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Previous Article | Next Article 
Journal of Virology, May 2000, p. 3948-3952, Vol. 74, No. 9
Institut National de la Santé et de la
Recherche Médicale U4631 and
Laboratoire de Virologie,2 Institut
de Biologie, 44093 Nantes Cedex 1, France
Received 10 November 1999/Accepted 29 January 2000
Cytotoxic T lymphocytes (CTLs) play a central role in the control
of persistent human cytomegalovirus (HCMV) infection in healthy virus
carriers. Previous analyses of the specificity of HCMV-reactive
CD8+ CTLs drawn from in vitro models in which
antigen-presenting cells were autologous fibroblasts infected with
laboratory HCMV strains have shown focusing of CTL responses against
the major tegument protein, pp65. By contrast, the 72-kDa major
immediate-early protein (IE1) was identified as a minor target for this
response. Here we have studied the fine specificity and T-cell-receptor
features of T-cell clones generated against autologous B lymphoblastoid cell lines stably transfected with HCMV cDNA coding for either pp65 or
a natural variant of IE1. This strategy allowed efficient generation of
T-cell clones against IE1 and pp65 and led to the identification of
several new IE1 and pp65 epitopes, including some located in
polymorphic regions of IE1. Such an approach may provide relevant
information about the characteristics of the CTL response to IE1 and
the effect of viral polymorphism on the immune response against HCMV.
Human cytomegalovirus (HCMV), the
largest of the betaherpesviruses, infects between 40 and 60% of
individuals in developed countries and nearly 100% in developing
countries. After primary infection, which is asymptomatic in
immunocompetent hosts, the virus persists lifelong under the control of
the immune system in latent sites, including cells of the myeloid
lineage (20, 32, 34, 35). The maintenance of this latent
state depends on the cellular immune response, and cytotoxic T
lymphocytes (CTLs) have been shown to play a major role in the control
of HCMV reactivation and replication (25, 29, 30, 38).
Due to inhibition of the major histocompatibility complex (MHC) class I
antigen presentation pathway by a set of viral proteins acting in a
cooperative and multistep process during the HCMV replication cycle
(1, 3, 13, 15-18, 39, 41, 42), the CTL response against
HCMV appears to be targeted against a small set of proteins (for a
review, see reference 5). Among these have been
identified, on the one hand, structural proteins which are delivered to
the cytosol by the infecting virion and rapidly presented to
CD8+ CTL by MHC class I before blockade of their processing
and, on the other hand, the immediate-early proteins, the presentation of which depends on de novo synthesis very early after virus infection (15).
Early analyses of the CTL response in seropositive individuals have
suggested that the 72-kDa immediate-early protein IE1 was a dominant
target for CD8+ CTLs (4). Nevertheless, more
recent observations, drawn from an in vitro experimental model with
autologous fibroblasts infected with the cytopathic AD169 viral strain
as a stimulator to propagate HCMV-specific CD8+ CTLs, have
designated the major tegument protein pp65 as a dominant target and
have shown a highly focused peptide-specific memory CTL response
against few dominant epitopes (21, 28, 40, 43).
In the present study, we have developed a novel strategy allowing
analysis of the composition and fine specificity of the CTL response of
HCMV-seropositive healthy donors against selected immunogenic HCMV
proteins. B lymphoblastoid cells (BLCs) were derived by Epstein-Barr
virus (EBV) transformation from peripheral blood mononuclear cells
(PBMCs) and stably transfected with plasmidic vectors coding for either
pp65 or IE1 proteins. These cells were then used as targets to
stimulate a specific autologous T-lymphocyte response and to amplify
reactive T-cell subsets. After cloning, the cells recognizing the
recombinant HCMV protein targets were selected, the characteristics of
their TCR repertoire was studied by sequencing their T-cell-receptor
Cells.
PBMCs were prepared from fresh heparinized peripheral
blood of three CMV+ healthy donors expressing known HLA
class I haplotypes (donor A, A1A1 B8B35 Cw4Cw7; donors B and C, A2A25
B18B44 Cw5Cw12) by Ficoll-Hypaque (Eurobio, les Ulis, France) density
gradient centrifugation. HLA class I genotyping was established in the
HLA typing Laboratory of Centre Régional de Transfusion Sanguine
de Nantes by PCR amplification techniques (6, 36). There was
no parental link between donors B and C. All donors carried antibodies
against EBV. BLC lines were established from PBMCs by EBV
transformation as previously described (24).
Expression vectors.
IE1 and pp65 full-length coding
sequences were amplified by reverse transcription (RT)-PCR from MRC-5
fibroblasts infected with a CMV strain selected from a clinical
isolate, cloned in KS+ vectors (Stratagene, La Jolla,
Calif.) and sequenced. The IE1 coding sequence used subsequently for
expression exhibited nucleotidic variation when compared to the
corresponding nucleotidic sequence of AD169 and Towne laboratory
strains. All of these mutations were also found in two other IE1 PCR
products independently amplified from the same clinical strain and then
were due to natural variation. Such a polymorphism was not found in the
pp65 coding sequence. IE1 and pp65 cDNAs were excised from
KS+ and subcloned in pRc/CMV expression vector (Invitrogen
Corp., San Diego, Calif.) under the control of CMV promoter.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Generation of Cytomegalovirus-Specific Human T-Lymphocyte Clones
by Using Autologous B-Lymphoblastoid Cells with Stable Expression of
pp65 or IE1 Proteins: a Tool To Study the Fine Specificity of the
Antiviral Response
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-chain junctions, and their fine specificity and HLA restriction
were determined. This study led to the identification of several new
class I MHC-restricted CTL epitopes against which were focused the
anti-pp65 and -IE1 responses. Since pp65 and IE1 cDNA used in this
study were cloned from a clinical strain, the IE1-expressed recombinant
protein was a genetic variant which diverged by several amino acids
from corresponding products of the laboratory strains Towne and AD169, classically used for analysis of memory CTL response to HCMV infection (4, 21, 28, 40, 43). Interestingly, several polymorphic residues were found within three of the four epitopes recognized by HCMV-specific T-cell clones generated against the clinical IE1
variant from a single donor (i.e., 22 of 22 clones restricted by HLA-A2
molecules and 23 of 25 characterized in the HLA-B18 context). Thus, the
present experimental system represents a powerful tool for
characterizing new target epitopes of the anti-HCMV T-cell response and
should allow in-depth analysis of the effect of viral polymorphism on
the modulation of the host repertoire.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Transfection of BLCs. Five million actively dividing BLCs were resuspended in 0.8 ml of complete medium, mixed with 15 µg of vector, incubated for 1 min at room temperature, and electroporated in an Easyject Plus apparatus (Eurogentec, Seraing, Belgium). One pulse of 250 V/cm using a 1,500-µF capacitor was delivered. The cells were immediately plated with 5 ml of fresh medium. The same protocol was repeated three times to obtain 1.5 × 107 electroporated BLCs for each transfection assay. Selection with G418 (0.8 mg/ml) started 2 days after electroporation. After 1 week, dead cells and debris were eliminated by centrifugation through a Ficoll-Hypaque gradient, and the cells from the same three electroporations were pooled.
Generation of specific T lymphocytes. Anti-pp65 and anti-IE1-specific T lymphocytes were amplified against BLCs expressing stably these viral recombinant proteins using the following protocol: donor PBMCs were plated at 2 × 106 cells per well in 24-well plates containing RPMI 1640 supplemented with 10% pooled human serum and 1 mM L-glutamine and then stimulated with 5 × 104 irradiated (30 Gy) autologous BLCs expressing either pp65 or IE1. After 9 days of coculture, 5 × 105 cells per well were restimulated with 1.25 × 105 autologous recombinant irradiated BLCs. Nine days later, recombinant interleukin-2 was added to the culture (20 IU/ml). Twenty days after the second stimulation, cells were cloned from the amplified lines by limiting dilution as previously described (37) at 0.3 cells per well. Growing colonies with a probability of monoclonality of >95% were kept for further analysis and expanded in vivo using standard conditions (9). Cells were maintained for 3 to 4 weeks without restimulation prior to functional analysis in order to decrease their spontaneous tumor necrosis factor (TNF) release and their nonspecific cytotoxic activity.
Flow cytometry analysis and functional assays. Expression of pp65 and IE1 recombinant proteins was checked by flow cytometry in stably transfected BLCs permeabilized and then fixed as described previously (14). (1C3 plus AYM-1)-fluorescein isothiocyanate (FITC) monoclonal antibodies (MAbs) (Argene) were used to detect pp65, and E13-FITC MAb (Biosys) was used to detect IE1.
T cells were phenotyped by indirect fluorescence as previously described (37). Anti-coreceptor and -TCR MAb were purchased from Immunotech-Coulter Corp. (Marseille, France). The proliferative and cytotoxic activity of T-cell clones against autologous irradiated BLCs stably transfected with pp65, IE1, or mock vector were estimated as previously described (8, 37).HLA restriction characterization. HLA restriction of T cells was characterized, as previously described (31), by testing their TNF content when added to COS cells cotransfected with an expression vector coding for their target viral protein and an expression vector coding for one of the HLA.
Characterization of target peptides. Synthetic peptides (23-mer overlapping by 12 residues) encompassing the whole primary amino acid sequence of pp65 and IE1 were obtained from Chiron Technologies (Pty Ltd., Clayton, Victoria, Australia). The pp65 peptides are numbered 1 to 50, and IE1 peptides are numbered 1 to 44 from the N terminus to the C terminus. Minimal 9- or 10-mer peptides corresponding to proposed MHC-restricted epitopes were obtained from Genosys (Pampisford, United Kingdom).
Peptides recognized by T-cell clones directed against pp65 and IE1 were characterized by determining the TNF release in an autopresentation assay. Ten-thousand T cells were incubated for 2 h with 10 µM pp65 or IE1 individual synthetic peptides. The quantification of TNF release was determined as described earlier (31).Analysis of TCR transcripts.
T-cell-receptor (TCR
) cDNAs were amplified by RT-PCR from total RNA of clones cDNAs as
previously described (31) and then directly sequenced.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Amplification and cloning of anti-pp65 or IE1 CTLs.
BLCs
expressing stably pp65 were generated from donors A and B. BLCs
expressing stably IE1 were generated from donor C (see, for example,
Fig. 1). They were subsequently used as
stimulators to amplify specific T cells from autologous PBMCs. The
presence of specific CTLs in bulk cultures was checked using a
technique in which their TNF release against COS cells cotransfected
with pRc/CMV-pp65 or -IE1, and the DNA coding for individual HLA class I alleles was measured. Cells amplified from donors A and B were reactive against COS cells coexpressing pp65 and HLA-A1 or -A2 molecules, respectively. Furthermore, the bulk CTL line from donor C
recognized COS cells expressing IE1 and either HLA-A2 or HLA-B18. Cell
lines were cloned by limiting dilution, and colonies were tested for
their reactivity against target cells expressing or not expressing
recombinant pp65 or IE1 proteins.
|
Selection of T-lymphocyte clones reactive against either pp65 or IE1 and characterization of their HLA-restricting allele. Two methods were used to screen for the specificity of T-cell clones derived from bulk cultures. Clones obtained from PBMCs stimulated against autologous BLCs expressing pp65 (donors A and B) were selected on the basis of their differential proliferative activities against autologous BLCs stably transfected with either pRc/CMV-pp65 or the empty pRc/CMV control. CD8+ CTL clones obtained from PBMCs stimulated against autologous BLCs expressing IE1 (donor C) were selected using a COS cell cotransfection assay which allowed us to characterize at the same time the HLA molecules restricting the presentation of HCMV peptides.
From 70 T-cell clones generated from donor A, a single clone (A18) proliferating against autologous targets expressing pp65 was isolated. Clone A18 recognized specifically pp65 when expressed in COS cells together with the HLA-A1 allele. From 75 clones generated from donor B, 2 clones (B3 and B20) were shown to recognize pp65-transfected BLCs. These two clones recognized pp65 in the HLA-A2 context, as indicated by COS screening assays. Of the 75 T-cell clones generated from donor C, 22 were reactive against IE1 in the HLA-A2 context and 26 were reactive in HLA-B18 context (Table 1).
|
+ and CD8+.
Analysis of the fine epitope specificity of pp65- and IE1-reactive
CTL clones.
The fine specificity of HCMV-reactive T-cell clones
was studied by screening their reactivity to synthetic peptides
encompassing the whole IE1 and pp65 proteins. The results from such
screenings are summarized in Table 1. The donor A-derived clone A18
recognized the pp65353-375 peptide in the HLA-A1 context.
The two clones B3 and B20 were specific for the HLA-A2-restricted
pp65485-507 peptide. The anti-IE1 response of donor
C-derived T cells was focused against the HLA-A2-restricted
IE1309-331 peptide and a dominant B18-restricted peptide
located within the IE1188-210-IE1199-221 overlapping peptidic region, which was recognized by 21 of 26 T-cell
clones. Nine-mer peptides recognized in the HLA-A2 and HLA-B18 contexts
by T-cell clones from donor C were further characterized through
screening of an additional set of peptides (Table
2).
|
TCR -chain features of IE1- and pp65-reactive T-cell
clones.
TCR V gene usage by anti-pp65 and -IE1 CTLs generated
from the three HCMV+ donors was first studied by flow
cytometry using a panel of 22 TCR V-specific MAbs (results are
summarized in Table 1). The clone generated from donor A against pp65
expressed the V1 segment. Of the two clones generated from donor B
against pp65, one expressed the V6 segment and the other expressed
the V14 segment. Of the 48 clones generated from donor C, 19 of 22 clones (i.e., 86%) recognizing the 9-mer IE1315-323
peptide in the HLA-A2 context expressed the TCR V20 gene segment and
17 of 26 clones (i.e., 63%) recognizing IE1 in the HLA-B18 context
expressed the V22 gene segment. All of these V22+
T-cell clones recognized the IE1199-207 epitope.
-chain sequencing of anti-pp65 and -IE1 CTL clones is presented
in Table 1. All V20 T-cell clones reactive against the IE1-HLA-A2
epitope exhibited identical junctional characteristics (i.e., J1.6
segment and identical CDR3 sequence), suggesting that they were the
progeny of a single clone. Similarly, of 17 CTL clones restricted by
the HLA-B18 molecule and expressing the V22 gene segment, 10 exhibited the J2.7 segment and identical junctional features. The
seven other clones used the J2.3 segment and showed identical
junctional sequences. All of these 17 clones exhibited conserved length
of their CDR3 loop and a shared NDN-encoded Gly at a conserved position
within the CDR3 loop.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this report, we present data on TCR -chain usage and the
fine specificity of anti-HCMV CD8+ CTL clones obtained
through stimulation of PBMCs from HCMV-seropositive individuals against
pp65- and IE1-BLC transfectants.
Although recombinant proteins were similarly expressed in the stably transfected BLC stimulator cells, generation of T-cell clones reactive against pp65 was less efficient than against IE1. Such a result is in disagreement with those obtained by other authors who used a fibroblast infection model to generate anti-CMV CTLs (21, 28, 40, 43), but it is consistent with more recent data (19, 31). In an infection model, (i) the substantial level of dense bodies that carry only tegument proteins in laboratory strains (for a review, see reference 23), (ii) blockade of IE1 processing and presentation due to its phosphorylation (12), and (iii) downregulation of MHC class I expression (1, 3, 13, 15-18, 39, 41, 42) may together lead to an underestimation of the importance of CTL response against IE1. However, we cannot exclude that, in our expression model, we underestimated the CTL response against pp65.
The TCR repertoire of IE1-specific T-cell clones identified in donor C was highly heterogeneous, showing that the response to CMV proteins is more diversified, as has already been suggested by others (40, 43). In our experiments, we only stimulated donors A and B with pp65 and donor C with IE1, but conclusive information should be obtained from further investigations by using our antigenic presentation strategy to generate CTLs against both IE1 and pp65 from PBMCs of an HCMV-seropositive individual.
The clones generated from donors A and B against autologous BLCs expressing pp65 that were not specific of this HCMV product proliferated mainly against autologous untransfected BLCs and were then probably directed against EBV antigens (data not shown). Such a result is consistent with a very recent report showing that BLCs transduced with a recombinant retrovirus encoding pp65 can be used as antigen-presenting cells to stimulate expansion of EBV- and CMV-specific CTLs simultaneously (33).
Amplification of anti-EBV CD4+ and CD8+ CTLs against BLC targets has been already described (reference 22 and our own results). Therefore, CD4+ anti-HCMV clones should be theoretically generated against BLCs expressing recombinant HCMV proteins. Other reports have shown a specific CD4+ T-cell response against IE1 in HCMV-seropositive individuals (2, 4, 10). Further investigations are necessary to obtain conclusive data regarding the efficiency of CD4+ T-lymphocyte-specific amplifications when the antigens are nuclear proteins synthesized by presenting BLCs.
Of the six epitopes characterized in this study, four were new. The pp65485-507 peptide recognized in the HLA-A2 context contained the 495-NLVPMVATV-503 9-mer epitope, which had been identified in previous studies as an immunodominant epitope (40, 43). This minimal epitope was recognized by the two clones generated from donor B against pp65 (data not shown). Concerning IE1, the 9-mer peptide 379-DEEDAIAAY-387, recognized in the B18 restricted context, was internal to a 12-mer epitope 378-SDEEEAIVAYTL-389 previously characterized in the same HLA context but which differed from its sequence by two residues (11).
The sequences of IE1 have long been assumed to be well preserved from genetic variation. Nevertheless, we and others recently described polymorphic regions in the coding sequence of this viral protein (7, 27, 44) which could interfere with presentation by MHC molecules and/or recognition by the immune system. Our data suggest that a large part of the memory CD8 CTL response against IE1 protein cloned from a clinical strain is directed against polymorphic epitopes restricted by two HLA molecules. A single residue exchange within a viral CTL epitope could later recognition or destruction by specific proteasomal cleavage, resulting in a lack of antigen presentation (26). Important information on the role possibly played by the IE1 natural polymorphism on the modulation of the anti-HCMV memory repertoire should be obtained from analysis of the T-cell-clone reactivity against target cells expressing IE1 proteins derived from various clinical strains or loaded with modified peptides.
In summary, the results presented here show that IE1-specific CTL clones can be efficiently generated through stimulation of PBMCs against autologous IE1 BLC transfectants. Study of the TCR and antigenic characteristics of CD8+ CTL clones obtained through this technique should help us analyze the role of IE1 in the control of viral reactivation and infection in HCMV-seropositive individuals with various HLA genotypes and to assess the impact of its peptidic polymorphism on the modulation of the host response against numerous HCMV isolates.
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ACKNOWLEDGMENTS |
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This work was supported by the Institute de la Santé et de la Recherche Médicale and by the Association pour la Recherche sur le Cancer.
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FOOTNOTES |
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* Corresponding author. Mailing address: INSERM U463, Institut de Biologie, 9 quai Moncousu, 44093 Nantes Cedex 1, France. Phone: (33) 02-40-08-47-12. Fax: (33) 02-40-35-66-97. E-mail: mmhallet{at}nantes.inserm.fr.
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