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* Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and
Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although side chain substitutions in PFRs rarely affect peptide binding affinity, the consequence of removing an entire residue is variable. For instance, structural studies have shown that MHC:peptide backbone interactions also involve PFRs (4, 5, 7) and that P-1 in particular appears to be important for peptide stability (21). In contrast, residues at P10 and P11 are frequently removed by Ag processing (7, 17, 22). It has also been noted that the identity of the PFR can contribute to the efficiency of Ag processing (20, 23).
Until recently, it was not clear whether PFRs could be recognized by
the TCR, modulate T cell responses, or influence immunogenicity
directly. We have shown that a significant percentage of hen egg
lysozyme (HEL) 48–63-specific T cells are dependent on the P11
COOH-terminal PFR W62, which results in a profound restriction of
TCR-V
usage (19, 24). What remains unclear is whether
PFR recognition is an infrequent occurrence or a common event
characteristic of TCR recognition of MHC class II:peptide complexes.
Invariably, the PFRs at P-1 and P11 of all of the MHC class II:peptide
complexes solved to date are solvent exposed and accessible to the TCR.
Thus, we would hypothesize that most T cell epitopes have the capacity
to generate PFR-dependent T cells and that PFR recognition may be
restricted to P-1 and P11.
Several factors have been shown to influence immunogenicity and immunodominance, including Ag processing, MHC binding, and the TCR repertoire (20, 25, 26, 27, 28). Our initial data suggested that PFRs may be another contributing factor because the substitution or removal of the HEL 48–63 W62 PFR was found to significantly reduce the immunogenicity of this epitope (19). We have also shown that PFR-dependent T cells can be potently antagonized by peptides that lack COOH-terminal PFRs and that these cells respond poorly to native protein or the immunogenic epitope delivered by a recombinant influenza virus (29). These data suggest that Ag processing can generate both stimulatory and antagonist peptides from a single immunogenic epitope. Interestingly, recent studies have shown that background genes in the nonobese diabetic (NOD) mouse, a model for type 1 diabetes, strongly influence Ag processing (30). Taken together, these observations suggest that the biology of PFRs may have considerable significance for immunogenicity, T cell immunoregulation, and autoimmunity.
In this study, we defined the peptide binding resister for nine epitopes in HEL and glutamate decarboxylase (GAD) using a novel approach. This analysis allowed us to formally define the flanking residues. We then analyzed T cell responses to these epitopes to address the following questions: 1) what proportion of MHC class II-restricted epitopes generate PFR-dependent T cells; 2) which PFRs are recognized; and 3) does PFR recognition always influence immunogenicity and could it affect immunodominance?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2aFc fusion proteins
The basic design of the MHC class II:Ig fusion proteins
used in the peptide binding assay is similar to that described by
Wucherpfennig and colleagues (31), with modifications.
Details of their construction, production, and purification can be
found elsewhere.4 Briefly, the extracellular MHC
class II 
- and -chains of H-2Ak,
H-2Ag7, or H-2Ek were
linked via a fos/jun leucine zipper to the hinge
and Fc domain of murine IgG2a. The constructs were transfected into
Drosophila melanogaster Schneider 2 cells according to the
manufacturer’s instructions (Invitrogen, Carlsbad, CA). After
hygromycin selection, the Drosophila melanogaster Schneider
2 transfectants were grown to a density of 1–2 x
107 cells/ml at 22°C and seeded at 3 x
106 cells/ml into serum-free medium, and protein
production was induced with 500 µM CuSO4. The
fusion protein was purified over a protein A column (Repligen, Needham,
MA) using a Biologic HR (Bio-Rad, Hercules, CA). After concentration
and buffer exchange into PBS/0.05% NaN3, the
fusion protein was adjusted to 2 mg/ml for storage at 4°C. Production
and quantitation were monitored by ELISA.
Peptides
Wild-type, PFR alanine-substituted, and biotinylated peptides used in functional and peptide binding assays were synthesized on a Rainin Symphony (Woburn, MA) purified by HPLC, and quantified by amino acid analysis. The arginine- and glutamic acid-substituted peptides used for binding register determination were produced by pin synthesis on an ACT 396 Omega (Advanced ChemTech, Louisville, KY) and quantified by absorbance at 210 nm (specific for peptide bonds), using the appropriate wild-type peptide described above as a reference. Amino acid analysis of selected pin peptides demonstrated a concentration variance of no more than 2-fold from the concentration determined by OD. Peptide biotinylation was performed on the resin in two steps: 1) addition of a caproic acid linker (spacer), and 2) addition of the biotin moiety. All peptides were produced by the Hartwell Center, St. Jude Children’s Research Hospital (Memphis, TN), and verified using analytical HPLC and mass spectrometry.
MHC class II peptide binding assay
Fusion proteins H-2Ak:2aFc (6 µg/ml),
H-2Ek:2aFc (15 µg/ml), or
H-2Ag7:2aFc (4 µg/ml) were incubated with
the appropriate biotinylated peptide, HEL 46–61 (400 nM), HEL 81–97
(2 µM), or GAD 220–236 (500 nM), respectively, and a titration of
mutant competitor peptide at the concentrations indicated in McIlvaine
citrate phosphate buffer, pH 5 (32), for 24–48 h in
96-well preblocked (5% BSA, TBS Tween 20) plates.
Na2HPO4 (20 µl, 1 M) and
BSA (30 µl, 10%) were added to the reactions and transferred to
Immulon 4 HBX ELISA plates (Dynex Technologies, Chantilly, VA) that
were previously coated with anti-mouse IgG2a (R11–89; BD
PharMingen, San Diego, CA) for 1 h at 37°C, washed, and blocked
for 1 h at 37°C. Plates were washed and probed with
streptavidin-HRP (1:5000 in 0.05% Tween 20 PBS; Zymed Laboratories,
San Francisco, CA), developed with one-step Turbo TMB-ELISA (Pierce,
Rockford, IL), and read at 450 nm.
Generation of murine T cell hybridomas
T cell hybridomas were generated as previously described (19). B10.BR and NOD mice (The Jackson Laboratory, Bar Harbor, ME) were used to generate T cell-specific HEL and GAD peptides, respectively. Some fusions were also performed with cells from mice immunized with either HEL protein (Sigma-Aldrich, St. Louis, MO) or rHEL.L56F/W62Y produced in Pichia pastoris yeast (29). Preliminary screening involved analysis of CD3 and CD4 expression by flow cytometry and of IL-2 production in response to the immunogen and native protein where appropriate.
Ag presentation assays
Assays were performed essentially as described elsewhere (19, 24, 33). Briefly, T cell hybridomas (5 x 104/well) were stimulated with LK35.2 (for HEL-specific hybrids) or M12C3.g7 (for GAD 65-specific hybrids; kindly provided by E. Unanue, Washington University, St. Louis, MO), APCs (2.5 x 104/well), and peptides at the concentrations indicated. Synthetic peptides were made using either standard Fmoc chemistry or Chiron pin technology in the Hartwell Center. Purity and quality were verified by HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Supernatants (100 µl) were removed after 24 h for estimation of IL-2 secretion by culturing with the IL-2-dependent cell line CTLL-2 (1 x 104 cells in 100 µl). After 24 h of culture, proliferation was determined by pulsing with [3H]thymidine (1 µCi/well; DuPont Pharmaceuticals, Wilmington, DE) for the final 6 h. These assays were used to determine the concentration of peptide (EC50) required to stimulate a 50% maximal CTLL response.
Flow cytometry
Analysis of T cell hybridomas for CD3 and CD4 expression was performed as described elsewhere (Abs from BD PharMingen) (24, 33).
Lymph node proliferation assays
Assays were performed as previously described (19). Briefly, B10.BR mice (The Jackson Laboratory) were immunized with 14 nmol of HEL, HEL peptide, or mutant peptide as described above. After 7–10 days, lymph node cells were removed and cultured in 96-well flat-bottom plates (6 x 105/well) with HEL protein or peptides in complete S-MEM with 10% FCS. Proliferation was measured by pulsing with [3H]thymidine (1 µCi/well) during the last 24 h of a 96-h culture.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of PFRs that are recognized by the TCR is dependent on the accurate establishment of the MHC class II peptide binding register for each epitope. The conventional and widely used approach employs truncated peptides or single amino acid substitutions to define MHC binding residues (33, 34, 35, 36, 37, 38, 39). However, this does not always lead to definitive answers. Here, we present a new approach using a standard MHC:peptide competition assay with peptides containing five amino acid substitutions at defined positions.
The crystallographic analysis of 14 MHC class II:peptide complexes has clearly shown that the periodicity of peptide binding is essentially conserved (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Thus, P1, P4, P6, and P9 always point toward the MHC, whereas P-1, P2, P5, P8, and P11 always point toward the TCR. Because most of the binding energy between MHC class II molecules and the peptide is usually confined to the peptide backbone, "removal" of a side chain by alanine substitution often has little or no effect on peptide binding. Indeed, some investigators have found that polyalanine (or most residues substituted for alanine) can bind to MHC class II molecules with reasonable affinity (2, 40). However, large bulky and/or charged residues are often not tolerated. We reasoned that substitution of all of the potential TCR binding residues with a large, charged residue, such as arginine or glutamic acid, would not significantly affect peptide binding. However, such substitutions in two or more side chains pointing toward the MHC are likely to disrupt binding.
On this premise, we synthesized three mutant peptides for each epitope
for use in an ELISA-based competition peptide binding assay using
soluble MHC class II dimers. First, the binding register for each
epitope was predicted on the basis of previous structural and
functional studies (2, 3, 4, 7, 36, 41, 42, 43, 44, 45, 46). See Fig. 1
for details of the peptides used.
Second, mutant peptides were produced with the spacing x, x + 2, x + 5,
x + 8, x + 11, which is the same as that between the TCR-exposed
residues at P-1, P2, P5, P8, and P11. One of the three mutant peptides
had substitutions at P-1, P2, P5, P8, and P11 for this predicted
register, whereas the other two peptides had these substitutions
"slid" one amino acid toward the NH2 or COOH
terminus. If the predicted binding register is correct, this would
result in the latter two peptides containing two or three
substitutions, respectively, of side chains that point toward the MHC.
We chose to use arginine as the substituting amino acid due to its size
and charge. For peptide residues that were arginine or lysine, glutamic
acid was used as the substituting amino acid.
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). In
almost all cases, only one of the three mutant peptides bound to its
respective MHC allele. For certain peptides (HEL 1–18 and HEL
85–100), more than one peptide bound. However, there was one peptide
for each epitope that was clearly superior to the other two in the set.
The only exception was for HEL 116–129, in which case two of the three
peptides bound comparably, albeit weakly. This may have been due to the
weak binding of these peptides and should be taken into account when
assessing data presented using this epitope. All of our initial
predictions were correct with the exception of GAD 284–300. Two
potential registers were proposed (2), and we chose to
make peptides for the NH2-terminal register.
However, our data clearly show that the COOH-terminal register is in
fact correct.
These experiments allowed us to define the epitopes within the nine
peptides examined as HELe24–32,
HELe52–60, HELe74–82,
HELe119–127, HELe6–14,
HELe88–96, GADe209–217,
GADe289–297, and
GADe527–535 (Fig. 2). To avoid confusion between core
epitopes and synthetic peptide sequences in this study, core MHC class
II binding epitopes (P1–P9) will be denoted by the suffix "e" and
superscripted (e.g., HELe52–60). Synthetic
peptides will be noted in the conventional way (e.g., HEL 48–63).
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We have previously shown that T cell hybridomas generated against
HELe52–60 were predominantly dependent on the
W62 PFR (19). To study T cell dependence on PFRs further,
we generated T cell hybridomas specific for six HEL epitopes defined
above by immunization with HEL protein or peptides. Only T cell
hybridomas (>90%
CD4+CD3+; 130 in total)
that responded in vitro to the native HEL protein (as measured by IL-2
production) were analyzed further. PFR dependency was determined using
a panel of peptides containing alanine substitutions at PFRs.
PFR-independent hybridomas were defined by their ability to respond to
all of the peptides, whereas PFR-dependent hybridomas responded to all
peptides except those that contained substitutions at a single PFR.
PFR-dependent T cell hybridomas (40% of total) were generated against
all of the H-2Ak- and
H-2Ek-restricted epitopes studied, except for
HELe6–14, which only generated 11 hybridomas
(Table I). The percentage of T cell
hybridomas that were dependent on a particular PFR varied from 12
to 83%.
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). Importantly, PFR-dependent T cell
hybridomas were obtained from mice immunized with HEL protein,
indicating that PFR-dependent T cells are generated from naturally
processed peptides. In almost all cases, PFR dependency was absolute in
that T cell hybridomas did not respond to the mutant peptides
containing the PFR alanine substitutions even at the highest
concentration, but they responded strongly to the wild-type peptide
(Figs. 3 and 4 and data not shown). In
contrast, PFR-independent hybridomas responded comparably to both
peptides. Dependence on PFR was not a consequence of reduced
sensitivity to Ag, because PFR-dependent and -independent T cells
responded with equal sensitivities to most wild-type peptides (Fig. 4).
The exception was HELe88–96-specific T cells
dependent on D87. It is possible that tolerance induction toward this
residue, which is conserved between HEL and murine lysozyme (ML), may
limit the sensitivity of these PFR-dependent T cells.
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and Fig. 5). Titration
analysis showed that the PFR-independent T cell hybridomas specific for
GADe209–217 responded equally to GAD 206–220
and GAD 206-E208A-220 (data not shown). All of the
GADe289–297-specific T cell hybridomas generated
were PFR-independent. It is noteworthy that the four P-1/P11 residues
to which PFR dependency was not observed were either small or
hydrophobic amino acids (V219, G288, V299, and L526).
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). Although HEL 71-R73A-86 bound
with slightly reduced affinity, this only had a small effect on the
ability of PFR-independent T cells to respond to the mutant vs
wild-type peptides (Fig. 3 and data not presented), suggesting that
this cannot explain the inability of PFR-dependent T cells to respond
to the mutant peptides.
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PFRs potentiate T cell responses and contribute to peptide immunogenicity
Previous studies in our laboratory have demonstrated that the PFR
W62 significantly enhances the immunogenicity of the HEL 48–63 peptide
(19). Therefore, we evaluated whether PFR recognition
always leads to increased immunogenicity. First, we determined the
immunodominance hierarchy of the various HEL peptides by in vitro
restimulation of lymph node T cells from B10.BR mice
(H-2k) immunized with HEL protein (Fig. 7). The T cell response was
dominated by the H-2Ak-restricted epitopes
HELe24–32, HELe119–127,
and HELe52–60, all of which generated a
significant percentage of PFR dependency (22, 57, and 68%,
respectively; see Table I). In contrast, most of the weaker epitopes
generated no or low percentages of PFR-dependent T cell hybridomas
(0–12%), with the exception of the HELe74–82
(83%). It is noteworthy that although the lymph node proliferation
response to this peptide was weak, a significant percentage of the
hybridomas generated from the HEL fusion were specific for this epitope
(37%). Thus, there may be a relationship between PFR dependency and
immunodominance.
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; compare 
in the center
graphs with 
in the right graphs). Indeed, wild-type
peptides derived from the epitopes that generated PFR dependency were
also more immunogenic than those that did not (e.g., HEL 1–18) (Fig. 8; compare in the center graphs). The most striking
observation was that two of the epitopes that generated the greatest
percentage of PFR-dependent T cell hybridomas
(HELe24–32 and
HELe119–127) also generated a much stronger
recall response to the native HEL protein, compared with epitopes that
generated few or no PFR-dependent T cells
(HELe88–96 and HELe6–14,
respectively) (Fig. 8; compare 
(HEL) in the center
graphs). In contrast, peptides lacking critical PFRs were
generally weak immunogens, inducing a poor in vitro recall response to
full-length and mutant peptides as well as to native HEL. The
relatively weak recall response to HEL after immunization with HEL
85–100 is consistent with the low percentage of T cell hybridomas
dependent on D87 (6%), which may be due to tolerance induced by this
HEL:ML conserved residue (Fig. 2). The only exception to this general
finding was HELe74–82, which generates a high
percentage of PFR dependency but is a weak immunogen (compare Table I
with Fig. 7). Taken together, these results demonstrate that T cell
recognition of PFRs is an important factor that contributes to peptide
immunogenicity and may influence immunodominance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Is there another explanation for our data other than direct recognition of PFRs by the TCR? It has been suggested that some peptides may bind in two alternate registers (i.e., the assignment of the P1 residues may shift by one or two residues either way) (2, 5). Although this is theoretically possible, no crystallographic data have been presented to directly support this idea. Indeed, H-2Ag7-GAD 65 221–235 has been speculated to bind with the core sequence of 224–232 or 226–234. However, only 224–232 was observed at P1–P9 in the crystal structure (2). Likewise, H-2Ad-OVA 323–339 may bind with the core sequence of 324–332, 329–337, or 326–334, but only 324–332 was observed at P1–P9 in the structure (5). Our binding experiments clearly showed that all but one epitope bound in a single register. The only exception was HELe119–127, which may bind in two registers. However, because these peptides bound poorly, an unequivocal answer could not be obtained. There are two further reasons why peptides are unlikely to bind in alternate registers or that PFR substitutions alter this register. First, if PFR substitution alters peptide binding register, then PFR-independent hybridomas would also fail to respond to mutant peptides because the key residue at P5 would no longer be accessible. Second, if some of the register assignments were incorrect, it is unlikely that only P-1 and P11 substitutions would have affected T cell function. For example, a single amino acid shift to the right in the register would move the residue in the P-1 position to P-2 and P11 to P10. This is not evident from our studies because we did not generate hybrids dependent on PFRs in other positions (P-3, P-2, P10, and P12 were analyzed in this study).
What factors influence whether residues at P-1 and P11 mediate PFR
dependency? There are three possibilities: 1) the type of amino acid,
2) tolerance induced by the murine homologue/molecule, and 3) Ag
processing. Interestingly, most of the residues at P-1 and P11 that do
mediate PFR dependency are capable of forming either a salt bridge or
hydrogen bond (Y, W, R, D, T, and E; Fig. 2 and Table II). The one exception is a methionine
(GAD.M537). In contrast, most of the residues that did not mediate PFR
dependency were small and/or hydrophobic (G, F, V, L, and I). Such
residues may not mediate as strong an interaction with the TCR, which
may be required for PFR dependency. One caveat, however, involves our
use of alanine substitutions to identify residues that mediate PFR
dependency. For some residues, alanine may be considered a conservative
substitution that fails to abrogate PFR dependency. A similar analysis
using peptides with different amino acid substitutions at PFRs would
have to be performed to rule out this possibility. Second, tolerance
induced by the murine homologue/molecule may also influence the
generation of PFR-dependent T cells. We found PFR dependency on the P-1
residues R73 and T118 (which are unique to HEL), yet we found no PFR
dependency on the P-1 residues R5 and T51 (which are conserved between
HEL and ML) (see Fig. 2). The only conserved HEL:ML residue that did
mediate PFR dependency was D87, although only four hybridomas were
identified and these were relatively insensitive (see Fig. 4). Lastly,
efficient Ag processing may limit the proportion of naturally processed
peptides that contain the P-1 or P11 residues (17, 19, 29). However, peptide elution studies performed to date suggest
that efficient processing is unlikely to result in all the naturally
processed peptides lacking either the P-1 or P11 residues
(14, 15, 16, 17, 18).
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One of the most striking features of PFR dependency is the fact that it
is absolute. All but 7 of the 105 PFR-dependent T cell hybridomas
(93%) examined to date completely failed to respond to the maximal
concentration (10 µM) of peptide containing the relevant PFR
substitution (this study and Ref. 19). Indeed, we have
previously shown that PFR-dependent T cells will not even respond to B
cells that express a single recombinant MHC:peptide complex, which
increases the effective ligand density 100-fold (19). Why
does the absence of the appropriate PFR have such a profound affect on
T cell function? One possible explanation is that an unusually large
proportion of the binding energy is directed toward this residue. The
TCR-VV domains are constrained by their requirement to interact
productively with the MHC molecule. Therefore, TCR selection of the
optimal TCR VV combination for recognition of P-1/P11 residues
may seem more difficult than for selection of the best CDR3 to
interact optimally with the peptide P5 residue. However, Ding et al.
(51) have shown that different TCRs recognizing the same
MHC:peptide complex can interact with almost completely different
residues, suggesting that there is great plasticity in TCR:MHC
recognition. Thus, the TCR repertoire may select the ideal V region to
interact with a particular PFR without compromising its ability to bind
MHC. This idea would be consistent with the profound restriction of
TCR-V usage observed among PFR-dependent T cells (19).
Additional structural and functional studies will be required to
resolve this important issue.
In this study, we have clearly shown that PFR recognition has a
significant effect on immunogenicity and thus may constitute an
important physiological role for PFRs. This is evident from a direct
comparison of the immunogenicity of wild-type and mutant peptides and
from the strength of the recall response to peptides after HEL
immunization or in response to HEL after peptide immunization. Although
five of the six H-2A/Ek-restricted, HEL-specific
epitopes generated PFR-dependent T cells, their proportion varied
(12–83%). Indeed, the contribution afforded by PFR dependency to the
immune response can be seen most clearly by comparing epitopes that
generated high and low numbers of PFR-dependent T cells. There was a
good correlation between the percentage of T cell hybridomas that were
PFR-dependent and the consequence of PFR substitution on diminution of
the T cell response to HEL protein or peptides (see Table I and
Fig. 8).
How could PFR dependency contribute to immunodominance? Although this
is difficult to assess conclusively from our data, it is evident that
the epitopes that generated the largest recall response after
immunization with HEL protein (HELe24–32,
HELe52–60, and
HELe119–127) all generate significant
percentages of PFR-dependent T cell hybridomas (22, 68, and 57%,
respectively; see Table I). This is in contrast with most of the weaker
epitopes (0–12%). The only exception was
HELe74–82, which induces a weak T cell response
after HEL immunization, yet 83% of the
HELe74–82-specific hybridomas were PFR
dependent. These data are consistent with previous studies suggesting
that the recall response to this epitope is weak/absent in the absence
of R73 (26). It is possible that this epitope is poorly
processed or binds weakly to H-2Ak and is thus a
poor immunogen as part of the native protein. The very high percentage
of PFR-dependent T cells observed with this epitope may be due to the
composition of the available TCR contact residues because the crucial
P5 residue and P8 are conserved between HEL and ML, and P2 and P11 are
leucines. This may make the nonconserved, charged arginine residue at
P-1 particularly "attractive" to the TCR repertoire. An accurate
assessment of the contribution afforded by PFR dependency to
immunodominance using MHC class II:peptide tetramers or analogous
staining reagents could prove enlightening and is worthy of further
investigation.
In summary, our data support the idea that PFR dependency is a common characteristic of MHC class II-restricted T cell responses. Indeed, a number of studies have presented data consistent with PFR dependency (23, 52, 53, 54, 55, 56, 57). Interestingly, most of these studies have involved the analysis of human T cell clones, suggesting that PFR dependency is not restricted to the mouse. We would speculate that the TCR repertoire is capable of recognizing the P-1 and P11 residues in any immunogenic epitope, whereby recognition is limited by the type of amino acid tolerance exerted to the host epitope and Ag processing. With this information, we may be able to predict whether PFR recognition is likely to occur for other T cell epitopes. It is particularly interesting that PFR dependency may be one of the most prominent and significant characteristics distinguishing TCR recognition of peptide-bound MHC class I and class II molecules. How might PFR dependency be relevant to MHC class II-restricted T cell responses? Given the large number of peptide residues in MHC class II:peptide complexes that are accessible to the TCR, it is possible that this results in an increased initial precursor frequency. Alternatively, we have previously speculated that PFR may play an immunomodulatory role by regulating the response of PFR-dependent T cells (29). It is also possible that the increased number of TCR:peptide vs TCR:MHC contacts may influence the efficiency of positive and negative selection for MHC class II-restricted T cells. Although the physiological benefit has yet to be fully elucidated, it is clear that PFR dependency significantly contributes to overall immunogenicity and may influence the immunodominance hierarchy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dario A. A. Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address: dario.vignali{at}stjude.org
3 Abbreviations used in this paper: PFR, peptide-flanking residue; HEL, hen egg lysozyme; NOD, nonobese diabetic; GAD, glutamate decarboxylase; ML, murine lysozyme.
4 P. Y. Arnold, K. M. Vignali, T. B. Miller, N. L. La Gruta, L. S. Cauley, L. Haynes, P. S. Adams, S. L. Swain, D. L. Woodland, and D. A. A. Vignali. Reliable generation and use of MHC class II:2aFc multimers for the identification of antigen-specific CD4+ T cells. Submitted for publication.
Received for publication June 5, 2001. Accepted for publication May 9, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. 19:5611.[Medline]
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 K. M. Williams and E. C. Bigley III Identification of an I-Ed-Restricted T-Cell Epitope of Escherichia coli Outer Membrane Protein F Infect. Immun., July 1, 2004; 72(7): 3907 - 3913. [Abstract] [Full Text] [PDF]
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L. M. Velloso, J. Michaelsson, H.-G. Ljunggren, G. Schneider, and A. Achour Determination of Structural Principles Underlying Three Different Modes of Lymphocytic Choriomeningitis Virus Escape from CTL Recognition J. Immunol., May 1, 2004; 172(9): 5504 - 5511. [Abstract] [Full Text] [PDF]
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M. W. Anderson and J. Gorski Cutting Edge: TCR Contacts as Anchors: Effects on Affinity and HLA-DM Stability J. Immunol., December 1, 2003; 171(11): 5683 - 5687. [Abstract] [Full Text] [PDF]
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M.-H. Jang, N. P. Seth, and K. W. Wucherpfennig Ex Vivo Analysis of Thymic CD4 T Cells in Nonobese Diabetic Mice with Tetramers Generated from I-Ag7/Class II-Associated Invariant Chain Peptide Precursors J. Immunol., October 15, 2003; 171(8): 4175 - 4186. [Abstract] [Full Text] [PDF]
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E. I. Buzas, A. Hanyecz, Y. Murad, F. Hudecz, E. Rajnavolgyi, K. Mikecz, and T. T. Glant Differential Recognition of Altered Peptide Ligands Distinguishes Two Functionally Discordant (Arthritogenic and Nonarthritogenic) Autoreactive T Cell Hybridoma Clones J. Immunol., September 15, 2003; 171(6): 3025 - 3033. [Abstract] [Full Text] [PDF]
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