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*
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and
Department of Neurology, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
-ß since the clones had the same V-J and Vß-Jß
rearrangements. The two clones had a similar fine specificity for the
MBP peptide, except for the P5 position of the peptide (lysine). In the
crystal structure of the HLA-DR2/MBP peptide complex, P5 lysine is a
prominent, solvent-exposed residue in the center of the DR2/MBP peptide
surface. Five microbial peptides with conservative or nonconservative
changes at the P5 position (lysine to arginine, serine, or proline)
activated one of these clones. In contrast, the other clone was
activated only by three of these peptides which had a conservative
lysine to arginine change at P5. The degree of specificity/degeneracy
in recognition of the P5 side chain was the key difference between
these TCRs since the Escherichia coli/Haemophilus
influenzae peptide stimulated both clones when the P5 position
was substituted from serine to arginine. These results demonstrate that
the complementarity-determining region 3 loops contribute to the degree
of degeneracy in peptide recognition by human MBP-specific
TCRs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies demonstrated that microbial peptides that have limited primary sequence homology to the self-peptide can activate myelin basic protein (MBP)4-specific T cell clones from multiple sclerosis patients (3, 7). Recognition of such diverse peptide sequences is due to the degenerate MHC class II peptide-binding motifs as well as a certain degree of "flexibility" in TCR recognition of MHC-bound peptides (3, 8). While the structural basis for degenerate peptide binding by MHC class II molecules has been well defined (9, 10, 11), the basis for the observed peptide cross-reactivity by human TCRs is only beginning to be understood (12, 13). In the crystal structure of a HLA-A2/tax peptide/TCR complex, approximately one-third of the TCR contact surface (326 Å2 of 998 Å2 of total MHC/peptide contact surface) covered the peptide, which was deeply buried in the MHC class I peptide binding. The TCR contact surface was relatively flat, except for a deep pocket that was occupied by the P5 side chain of the peptide (tyrosine in tax(11, 12, 13, 14, 15, 16, 17, 18, 19)) (12, 14).
Structural features of TCRs that contribute to the recognition of
diverse peptide sequences were examined using two human MBP-specific T
cell clones that differed only in the sequences of the
complementarity-determining 3 (CDR3) regions of and ß. Analysis
of a large panel of single-amino acid analogue peptides indicated that
the clones had a similar fine specificity for the MBP(85–99) peptide,
except for lysine 93 (P5 position). The T cell clone that had a broader
specificity for the P5 side chain was activated by five different
microbial peptides which had conservative (arginine) or nonconservative
(serine, proline) substitutions of lysine 93. The other T cell clone
was activated only by those peptides that had a conservative lysine to
arginine substitution at P5. These results indicate that sequence
differences in the TCR CDR3 loops can determine the degree of
cross-reactivity between a self-peptide and diverse microbial peptides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The L243 hybridoma was obtained from the American Type Culture Collection (ATCC HB55).
Database search
The GenPept protein database was searched for viral and bacterial sequences using the program "stringsearch." The generated sequence files were searched with the T cell recognition motif using the program "findpatterns" of the Genetics Computer Group software (University of Wisconsin, Madison, WI).
Peptide synthesis
Single-amino acid analogue peptides of MBP(85–101) were synthesized on pins at a 1-mg scale by Chiron Mimotopes, San Diego, CA. Peptides M48–M66 and S1–S52 were synthesized by Quality Controlled Biochemicals, Hopkinton, MA. These peptides were subjected to quality control by reverse-phase HPLC and mass spectrometry. Peptides were dissolved in water or 40% acetonitrile, 100 mM HEPES, pH 7.4. Previous experiments had demonstrated that addition of acetonitrile to a final concentration of 1% did not affect T cell proliferation.
T cell proliferation assays
Human MBP(85–99)-specific T cell clones (15, 16) were maintained by weekly restimulation with 1 µg/ml PHA (Murex Diagnostics, Norcross, GA) in RPMI supplemented with 10% human serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM glutamine, and 5 U/ml recombinant human IL-2 (Boehringer Mannheim, Indianapolis, IN) using irradiated human PBMC as feeder cells. Analysis of a panel of MBP analogue peptides indicated that the T cell clones maintained their fine specificity for several years under these culture conditions.
T cell proliferation assays were set up in triplicates in 96-well U-bottom plates with 5 x 104 T cells and 5 x 104 APCs per well in serum free AIM-V medium (Life Technologies, Gaithersburg, MD). An EBV-transformed B cell line homozygous for the HLA-DR2 haplotype (MGAR, DRB1*1501) was used as APCs; these cells were treated with mitomycin C (50 µg/ml, 20 min), washed, and irradiated (6000 rad). Peptides were added to triplicate cultures at concentrations ranging from 5 nM to 50 µM. After 48 h of culture, [3H]thymidine was added (1 µCi/well), and after an additional 16 h cells were harvested onto glass fiber filters (Wallac, Gaithersburg, MD) and radioactivity was counted in a ß scintillation counter (Wallac).
Peptide presentation by DR2 transfectants
The MHC restriction was determined using L cells transfected with either DR2a (DRA, DRB5*0101) or DR2b (DRA, DRB1*1501) as APC. APC were incubated overnight in a six-well plate in the presence or absence of 50 µM MBP(85N99) peptide. Cells were then harvested, washed three times, irradiated (3,000 rad), and seeded into a 96-well U-bottom plate at 5 x 104 cells/per well. T cells were added at 5 x 104 cells/well, and T cell proliferation was determined as described above.
Peptide binding assay
Peptide binding was examined using soluble DR2 (DRA, DRB1*1501)
expressed in Drosophila Schneider cells (17) (L. Gauthier et
al., unpublished data). Peptide binding assays were set up in a 50-µl
volume with 0.2 µM DR2 and 1.0 µM biotinylated MBP peptide in PBS,
1 mM EDTA, 1 mM PMSF. The peptide carried a biotin moiety and a
four-amino acid spacer N-terminal to the MBP (85–99) sequence
(biotin-SGSG-ENPVVHFFKNIVTPR). Unlabeled competitor peptides were
added at concentrations ranging from 0.1 µM to 100 µM. Following an
overnight incubation at 37°C (
18 h), the amount of DR2-bound
biotinylated peptide was quantitated.
A 96-well flat-bottom plate was coated overnight at 4°C with 200 ng/well anti-DR mAb (L243) in 50 µl of 100 mM bicarbonate, pH 9.6. Following four washes with PBS, 0.05% Tween 20 (PBS-Tween), nonspecific binding sites were blocked at room temperature for 2 h using 3% BSA in PBS-Tween. Plates were then washed, and 50 µl of 3% BSA, PBS-Tween were added to each well, followed by 50 µl of each peptide binding reaction and 50 µl of 3% BSA, PBS-Tween which were used to rinse the tubes from the peptide binding reaction. Following a 1-h incubation at room temperature, plates were washed four times with PBS-Tween and 100 µl of europium-labeled streptavidin (diluted 1/2000 in 0.5% BSA, PBS) (Wallac) were added. Following a 1-h incubation, plates were washed six times, and 180 µl of Delfia enhancement solution (Wallac) were added. The fluorescence was quantitated after a 30-min incubation in a Delfia plate reader (Wallac).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study, viral and bacterial peptides were identified
that activated three of seven MBP(85–99)-specific T cell clones that
were examined (3). To determine whether cross-reactive microbial
peptides could also be identified for some of the other clones, the TCR
recognition motif was further defined using a panel of analogue
peptides in which individual peptide residues were substituted by all
naturally occurring amino acids, except cysteine (Fig. 1
). A human B cell line homozygous for
the HLA-DR2 haplotype (MGAR, DRB1*1501) was used as APCs; peptides were
used at a concentration of 1 µM. The analysis focused on two
MBP(85–99)-specific T cell clones (Ob.1A12 and Ob.2F3) from a multiple
sclerosis patient with the HLA-DR2 haplotype (DRB1*1501) (15, 16).
Sequence analysis of TCR - and ß-chains had demonstrated that
these clones used the same V-J and Vß-Jß rearrangements and
differed at a single position in the CDR3 region of (threonine or
alanine) and at two positions in the CDR3 region of ß
(alanine-asparagine or serine-leucine) (Table I).
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). The
93K to Q analogue had no activity on clone Ob.2F3, not even at high
peptide concentrations, while peptides with substitution by M, S, or T
had some activity at high peptide concentrations (data not shown). For
both clones, substitution of 93K by aromatic or negatively charged
residues resulted in a major reduction or complete loss of T cell
activation. Residues 90H and 91F (P2 and P3 position relative to the P1 anchor) were major TCR contact residues for both T cell clones since substitutions at these positions either greatly reduced or abolished T cell activation. Some of the 90H analogue peptides (i.e., 90H to F) induced a certain degree of T cell activation at higher peptide concentrations. The observation that substitution of 91F by some charged residues (D or K) induced T cell proliferation was surprising but reproducible.
In contrast to the strong preferences observed for 90H, 91F, and 93K,
many substitutions of residues 88V and 94N were tolerated. The two
clones differed in the recognition of the proline analogue of 94N;
since proline can affect the conformation of the peptide backbone, it
may affect the neighboring P5 position. All analogues of 95I (Fig. 1)
and 96V (data not shown) induced proliferation by both T cell clones.
These results indicated that only a limited number of peptide residues
were critical for TCR recognition by these MBP(85–99)-specific T cell
clones.
TCR CDR3 loops contribute to cross-reactivity with microbial peptides
Based on the recognition motif for clone Ob.1A12, which had a
broader specificity for 93K (P5 position) than Ob.2F3, a database
search of microbial antigens was performed (Table II). Two sets of peptides were
synthesized (M48–M65 and S1–S52) and tested; data for the first set
(M48–M65) are shown in Table III. In
addition, a peptide from Mycoplasma genitalium (M66)
that had strong sequence homology to MBP(85–99) was synthesized, even
though it did not precisely match the search motif. This peptide did
not stimulate the T cell clones, even though it had the highest degree
of sequence identity (seven amino acids) with MBP(85–99).
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). The P2 and P3 positions (90H and
91F) of these peptides were conserved with MBP(85–99); otherwise, the
sequences were quite distinct from the MBP peptide and from each other.
Two peptides from the first set activated clone Ob.1A12; these peptides
were derived from Mycobacterium avium (M56) and
Escherichia coli/Haemophilus influenzae
(M53) (Table III). The M. avium peptide that had
a conservative lysine to arginine substitution at P5 also activated
clone Ob.2F3; the other peptide had a nonconservative lysine to serine
substitution and activated only clone Ob.1A12 (Table III). In addition,
several peptides (i.e., M50, M57) induced some T cell proliferation at
the highest peptide concentration tested (50 µM); these peptides were
not included in Table IV.
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, Fig. 2). The peptides from M.
tuberculosis and M. avium were derived
from a homologous segment of transposase but differed at 5 positions in
the 15-amino acid segment. The five peptides that activated clone
Ob.1A12 had conservative (arginine) and nonconservative (proline or
serine) substitutions of lysine 93. Only the three microbial peptides
with the conservative lysine to arginine substitution activated clone
Ob.2F3 (Table IV); proline at P5 was tolerated by clone Ob.2F3 only in
the context of the MBP peptide but not in the B.
subtilis peptide. Dose-response experiments indicated that
peptides derived from S. aureus and M.
avium activated both T cell clones at concentrations of
<100 nM; the MBP(85–99) peptide was effective at even lower peptide
concentrations (Fig. 2). Stimulation by the three other bacterial
peptides required higher peptide concentrations.
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).
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).
Recombinant, soluble HLA-DR2 (DRA, DRB1*1501) that was affinity
purified from supernatants of Drosophila Schneider cell
transfectants was used for these experiments. In the binding assay,
unlabeled microbial peptides were used as competitors of a biotinylated
MBP peptide. Following the binding reaction, DR2 molecules were
captured with an anti-DR mAb and the amount of DR2-bound
biotinylated peptide was quantitated with europium-labeled
streptavidin. The M. avium and M.
tuberculosis peptides competed for MBP peptide binding at
the lowest peptide concentrations, followed by the S.aureus, B. subtilis, and E.
coli/H. influenzae peptides. The MBP(NOREF>85–99)
peptide with an aspartic acid substitution at the P4 position (85–99,
92D) did not compete for MBP peptide binding and was used as a negative
control.
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). Clone Ob.2F3 responded to only three
of the five microbial peptides that activated clone Ob.1A12, due to
differences in the TCR CDR3 sequences. The HLA-DR2a transfectant was
also functional since it presented a tetanus toxoid peptide to
HLA-DR2-restricted T cell clones from a normal subject (data not
shown). These results indicate that activation of these human T cell
clones by MBP(85–99) or cross-reactive microbial peptides does not
require human costimulatory or accessory molecules that are expressed
by EBV-transformed human B cells but not by murine L cells transfected
with HLA-DR2 cDNA constructs. Taken together, these results demonstrate
that the TCR CDR3 regions of these MBP-specific T cell clones determine
the degree of cross-reactivity with microbial peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-J and
Vß-Jß segments, allowing the contribution of the TCR CDR3 regions
to be analyzed. Both T cell clones were activated by three microbial
peptides that had a conservative lysine to arginine substitution at the
P5 position, while two other microbial peptides that had
nonconservative substitutions of the P5 position (lysine to proline or
serine) only activated the T cell clone with the broader specificity
for the P5 side chain. Another important structural element may be the
TCR V segment, since the V CDR1 loop made extensive contacts with
the HLA-A2-bound tax (11, 12, 13, 14, 15, 16, 17, 18, 19) peptide in the crystal structure (12).
V usage (V3.1) was conserved among human T cell clones that
recognized the MBP peptide in the context of different HLA-DR2
subtypes. Shared V3.1 usage may account for the observation that the
P2 and P3 positions of the MBP peptide (H90 and F91) were important for
TCR recognition (18). The crystal structure of the HLA-DR2/MBP peptide
complex demonstrated that 90H, 91F, and 93K (at the P2, P3, and P5
positions) were prominent, solvent-exposed residues that could
be contacted by the TCR (22). 89V and 92F, which were previously shown
to be important for HLA-DR2 binding, were located in the P1 and P4
pockets of the HLA-DR2 binding site. The data also demonstrate the importance of combinatorial effects in shaping peptide surfaces that can be recognized by a TCR. Algorithms that are based on single amino acid substitutions can be used to predict peptides that will bind with high affinity to particular MHC class II molecules (23). In contrast, particular amino acid combinations are important in shaping peptide surfaces that can be recognized by a TCR. This notion is supported by the observation that the majority of microbial peptides that perfectly matched the MHC binding/TCR recognition motif did not stimulate MBP-specific T cell clones. Identification of a complete set of peptide sequences that act as agonists for a TCR will require analysis of combinatorial peptide libraries. At present, such an analysis represents a technical challenge due to the large number of peptides that may need to be sequenced from phage display libraries or peptide libraries on beads. However, the complexity of the peptide repertoire recognized by an individual TCR may be greatly underestimated unless the combinatorial nature of peptide recognition by the TCR is taken into consideration. The observation that some T cell clones are activated by randomized peptides in which each position was synthesized with a mixture of naturally occurring amino acids supports this notion (7).
Stimulatory microbial peptides were previously not identified for the two T cell clones described in this report, even though a large number of peptides had been synthesized and tested. The three bacterial peptides that activated both clone Ob.1A12 and clone Ob.2F3 actually matched the original search criteria, illustrating how recent progress in the sequencing of microbial genomes has facilitated the identification of such peptides. These peptides (derived from S. aureus, M. avium, and Mycobacterium tuberculosis) all had a conservative lysine to arginine substitution of 93K. The other two peptides did not match the original search motif since nonconservative substitutions of 93K were not considered; these residues had not been included in the original search criteria because some of the other T cell clones had a strong preference for a positive charge (lysine or arginine) at P5. Since many microbial genomes have not yet been sequenced, other stimulatory peptides are likely to exist. Also, certain sequences may be overlooked by search criteria that utilize a motif based on single-amino acid substitutions.
The two T cell clones studied (Ob.1A12 and Ob.2F3) were not as degenerate in TCR recognition of the MBP(85–99) peptide as other T cell clones. For example, every position of the MBP peptide could be individually substituted by at least one structurally related amino acid for clones Hy.2E11 and Hy.1G11 (data not shown). Also, a MBP(87–97)-specific T cell clone was recently reported that was even more degenerate. This T cell clone was activated by an 11-mer peptide in which all peptide positions were randomized (synthesized with a mixture of all naturally occurring amino acids) (7). Clone Ob.1A12 and other MBP(85–99)-specific T cell clones were not activated by such randomized peptides (even at high peptide concentrations), indicating that such a degree of degeneracy is not required for the activation of MBP-specific T cell clones by microbial peptides. Taken together, these findings suggest that TCRs differ in terms of the degree of specificity/degeneracy of peptide recognition (3, 7, 24, 25). T cells with a higher degree of degeneracy are more likely to be engaged in an immune response and to cross-react with a self-peptide.
While the activation of autoreactive T cells is required for the development of T cell-mediated autoimmunity, autoimmune disease following T cell activation by cross-reactive microbial peptides may depend on a number of additional factors: 1) the inheritance of particular alleles of MHC and other genes that confer susceptibility/resistance to autoimmunity; 2) a sufficient degree of clonal expansion of a pathogenic T cell population; 3) induction of functional properties (i.e., cytokine profile) that make such expanded T cell clone(s) pathogenic; 4) access of T cells to the target organ (26). The observation that some autoreactive T cell clones can be activated by several microbial peptides indicates that different peptides could be involved in the initial activation of autoreactive T cells and in triggering relapses.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Kai W. Wucherpfennig, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston MA 02115. E-mail address:
4 Abbreviations used in this paper: MBP, myelin basic protein; CDR, complementarity-determining region.
Received for publication July 10, 1998. Accepted for publication September 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cell receptor structure at 2.5Å and its orientation in the TCR-MHC complex. Science 274:209.ß with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2. J. Immunol. 157:5403.[Abstract]
ß heterodimers. J. Biol. Chem. 271:20156.This article has been cited by other articles:
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  Z. Kato, J. N. H. Stern, H. K. Nakamura, K. Kuwata, N. Kondo, and J. L. Strominger Positioning of autoimmune TCR-Ob.2F3 and TCR-Ob.3D1 on the MBP85-99/HLA-DR2 complex PNAS, October 7, 2008; 105(40): 15523 - 15528. [Abstract] [Full Text] [PDF]
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 A. D. Gritzapis, L. G. Mahaira, S. A. Perez, N. T. Cacoullos, M. Papamichail, and C. N. Baxevanis Vaccination with Human HER-2/neu (435-443) CTL Peptide Induces Effective Antitumor Immunity against HER-2/neu-Expressing Tumor Cells In vivo. Cancer Res., May 15, 2006; 66(10): 5452 - 5460. [Abstract] [Full Text] [PDF]
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 A. M. Ercolini and S. D. Miller Mechanisms of Immunopathology in Murine Models of Central Nervous System Demyelinating Disease J. Immunol., March 15, 2006; 176(6): 3293 - 3298. [Abstract] [Full Text] [PDF]
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M. P. Mycko, H. Waldner, D. E. Anderson, K. D. Bourcier, K. W. Wucherpfennig, V. K. Kuchroo, and D. A. Hafler Cross-Reactive TCR Responses to Self Antigens Presented by Different MHC Class II Molecules J. Immunol., August 1, 2004; 173(3): 1689 - 1698. [Abstract] [Full Text] [PDF]
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Y. Uemura, S. Senju, K. Maenaka, L. K. Iwai, S. Fujii, H. Tabata, H. Tsukamoto, S. Hirata, Y.-Z. Chen, and Y. Nishimura Systematic Analysis of the Combinatorial Nature of Epitopes Recognized by TCR Leads to Identification of Mimicry Epitopes for Glutamic Acid Decarboxylase 65-Specific TCRs J. Immunol., January 15, 2003; 170(2): 947 - 960. [Abstract] [Full Text] [PDF]
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A. Amrani, P. Serra, J. Yamanouchi, J. D. Trudeau, R. Tan, J. F. Elliott, and P. Santamaria Expansion of the Antigenic Repertoire of a Single T Cell Receptor upon T Cell Activation J. Immunol., July 15, 2001; 167(2): 655 - 666. [Abstract] [Full Text] [PDF]
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P. J. Costello, R. J. Winchester, S. A. Curran, K. S. Peterson, D. J. Kane, B. Bresnihan, and O. M. FitzGerald Psoriatic Arthritis Joint Fluids Are Characterized by CD8 and CD4 T Cell Clonal Expansions that Appear Antigen Driven J. Immunol., February 15, 2001; 166(4): 2878 - 2886. [Abstract] [Full Text] [PDF]
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J. K. Sandberg, L. Franksson, J. Sundback, J. Michaelsson, M. Petersson, A. Achour, R. P. A. Wallin, N. E. Sherman, T. Bergman, H. Jornvall, et al. T Cell Tolerance Based on Avidity Thresholds Rather Than Complete Deletion Allows Maintenance of Maximal Repertoire Diversity J. Immunol., July 1, 2000; 165(1): 25 - 33. [Abstract] [Full Text] [PDF]
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J. L. Grogan, A. Kramer, A. Nogai, L. Dong, M. Ohde, J. Schneider-Mergener, and T. Kamradt Cross-Reactivity of Myelin Basic Protein-Specific T Cells with Multiple Microbial Peptides: Experimental Autoimmune Encephalomyelitis Induction in TCR Transgenic Mice J. Immunol., October 1, 1999; 163(7): 3764 - 3770. [Abstract] [Full Text] [PDF]
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