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*
Virginia Mason Research Center, Seattle, WA 98101;
R. H. Williams Laboratory and Molecular and Cellular Biology Program and
Department of Immunology, University of Washington, Seattle, WA 98195; and
§
Virology Division, Harborview Medical Center, Seattle, WA 98104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Recently, MHC tetramer technology has been developed to detect T cells. This technology utilizes specific, labeled MHC-peptide complexes to detect Ag-specific T cells by direct binding of the TCR complex, with visualization by fluorescence flow cytometry. This has been successful in a variety of studies focused on the identification of CD8+ class I-restricted T cells (5, 6, 7, 8, 9, 10), whereas analysis of CD4+ class II-restricted T cells has been much more elusive. Most class II MHC tetramers studies are limited to murine class II molecules (11, 12, 13). Recently, we utilized DR tetramers to analyze human DR-restricted T cell responses to influenza A (14). The low frequency of Ag-specific peripheral T cells in humans and concerns related to the stability of soluble class II molecules have challenged the development of this technology, particularly with HLA-DQ molecules, in which the molecules are unstable compared with HLA-DR (15, 16). We now report the development of class II peptide-tetramers that detect human peripheral blood T cells restricted by HLA-DQ molecules in Ag-specific immune responses to HSV-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Patients were HSV-2-infected individuals who visit the outpatient sexually transmitted disease clinics of the University of Washington Medical Center. HSV-2 infection was confirmed by HSV type-specific immunoblot (17). All patients were asymptomatic when their PBMC were obtained for these studies. HLA-DR and DQ typing were performed by reverse dot blot hybridization (18).
Isolation and characterization of DQ0602-restricted T cells
PBMC from HSV-2-seropositive individual 5491 (HLA-DRB1*1501, *0301 and DQB1*0602, *0201) were plated at 2 x 106 cells/well in a 24-well plate in medium consisting of RPMI and 15% heat-inactivated pooled human serum. Cells were stimulated with VP16 protein (gift from Chiron, Emeryville CA) at 2 µg/ml. Starting on day 5, cells were fed every other day with fresh medium and IL-2 (Hemagen, Worcester MA) at a concentration of 16 U/ml. On day 12, cells were washed and plated at 1 cell/well in a 96-well U-bottom plate which also contained 1.5 x 105 irradiated (3300 rad) autologous PBMC and 0.8 µg/ml PHA. IL-2 was added 48 h later. At day 26, cells from each positive well were evaluated for Ag-specific proliferation in the presence of 2 µg/ml VP16 and L-243 (anti-DR) or SPVL-3 (anti-DQ) at 5 µg/ml, with 1 x 105 irradiated PBMC from an HLA haplotype-matched individual as APC (HLA-DRB1*0101, *1501 and DQB1*0501, *0602). After 48 h, [3H]thymidine (1 µCi/well) was added for 16 h, and cells were then harvested and counted. Cells that proliferated in the presence of L-243 and that were inhibited by SPVL-3 were selected for epitope specificity.
For epitope studies, a panel of 60 peptides was used, each 20 aa long, which corresponded to the entire VP16 protein with a 12-aa overlap between adjacent peptides. The peptides were synthesized by Multipin peptide technology (Chiron Technologies, Clayton, Australia). For the proliferation assays, triplicate wells contained 104 T cells, 105 irradiated PBMC (DRB1*0101/1501, DQB1*0501/0602), and 1 µM peptide in 200 µl T cell medium in 96-well U-bottom plates. After 48 h, 1 µCi [3H]thymidine was added, and the cells were harvested and counted after an additional 16 h.
Construction of DQ0602-leucine zipper-biotinylation site expression vectors
Chimeric cassettes containing the coding regions for the HLA-DQ
extracellular domain/leucine zipper
(LZ)3 molecules were
made using a PCR-mediated splicing overlap extension technique
(19). Plasmids pN15LZ
and pLN15LZß were gifts from
Dr. D. Ostrov and Dr. S. Nathenson, Mt. Sinai Medical
School (New York, NY). For generation of the soluble DQA1 chain, cDNA
of DQA1*0102 was amplified in the first round using the primer pair,
(+) 5'-GGAATTCATGATCCTAAACAAAGCTCT-3' and (-)
5'-CCAGGTCTGCTGACGACTCTGTGAGCTCTGACATAG-3' (sharing homology with the
5' end of basic LZ) at a concentration of 4 nM for each primer. For the
second round amplification, the first round product was used as the
initial (+) primer on the pN15LZ template containing the basic LZ
cDNA motif at a concentration of 10 pM to form a DQA1*0102/LZ chimera.
The primer pair, (+) 5'-GGAATTCATGATCCTAAACAAAGCTCT-3' and
(-) 5'-CTGGTACCATCCTACTGGGCGAGTT-3' (sharing homology with
the 3' end of LZ), was then used to amplify the chimera. The fragment
was TA cloned into pCR2.1Topo (Invitrogen, San Diego, CA) sequenced and
then subcloned into the metal inducible Drosophila
expression vector pRmHa-3 (gift from Dr. L. S. B. Goldstein,
Howard Hughes Medical Institute, La Jolla, CA) using EcoRI
and KpnI sites engineered into the second round primer pair
(underlined above).
For generation of the soluble DQB1 chain, cDNA of DQB1*0602 was initially amplified using the primer pair, (+) 5'-ACCTCGAGGTCTCAATTATGTC-3' and (-) 5'-CCAGGTCTGCTGACGACTTGCTCTGGGCAGA-3' (sharing homology with the 5' end of acidic LZ) at a concentration of 4 nM. For the second round amplification, the first round product was used as the initial (+) primer on the pN15LZß template containing the acidic LZ cDNA motif at a concentration of 10 pM to form a DQB1*0602/LZ chimera. The primer pair, (+) 5'- ACCTCGAGGTCTCAATTATGTC-3' and (-) 5'-ACAAGCTTCCCTGAGCCAGTTCCTTTTCC-3' (sharing homology with the 3' end of the acidic LZ), was then used to amplify the chimera. This DQ cassette was then cloned into vector pAC1 (Avidity, Denver, CO), using the XhoI and HindIII sites (underlined above), 5' of the biotinylation sequence; the complete DQB1*0602-LZ biotinylation site cassette was then recovered by PCR amplification with the primer pair, (+) 5'-AGAATTCGTCTCAATTATGTCTTGGAA-3' and 5'-CTGGTACCTTAGTGCCATTCGATTTTCTG-3', and TA was cloned into pCR2.1Topo, sequenced, and then subcloned into the Drosophila expression vector pRmHa-3 using EcoRI and KpnI (sites underlined).
Generation of DQ0602 tetramers
The DQA1 and DQB1 chimeric cDNAs in the Schneider expression vector pRmHa-3 were cotransfected with the phshsneo plasmid (gift from Dr. M. McKeown, Salk Institute, San Diego, CA) into S-2 Schneider cells (gift from Dr. D. Zaller, Merck Research Laboratories, Rahway, NJ) by standard calcium phosphate transfection techniques. Cells were selected with G418 at 2 mg/ml (Life Technologies, Gaithersburg, MD). Cells were expanded and were grown to a density of 1 x 107 cells/ml. CuSO4 was added to a concentration of 1 mM to induce the production of class II molecules. The DQ0602 molecules from the supernatant were purified by affinity chromatography using SPVL-3 as previously described (20).
The class II molecules were concentrated to 1–2 mg/ml and then dialyzed in 10 mM Tris (pH 8), 10 mM NaCl. The protein was then biotinylated by using the Bir A enzyme according to the manufacturer’s conditions (Avidity, Denver, CO). The excess biotin was removed by dialysis. The biotinylated DQ0602 molecules were then loaded with the appropriate peptides at a 10-fold molar excess of peptide over class II molecules. Loading was conducted at 37°C for 72 h in a 100 mM sodium phosphate buffer, pH 6, with 0.25% n-octylglucopyranoside (20). Class II tetramers were generated by incubating an 8-fold molar excess of loaded biotinylated class II molecules with PE-streptavidin (BioSource International, Camarillo, CA) at room temperature for 4 h.
Tetramer analysis of T cell clones and PBMC
T cell clones were incubated at 37°C for 1–2 h in 50 µl media with 20 µg/ml tetramers/5 x 105 cells. CD4-Cychrome or CD4-PerCP (PharMingen, San Diego, CA) was added at the end of tetramer staining and incubated for an additional 15 min at room temperature. The cells were washed twice with PBS + 1% FBS before analysis by a Becton Dickinson FACSort. For staining of PBMC, 2 x 106 nylon wool-purified T cells were stimulated with VP16 protein at 2 µg/ml with autologous adherent PBMC as APC. Adherent PBMC were prepared by plating out PBMC at 5 x 106 cells/well in a 24-well plate. Cells were allowed to adhere overnight, and nonadherent cells were removed with a plastic pipette. The nonadherent cells were passed through a nylon wool column, and cells eluted from the column were used as nylon wool-purified T cells. Starting at day 5, and every other day, IL-2 was added to a final concentration of 10 U/ml. The T cells were stained with DQ0602/peptide tetramers 10 days later.
Measurement of cell divisions and precursor frequency
PBMC were stained with 0.8 µM 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) for 10 min at 37°C. Staining was stopped by adding 100% FBS and subsequently washing the cells twice in RPMI culture media. Cells were then plated at 3 x 106 cells/well in 24-well plates and stimulated with 2 µg/ml VP16 protein overnight. Cells were stained for surface markers and with DQ0602/369–380 tetramers or control tetramers and analyzed by FACS after 10 days in culture. A portion of the CFSE-stained cells was polyclonally stimulated with 2.5 µg/ml PHA and 10 U IL-2 and also analyzed by FACS on day 10. Stimulation of T cells with PHA and IL-2 results in cell division with distinct CFSE fluorescence peaks, allowing determination of the mean CFSE fluorescence for each generation. These values were used to calculate the average number of cell divisions in cells stimulated with specific Ag. Precursor frequency was estimated by dividing the fraction of tetramer-positive cells by 2x, where x is the average number of cell divisions as calculated by CFSE fluorescence.
Peptide binding assays
Competition peptide binding assays with purified DQ0602 were conducted as previously described (20). The reaction mixtures consisted of 25 nM purified DQ0602, 0.1 µM biotinylated IA-2 499–509 peptide, and various concentrations of nonbiotinylated VP16 369–380 or VP16 33–52 peptides. The peptide/class II complexes were captured on a SPVL-3-coated plate and measured by a europium-labeled streptavidin assay using a Delfia 1232 fluorometer (Wallac, Gaithersburg, MD). The IC50 was determined by plotting a curve and extrapolating the concentration at which 50% inhibition occurs.
Analysis of TCR genes
mRNA was prepared from T cell clones using the Quick Prep Micro RNA Purification Kit (Pharmacia Biotech, Piscataway, NJ). cDNA synthesis was conducted using the Superscript Preamplification system (Life Technologies). TCR Vß gene usage was determined by PCR amplification of cDNA using a set of 5' Vß 1–23 primers and 3'-constant region primers as described (21).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The tegument protein VP16 of HSV-2 virus is one of the major
proteins recognized as a viral Ag in HSV-2-infected subjects
(22). Forty-eight T cell clones were isolated from
peripheral blood of an HSV-2-infected individual that were specific for
the HSV-2 VP16 protein. Proliferation assays in the presence of either
an anti-DR or anti-DQ Ab indicated that seven of these clones
were restricted by DQ0602, the only DQ molecule in common to both the
responders and the APC (see Materials and Methods). Fig. 1
illustrates the proliferation of two of
these T cell clones, c5 and c44, in the presence of VP16 protein and
BLS-DQ0602 B-lymphoblastoid cell line (LCL) as APC. The BLS-DQ0602 LCL
is an EBV-transformed human B cell line that was transfected with DQ
genes and had DQ0602 as the only expressed class II molecule
(16), confirming that DQ0602 was the restriction element
for these T cell clones. To determine the dominant DQ0602-restricted
epitope for the VP16 protein, all 7 T cell clones were screened with a
panel of 60 overlapping peptides that covered the entire
VP16 protein. Six of the seven DQ-restricted clones, c5, c8, c31, c48,
c68, and c73, were specific for both peptides 361–380
(AYSRGRTRNNYGSTIEGLLD) and 369–388 (NNYGSTIEGLLDLPDDDDAP). Truncation
analysis of the peptides indicated that all 6 clones recognize peptide
369–380 (NNYGSTIEGLLD). The other clone, c44, was shown to recognize
the VP16 peptide 33–52 (PPLYATGRLSQAQLMPSPPM). The reactivity profiles
of clone c5 and c44 to the panel of overlapping VP16 peptides are shown
in Fig. 2. TCR Vß transcripts in clones
that recognized 369–380 were identified by RT-PCR analysis using a set
of Vß primers. Two different Vß-chains were identified: clones c5,
c8, c31, and c68 used Vß 6.5; and clones c48 and c73 used Vß
13.2.
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, tetramer staining was specific
for T cells with corresponding functional specificity: Clone c5 T
cells, which proliferated to peptide 369–380, could be stained only by
the DQ0602 tetramers loaded with peptide 369–380 but not by the
tetramers loaded with peptide 33–52, whereas clone c44 T cells, which
proliferated to peptide 33–52, could be stained only by tetramers
loaded with peptide 33–52.
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Peripheral blood lymphocytes from individual 5491 were incubated
with VP16 protein in vitro and stained with the DQ0602/peptide
tetramers 10 days later. T cells identified by both the DQ0602/369–380
and DQ0602/33–52 tetramers were detected (Fig. 4, A and B). T
cells that stained with the DQ0602/369–380 tetramers were observed at
a higher frequency (3.3%) than the T cells stained with the
DQ0602/33–52 tetramers (0.6%). This observation was consistent with
the observation in T cell cloning that clones specific for epitope
369–380 were observed at a much higher frequency than the 33–52
epitope. Similar staining patterns with the DQ0602/369–380
tetramers were also observed with peripheral blood T cells
from two additional HSV-2 infected individuals who carried the DQ0602
haplotype: 5624 (DRB1*1501/1101, DQB1*0602/0301) and 5633
(DRB1*1501/0801, DQB1*0602/0402) (Fig. 4, C and
D). T cells corresponding to the 33–52 epitope were not
detected in these two individuals.
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, E
and F). No staining was observed with tetramers that were
loaded with a DQ0602 high binding affinity peptide, insulin B 5–15
(20) (data not shown). Precursor frequency of DQ0602-restricted T cells in peripheral blood
To estimate the initial frequency of DQ0602/369–380 T cells
present in the peripheral blood of the HSV-2-infected individuals, we
combined the use of specific tetramer staining with another
fluorescent-based technique that quantitates cell divisions. As shown
in Fig. 5, after 10 days of culture, T
cells from individual 5491 staining positive with DQ0602/369–380
tetramer are found predominantly in the CFSE-dull quadrant of the
fluorescence profile. The mean CFSE fluorescence of these cells
corresponds to 
10 cell divisions. The observed frequency of 2.1%
for tetramer-positive cells after 10 cell divisions indicates an
initial precursor frequency of 0.002%.
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Peptide binding studies for the DQ0602 molecule were performed to
compare the class II-peptide avidity of the two VP16 epitopes, 369–380
and 33–52. As shown in Fig. 6, peptide
369–380 has an IC50 of 2 µM, whereas peptide
33–52 has an IC50 of 0.4 µM. Because this
binding avidity is inversely related to the frequency of
epitope-specific T cells observed, this indicates that the relative
higher frequency of 369–380 T cells was not directly due to a high
affinity of 369–380 for DQ0602 but likely results from a more complex
set of parameters associated with selection of immunodominant epitopes,
possibly including differential T cell selection or variable processing
of the VP16 protein, enabling the 369–380 epitope to be preferentially
processed compared with the 33–52 epitope. This is compatible with
x-ray crystallography structural studies in which the carboxyl-terminal
region of the VP16 protein has a disordered structure
(23), potentially allowing this region easy access to
protease and capture by class II molecules. This is also consistent
with a general property of this part of the VP16 molecule, rather than
specific for DQ0602 determinant selection, because previous studies
have also demonstrated that the carboxyl terminal of the VP16 contained
numerous T cell epitopes (22).
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The specificity of T cells detected with the DQ0602/369–380
tetramer was further confirmed by isolating the cells sorted by flow
cytometry. T cells positively stained with the DQ0602/369–380 tetramer
were FACS sorted and cloned at 3 cells/well; the sorted T cells were
then expanded by PHA in the presence of IL-2 to generate 47 cell lines.
Staining with tetramers and proliferation assays with the 369–380
peptide are summarized in Table I.
Thirty-six cell lines were positive for the staining and the
proliferation assays; 11 lines were negative for both staining and the
proliferation assays. Thus, 77% of the sorted T cells were specific
for the VP16 369–380 peptide as assayed by tetramer staining and
proliferation, compared with 2–3% T cells staining with the tetramer
in the original unsorted sample. This confirms the Ag-specific
properties of peripheral blood T cells detected using tetramer
staining. The less then perfect correlation between the sorted cells
and reactivity to the VP16 peptide was probably due to nonstringent
fluorometry settings for the T cell population, which was gated for 1%
positive cells. The overall fluorescence intensity as shown in Table I
for these cell lines was variable, due to heterogeneity in the cell
population. There was no correlation between the extent of
proliferation and the intensity of the fluorescence signal.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William Kwok, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle WA 98101-2795.
3 Abbreviations used in this paper: LZ, leucine zipper; CFSE, 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester; LCL, lymphoblastoid cell line.
Received for publication December 27, 1999. Accepted for publication February 9, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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/ß heterodimer. J. Exp. Med. 169:345.ß protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus. J. Immunol. 161:6439.1-w29/Vß1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur. J. Immunol. 22:1261.[Medline]
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