Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0804456 on May 27, 2005

Published online before print May 27, 2005
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(Journal of Leukocyte Biology. 2005;78:372-382.)
© 2005 by Society for Leukocyte Biology

Hypervariable region 1 variant acting as TCR antagonist affects hepatitis C virus-specific CD4+ T cell repertoire by favoring CD95-mediated apoptosis

Cristiano Scottà*, Loretta Tuosto*, Anna Maria Masci{dagger},{ddagger}, Luigi Racioppi{dagger}, Enza Piccolella* and Loredana Frasca*,1

* Department of Cellular and Developmental Biology, La Sapienza University, Rome, Italy;
{dagger} Department of Cellular and Molecular Biology and Pathology, Federico II University, Naples, Italy; and
{ddagger} IRCCS San Raffaele, Rome, Italy

1 Correspondence: Department of Cellular and Developmental Biology, La Sapienza University, Rome, 00185, Italy. E-mail: lfrasca{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have described previously that hypervariable region 1 (HVR1) variants of hepatitis C virus (HCV) frequently act as T cell receptor (TCR) antagonists for HVR1-specific helper T cells. These naturally occurring HVR1-antagonistic sequences interfered with the effects of HVR1-agonistic sequences such as TCR down-regulation and early activatory signals. By taking advantage of these findings, in this paper, we have analyzed the fate of these HVR1-specific antagonized CD4+ T cells. We present the evidence that TCR antagonism renders agonist-activated T cells susceptible to bystander CD95-mediated killing by suppressing the expression of cellular Fas-associated death domain-like interleukin-1ß-converting enzyme-like inhibitor proteins. To verify whether the TCR repertoire of a HVR1-specific T cell population could be modified consequently, we used a HVR1-agonistic sequence to induce in vitro CD4+ T cells and another HVR1 sequence with antagonistic property to mediate suppressive phenomena. HVR1-specific T cells were cultured with the agonist alone or with the agonist plus the antagonist. HVR1 specificity and T cell repertoires were followed over time by analyzing TCR ß-variable gene segment by "spectratyping". The results showed that the specificity for the agonist was rapidly spoiled after culture in the presence of the antagonist, and the TCR repertoire was strongly modified as a result of CD95-mediated apoptosis of agonist-specific clonal expansions. These data support the hypothesis that in HCV infection, the generation of TCR antagonists may reshape the T cell repertoire, representing an efficacious immune evasion strategy of a highly mutant pathogen.

Key Words: HCV infection • T cell deletion • altered peptide ligands • spectratyping


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that highly variable infectious agents can induce persistent infections, probably through generation of escape mutants [1 2 3 4 ]. An important example is represented by hepatitis C virus (HCV) infection, which evolves to chronicity in the majority of infected patients [5 ]. The inability of the immune system to eliminate this virus has been ascribed to lack of immune recognition, insufficient CD4+ T cell help, induction of unresponsiveness in HCV-specific T cells, immune deviation, and activation of incomplete immune cell functions [6 , 7 ]. However, phenomena of T cell receptor (TCR) antagonism, evidenced in vitro studies, may also play a role in vivo to determine HCV immune escape, as better documented in other infections such as malaria [8 9 10 11 12 13 14 15 ]. We have demonstrated that natural variants of the HCV hypervariable region 1 (HVR1) sequence can frequently act as TCR antagonists for HVR1-specific helper T cells derived from infected and healthy individuals in vitro [13 , 14 ]. In this system, TCR antagonists were shown to suppress proliferation and cytokine secretion by interfering with TCR down-regulation and early signal transduction events generated by the agonist [13 ]. Here, we wondered about the fate of T cells susceptible to TCR antagonism in terms of survival capacity. Indeed, it is well known that a correct antigenic stimulation leads to the up-regulation of antiapoptotic molecules by T cells [16 ]. Among these, the cellular Fas-associated death domain-like interleukin-1ß (IL-1ß)-converting enzyme-like inhibitor proteins (c-FLIP) block CD95 signal transduction [16 17 18 19 ]. However, it is also clear that when agonists and antagonists are offered together by an antigen-presenting cell (APC), antagonized T cells are still recruited to the APC surface to form T-APC synapse, but this interaction does not induce proper T cell signaling [13 , 20 21 22 23 24 ].

In this scenario, it was tempting to speculate that antagonized T cells, blocked in their functions, also received nonphysiological stimuli, which impair the induction of survival factors and alter their capacity to resist environmental death signals. If occurring, T cell death, as a result of TCR antagonism, may modify the agonist-primed T cell repertoire, a phenomenon that may have a great impact in vivo. To assess whether apoptotic phenomena are favored by TCR antagonism and concur to change the agonist-induced immune cell repertoire, we used, as an in vitro system, human HVR1-specific CD4+ T cell clones and ex vivo-primed, HVR1-specific T cells [13 , 14 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents, peptides, antibodies
Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma Chemical Co. (St. Louis, MO); ionomycin was from Calbiochem (La Jolla, CA). Synthetic peptide corresponding to residues 307–319 of influenza hemagglutinin (HA) was synthesized as described [13 ]. Linear 18-mer peptides 295 GFATQRLTSLFALGPSQK and 266 GHVTSTLTSLFRPGASQK, corresponding to residues 393–410 of the HCV polyprotein, were also synthesized as described [13 ]. Fluorescein isothiocyanate (FITC)-conjugated anti-TCR {alpha}ß monoclonal antibody (mAb; WT31), phycoerythrin (PE)-conjugated anti-CD4 mAb (Leu-3a), and a peridinin chlorophyl protein (PerCP)-conjugated anti-CD20 mAb (Leu-16) were purchased from Becton Dickinson (San Diego, CA). The agonistic anti-human CD95 antibody (CH11) was purchased from Upstate Biotechnology (Lake Placid, NY). Dr. David H. Lynch (Immunex, Seattle, WA) kindly provided antagonistic anti-human CD95 antibody (M3). Hamster anti-human CD95L antibody 4H9 was purchased from Immunotech (Cedex, France). FITC-conjugated anti-hamster antibody was from BD Biosciences (Heidelberg, Germany). H. Walczak [25 ] kindly provided anti-human c-FLIP (NF6). The FITC-conjugated goat anti-mouse antibody was purchased from Dako Cytomation (Denmark).

T cell clones and APC
CD4+ T cell clones F17, HA307-19-specific and DRB1*1101-restricted, were derived from a DRB1*0101/DRB1*1101 individual [13 ]. CS1 and L12 CD4+ T cell clones were obtained from a HCV-infected and a healthy individual, respectively. CS1 clone, specific for HVR1 variant 295 and DRB1*1101-restricted, was characterized previously [13 ]. L12 clone, specific for variant 295 and DRB1*1101-restricted, was derived from a line obtained from a healthy donor described previously [14 ] and cloned by limiting dilution (see below). T cell clones F17, CS1, and L12 were maintained in culture by weekly stimulation in the presence of allogenic peripheral blood mononuclear cells (PBMC), 0.5 µg/ml phytohemagglutinin (Murex, Dartford, UK), and 20 U/ml human recombinant IL-2 (Boehringer Mannheim, Mannheim, Germany) as described [13 ]. Epstein Barr Virus (EBV)-transformed B cells [B lymphoblastoid cells (B-LCL)] used as APC were generated by incubation of 5 x 106 PBMC with EBV, obtained from the Marmoset lymphoblastoid cell line B95-8 as described previously [13 ]. B-LCL used as autologous APC for Li295 were generated from a DRB1*0101/1101-expressing healthy donor (Li). B-LCL, used to stimulate clones CS1 and L12, were derived from an infected and a healthy donor, respectively [13 , 14 ]. The following homozygous B-LCL were also used as APC: Sweig (DRB1*1101) and SA (DRB1*0101).

Induction of HVR1-specific T cell line, cloning by limiting dilution, and "semi-cloning" assay
HVR1-specific T cell line Li295 was obtained by culturing 2 x 106 PBMC of a healthy donor in RPMI 10% human serum (HS) without IL-2 in the presence of 10 µg HVR1 peptide 295. After 7 days, cells were restimulated with irradiated, autologous PBMC pulsed with peptide and 5 U/ml human recombinant IL-2. Cells were maintained in culture with irradiated, autologous PBMC or mitomycin-C (mit-C)-treated, autologous B-LCL pulsed with peptide in RPMI 10% HS and 10 U/ml IL-2. Proliferation assays to assess specificity were performed after two rounds of in vitro stimulation. At the same time, Li295 was cloned by limiting dilution in Terasaky plates in the presence of autologous, irradiated PBMC pulsed with agonist variant 295 (15 µg/ml) and 20 U/ml IL-2 at 0.3 cells per well [13 ]. For semi-cloning assay, we followed a modified version of the assay described previously by Plebanski et al [15 ]. Li295 was cultured at 5 x 103 cells per well in the presence of 5 x 104 autologous B-LCL prepulsed with peptides in 96-well flat-bottomed microtiter plates in the presence of 10 U/ml IL-2. A week later, the cells were tested in proliferation assays by using autologous B-LCL as APC.

T cell proliferation assay
Proliferation of T cell lines and clones was assessed as described previously [13 , 14 ]. Responder T cells (1.5x105) were cultured in triplicates with 4 x 104 mit-C-treated cognate B-LCL, prepulsed or not with the specific peptide in 96-well flat-bottomed microtiter plates (Costar, Corning, NY) for 2 days. Cells were labeled with 1 µCi 3H-thymidine and harvested 18 h later. 3H-Thymidine incorporation was assessed as described [13 ]. Standard deviations of the mean counts per minute (cpm) of triplicate cultures were consistently below 10%.

TCR antagonism assay
TCR antagonism was tested as described [13 ]. Briefly, autologous B-LCL were prepulsed overnight at 37°C with different doses of agonist peptides (usually between 10 and 30 µg/ml). After washing, APC were treated with mit-C and plated out in 96-well flat-bottomed microtiter plates, and variant peptides were added directly into the wells at various concentrations. After further 5 h of incubation, T cells were added into the wells, and proliferation was measured as described above.

Analysis of TCR down-regulation
TCR down-regulation was assessed as previously reported [13 ]. Briefly, autologous B-LCL (2x105), pulsed overnight with 15 µg/ml agonist or antagonist peptides and washed to eliminate unbound peptide, were mixed with resting 105 T cells 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 APC, previously pulsed with the agonist. The plates were centrifuged at 1000 rpm for 3 min and cultured at 37°C for 18 h. After incubation, the cells were washed with phosphate-buffered saline (PBS), 0.5 mM EDTA, to disrupt conjugates. Cells were stained with a FITC-conjugated anti-TCR {alpha}ß antibody (WT31), a PE-conjugated anti-CD4 antibody (Leu-3a), and a PerCP-conjugated anti-CD20 antibody (Leu-16) to discriminate between B and T cells in the cell population. The relative TCR and CD4 expression was assessed by using flow cytometric analysis.

Detection of apoptotic T cells
T cell line or clones were activated by autologous B-LCL, pretreated with peptides in 96-well flat-bottomed microtiter plates in 200 µl medium for 24 or 48 h. Cells were washed and stained with the PerCP-conjugated anti-CD20 antibody (Leu-16) to gate out APC, and apoptosis was measured in the CD20– population by staining with FITC-conjugated Annexin V and propidium iodide (PI), according to the protocol of the Apotest kit (Dako Cytomation). Flow cytometric analysis was performed on a Becton Dickinson FACScalibur flow cytometer.

Flow cytometric analysis of CD95L and c-FLIP
T cells (105) were cultured with 2 x 105 B-LCL, prepulsed or not, with peptides for 24 h, washed, and stained, first with the hamster anti-CD95L antibody (or isotype-matched antibody), followed by staining with the goat anti-hamster FITC-conjugated antibody and then with the PE-conjugated anti-CD4 antibody. For detection of c-FLIP, T cells were fixed with PBS, 2% paraformaldehyde, and permeabilized in PBS, 0.5% bovine serum albumin, 0.02% sodium azide, and 0.5% saponin. Cells were incubated for 15 min at room temperature with anti-human c-FLIP (NF6) or isotype-matched antibody, followed by incubation with goat anti-mouse FITC-conjugated antibody (Dako Cytomation). Data are presented as fluorescence intensities per number of cells over irrelevant control staining.

Cytotoxic assay
F17 effector cells were activated with a mixture of 0.05 µM PMA and 0.5 µM ionomycin for 4 h and mixed with target T cell clones previously labeled with 51Cr (Amersham, Piscataway, NJ) for 1 h in the presence of peptide-pulsed B-LCL at different ratios [26 ]. After 4 h, 51Cr release in the supernatants was determined on an ME Plus {gamma}-scintillation counter (Micromedic Systems, Huntsville, TN). The percentage of specific lysis was calculated as follows: % specific lysis = 100 x (experimental release–spontaneous release)/(maximum release–spontaneous release).

Analysis of the TCR repertoire
RNA was extracted by using the guanidium hydrocloride-containing Trizol reagent (Life Technologies, Gibco-BRL, Gaithesburg, MD). First-strand cDNA synthesis was performed by using oligo (dT) as a primer for reverse transcription (RT) of 1 µg total RNA (Superscript II RT, Life Technologies). Polymerase chain reaction (PCR) amplification was performed according to Yassai et al. [27 ]. Briefly, cDNA was amplified for 30 cycles under nonsaturating PCR conditions with a panel of 25 TCR ß-variable gene-segment (BV), family-specific primers and a ß-constant primer in duplex. Simplex reactions were used for BV 6.1 and 6.2 amplifications. The common constant primers were labeled at the 5' end with 5' 6-carboxyfluorescein. To normalize the results, the amounts of templates from different samples were titrated at different dilution points of starting material by amplifying the TCR ß-chain constant cDNA. TCR "spectratyping" was performed as described by Yassai et al. [27 ]. Briefly, an equivalent volume of PCR-labeled product was mixed with formamide dye-loading buffer and in the presence of tetramethylrhodamine-labeled size markers (Applied Biosystems, Foster City, CA), heated at 94°C for 2 min and applied to a prerun 5% acrylamide-urea sequencing gel. Gels were run on 377 ABI automatic DNA sequencer for 110 min at 40 W. After resolution on the gel, the labeled PCR products were analyzed by Gene Scan software (Applied Biosystems). TCR spectratyping of a healthy PBMC repertoire typically results in a banding pattern composed of between seven and eight bands at three nucleotide base intervals, reflecting the correct "in frame" nature of functionally rearranged BV-chain TCR products. The limited number of PCR cycles used leads to generation of PCR products with a distribution representative of the starting material, i.e., a Gaussian distribution.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antagonized T cell clones are susceptible to bystander CD95-mediated apoptosis
It is well known that correctly stimulated T cells up-regulate death receptors such as CD95L but at the same time, are protected from CD95-mediated apoptosis through up-regulation of survival factors such as c-FLIP [16 17 18 19 ]. TCR antagonists could interfere with induction of such protective signals and impair survival. To verify this, we used the two previously characterized, HVR1-specific T cell clones, CS1 and L12, specific for variant 295 and DRB1*1101-restricted. For both clones, the HVR1 sequence of HCV-Envelope-2 protein variant 266 exerts strong TCR antagonism when copresented with the agonist variant 295, as reported in Figure 1 . In both cases, the antagonist variant 266 acted by inhibiting TCR down-regulation induced by the agonist (ref. [13 ] and data not shown). As c-FLIP has been described to block CD95-CD95L-mediated apoptosis [16 , 17 ], we looked at its expression by antagonized T cells. Intracellular staining of T cells cultured for 24 h with autologous B-LCL, presenting the agonist alone or the agonist plus the antagonist ligand (ratio 1:3), demonstrated that c-FLIP was up-regulated in CS1 and L12 T cell clones, only upon recognition of the agonist (Fig. 2A ). Next, we addressed direct and bystander induction of apoptosis during TCR antagonism phenomena. First, we cultured each clone with autologous B-LCL, pulsed with the agonist alone or the agonist/antagonist pair (ratio 1:3). Peptide HA307-19, used as control in this setting, was previously shown to act as an irrelevant peptide for both clones in antagonist assays (ref. [13 ] and Fig. 1 ). Apoptosis was measured by fluorescein activated cell sorter (FACS) analysis on the Annexin V- and PI-stained CD20– cell population (see Materials and Methods). Figure 2b and 2C , reports a representative experiment performed with CS1 T cell clone. We failed to observe a significant apoptosis in all conditions in the first 24 h of culture (B). A slight induction of apoptosis was observed after 48 h of culture (C) and was not inhibited by the antagonistic anti-CD95 antibody M3. Similar results were obtained with L12 T cell clone (data not shown).



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  		Figure 1. HVR1-variant 266 exerts TCR antagonism on HVR1-specific T cell clones. CS1 (A) and L12 (B) T cell clones were cultured with autologous B-LCL pulsed with 15 µg/ml of the agonist variant 295 in the absence or presence of the antagonist variant 266 or the control peptide HA307-19 (HA) at the indicated ratios. Proliferation was assessed as 3H-thymidine incorporation and is expressed as cpm of triplicate cultures. Data are from one out of 10, and three independent experiments, respectively.

 


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  		Figure 2. (A) c-FLIP is not up-regulated during TCR antagonism in T cell clones. CS1 and L12 T cell clones were cultured for 24 h with autologous B-LCL, unpulsed (Ctr) or pulsed with 15 µg/ml agonist variant 295 in the absence (295) or presence of the antagonist variant 266 (295+266) at a 1:3 ratio. Expression of c-FLIP was assessed on the gated, PE-conjugated, anti-CD4, antibody-stained population by intracellular staining. Data are reported as fluorescence intensity per number of cells. Data are from one representative out of two independent experiments performed with each clone. (B and C) Antagonized T cell clones do not undergo "fratricide killing." CS1 T cell clone was cultured for 24 (B) or 48 (C) h in different conditions, as indicated. Agonist/antagonist or agonist/control peptide HA307-19 ratio was 1:3. The percentage of Annexin V+ and PI+ T cells in the CD20– population is reported. (C) Solid bars represent the percentage of apoptosis measured in the presence of the antagonistic anti-CD95 antibody M3. Data are from one representative out of three independent experiments.

 
Failure to up-regulate c-FLIP may render antagonized T cells susceptible to bystander killing operating through CD95. To verify this, we repeated the experiment in Figure 2 in the absence (data not shown) or in the presence of the agonistic anti-CD95 antibody CH11. When this antibody was added to the cultures, we already detected increased apoptosis in antagonized CS1 and L12 T cell clones after only 24 h of culture (Fig. 3A ). A hypothetical scenario sees antagonized T cells killed by corecruited, activated CD95L+ T cells of the same or different specificity. With this in mind, we cultured the CS1 T cell clone in the simultaneous presence of autologous B-LCL and T cell clone F17, specific for peptide HA307-19 and DRB1*1101-restricted [13 ]. B-LCL were unpulsed or pulsed with the agonist alone or the agonist/antagonist pair (ratio 1:3). F17 was preactivated with PMA and ionomicin or stimulated directly into the well by its antigenic peptide HA307-19, displayed on the same APC bearing the agonist/antagonist pair. Both treatments induced CD95L up-regulation as expected in the F17 clone (data not shown and ref. [25 ]). Apoptosis of 51Cr-labeled, antagonized T cells was determined in a classical cytotoxic assay, results of which are reported in Figure 3B . Again, more antagonized T cells were killed in comparison with the correctly activated (cultured with the agonist variant 295 alone) cells, and the killing was abolished in the presence of the antagonistic anti-CD95 antibody M3 (data not shown and ref. [26 ]).



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  		Figure 3. Antagonized T cells are susceptible to CD95-mediated bystander killing. (A) CS1 and L12 T cell clones, treated as reported in Figure 2 , were cultured in the presence of the agonistic anti-CD95 antibody CH11 (100 ng/ml). Apoptosis, evaluated as in Figure 2 , is reported as histograms of cells stained with Annexin V. One representative, out of two independent experiments, performed with each clone is shown. (B) 51Cr-labeled CS1 T cell clone (target cells) was cultured for 16 h with effector F17, prestimulated with PMA + ionomicin (PI F17) or stimulated into the assay with peptide HA307-19 (F17+HA307). Target and effectors were cultured in the presence of B-LCL, unpulsed (–) or pulsed with agonist 295 or agonist 295 plus antagonist 266. The agonist/antagonist ratio was 1:3. Values are reported as percentage of specific 51Cr release and are derived from one out of two independent experiments.

 
TCR antagonism phenomena can modify the agonist-induced TCR repertoire
The results presented above suggest that a single T cell encountering an APC presenting an agonist plus an antagonist may be killed by a bystander-corecruited CD95L+ T cell. However, the outcome of apoptotic phenomena determined by TCR antagonism may be different in a T cell population where T cells (susceptible or unsusceptible to a given antagonist) coexist [28 , 29 ]. To dispose of an in vitro system suitable for studying the effect of TCR antagonism on the shape of polyclonal T cell populations, we induced in vitro HVR1-specific T cells [14 ]. Line Li295 (CD4+ phenotype, data not shown), obtained from a healthy individual expressing the DRB1*0101/1101 alleles, recognized variant 295 well and variant 266 poorly. Proliferative response and restriction of this line are reported in Figure 4A and 4B , respectively. Of note, response of Li295 was restricted by both human leukocyte antigen (HLA)-DR alleles expressed by the individual. Variant 266 exerted TCR antagonism on cell proliferation, mainly at the agonist/antagonist ratio of 1:3 and in this condition, also interfered with TCR down-regulation induced by the agonist (Fig. 4C and 4D) , as measured by FACS. Next, we looked at apoptosis after 24 h of culture of Li295, and autologous B-LCL presenting the agonist 295 plus the antagonist 266 or as control, presenting with the agonist alone. In contrast with the result in Figure 2 , apoptosis of antagonized T cells was detected after 24 h of culture and was inhibited significantly by the antagonistic anti-CD95 antibody M3 (Fig. 4E) . The percentage of T cells expressing c-FLIP was 0.1% (MFI=2) in unstimulated T cells and 13% (MFI=3) and 1.5% (MFI=2.4) in T cells stimulated with variant 295 alone or with the agonist/antagonist pair, respectively. CD95L up-regulation was mainly detected during stimulation with the agonist variant 295 (89% with MFI=123 vs. 20% with MFI=17 of control, unstimulated cells). However, expression of CD95L was up-regulated in a small percentage of agonist plus antagonist-stimulated cells (25% with MFI=31 vs. 20% with MFI=17 of control, unstimulated cells). These data suggest that the fraction of T cells becoming CD95L+ can influence cellular killing when TCR antagonism operates. To define in more detail the clonotypic composition of line Li295, we cloned it after 1 week from the first activation with antigen. We obtained 11 clones, whose BV use and reactivity are resumed in Table 1 , in comparison with BV use and reactivity of the parental line Li295. The analysis of the BV use of Li295 cell population shows the presence of one major clonal expansion, BV3 (47% of the total BV repertoire), and two other important BV, BV6.1 (33%) and BV16 (13%). BV1, BV4, BV5.3, BV11, and BV13 were far less represented segments (% <5). The reactivity of the four clones, Li7, Li13, Li18, and Li22, was similar to that of the parental Li295, although Li7 possessed the lowest reactivity for variant 295. The other agonist-specific seven clones were marginally susceptible or unsusceptible to TCR antagonism and/or variably cross-reacting toward variant 266. These results suggested that despite its globally scant response to variant 266, Li295 contained T cells with heterogeneous behavior with respect to recognition of variants 295 and 266. In this regard, we decided to repeat the experiments performed with CS1 and L12 clones by using Li295-derived clones that were completely susceptible to the antagonist of the parental line (variant 266) or cross-reacting with variant 266 and unsusceptible to antagonism. A representative experiment performed to measure different responses of clones Li12 and Li22 to the agonist, the antagonist, and the agonist plus the antagonist is shown in Figure 5A and 5B , which reports the proliferative responses for each clone. Figure 5C and 5D , reports apoptosis in the presence of the anti-CD95 antibody CH11, and Figure 5E and 5F , reports c-FLIP expression. The data clearly show that again, when antagonism operated, T cells were not protected from bystander CD95-mediated apoptosis (i.e., Li22, Fig. 5C ). Similar results were obtained with Li13 and Li18 T cell clones (data not shown). In contrast, a clone (Li12), which did not recognize variant 266 as an antagonist, but could respond to this variant, was more resistant to bystander CD95-mediated killing (Fig. 5D) . Measurement of c-FLIP expression shows that the copresentation of variant 266 with variant 295 abolished c-FLIP up-regulation in a clonal population susceptible to TCR antagonism (i.e., Li22).



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  		Figure 4. Characterization of ex vivo-induced, HVR1-specific T cell Li295, which was cultured with autologous B-LCL (A) or partially matched homozygous B-LCL (B), pulsed with the indicated concentrations of HVR1 variants (peptides 295 and 266). Proliferation was estimated as in Figure 1 . (C) Variant 266 acts as a TCR antagonist for the Li295 T cell line, which was cultured with autologous B-LCL, prepulsed with 15 µg/ml agonist variant 295 in the presence of antagonist variant 266 or control peptide HA307-19 (HA307) at the indicated ratios. T cell proliferation is reported as in A + SD, and data are from one out of three independent experiments. (D) Variant 266 inhibits agonist-mediated TCR down-regulation. Li295 was cultured with unpulsed (–), 295-pulsed, and 295 plus 266 (ratio 1:3)- or 295 plus HA307 (ratio 1:3)-pulsed, autologous B-LCL. TCR down-regulation was assessed by FACS, and the results are reported as mean of fluorescence intensity (MFI) of TCR expression + SD. Data are from one out of two independent experiments. (E) CD95-mediated apoptosis occurs in Li295 during antagonism. The Li295 T cell line was cultured with autologous B-LCL pulsed with 295 and 295 + 266 variants at the indicated ratio. Apoptosis was determined, as reported in Figure 2 , after 24 h of culture in the absence (hatched bars) or in the presence (solid bars) of the antagonistic anti-CD95 antibody M3. Data are reported as mean values of two independent experiments + SD.

 

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  		Table 1. BV Use and Reactivity of HVR1-Specific T Cell Line and Clones

 


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  		Figure 5. (A and B) Characterization of Li295 at clonal level. Li22 (A) and Li12 (B) T cell clones were cultured with B-LCL, prepulsed with the agonist variant 295 (15 µg/ml), the antagonist variant 266 (30 µg/ml), or the agonist variant 295 (15 µg/ml) plus the antagonist variant 266 at a 1:3 ratio. Data are from one out of two independent experiments performed with each clone, and proliferation was estimated as in Figure 1 . (C and D) Only antagonized T cell clones are susceptible to CD95-mediated bystander killing. Clones Li22 (C) and Li12 (D) were cultured in the same conditions as A and B in the presence of the agonistic anti-CD95 antibody CH11 (100 ng/ml). Apoptosis, evaluated as in Figure 2 , is reported as percentage of cells positively stained with Annexin V. One representative out of two independent experiments is reported for each clone. (E and F) c-FLIP is not up-regulated in antagonized T cell clones. Clones Li22 (E) and Li12 (F) were cultured for 24 h with autologous B-LCL in the same conditions as A and B. Expression of c-FLIP was assessed as in Figure 2 . Data are reported as percentage of T cells positively stained with anti-c-FLIP antibody NF6. Data are from one representative out of two independent experiments performed with each clone.

 
To establish whether death favored by TCR antagonism changes the composition of the agonist-primed TCR repertoire, Li295 cells were cultured in the presence of the agonist/antagonist pair (ratio 1:3) for 7 days (first stimulation), restimulated, and then cultured for a further 7 days (second stimulation). Cell death with respect to T cells cultured with the agonist alone was measured after 24 h from the beginning of each stimulation. The results of both stimulations showed an increment of cell death in agonist plus antagonist-stimulated T cells as compared with agonist-stimulated T cells measured, as reported in Figure 5 . Indeed, in the first stimulation, the percent of apoptosis of antagonized T cells was 42 versus eight of control agonist-stimulated cells, and in the second stimulation, the percent of apoptosis of antagonized cells was 38 versus 12 of control agonist-stimulated T cells. TCR repertoires of T cells, before and after culture with the antagonist (collected at the end of each stimulation), were analyzed by spectratyping. Figure 6A reports the composition of the TCR repertoire of Li295 immediately before culture in the presence of the agonist/antagonist pair, only in relation to the most represented BV segments, whereas Figure 6B and 6C , shows the effect of the antagonist. After 7 days of culture in the presence of the agonist plus the antagonist variants (first stimulation, Fig. 6B ), the largest clonal expansion (BV3) decreased and completely disappeared 7 days after the second stimulation (Fig. 6C) . In contrast, BV3 clonal expansion in control cultures (in the presence of the agonist alone) still persisted after the first and the second stimulation (data not shown). Moreover, culture with the agonist/antagonist pair induced a particular expansion of the BV6.1 and BV16 family. To further verify the change in the composition of the agonist-primed TCR repertoire, we measured the proliferation to the agonist and the antagonist variants. Reactivity to the agonist variant 295 decreased after the first culture in the presence of the antagonist (Fig. 6E) and tended to disappear after the second stimulation (Fig. 6F) , whereas in the presence of the agonist alone, reactivity was maintained (data not shown).



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  		Figure 6. Culture of Li295 in the presence of the antagonist variant 266 changes the TCR repertoire and abolishes response to the agonist variant 295. 295-Primed T cells [Li295(295), A and D] were stimulated every 7 days (first stimulation, B and E; second stimulation, C and F) with autologous B-LCL, pulsed with the agonist 295 (15 µg/ml) plus the antagonist 266 (ratio 1:3). The composition of the TCR repertoire (A–C) was analyzed by spectratyping, and pie diagrams show BV composition (Others, sum of BV segment % whose expression was <5%). The reactivity to the agonist variant 295 (D–F) was measured as in Figure 1 . Spectratyping and proliferation data are from one out of two independent experiments.

 
A semi-cloning assay, in which line Li295 was divided into 50 wells (see Materials and Methods), was performed by culturing half in the presence the agonist alone (Fig. 7A ) and the remainder in the presence of agonist plus antagonist (1:3 ratio, B). Again, 1 week of culture with the agonist plus the antagonist variants was sufficient to negatively select agonist 295-specific micro-lines (Fig. 7B) , which were easily obtained following culture with the agonist 295 alone (Fig. 7A) . Figure 7A also shows that the agonist-specific micro-lines were susceptible to TCR antagonism exerted by variant 266. Of note, the unique HVR1-specific micro-line obtained after culture with the agonist/antagonist pair was cross-reacting toward the antagonist variant 266 and escaped antagonistic phenomena (Fig. 7B) .



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  		Figure 7. Semi-cloning of Li295, which was divided into 25 micro-lines cultured with autologous B-LCL, pulsed with the agonist variant 295 (A) and 25 micro-lines cultured in the presence of the agonist plus antagonist variants (ratio 1:3, B). After 7 days, growing cells (11 and 12 micro-lines in A and B, respectively) were collected and tested for their reactivity to 15 µg/ml agonist variant 295 [295 (15)], 15 or 45 µg/ml antagonist variant 266 [266 (15) and 266 (45), respectively], and 15 µg/ml variant 295 plus 45 µg/ml variant 266 [295+266 (15+45)], as indicated. Proliferation is reported as in Figure 1 . Data are from one representative out of two independent, semi-cloning assays.

 
The antagonist variant 266 acts as an agonist in the induction phase of HVR1-specific T cells derived from the same donor
The latter findings of selection of weakly antagonist-specific cells (Fig. 7B) after culture with the agonist/antagonist pair and the results reported in Figure 6 raised the question as to whether variant 266 alone could be able to induce in vitro HVR1-specific T cells. Therefore, we used variant 266 as an agonist to elicit a HVR1-specific response in T cells derived from the same individual used to induce line Li295 (Fig. 8 ). We obtained line Li266 (CD4+ phenotype, data not shown), specific for variant 266 and cross-reacting with variant 295 (Fig. 8B) . It is more important that the analysis of the BV use of Li266 revealed consistent expression of BV6.1, BV16, and BV18 (A). These results are in agreement with the notion that BV6.1 and BV16 (expanded at the expense of the BV3 family in Li295 when antagonism operated, Fig. 6 ) are BV segments that confer cross-reacting nature to HVR1-specific TCRs of the analyzed individual.



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  		Figure 8. Characterization of ex vivo-induced, HVR1-specific T cell Li266. (A) The composition of the TCR repertoire was analyzed by spectratyping, and the pie diagram shows BV composition of Li266 (Others, sum of BV segment % whose expression was <5%). (B) Li266 T cell line was cultured with autologous B-LCL, pulsed with increasing concentrations of HVR1 variants (295 or 266 as indicated). Proliferation was estimated as in Figure 1 . Spectratyping and proliferation data are from one out of two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work provides evidence that TCR antagonists can modify the agonist-specific TCR repertoire by interfering with pathways that serve to protect T cells from deletion mechanisms acting through the CD95-CD95L system. This phenomenon leads to specific elimination of T cells narrowly focused to the agonist. The data supporting this assumption have been produced by using human HVR1-specific CD4+ T cell clones and lines, as an in vitro system of TCR antagonism [13 , 14 ].

By using monoclonal HVR1-specific CD4+ T cells, we have shown that suppression of c-FLIP expression was associated with susceptibility of antagonized T cells to CD95-mediated apoptosis, supporting the protective role of c-FLIP in regulating the viability of correctly antigen-activated T cells [16 17 18 19 , 30 ]. In spite of the absence of fratricide killing during antagonism (Fig. 2) , we clearly demonstrated that the agonistic anti-CD95 antibody CH11 could kill antagonized T cells selectively and not agonist-activated T cells, implying that the former became susceptible to bystander CD95-mediated apoptosis. Reinforcing this issue, we demonstrated (Fig. 3B) that when neighboring T cells of clone F17 recognized antigen on the same APC as the antagonized CS1 clone, this latter was more efficiently killed than agonist-stimulated and unstimulated T cells. We believe this result shows convincingly that the vicinity of antagonized and activated T cells, in the milieu of antigen recognition, can favor bystander killing, phenomena-specific for the antagonized cells. Knowing that the total T cell repertoire expanded by an agonist may contain clones displaying different degrees of cross-reactivity and/or susceptibility to a TCR antagonist [28 , 29 ], we looked at the effect of apoptotic phenomena on the TCR repertoire of an ex vivo-primed, HVR1-specific T cell population (Fig. 4) . The line obtained (Li295) was focused toward variant 295. However, at the clonal level, it manifested a heterogeneous reactivity in that T cell clones, cross-reacting or escaping antagonism, were also present (Table 1) [30 ]. In this regard, we have demonstrated that clones susceptible to antagonism exerted by variant 266 (Li22 in Fig. 5 and Li13 and Li18, data not shown) manifested the same susceptibility to bystander CD95-mediated apoptosis observed in CS1 and L12 T cell clones, although derived from a different individual. In contrast, clones that escaped antagonism were more resistant to bystander CD95-mediated killing (i.e., Li12 in Fig. 5 ). This strongly suggests that in a heterogeneous cell population, where T cell clones susceptible and unsusceptible to antagonism coexist, unsusceptible clones can kill the susceptible ones. Supporting this and in contrast with the results obtained with monoclonal T cells, Li295 underwent significant CD95-induced apoptosis during antagonism (Fig. 4E) . The partial up-regulation of CD95L and c-FLIP supports the hypothesis that part of Li295 T cells became activated and killed the antagonized counterpart. Although we do not present direct evidence that only the agonist-specific T cell clones die, a dramatic change of the TCR repertoire is demonstrated clearly. Indeed, our data show that when the BV3 family was eliminated completely, the response to the agonist variant 295 was spoiled. Moreover, our data tend to exclude that the disappearance of the BV3 family was simply a result of death by neglect rather than to CD95-mediated apoptosis. This is shown first in that CD95L was partially up-regulated in Li295 when antagonism occurred and second, in that the antagonistic anti-CD95 antibody M3 rescued Li295 from death (Fig. 5) . In addition, we provided IL-2 at the beginning of each culture (and in a semi-cloning assay) in all culture conditions. This cytokine, which can delay death as a result of growth factor withdrawal, is ineffective in rescuing T cells dying via the CD95 pathway [31 , 32 ].

The analysis of the TCR repertoire of Li295-derived clones revealed that the two clones expressing BV3+ TCR were reactive for variant 295 and strongly susceptible to TCR antagonism exerted by variant 266. In contrast, the clones Li4 and Li12, recognizing variant 266 and unsusceptible to TCR antagonism, expressed BV6.1 and BV16, respectively (Table 1) . As in the experiment shown in Figure 6 , BV6.1+ and BV16+ TCRs are largely amplified in the presence of the antagonist variant 266, and the BV3 family disappears; these data support the hypothesis that antagonist-specific cells are those that become activated during antagonism and are likely to kill the antagonized counterpart. This is further supported by the evidence that primary culture of T cells derived from the same individual with the antagonist variant 266 alone could induce an antagonist-specific CD4+ T cell population expressing BV6.1 as dominant BV (and BV16) and with no detectable levels of BV3 expansion (Fig. 8) .

TCR antagonists have conventionally been considered peptide reagents that induce a block of T cell activation and functions [8 9 10 ]. More recently, it has been suggested that they could induce incomplete signals that may ultimately lead to induction of anergy [33 ]. Some authors have also shown nicely how TCR antagonism can interfere with activation of cytotoxic T cell responses in the priming phase, narrowing the repertoire of immune cells and inducing the activation of partial immune cell functions [11 12 13 14 15 ]. Our novel hypothesis is that TCR antagonism not only blocks the T cell response to a given epitope but also modifies the initial agonist-specific T cell repertoire through deletion, so that the original reactivity will never be restored, even if the antagonist is eliminated subsequently. Our in vitro system does not address definitely whether further culture of the HVR1-specific T cell population Li295 in the presence of the agonist plus the antagonist variant eventually yields T cells with high avidity for the antagonist. The fact that we successfully obtained an antagonist-specific T cell line (Li266) by in vitro stimulation with the antagonist variant 266 suggests that in physiological conditions, antagonist-specific clones may arise, allowing restoration of the HVR1-specific response. This is likely in that variant 295 and variant 266 possess equally suitable "motifs" for binding to HLA-DR11 [13 ]. However, we emphasize that each time an antagonist is generated, the immune response could be shut off, requiring reprogramming. For the immune response to become efficient again, new, specific T cells must arise, and this will take time, leaving a time window for the virus to overgrow and making TCR antagonism an efficient strategy of escape. It should be pointed out that the analysis of the BV families expressed by T cells induced in vitro with variant 266 (derived from the same individual used to induce the Li295 T cell line) suggests that deletional mechanisms could also be operative during the induction phase of HVR1-specific responses if simultaneous infection by multiple viral variants with antagonistic properties occurs.

Whether deletion mechanisms are genuinely favored in vivo remains to be established. Clarification of this aspect will help to find strategies to contrast TCR antagonism phenomena induced by mutant pathogens and help to shed light on how to set up peptide-based immune therapies.


   ACKNOWLEDGEMENTS
 
The present study was financially supported by COFIN and Ateneo projects. The authors thank H. Walczak and P. H. Krammer for providing the anti-c-FLIP NF6 antibody, A. Nicosia for providing HVR1 peptides, and The Italian Red Cross in Bergamo for providing buffy coats.

Received August 16, 2004; revised March 14, 2005; accepted April 7, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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