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and an Antigenic Peptide Bound to MHC Class I1
Departments of
*
Cell Biology and
Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain transgenic mice using a
TCR derived from a T cell clone specific for the immunodominant peptide
of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by
H-2Kb. We immunized these mice with VSV8 or analogs
substituted at TCR contact residues (positions 1, 4, and 6) and
analyzed the CDR3
sequences of the elicited T cells. In
VSV8-specific CTLs, we observed a highly conserved residue at position
93 of CDR3 and preferred J usage, indicating that multiple
residues of CDR3 are critical for recognition of the peptide.
Certain substitutions at peptide position 4 induced changes at position
93 and in J usage, suggesting a potential interaction between
CDR3 and position 4. Cross-reactivity data revealed the foremost
importance of the J region in determining Ag specificity.
Surprisingly, substitution at position 6 of VSV8 to a negatively
charged residue induced a change at position 93 of CDR3 to a
positively charged residue, suggesting that CDR3 may interact with
position 6 in certain circumstances. Analogous interactions between the
TCR -chain and residues in the C-terminal half of the peptide have
not yet been revealed by the limited number of TCR/peptide-MHC crystal
structures reported to date. The transgenic mouse approach allows
hundreds of TCR/peptide-MHC interactions to be examined comparatively
easily, thus permitting a wide-ranging analysis of the possibilities
for Ag recognition in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR is a disulfide-linked heterodimer. The - and
-chains each contain a constant region, assumed to be associated
with CD3 accessory molecules that trigger downstream signaling, and a
variable region involved in specific Ag
recognition. The variable region of the -
and -chains includes three Ag-binding loops or
complementarity-determining regions (CDRs),7
referred to as CDR1, CDR2, and CDR3, which are analogous to those found
in Igs. CDR1 and CDR2 are encoded by V or V gene segments present
in the germline. For the -chain, the CDR3 loop is encoded
following the joining of a V gene to one of a number of J genes,
whereas for the -chain, V-D-J joining encodes CDR3 (1, 2). This gene recombination is partly responsible for creating
the diversity in CDR3 loop sequences, and further diversity is
generated by deletion or addition of nucleotides at the junctions. It
was recently demonstrated for Abs that diversity only in the H chain
CDR3 is sufficient to develop responses to a variety of protein and
hapten Ags (3).
Extensive structural and functional studies exploring the specificity
of TCR recognition for peptide-MHC (pMHC) have been conducted. Recently
reported crystal structures of TCR/pMHC complexes have revealed that
TCRs bind to pMHC complexes with a generally diagonal footprint in
which the -chain is over the N-terminal half of the peptide and the
-chain over the C-terminal half (4, 5, 6, 7, 8, 9, 10, 11). The crystal
structures, along with functional analyses using TCR single-chain
transgenic mice (12, 13, 14) or site-directed mutagenesis of
CDR3 loops (15, 16), have all demonstrated the existence
of interaction sites between the CDR3s and the antigenic peptide, thus
identifying the CDR3 loops as important determinants for peptide
recognition.
Although the overall diagonal orientation of the TCR over pMHC class I appears to be general, the particular contact sites vary for different TCRs bound to the same ligand (7), as well as for the same TCR complexed with different ligands (8, 10). For a fuller understanding of the general rules for TCR/pMHC interactions, a much more extensive functional and structural database is needed. Over the years, we have used a variety of structural and functional strategies to elucidate the interactions that occur between TCRs and the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2Kb (17). In early studies, we identified the residues at positions 1, 4, and 6 of VSV8 as potential TCR contacting sites by using x-ray crystallography (18) and a biological T cell recognition assay (19). Next we used a series of H-2Kb mutants to map the TCR binding surface on the MHC, work that allowed us to first propose the diagonal orientation of TCR over pMHC (20). Structural information regarding the interaction of TCRs with VSV8/H-2Kb is limited to a single low-resolution structure that is nonetheless sufficient to confirm the overall diagonal orientation observed in the TCR/pMHC structures that have been reported in other systems (21).
To better "visualize" the interactions that occur between TCRs and
VSV8, and to observe a large number of such interactions comparatively
easily, we have used the technique of variant peptide immunization of
TCR single-chain ( or ) transgenic mice. In our previous efforts
to identify the residues in CDR3 that are important for the
recognition of VSV8 and its variants (14), we introduced
the V2 TCR -chain of a VSV8-specific CTL clone (N30.7) into
TCR-deficient mice, so that all the T cells would express an
identical -chain. These TCR -chain transgenic (Tg) mice were
immunized with VSV8 peptide analogs with a substitution at position 6,
and the CDR3 sequences of peptide-specific CTL were analyzed. We
found that a substitution at position 6 of VSV8 can induce a
compensatory change at position 98 of CDR3 as well as a change in
CDR3 loop length. It was then concluded that position 98 of CDR3
is a key residue determining the specificity of TCR recognition of
position 6 of the peptide and that an optimal length of CDR3 is
required to facilitate interaction. Site-directed mutagenesis also
confirmed the importance of position 98 in
VSV8/H-2Kb recognition (22, 23).
Regarding the interactions between TCRs and VSV8, several questions
remained to be answered, including: 1) Which positions of the peptide
interact with CDR3? 2) How does CDR3 loop length and composition
change to recognize different peptides? 3) Are these changes
qualitatively different from those observed in CDR3? and 4) Does
position 4 of VSV8 contact CDR3 and/or CDR3? To approach these
questions, we recently derived TCR -chain transgenic (Tg) mice
using the V13 TCR -chain from N30.7, the same VSV8-specific CTL
clone whose -chain was used to generate the Tg mice used in our
previous work (14). In these Tg mice, the presence of
the transgene inhibits the rearrangement and expression of endogenous
TCR -chain genes due to allelic exclusion. After immunization of the
mice with VSV8 and variants bearing substitutions at potential TCR
contact residues, the CDR3 loop sequences of peptide-specific T
cells were analyzed to see which, if any, amino acid substitutions
could induce compensatory changes in CDR3 composition and/or length.
Here we show that this approach, in which hundreds of TCR/pMHC
interactions could be examined, has revealed previously undescribed
interactions between TCRs and VSV8 and has highlighted the general
importance of the TCR J region in determining Ag specificity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mice
Derivation of the Tg mice has been described in detail
previously (14). These mice are transgenic for the TCR
-chain of the VSV8-specific CTL clone N30.7 (24). They
also carry a targeted disruption of both alleles of the endogenous
TCR C locus and are therefore unable to express
their endogenous TCR -chain genes.
Generation of N30.7 Tg mice
The VDJ segment of the TCR -chain cDNA (V13) of the
VSV8-specific CTL clone N30.7 was amplified by PCR (using primers
containing appropriate intronic sequences, splice acceptor/donor sites,
and restriction sites) and cloned into the
ClaI/NotI sites of the TCR shuttle vector
(Ref. 25 ; provided by M. Davis, Stanford University,
Stanford, CA). After removal of prokaryotic sequences by digestion with
PvuI/SalI, the transgene construct was injected
into (CBA x C57BL/6)F2 zygotes. Mice
carrying the V13 transgene were identified by PCR analysis of tail
DNA using the primer set 5'-CTTTGTCTCCTGGGTGCAGGT-3' and
5'-TTCACCACCCACCCAGTGCAT-3'. Transgene carriers were crossed for
several generations with C57BL/6 mice to obtain transgenic mice
homozygous for H-2b. MHC haplotype was monitored
by PCR using primer sets specific for H-2Kb and
H-2Kk.
Generation of peptide-specific CTLs
Peptides (Table I
) were
synthesized and purified to >98% homogeneity by HPLC before being
used for immunization. Tg or Tg mice were immunized in their hind
footpads with 20 µg of peptide emulsified in CFA and boosted 1 wk
later with 20 µg of peptide emulsified in IFA. One week after the
boost, spleen cells were cultured at 5 x
106/ml with 1 µM immunizing peptide in IMDM
supplemented with 10% heat-inactivated FBS (HyClone Laboratories,
Logan, UT). Cells were restimulated once in vitro, and cell sorting was
performed after 13 days of culture.
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CTL activity was measured in a standard 4-h 51Cr-release assay as described (31). The percentage of specific lysis was calculated as [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100%.
FACS analysis and cell sorting
To detect surface expression of the V13 transgene,
dissociated lymph nodes of 6- to 8-wk-old mice were stained with an
anti-V13 (MR12-3; BD PharMingen, San Diego, CA) Ab. To sort
Tg+ CTLs from in vitro cultures, cells were
stained with anti-CD8 (53-6.7; BD PharMingen) and anti-V13
Abs and CD8+ V13+ cells
were collected. FACS analysis and cell sorting of stained cells were
performed on a FACScan and FACStar (BD Biosciences, Mountain View, CA),
respectively.
Preparation of mRNA and cDNA and V family usage typing
mRNA was extracted from 5 x 105 to
2 x 106 sorted Tg+
CTLs, and single-stranded cDNA was made as described (14).
V family usage was determined by PCR using a TCR C primer paired
with 1 of 20 V primers, each specific for a particular V gene
family and designed to recognize all known members of that family
(32). Each culture was found to express a limited number
(i.e., 2–4) of V families (data not shown).
Analysis of TCR CDR3 sequences
Double-stranded TCR -chain cDNA was prepared by PCR as
described above from sorted
CD8+V13+ cells. PCR
products were cloned into pCR2.1 (Invitrogen, San Diego, CA) and the
TCR CDR3 sequences in the resulting plasmids were determined. At
least two Tg mice were immunized with each peptide variant and the
sequencing data pooled.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains of VSV8-specific CTLs from Tg mice exhibit
conserved J usage and a conserved residue at position 93 of the CDR3
loop
Mice transgenic for the TCR -chain (V13) of the
VSV8-specific CTL clone N30.7 were derived as described in
Materials and Methods. FACS analysis revealed that virtually
all peripheral CD8+ T cells in these Tg mice
expressed the V13 transgene on their surface, and V family usage
typing by RT-PCR (32) indicated that the presence of
the rearranged V13 transgene eliminated the rearrangement and
expression of endogenous TCR -chain genes (data not shown). To
investigate the features of CDR3 that are important for interaction
with VSV8/H-2Kb, Tg mice were immunized with
VSV8, and the CDR3 sequences of VSV8-specific CTLs were
determined.
Cytotoxicity assays showed that VSV8-specific CTLs could be
induced in Tg mice (Fig. 1),
indicating that the transgenic TCR -chain could pair with endogenous
TCR -chains to allow recognition of
VSV8/H-2Kb. TCR V family usage typing
indicated that the V1, V2, V5, and V15 families were used
in VSV8-specific CTLs. As the N30.7 TCR -chain is a member of the
V2 family (24), we first compared the CDR3 loop
sequences of the V2 TCRs with that of the N30.7 TCR (Table II). We found that the CDR3 loop
sequences from VSV8-specific CTLs elicited from Tg mice showed
striking similarity to that of the N30.7 TCR. Among the CDR3
sequences from the V2 family, position 93 (Pro) and the J15
region (YQGGRALI) are highly conserved. (Note that position 93 occurs
at the V-J junction and is generally not germline-encoded.) These data
suggest that the residue at position 93 and the J region of the
CDR3 loop are important elements for interaction with VSV8. We also
found that the predominant CDR3 length was 10-aa long (Table III),
but that the CDR3 length was less restricted as compared with that
previously observed for CDR3 (14).
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were also associated with usage of a specific V family, CDR3
loop sequences from VSV8-specific CTLs expressing different V
families were examined and compared (Table II
). We found that for three
other V families (V1, V5, or V15), the residue at position
93 and the J region of CDR3 were highly conserved. However, for
these V families, the residue at position 93 was Ser/Thr instead of
Pro, which was found in V2 CTLs. Yet, J15 was again the
predominant J family among sequenced CDR3 loops for all of the
V families examined. Our data suggest that in VSV8-specific CTLs,
the specific J usage is induced by the peptide independently of the
V family, whereas the identity of the conserved residue at position
93 is determined by both the antigenic peptide and the V family.
In the crystal structure of the 2C TCR (4, 6), a
portion of the J region lies adjacent to the CDR3 loop. This
observation brought up a question: do J15 TCR -chains
preferentially pair with the transgenic TCR -chain, resulting in
predominant J15 usage even in naive Tg mice? To address this
issue, CDR3 loops of TCRs from naive (i.e., unimmunized) Tg mice
were sequenced (data not shown). We found that in naive mice, there is
no predominant J usage and the occurrence of J15 among all CDR3
sequences is extremely low (1/22). Thus, the predominant J15 usage
observed in VSV8-specific CTLs is specifically induced by the
immunizing peptide and is not caused by preferential pairing with the
transgenic TCR -chain.
Position 4 variants elicit CTLs in Tg mice, but not in Tg
mice
Although immunization of N30.7 Tg mice with VSV8 permits
induction of peptide-specific CTLs (Ref. 14 , and Fig. 1),
immunization with the position 4 variants D4 (Val
Asp), E4
(ValGlu), K4 (ValLys), R4 (ValArg), or Y4 (ValTyr) was
unable to elicit a CTL response, whereas I4 (ValIle) induced only a
weak response (Fig. 1 and data not shown). A possible explanation for
these results is that position 4 of VSV8 interacts with the TCR
-chain, and that the fixed Tg chain cannot tolerate any change at
this position of the peptide. To determine whether position 4 might
also interact with the -chain, N30.7 Tg mice were immunized with
these same peptide variants. A cytotoxic response was obtained in all
cases (Figs. 1 and 2), supporting the
idea that position 4 of the peptide interacts principally with the
TCR -chain and not with the -chain.
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at position 93 and in J usage
To map potential interaction sites between the TCR -chain and
position 4 of the peptide, CDR3 sequence analysis was performed on
CTLs from Tg mice responding to VSV8 variants in which Val was
substituted by a positively charged (Lys in K4 or Arg in R4) or bulky
(Tyr in Y4) residue. Comparison of CDR3 sequences within a specific
V family showed that nonconservative substitution of Val to Lys,
Arg, or Tyr induced compensatory changes in the CDR3 sequence (Table IV), suggesting a direct interaction
between CDR3 and position 4 of the peptide. Consistent with the
results for VSV8-specific CTLs, TCRs of K4-, R4-, or Y4-specific CTLs
had a highly conserved residue at position 93 and a conserved J
segment when a specific V family was considered. When position 4 was
changed to Lys, the residue at position 93 of CDR3 was changed to
Arg or Glu, the J15 gene segment used by VSV8-specific CTLs was
replaced by J18 or J22, and changes in the CDR3 length were
also observed. For the ValArg substitution, we observed changes in
the residue at position 93 and J usage, but not in CDR3 length,
whereas the ValTyr substitution induced compensatory changes in both
the CDR3 motif and length. These CDR3 sequence data indicate that
certain position 4 substitutions can induce changes both at position 93
and in the J region, leading to alterations in the nature of the
CDR3 loop that have functional consequences, as revealed by the
finding that K4-, R4-, and Y4-specific CTLs were unable to cross-react
with VSV8 (Fig. 2).
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mice (Fig. 1 and data
not shown), and A4- or I4-specific CTLs could cross-react with VSV8
(Fig. 2 and data not shown). The predominant CDR3 sequence used by
V15 A4-specific CTLs (SXYQGGRALI; Table IV), with Ser at position 93
and usage of J15, was the same as that of VSV8-specific CTLs using
the V15 family. For I4-specific CTLs using the V5 family, a
substitution of Ile for Val also did not induce changes of the residue
at position 93 or in the J15 usage, although the N-terminal residue
(Y) of the J15 portion of CDR3 (YQGGRALI) was replaced with a random
residue added at the VJ junction, thus maintaining a predominant
CDR3 length of 10 (Table IV). These findings suggest that both the
CDR3 sequence and the CDR3 length are important in recognizing
the antigenic peptide. Overall, VSV8 peptide variants with a position 4
substitution of Val to Ala or Ile induced a similar CDR3 motif in
the responding T cell population, which is consistent with the finding
that the A4- and I4-specific CTLs were able to cross-react with
VSV8.
Finally, when Tg mice were immunized with VSV8 variants with a
change at position 4 from Val to negatively charged residues (Glu in E4
or Asp in D4), CTL responses could be elicited and, surprisingly, E4-
or D4-specific CTLs showed strong cross-reactivity with VSV8 (Fig. 2).
Although we expected that a ValGlu or Asp substitution at position 4
of VSV8 would change the CDR3 motif (because Glu and Asp have very
different electrostatic properties from Val), we found instead that
TCRs of E4- or D4-specific CTLs had similar CDR3 motifs to those of
VSV8-specific CTLs (Table IV) in that J15 usage was maintained.
However, although position 93 of CDR3 was conserved for a specific
V family, for V5 E4-specific CTLs, Thr was found at position 93
rather than the Ser seen in V5 VSV8-specific CTLs. Also, although
the J15 usage was maintained, two residues (YQ) at the N terminus of
the J region were not present, and the predominant CDR3 loop
length was 9 residues rather than 10. Similarly, when Val at position 4
of VSV8 was substituted with Asp, V1 D4-specific CTLs exhibited Leu
at position 93 instead of the Ser seen there in V1 VSV8-specific
CTLs, one residue (Y) was absent from the N terminus of J15, and the
CDR3 length was changed from 10 to 9 residues. Taken together, the
data showed that the ValAsp/Glu substitution can induce CTLs having
compensatory changes of the CDR3 sequence at position 93 and
deletions at the N terminus of the J region, again suggesting that
position 93 and the N-terminal region of J may interact with
position 4 of the peptide. Moreover, the CDR3 length was altered,
presumably to allow interaction between CDR3 and the substituting
residue at position 4 of the peptide.
Substitution at peptide position 6 to Asp or Glu induces a
compensatory change at position 93 of CDR3
Previous results from Tg mice showed that changes at
position 6 of VSV8 could induce compensatory changes at position 98 of
CDR3, suggesting that position 6 of the peptide specifically
interacts with position 98 (14). From the crystal
structures of TCR/pMHC class I complexes (4, 5, 6, 7, 8, 10, 11),
residues close to the C terminus of the peptide interact with the TCR
-chain and not with the TCR -chain. We would hypothesize that: 1)
a nonconservative substitution for Gln at position 6 of VSV8 would not
induce a CTL response in Tg mice; and 2) even if substitutions of
Gln to certain residues could induce a CTL response, these
substitutions would not induce compensatory changes in the CDR3
loop. To test these hypotheses, Tg mice were immunized with position
6 variants of VSV8. As expected, K6 (GlnLys) and R6 (GlnArg)
peptide variants could not elicit CTL responses (data not shown).
However, we found that D6 (GlnAsp) or E6 (GlnGlu) could induce
very strong CTL responses in Tg mice, and that D6- or E6-induced
CTLs could cross-react with VSV8 (Fig. 2). Like position 4
variant-specific CTLs, we found that in the TCR -chains of D6- or
E6-specific CTLs, position 93 and J usage were highly conserved
(Table V). Surprisingly, we found that
position 93 is compensatorily changed to a positively charged residue
when Gln at position 6 is changed to the negatively charged Asp (D6) or
Glu (E6). These data suggest the possibility of a direct interaction
between CDR3 and position 6 of the peptide. Such an interaction has
not yet been revealed by the limited number of TCR/pMHC class I crystal
structures reported to date (4, 5, 6, 7, 8, 10, 11).
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Our previous data from Tg mice, as well as the crystal
structures of TCR/pMHC complexes, suggested that position 1 of VSV8
might interact with the TCR -chain (14, 33). To
determine whether position 1 of VSV8 interacts with the CDR3 loop,
Tg mice were immunized with VSV8 peptide variants bearing a
substitution at position 1, and the CDR3 loop sequences of these
peptide variant-specific CTLs were examined (Table V). It was found
that all position 1 peptide variants examined (i.e., K1, I1, and E1)
elicited CTLs, all of which cross-reacted with VSV8 (Fig. 2, and data
not shown). CDR3 sequence analysis showed that position 1
substitution of Arg to Lys (K1), Ile (I1), or Glu (E1) did not induce
changes at position 93 or in J usage. Consistent with the reported
crystal structures of TCR/pMHC class I complexes (4, 5, 6, 7, 8, 10, 11), these data indicated that position 1 of the peptide is not
specifically contacted by CDR3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, rather than the -chain, interacts with
position 4 of VSV8 in vivo
In our previous (14, 31, 33) and present
studies, we used Tg and Tg mice as tools to define TCR
interactions with VSV8/H-2Kb. Although VSV8
elicits strong CTL responses in both Tg and Tg mice, a wide
variety of position 4 VSV8 variants elicit strong responses only in
Tg mice (Figs. 1 and 2), suggesting that position 4 of VSV8
interacts with the TCR -chain. Alteration of position 4 of the
peptide apparently disrupts interaction with the Tg chain, rendering
position 4 variants unable to induce CTL responses in Tg mice.
However, in Tg mice, the TCR -chain can vary, so a position 4
peptide variant can induce T cells expressing compatible -chains.
Our finding provides biological evidence that the central residue of
the antigenic peptide primarily contacts the TCR -chain rather than
the -chain. Crystal structures of TCR/pMHC class I complexes are
consistent with this idea, despite the significant differences in
details among the different structures (4, 5, 6, 7, 8, 10).
Position 93 and the J region of CDR3 are important elements
for interaction with position 4 of the peptide
Analysis of CDR3 sequences of TCRs of VSV8-specific CTLs from
Tg mice (Tables II and III) showed that the residue at position 93
and the J region are highly conserved for one specific V family.
Even for different V families, we discovered a predominant J15
usage. Taken together, we conclude that the residue at position 93 and
the J region of the CDR3 loop are critical for recognition of
VSV8. Because in VSV8-specific CTLs derived from Tg mice, only two
conserved residues were found in the CDR3 loop (14),
our current data from Tg mice immunized with VSV8 suggest that a
greater number of CDR3 residues may make critical contacts with the
peptide in vivo, a conclusion not inconsistent with the crystal
structures of certain TCR/peptide-H-2Kb complexes
(6).
Nonconservative substitution of Val at position 4 of VSV8 to Lys, Arg,
or Tyr, which are distinct residues from Val in terms of size and other
characteristics such as hydrophobicity and electropolarity, induces
dramatic conserved changes at position 93 of CDR3 and in J usage
(Table IV), indicating that these elements are important for
interaction with position 4 of the peptide. The negatively charged
residue Glu is present at position 93 in most K4- and R4-specific CTLs,
suggesting that this Glu may interact through a salt bridge with
the positively charged side chain at position 4. In addition, the
change of J usage indicates the importance of this region for
interaction with position 4 of the peptide. Our data indicate that,
unlike the CDR3 loop in which position 98 is mostly involved in the
interaction with position 6 of VSV8, residues at position 93 and in the
J segment of the CDR3 loop may contact peptide position 4,
providing strong biological evidence for the complexity of the
interaction of the CDR3 loop with position 4 of the peptide in vivo.
Crystal structures of TCR/pMHC complexes (4, 5, 6, 7) have
revealed that position 93 and the J region of the CDR3 loop have
extensive contacts with the central residue of the antigenic peptide.
Based on the crystal structure of the 2C TCR complexed with its ligand
(dEV8/H-2Kb), we have modeled the interaction
between the VSV8-specific TCR N15 and the VSV8 peptide (Fig. 3). The model supports the likelihood of
interaction between the J region of CDR3 and position 4 of
VSV8.
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interacts with position 4 of VSV8 and
its variants, we were initially surprised to find that immunization of
Tg mice with the position 4 variants A4 and I4 induces CDR3
sequences that are quite similar to those elicited by VSV8 in terms of
length, the residue at position 93, and the J sequence (Tables II–IV). However, cytotoxicity assays then revealed that A4- and
I4-induced CTLs could cross-react with VSV8 and that VSV8-induced CTLs
could cross-react with A4 and I4 (data not shown). Because Val, Ala,
and Ile all have hydrophobic side chains, we propose that the same
types of interaction (possibly van der Waals) may be involved between
these hydrophobic residues and the CDR3 loop.
When the hydrophobic Val at position 4 of VSV8 was substituted with a
negatively charged residue (Asp or Glu), we found that D4- or
E4-induced CTLs also use the same J15 segment as VSV8-specific CTLs
(Table IV), indicating that Glu or Asp can apparently form similar
contacts with the J region as does VSV8. Thus, common structural
features, rather than the charge, may be the determinant of the
specificity of Glu or Asp interaction with the J region of the
CDR3 loop. Consistent with this is our finding that D4- and
E4-specific CTLs from Tg mice can cross-react with VSV8. Although
the change of Val to Asp or Glu at position 4 of VSV8 does not alter
J usage, it does induce changes at position 93. In previous studies
with Tg mice (14), a substitution of Gln to Glu or Asp
at position 6 of VSV8 induced a change of Val or Thr at position 98 of
CDR3 to the positively charged residue Lys, suggesting that there is
a salt bridge between position 98 of the CDR3 loop and Glu at
position 6 of VSV8. Here we observed that when Val at position 4 of
VSV8 was changed to Glu or Asp, a positively charged residue was not
induced at position 93, indicating the absence of a salt bridge with
position 4 of the peptide variant. Instead, interaction between
position 4 and Thr at position 93 might be mediated by a hydrogen
bond.
The J region is an important determinant of TCR specificity
Our current data reveal the importance of the J region in
recognizing a specific antigenic peptide in vivo. First, we found that
VSV8 and its variants each induce a specific, predominant J usage
(Tables II–V). Second, for particular peptide-specific CTLs, J
usage is even conserved across different V families, whereas
position 93 tends to be conserved only within a particular family
(Tables II and III). Third, substitutions for Val at P4 can trigger
compensatory changes not only at position 93 but also at the J
region by deletion from the N terminus or replacement with a different
J segment (Table IV). Fourth, we found a correlation of J usage
with cross-reactivity of peptide variant-specific CTLs with
VSV8: peptide variant-induced CTLs that could cross-react with
VSV8 used J15. No cross-reactivity was seen when a different J
was used (Tables IV and V). It is noteworthy that even among a panel of
VSV8-specific CTL clones previously derived from standard C57BL/6 mice
(24), a significant proportion (3 of 12) used J15
(referred to as A10 in that earlier report), a striking finding
considering the fact that the murine germline contains >60 different
J gene segments (36).
The crystal structures of TCR/pMHC class I complexes showed that the J
region of CDR3 does not contact the antigenic peptide
(4, 5, 6, 7). Our previous studies of CDR3 loop sequences
from position 6 variant-specific CTLs elicited from Tg mice showed
that J usage is not highly conserved (14), suggesting
that the J region does not specifically interact with the peptide in
vivo either. Instead, CDR3 residues that contact peptide are encoded
by the D segment and N addition. In contrast, structural studies
(4, 5, 6, 7) reveal that the N terminus of J is located at
the apex of the CDR3 loop, enabling the J region to make multiple
contacts with a central peptide residue. Our current data provide
biological evidence for the importance of these J interactions in
determining TCR specificity in vivo. Given this difference between the
TCR - and -chains, it is relevant to note that the murine
germline contains >60 J gene segments (36) but only 14
J gene segments (37, 38).
Changes in CDR3 loop length are induced by substitutions at
position 4 of VSV8
Our previous studies in Tg mice showed that nearly all
sequenced CDR3 loops for a particular position 6 variant-specific
CTL population had an identical length, and that substitution at
position 6 of VSV8 changed the CDR3 loop length (14).
These results suggest that the loop length could be very important for
interactions between CDR3 and position 6 of the peptide, and that
loop length may facilitate the interaction between position 98 of the
CDR3 loop and position 6 of VSV8 or its variants.
Our data in Tg mice now indicate that the length of the CDR3 loop
could also be important for TCR recognition of VSV8 and its variants.
In Tg mice, sequenced CDR3 loops of TCRs of peptide-specific CTLs
have a favored length (Tables II–V). A substitution of Val at position
4 to the large residues Tyr or Lys not only changed the CDR3 loop
sequence, but also shortened the CDR3 loop length. Recently, we have
found that trinitrophenyl-labeled-K4-specific CTLs from Tg mice also
have shorter CDR3 loops than do VSV8-specific TCR -chains
(39). One possible explanation is that a CDR3
loop of shorter length may create a bigger cavity at the TCR and
interface, allowing bulky side chains or hapten groups to be
accommodated.
The TCR -chain may contact position 6 of VSV8 under certain
circumstances
Using Tg mice, we showed previously that interaction between
position 98 of CDR3 and peptide position 6 is critical for
recognition of VSV8 and its variants (14). Thus, we
predicted that K6 and R6 peptides would be unable to induce CTL
responses in Tg mice, as substitution of Gln at position 6 to Lys or
Arg would likely abolish this critical contact with the Tg chain. We
found that this was indeed the case, a result compatible with the
finding that VSV8-specific CTLs, including N30.7, are generally unable
to recognize position 6 variants of VSV8 (22, 31, 33).
However, we were surprised to find that both D6 and E6 could elicit
strong CTL responses in Tg mice, and that the residue at position 93
of CDR3 was changed from a Ser to a positively charged residue
(Table V), suggesting the possibility of an interaction between
position 6 of the peptide and the TCR -chain in the special
situation where the -chain may be somewhat incompatible with the
residue at position 6. One possible explanation is the following:
Because Gln, Asp, and Glu have similar structures, Asp/Glu can still
interact with the Tg chain CDR3, but with weaker affinity.
Therefore, the residue at position 93 of CDR3 is reciprocally
changed to Lys/Arg to enhance the binding of position 6 of the peptide
to the TCR. This hypothesis is supported by the crystallographic
finding that the VSV8/H-2Kb and
E6/H-2Kb complexes are very similar in structure
(30). Further support for the idea that the TCR -chain
can interact with position 6 in certain circumstances comes from
our finding that immunization of Tg mice with a VSV8 variant in
which Gln at position 6 is changed to trinitrophenyl-labeled-Lys can
induce alterations both at position 93 and in J usage
(39).
An analogous interaction between the TCR -chain and a residue toward
the C terminus of the peptide has not yet been seen in the limited
number of TCR/pMHC class I crystal structures published to date
(4, 5, 6, 7, 8, 10, 11). However, the extraordinary structure of
the BM3.3 TCR complexed with pBM1/H-2Kb, in which
the CDR3 loop bends away from the MHC peptide-binding groove and
makes absolutely no contacts with the peptide (11), is an
indication that much remains to be discerned, using a combination of
x-ray crystallography and various biological strategies, regarding the
possibilities for interaction between the TCR and its ligand.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
3 Current address: Department of Immunology, Tsukuba University, 1-1-1 Tennodai, Tsukuba City, Ibaraki, Japan 305-8575.
4 Current address: Infrastructure Services, Department of Engineering, InfoSpace Inc., Bellevue, WA 98004.
5 Current address: Laboratory of Molecular Genetics and Immunology, Rockefeller University, 1230 York Avenue, Box 98, New York, NY 10021.
6 Address correspondence and reprint requests to Dr. Stanley G. Nathenson, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: nathenso{at}aecom.yu.edu
7 Abbreviations used in this paper: CDR, complementarity-determining region; pMHC, peptide-MHC; Tg, TCR -chain transgenic; Tg, TCR -chain transgenic; VSV, vesicular stomatitis virus.
Received for publication May 14, 2001. Accepted for publication August 14, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I/peptide complex over a broad interface area. Mol. Immunol. 35:593.[Medline]
chain gene rearrangement. Proc. Natl. Acad. Sci. USA 95:12538. repertoire of the responding CD8+ cytotoxic lymphocyte population. J. Immunol. 162:7263. T cell receptor (TCR) heterodimer in complex with an anti-TCR Fab fragment derived from a mitogenic antibody. EMBO J. 17:10.[Medline]
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) the immunizing peptide (0.1 µM) as
targets.
