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Journal of Virology, November 2000, p. 10240-10244, Vol. 74, No. 21
Institute of Virology, Erasmus University
Hospital, 3000 DR Rotterdam,1 and
Laboratory for Bacterial Vaccine Research, National Institute
of Public Health and the Environment, 3720 BA
Bilthoven,2 The Netherlands
Received 30 May 2000/Accepted 1 August 2000
Virus-specific cytotoxic T lymphocytes (CTL) play a major role in
the clearance of respiratory syncytial virus (RSV) infection. We have
generated cytotoxic T-cell clones (TCC) from two infants who had just
recovered from severe RSV infection. These TCC were functionally
characterized and used to identify HLA class I (B57 and C12)-restricted
CTL epitopes of RSV.
Respiratory syncytial virus (RSV) is
a common cause of upper respiratory tract infections but
may Peripheral blood mononuclear cells (PBMC) were collected from two
infants 4 weeks after a severe, laboratory-confirmed RSV infection. At
the time of infection, they were 1 and 2 months old and had both been
admitted to the intensive care unit. B-lymphoblastic cell lines (B-LCL)
were generated by Epstein-Barr virus transformation (22),
infected with RSV A2 (ATCC VR1322), and UV irradiated (24)
to serve as autologous antigen-presenting cells (APC). PBMC (3 × 104/well in 96-well round-bottom plates) were stimulated
with APC (104/well) and expanded in RPMI 1640 medium
supplemented with antibiotics, 10% pooled and heat-inactivated human
serum, and recombinant human interleukin-2 (IL-2; 50 IU/ml). After 2 weeks, T cells were harvested and cloned by limiting dilution using
phytohemagglutinin stimulation as previously described (25).
TCC thus generated were expanded and tested for RSV specificity by
[3H]thymidine incorporation assays as previously
described (22). All of the RSV-specific TCC proved to be of
the CD8+ phenotype in fluorescence-activated cell sorter
analysis. TCC whose specificity for RSV was confirmed by a second
proliferation assay were tested for protein specificity by a gamma
interferon (IFN- Thirty-four RSV-specific TCC were generated from the PBMC of patient 1, as detected in a [3H]thymidine incorporation assay. Of
these, 27 proved to be RSV F specific and 7 were RSV 1B specific in an
IFN-
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HLA Class I-Restricted Cytotoxic T-Cell Epitopes of
the Respiratory Syncytial Virus Fusion Protein
ABSTRACT
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especially in young infants, the elderly, or immunocompromised
individualscause severe lower respiratory tract infections
(12). In rodent models, CD4+, as well as
CD8+, RSV-specific T lymphocytes proved to be involved in
both recovery from and immune pathogenesis of the infection (1, 2,
8, 19). Therefore, a fine balance must exist in these models
between protective and disease-enhancing effects of virus-specific T
lymphocytes. RSV-specific CD8+ cytotoxic T lymphocytes
(CTL) against virtually all RSV proteins have been demonstrated to
circulate in humans after RSV infection (4, 7, 9). CTL may
be expected to play a crucial role in the clearance of RSV infections,
but their role in protection and immune pathology remains unclear.
Therefore, the identification of CTL epitopes of human RSV may
contribute to future studies concerning the role of CTL in pathogenesis
and protection from RSV infection. Here we describe the functional
characterization of CD8+ cytotoxic T-cell clones (TCC)
generated from two infants who had just recovered from severe RSV
infection. These TCC were also used to identify, for the first time,
HLA class I-restricted CTL epitopes of RSV.
) ELIspot assay (Mabtech AB, Stockholm, Sweden). In
this test, paraformaldehyde-fixed autologous B-LCL that had been
infected with recombinant vaccinia viruses (rVV) expressing different
RSV proteins (F, G, N, P, M2, SH, M, 1B, or 1C) were used as APC. Briefly, TCC (5 × 103/well) were incubated with APC
(104/well) for 4 h, transferred to anti-human
IFN--coated plates, and incubated for another 18 h. The ELIspot
assay was further performed in accordance with the kit
manufacturer's instructions. The results are shown as numbers of
IFN--producing cells (spots) per well.
ELIspot assay (Fig. 1A).
Twenty-four RSV-specific TCC were generated from the PBMC of patient 2 as detected by a [3H]thymidine incorporation assay. Of
these, 10 were RSV F specific and 14 proved to be RSV 1C specific in an
IFN- ELIspot assay (Fig. 1B). None of the clones detected by the
[3H]thymidine incorporation assay was found to be
negative by the IFN- ELIspot assay. Since the F protein is
considered to be a major CTL target (4, 7, 9), further
efforts were focused on the identification of CTL epitopes in the F
protein. To this end, 18-mer peptide amides overlapping by 12 amino
acids, together spanning the entire F protein of RSV A2 (11,
18) (n = 94), were generated in an automated
multiple-peptide synthesizer as previously described (26).
The purity of the peptides varied between 50 and 90%, as determined by
analytical reverse-phase high-performance liquid chromatography
(C8 column; gradient of 0.1% trifluoroacetic acid in water
to 0.1% trifluoroacetic acid in acetonitrile). Autologous B-LCL of
patients 1 and 2 were pulsed overnight with 1 and 3 µM peptide,
respectively. RSV F-specific TCC of patient 1 reacted with peptides 17 and 18 (Fig. 2A). All of the RSV F TCC of
patient 2 reacted with peptide 91 and marginally with peptide 90 (Fig.
2B). Subsequently, two additional sets of partially overlapping 8- to
12-mer peptides (80 to 95% purity) were generated to determine the
respective minimal epitopes. Autologous B-LCL of patients 1 and 2 were
pulsed for 1 h with the different peptides at 1 and 3 µM,
respectively. All of the RSV F-specific TCC of patient 1 reacted with
one nine-mer peptide (RARRELPRF) spanning residues 118 to 126 of the F
protein (Fig. 2C). The RSV F TCC of patient 2 all reacted with one
nine-mer peptide (IAVGLLLYC) spanning residues 551 to 559 of the F
protein (Fig. 2D).
View larger version (25K):
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FIG. 1.
Responses of RSV-specific TCC from patients 1 (A) and 2 (B) measured by an IFN- ELIspot assay using autologous B-LCL
infected with RSV A2 or rVV expressing the RSV F, 1B, or 1C protein.
Results are indicated as numbers of spots per well. No responses
against the B-LCL infected with the other rVV were found (data not
shown).
View larger version (27K):
[in a new window]
FIG. 2.
Fine mapping of T-cell epitopes on the RSV F protein.
Responsiveness of TCC from patients 1 (A) and 2 (B) to partially
overlapping 18-mer peptide amides (n = 94) spanning the
F protein was measured by an IFN- ELIspot assay. Minimal peptide
activation of TCC from patients 1 (C) and 2 (D) was measured in an
IFN- ELIspot assay using APC pulsed with two additional sets of
overlapping 8- to 12-mer peptides. med, medium.
The HLA restriction of the recognition by the RSV F TCC was also
determined by the IFN- ELIspot assay using a set of allogeneic B-LCL
with partially matched HLA types loaded with the respective minimal
peptides, as shown in Fig. 3. Recognition
of the RSV F epitope of patient 1 proved to be HLA B57 restricted.
Recognition of the epitope of patient 2 proved to be C12 restricted.
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Well-growing TCC of both specificities from each of the patients were
arbitrarily selected and tested for cytotoxic activity by a chromium
release assay as previously described (23). Briefly, autologous 51Cr-labeled, RSV-infected B-LCL and control
B-LCL or, in the case of the F-specific TCC of patient 2, minimal-peptide-loaded B-LCL and control B-LCL were incubated with TCC
for 4 h at 37°C at an effector-to-target cell ratio of 10:1,
which was found to be the most discriminative ratio in preliminary
experiments. Spontaneous 51Cr release (target cells plus
medium) and maximum 51Cr release (target cells plus Triton
X-100) were determined in 12 identical wells. Supernatants were
harvested and analyzed in a gamma counter. Assay results were accepted
only when the spontaneous-to-maximum release ratio was <25%. All of
the TCC tested showed RSV-specific lysis (Fig.
4A). Virus-specific production of
cytokines in cell-free culture supernatant was measured as previously
described (6). TCC were stimulated with RSV-infected B-LCL
or control B-LCL or, in the case of the F-specific TCC of patient 2, with minimal-peptide-loaded B-LCL or control B-LCL for 48 h. The
concentrations of the following cytokines were measured using
commercially available sandwich enzyme-linked immunosorbent assay
systems in accordance with the manufacturers' instructions: IL-4 (CLB,
Amsterdam, The Netherlands; detection limit, 7 pg/ml), IFN-
(Medgenix Diagnostics, Fleurs, Belgium; detection limit, 25 pg/ml),
IL-10 (Pharmingen, San Diego, Calif.; detection limit, 20 pg/ml). All
of the TCC produced predominantly IFN-, indicating a type 1 phenotype in vitro (Fig. 4B).
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In the present study, we identified two nine-mer CTL epitopes on the RSV fusion protein. To our knowledge, these are the first HLA class I-restricted CTL epitopes described for RSV in humans. We found CTL epitopes with an HLA restriction with a low prevalence in the population. But using the techniques described here on samples of more children with a recent RSV infection, CTL epitopes with more prevalent HLA restrictions may be identified.
The TCC found in the children studied were all CD8+ CTL with a type 1-like cytokine profile. No RSV-specific CD4+ T cells were detected in the samples of these infants, while the use of similar stimulation protocols did result in the identification of such cells in other systems (14, 17, 20, 22). Virus-specific CTL responses play a major role in the clearance of RSV infections but may also be involved in pathogenesis (1, 3, 5, 8, 15). In mice, enhanced lung pathology induced by priming with formalin-inactivated RSV has been associated with the absence of a CTL response (13, 21). In young children, a CTL response against RSV has been described but poor responses were found in younger and more severely ill patients (10, 16). The poor CTL response in young children has been suggested to be one of the possible causes of more severe disease. In these two patients, it was possible to detect RSV-specific cytotoxic TCC, showing that also in very young children with clinically severe infections, priming of a CTL response does occur, although we cannot say anything about the quantitative responses.
In conclusion, use of the IFN- ELIspot assay to determine epitope
specificity proved to be sensitive and convenient, since only small
numbers of T cells and APC were needed. This and similar studies may be
important for future studies concerning the role of CTL in the
pathogenesis of RSV infection in children.
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ACKNOWLEDGMENTS |
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This study was financially supported by the Sophia Foundation for Medical Research, Rotterdam, The Netherlands (grant 214).
We thank Humphrey Brugghe and Bea van't Land for technical assistance and Conny Kruyssen for assistance in preparing the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Virology, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: 31-10-4088066. Fax: 31-10-4089485. E-mail: osterhaus{at}viro.fgg.eur.nl.
Present address: LVF, 8917 EN Leeuwarden, The Netherlands.
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Journal of Virology, November 2000, p. 10240-10244, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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