| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
*
Virginia Mason Research Center,
University of Washington School of Medicine, and
Fred Hutchinson Cancer Research Center, Seattle, WA 98101
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-helical portion of the DRß polypeptide, but not when
presented by other DR4, DR11, and DR13 alleles that are negative for
this sequence. Using a panel of APCs expressing DR4 molecules that were
mutagenized in vitro at individual residues within this shared epitope
and using peptide analogues with single amino acid substitutions of
predicted MHC and TCR contact residues, a unit of recognition was
identified dependent on DRß residues 67–71 and relative position 4
(P4) of the VP16 393–405 peptide. The interactions of this portion of
the peptide-DR ligand with the ESL4.34 TCR support a structural model
for MHC-biased recognition in some Ag-specific and alloreactive T cell
responses and suggest a possible mechanism for autoreactive T cell
selection in rheumatoid arthritis. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The interaction between ß TCRs and peptide-MHC class II ligands
involves direct contact between the TCR and residues on both the MHC
molecule and the bound peptide (9, 10). Analyses of the crystal
structures of MHC class II molecules complexed with antigenic peptides
(11, 12, 13, 14, 15) and studies using substituted peptides and mutated MHC
molecules (6, 16, 17) have identified putative TCR contact residues on
both the antigenic peptides and the -helical regions of the MHC
class II molecules. Two recent crystal structures of class I
MHC-peptide-TCR complexes (18, 19, 20) have demonstrated that the ß
TCR recognizes its ligand through three variable loops
(complementarity-determining regions
(CDRs4)) on each polypeptide
that form a relatively flat surface that fits diagonally over the
MHC-peptide complex. All three CDRs of the TCR -chain and CDR3 of
the ß-chain contact the MHC helices, while the CDR1 and CDR3
loops of both the TCR - and ß-chains contact the bound peptide.
Based on sequence and structure comparisons between peptides complexed
with MHC class I and class II molecules, a similar mode of TCR
interaction with peptide-MHC class II ligands has been predicted (19, 21).
While the overall conformation of the peptide-MHC-TCR ternary complex
is thought to be similar for all MHC class I and class II molecules and
all ß TCRs (19, 21), the nature of the signal generated upon TCR
ligation can vary greatly, leading to activation, apoptosis, or
nonresponsiveness of the T cell and positive or negative selection in
the thymus (22, 23). TCR activation can, in turn, result in a range of
different effector functions, including T cell proliferation,
cytolysis, and cytokine secretion of the Th0, Th1, or Th2 profiles.
Factors that contribute to the outcome of TCR ligation include the
nature and cell surface density of the peptide-MHC ligand, the avidity
of its interaction with the TCR, the presence or absence of other
costimulatory molecules, and the local cytokine milieu (22, 24, 25, 26).
Specific contacts between amino acid residues on the peptide, MHC, and
TCR molecules have profound effects on the nature of T cell
stimulation. Single substitutions of MHC or peptide residues predicted
to contact the TCR can enhance, antagonize, or anergize a T cell
response or selectively inhibit some but not all effector functions
(27, 28, 29, 30, 31, 32).
The TCRs of human CD4+ T cells primarily recognize
antigenic peptides presented by MHC class II (HLA-DR, DQ, or DP)
molecules. HLA-DR molecules are heterodimeric glycoproteins composed of
a nonpolymorphic DR and a polymorphic DRß chain. The polymorphic
amino acid residues in DRß polypeptides are mostly clustered in three
hypervariable regions (HVRs) which line the peptide binding groove
(33). HVR1 (residues 9–14) and HVR2 (residues 26–33) are located on
the floor of the groove and predominantly influence the specificity of
peptide binding, while HVR3 (residues 67–74) is located on the
-helical portion of the DRß molecule and can affect both peptide
binding and TCR interaction through direct contacts with both molecules
(11, 13, 14). HVR3 amino acid sequences can vary greatly within
serologically related DR types but are conserved among serologically
distinct alleles. For example, the ILEDE amino acid sequence at DRß
positions 67–71 is present on some but not all allelic variants of the
DR1, DR4, DR11, and DR13 specificities. Another sequence at this
position, LLEQRRAA, which is encoded by certain DR1, DR4, and DR14
alleles, constitutes the "shared epitope" thought to play a role in
the development of rheumatoid arthritis (RA) (34, 35).
In this study, we examined the molecular basis of the interactions between the TCR, MHC class II, and peptide molecules, using a herpes simplex type 2 virus (HSV-2)-specific human CD4+ T cell clone, ESL4.34, which recognizes a peptide determinant corresponding to residues 393–405 of the virion protein VP16, in the context of several HLA-DR alleles. This T cell, which elicits strong proliferative, cytolytic, and cytokine secretion responses, recognizes the VP16 393–405 peptide with a pattern of restriction that correlates with a specific amino acid sequence at positions 67, 70, and 71 of HVR3 of the DRß polypeptide. By a combination of molecular modeling and assays of peptide binding and T cell stimulation using in vitro mutagenized DR molecules and substituted peptide analogues, we have dissected the structural interactions between DRß HVR3, the peptide, and the TCR. The DRß HVR3 was found to have a direct effect on both peptide binding and TCR recognition, suggesting an important role for this epitope in alloreactivity, in T cell repertoire selection, and in autoimmune disease.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HSV-1 strain E115, HSV-2 strain 333, and a recombinant virus
consisting of VP16 of HSV-2 in an HSV-1 background, RP-2, were grown in
human diploid cells and titered by plaque assay on Vero cells as
previously described (36). Crude viral Ags were prepared from virus
stocks by exposure to UV light for 30 min at 10 cm from a GT038 bulb
(General Electric, Cleveland, OH), eliminating infectious virus, and
used at a final dilution of 1:100 which corresponds to 106
to 107 plaque-forming units per ml before UV treatment.
Peptides corresponding to amino acids 1–409 of VP16 of HSV-2, 13 amino
acids long and overlapping by 9 amino acids, were obtained from Chiron
Mimotopes, Clayton, Australia. The VP16 393–405 peptide
(LVAPRMSFLSAGQ) and its analogues (see legend to figures and tables)
were synthesized with an Applied Biosystems 432 Peptide Synthesizer
(Foster City, CA). Peptides were biotinylated by amino-terminal
addition of biotin onto 
-aminocaproic acid, as described by Kwok et
al. (37), and used at the concentrations shown in the figure legends.
T cell cloning
Mononuclear cells were prepared from the herpetic vesicle fluid
from a DRB1*0402/*1301-positive patient with culture-proved recurrent
HSV-2 infection by Ficoll-Hypaque density gradient centrifugation. The
cells were stimulated with PHA (0.4 µg/ml PHA-P; Murex Diagnostics,
Dartford, U.K.) and an equal number of irradiated (3300 rads
-irradiation) allogeneic PBMC in T cell medium (RPMI 1640 containing
25 µM HEPES, 2 µM L-glutamine, 50 µg/ml streptomycin,
50 U/ml penicillin, and 10% heat-inactivated human male serum).
Acyclovir (50 µM) was added for the first 2 weeks of culture to
prevent viral replication (38). Human natural IL-2 (50 U/ml;
Schiaperelli Biosystems, Columbia, MD) was added on day 3, and the
cells were subsequently fed with IL-2 every 2 to 3 days. After 16 days
of growth, 5 x 105 cells were restimulated with crude
HSV-2 Ags and 5 x 105 autologous irradiated PBMC as
APC and maintained as above. After 12 days, the cells were cloned at 1
cell/well using PHA, IL-2, and allogeneic feeders and subsequently
restimulated every 12 to 14 days.
Analysis of TCR genes
mRNA was prepared from T cell clones that were restimulated with
lymphoblastoid cell lines (LCLs) as APC instead of PBMC using the
QuickPrep Micro RNA Purification Kit (Pharmacia Biotech, Alameda, CA)
and used for first strand cDNA synthesis using the Superscript
Preamplification system (Life Technologies, Gaithersburg, MD). TCR V
and Vß gene segment usage was determined by PCR amplification of
first strand cDNA (RT-PCR) using a set of subfamily specific 5'-primers
(V1–29 and Vß1–23) and constant region 3'-primers (kindly
provided by Dr. Christine Vissinga, Virginia Mason Research Center,
Seattle, WA) essentially as described by Choi et al. (39). Vß typing
was confirmed using the Clontech (Palo Alto, CA) TCR Amplimer Kit.
Nucleotide sequences of the V-(D)-J junctions were determined by cycle
sequencing of both strands of the PCR products obtained as above using
the Prism Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit
(Applied Biosystems) and analyzed on an Applied Biosystems 373 DNA
Sequencer. Two different V7 genes were detected by sequence
analysis; therefore, the V7 PCR products were cloned into the pCRII
vector (Invitrogen, San Diego, CA), used to transform One Shot
(INVF') cells (Invitrogen) and selected on antibiotic-containing
L-agar plates. Plasmid DNA was prepared from overnight cultures of
isolated colonies using the QIAprep Spin Miniprep Kit (Qiagen, Santa
Clarita, CA) and amplified as above by PCR. The nucleotide sequences of
both strands of the PCR products were determined by cycle sequencing as
above.
Lymphoblastoid cell lines and Abs
The EBV-transformed LCLs YAR (DRB1*0402), MT (*0404), HHKB
(*1301), JVM (*1102), TISI (*1103), and MAT (*0301) were obtained from
the VIIIth and Xth International Histocompatibility Workshop Panels.
The LCL 8854 (*1303) was generated by EBV transformation of locally
characterized PBMC. The MHC class II-deficient BLS-1 line was kindly
donated by Dr. Janet Lee, Sloan-Kettering Memorial, New York, NY. LCLs
were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS
(HyClone, Logan, UT) and sodium pyruvate, HEPES,
L-glutamine, streptomycin, and penicillin as above. mAbs
used were L243 (anti-DR; American Type Culture Collection,
Rockville, MD), SPV-L3 (anti-DQ; kindly provided by Dr. Hans Yssel,
DNAX, Palo Alto, CA), B7/21 (anti-DP; Becton Dickinson, San Jose,
CA), anti-ß TCR and anti-Vß8 (PharMingen, San Diego,
CA), NFLD.D1, and NFLD.D11 (anti-DR4 and -DR4w4; kindly provided by
Dr. Sheila Drover, University of St. Johns, St. Johns, Canada). Cell
surface expression of HLA-DR and TCR was assayed by mAb staining as
described previously (40) and analyzed on a Becton Dickinson FACSort
flow cytometer.
Retroviral gene transfer
HLA-DR genes and mutagenized constructs were introduced into MAT LCLs by retroviral infection using the methods of Kwok et al. (41). The construction of the retroviral vectors containing wild-type and mutagenized DRB1*0404 genes which were altered at codons 67 (L to I), 70 (Q to D), 71 (R to E), and both 67 and 71 (L67 to I67 and R71 to E71) has been described previously (40). For the purpose of clarity, these mutants are named according to their differences from the DRB1*0402 amino acid sequence in this report, i.e., DRB1*0402-Q70,R71, DRB1*0402-L67,R71, DRB1*0402-L67,Q70, and DRB1*0402-Q70, respectively. Surface expression of the transgenes was monitored by flow cytometry using the NFLD.D1 and NFLD.D11 mAbs.
T cell proliferation assays
Stimulator cells (PBMC irradiated with 3,300 rads or LCL
irradiated with 20,000 rads -irradiation) were pulsed with Ag
(peptide concentrations shown in figure legends) for 2 to 4 h at
37°C, 5% CO2, followed by three washings with
supplemented RPMI medium. PBMC (105) or LCL (2.5 x
104) were plated in 96-well U-bottom plates in T cell
medium containing 104 cloned T cells or bulk lesion-derived
cells as responders in a final volume of 150 µl. All assays were
performed in triplicate. After incubation for 48 h,
[3H]thymidine (1 µCi/well; NEN, Boston, MA) was added
for 12 to 16 h, the cells were harvested using a Tomtec 96 Mach
III Harvester (Hamden, CT), and 3H incorporation was
measured on a 1450 Microbeta Plus Liquid Scintillation Counter (Wallac,
Gaithersburg, MD). Stimulation indices were calculated as (cpm of
sample - cpm in absence of responders)/cpm in absence of
stimulators. To determine restricting HLA molecules, mAbs L243, SPV-L3,
and B7/21, which recognize HLA-DR, DQ, and DP, respectively, were used
as previously described (38).
T cell cytotoxicity assays
Target LCLs (5 x 105) were pulsed with peptide (concentrations shown in figure legends) and simultaneously loaded with 51Cr (25 µCi; NEN) for 8 to 12 h at 37°C, 5% CO2, followed by gentle washing three times with supplemented RPMI medium. Labeled targets (1–2 x 103) were incubated with effector T cells at various E:T ratios in a final volume of 150 ml in T cell medium in 96-well U-bottom plates. After 4 h, cell supernatants (50 µl) were assayed for 51Cr release in a 1450 Microbeta Plus Liquid Scintillation Counter. Percent specific lysis was expressed as (cpm of sample - cpm of spontaneous release)/(cpm of maximum release - cpm of spontaneous release).
Peptide binding assays
Binding of biotinylated peptides to HLA-DR on whole cells, with subsequent capture of the peptide-class II complex, was performed using a europium-labeled streptavidin assay as described previously (37), except that the L243 Ab was used to immobilize the HLA-DR molecules. A 10 µM peptide concentration was used in all experiments, this concentration having been previously determined to be submaximal for binding. The class II-deficient LCL, BLS-1, served as a negative control for nonspecific binding.
Modeling of peptide-HLA-DR complexes
Molecular models of DRB1*0402, *1102, and *1301 were constructed from the crystal structure coordinates of the HLA-DR1 molecule complexed with the influenza hemagglutinin peptide 306–318 (11) as previously described (42). Using the interactive graphics program PSSHOW (43), amino acid side chains were replaced and assigned to the preferred conformer from the Ponder and Richards rotamer database (44) that was closest to the side chain conformer in the crystal structure template. The HSV VP16 393–405 peptide was constructed from the hemagglutinin peptide coordinates (11), and several different alignments of the anchor residues with the VP16 393–405 sequence were examined to predict the binding motif. Peptide binding experiments were designed to test the predicted binding motif.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recovery of cells from herpetic vesicle fluid and subsequent
stimulation with whole HSV-2 Ag followed by cloning yielded a T cell
clone, ESL4.34, that responded to HSV-2 and RP-2 but not HSV-1 in
proliferation assays (Fig. 1
), indicating
a specificity for VP16 of HSV-2 but not HSV-1. The fine specificity of
ESL4.34 was determined by testing its proliferative response to a set
of 10 pools of overlapping peptides corresponding to amino acids 1–409
of VP16 of HSV-2. Only a pool spanning amino acids 361–409 (pool 10)
gave a positive response (Fig. 1). Assay with individual peptides of
this pool identified the epitope as one contained in overlapping
peptides 389–401 and 393–405 (Fig. 1). Peptide VP16 393–405 was used
in subsequent studies to characterize the residues required for MHC
binding and T cell recognition.
|
shows that both DRB1*0402 and DRB1*1301
presented VP16 393–405 to ESL4.34, eliciting both proliferative and
cytolytic responses.
|
- and ß-chain gene usage was determined by PCR
amplification of cDNA using primers specific for V1–29 and
Vß1–23. PCR products corresponding to V7 and Vß8 only were
obtained. Flow cytometric analysis of ESL4.34 using anti-ß TCR
and anti-Vß8 mAbs confirmed that all ß TCR+
cells expressed Vß8. Nucleotide sequence analysis of the Vß8 PCR
product in both directions revealed a unique V-D-J
junctional sequence that encodes a TCR ß-chain CDR3 (residues
92–117) amino acid sequence CASSERGDTDTQYFGPGTRLTVLEDL, which
corresponds to Vß8, Dß (ERGD), Jß2.3 (TDTQYFGPGTRLTVLEDL),
and Cß2. Sequencing of the V7 PCR product revealed two V7 TCR
gene sequences, and cloning and sequencing of these two products
identified distinct V7J junctional sequences. One of these
contained a G to C nucleotide substitution which, if translated, would
result in the substitution of the conserved cysteine residue at
position 90 with a serine residue, resulting in a nonfunctional TCR
-chain (45). The viable TCR -chain CDR3 region was encoded by
V7.2, Jß2.3, and a V-J junctional sequence (residues 90–106),
which code for the amino acid sequence CAPRGAGRRALTFGSGT. Pluriallelic restriction of ESL4.34 responses to VP16 393–405
The above experiments indicate that ESL4.34 is a
V7+Vß8+ T cell clone specific for VP16
393–405 presented by both autologous DR alleles, DRB1*0402 and
DRB1*1301, in both proliferation and cytotoxicity assays (Fig. 2
). This
clone, however, does not respond to the peptide presented by the
allogeneic DRB1*0404 allele, which differs from *0402 only at three
amino acid positions (residues 67, 70, 71) in the DRß helix (Fig. 2). To further investigate the pluriallelic restriction of ESL4.34, a
set of LCLs expressing DRB1 allelic variants of three serologically
defined DR types, DR4, DR11, and DR13, which differ in their
-helical amino acid sequences, were used as APCs for VP16 393–405
in proliferation and cytotoxicity assays. Figure 3, A and B, shows
that VP16 393–405 was recognized by ESL4.34 in proliferation and
cytotoxicity assays, when presented on LCL expressing DRB1*0402,
DRB1*1301, and DRB1*1102. Each of these DRB1 alleles encode the same
sequence at DRß residues 67–71 (HVR3), namely ILEDE, whereas the
other DR4, DR13, and DR11 alleles that did not support ESL4.34
responses had different HVR3 sequences, as summarized in Table I. Even the single isoleucine to
phenylalanine substitution at DRß position 67, that distinguishes
DRB1*1102 and DRB1*1103, is sufficient to abolish ESL4.34 responses.
Further analyses of ESL4.34 responses VP16 393–405-pulsed LCLs
expressing other DRß 67–71 ILEDE-negative DR4 (DRB1*0401 and *0405)
and DR11 (DRB1*1101) alleles (data not shown) were consistent with the
hypothesis that this motif at HVR3 is required for ELS4.34 recognition.
|
|
C shows that the
relative binding of VP16 393–405 to DR expressed on the various APCs
varied greatly for the different DRB1 alleles and showed no correlation
with the proliferative and cytolytic responses of ESL4.34 to VP16
393–405. While DRB1*1303 and DRB1*1103 bound the peptide as well as,
or better than, their stimulatory DRB1*1301 and DRB1*1102 counterparts,
no proliferative or cytolytic responses were observed using these
ligands. This suggests that the ability of ESL4.34 to respond to its
peptide-MHC ligand is controlled in part by the presenting MHC molecule
itself, through specific interactions between residues on the TCR and
the DR molecule. This hypothesis, however, does not exclude the
involvement of the peptide in ESL4.34 responses, since no proliferation
or cytotoxicity was observed in the absence of VP16 393–405, and the
responses increased proportionately with peptide concentration (Fig. 2). Specific HVR3 residues on HLA-DRß control ESL4.34 responses
The three DRB1 alleles (DRB1*0402, DRB1*1102, and DRB1*1301) that
could present VP16 393–405 to ESL4.34 in proliferation and
cytotoxicity assays are serologically and structurally distinct but
share a common amino acid sequence, ILEDE, at DRß positions 67 to 71,
whereas the other DRB1 alleles tested in the present study encode
different amino acid sequences at this region. This suggests a role for
this "shared epitope" in association with VP16 393–405 in
eliciting ESL4.34 responses. To test this hypothesis, we generated a
panel of LCLs expressing mutagenized DRB1 molecules that distinguish
the stimulatory DRB1*0402 allele from the nonstimulatory DRB1*0404
molecule, which differ only at the three polymorphic residues in this
epitope, residues 67, 70, and 71. Surface expression of these mutant
DR4 molecules was comparable with that of wild-type DRB1*0402 and
DRB1*0404 in YAR and MT LCLs, respectively (Fig. 4). Figure 5 shows the proliferative and cytolytic
responses of ESL4.34 to VP16 393–405 presented by these mutant APCs
and the relative binding affinities of this peptide for the mutant DR
molecules. Binding was drastically reduced by substitutions at 67 and
71 (DRB1*0402-L67, R71) and substitutions at 70 and 71 (DRB1*0402-Q70,
R71), but changes at residues 67 and 70 (*0402-L67,Q70, and *0402-Q70)
had less significant effects on VP16 393–405 binding (Fig. 5C). In contrast, substitution of any of the DRB1*0402
residues 67, 70, or 71 completely abrogated ESL4.34 proliferative and
cytolytic responses to VP16 393–405. These data, together with the
responses of ESL4.34 to the DR4, DR11, and DR13 allelic variants (Fig. 3), confirm that DRß residues I67, D70, and E71 are essential (and
possibly sufficient) for presentation of VP16 393–405 to ESL4.34. The
involvement of HVR3 in peptide binding and ESL4.34 responses does not
exclude less significant contributions by the first and second HVRs of
DRß and/or the V/G dimorphism at position 86. Significant influences
of the DRB3 and DRB4 gene products, which are encoded on DR11, DR13,
and DR4 haplotypes, however, are unlikely, because these molecules have
distinct HVR3 sequences.
|
|
Molecular modeling of the VP16 peptide 393–405 complexed
with DRB1*0402, DRB1*1102, and DRB1*1103 suggested a binding motif such
that V394 occupies pocket 1 and R397 occupies pocket 4. This predicted
binding motif was tested by synthesizing peptides with nonconservative
substitutions at these anchor positions and measuring their relative
binding to LCLs expressing DRB1*0402, DRB1*0404, DRB1*1301, DRB1*1303,
DRB1*1102, and DRB1*1103 (Fig. 6).
Substitution of valine at position 394 with lysine (P1 [V
K])
dramatically decreased the binding of this peptide to DRB1*0402,
DRB1*1102, and DRB1*1103, whereas replacing leucine at position 393
with lysine (P-1 [LK]) slightly increased the level of binding to
these alleles (Fig. 6). This indicates that V394 is the first anchor
residue. The increase in binding observed for L393K (P-1 [LK]) may
be due to favorable interactions between K393 and DR E53.
Substitution of arginine at VP16 393–405 position 397 with the
negatively charged residue glutamic acid (P4 [RE]) reduced peptide
binding to the DRß alleles containing negatively charged residues at
positions 70 or 71, whereas binding to DRB1*0404 which has arginine at
DRß position 71 was increased by this substitution (Fig. 6). This
clearly demonstrates the importance of the negatively charged DRß
residues D70 and E71 of DRB1*0402, DRB1*1301, DRB1*1102, and DRB1*1103
in influencing the nature of pocket 4. Substitution of this P4 residue
with a nonpolar alanine (P4 [RA]) resulted in a less dramatic
reduction in binding to DRB1*0402, and surprisingly, an increase in
binding to DRB1*0404 compared with that of the P4 (RE) analogue
peptide. The minimum binding epitope of VP16 393–405 was tested using
truncated analogues with two, three, or four residues removed from the
C terminus. The VP16 peptides 393–401 and 393–402 did not bind or
bound very poorly to DRB1*0402 (data not shown), but VP16 393–403 did
bind although with a lower affinity than the wild-type VP16 393–405
peptide (Fig. 6). These observations suggest that the two C-terminal
residues of VP16 393–405 are not anchor residues but that they
may further stabilize the association by interacting with residues on
the DR molecule.
|
compares the DRB1*0402-binding and
ESL4.34-stimulatory capacities of the minimum VP16-binding epitope VP16
393–403 and two analogues of this peptide with substitutions at the
predicted TCR contact positions 5 (P5 [MNL]) and 7 (P7 [FI])
of the truncated peptide. Although VP16 393–403 bound to DRB1*0402
more weakly than the full length VP16 393–405 peptide, the
proliferation and cytolytic responses of ESL4.34 to the two peptides
were similar, indicating that the two C-terminal residues of VP16
393–405 are not involved in T cell responses. The binding of VP16
393–403 analogues with a phenylalanine to isoleucine substitution at
position 400 (P7 [FI]), or a methionine to norleucine substitution
at position 398 (P5 [MNL]), was better or slightly reduced
compared with that of the VP16 393–403 peptide. However, the responses
of ESL4.34 to these APCs were greatly diminished or completely
abrogated (Table II), indicating that these peptide residues are TCR
contact residues that can modulate T cell recognition while playing
only a minor role in binding to DR.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß TCR
molecules (19, 21), the relative contributions of peptide and MHC to T
cell specificity can vary greatly. We therefore have investigated the
structural interactions between the TCR, MHC, and peptide for a
CD4+ T cell clone, ESL4.34, which recognizes the VP16
393–405 peptide epitope of HSV-2 in the context of multiple distinct
HLA-DR alleles. This clone, which was isolated from a DRB1*0402/*1301
heterozygous individual, responds to the peptide presented by both
parental DR alleles, as well as the allogeneic DR11 allele, DRB1*1102,
in proliferation and cytotoxicity assays. These three alleles are
serologically and structurally distinct, but they share a common amino
acid sequence motif, ILEDE, at positions 67–71 of the DRß
polypeptide. The role of this sequence motif in supporting ESL4.34
stimulation was confirmed by testing an extended panel of LCLs
expressing DR4, DR13, and DR11 variants which were negative for this
sequence and by site-directed mutagenesis experiments in which
DRB1*0404-specific substitutions were introduced into the
DRB1*0402 gene at codons 67, 70, and 71. We found that
proliferative and cytolytic responses of ESL4.34 to VP16 393–405
exhibited an absolute dependence on the presence of DRß I67, D70, and
E71 (summarized in Table I). This restriction of T cell recognition
appears to be due to a direct interaction between these residues and
the TCR, since no correlation was found between the relative binding
affinities of VP16 393–405 for HLA-DR and ESL4.34 reactivity, and some
DRß 67–71 ILEDE-negative alleles strongly bound the peptide but
failed to stimulate the T cells. Thus, amino acid substitutions that
had little or no effect on relative peptide binding, such as the single
I67 to F67 substitution that converts DRB1*1102 to *1103; mutations
that change DRB1*0402 to DRB1*0402-Q70 and DRB1*0402-L67,Q70; and
substitutions that distinguish DRB1*1301 and DRB1*1303, totally
abolished ESL4.34 recognition. This suggests that the responses of
ESL4.34 are solely directed against HVR3 of the DRß molecule, similar
to allospecific responses, but our observations that no T cell
recognition occurred in the absence of VP16 393–405 and the responses
increased proportionately with peptide concentration (Fig. 2) confirm
the Ag-specific nature of ESL4.34 stimulation.
Numerous studies using allelic variants of class II and in vitro
mutagenized class II molecules with substituted peptide analogues or
using sequence analysis of naturally processed peptides presented by
class II, have identified residues on MHC molecules that are critical
for peptide binding (2, 5, 6, 7) and T cell activation (9, 16, 17, 50) and
have shown that TCR recognition is dependent both on residues necessary
for peptide binding and residues with little or no effect on binding.
Our results indicate that DRß E71 is critical for the binding of VP16
393–405, since substitution of this residue with R in DR4 variants and
in DRB1*0402 mutants (DRB1*0402-Q70,R71, and DRB1*0402-L67,R71)
drastically reduced binding of the peptide. Less significant reductions
in relative peptide binding were associated with substitutions of DRß
D70, while changes at position 67 alone had little or no effect on
binding. This hierarchy of peptide binding dependence in the order
E71 > D70 > I67 is consistent with the location of these
residues in the crystal structures of HLA-DR molecules, which place
residues 67, 70, and 71 at the highest point of the DRß helix,
with residue 71 pointing "up" toward the TCR and into the peptide
binding cleft interacting with peptide position 4, residue 70 pointing
up, and residue 67 pointing up and away from the peptide binding cleft
(11, 14). There is precedent for the importance of HVR3 in both peptide
binding and Ag-specific T cell responses to DRB1*0402 (51, 52),
DRB1*0401 (17, 50, 53, 54), DR13 (55), and DR11 (47, 56) and in
alloreactive T cell responses (40, 57, 58).
The peptides present in the five MHC class II molecules for which
crystal structures have been determined to date, which include DR1,
DR3, DR4, and I-Ek, have remarkably similar conformations
(11, 12, 13, 14, 15). In every case, the peptide binds in a extended conformation
with residues at relative peptide positions P1, P4, P6, P7, and P9
projecting into pockets in the MHC class II molecule, and P-2, P-1, P2,
P3, P5, P8, P10, and P11 exposed to the solvent. Molecular modeling of
VP16 393–405 complexed with DRB1*0402 predicts a binding motif such
that V394 occupies pocket 1 and R397 occupies pocket 4, as follows:
 |
 |
In the diagonal binding mode observed in the class I MHC-peptide-TCR
crystal structures, the CDR1 loops interact with the peptide termini,
the CDR2 loops interact predominantly with the MHC helices, and the
CDR3 loops cover the central portion of the MHC-peptide ligand
(18, 19, 20). In the absence of crystallographic data for class II
MHC-peptide-TCR complexes, a similar diagonal binding orientation has
been predicted based on shared properties of the class I and class II
MHC molecules (19, 21). It is interesting to note the presence of three
arginine residues in the CDR3 sequence of the ESL4.34 TCR chain and
an arginine at position 97 in CDR3 of the TCR ß chain. A binding
configuration analogous to the crystal structures of the class I
MHC-peptide-TCR complexes would position the ESL4.34 CDR3 loops such
that these arginines could potentially interact with residues of the
negatively charged ILEDE epitope.
Our data indicate that although ESL4.34 displays promiscuous recognition of MHC class II molecules, its activation requirements are highly specific and depend on both the presence of the DRß 67–71 ILEDE sequence and the VP16 393–405 peptide. Although multiple DR alleles expressing the ILEDE epitope were able to present this peptide to ESL4.34, conservative substitutions at this HVR eliminated T cell reactivity. Although the presence of the DRß 67–71 ILEDE epitope has an influence on peptide binding, the relative affinity of the peptide-DR interaction does not have a major effect on the capacity of the ligand to stimulate ESL4.34. Instead T cell recognition appears to depend on specific interactions between the TCR and DR residues 67, 70, and 71, and peptide residues such as methionine at P5 and phenylalanine at P7, as discussed above. Other workers have also found that T cell responses do not depend on the affinity of peptide for MHC (59) and conservative substitutions in antigenic peptides and MHC molecules can have more dramatic effects on T cell responsiveness than nonconservative substitutions or unrelated peptides (27, 28, 29, 30, 31, 32). In this respect, the pluriallelic T cell recognition by clone ELS4.34 is reminiscent of recognition patterns of alloreactive T cells, in which specific recognition of particular DRß HVR3 epitopes has been shown to be essential for the activation of many alloreactive T cell clones (40, 58, 59).
The peptide-self-MHC ligands recognized by developing thymocytes have an important influence on the selection of the peripheral T cell repertoire. In particular, the DRß HVR3 residues have been shown to determine the specificity of positive selection and exert a strong bias on the nature of ligands recognized by T cells selected in an individual (60, 61). Promiscuous DR-restricted T cell recognition of Ag has been seen in the context of both parental alleles (48), consistent with the finding that ESL4.34 recognizes VP16 393–405 in the context of the parental alleles, DRB1*0402 and *1301. The ILEDE epitope, present on both alleles, likely amplified the selection bias for this (and related) specificities.
This observation may have important implications for the selection of autoreactive T cells. The third HVR of DRß is thought to have a central role in susceptibility to autoimmune diseases including RA (34, 35) and autoimmune hepatitis (62). In RA, the genetic element most highly associated with this disease is a shared epitope consisting of the amino acid sequences LLEQRRAA or LLEQKRAA at DRß positions 67–74, which are present on HLA alleles that are associated with RA (DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0101, and DRB1*1402) in different populations (35). Interestingly, DRB1*0401/*0404-positive individuals, who are heterozygous for these two HVR3 sequences, have an increased risk of developing RA compared with the risk associated with possession of either HVR3 alone. This might reflect the influence of the HVR3s of both parental alleles in biasing the selection of T cells that exhibit promiscuous recognition of Ags in the context of both alleles, potentially contributing to the specificity of T cell responses that result in autoimmune activation in RA. We hypothesize that the HVR3 portion of the peptide-MHC ligand is a discrete site of T cell recognition that may itself function as a dominant selection element for autoreactive T cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Education and Research Centre, St. Vincent’s Hospital, Elm Park, Dublin 4, Ireland.
3 Address correspondence and reprint requests to Dr. Gerald T. Nepom, Virginia Mason Research Center, 1000 Seneca Street, Seattle, WA 98101-2744.
4 Abbreviations used in this paper: CDR, complementarity-determining region; HVR, hypervariable region; RA, rheumatoid arthritis; HSV-2, herpes simplex type 2 virus; HSV-1, herpes simplex type 1 virus; LCL, lymphoblastoid cell lines.
Received for publication April 27, 1998. Accepted for publication June 5, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
,ß1*0401) molecule is a major determinant of T cell recognition of peptide. J. Exp. Med. 181:915.ß T cell receptor structure at 2.5 angstrom and its orientation in the TCR-MHC complex. Science 274:209.-ß heterodimers of mixed allotype. J. Immunol. 141:3123.[Abstract]
-helix motif shared by DR11 and DR8 alleles is implicated in the pluriallelic restriction of peptide-specific T cell lines. Hum. Immunol. 40:279.[Medline]
,ß1*0401)-restricted T cell antigen recognition. J. Immunol. 155:1951.[Abstract]
,ß1*0402)-restricted T-cell clone. Hum. Immunol. 41:193.[Medline]
ß sequences of crossreactive anti-DR alloreactive T-cell clones: identification of possible contact residues based on charge complementarity between TCR chains and DR determinants. Eur. J. Immunogenet. 19:21.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. Kudela, B. Janjic, J. Fourcade, F. Castelli, P. Andrade, J. M. Kirkwood, T. El-Hefnawy, M. Amicosante, B. Maillere, and H. M. Zarour Cross-Reactive CD4+ T Cells against One Immunodominant Tumor-Derived Epitope in Melanoma Patients J. Immunol., December 1, 2007; 179(11): 7932 - 7940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sospedra, P. A. Muraro, I. Stefanova, Y. Zhao, K. Chung, Y. Li, M. Giulianotti, R. Simon, R. Mariuzza, C. Pinilla, et al. Redundancy in Antigen-Presenting Function of the HLA-DR and -DQ Molecules in the Multiple Sclerosis-Associated HLA-DR2 Haplotype J. Immunol., February 1, 2006; 176(3): 1951 - 1961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Voo, G. Peng, Z. Guo, T. Fu, Y. Li, L. Frappier, and R.-F. Wang Functional Characterization of EBV-Encoded Nuclear Antigen 1-Specific CD4+ Helper and Regulatory T Cells Elicited by In vitro Peptide Stimulation Cancer Res., February 15, 2005; 65(4): 1577 - 1586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Maccalli, Y. F. Li, M. El-Gamil, S. A. Rosenberg, and P. F. Robbins Identification of a Colorectal Tumor-Associated Antigen (COA-1) Recognized by CD4+ T Lymphocytes Cancer Res., October 15, 2003; 63(20): 6735 - 6743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.-G. Lim, J. M. Slavik, K. Bourcier, K. J. Smith, and D. A. Hafler Allelic Variation of MHC Structure Alters Peptide Ligands to Induce Atypical Partial Agonistic CD8+ T Cell Function J. Exp. Med., July 7, 2003; 198(1): 99 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, R. Turner, B. M. Baker, and W. E. Biddison MHC Allele-Specific Molecular Features Determine Peptide/HLA-A2 Conformations That Are Recognized by HLA-A2-Restricted T Cell Receptors J. Immunol., September 15, 2002; 169(6): 3146 - 3154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Gebe, E. J. Novak, W. W. Kwok, A. G. Farr, G. T. Nepom, and J. H. Buckner T Cell Selection and Differential Activation on Structurally Related HLA-DR4 Ligands J. Immunol., September 15, 2001; 167(6): 3250 - 3256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Joshi, P. R. Suresh, and V. S. Chauhan Flexibility in MHC and TCR Recognition: Degenerate Specificity at the T Cell Level in the Recognition of Promiscuous Th Epitopes Exhibiting No Primary Sequence Homology J. Immunol., June 1, 2001; 166(11): 6693 - 6703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Doherty, S. Norris, L. Madrigal-Estebas, G. McEntee, O. Traynor, J. E. Hegarty, and C. O'Farrelly The Human Liver Contains Multiple Populations of NK Cells, T Cells, and CD3+CD56+ Natural T Cells with Distinct Cytotoxic Activities and Th1, Th2, and Th0 Cytokine Secretion Patterns J. Immunol., August 15, 1999; 163(4): 2314 - 2321. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
