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CUTTING EDGE |
*
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38101; and
Department of Pathology, University of Tennessee Medical Center, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We have recently shown that T cells can directly recognize and are highly dependent on this PFR, profoundly effecting TCR-Vß usage and peptide immunogenicity (5). Numerous studies have shown that altered peptide ligands can prevent or modulate T cell function (6, 7, 8). Substitution of peptide residues can alter recognition by the TCR and generate peptides that either inhibit T cell responses to the agonist peptide (antagonist peptides) or have no effect on responses to the agonist peptide (null peptides). Given that Ag processing of HEL can simultaneously generate peptides that contain and lack PFR, can exposure of PFR-dependent T cells to this naturally processed mixture of peptides modulate their function?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Wild-type HEL and mutants were generated by recombinant PCR and cloned into pPIC-9 (Invitrogen, San Diego, CA). rHEL was made in Pichia pastoris yeast according to the manufacturers instructions (Invitrogen). This gave rise to a soluble, secreted product with six additional amino acids (EAEAYG) at the N terminus before the start of the native protein. T cell responses to the recombinant and native HEL (L6876; Sigma, St. Louis, MO) were indistinguishable. rHEL protein was purified from yeast broth by conventional chromatography using SP Sepharose (Pharmacia, Piscataway, NJ), CHT (Bio-Rad, Richmond, CA), and Superdex 75 (Pharmacia) run on a BioLogic Chromatography System (Bio-Rad). The purified protein gave a single band on silver-stained SDS-PAGE gels and a single species of the correct m.w. by mass spectrometry. A detailed production and purification protocol is available on request (dario.vignali@stjude.org). In addition to the wild-type rHEL (rHEL.wt), two mutants were made. rHEL.W62/63A is the complete HEL protein with the two PFR W62 and W63 replaced with alanine. rHEL.L56F.W62Y has the two TCR contact residues, L56 and W62, which are the only residues in this epitope that differ between HEL and ML replaced with the analogous ML residues phenylalanine and tyrosine, respectively.
Ag presentation and antagonism assays
Ag presentation assays were performed essentially as described elsewhere (5, 9). Briefly, T cell hybridomas were stimulated with synthetic peptides (Center for Biotechnology core facility at St. Jude Children’s Research Hospital, or Chiron Technologies, Emeryville, CA (Mimotopes)) at the concentrations indicated, together with LK35.2 as APC (murine B cell lymphoma; H-2Akd). After 24 h, supernatants were removed for the estimation of IL-2 secretion against a recombinant murine IL-2 standard (Genzyme, Cambridge, MA) by culturing with the IL-2-dependent T cell line CTLL-2. Antagonism assays were set up in the same way except that the APC were first prepulsed with the agonist, washed three times, and then put into the assay with the concentration of antagonist peptide indicated.
In some experiments, the following reagents were also used: 1) Various concentrations and combinations of the rHEL proteins described above. 2) LK.HEL is the LK35.2 B cell lymphoma transfected with HEL 1–80 attached to H-2Kk transmembrane and cytoplasmic tail (10). It was used instead of the LK35.2 cells as APC with or without the addition of 3 µM of rHEL.W62/63A. 3) HEL-flu is HEL 46–63 inserted into the neuraminidase stalk of influenza A virus (A/WSN/33 (H1N1)) (11). LK35.2 cells plus HEL-flu was added per well, with or without the addition of 3 µM of rHEL.W62/63A.
CD3
tyrosine phosphorylation analysis
CD3 tyrosine phosphorylation analysis was
determined as previously described (12). Briefly, LK35.2 cells pulsed
with 3 µM peptide were mixed with T cell hybridomas and incubated at
37°C for 5 min. The cell pellet was lysed in 1% Brij 97
(polyoxyethylene 10 oleyl ether; Sigma) at room temperature (RT) for
1 h and immunoprecipitated with a rabbit anti-CD3 antisera
(2 µl, No. 387, gift from Larry Samelson, National Institutes of
Health, Bethesda, MD) for 2 h at RT followed by 25 µl of protein
A-Sepherose (Pharmacia) for 1 h at RT. Eluted proteins were
resolved by SDS-PAGE and transferred onto poly(vinylidene difluoride)
membrane (Schleicher & Schuell, Keene, NH). Blots were blocked with 5%
BSA (Boehringer Mannheim, Indianapolis, IN) in TBS-T, and tyrosine
phosphorylation was detected with biotinylated 4G10 for
60 min at RT (0.1 µg/ml; Upstate Biotechnology, Lake Placid,
NY), followed by 1:12,000 dilution of streptavidin-horseradish
peroxidase preformed complexes (60 min at RT) (Amersham, Arlington
Heights, IL). Blots were developed using ECLplus (Amersham). To detect
the original protein, blots were stripped in 100 mM of 2-ME (Bio-Rad),
2% SDS, and 62.5 mM of Tris-HCl (pH 6.7) for 30 min at 50°C; washed
three times; and blocked with 5% nonfat dry milk in TBS-T at 4°C
overnight. Blots were probed with the rabbit anti-CD3 antisera
for 90 min at RT (1:200), followed by protein A-horseradish peroxidase
for 60 min at RT (1:10,000) (Amersham). Blots were developed as
described above.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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8J33/V16J33.Vß8.3] vs WW236-
[TCR.V8J22.Vß8.3], and WW157+ [TCR.V4J23.Vß14] vs
WW221- [TCR.V4J45.Vß8.3], where the suffix indicates
their ability (+) or inability (-) to be antagonized) (5). Initially,
the ability of a panel of analogue peptides to antagonize T cell
responses to the agonist HEL 48–63 was tested (these included:
48-L56X-63 (where X was replaced with either N, D, Q, E, I, K, F, S, T,
Y, or V), 48-L56A-61AA, and 48-Q57A-63; Fig. 1
). HEL 48-Q57A-63 was identified as the
best analogue peptide antagonist for the T cell hybridomas WW241+ and
WW157+ (Figs. 1 and 2A). HEL
48-L56A-63 and 48-L56A-61AA, which bind to H-2Ak with an
affinity comparable to the agonist, had no effect on T cell function
and are thus null peptides. In contrast, WW236- and WW221- were not
antagonized by any of the peptides tested. Strikingly, WW241+ and
WW157+ were strongly antagonized by HEL 48–61 in a dose-dependent
manner (Fig. 2A). An equimolar concentration of the agonist
and the 48–61 antagonist gave a 29.0% and 81.5% reduction in IL-2
response, respectively (compared with 0% and 43.1%, respectively,
when 48-Q57A-63 was used as an antagonist). Thus 48–61, a naturally
processed HEL peptide (2, 3), was a much more potent antagonist than
the peptide analogue 48-Q57A-63. In contrast, the control T cell
hybridomas, WW236- and WW221-, were not antagonized by 48–61 (Fig. 2A).
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, a lower pp21 band and an upper pp23 band. Differential CD3
phosphorylation, in which only the lower band is
produced, has been shown to correlate with the delivery of a partial
signal and the induction of antagonism and anergy (12, 15, 16). Clear
differences were observed in the CD3 phosphorylation
pattern following stimulation of the hybridomas that were either
sensitive or resistant to antagonism (Fig. 2
B). While HEL
48-L56A-63 failed to induce CD3 phosphorylation, HEL
48–61 induced the lower pp21 tyrosine phosphorylated form
of CD3, but essentially none of the upper pp23 form in the
antagonizable hybridomas (WW241+ and WW157+). In contrast, HEL 48–61
did not induce any CD3 phosphorylation in the two
nonantagonizable hybridomas (WW236- and WW221-) above that
observed with the control peptide, HEL 48-L56A-63. These data indicate
that there are clear biochemical differences in the way these
hybridomas respond to antagonist peptides, which may underlie their
functional phenotype.
Since PFR-dependent T cells were antagonized by short peptides, can
naturally processed peptides generated from the native HEL protein that
lacked C-terminal PFR antagonize T cell responses to agonist peptides
generated from the same epitope? One of the two antagonizable T cell
hybridomas (WW157+) responded more weakly to HEL than its control
(WW221-) despite both producing identical IL-2 responses to HEL 48–63
(Fig. 3A). Could further
antagonism be observed in both WW241+ and WW157+ if the ratio between
naturally processed peptides containing and lacking the PFR Trp62 was
altered? This was achieved by the use of rHEL produced in P.
pastoris yeast. Three versions were generated: the wild-type
protein (rHEL.wt) and two mutants. One has the PFR W62 and W63 replaced
with alanine (rHEL.W62/63A), while the other has the two TCR contact
residues, L56 and W62, replaced with the analogous mouse lysozyme
residues, phenylalanine and tyrosine, respectively (rHEL.L56F.W62Y).
Differential processing of the recombinant mutant protein rHEL.W62/63A
can produce antagonist, but not agonist peptide. By increasing the
concentration of rHEL.W62/63A while keeping the concentration of
rHEL.wt fixed, the ratio of peptides generated that lack W62 (the
antagonists) over the concentration of peptides that contain the W62
(the agonists) would increase. Our data show that both of the T cell
hybridomas that were susceptible to antagonism (WW241+ and WW157+)
could be significantly inhibited with modest amounts of mutant protein
(Fig. 3B). For instance, the ratio of antagonist over
agonist peptides generated from native HEL is 
1:1 (2), so addition
of an equimolar concentration of mutant rHEL.W62/63A can reasonably be
predicted to change this ratio to 3:1. This relatively small change
gave rise to a 53.7% and 70.4% reduction in IL-2 production by WW241+
and WW157+, respectively. We reasoned that this could not be due to
peptide competition for MHC binding because neither WW236- nor WW221-
could be inhibited by addition of these mutant proteins, and the
control mutant protein, rHEL.L56F.W62Y, which restores this epitope to
the murine lysozyme sequence and generates peptides that bind to
H-2Ak comparably (17), had no effect on WW241+ and WW157+.
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C). Furthermore, the response of WW241+ could be
antagonized by the addition of rHEL.W62/63A. Differences in the ability
of WW241+ and WW157+ to respond to HEL, LK-HEL, and HEL-flu relative to
their control hybridomas is likely due to differences in the amount of
48–61 required to induce antagonism (Fig. 2A; molar ratio
of agonist:antagonist peptides required to give 50% reduction in IL-2
secretion is 1:1.57 for WW241+ and 1:0.28 for WW157+). Taken together, our data demonstrate that natural processing by APC can generate both agonist and antagonist peptides from the same antigenic epitope, which can modulate the response of PFR-dependent T cells. Given that over half of the T cells generated to the HEL 52–61 epitope are PFR-dependent (5), these data raise the intriguing possibility that this process could play an important role in regulating T cell responses. Indeed, previous studies have suggested that endogenous altered peptide ligands can affect peripheral T cell responses (20). Our data clearly show that HEL 48–61 was a more potent antagonist than any peptide containing substitutions at either L56 or Q57. The amino acid substitutions made to generate altered peptide ligands are often subtle, so it is striking that the complete removal of a residue would generate such a potent T cell antagonist. The alteration of PFR may therefore provide an alternative approach to producing peptide antagonists for immunotherapy.
Can normal murine or human T cells be PFR dependent? If so, can they be antagonized by peptides that lack PFR? While our current knowledge of TCR and MHC class II structure would suggest that any TCR could recognize PFR, and that any PFR-dependent T cell could be antagonized by "short" peptides, further studies will clearly be required to confirm these assumptions. We have recently isolated a T cell clone that is partially PFR-dependent. Although it responds to low concentrations of HEL 48–63, it is only stimulated by the highest concentration of HEL 48–61. Interestingly, the response of this clone is still antagonized by substimulatory concentrations of 48–61. In addition to these studies, the generation of transgenic mice expressing PFR-dependent TCR will allow us to study this further.
Previous studies have suggested that distinct peptides may be generated if the proteins are either derived from different intracellular and extracellular sources, or processed by different cell types (10, 18, 19). Although we could not detect any significant difference in the response to exogenously- and endogenously-derived HEL peptides, a more detailed investigation of this issue is warranted. What influence might such differences have on tolerance induction and autoimmunity? With few exceptions, only MHC class II molecules present peptides containing PFR (1). It is therefore tempting to question whether this feature might be linked to the generation of autoimmune disease, the vast majority of which are MHC class II restricted. It had previously been proposed that a distinct set of peptides are presented by the thymic cortical epithelial cells that mediate positive selection (21, 22). Peptide elution studies have failed to define clear differences between MHC class II-bound peptides from the thymus vs the spleen (23). However, the recent finding that thymic cortical epithelial cells exclusively utilize the cysteine proteinase cathepsin L, while bone marrow-derived APC use cathepsin S for MHC class II-associated invariant chain degradation, suggests that the proteolytic environment in cells that mediate positive and negative selection may be different (24). If proportionally more peptides lacking PFR were generated in the thymic medulla, the site of negative selection, than in the thymic cortex, the site of positive selection, then autoreactive, PFR-dependent T cells may be inefficiently deleted and hyperstimulated in the periphery. Under normal conditions, professional APC might be expected to present a similar array of peptides as those presented by the bone marrow-derived cells that mediate negative selection in the thymus. Therefore, peptides presented from a self epitope that lack PFR may help to maintain peripheral tolerance by inducing anergy in the PFR-dependent T cells. However, if different APC that express MHC class II at the site of an autoimmune reaction generate more agonist than antagonist peptides, then this may be sufficient to break tolerance. While it remains to be determined if PFR-dependent T cells initiate autoimmunity, they may exacerbate an autoimmune response.
In summary, our data demonstrate that the natural processing of an immunogenic epitope can give rise to both agonist and antagonist peptides that can modulate T cells specific for this epitope. While this may provide an additional mechanism for controlling the response of PFR-dependent T cells, it also highlights how small changes in the ratio of agonist vs antagonist peptides generated from a protein either by different cell types or by different processing pathways could lead to deleterious responses. These data are likely to have important implications for vaccine development, peptide immunotherapy, and autoimmunity.
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Acknowledgments |
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antisera 387,
William Walker for HEL-flu, Jose Moreno for LK.HEL (LK.G207), and
Marcia Blackman, Mary Ellen Conley, and Jose Moreno for their critical
review of the manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Dario Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. E-mail address:
3 Abbreveations used in the paper: HEL, hen egg lysozyme; PFR, peptide flanking residues.
Received for publication August 25, 1998. Accepted for publication October 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß TCR. J. Immunol. 159:2563.[Abstract]
Phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515. and lack of ZAP-70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
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