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Departments of
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Microbiology and Immunology and
Cell Biology, 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 from a VSV
peptide-specific CD8+ T cell clone. These mice showed an
even more restricted TCR Vß usage consequent to peptide immunization.
However, in both C57BL/6 and TCR transgenic mice, single amino acid
replacements in TCR-contact residues of the VSV peptide could alter the
TCR Vß usage of the responding CD8+ T lymphocytes. These
results provide in vivo evidence for an interaction between the
antigenic peptide and the germline-encoded complementarity-determining
region-ß loops that can influence the selection of the responding TCR
repertoire. Furthermore, only replacements at residues near the C
terminus of the peptide were able to alter the TCR Vß usage, which is
consistent with the notion that the TCR ß-chain interacts in vivo
preferentially with this region of the MHC/peptide
complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chains (4, 5). The variable
regions of the TCR - and ß-chains are encoded by V/J and V/D/J
transcriptional elements, respectively (4, 5). These genetic elements
are rearranged as a result of somatic recombination in the thymus
during early steps of T cell ontogeny (5).
As is found for Abs, the portions of the TCR variable region predicted
to interact with the Ag have been called complementarity-determining
regions (CDRs)5 (5, 6). While
the CDR1 and CDR2 are germline encoded by each variable gene segment,
the CDR3 results from the V(D)J junction. Since the CDR3 shows the
highest diversity within the TCR structure, it was initially proposed
that this portion of the TCR would interact with the antigenic peptide,
while the less diverse CDR1 and CDR2 would contact the MHC -helices
(7). However, the recently solved crystal structures of three MHC class
I-restricted TCRs complexed with their cognate ligands have revealed
that in addition to the CDR3, the CDR1 could also interact with the
peptide (8, 9, 10, 11). In these structures, the CDR3 of the - and
ß-chains come in close apposition, interfacing the center of the
peptide, while the CDR1 of the - and ß-chains contact residues of
the antigenic peptide near the N and C termini, respectively (8, 9, 10, 11).
It is interesting that all the structures show the same overall
orientation, in which the TCR interfaces the MHC class I/peptide
complex in a diagonal orientation parallel to the ß sheets of the MHC
Ag binding groove, with the - and ß-chains flanking the amino and
carboxyl termini of the peptide, respectively (8, 9, 10, 11, 12). This
diagonal orientation revealed by the crystal structures agrees with
that previously predicted by in vivo studies performed with CTL clones
and transgenic mice (13, 14).
However, despite structural and biochemical data addressing the role that the different regions of the TCR play in the interaction with the MHC/peptide complex, still largely unknown are the structural features that determine which members of the highly diverse TCR repertoire are expanded during the onset of a T cell response triggered by a particular Ag. In this regard, it has been observed in a variety of systems that the T cell response to an Ag is biased toward the usage of a limited set of TCR variable gene families (15, 16, 17, 18). These observations have revealed that the T cell responses are preferentially oligoclonal, involving the activation of a restricted set of T cell clones (17). This oligoclonality is presumably determined by the structure of the antigenic peptide and its interaction with the responding TCR molecules.
To study the role that the structure of the antigenic peptide plays in the expansion of the responding T cells during an immune response, we analyzed the effect that single amino acid replacements in the sequence of an antigenic peptide have on the repertoire of activated T cells. As a system, we have used an eight-amino acid-long peptide corresponding to residues 52–59 from the nucleocapsid of the vesicular stomatitis virus (VSV) (19). The VSV peptide binds to H-2Kb, triggering a strong immune response mediated by cytotoxic CD8+ T cells. Previous analysis of VSV-specific CTL clones revealed the preferential usage of the Vß13 and Vß8 elements (20). Functional studies and analysis of the crystal structure of the H-2Kb/VSV complex have allowed the potential TCR-contact residues of the VSV peptide to be identified (21, 22, 23). In the present work, we asked how single amino acid replacements in TCR-exposed positions of the VSV peptide would affect the TCR repertoire of the responding CD8+ T cell population.
When C57BL/6 (B6) mice were immunized with the VSV peptide, the
predominant TCR Vß element used by the responding CD8+ T
cell population was Vß13. Immunization of these mice with certain
singly substituted VSV-derived peptide variants caused a change in the
predominant TCR Vß usage. To focus our study on changes in the TCR
ß-chain resulting from alterations in the sequence of the VSV peptide
independently of the TCR -chain, we created a transgenic mouse that
expressed exclusively the TCR -chain of a VSV-specific,
H-2Kb-restricted T cell clone (N30.7, V2/Vß13) (20, 24). When the transgenic mice were immunized with the VSV peptide,
Vß13 was again the predominant Vß element present in VSV-specific
CTL populations. However, the TCR Vß usage was profoundly altered
when these transgenic mice were immunized with peptides carrying single
replacements near the C terminus of VSV. Peptides with replacements at
the N terminus of VSV were able to induce a strong cytotoxic response
but did not alter the TCR Vß usage. Our results are discussed with
regard to the orientation of the TCR/ligand interaction in vivo and the
effects that point mutations in the epitope sequence can have on the
nature of responding cytotoxic T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wild-type VSV peptide (RGYVYQGL) and singly substituted variants were synthesized by a solid-phase method using Fmoc chemistry on an automated 433A peptide synthesizer (Applied Biosystems, Foster City, CA) at the Peptide Synthesis Facility of the Albert Einstein College of Medicine. All peptides were purified to >98% homogeneity by reversed-phase HPLC on a Vydac C-18 column (2.1 or 4.6 mm x 25 cm, 300 Å) using HP-1090M HPLC (Hewlett Packard). The identity of the purified peptides was determined by a tandem quadrupole mass spectrometer (TSQ700, Finnigan MAT, San Jose, CA).
Animals and immunizations
B6 mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). The N30.7 V2 transgenic mice were designed as described
previously by Wang et al. (24). These mice carry the TCR -chain of
the H-2Kb/VSV-specific CTL clone N30.7 (20) as a transgene
in a TCR-/- background. All animals were maintained
under approved institutional guidelines in the Animal Institute of the
Albert Einstein College of Medicine.
Mice were immunized by injecting 15 µg of peptide emulsified in CFA in the hind footpads. Seven days later, animals were boosted with 15 µg of peptide emulsified in IFA. One week after the booster, animals were sacrificed and spleens removed for culture and generation of peptide-specific CTL populations.
Culture of peptide-specific CTL populations and cytotoxicity assays
Splenic cells (5 x 107) were cultured for 7
days in 10 ml of IMDM, supplemented with 2 mM glutamine, 50 U/ml
penicillin, 50 µg/ml streptomycin, 50 µM 2-ME, and 10%
heat-inactivated FCS (HyClone, Logan, UT), in the presence of 1 µM of
the peptide used for immunization. At day 7, cells were restimulated
with the same peptide (1 µM) presented by mitomycin C-treated
syngeneic B6 splenic cells. Cultures were supplemented with 50 U/ml
rhIL-2. At day 14 of culture, cells were harvested for cytotoxic assays
and Vß family usage analyses. This protocol was followed for
generation of peptide-specific CTL populations from both wild-type B6
and N30.7 V2 transgenic mice.
Peptide-specific cytotoxicity mediated by CTL populations was determined by 51Cr release assay. RMA/s cells were incubated for 1 h at 37°C with 1.85 MBq of Na251CrO4 and 1 µM of specific peptide. These target cells (3 x 103) were mixed with CTL effector cells in E:T ratios ranging from 30 to 50. After a 4-h incubation at 37°C and 8% CO2 in air, supernatants were collected and the amount of 51Cr released was measured in a gamma counter. The percentage of specific lysis was determined as 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). Experimental release is the radioactivity in the supernatant of target cells mixed with CTLs, spontaneous release is that in the supernatant of target cells incubated alone, and maximum release is that in the supernatant after complete lysis of target cells with 1% Triton X-100 in PBS.
Preparation of the H-2Kb/VSV tetramers and binding to VSV-specific CD8+ CTL populations
The detailed procedure for preparation and characterization of H-2Kb/VSV tetramers will be described elsewhere (A. M. Kalergis, E. Palmieri, E. C. Goyarts, S. Honda, and S. G. Nathenson, manuscript in preparation). Briefly, using a form of the H-2Kb molecule carrying a single unpaired cysteine residue at the C terminus, H-2Kb/ß2-microglobulin (ß2m)/VSV complexes were obtained from the folding of rß2m and H-2Kbcys in the presence of VSV peptide as previously described (21). After purification by gel filtration (G-75, Pharmacia), H-2Kb/ß2m/VSV complexes were treated with 1-biotinamido-4- (4'-(maleimidomethyl)-cyclohexanecarboxamido)-butane (Pierce, Rockford, IL), a biotinylating reagent specific for free –SH groups. This procedure ensures the incorporation of biotin only at the free cysteine residue engineered at the C terminus of H-2Kb. Tetramerization of the biotinylated H-2Kb/VSV complexes was obtained by incubation with streptavidin-PE at a molar ratio equal to 8:1, ensuring the saturation of the four biotin-binding sites of streptavidin. H-2Kb/VSV tetramers were purified from the excess of H-2Kb/VSV monomers by gel filtration in a G-200 column (Pharmacia).
VSV-specific CD8+ CTL populations from B6 and N30.7 V2
transgenic mice were double stained with 0.5 µg of
H-2Kb/VSV tetramers-PE and 0.5 µg of anti-CD8-FITC
mAb (PharMingen, San Diego, CA) for 1 h in the presence of 0.1%
BSA (fraction V, Sigma, St. Louis, MO). Cells were washed three times
with 0.1% PBS-BSA and immediately analyzed using FACScan (Becton
Dickinson, Mountain View, CA). Splenocytes from naive B6 and N30.7
V2 transgenic mice were included as controls in all of these
experiments.
Flow cytometric analysis of TCR Vß usage in peptide-specific CD8+ CTL populations
At day 14 of culture, 1 x 105 cells from the
peptide-specific CTL populations were double stained for CD8 and for
TCR Vß with a panel of FITC-conjugated Abs specific for different TCR
Vß families. Those Abs included: anti-Vß2 (B20.6),
anti-Vß4 (KT4), anti-Vß5 (MR9-4), anti-Vß6 (RR4-7),
anti-Vß7 (TR310), anti-Vß8.1/8.2 (MR5-2), anti-Vß8.3
(1B3.3), anti-Vß9 (MR10-2), anti-Vß10 (B21.5),
anti-Vß11 (RR3-15), anti-Vß12 (MR11-1), anti-Vß13
(MR12-3), and anti-Vß14 (14-2). All of the Abs were purchased
from PharMingen. Cells were incubated for 30 min on ice with 0.1 µg
of anti-CD8-PE and 0.1 µg of the specific anti-Vß mAb in
the presence of 0.1% BSA (fraction V, Sigma). After incubations, cells
were washed three times with 0.1% PBS-BSA and immediately analyzed
using FACScan (Becton Dickinson). Data are reported as the fraction of
total CD8+ cells expressing a particular Vß family.
Statistical analyses were performed using the Student t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously observed that mice mount a cytotoxic immune
response after immunization with peptides of the proper length and
sequence for MHC class I binding (25, 26). In this study, immunization
of B6 mice with the VSV peptide (Fig. 1
),
which binds to H-2Kb, triggered a strong peptide-specific
cytotoxic response (Fig. 2A).
The responding CD8+ CTL populations were strongly biased to
the participation of T cells that showed the preferential usage of the
Vß13 and Vß8.1/8.2 (8.1 or 8.2) elements (Table I). These Vß families represented up to
50% of the total CD8+ CTL population, with the Vß13 gene
element being always predominant and significantly expanded as compared
with unimmunized mice (p < 0.001) (Table I).
No significant expansion of any of the other Vß families tested was
consistently observed after immunization with VSV peptide (Table I).
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B, the majority of
CD8+ T cells responding to VSV peptide bound to
H-2Kb/VSV tetramers, indicating that this MHC/peptide
complex is the specific ligand for those CD8+ lymphocytes.
As control, splenocytes obtained from naive B6 mice were also stained
with H-2Kb/VSV tetramers and anti-CD8 mAb; however,
CD8+ T cells from these naive animals did not bind
H-2Kb/VSV tetramers (Fig. 3A).
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The H-2Kb/VSV crystal structure shows that positions 1 (N terminus) and 6 (toward the C terminus) of the VSV peptide are solvent exposed and available to contact the TCR (21, 22). Functional studies from our laboratory have suggested proximity between the TCR ß-chain and position 6 of the VSV peptide (24, 28). We therefore evaluated whether single amino acid replacements at position 6 of the VSV peptide could alter the TCR Vß repertoire of the responding CD8+ CTL populations.
Like the VSV peptide, VSV-derived peptide variants carrying single
substitutions at position 6 (Fig. 1
) were able to elicit cytotoxic
responses in B6 mice (Fig. 2A). However, the TCR Vß usage
of the CD8+ T cells expanded by immunization with position
6 peptide variants was significantly altered compared with the Vß
usage of anti-VSV CTL populations. For example, in the case of the
M6 peptide, in which the glutamine residue at position 6 was replaced
by methionine, CD8+ CTL populations were induced that
showed a usage of Vß13 similar to those induced by VSV, but M6 also
induced abundant Vß11+ CD8+ T cells (Fig. 4A). The Vß11 element was
expressed by 20% of the M6-induced CTLs, a frequency significantly
higher than the 6% of Vß11+ cells present in the
VSV-induced CTL populations (p < 0.0001).
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A). The Vß8.1/8.2
usage of these cells (24%) was significantly higher than that observed
in VSV-specific CD8+ T cells (p <
0.0086). The Vß13 element was practically absent in K6-specific CTL
populations from these mice (5.7% compared with the 25.2% observed in
VSV-specific CTLs, p < 0.0002).
Two other peptides carrying replacements at position 6 of VSV were used
for immunization of B6 mice; these were E6 and I6, in which glutamine 6
was replaced by glutamic acid or isoleucine, respectively. The
preferential TCR Vß usage observed in E6-specific CD8+
CTL populations was Vß8.1/8.2 (14%, Fig. 4A). The Vß13
element (7.6%) was significantly reduced when compared with
VSV-specific CD8+ CTLs (25.2%, p <
0.005). In the case of I6 peptide-specific CD8+ CTL
populations from B6 mice, the frequency of the Vß13 gene element was
also decreased (4.3%, p < 0.01) (Fig. 4A).
Single amino acid replacements at the N terminus of the VSV peptide do not alter the TCR Vß repertoire of the responding peptide-specific CD8+ CTL populations in B6 mice
Two replacements were made at position 1 of VSV, where the
wild-type arginine residue was either changed to lysine (a conservative
change) or isoleucine (a nonconservative change). These K1 and I1
peptide variants (Fig. 1) were able to trigger peptide-specific
cytotoxic responses in B6 mice similar to that observed for the VSV
wild-type peptide (Fig. 2A). When the TCR Vß usage was
analyzed, the CD8+ CTL populations specific for these two
peptides showed a pattern similar to that observed in VSV-induced
CD8+ CTL populations (Fig. 4B). Thus, the
predominant Vß elements used by K1- or I1-induced CD8+
CTL populations were Vß13 and Vß8.1/8.2. In the case of K1-induced
CTL populations, there appeared to be a decrease in Vß13 usage when
compared with VSV-induced CTL populations, but this difference was not
statistically significant.
Immunization of N30.7 V2 transgenic mice with VSV peptide
triggers a CD8+ T cell-mediated cytotoxic response biased
to the contribution of a single TCR Vß element
To focus our study only on the repertoire of TCR ß-chains
independently of variations in the TCR -chain, we created a
transgenic mouse expressing the TCR -chain of the
H-2Kb/VSV-specific CTL clone N30.7, in an endogenous
TCR-deficient background. T cells obtained from these mice express
only the transgenic TCR -chain (V2, Fig. 5), since they carry a mutation in the
endogenous C gene (24, 29). When N30.7 TCR transgenic mice were
immunized with the VSV peptide, a strong, specific cytotoxic response
was obtained (Fig. 2B). In these mice, Vß13+
CD8+ cytotoxic T cells represented up to 70% of the VSV
peptide-specific CTL population (Table I and Fig. 6A). Neither Vß8.1/8.2 nor
any other of the tested Vß elements were observed to predominate in
VSV-specific CTL populations from these mice (Table I).
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2 transgenic mice bound H-2Kb/VSV tetramers
(Fig. 3D). Thus, those CD8+ lymphocytes are
specific for the H-2Kb/VSV complex. Naive CD8+
T cells from spleens of unimmunized N30.7 V2 transgenic mice did not
bind H-2Kb/VSV tetramers (Fig. 3C).
Single amino acid replacements toward the C terminus of VSV peptide
cause an alteration in the TCR Vß repertoire of the responding
peptide-specific CD8+ CTL populations in N30.7 V2
transgenic mice
As was observed in B6 mice, immunization of N30.7 V2 transgenic
mice with peptide variants carrying certain replacements at position 6
of the VSV peptide also resulted in significant alterations of the TCR
Vß usage. R6-specific CD8+ CTL populations induced in
N30.7 V2 transgenic mice show two major TCR ß-chain families (Fig. 6A): Vß11 (48.7%) and Vß13 (38.2%). These frequencies
differ significantly from those observed in VSV-specific CTL
populations (Vß11, 1.8%, and Vß13, 69%; p <
0.001 in each case), indicating that this single amino acid replacement
in the peptide favors the expansion of Vß11+ T cells. In
contrast, the K6 and I6 peptides maintained Vß13 as the major Vß
element present in the responding CD8+ CTL populations
(Fig. 6A). This result contrasts with the observed reduction
in Vß13 usage in B6 mice immunized with those peptides (Fig. 4A; see Discussion).
As seen in Fig. 6A, a negative charge in position 6 of the
VSV peptide induced the expansion of Vß7+
CD8+ T cells. Thus, D6 peptide-specific CTL populations
show Vß7 (27.5%) and Vß13 (24.6%) as the two major Vß elements
involved in D6 recognition. These values differ significantly from
VSV-induced CTL populations, in which Vß7 is expressed in only 5% of
the CD8+ T cells (p < 0.001).
Furthermore, E6 peptide-induced CD8+ CTL populations show
Vß7 as the only significant Vß element (73%), contrasting the low
frequency of this Vß family in VSV-induced CTL populations (5%,
p < 0.0001).
Single amino acid replacements at the N terminus of VSV peptide do
not alter the TCR Vß repertoire of the responding peptide-specific
CD8+ CTL populations in N30.7 V2 transgenic mice
N30.7 TCR transgenic mice mounted a strong cytotoxic response
to the K1 and I1 peptides (Fig. 2B) and showed Vß13 as the
predominant Vß element present in the peptide-specific
CD8+ CTL populations (Fig. 6B). None of the
other Vß elements tested were significantly abundant in these
peptide-specific CTL populations. Thus, while peptides carrying single
substitutions at position 1 of the VSV peptide were able to trigger a
cytotoxic immune response in both wild-type B6 and N30.7 V2
transgenic mice, they did not significantly alter the TCR Vß usage in
CD8+ CTL populations expanded after peptide immunization.
VSV peptide variants that alter the TCR Vß repertoire are not recognized by VSV-specific CTL populations
Since certain VSV peptide variants carrying mutations at position
6 of the VSV peptide abolished the predominance of Vß13 in the
responding CD8+ T cell populations, it might be expected
that those peptide variants would not be recognized by VSV-specific CTL
populations that are enriched in Vß13 TCRs. Indeed, VSV-specific CTL
populations from B6 mice cross-reacted only with those peptide variants
that did not alter the Vß usage (Table II), namely, K1, I1, and M6 peptides.
These three peptides induced an equivalent expansion of
Vß13+ CD8+ T lymphocytes upon immunization as
did the wild-type VSV peptide (Fig. 4, A and B).
These results support a correlation between TCR Vß usage and the Ag
specificity of CTL populations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported previously that T cell responses to specific Ags
can be biased, as shown by the expansion of a limited number of TCR
V and Vß elements (15, 16, 17, 18). We report here that the
H-2Kb-restricted cytotoxic T cell response to the VSV
peptide in B6 mice is mediated mainly by CD8+ T cells
expressing Vß13 or Vß8.1/8.2 TCR molecules (Table I, Fig. 4A). That H-2Kb/VSV is the specific ligand for
those CD8+ T cells is demonstrated by the fact that they
bind to H-2Kb/VSV tetramers (Fig. 3). We have also found
that single amino acid substitutions at TCR-contact residues of the VSV
peptide can alter the TCR Vß usage of a peptide-specific CTL
population. Furthermore, only substitutions near the C terminus of the
peptide altered the Vß usage, a finding consistent with the notion of
an interaction between this region of the peptide and the TCR Vß
domain (Fig. 4A). However, it is possible that the point
mutation in the peptide could affect the V usage in the B6 mice and
that the observed alteration in the Vß usage could be a secondary
effect due to VVß pairing restrictions. This could be a feasible
alternative, especially in view of previously reported restrictions on
the pairing of specific TCR V and Vß elements (30, 31, 32, 33).
To evaluate whether the effect of the peptide mutation on the TCR Vß
usage is a direct or indirect effect on the ß-chain, we fixed the TCR
-chain as a transgene, allowing the analysis of TCR ß-chains
independently of the TCR V usage. With this aim, a transgenic mouse
was engineered expressing the TCR -chain from a VSV-specific clone
(N30.7) in an endogenous C-deficient background (24, 29). Although
these mice express only the transgenic TCR -chain and no endogenous
-chains (Fig. 5), they potentially can express any of the endogenous
TCR Vß elements (Table I). When these mice were immunized with the
VSV peptide, only Vß13 TCRs were significantly expanded. In agreement
with what was observed in the B6 mice, only amino acid replacements
near the C terminus of the VSV peptide altered the Vß usage in
peptide-immunized N30.7 V2 transgenic mice (Fig. 6A).
Since these mice express the transgenic TCR -chain in the absence of
any endogenous -chain, the alterations of the TCR Vß usage
resulting from replacements near the C terminus of VSV peptide are not
an indirect effect due to alterations of the TCR -chain, but are a
direct effect on the TCR ß-chain. Such data are consistent with an
orientation in which the TCR ß-chain is interacting in vivo with the
C-terminal region of the VSV peptide; furthermore, since the observed
alterations in the TCR Vß usage affected entire CTL populations, it
is highly probable that this TCR/MHC-peptide orientation is common for
most of the T cell clones involved in the recognition of
Kb/VSV complex.
Despite the similarities between B6 and TCR transgenic mice in the
response to Kb/VSV, some important differences were
observed after peptide immunization. It is noteworthy that Vß8.1/8.2,
Vß elements that were abundant in VSV-specific CTL populations
derived from B6 mice, were significantly reduced in VSV-specific CTL
populations derived from N30.7 V2 transgenic mice. Moreover, in
these transgenic mice there was no significant Vß8.1/8.2 usage after
immunization with any of the peptides tested. In contrast, these TCR
Vß elements were abundant in most of the peptide-specific CTL
populations derived from B6 mice and even predominant in the case of
the K6 peptide. Furthermore, TCR transgenic mice responded to K6 and
I6 peptides by expanding Vß13 TCRs, while B6 mice responded to the
same peptides by preferentially expanding Vß8.1/8.2 TCRs, with no
significant Vß13 expansion. These observations could be the result of
structural restrictions imposed by the transgenic TCR -chain on the
universe of Vß elements with which it can pair to produce a
functional TCRß heterodimer able to recognize
H-2Kb/VSV. VVß pairing restrictions have been observed
previously in other systems in which structural constraints on TCR
formation could limit the association between different TCR V and
Vß elements (30, 31, 32, 33).
The molecular basis for the changes of the TCR Vß usage in response to small alterations in the antigenic peptide sequence described here is currently unknown. However, one or more of the following three explanations could account for this phenomenon: 1) a direct contact between the CDR1 of the TCR ß-chain and position 6 of the VSV peptide, 2) the requirement for particular CDR1 and/or CDR2 structures to position a given CDR3 sequence during recognition of the VSV peptide, or 3) a conformational change in the structure of the TCR binding domain of the MHC due to the single mutation in the peptide.
The first explanation implies a direct contact between the CDR1 of the TCR ß-chain and position 6 of the VSV peptide. Thus, single replacements at that position of the peptide could impose restrictions on the universe of TCR Vß elements that would be expanded upon peptide immunization. This hypothesis suggests a role for CDR1ß in recognition of the peptide bound to the MHC, a notion supported by the recently reported high-resolution crystal structures of TCRs complexed with their cognate ligands. In both human and mouse systems, the CDR1 of the TCR ß-chain interacts with residues toward the C terminus of the antigenic peptide bound to the MHC groove (8, 9, 10). In fact, in the 2C/H-2Kb-dEV8 structure reported by Garcia et al., 2C-CDR1ß makes 6 contacts with position 6 of the dEV8 peptide as compared with CDR3ß, which makes only one (10). In addition, site-directed mutagenesis studies have provided evidence for a role of CDR1ß loops in recognition of MHC class I-peptide complexes (34, 35), although those experiments could not determine whether the CDR1ß loop was interacting with residues of the peptide or of the MHC. Thus, a direct interaction between the CDR1ß loop and the antigenic peptide is a feasible explanation for the changes in Vß usage reported here.
Alternatively, the second possibility is that particular CDR1 and CDR2 structures would be required to accommodate or position a CDR3 loop of a specific sequence during Ag recognition. We have recently reported that single replacements in P6 of the VSV peptide induced charge-compensatory changes at position 98 of the TCR CDR3ß loop (CDR3ß98) (24). While negatively charged residues at P6 expanded TCRs with a positive charge at CDR3ß98, the opposite finding was observed with positively charged residues at P6 of VSV (24). This requirement for certain CDR3 sequences could limit the germline-encoded CDR1 and CDR2 loop structures that would allow Ag recognition. Further support for this view comes from the observation that in hybridomas recognizing MHC class II/peptide complexes, the use of a particular Vß gene segment correlated with the presence of a given CDR3ß sequence motif (36).
Finally, the third explanation suggests that a single change in the
peptide sequence could induce a conformational change in the MHC
structure that would alter its binding domain for TCRs. Such an
alteration could then be responsible for the expansion of CTLs carrying
TCRs with different variable gene elements. To explore this possibility
for the E6 peptide, which most profoundly alters Vß usage in the TCR
-chain transgenic mice (Fig. 6A), we have recently solved
the structure for the H-2Kb/E6-peptide complex and compared
it with the H-2Kb/VSV structure (C. T. Thomson,
A. M. Kalergis, S. G. Nathenson, and J. C. Sacchettini,
manuscript in preparation). The only conformational changes observed
between these two MHC/peptide complexes were localized to the peptide.
Therefore, in the case of the E6 peptide, the alteration in Vß usage
is not due to conformational changes in MHC residues. However, in two
previous crystallographic studies, peptide variants carrying single
substitutions at TCR-contact residues were observed to induce changes
in the conformation of at least one MHC residue (37, 38). These two
studies raise the possibility that for peptides other than E6,
conformational changes in MHC residues could be involved in causing the
changes in the TCR Vß gene usage observed as a result of single
substitutions at position 6 of the VSV peptide.
When considered together with previous observations made in MHC class II-restricted responses (36, 39, 40), our results reveal that alterations in the TCR repertoire of the responding T cell population due to minor changes in the Ag are a general and biologically significant phenomenon affecting both CD4+ and CD8+ compartments. Previous studies performed in CD4+ T cell-mediated responses specific for antigenic peptides bound to MHC class II molecules have shown that replacements at positions toward the C terminus of the peptide can alter the TCR Vß usage of the responding CD4+ T cells (36, 39, 40). By studying entire peptide-specific CD8+ cytotoxic T cell populations, we have observed that replacements at TCR-contact residues of the antigenic peptide can alter the TCR repertoire, supporting the notion that this is a common feature shared by both MHC class I- and II-restricted immune responses.
We demonstrate here that cytotoxic immune responses are particularly sensitive to minor epitope alterations. It is striking that only a single replacement in a TCR-contact residue of the VSV peptide can completely modify the TCR repertoire of the responding CTL population. In addition, those peptide variants that altered the TCR repertoire are not recognized by VSV-wild-type peptide-specific CTL populations. Such a phenomenon could be relevant for understanding the strategies used by pathogens to evade the immune system. It is well known that antigenic variation is a common strategy employed by a number of different microorganisms, including viruses, bacteria, and protozoans to escape recognition by immune cells (41). Our findings show that a change in a single amino acid residue of an immunodominant T cell epitope would require the host organism to generate an entirely new immune T cell response.
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Footnotes |
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2 Current address: Department of Parasitology and Immunology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan.
3 Current address: Corixa Corporation, 1124 Columbia Street, Suite 225, Seattle, WA 98104.
4 Address correspondence and reprint requests to: Dr. Stanley G. Nathenson, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461. E-mail address:
5 Abbreviations used in this paper: CDR, complementarity-determining region; VSV, vesicular stomatitis virus; B6, C57BL/6; ß2m, ß2-microglobulin.
Received for publication January 21, 1999. Accepted for publication March 29, 1999.
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, p < 0.001
when compared with VSV peptide). In K6-specific CTL populations,
Vß8.1/8.2 was increased as compared with VSV peptide (*,
p < 0.0086). B, Peptide variants
carrying mutations at P1 (N terminus of VSV) do not significantly alter
the TCR Vß usage of peptide-specific CTL populations. Groups of five
mice were immunized with each peptide. Each bar represents the average
of three to five independent experiments.
