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The Journal of Immunology, 1998, 161: 594-601.
Copyright © 1998 by The American Association of Immunologists

The Clonal Composition of a Peptide-Specific Oligoclonal CTL Repertoire Selected in Response to Persistent EBV Infection Is Stable Over Time1

Victor Levitsky2, Pedro-Otavio de Campos-Lima3, Teresa Frisan and Maria G. Masucci

Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V{alpha} and Vß chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor’s blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunologic memory is usually understood as the ability of the immune system to respond efficiently and with fast kinetics to previously encountered Ags. The most common manifestations of the response are production of Abs and proliferation of different subsets of Ag specific T cells (reviewed in Refs. 1–4). The development of T cell memory is accompanied by phenotypic changes and an increase in the number of the specific cells. The latter phenomenon appears to be accounted for by the high burst of T cell proliferation triggered upon the first contact with specific Ag (5). The population of Ag-specific T cells expanded during the primary response shrinks in size as soon as the Ag is cleared or its load is significantly decreased. Surviving cells enter the pool of memory T cells that can persist for extended periods of time. The above sequence of events was observed on many experimental models of T cell memory, but a number of important issues concerning the mechanisms of selection and maintenance of memory remain unclear.

It is generally agreed now that the specific Ag is not essential for the maintenance of memory cells (5, 6, 7, 8). However, only T cells that are activated by the persistent specific Ag can ensure protection against pathogens entering nonlymphoid organs (9). The suggestion that populations of memory T cells are functionally different and maintained by different mechanisms in the presence or the absence of Ag is also supported by a number of studies on the in vivo kinetics and phenotype of these cells (reviewed in 1 . T cell memory "without Ag" has attracted considerable interest, and several models have been suggested to explain the maintenance of memory in this situation (10, 11, 12, 13, 14). It is quite obvious, however, that the "infectious memory" that is associated with chronic antigenic stimulations in the course of persistent viral infections and autoimmune diseases may be more relevant in a clinical perspective. Conceivably, the specific Ag may exert several contrasting effects on the selection and maintenance of memory T cell repertoires. Chronic antigenic stimulation may result in clonal exhaustion of high affinity T cells (15), but it may also trigger the preferential expansion of high affinity clones, thereby narrowing the specific repertoire selected during the primary response. Moreover, it cannot be ruled out that new clones that were not involved in the primary response are recruited by the persistent Ag into the memory repertoire at latter stages. Experimental data concerning the composition and dynamics of memory repertoires in the presence of specific Ag are scarce, especially in humans, and do not allow estimation of the impact of the above scenarios in the course of natural immune responses. Data from various mouse models have shown that memory T cells can be maintained in vivo for long periods of time without apparent signs of clonal exhaustion and that a significant proportion of primary Ag specific T cell repertoires is preserved in memory response. However, these studies used either TCR transgenic mice (16) or unusual T cell responses with highly restricted usage of TCR V{alpha} or Vß chains (17, 18). It is unclear, therefore, to what extent these results are relevant for human immune responses that are characterized by much higher longevity and are usually composed of T cell clones that exhibit heterogeneous usage of TCR-variable polypeptide chains and presumably have various affinities and avidities for the specific Ag. Several groups have reported long term persistence of a single Ag-specific clone under conditions of constant antigenic stimulation in humans, but it remained unclear whether this stability reflected the ongoing selection of the most efficient clone or the general stability of polyclonal repertoires (19, 20). None of these studies has investigated the role of TCR avidity/affinity in the maintenance of memory T cell responses.

EBV provides a textbook case of persistent viral infection in humans (21). Up to 95% of humans worldwide are life-long carriers of the virus. EBV-infected individuals mount a strong EBV-specific CTL response restricted through different HLA alleles. This memory response is directed mainly against EBV proteins expressed in latently infected B cells that grow in culture as lymphoblastoid cell lines (LCLs)4 (reviewed in 22 . One of the immunogenic EBV proteins, the EBV nuclear Ag 4 (EBNA4), contains several antigenic epitopes recognized by HLA A11-restricted cytotoxic T cells (23, 24). The CTL response against a peptide mapped to the 416 to 424 residues of the protein (IVTDFSVIK, designated IVT) has been characterized in several EBV-infected HLA A11-positive individuals. In most of the cases, the IVT-specific response was shown to be oligoclonal and composed of T cell clonotypes expressing structurally heterogeneous TCRs (25). Our previous studies have demonstrated that the IVT epitope is highly conserved among EBV strains isolated from Caucasian individuals, indicating that the variability of the IVT-specific CTL repertoire is not influenced by the probable polymorphism of epitope sequences (23, 26).

In this study we present a 5-yr follow-up of the IVT response in one HLA A11-positive EBV carrier. Our results show that the characteristic oligoclonality of the response is stably preserved in this individual over time and suggest that, even in the continuous presence of Ag, the avidity/affinity of interaction with specific Ag is not the major driving force of the selection and maintenance of memory T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

EBV-transformed LCLs were obtained by infection of lymphocytes from HLA class I-typed donors with culture supernatants of the virus producer B95.8 cell line (27). All cell lines were maintained in RPMI 1640 supplemented with 100 µg/ml streptomycin, 100 IU/ml penicillin, and 10% FCS (complete medium).

Synthetic peptides

Peptides, synthesized by the Merrifield solid phase method (28), were purchased from Alta Bioscience (Alta Bioscience, University of Birmingham, School of Biochemistry, Birmingham, U.K.). The peptides were dissolved in DMSO at a concentration of 10-2 M and further diluted in PBS to obtain the indicated concentrations before the assays. The protein concentration of the DMSO stock solutions was determined in a Biuret assay (29). An alanine replacement set of the IVT peptide was produced by consecutive substitution of the amino acids in each position of the peptide with alanine.

Generation of CTL cultures and clones

EBV-specific CTLs were obtained as previously described (30) by stimulation of lymphocytes from the EBV-seropositive donor BK (HLA A2, 11 B7, 35) with the autologous B95.8 virus-transformed LCL. After two or three consecutive restimulations the cultures were expanded in complete medium supplemented with 10 U/ml rIL-2 and 30% (v/v) culture supernatant from the gibbon lymphoma line MLA144 (31). The same protocol was used to generate EBV-specific CTL minicultures that were initiated from 104, 3 x 104, or 6 x 104 PBLs in U-shaped 96-microwell plates. Single cell cloning was performed by limiting dilution in 96-well plates in 200 µl of IL-2-supplemented medium containing 105 irradiated (3000 rad) allogeneic PHA-pulsed PBLs as feeders. Growing cultures were transferred into 24-well plates and were fed twice a week by replacing half the medium. The EBV specificity and class I restriction of the clones were investigated by testing their cytotoxic activity against a panel of EBV-positive and -negative targets, including the autologous LCLs, allogeneic LCLs matched through single class I alleles, at least two cell lines for each allele, PHA (1 µg/ml/107 cells)-activated blasts, HLA-mismatched LCLs, and the prototype NK-sensitive target K562.

Cytotoxicity tests

Cytotoxic activity was measured in standard 4-h 51Cr release assay (30). The targets were labeled with Na51CrO4 (0.1 mCi/106 cells) for 1 h at 37°C. For polyclonal CTL cultures, the cytotoxicity tests were routinely run at 10:1, 3:1, and 1:1 E:T cell ratios in triplicate. CTL clones were tested at E:T cell ratios of 3:1 or 5:1. Peptide pulsing experiments were performed by adding 15 µl of the indicated peptide preparations diluted in PBS to triplicate wells of 96 V-shaped well plates containing 4 x 103 labeled targets in 25 µl of complete medium. The plates were preincubated for 1 h at 37°C, and the CTLs were then added in 100 µl of complete medium. Peptide toxicities were checked in each assay and were always <3%. To calculate the amount of peptide (P50) required for half-maximal lysis of peptide-pulsed targets, titration curves were normalized to the level of specific lysis induced at 10-7 M of the IVT peptide according to the formula: normalized lysis at titration point X = % specific lysis at titration point X/% specific lysis 10-7 M x 100. The mean normalized values of specific lysis (three to seven experiments for each CTL clone) were used to build integrated normalized titration curves from which the P50 values were derived.

RNA extraction, first strand cDNA synthesis, PCR amplification, and sequencing

Total RNA was extracted from 2 to 10 x 106 cells by the single-step acid guanidinium thiocyanate-phenol-chloroform method described by Chomczynski (32). First-strand cDNA was prepared as previously described (25). PCR primers (Clontech, Palo Alto, CA) specific for the variable domains and the constant region of TCR (Clontech) were used to amplify 22 {alpha}-chain and 24 ß-chain TCR families. Each PCR reaction contained 0.5 mM of each primer, 2% of the product of first-strand cDNA synthesis, 0.2 mM dNTP, 2 U of Taq polymerase (Amersham, Aylesbury, U.K.), 10 mM Tris-HCl (pH 8), 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin in a final volume of 50 µl. The reaction was overlaid with 50 µl of mineral oil and run for 30 cycles of 1-min denaturation at 95°C, 1-min annealing at 55°C, and 1-min extension at 72°C. Ten percent of each PCR reaction was analyzed in a 1.8% agarose gel containing 0.5 mg/ml ethidium bromide. Amplified fragments were then blunt end cloned into the EcoRV site of the pGEM plasmid (Promega, Madison, WI). Ten micrograms of recombinant plasmid DNA was denatured with NaOH for 10 min, neutralized by sodium acetate (pH 4.8), ethanol precipitated, and used for direct double strand sequencing with fluorescein-labeled M13 universal (5'-CGACGTTGTAAAACGACGGCCAGT-3') and M13 reverse (5'-CAGGAAACAGCTATGAC-3') primers using an ALF automated DNA sequencer (Pharmacia, Uppsala, Sweden).

CD8 blocking experiments

The OKT8 mouse mAb (American Type Culture Collection, (ATCC), Manassas, VA; CRC 8014) of the IgG2a subclass was used as anti-CD8. The CD4 (ATCC CRL 8002)-specific OKT4 (IgG2b) and the MHC class II-specific 9.3F10 (ATCC HB 180, IgG2a) Abs were used as a negative control. The Abs were purified from ascites induced in pristane-pretreated mice as described previously (33). To assess their effect on CTL lysis, preparations of purified Abs were diluted in PBS, and 25 µl of each dilution was added in triplicate to the effector cells resuspended in 25 µl of complete medium in V-shaped 96-well plates. The plates were preincubated for 30 min at 37°C before adding 4 x 103 51Cr-labeled targets in 50 µl of complete medium, and the assay was then run for additional 4 h.

Surface marker analysis

Indirect immunofluorescence staining was performed using the OKT3 and OKT8 mAbs and FITC-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark) as second Ab. The percentage of positive cells and the mean fluorescence intensity were determined with a FACSort analyzer (Becton Dickinson, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of IVT-specific clones from donor BK

EBV-specific CTL cultures were generated on five different occasions during a period of 57 mo by stimulation of PBLs from the EBV-seropositive donor BK (HLA A2, A11, B7, B60) with the autologous B95.8 virus-transformed LCL (Table IGo). CTL clones were obtained by limiting dilution from polyclonal cultures or minicultures established as described in Materials and Methods, and their cytotoxic activity was tested against a panel of targets including the autologous LCL, allogeneic LCLs sharing single HLA class I allele, mismatched LCLs, and the K562 cell line. EBV-specific A11-restricted clones were further tested for recognition of A11-positive PHA blasts preincubated with 10-7 M of the IVT peptide. Most of the clones isolated in each stimulation lysed the autologous LCL in an A11-restricted fashion. Between 55 to 88% of the cytotoxic clones were specific for the IVT peptide (Table IGo), while the remaining cytotoxic clones were either specific for the subdominant A11-restricted EBNA4-derived epitope AVFDRKSVAK (designated AVF) (34) or exhibited EBV-specific lysis restricted through other class I alleles expressed by this donor. These results are in good agreement with previous data that have demonstrated the immunodominant nature of the IVT epitope (24, 34).


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  		Table I. Representation of IVT-specific clones in five independent stimulations of PBLs from donor BKa

 
Composition and longitudinal analysis of IVT-specific CTL response

We have previously shown that the IVT-specific response of donor BK is composed of T cells expressing several structurally different TCRs (25). We have now extended this analysis to a panel of 83 clones isolated from the five reactivation experiments. Each clone was tested for recognition of IVT analogues in which each position of the peptide was consecutively substituted with alanine and was tested in standard chromium release assays using as targets A11-positive PHA blasts prepulsed with 10-9 or 10-10 M of the synthetic peptide. The tests were repeated between 2 and 10 times for each CTL clone with highly reproducible results. Only five patterns of reactivity were revealed by this analysis (Fig. 1Go). Alanine substitutions at positions 4 (P4) and P5 of the IVT peptide abrogated recognition, whereas substitutions at P2 and P3 were fully compatible with recognition by all clones. Recognition of the P1, P6, P7, and P8 analogues discriminated among five functionally distinct groups of clones. Sensitivity to substitutions in all four positions characterized the group 1 clones, while group 2 and 3 clones tolerated the P6 and P1 substitutions, respectively. A less stringent recognition of P7 and P8 or P6, manifested by the significant lysis of cells pulsed with 10-9 M of these analogues, distinguished group 3 clones from group 4 and 5 clones.



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  		FIGURE 1. Patterns of fine peptide specificity of IVT-specific CTL clones revealed by ASM. The CTL clones were tested for recognition of a set of IVT analogues in which each position of the peptide was consecutively substituted with alanine. Roman numbers on the x-axis indicate the modified position of the analogue. Black and striped bars show the level of specific lysis of target cells preincubated with 10-9 and 10-10 M of the peptides, respectively. Killing of the untreated targets (-) is shown by white bars. Each CTL clone was tested at least twice and up to 10 times with highly reproducible results.

 
CTL clones derived from the first four stimulation procedures were isolated by limiting dilution of polyclonal CTL cultures. CTL clones exhibiting group 1 and 2 reactivity were obtained on all four occasions, while CTL clones of the other specificities were generated only from stimulation III (Fig. 2Go). In the latter case, polyclonal CTL culture was made by combining several minicultures expressing IVT-specific reactivity. The diversification of the repertoire observed in stimulation III could reflect actual changes in the composition of IVT-specific CTLs in vivo or be an artifact of in vitro manipulations performed during stimulation and/or cloning procedures. To distinguish between these possibilities, minicultures were generated starting from different amount of PBLs. The majority of minicultures established from 1 x 104 or 3 x 104 PBLs did not exhibit specific reactivity against the IVT or AVF peptide, while both reactivities were detectable in 80% of the minicultures initiated from 6 x 104 PBLs/well (data not shown). This is consistent with the results of limiting dilution analysis that set the frequency of IVT-specific CTLs in donor BK in the range of one precursor cell per 4.5 to 5.5 x 104 PBLs (T. Frisan et al., unpublished observations). Eleven CTL minicultures exhibiting IVT-specific reactivity were tested for their ability to recognize the A1, A6, and A8 analogues that would allow classifying the reactivity as representative of one of the functional groups. The results of this test are presented in Table IIGo. One CTL culture exhibited the pattern of reactivity characteristic of group 1 clones, four cultures had reactivity exhibited by group 2 clones, and group 4 and 5 reactivities were represented by two minicultures each. Two additional minicultures exhibited a mixed pattern of reactivity. The results have shown that stimulation of CTLs in minicultures allowed an enrichment of the CTL population for a certain group of reactivity. CTL clones generated from representative minicultures obtained in stimulation V were tested by ASM and were shown to belong to group 1, 2, 4, or 5. None of the clones exhibited the group 3 recognition pattern (Fig. 2Go). As was expected, the majority of CTL clones generated from a particular miniculture exhibited only one of the four reactivities.



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  		FIGURE 2. Number of IVT-specific clones belonging to different groups of reactivities tested by ASM in each stimulation. Clones were obtained from CTL bulk cultures (stimulations I, II, and IV) or from separate minicultures (stimulations III and V) by limiting dilution and were tested by ASM.

 

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  		Table II. Reactivity of IVT-specific minicultures with analogues of IVT peptidea

 
CTL clones with similar fine peptide specificities, as determined by ASM, may have different TCR structures. The TCR V{alpha} and Vß usage and the CDR3 sequences of 18 representative IVT-specific clones derived from the five stimulations were determined by RT-PCR amplification and sequencing of the PCR products. Four IVT-specific TCRs, each composed of a unique combination of V{alpha} and Vß chains and characterized by unique CDR3 sequences were identified in this screening. All clones expressing a given V{alpha}/Vß combination had identical nucleotide sequence and represented, therefore, the progeny of the same precursor cell (Fig. 3Go). A perfect match was observed between the ASM reactivity of the clones and the expression of one of the four IVT-specific TCRs identified by sequencing. Thus, all clones in group 1 expressed the AV21S1/BV2S1 TCR, clones in group 2 expressed the AV25S1/BV21S1 TCR, clones in group 4 expressed the AV1S2/BV1S1 TCR, and clones in group 5 expressed the AV1S1/BV22S1 TCR. The only clone belonging to group 3 was lost before the characterization of its TCR could be performed. These results strongly suggest that all other clones characterized by ASM express one of the four TCRs. Clones belonging to groups 1 and 2 were represented in all four stimulations and were the only components isolated from stimulations I, II, and IV. Clonotypes 4 and 5 were isolated in stimulations III and V. Therefore, CTL clones expressing TCR of clonotypes 1 and 2 persisted for at least 57 mo, while CTL clones expressing two other TCRs persisted for at least 43 mo.



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  		FIGURE 3. Nucleotide and deduced amino acid sequences of the CDR3 of {alpha}- and ß-chains of IVT-specific clones derived from different stimulations. For each TCR, the nucleotide and deduced amino acid sequence (the last shown in single letter code) were defined according to the method of Chothia et al. (56). The variable (V) and joining (J) genomic segments of the {alpha} (A) and ß (B) chains are indicated according to nomenclature of Arden et al. (57).

 
Functional and phenotypic characterization of IVT-specific CTLs

T cell avidity and TCR affinity for specific Ag are believed to play an important role in selection of memory T cells. To evaluate the impact of these parameters in the selection of IVT-specific CTL repertoire, clones belonging to the five groups were compared for 1) lysis of peptide-pulsed A11-positive PHA blasts in peptide titration experiments, 2) lysis of an HLA A11-positive LCL, 3) expression of the TCR-associated CD3 and the CD8 coreceptor, and 4) sensitivity to blocking by CD8 specific Abs. The amount of IVT peptide required for half-maximal lysis of A11-positive blasts (P50) was calculated for 13 clones representative of the different groups (Table IIIGo). Although the P50 values fell in a relatively narrow range of 5 to 25 pg, the average P50 value of group 1 clones was twice as high as the P50 value determined for group 2 clones (p < 0.05). Group 1 clones appeared to be less efficient also in recognition of HLA A11-positive LCL EA-B1 (Table IIIGo). This difference attracted our attention, since clones of the two groups were detected in all stimulations, while differences in avidity to specific peptide:MHC complex would be expected to lead to the disappearance of low avidity clones. To confirm our observation, another set of clones representative for the two groups was tested for recognition of a panel of four HLA A11-positive LCLs that included two different cell lines derived from donor BK. As shown in Figure 4Go, clones belonging to group 1 were less efficient in recognition of all four LCLs. This could not be attributed to the low activity of cytolytic machinery of these clones, since they killed the same LCLs prepulsed with synthetic IVT peptide as efficiently as CTL clones from other groups (data not shown).


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  		Table III. Cytotoxic activity and phenotype of IVT-specific CTL clones

 


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  		FIGURE 4. Lysis of HLA A11-positive LCLs by CTL clones belonging to different functional groups. Three CTL clones exhibiting group 1 (BK 10-9-44, BK 10-9-20, BK 60-9-5) or group 2 (BK 60-7-132, BK 60-3-3, BK 60-2-14) ASM reactivity were tested for their ability to lyse four HLA A11-positive LCLs: BK-B5, BK-S1, EA-B1, and SI-B1. Columns and error bars represent the means and SDs of the values of specific lysis obtained with each individual clone from group 1 or 2.

 
The levels of CD3 and CD8 expression were investigated by indirect immunofluorescence and FACS analysis. A compilation of the data is presented in Table IIIGo. Similar levels of CD3 and CD8 expression were detected in different CTL clones and in repeated experiments performed with the same clone.

In the final set of experiments CD8 blocking was performed to evaluate the relative TCR affinities of the IVT-specific CTL clones. The cytotoxic activity against an HLA A11-positive LCL was tested in chromium release assays performed in the presence of increasing concentrations of the CD8-specific mAb OKT8 (Fig. 5Go and Table IVGo). The CTL clones exhibited different sensitivities to CD8 blocking. The cytotoxic activity of clones from group 1 was reduced by >70% at all concentrations of anti-CD8 tested, whereas only 30 to 40% inhibition was achieved with clones from groups 4 and 5 even at the highest concentration of anti-CD8. Fifty percent inhibition of cytotoxic activity was obtained by addition of 150 µg/ml OKT8 with clones from group 2, while the only group 3 clone (BK 219) exhibited an intermediate sensitivity to blocking with 50% inhibition at 17 µg/ml OKT8.



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  		FIGURE 5. Inhibition of cytotoxic activity by treatment with CD8-specific Ab (anti-CD8). The cytotoxic activity of CTL clones untreated or preincubated with anti-CD8 was tested against an A11-positive LCL. The effectors were preincubated for 1 h at 37°C with the indicated concentrations of the mouse anti-human CD8 mAb OKT8 before addition of the target cells. One representative experiment of five performed for each clone is shown in the figure.

 

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  		Table IV. Sensitivity of CTL clones from group I and II to CD8 blocking1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to characterize an oligoclonal peptide-specific CTL repertoire over time and to understand how the avidity/affinity of interaction with the specific persistent Ag affects the maintenance of individual clonal components of T cell memory.

The follow-up of the IVT-specific response revealed four clonotypes that accounted for almost all the IVT-specific CTL reactivity detected in PBLs from donor BK after their in vitro reactivation with autologous LCL. The clonotypes expressed structurally different TCRs (Fig. 3Go) and had different fine peptide specificity as determined by ASM (Fig. 1Go). Clonotypes 1 and 2 were reactivated in all in vitro stimulation procedures performed in the course of the study, while two other clonotypes (4 and 5) were detected on two different occasions separated by the interval of 47 mo. The relatively rare appearance of CTL clones from clonotypes 4 and 5 was most likely due to the poor ability of these clones to proliferate in vitro. Stimulation in minicultures was necessary for efficient reactivation of these clones, and it was generally more difficult to expand them in vitro. However, the analysis of minicultures obtained in stimulation V suggested that CTLs belonging to clonotypes 4 and 5 persisted in vivo at a relatively high frequency, since the relevant reactivities were not under-represented compared with reactivity 1 or 2 (Table IIGo). Thus, the protocol of in vitro reactivation of memory cells can significantly affect the size of the detected repertoire. Nevertheless, our results clearly show that at least the part of the IVT-specific repertoire that can be reactivated in vitro is remarkably stable in time. Long time persistence of human T lymphocytes with phenotypic characteristics of memory cells was first demonstrated by the analysis of T cells carrying irradiation-induced chromosomal damage (35). Later, the long time persistence of individual Ag-specific memory T cells was documented in autoimmune and antiviral responses (16, 19, 20). However, it was not directly shown at the clonal level that an entire heterogeneous peptide-specific TCR repertoire or at least a proportion of it can be stable in time. This demonstration is particularly interesting in the context of the chronic antigenic stimulation that is very likely to be provided to IVT specific T cells by persistent EBV infection. Although EBNA4-expressing cell are not detectable in the peripheral blood of normal virus carriers (36, 37), several lines of evidence suggest that in vivo, such cells emerge and are presented to the immune system in a regular manner. EBV-associated lymphoma expressing the full set of EBNAs (EBNA 1–6) develops with a very high frequency in severely immunocompromised individuals. This tumor can be efficiently controlled by adoptive transfer of EBV-specific CD8+ CTLs (38, 39), suggesting that in normal individuals the CTLs are responsible for keeping the EBV-positive immunoblasts below the threshold of detection. Moreover, infectious EBV is always detected in saliva of normal virus carriers (40), which presumably provides a reservoir for constant infection of new B cells in the same donor. Primary EBV infection is known to result in the blasts transformation of B cells both in vitro and in vivo and is accompanied by the expression of the full set of immunogenic EBNAs (27, 41). Our results indicate that the chronic antigenic stimulation associated with EBV persistence does not result in any apparent changes in the composition of the IVT-specific repertoire analyzed in this study. This raises the possibility of using individual T cells expressing a particular TCR for longitudinal monitoring of immune responses against persistent Ags. The composition of Ag-specific repertoires involved in such responses can be correlated with reactivation of persistent viral infections and progression of autoimmune diseases. Tools recently developed for ex vivo analysis of Ag specific T cells will be instrumental for such studies (42).

T cell affinity for specific Ag is generally assumed to be an important factor driving the selection of memory T cells. It was known for a long time that affinity maturation occurs during the establishment of B cell memory and the selection of high affinity cells is favored by a decrease in antigenic load (43, 44). A similar process of Ag-driven affinity selection was believed to happen during the selection of T cell memory. This idea was supported by a long standing observation that Ag-specific memory CTLs are more resistant to inhibition by CD8-specific Abs than cells of the same specificity detected during the primary response (45, 46). It is conceivable that, by analogy with B cells, low amounts of persistent Ag may promote even further selection of high affinity T cells and restrict the clonal heterogeneity of memory repertoires. This scenario was suggested by various investigators, including ourselves (25), to explain the high level of TCR conservation or restriction observed in a number of T cell responses.

To characterize the role of avidity in the selection of IVT-specific memory repertoire we have tested the recognition of IVT-pulsed HLA A11-positive blasts and HLA A11-positive LCLs by CTL clones representative for different clonotypes in cytotoxicity assays. The amount of IVT peptide sufficient to induce 50% of maximal lysis (P50) of target cells was calculated for each representative clone. P50 values determined for different IVT-specific clones were in the range of 5 to 25 pg. A reproducible and statistically significant difference was detected in P50 values of clones belonging to clonotypes 1 and 2 (Table IIIGo). Clonotype 1 clones required twice as much peptide as clonotype 2 clones for half-maximal triggering. The observation was confirmed by testing a new set of clones representative for these two clonotypes for recognition of a panel of HLA A11-positive LCLs (Fig. 4Go). Again, significantly lower levels of specific lysis were achieved with clonotype 1 CTLs. Overall, our results indicate that clones expressing type 1 TCR possess relatively lower avidity for the specific MHC:peptide complex that results in the less efficient recognition of both peptide-pulsed and virus-infected cells.

To correlate the differences in T cell avidity with the affinity of TCRs, we have tested the effects of increasing amounts of anti-CD8 on the cytotoxic activity of clones expressing different TCRs. Extensive functional and structural data indicate that in the presence of equal ligand density, TCR affinity correlates with resistance to blocking of the CD8 coreceptor (47, 48, 49, 50). IVT-specific clones belonging to different clonotypes exhibited strikingly different sensitivity to CD8 blocking. Cytotoxic activity of CTL clones expressing type 1 TCR was inhibited by >50% at all concentrations of anti-CD8 tested (Fig. 5Go and Table IVGo), while clones of other clonotypes could be inhibited at the same level only with high concentrations of anti-CD8 or exhibited <50% inhibition at all tested concentrations. Independently of the expression of a particular TCR, all CTL clones expressed comparable levels of CD8 and CD3, indicating that differences in sensitivity to anti-CD8 did not result from the different amounts of TCR or CD8 coreceptor on the surface of these cells.

Taken together, our results demonstrate that compared with the other clonotypes, CTL clones expressing V{alpha}21S1/Vß2S1 TCR have low T cell avidity for the specific Ag that correlates to low affinity of the TCR. The persistent recovery of this clonotype in CTL reactivation experiments performed over a period of almost 5 yr demonstrates that a memory repertoire can stably accommodate a range of TCR affinities and T cell avidities. The generality of this phenomenon for EBV and other persistent infections remains to be established. It is noteworthy, however, that the IVT-specific response of another HLA A11-positive EBV carrier analyzed in our previous study was also composed of CTL clones with different sensitivities to CD8 blocking (25). This supports the contention that long time persistence of CTLs with different TCR affinity is not an unusual feature of the EBV-specific response. Unfortunately, longitudinal analysis of the repertoire and detailed avidity data are not available for that donor.

Another interesting question raised by our data is the effect of antigenic load on the range of avidities and TCR affinities selected into a memory repertoire. Due to the intrinsic limitations of the human model, this question is difficult to address experimentally. Presumably, the amount of Ag may affect the selection of specific T cell repertoire both at the time of the first antigenic challenge and after the establishment of persistent infection. For primary response, the effect of different antigenic loads could be investigated by comparing the heterogeneities of peptide-specific repertoire in virus carriers with or without previous history of infectious mononucleosis. In these two situations the antigenic load should differ dramatically. Monitoring of antigenic load in chronic EBV carriers is hampered by the constant elimination of immunogenic EBV-infected blasts by specific CTLs. However, it may be pertinent for this discussion that the IVT peptide is presented at a relatively high number of copies at the surface of EBV-infected cells (34). This could make possible the persistence of specific T cells with different avidities by decreasing the competition for resource Ag and at least partly explain our finding. On the other hand, a long time antigenic stimulation should lead to the preferential selection of the best performing clones. Therefore, our data may be viewed in line with the idea that the maintenance of memory T cells, regardless of their avidity or TCR affinity, may be promoted by some type of cross-reactive interactions or nonspecific stimulation (10, 11, 12, 13, 14). Primary response or transition to memory may represent the only window in the peripheral development of T cells in which specific repertoires are selected on the basis of avidity (18, 51). This would also imply a major role of the first antigenic challenge in determining the overall size and composition of Ag-specific T cell repertoires selected during natural or vaccination-induced immune responses. The above model is in agreement with recent data that demonstrated different requirements of naive vs memory T cells for expansion and long term survival (52) and the dispensability of the specific Ag for the maintenance of T cell memory (5, 7, 8, 52). A number of signals that can promote the maintenance of memory T cells in an Ag-independent manner have been recently defined (11, 53, 54). This model does not exclude, however, that periodical exposure to the persistent Ag affects functional and phenotypic characteristics of memory cells (9) or that a significant imbalance in the antigenic load can eventually change the clonal composition of memory (55).

To our knowledge this study represents the first longitudinal analysis of a polyclonal, structurally heterogeneous, peptide-specific T cell repertoire combined with the analysis of avidity and TCR affinity of its individual components. Our results demonstrate that in the presence of the specific Ag, the composition of memory repertoire is remarkably stable over time and accommodates a range of T cell avidities and TCR affinities for the specific antigenic peptide.


   Footnotes
 
1 This work was supported by grants from the Swedish Cancer Society, the Pediatric Cancer Foundation, the Karolinska Institute, the Magnus Bergwal Stiftelse (Stockholm, Sweden), and in part by a fellowship (to V.L.) from the Cancer Research Institute (New York, NY) and the Concern Foundation (Los Angeles, CA). Back

2 Address correspondence and reprint requests to Dr. Victor Levitsky, Microbiology and Tumorbiology Center, Box 280, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail address: Back

3 Current address: Cold Spring Harbor Laboratory, 1 Bungtown Road, P.O. Box 100, Cold Spring Harbor, NY 11724. Back

4 Abbreviations used in this paper: LCL, lymphoblastoid cell line; EBNA, EBV nuclear antigen; IVT, IVTDFSVIK; ASM, alanine scanning mutagenesis; P50, amount of peptide sufficient to induce half-maximal lysis; AVF, AVFDRKSVAK; P, position. Back

Received for publication January 20, 1998. Accepted for publication March 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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