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Immunology |
Department of Medicine, University of Wales College of Medicine, Cardiff CF4 4XX, United Kingdom [S. J. Y., E. M. L., L. K. B., S. M.]; Department of Obstetrics and Gynaecology, Llandough Hospital, Penarth, CF64 2XX, United Kingdom [A. N. F.]; Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom [P. R. D., V. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently several groups, using peptides (17 , 18) , tumor cells (19) , soluble proteins (20) , or recombinant adenoviruses (21 , 22) , have demonstrated that human CTLs against HPV 16 restimulate PBMCs from patients in vitro. The use of different patient groups (CaCx or CIN3) and different techniques for in vitro restimulation of HPV-specific CTLs have led to detection rates of 0–80%. Even when the same HLA-A*0201-restricted peptide epitope (HPV 16 E711–20) has been used to restimulate CTLs, there has been considerable variation in detection of CTLs among patients (15 , 17 , 18) . Despite this variation, HPV-specific CTLs cannot be detected in the PBMCs of normal controls unless dendritic cells are used as antigen-presenting cells in vitro (18) . Collectively, these studies suggest that memory CTLs against HPV can be detected in patients but that they are at low frequencies compared with systemic CTLs against viruses such as influenza A. The variation in CTL responses detected may reflect either an individual variation in response against HPV or the efficiency of in vitro restimulation/detection methods (23) . There is a need to increase the sensitivity of HPV-specific CTL detection both in blood and at sites of disease for immune monitoring in epidemiological and clinical trial studies.
Novel technologies such as ELISPOT assays, intracellular cytokine staining, and fluorogenic MHC-peptide complexes (tetramers) have been used to greatly increase the sensitivity of antigen-specific CD8+ T-cell detection (24, 25, 26, 27, 28) . The CD8+ T-cell frequencies obtained with these techniques suggest that conventional techniques may greatly underrepresent the actual number of antigen-specific CD8+ T-cell effectors (23 , 25) . The use of tetramers is particularly attractive because it allows direct quantitation of antigen-specific T cells from blood or sites of disease without the need for in vitro restimulation. Furthermore, the CD8+ T cells detected by tetramers are largely cytotoxic T-cell effectors (25 , 29) . To investigate whether tetramers could be used to identify HPV-specific CTLs, we constructed a tetramer containing HLA-A*0201 complexed to the best studied HPV CTL epitope, HPV 16 E711–20. In this report, we describe the use of this HPV tetramer to quantify HPV-specific CTLs in peripheral blood and as a tool to isolate high-affinity CTLs capable of killing HPV 16+ tumor cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
The C1R-A2 cell line, a transfectant expressing the HLA
A*0201 allele (30)
was maintained in
RPMI 1640 containing 10% FCS (Life Technologies) and 400 µg/ml G418
(Life Technologies). CaSki, an HLA-A*0201 cervical carcinoma
cell line expressing HPV-16 E6 and E7 proteins (obtained from ATCC) was
maintained in RPMI containing 10% FCS (31)
. MDA-231, an
HLA-A*0201 breast epithelial carcinoma cell line (kind gift
from Linda Sherman, Scripps Clinic, La Jolla, CA) was maintained
in RPMI containing 10% FCS (32)
.
Patients and Controls.
CaCx patients were recruited (with informed consent) from patients
presenting for surgical treatment at the University Hospital of Wales,
Cardiff (18)
. All CIN3 patients had histologically
confirmed CIN3 and were recruited when they attended colposcopy clinics
at the University Hospital of Wales or presented for surgery at
Llandough Hospital (22)
. For tetramer analyses, PBMCs from
10 HLA-A*0201+ patients were used, with
samples from nine CIN3 patients and one CaCx patient. For controls,
nine HLA-A*0201+ laboratory volunteers
(ages, 26–50 years) were used: four males and five females. None of
the females had any history of abnormal cervical cytology, and none of
the controls were in high-risk groups for acquisition of sexually
transmitted diseases.
PBMC Isolation.
PBMCs were separated from heparinized blood samples (8.5–50 ml) by
centrifugation on a Histopaque density gradients (Sigma) and washed
three times with RPMI 1640 before use. Patient PBMCs were frozen in
aliquots containing 5–10 x 106
cells, stored in liquid nitrogen, and thawed before use for CTL
generation (18)
and tetramer staining.
Peptides.
HPV 16 E711–20 (YMLDLQPETT; Ref.
(33)
was synthesized by Immune Systems Ltd., Paignton,
United Kingdom. As controls, two other HLA-A*0201-presented
peptides were used; TP (GLLGFVGTL), derived from the TAP2 protein
(34)
, synthesized by Immune Systems Ltd.; and CP36
(YLKTIQNSL) from Plasmodium falciparum
(35)
, synthesized by the Peptide and Protein Facility of
the University of Wales College of Medicine.
Tetramer Synthesis.
MHC class I/peptide complexes were synthesized and tetramerized as
described previously (24)
. Briefly, purified
HLA-A2.1 heavy chain and human ß2-microglobulin were
synthesized using a prokaryotic expression system (pET; R+D Systems,
Abingdon, United Kingdom). The heavy chain was modified by deletion of
the transmembrane region and cytosolic tail and addition at the COOH
terminus of a sequence containing the biotinylation site recognized by
the enzyme BirA. Heavy chain and ß2-microglobulin (Sigma)
were refolded by dilution around peptide 11–20 from the E7 protein of
HPV-16 (YMLDLQPETT). Refolded complexes (45 kDa) were purified by gel
filtration and biotinylated using BirA in the presence of
biotin (Sigma), ATP (Sigma), and Mg2+(Sigma).
Biotinylated complexes were purified by gel filtration and ion
exchange, using fast protein liquid chromatography, and then
PE-conjugated streptavidin (Sigma) was added at a 1:4 molar ratio to
form fluorogenic HPV-16 E711–20 tetramer.
In Vitro CTL Induction Using Peptide Stimulation.
Thawed PBMCs from patients were stimulated as described previously
(18)
. Briefly, 2 x 106/ml PBMCs in RAB were cultured with peptide at
a concentration of 10 µg/ml. On day 4, 1 ml of RAB containing 25
units/ml IL-2 (Chiron UK Ltd, Harefield, Middlesex, United
Kingdom) was added. On day 6, 1 ml of medium was aspirated from each
well and replaced with 1 ml of fresh medium containing 10 units/ml
IL-2. On day 7, fresh or thawed irradiated autologous PBMCs were
resuspended at 3 x 106/ml in RAB
containing 10 µg/ml peptide and 3 µg/ml
ß2-microglobulin. Antigen-presenting cells were
allowed to adhere for 2 h, and then were washed before the
addition of 1–2 x 106/ml
effectors. On day 9, 1 ml of RAB containing 25 units/ml IL-2 was added
to each well. On day 13, the contents of the wells were split and
topped up with medium containing 10 units/ml IL-2. The cells were used
in a cytotoxicity assay on day 14.
FACS Staining, Sorting, and Generation of Monoclonal and
Polyclonal CTL Lines.
Thawed PBMC samples were stained with PE-labeled
HPV-E711–20 tetramer for 15 min at 37°
before the addition of Tricolour-anti-CD8 (Caltag, Burlingame, CA) or
FITC-anti-CD8 (DAKO AS, Glostrup, Denmark) for 15 min on ice, followed
by extensive washes with PBS containing 1% FCS. The cells were fixed
in PBS containing 2% paraformaldehyde and 1% FCS before analysis on a
FACScan (Becton Dickinson, Mountain View, CA). Small lymphocytes were
gated by forward and side scatter profiling, with up to 1 x 106 cells being collected for analysis of
PBMC samples. For some individuals, tetramer staining was performed
after 1 week of in vitro culture with the HPV 16
E711–20 peptide.
The line from the patient JJ, stimulated as described (18) , was stained with PE-labeled HPV-E711–20 tetramer for 15 min at 37° before the addition of Tricolour-anti-CD8 (Caltag) for 15 min on ice, followed by extensive washes, and then was sorted using a FACSVantage (Becton Dickinson). Small lymphocytes were gated by forward and side scatter profiling and then sorted according to tetramer/CD8 double staining. Single cells, or 500 cells for the monospecific, polyclonal line D4, were sorted directly into a U-bottomed 96-well plate. Each well had been coated overnight at 4°C with anti-CD3 and anti-CD28, both at 100 ng/ml in PBS, and contained 105 irradiated B cells (LG2) in CTL medium [Iscove’s medium (Sigma) with 5% human serum containing 100 units/ml IL-2]. Plates were incubated at 37° in 5% CO2 for 7–14 days without any manipulation, and then proliferating blasts were expanded in CTL medium, followed by restimulation using 5 µg/ml phytohemagglutinin with irradiated allogeneic peripheral blood lymphocytes and B cells as feeders (36) .
Cytotoxicity Assays.
Cytotoxicity was measured in a standard 4-h
51Cr-release assay as described previously
(18)
. C1R-A2 target cells were pulsed with 10 µg/ml
peptide for 2 h after labeling with 51Cr
(Na251CrO4;
Amersham International, Little Chalfont, United Kingdom). Cytotoxicity
against HPV 16 and 18 E6 and E7 was measured using a recombinant
vaccinia virus, TA-HPV (gift of Cantab Pharmaceuticals, Cambridge,
United Kingdom; Ref. 37
). TA-HPV has been shown to express
HPV antigens by both Western blot (37)
and by recognition
by HPV-specific CTLs (18
, 22)
. C1R-A2 cells were infected
with the vaccinia viruses (multiplicity of infection, 15) for a maximum
of 12 h before 51Cr labeling. After a 4-h
incubation, radioactive counts were obtained by ßeta plate liquid
scintillation counting (Wallac, Turku, Finland).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ref. 18
). Clear populations of
CD8+ tetramer+ cells could
be distinguished in all CTL lines tested (Fig. 1, F–J
), and
this varied between 2 and 12% of the T cells analyzed. No
CD8+ tetramer+ cells could
be detected in an HLA-A*0201-restricted CTL line recognizing
influenza M158–66 peptide (data not shown).
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. Previous work has shown that tetramers can be used directly to
visualize and clone human T cells constituting at least 0.01% of
CD8+ T cells (36
, 39)
. Using this
criterion, CD8+tetramer+ T
cells could be detected in PBMCs of both the patient and control
groups. However, there was variation in the frequencies of
CD8+tetramer+ cells
detected (Table 1)
. A range of 0.079–0.005% (1 of 1,260 to 1 of
19,073) was found in patients, with a similar range, 0.054–0.002% (1
of 1,855 to 1 of 42,004) in the normal control group (Table 1)
. These
low frequencies are similar to those found for melanoma-specific CTLs
in PBMCs (36
, 39
, 40) but are an order of magnitude lower
than the frequencies detected for systemic antiviral CD8 T cells
(24
, 29
, 41)
. In some patients and controls,
e.g., P6–P10 and C5–C9 in Table 1
, the number of
CD8+tetramer+ cells was
close to the limits of detection.
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and Fig. 2
). In contrast, no expansions of similar magnitude were seen for the
eight control PBMC samples (Table 1)
. These results are consistent with
previous reports that document a failure to generate HPV 16
E711–20-specific CTL in normal controls after
short-term in vitro peptide restimulation (17
, 18)
.
|
; Ref. 18
). To further investigate the fine
specificity of HPV 16 E711–20 CTLs, we used the
HPV 16 E711–20 tetramer to isolate purified
populations of CD8+ CTLs (Fig. 3A
) from the JJ CTL line (Fig. 1, D and I
). The resulting polyclonal CTL line D4, and clone C6
stained with HPV 16 E711–20 tetramer at high
levels (Fig. 3, B and C
) and were phenotypically
stable (92 and 99% TCR Vß6, respectively) after prolonged tissue
culture (data not shown).
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, 3D
, and 3E
). Furthermore, both D4 and C6 were
able to recognize HLA-A*0201+ target cells
infected with TA-HPV vaccinia but not cells infected with control
vaccinia (Fig. 3, D and E
). However, lysis of
TA-HPV-infected targets was lower than HPV 16
E711–20-pulsed target cells (Fig. 3, D and E
). Both D4 and C6 populations were also
able to lyse HLA-A*0201+, HPV
16+ CaSki cervical epithelial cell targets but
not HLA-A*0201+, HPV
16- breast epithelial target cells (MDA-231).
Pretreatment of CaSki and MDA-231 targets with IFN-
prior to use in
CTL assays (44)
did not increase the level of CTL lysis
despite increases in HLA class I expression (data not shown).
Furthermore, there was no CTL recognition of HPV 16-negative target
cells that were HLA matched with CaSki for one or more HLA class I
alleles (data not shown). These results confirm that both clonal and
polyclonal populations of HPV 16
E711–20-specific CTLs are able to recognize
endogenously processed HPV 16 E7 antigens. |
DISCUSSION |
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10 pM
for both
CTL.7
This is similar to the values obtained with melanoma-specific CTLs
capable of recognizing endogenously processed tumor antigens
(48)
. Tetramer-directed cell sorting offers a precise method to generate both monospecific polyclonal lines (D4) and more importantly, clones such as C6. Both have been propagated for up to 10 months,8 maintaining phenotype (CD8, TCR Vß6) and specificity, thus suggesting that these CTLs could easily be propagated for adoptive immunotherapy. By contrast, the use of conventional limiting dilution methods to isolate CTLs from peptide-specific CTL lines is highly inefficient, with only 1% of CTL clones being able to recognize endogenous HPV antigens (49) . Another drawback to the limiting-dilution cloning approach is the possibility that so-called "CTL clones" may actually be derived from mixed populations of CD8+ and CD4+ T cells, which may not be stable over time. Thus, the use of the HPV 16 E711–20 tetramer has efficiently isolated purified populations of stable, high-affinity HPV-specific CTLs not obtainable by conventional methods.
Both D4 and C6 CTLs were obtained from
CD8hitetramerhi populations
from the JJ CTL line (Fig. 3A
). This CTL line had a
heterogeneous pattern of tetramer staining (Fig. 1I
)
compared with other HPV 16 E711–20-specific CTL
lines (Fig. 1, F, H, and J
). This
might reflect the presence of both tetramerlo or
tetramerhi CTL subpopulations within the
polyclonal CTL line as has been described recently for
melanoma-specific CTLs (50)
. However, further in
vitro culturing of the parental JJ CTL line in the absence of
antigen resulted in a more homogeneous population of predominantly
CD8lotetramerhi T
cells.9
This might reflect the presence of nonsynchronously activated CTL
subpopulations (with differing levels of TCR expression) rather than
distinct tetramerlo or
tetramerhi populations. Alternatively,
tetramerhi T cells may have a longer life span
in vitro than tetramerlo T cells.
The results discussed above established the specificity of the tetramer for detecting effector CTLs from in vitro restimulated PBMCs. A goal of this study was to develop tetramer reagents that allow direct quantitation of HPV 16-specific CTLs from ex vivo blood or tissue biopsy samples, as has been demonstrated for melanoma (28 , 29 , 51) and viral antigens (29 , 38 , 52) . This would be particularly beneficial for rapid monitoring of HPV-specific CD8+ T-cell frequencies either in epidemiological studies in developing countries (53) or for vaccine trials (21) . Analysis of ex vivo PBMC samples revealed that staining of CD8+ T cells with the HPV 16 E711–20 tetramer was rare (1 of 1,260 to 1 of 42,000) in both CIN3 patients and controls. On the basis of epidemiological data, HPV would be detected in 65% (54) and 5% (55) of CIN3 patients and controls respectively. Although only small numbers of patients and controls were studied, there was no correlation between the numbers of tetramer+ cells detected and HPV-associated disease. It was not possible to examine the HPV 16 E711–20 cells for phenotypic markers associated with T-cell memory because of the low frequency of cells detected and the limited PBMC samples available. However, the enhanced detection of tetramer+ cells after peptide stimulation of patient samples suggests that these were more readily able to proliferate in vitro. This observation is consistent with previous findings that HPV 16 E711–20-specific CTLs can be generated in patients, but not controls, by standard restimulation protocols (17 , 18) .
The staining patterns of ex vivo PBMCs were not as clear as
for established HPV 16 E711–20-specific CTL
lines (Fig. 1
). This is particularly problematic where the numbers of
CD8+tetramer+ cells are
close to the limit of detection by FACS (Table 1)
. However, it is not
surprising that such low frequencies were seen in PBMCs in the absence
of in vitro restimulation (Table 1)
because similar results
have been observed in melanoma (28)
. It may be that for
HPV-specific CTLs, as for melanoma, PBMCs will not be the best
compartment for study. Higher frequencies of tumor-specific CTLs may be
detected among tumor-infiltrating lymphocytes (18)
or in
lymph nodes that have been infiltrated with tumor (28)
.
The frequencies detected for HPV 16 E711–20 CD8+ cells (1 of 1,260 to 1 of 42,000) are an order of magnitude lower than found for other viral antigens and tumor antigens. For example, memory CTLs recognizing influenza A M158–66 can be found in 1 of 500 small lymphocytes (29) , whereas for EBV EBNA3C325–333, up to 1 of 25 T cells can be detected using tetramer (41) . However, it should be noted that both of these viruses generate strong systemic CTL responses, whereas HPV may have evolved mechanisms to avoid immune recognition (56) . Furthermore, the immunodominant peptide epitopes of influenza A and EBV were used to construct tetramers. It is not clear whether HPV 16 E711–20, which was defined using the reverse immunogenetic approach (33) , is immunodominant. Even for immunodominant viral epitopes, at least 10-fold variation has been observed in the frequency of CD8 T cells detected by tetramer (29 , 41) . The variation in the frequency of CD8+ HPV 16 E711–20 tetramer+ T cells among patient PBMCs in this study might explain the contrasting results obtained for HPV 16 E711–20-specific CTL detection (15 , 17 , 18) . However, the current study is limited by the use of tetramers incorporating a single peptide epitope from HPV, and additional HPV CTL epitopes need to be defined. In the future, multiple tetramers comprising different CTL epitopes may allow more precise assessment of the role of CTL in cervical disease, including the possibility that HPVs can induce immunological tolerance (56) . Nevertheless, the HPV tetramer we have characterized may be beneficial for monitoring patients who have been immunized with the HPV 16 E711–20 peptide as part of ongoing clinical trials (47) .
Recently, tetramer-driven sorting has been used to derive melanoma-specific CTLs suitable for use in adoptive immunotherapy (36 , 40 , 50) . Regardless of the natural role of HPV-specific CTLs, our current studies demonstrate that it is possible to use a HPV tetramer to select high-affinity HPV-specific CTLs, which have potential application in the immunotherapy of CaCx. However, the frequent down-regulation of HLA class I molecules in cervical tumors (57) and the low levels of HPV E6 and E7 expression (58) may confound attempts at CTL-based immunotherapy. The use of the HPV 16 E711–20 tetramer to derive large numbers of stable HPV-specific CTL clones will now allow detailed investigation of the factors influencing CTL recognition of HPV-transformed tumor cells.
ACKNOWLEDGMENTS
This paper was presented in part at the
17th International Papillomavirus Conference
1999, Charleston, SC.
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FOOTNOTES |
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1 This work was supported by grants from the
Medical Research Council, HEFC(W), The Cancer Research Campaign, and
Ligue National Contre le Cancer. 
2 To whom requests for reprints should be
addressed, at University of Wales College of Medicine, Tenovus
Building, Heath Park, Cardiff CF4 4XX, United Kingdom. Phone:
44-1222-745004; Fax: 44-1222-745003.
3 These authors contributed equally to this
work.
4 P. R. Dunbar is the Girdlers Research
Fellow at Green College, University of Oxford.
5 S. Man is a Royal Society University Research
Fellow.
6 The abbreviations used are: CaCx, cervical
cancer; CIN, cervical intraepithelial neoplasia; HPV, human
papillomavirus; CD, cluster of differentiation; PBMC, peripheral blood
mononuclear cell; RAB, RPMI 1640 supplemented with 10% pooled human AB
serum; PE, phycoerythrin; IL-2, interleukin 2; FACS,
fluorescence-activated cell sorting; TCR, T-cell receptor; CTL,
cytotoxic T lymphocyte.
7 S. Youde, unpublished observations.
8 S. Youde and P. R. Dunbar, unpublished
observations.
9 P. R. Dunbar, unpublished observations.
Received 7/27/99. Accepted 11/12/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) cells sensitized with either E711–20 (
) or
as controls, TP from TAP2 (
), or CP38 from P.
falciparum (x). The recognition of endogenously processed
antigens was measured after infection of C1R-A2 cells with the
recombinant vaccinia virus, TA-HPV (
), or the wild-type vaccinia
virus Wy-vac (
). These CTL lines were then double-stained with the
HPV 16 E711–20 tetramer (X axis) and
CD8-tricolour or CD8-FITC (Y axis). Each flow cytometric
plot (F–J) corresponds to the CTL line above
(A–E). The percentages of
CD8+tetramer+ cells in the gated T-cell
populations are shown in the upper corners of
panels F–J.
). Percentage of lysis
was calculated as follows: 100 x [(specific
release - spontaneous release)/(maximal
release - spontaneous release)].