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,
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Virginia Mason Research Center, Benaroya Research Institute, Seattle, WA 98101;
R. H. Williams Laboratory, Molecular and Cellular Biology Program, and
Departments of Medicine and Laboratory Medicine, University of Washington, Seattle, WA 98195; and
Fred Hutchinson Cancer Research Center, Seattle, WA 98109
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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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A number of recent studies have used soluble MHC multimers to directly
identify T cells restricted to specific peptide epitopes. This
technology has been used to track T cells specific for both viral Ags
(6, 7, 8, 9) and tumor Ags (10, 11, 12, 13) in both animal
models and in humans when the peptide epitope is known. The majority of
these studies have focused on class I-restricted T cells, as initial
efforts in producing class II molecules were hampered by difficulties
in generating stable soluble forms of the molecules and inefficiency in
biotinylation of these molecules. One approach to stabilizing the
soluble class II molecules has involved covalently tethering the
peptide to the 
-chain of the class II molecule (14, 15). However, tethering of peptide to class II molecules does
not appear to be essential for the stability of soluble class II
molecules, as demonstrated by the successful detection of Ag-specific T
cells in mice and humans by using soluble MHC molecules with
exogenously loaded peptide (16, 17, 18, 19).
Efficient production of class II MHC tetramers containing soluble peptide offers the potential to detect and isolate T cells specific to Ags in which the T cell epitopes are not previously known. In this approach, tetramers are loaded with a mixture of overlapping peptides that cover the entire Ag. These tetramers then are used to identify and isolate epitope-specific T cells from PBMC that have been stimulated with the whole Ag. With this approach, we have identified four DRA1*0101/DRB1*0401 (DR0401)3-restricted T cell epitopes and two DRA1*0101/DRB1*0404 (DR0404)-restricted T cell epitopes of the HSV-2 tegument protein VP16. Earlier studies have demonstrated that VP16 epitopes are recognized by CD4+ T cells that infiltrate genital lesions in individuals with HSV-2 infection, suggesting that the protein serves as an important Ag in the natural immune response to HSV-2 virus (20, 21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction of the expression vectors for generation of
soluble DR0401 has been described previously (18). A
similar approach using the same primers was used to generate the
DRB1*0404-leucine zipper-biotinylation site expression vector from
DRB1*0404 cDNA. DRB1*0404 cDNA was isolated from the EBV-transformed B
lymphoblastoid cell line Bin-40, and was a gift from Dr. P.
Gregersen (North Shore University Hospital, Manhasset, NY).
Briefly, a chimeric cassette containing the extracellular coding region
for the DRB1*0404 chain appended to the acidic leucine zipper motif was
generated from DRB1*0404 and leucine zipper cDNA by using the
PCR-mediated splicing overlap technique (22, 23). A
site-specific biotinylation sequence then was added to the 3' end of
the DRB1*0404/leucine zipper cassette. The chimeric cDNA was subcloned
into the Cu-inducible pRmHa-3 Drosophila expression vector.
For class II production, DR
and DR expression vectors were
cotransfected into Schneider S-2 cells. Soluble class II molecules
produced after addition of CuSO4 were purified by
affinity chromatography, concentrated, and subsequently biotinylated
with the Bir A enzyme (Avidity, Denver, CO; Ref. 24).
For generation of tetramer pools, a panel of 60 overlapping VP16 peptides, p3 to p62, were used. These peptides, each 20 aa in length, corresponded to the entire VP16 protein with a 12-aa overlap between adjacent peptides. Peptides were synthesized on polyethylene pins with 9-fluorenylmethoxycarbonyl chemistry by Chiron Technologies (Clayton, Australia). Individual peptides were weighed out and dissolved in DMSO to achieve the appropriate peptide concentration. The peptides were divided into 12 pools, each containing 5 different overlapping peptides. Five peptides per pool was found to preserve sensitivity in identifying individual peptide epitopes. A similar range of peptides per pool has been used in earlier epitope mapping studies (25, 26). The biotinylated class II molecules DR0401 and DR0404 at a concentration of 1 mg/ml were each loaded with the 12 different peptide pools by incubation for 48 h at 37°C with 25-fold molar excess of peptides (total) in 100 mM sodium phosphate, pH 6.0, and 0.2% n-octyl-D-glucopyranoside. Tetramers were formed by incubating class II molecules with PE-labeled streptavidin (BioSource International, Camarillo, CA) for 6 h at room temperature at a molar ratio of 8 to 1. For single peptide tetramers, the peptide was loaded at a concentration of 5-fold molar excess over the class II concentration.
Staining and isolation of VP-16-specific T cells
PBMC from a DRB1*0401, DRB1*0404 HSV-2-positive individual were stimulated with VP16 protein at 2 µg/ml (a gift from Chiron Corporation). IL-2 was added (10 U/ml final concentration) every other day starting on day 5. T cells were stained with tetramer pools on day 11 or 12. For each pool, 2 x 105 cells were incubated with 0.5 µg of PE-labeled tetramer in 50 µl of culture medium (10 µg/ml) at 37°C for 1 to 2 h, and then stained with anti-CD4-FITC (BD PharMingen, San Diego, CA) for 15 min at room temperature. Near maximal staining of T cell clones was observed with 0.05 µg of tetramer reagent, indicating that 10 µg/ml provides an excess of MHC molecules for the staining reaction. Cells were washed and analyzed with a Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Tetramers loaded with the corresponding single peptides were generated for those pools that gave positive staining, and analysis was done on day 14 or 15. Levels of background staining, generally around 0.1%, were determined by using tetramers loaded with an irrelevant peptide, HA307–319 (influenza A hemagglutinin protein, residues 307–319). Cells that were positive for a particular tetramer were single-cell sorted into 96-well U-bottom plates by using a Becton Dickinson FACSVantage (San Jose, CA) on the same or following day. Sorted cells were expanded with 1.5 x 105 unmatched, irradiated (5000 rad) PBMC per well as feeders with 2.5 µg/ml PHA and 10 U/ml IL-2 added 24 h later. Specificity of cloned T cells was confirmed by staining with tetramers (loaded with cognate peptide or control peptide, HA307–319) and T cell proliferation assays with 10 µg/ml of specific peptide and DR-transfected bare lymphocyte syndrome (BLS) line BLS-DRA1*0101/DRB1*0401 (BLS-DR0401) or BLS-DRA1*0101/DRB1*0404 (BLS-DR0404) as APCs (18, 27). Competition binding assays of identified peptides were performed with purified DR0401 and DR0404 protein as previously described (28).
Measurement of cytokine secretion
In experiments that used IFN-
secretion as an indicator of T
cell reactivity, PBMC were stimulated with VP16 protein as described
above and assayed on day 11 or 12. PBMC
(1 x 105) were incubated together with an
equal number of BLS-DR0401 or BLS-DR0404 APCs that had been pulsed with
50 µg/ml of each peptide pool. After 3 h of incubation, IFN-
secretion was determined by use of a cytokine secretion capture assay
following the protocol supplied by the manufacturer (Miltenyi Biotec,
Auburn, CA; Ref. 29). Briefly, cells were washed once in
PBS and incubated in 100 µl of medium on ice for 5 min with an Ab-Ab
conjugate directed against both CD45 and IFN-. Prewarmed medium was
added to a final volume of 2 ml, and cells were incubated at 37°C for
45 min under gentle rotation to allow cell surface capture of secreted
IFN-. Cells were washed once in PBS and then stained for 15 min on
ice with a second PE-conjugated Ab directed against IFN- as well as
a FITC-conjugated Ab directed against CD4. Cells were washed once in
PBS and analyzed by flow cytometry as described above. For pools that
showed significant IFN- staining, reactivities of individual
peptides were assayed by the same approach.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the class II-restricted epitopes of the HSV-2 VP16 protein, peptides of 20 aa in length spanning the entire sequence of the VP16 protein, with a 12-aa overlap between consecutive peptides, were used to generate two tetramer panels, one for the DR0401 molecules and the other for the DR0404 molecules. Each panel included 12 different tetramer pools, designated as pool 1 to pool 12. Each pool contained the soluble class II molecules together with five different peptides of the VP16 protein.
With these two tetramer panels, we analyzed PBMC from a DRB1*0401,
DRB1*0404 HSV-2-positive individual. Cells were stimulated with 2
µg/ml of VP16 protein and 11 or 12 days following stimulation were
stained with each tetramer panel. Four pools, 2, 5, 6 and 12, gave
significant staining above background with the DR0401 tetramer panel
(Fig. 1
A). Staining with the
DR0404 tetramer panel identified two pools, 6 and 12, as having
significant staining (Fig. 1B). We then generated tetramers
loaded with the individual peptides of the positive pools and used
these to determine which peptide epitopes in the pool were recognized
by responding T cells. For DR0401/pool 2, peptides p9, p10, and p12
gave positive staining (Fig. 2A); for DR0401/pool 6,
peptide p32 gave positive staining; and for DR0401/pool 12, peptides
p61 and p62 gave positive staining. We were unable to identify any
individual peptides for pool 5 despite repeated attempts. We speculate
that this may reflect T cells of low avidity, which are difficult to
consistently stain using tetramers. For DR0404/pool 6, peptides p31 and
p32 gave positive staining (Fig. 2B); and for DR0404/pool
12, peptide p58 showed positive staining.
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production as an
indicator for epitope-specific cells. Cells were stimulated with 2
µg/ml of VP16 protein and 11 or 12 days following stimulation were
restimulated for 3 h with BLS-DR0401 or BLS-DR0404 cell lines
pulsed with 50 g/ml of each peptide pool. For PBMC stimulated with the
BLS-DR0401 cell line, pools 2, 5, and 12 were positive for IFN-
secretion as measured by flow cytometry (Fig. 3). As seen in Fig. 1
A, both
pools 2 and 12 were positive in the DR0401 tetramer analysis. Pool 5
was positive in the tetramer screen; however, as noted, we were unable
to see tetramer staining with the individual peptides of pool 5.
Interestingly, pool 6, which was positive in the tetramer screen, did
not show appreciable IFN- production. This suggests that although
the two methods generally coincide, differences in what each approach
measures, TCR/MHC avidity with tetramers and specific effector function
with IFN- production, allows each method to identify occasional
cells that would be undetectable by the alternate approach.
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T cells that were specific for peptides p10 and p61 for DR0401 and
peptide p58 for DR0404 were isolated and tested for specificity.
Tetramer-positive T cells were single-cell sorted and expanded, and
>20 clones specific for each DR/peptide complex were identified. All
of the clones obtained demonstrated Ag specificity as shown by
proliferation assays and tetramer staining. Tetramer staining and
proliferation data are shown for three different clones for each
specificity in Fig. 4. Although strong
staining and Ag-specific proliferation were observed for most clones,
there existed variations in staining intensity and degree of
proliferation for a number of clones. Some of these atypical patterns
are illustrated in Fig. 4C with DR0401-restricted clones
specific for p10. Both clones p10–1 and p10–2 consistently showed a
broader range of staining intensity compared with the majority of
clones, which showed more focal staining. In contrast, clone p10–3
consistently showed poor tetramer staining despite vigorous
proliferation to specific peptide. We speculate that these different
staining patterns reflect different avidities of the TCR for the
MHC/peptide complex in the different clones. These atypical staining
patterns were not unique to clones restricted to p10; a few clones
specific for p58 and p61 also showed weak staining, whereas clones
specific for p10 with strong staining were also observed.
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A with the DR0401/p9
tetramers was likely attributable to the overlapping of peptide p9
(VP1649–68) and peptide p10 (VP1657–76), as
both peptides contain the shorter epitope.
Similar examination of peptide p61 (VP16465–484)
identified a 13-mer peptide, VP16472–484, which stimulated
DR0401-restricted T cell clones directed against the p61 epitope (data
not shown). Therefore, the positive staining seen with the DR0401/p62
tetramer in Fig. 2A was likely attributable to the
overlapping of peptides p61 and p62. These results also demonstrate
that tetramer staining with overlapping peptides provides an
alternative approach to directly mapping minimal epitopes. Truncation
studies with peptide p58 refined the DR0404-restricted epitope to the
132-mer VP16443–455, (data not shown).
The DR0401-restricted and the DR0404-restricted epitopes as
characterized in the truncation studies are listed in Table I. Interestingly, the DR0404-restricted
443–455 epitope is not found as a DR0401-restricted epitope, nor are
the 58–69 and 472–484 DR0401-restricted epitopes found as
DR0404-restricted epitopes. In vitro peptide binding was analyzed with
purified DR0401 and DR0404 molecules. Peptides from the
DR0401-restricted epitopes (VP1658–69 and
VP16472–484,) bind with a much lower affinity to
DR0404 compared with the DR0404 restricted peptide
VP16443–455 (Table I). In contrast, the affinity of
the DR0404443–455 epitope for DR0401 is lower when
compared with peptides 58–69 and 472–484, although the magnitude of
this difference is not as great with peptide 58–69 (Table I). These
experimental observations are in agreement with previous reports that
the limited polymorphism between these alleles at codons 86 and 71 of
the DRB1 chain may dictate unique binding patterns, although factors
other than peptide binding also are likely important in dictating which
epitopes become immunodominant for each allele (33, 34).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion; however, some epitopes were
identified only by one or the other method, likely reflecting the
different T cell characteristics each approach measures. Earlier
studies examining Ag-specific CD8+ T cells with
class I tetramers have likewise demonstrated this incomplete
concordance between Ag-specific cytokine production and tetramer
staining (10, 35). The MHC class II tetramers in this study were generated by using a peptide mixture. Notably, the current results demonstrate that tetramers generated with peptide mixtures are capable of staining Ag-specific T cells. We speculate that within a given peptide mixture, only one or two peptides can bind to the MHC with high affinity. These high-affinity peptide complexes likely ensure the formation of sufficient numbers of tetramers containing identical MHC/peptide complexes. Data from our laboratory and others suggests that 10–50% of the soluble MHC molecules are loaded when incubated with exogenous peptide (Ref. 36 and W. Kwok, B.McFarland, C. Beeson, unpublished observations). Applying these observations to tetrameric molecules suggests that with exogenous peptide loading, less than 6% of the tetramers have all four MHC molecules loaded with peptide, assuming a lack of cooperative interactions. Staining with dimeric or trimeric MHC/peptide complexes previously has been reported (37). Therefore, the T cell staining observed in our study is likely at least partly attributable to binding of MHC tetramer complexes with only two or three sites occupied by the cognate peptide.
One of the concerns of the TGEM approach is that the relevant peptides will be outcompeted by irrelevant peptides present in the pool. For this reason, we purposefully loaded DR0401 molecules with different molar ratios of cognate peptide (VP16 p61) to competitor peptide (HA307–319), and these mixed tetramers were used to stain a DR0401/p61 clone. In competition experiments, these two peptides possess similar affinities for the DR0401 molecule (data not shown). There was no significant difference in staining pattern when the molar ratio of p61 to HA307–319 was 1:1. We observed an appreciable decrease in staining intensity of the DR0401/p61 clone with a 1:10 molar ratio; nonetheless, staining of clones with these mixed tetramers was still 10-fold above background (data not shown). Likewise, we were able to obtain similar staining above background by using cognate tetramer diluted 100-fold, suggesting that an excess of class II molecules are present during the staining reaction. These findings suggest that loading of peptide mixtures onto class II within the parameters described should not preclude the TGEM approach from detecting Ag-specific T cells of interest. Earlier studies that used pooled peptides to stimulate cells have shown similar results (25, 26, 38), and studies that used combinatorial peptide libraries have demonstrated that HLA competition only becomes a contributing factor in T cell stimulation when competitor peptides are present in several orders of magnitude excess (39). Nonetheless, it is conceivable that the pooled peptide approach may have difficulty in detecting peptide epitopes with very low MHC binding affinity in the presence of other peptides in the same peptide pool with high MHC binding affinity.
The standard approach for cloning T cells and mapping out antigenic
epitopes involves Ag challenge of PBMC followed by plating individual
cells into 96-well plates. Cells then are expanded and assayed for MHC
restriction and peptide specificities by screening clones with
overlapping peptides that cover the Ag, a labor-intensive and
time-consuming process. Alternatively, epitopes can be identified by
using a recently described flow cytometry-based approach that uses
IFN- production as a marker of reactivity. Although this approach
simplifies isolation of epitope-specific clones, the task of
identifying individual MHC restriction elements remains. With TGEM, the
HLA restriction and peptide specificity of the T cells are known
because of the specificity of tetramer staining. Tetramer staining also
offers the opportunity for straightforward cloning of the Ag-specific T
cells through single-cell sorting. As a consequence, large numbers of T
cells clones with known Ag specificities and MHC restrictions can be
obtained easily. In addition, we have found that the Ag-dependent
expansion of T cells in the TGEM procedure can be performed without
purified Ag. For assay of viral antigenic epitopes, we have previously
shown that stimulation with UV-inactivated virus is effective
(19). Similarly, peptide pools can be used for initial
stimulation in the event that whole Ag is not available.
Knowledge of specific Ag epitopes and their associated MHC restrictions provides a valuable foundation for directed immunotherapies in a number of different areas including autoimmune and infectious diseases and cancer (1, 2, 3, 4, 5). TGEM allows rapid identification of CD4+ T cell epitopes and can easily be applied to a variety of Ags, especially as tetramer reagents become more widely available.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William W. Kwok, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101-2795. E-mail address: bkwok{at}vmresearch.org
3 Abbreviations used in this paper: DR0401, DRA1*0101/DRB1*0401; DR0404, DRA1*0101/DRB1*0404; TGEM, tetramer-guided epitope mapping; HA307–319, influenza A hemagglutinin protein, residues 307–319; BLS, DR-transfected bare lymphocyte syndrome.
Received for publication November 1, 2000. Accepted for publication March 20, 2001.
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References |
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and T-cell receptor extracellular segments. Proc. Natl. Acad. Sci. USA 91:11408.This article has been cited by other articles:
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