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Department of Cellular and Developmental Biology, "La Sapienza" University, Rome, Italy;
Department of Clinical Medicine, "La Sapienza" University, Rome, Italy; and
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia, Rome, Italy
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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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It has recently been shown in various human infectious diseases that the appearance of mutated epitopes displaying TCR antagonistic activity correlates with the severity and persistence of infection (7, 8). In fact, natural antagonist variants of hepatitis B virus (9), HIV (10, 11), hepatitis C virus (HCV)4 (7, 8, 12), and more recently Plasmodium falciparum Ags have been identified (13). Functional studies have shown that they can inhibit the cytotoxic activity of CD8+ T cells (7, 8, 9, 10, 11, 12, 13). Worthy of note is that these naturally occurring T cell epitopes antagonize TCR with an efficiency much higher than that of in vitro-produced altered peptide ligands, suggesting that their appearance is regulated by the selective pressure of the immune system. Hence, these natural variants represent a more suitable tool to characterize the mechanisms involved in TCR signaling by peptide-MHC complexes with agonistic or antagonistic properties.
Because of the pivotal role of cytotoxic T cells in the clearance of viruses, antagonism has mostly been studied in relation to the capacity of highly variable viruses to produce mutated epitopes that inhibit CD8+ T cell responses. Nevertheless, an increasing body of evidence indicates that CD4+ T cells are crucial to prime and maintain virus-specific CTLs (14). Thus TCR antagonism of CD4+ Th cell functions may be another important strategy by which mutant viruses can elude a protective response. It has recently been shown that HIV-specific vaccine-induced CD4+ CTLs isolated from an infected patient could be antagonized by an epitope sequence of the infecting isolate variant strain in vitro (15). Therefore, we wondered whether TCR antagonism of CD4+ T cell responses was a phenomenon evocated by highly mutant viruses. To address this issue, we examined the effect of sequence variation of CD4+ T cell epitopes in HCV infection.
HCV is a major cause of chronic hepatitis worldwide (16). Similarly to HIV, HCV establishes persistent infection in >50% of the cases, despite a wide array of B and T cell responses being induced (8, 17). The ability of the virus to undergo rapid and substantial Ag modifications during replication is thought to be a major factor in this process. In fact, HCV exists in the bloodstream of infected patients as quasispecies as a result of the combined action of viral mutation and variants selection by the host immune response (18, 19). Within the HCV genome, a hypervariable region (HVR1), consisting of the N-terminal 27 aa of the putative Envelope 2 protein (E2), has been identified. This region contains both B and T cell epitopes (8, 20, 21, 22). We examined the response of CD4+ T cell lines and clones derived from HCV-infected patients to different peptides reproducing HVR1 sequences of many viral isolates. Our results show that powerful TCR antagonism does occur in HVR1-specific CD4+ T cells in the presence of HVR1 variants. In fact, the total inhibition of cytokine secretion as well as T cell proliferation at low antagonist/agonist ratios were observed. The mechanisms involved in TCR antagonism were addressed in terms of the antagonist capacity to inhibit T cell activation at the level of TCR down-regulation and early signal transduction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen different natural HVR1 variants (residues 384–410) available in the databank were synthesized as multiple antigenic peptides (MAP) (23). These sequences were chosen among a larger set of HVR1 sequences from natural HCV isolates collectively representing most of the observed viral variability as previously described by Punturiero at al. (23). Variant numbers, GenBank accession numbers and sequences of these HVR1 MAPs are reported below: 295, D10687, NTHTVGGTEGFATQRLTSLFALGPSQK (1180–1260 bp); 291, S35631, ETHSVGGSAAHTTSRFTSLFSPGPQQN (580–660 bp); 269, U24616, ATYTTGGSAAKTAHRLASFFTVGPKQD (22–102 bp); 266, D00574, HTRVTGGVQGHVTSTLTSLFRPGASQK (1240–1320 bp); 293, S70291, QTRTVGGANARNTYGLTTLFTTGPKQN (1–81 bp); 268, M62381, ETHVTGGSAGRTTAGLVGLLTPGAKQN (1426–1506 bp); 302, M62382, ETHVTGGSAGRSVLGIASFLTRGPKQN (1426–1506 bp); 299, D30613, GTHVTGGKVAYT TQGFTSFFSRGPSQK (1491–1571 bp); 296, D43651, NTHVTGGVVARNAYRITTFLNPGPAQN (39–119 bp); 292, S62395, ETHVTGGSAASTTSTLTKLFMPGASQN (43–123 bp); 284, X79672, NTRVTGGVQSRTTGTFVGLFTPGPSQR (1–81 bp); 294, D88472, GTTTVGSAVSSTTYRFAGMFSQGAQQN (1485–1565 bp); 303, D88474, ETYIIGAATGRTTAGLTSLFSSGSQQN (1488–1568 bp). Two additional sequences, derived from the pedigreed inoculum H77 (24), were also synthesized as MAPs: 304 (H77–1) ETHVTGGNAGRTTAGLVGLLTPGAKQN (1–81 bp); 305 (H79) ETHVTGGSAGHTAAGIASFFAPGPKQN (1–81 bp).
Linear 18-mer peptides corresponding to the C-terminal region of MAPs 266 and 295 (residues 393–410 of the HCV polyprotein), and the corresponding leucin to glutammic acid substitution mutants were also synthesized (266C, 295C, 266E, and 295E). Control peptides HCV-core-32-49 (Primm, Ulilau Italy) and HA-306–318 (Neosystem Laboratoire, Strasbourg, France) were synthesized in a linear form.
Generation of T cell lines, T cell clones, and B cell lines
The T cell lines used in this study were generated by stimulating PBMC of chronically infected patients with 10 µg/ml of synthetic peptide for 10 days. Activated T cells were maintained in culture with irradiated autologous PBMC, peptide, and 20 U/ml of human recombinant IL-2 (hrIL-2; Boerhinger Mannheim, Mannheim, Germany) and were used after at least 3 wk from the first stimulation. HVR1-specific T cell lines were cloned by limiting dilution in Terasaky plates (0.3 cell/well) in the presence of allogenic PBMC, 0.5 µg/ml PHA (Murex, Dartford, U.K.), and 20 U/ml hrIL-2. The peptide-specificity of T cell lines and clones was assessed by using a proliferation assay. Homozygous lymphoblastoid cell lines used as APC were: PE117 (DRA*0101, DRB1*0404, DRB4*0101, DQA1*03, DQB1*0302, DPA1*01, DPB1*0401), MGAR (DRA*0102, DRB1*1501, DRB5*0101, DQA1*0102, DQB1*0602, DPA1*01, DPB1*0401), MZ070782 (DRA*0101, DRB1*0102, DRB6*0101, DQA1*01, DQB1*0501, DPA1*01, DPB1*0401), and Sweig (DRB1*1101, DQA1*0501, DQB1*0301). T cell lines T7 and T2 derived from a patient expressing DRB1*0404 and DRB1*1501 and DRB5*0101 were specific for the HVR1 variant 291 and 269, respectively, and restricted by HLA-DRB1*0404. HVR1-specific CS1 and CS4 T cell clones were obtained from a patient expressing DRB1*1101 and DRB1*0102, and were specific for the HVR1 variant 295. Two T cell clones, F17 and 2.3C9, specific for peptides HA-306-318 and HCV-core-32-49, respectively, and restricted by DRB1*1101 were used as controls. EBV-transformed B cells (B lymphoblastoid cell lines (B-LCL)) were generated by incubation of 5 x 106 PBMC with EBV obtained from the Marmoset lymphoblastoid cell line B95-8 as previously described (25).
The IL-2-dependent murine T cell line CTLL-2 (European Collection of Animal Cell Cultures, Salisbury, U.K.) was cultured in RPMI 1640 medium, supplemented with 2 mM L-glutamine, 50 UI/ml penicillin, 50 µg/ml streptomycin, 10 U/ml hrIL-2, and 10% FCS. The cells were cultured in 25-cm2 flasks and were subcultured every 3 days. Before use in a proliferation assay, the CTLL-2 cells were washed twice and cultured overnight in normal culture medium, but without added hrIL-2.
Isolation of RNA and RT-PCR
The mRNA from T cell clones (2.5 x 105) was isolated by QuickPrep Micro mRNA purification kit (Pharmacia Biotech, Uppsala, Sweden). cDNA was synthesized from oligo(dT)-primed RNA in 20 µl of reverse transcriptase buffer and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Rockville, MD). The Vß usage of T cell clones was determined by PCR amplification using 24 Vß subfamily-specific primers and the Cß primer 5'-CGGGCTGCTCCTTGAGGGGCTGCG-3' as described by Genevée et al. (26). Thirty cycles of denaturation (94°C, 30s), annealing (60°C, 30s), and extension (72°C, 30s) were conducted in the automated DNA thermal cycler GeneAmp PCR System 2400 (Perkin-Elmer Cetus, Norwalk, CT). PCR products were size fractionated by agarose electrophoresis.
Proliferation assays
Proliferative response of T cell lines and clones was assessed by incubation of 1–2 x 104 T cells with 4 x 104 mitomycin C (Mit-C)-treated autologous B cell lines pulsed with different concentration of synthetic peptides (usually between 1 and 30 µg/ml). After 2 days, the cells were pulsed with 1 µCi of [3H]thymidine and incubated for 18 h before cell harvesting. The results are expressed as the mean cpm of triplicate determinations. SD was always <10%. Data are representative of at least three independent experiments.
TCR antagonism assay
To test the HVR1 variant peptides for TCR antagonism, we used a modified version of the method described by De Magistris et al. (1). Briefly, autologous APCs (B-LCLs) were prepulsed overnight at 37°C with different doses of agonist peptide (usually between 10 and 30 µg/ml). The choice of the agonist dose was dictated by the evidence that although T cells proliferated significantly to 1 µg/ml of agonist peptide, optimal proliferation was obtained with concentration ranging between 10 and 30 µg/ml. After washing, the APCs were treated with Mit-C, plated in flat-bottom microtiter plates, and the variant peptides were added directly into the wells at various concentrations. After a further 5 h of incubation, T cells were added into the wells and proliferation was measured as described.
Evaluation of agonist and antagonist induced TCR down-regulation
Autologous B-LCLs (2 x 105) pulsed
overnight with 15–30 µg/ml of either agonist or antagonist peptides
and washed to eliminate unbound peptide, were mixed with resting
105 CS1 or CS4 T cell clones in round-bottom
96-well plates. In the antagonism experiments, the antagonist peptide
and the control peptide were added directly into the assay during
incubation of the T cells with the APCs previously pulsed with the
agonist. The plates were centrifuged at 1000 rpm for 3 min and cultured
at 37°C for either 6 or 18 h. After incubation, the cells were
washed with PBS 0.5 mM EDTA to distrupt conjugates. Cells were stained
with a FITC-conjugated anti-TCR 
ß Ab (WT31; Becton Dickinson,
San Diego, CA), a PE-conjugated anti-CD4 Ab (Leu-3a; Becton
Dickinson), and a peridinin chlorophyl protein-conjugated anti-CD20
Ab (Leu-16; Becton Dickinson) to discriminate between B and T cells in
the cell population. The relative TCR and CD4 expression was assessed
by using flow cytometric analysis.
Measurement of IL-2, IL-4, and IFN-
production
T cells (4 x 104 cells/well) were
cultured with Mit-C-treated B-LCLs (8 x 104
cells/well) prepulsed or not with the antigenic preparation. The
experiment was performed in flat-bottom microtiter plates as described
above for the TCR antagonism assay. After 48 h, culture
supernatants were collected and stored at -20°C until used. IL-4 and
IFN- production by the HVR1-specific T cell clones and lines was
measured by ELISA using a quantitative sandwich enzyme immunoassay kit
(R&D Systems, Minneapolis, MN). IL-2 production was measured by a
biological assay. Briefly, culture supernatants were transferred into
96-well round-bottom microtiter plates as triplicate cultures and the
plates stored at -20°C until used. In each well, 3 x
103 CTLL-2 cells were added. In each experiment,
a standard titration for hrIL-2 was included. After 8 h of
incubation, proliferation of CTLL-2 was measured as
[3H]TdR incorporation by liquid scintillation
spectroscopy. The results are expressed as the mean of triplicate
cultures. SEs were routinely <10%.
T cell clone stimulation and immunoblotting
T cell clones (106 cells) were activated for 2 min at 37°C with 3 x 105 B-LCLs (T:APC ratio 3:1) prepulsed overnight with different concentrations of agonist peptide or 100 µg/ml of antagonist peptide in a final volume of 50 µl. At the end of incubation, cells were harvested and lysed for 30 min on ice in 1% Nonidet P-40 lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM EGTA in the presence of inhibitors of proteases and phosphatases: 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM pefabloc-sc, 50 mM NaF, 10 mM Na4P2O7, and 1 mM NaVO4. For antagonist experiments, APCs were pulsed for 10 h with 30 µg/ml of agonist peptide, washed, and incubated overnight with medium alone or 100 µg/ml of the antagonist peptide or 100 µg/ml of the control peptide HA-306-318. Postnuclear lysates, obtained after centrifugation at 14,000 rpm for 10 min at 4°C, were boiled in SDS sample buffer before gel electrophoresis. After equilibration in transfer buffer (25 mM Tris-base, 192 mM glycine, 20% ethanol), gels were transferred to nitrocellulose membranes. Blots were blocked in PBST (10 mM phosphate buffer, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 0.1% Tween 20) in the presence of 1% gelatin for at least 1 h and incubated overnight at 4°C with anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) in PBST 0.1% gelatin. At the end of the incubation, blots were extensively washed, incubated with 1/2000 dilution of peroxidase conjugated goat anti-mouse (Amersham International, Bucks, U.K.), and developed with the enhanced chemiluminescence detection system (Amersham International).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify HVR1 variants that act as TCR antagonists, a panel of
HVR1 sequences were tested for their ability to stimulate CS1 and CS4 T
cell clones specific for variant 295 (Table I
). Six variants of 15 tested with
nonimmunogenic activity (Fig. 1,
a and b) were chosen and tested as TCR
antagonists by using a modified version of the method described by De
Magistris et al. (1). The results reported in Fig. 1, c and d, clearly show that variant 266 powerfully
inhibited the proliferation of both clones in a dose-dependent manner.
It should be underlined that an agonist/antagonist ratio of 1:1 was
sufficient to induce a 100% decrease of DNA synthesis in CS1 T cell
clone. The variants 268 and 299 also behaved as antagonists, but their
effect was weaker and limited to CS1 T cell clone only. To further
characterize the HVR1-epitope with antagonistic activity, we
synthesized two 18-mer peptides encompassing the C-terminal region of
the agonist and antagonist variants 295 and 266 (295C and 266C). This
choice was determined by previous findings showing that the reactivity
of HVR1-specific CD4+ T cells was directed toward
the C-terminal part of the HVR1 (residues 393–410; data not shown).
266C was used to antagonize the proliferation of CS1 and CS4 clones
induced by peptide 295C. Peptides HCV-core-32-49 and HA-306-318, known
to bind the restriction allele DRB1*1101 used by CS1 and CS4 T cell
clones, were used as controls in similar experiments. The data reported
in Fig. 2, a and b
clearly demonstrate that the antagonistic effect of variant 266 resided
in its C-terminal region. Moreover, the lack of inhibition by the
control peptides ruled out the possibility that the antagonistic effect
was due to mere competition for MHC binding between agonist and
antagonist peptides. T cell clones 2.3C9 and F17 specific for peptide
HCV-core-32-49 and for HA-306-318, respectively, and also restricted by
DRB1*1101, were not inhibited by peptide 266C (Fig. 2, c and
d), confirming that the antagonistic activity of variant 266
is restricted to TCRs specific for peptide 295C.
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In the mouse system, TCR antagonism can result not only in
inhibiting proliferation, but also in blocking of release of both Th1-
and Th2-type cytokines by T cells (27). Therefore, we
decided to establish whether inhibition of proliferation by the
strongest antagonist, peptide 266C, was accompanied by inhibiting
cytokine release in both CS1 and CS4 T cell clones. These cells can be
classified as Th0 in that they produce IL-4, IL-2, and IFN- in
response to antigenic stimulation (data not shown). Inhibition of the
proliferative response of CS1 and CS4 T cell clones by the antagonist
266C paralleled a proportional decrease of secretion of all three
cytokines tested (Fig. 3).
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It has recently been reported that pure antagonists fail to induce
TCR down-regulation in responding T cells (28, 29). Thus,
we tested the ability of both agonist and antagonist peptides to
cause TCR and CD4 down-regulation in the HVR1-specific T cell
clones. The results reported in Fig. 4,
a and b demonstrate that peptide 266C failed to
induce either TCR or CD4 down-regulation, confirming that it acts as a
pure antagonist. Because antagonist peptides are able to interfere with
agonist-induced TCR serial triggering (28, 29), we next
verified whether peptide 266C could block TCR down-regulation in
response to the agonist peptide 295C. APC previously pulsed with the
agonist peptide were pulsed with an equal amount of the antagonist
peptide 266C. The agonist/antagonist ratio of 1:1, known to induce
complete inhibition of T cell clone proliferation, was used. We found
that agonist-induced TCR internalization was completely blocked by the
antagonist (Fig. 4c), while the addition of the control
peptide HCV-core-32-49 did not affect TCR down-regulation.
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We next asked whether TCR antagonism mediated by peptide 266C
would also occur when the agonist and the antagonist peptides were
presented by different APCs. For this purpose APCs were separately
pulsed with the same amount of agonist and antagonist peptides and
subsequentely mixed before incubation with HVR1-specific T cell clones.
The results in Fig. 5 show that when the
agonist and the antagonist peptides were presented by different APCs, T
cell antagonism did not occur. This finding suggests that an antagonist
can inhibit TCR triggering only when offered to the responding T cells
on the same APC as the agonist.
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Several reports have described the effect of TCR engagement by
class II-restricted antagonist peptides on the early signaling cascade.
However, most of these results were obtained following TCR stimulation
by agonist and antagonist peptides separately (30, 31). We
thus investigated the effect of the copresentation of agonist and
antagonist peptides on the early TCR-induced tyrosine phosphorylation
events. As shown in Fig. 6a,
TCR engagement by peptide 295C induced the dose-dependent tyrosine
phosphorylation of a series of cellular substrates probably
representing p95vav, SLP-76, ZAP-70, and the
recently cloned p36 LAT. In contrast, the maximal concentration of
antagonist peptide 266C failed to increase the level of phosphorylation
observed following stimulation of T cells with unpulsed APCs. We next
verified whether the antagonist peptide was able to interfere with the
agonist-induced tyrosine phosphorylation signals. As shown in Fig. 6b, the antagonist peptide 266C strongly inhibited the
tyrosine phosphorylation events induced by the agonist 295C. In
particular, tyrosine phosphorylation of proteins likely to be SLP-76
and p36 LAT was mostly affected. In contrast, the copresentation of the
control peptide HA-306–318 did not have any significant effect.
Altogether, these results suggest that the antagonist peptide 266C
interferes with the agonist-induced T cell activation by blocking the
early events of the TCR signaling cascade.
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A comparison between the sequences of the C-terminal region of
both peptides 295C and 266C reveals a total homology between residues
399–403. This led us to speculate that this region might contain
crucial residues involved in MHC binding, further sustained by
comparison of the carboxyl-terminal sequences of many HVR1 variants
(23), indicating that the amino acidic positions at
residues 399 and 402 were those highly conserved. In addition, the
presence of these residues at these sites is compatible with their
possible role as P1 and P4 anchors for binding to DR molecules
(32). In an attempt to verify the hypothesis that position
399 was the primary anchor, we introduced a nonconservative
substitution at this residue (L
E) in both agonist and antagonist
peptides (295E and 266E). Substitution of L399 with E gave rise to loss
of function of both the agonist and antagonist peptides. In fact, while
peptide 295E was unable to induce proliferation of the CS1 T cell clone
(Fig. 7a), the antagonist
mutant 266E was severely impaired in its ability to block proliferation
of this clone (Fig. 7b). Similar results were obtained with
the CS4 T cell clone (data not shown). These results suggest that
residue 399 may be an important anchor for MHC class II binding.
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We also verified the possibility that TCR antagonism was a common
phenomenon among HVR1 variants and not only a prerogative of the
variants analyzed and CS1 and CS4 T cell clones. Thus, we analyzed the
effect of different HVR1 variants by testing the response of
HVR1-specific T cell lines derived from a different HCV-infected
patient. The use of lines instead of clones would probably reflect a
more physiological system in vitro, because T cell responses in vivo
are either polyclonal or oligoclonal. To this aim, two lines were
obtained from a patient expressing DRB1*0404 and DRB1*1501 and
DRB5*0101. These lines, T7 and T2, were specific for the variants 291
and 269, respectively, and were restricted by HLA-DRB1*0404 (Table I
).
Again, nonstimulatory HVR1 variants were selected and then tested for
TCR antagonism in a preliminary screening where the inhibition of IL-2
release and [3H]thymidine incorporation at an
agonist/antagonist ratio of 1:1 were determined (data not shown). The
results in Fig. 8 clearly show that HVR1
antagonist variants are frequently generated. In fact, both T cell
lines were inhibited by at least two HVR1 variants with an efficiency
ranging between 60 and 100% of the proliferative response at the
agonist/antagonist ratios of 1:2.5 and 1:5. A slightly higher
agonist/antagonist ratio was required to achieve a relevant inhibition
of the proliferative response with these lines as compared with the T
cell clones described above. This was expected in that it is more
difficult to inhibit a polyclonal T cell response by TCR antagonism.
However, with the T7 T cell line an inhibition ranging from 50 to 70%
of the [3H]thymidine incorporation has been
found at the agonist/antagonist ratio of 1:1 with variants 293, 296,
and 298 (data not shown). As shown in Fig. 8, the three HVR1 variants
293, 296, and 298 acted as powerful TCR antagonists for the T7 T cell
line (Fig. 8a), while the other two variants (292 and 304)
acted as powerful TCR antagonists for the line T2 (Fig. 8b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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From a panel of 16 HVR1 variants we found that at least one functioned
as a potent TCR antagonist for each of the different
HVR1-specific CD4+ T cell lines and clones
analyzed. This antagonistic effect was always found at a low
agonist/antagonist ratio. Such strong antagonistic activity of a
natural epitope has not been described before for Th cells, although
potent peptide antagonists have been discovered for cytotoxic
CD8+ T cell responses to HIV (10, 11) and to P. falciparum (13). In
keeping with results reported in the mouse system (27), we
found that inhibition of the proliferative response of HVR1-specific T
cell clones was accompanied by inhibiting the secretion of IL-2,
IFN-, and IL-4 cytokines. Because it has been shown that different
thresholds of activation signals are required to induce different
cytokine patterns (33), our results suggest that HVR1
antagonists may completely inhibit the signal transduction cascade
involved in cytokine release.
It is commonly accepted that TCR down-regulation is an important mechanism that allows T cells to calibrate their response to antigenic stimulation. Very recent works have shown in both mouse (29) and humans (28) that an antagonist suppresses T cell activation by competitively engaging the specific TCR in unproductive interactions that do not result in down-regulation. Moreover, when agonists and antagonists were offered together, the antagonist inhibits agonist-induced TCR down-regulation. We verified this in our system and our data show that peptide 266C acts as a typical antagonist. It is noteworthy that this peptide completely inhibited agonist-induced TCR down-regulation at the agonist/antagonist ratio of 1:1, whereas previous studies with antagonists of human CD4+ T cells suggested that a much higher ratio (104 antagonist to agonist molar excess) was required to obtain 50% inhibition of TCR down-regulation (28).
We also showed that agonist and antagonist peptides must be presented by the same APC for inhibition to occur. These data are in agreement with the results of a previous report, also obtained with CD4+ T cells, where an antagonist of the antigenic peptide of influenza hemagglutinin (HA-307-319) was used (34). This can be explained by the fact that an essential step in T cell activation is the formation of a contact zone (synapse) between T cells and APCs, where multiple TCR/peptide-MHC complexes are recruited (35, 36, 37) and where TCR serial triggering occurs (38). Therefore, serial triggering can only be inhibited if the TCR encounters the agonist and the antagonist in this particular contact area and not when they are offered on different APCs. In contrast, in cytotoxic assays where APCs pulsed with the antagonist peptide were present in considerable excess with respect to APCs loaded with agonist, inhibition of target lysis was observed (15, 29). In these particular experimental conditions, because of the high concentration of cells pulsed with the antagonist, the TCR might be forced to engage the antagonist peptide-MHC complexes instead of interacting with the agonist-loaded APCs. Thus, this phenomenon cannot be considered a truly antagonistic effect, but rather may reflect a straighforward competition for TCR binding between MHC/agonist and MHC/antagonist complexes.
It has been shown in the mouse system that engagement of the TCR
by antagonistic MHC-peptide complexes results in incomplete
intracellular signals characterized by a distinct pattern of 
-chain
phosphorylation and failure to activate associated ZAP-70 kinase
(30, 31). In human CD8+ T cells, the
-chain was found to be constitutively phosphorylated and the
antagonist ligands were unable to induce significant modifications,
suggesting that the antagonists did not affect protein tyrosine
phosphorylation (39). Our evidence that stimulation with
antagonist peptides alone failed to induce TCR down-regulation opened
up at least two possibilities: either the antagonist induced an
incomplete phosphorylation pattern or it did not induce any
phosphorylation event at all. Our demonstration that the
antagonist-exposed T cells did not show any early phosphorylation
pattern (Fig. 6a) and did not become anergic (data not
shown) strongly supports the second hypothesis. However, evidence that
the antagonist peptides totally abrogated TCR down-regulation mediated
by the agonist implied that, although unable to mediate intracellular
signal transduction, the antagonist might influence the signals
delivered by the agonist. In fact, our results demonstrate that
TCR-mediated early signal transduction in human
CD4+ T cells was strongly inhibited by the
antagonist. This is the first piece of evidence in humans that class
II-restricted antagonist peptides inhibit Ag-mediated early signal
transduction, as observed in class I-restricted antagonists
(39). One possible explanation of these findings is that
the antagonist may interfere with the productive clustering of the
MHC/agonist complexes and TCRs necessary for complete phosphorylation
events to occur (36, 39, 40). Alternatively, the low
stability of TCR/antagonist complexes (41) may reduce the
occupancy of TCRs by their ligands, which is crucial to reaching the
time threshold for induction of any intracellular signals (28, 38). The latter hypothesis is supported by recent findings on
the importance of the duration of antigenic stimulation in determining
the fate of naive and effector T cells (42).
Two important prerequisites for altered peptide ligands to
function as TCR antagonists are the maintainance of MHC binding
capacity and the presence of conservative mutations at the TCR contact
residues; HVR1 variants appear to satisfy both requirements. In fact,
the comparative analysis of 234 unique HVR1 sequences extracted from
databases showed a pattern of conserved residues important for the
binding to DR molecules in 80% of the natural viral variants, in
particular at positions 399 and 402 (23). The hydrophobic
amino acids present at position 399 may, in fact, represent the primary
anchor for binding to multiple DR alleles (32). This
assumption is supported by our evidence that introducing a
nonconservative substitution at residue 399 dramatically abolished both
agonistic and antagonistic activity (Fig. 7). Moreover, the aliphatic
amino acids L, I, and M, known to act as P4 anchor for DR4 and DR11
binding, are present at position 402 in the HVR1 peptides used to
generate the T cell clones CS1 and CS4 and the T cell lines T7 and T2
as well as in HVR1 variants working as antagonists (43, 44). Because these T cells are restricted precisely by DR11 and
DR4, respectively, we can presume that residue 402 represents the
secondary anchor (P4). Interestingly, in a recent report, decapeptides
located in the carboxyl-terminal portion of HVR1 were described as
binding to the highly expressed HLA-class I molecule HLA-A2, and some
of these variants antagonize CTL activity (8). Mutated
epitopes that still bind the restriction elements have also been
identified in the case of other infective agents such as P.
falciparum (13), where TCR antagonism also
occurs.
With regard to the possible amino acidic residues responsible for signaling through the TCR, many studies (45, 46, 47) suggest that these are usually the 3–5 aa residues that protrude upwards from the peptide sequence bound to MHC molecules. Conservative substitutions at these putative TCR contact sites usually allow altered peptide ligands to maintain interaction with the TCR, but at the same time may interfere with the results of this interaction, converting an agonist into a partial agonist or an antagonist ligand (1, 48). On the other hand, it is possible that amino acid substitution at the MHC anchor residues, which still allows MHC binding, induces subtle changes in the peptide conformation, which in turn influences the residues involved in TCR engagement (49). Because HVR1 variant peptides present conservative substitutions at residues close to the putative P1, P4, and P6 anchors, as well as at the possible MHC binding residues, both conditions are satisfied.
Our data on the antagonistic effect of HVR1 variants support the thesis
that high viral variability can suppress a Th type of response, which
is of particular importance in HCV infection (50, 51, 52). It
is well known that vigourous and sustained Th responses directed
against several HCV Ags are associated with a self-limited course of
infection (50, 51, 52, 53). In chronically infected patients, a
correlation between CD4+ T cell response and
viral clearance after IFN- treatment has been established (51, 54), and our recent data5 on the
immunogenicity of HVR1 sequences are in agreement with these
observations. Because HCV exists in bloodstream of infected patients as
quasi-species (55), T cell antagonism could influence the
priming of a CD4+ T cell response toward HVR1
immunogenic variants, which will be presented together during
infection.
Althought there is evidence that HCV-specific Abs (56, 57, 58) and CD8+ T cells (8, 59) can exert selective pressure for the generation of escape mutants, whether CD4+ T cell responses provide analogous pressure is not evident. However, in HIV infection the variation of CD4+ Th epitopes has been described as an important cause of viral persistance (60). Because CD4+ T cells allow the activation of both B and CD8+ T cell populations and produce the necessary factors needed to support their functional differentiation and survival (14, 61), we can hypothesize that also in HCV infection the antagonism of CD4+ T cell help could favor HCV persistence. Only an accurate analysis of HVR1 variants with agonistic and antagonistic activity arising in infected patients may validate our hypothesis. To this aim, HVR1 sequencing from serum samples of infected individuals is in progress.
Our study lends strong support to the idea that CD4+ T cell antagonism may be a phenomenon common to infection mediated by highly mutant viruses, thus representing an important escape mechanism that may favor chronicity.
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Acknowledgments |
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Footnotes |
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2 L.F. and P.D.P. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Enza Piccolella, Dipartimento di Biologia Cellulare e dello Sviluppo, Università "La Sapienza", Via degli Apuli 1, 00185 Roma, Italy. E-mail address:
4 Abbreviations used in this paper: HCV, hepatitis C virus; HVR, hypervariable region; E2, envelope 2 protein; MAP, multiple antigenic peptide; hrIL-2, human recombinant IL-2; B-LCL, B lymphoblastoid cell line; Mit-C, mitomycin C.
5 Del Porto P., C. Scottà, G. Puntoriero, A. Nicosia and E. Piccolella. Cross-reactive CD4+ T cell response to the hypervariable region 1 in HCV-infected patients. Submitted for publication.
Received for publication February 2, 1999. Accepted for publication April 28, 1999.
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) or the unrelated peptides HCV-core-32-49 (core, 
, 
)
and HA-306-318 (HA306, 
) were added directely into the wells at
increasing concentrations. The antagonism assay with CS1
(a) and CS4 (b) T cell clones was
performed as described above. The antagonist peptide concentrations are
reported in two decimal scales: the upper scale refers to continous
lines, while the lower scale to dashed lines. Proliferation of CS1 T
cell clone to 15 µg/ml and 30 µg/ml of agonist peptide (295C) were
28,800 and 22,700 cpm, respectively; proliferation of CS4 T cell clone
to 15 µg/ml and 30 µg/ml of agonist peptide (295C) were 14,818 and
21,807 cpm, respectively. Control peptides fail to induce antagonistic
effects (c and d). Clone F17
(c) or clone 2.3C9 (d) were cultured with
APCs prepulsed with 10 µg/ml of peptide HA-306-318 (H306) and with an
equal amount of peptide HCV-core-32-49 (core), respectively, in the
presence of 30 µg/ml of peptide 266C.
) and IFN-
) and CS4 (
