V
12
can only be detected by testing individual clones and these
V
phenotypes do not expand in polyclonal cultures exposed to MAM (18). However, TCRs of the minor phenotype also appear to use the J
-encoded tyrosine for
MAM recognition (Fig. 2).
It is possible that J usage can affect low affinity interactions of TCR with other superantigens. Incomplete deletion of V
8+ or V
6+ cells in mice carrying Mls-1a (Mtv-7)
revealed that TCRs that escaped deletion used distinct J
regions (53, 54). In this experimental system, J
1.2 seemed
to protect V
6+ TCR from interaction with a retroviral
superantigen, Mls-1a. However, these studies did not find
any conserved motifs in the CDR3 region that could be
implicated in unresponsiveness to Mls-1a (54). Staphylococcal superantigens seem to be CDR3-independent (Fig.
4 and reference 55). However, in the case of SEB and Urtica dioica superantigens, it was suggested that the J
segment, but not the CDR3 region, can affect superantigen
binding by influencing the quaternary structure of the
TCR-
chain (55).
The major MAM-responsive V phenotype is strikingly
dependent on discrete residues of CDR3. This fact clearly
distinguishes MAM from other superantigens. Thus, recognition of MAM is dependent on junctional diversity of the
TCR
chain.
Mycoplasma arthritides is a microorganism
that causes a disease in rodents that is remarkably similar to
human rheumatoid arthritis (RA). It is also the only mycoplasma that produces MAM. It should be emphasized that
Mycoplasma arthritides is not a human pathogen. Surprisingly, preferentially expanded T cell clonotypes found in
human RA often belong to the MAM-reactive TCR phenotype. Dominant clones from RA patients, characterized
in three independent studies, were found to use V17 followed by isoleucine at position 94 (50, 56, 57). In one of
the studies, these T cells were shown to be autoreactive
against a lymphoblastoid cell line expressing HLA-DR4
(50). Similar autoreactivity was reported in a TCR transgenic mouse model of RA (58). Inflammatory sinovitis in
mice was triggered by the transgenic TCR recognition of
host MHC class II (Ag7). This transgenic TCR-
chain
used a mV
6 followed by isoleucine (CASSI) (D. Mathis,
personal communication). Murine V
6 is the closest homologue of human V
17, and mouse V
6+ T cells are also
MAM-reactive (15). Thus, both murine and human TCRs
associated with RA appear to express a conserved isoleucine at the COOH-terminal end of the V
region. The
role of the conserved isoleucine in the CDR3-
region of
arthritogenic TCRs remains to be determined. Is it necessary for interaction with MHC class II or a putative joint-specific peptide? Is it possible that tolerized self-reactive
clones can be reactivated by MAM in mice infected by Mycoplasma arthritides?
There is a strong genetic link between certain DR alleles
(class II MHC) and RA in humans (59, 60). Position 71 of
the particular DR4 or DR1 chains, a lysine residue, is associated with susceptibility for RA (61). This residue determines the nature of the peptides in the class II groove by
interacting with P4/P5 of the peptide (for review see reference 62). Conserved CDR3-
motifs that include isoleucine 94 have been proposed as evidence for antigen-driven
immune response in RA (50). Whether superantigens like
MAM have a role in RA pathogenesis is still an open question.
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Footnotes |
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Address correspondence to Andrew S. Hodtsev, Mount Sinai School of Medicine, Ruttenberg Cancer Center, 1 Gustave Levy Place, New York, NY 10029. Phone: 212-824-8123; Fax: 212-849-2446; E-mail: andrew_hodtsev{at}smtplink.mssm.edu
Received for publication 6 November 1997 and in revised form 2 December 1997.
1 Abbreviations used in this paper: MAM, Mycoplasma arthritidis mitogen; MMTV, mouse mammary tumor virus; SE, staphylococcal enterotoxin.We are grateful to N. Bhardwaj for her clones of human T cells, B.C. Cole for purified MAM protein, J.P. Morgenstern for the pBabe vector, W.S. Pear for the BOSC cell line, and D. Kostyu for HLA typing. We also thank G. Kelsoe, A. Chervonsky, and S. Santagata for discussion of the manuscript.
This work was supported by an Aaron Diamond Foundation fellowship to A.S. Hodtsev and by National Institutes of Health grants to D.N. Posnett (AI-31140 and AI-33322) and to Y. Choi (CA-59751).
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