International Immunology Advance Access originally published online on September 30, 2007
International Immunology 2007 19(11):1291-1301; doi:10.1093/intimm/dxm099
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Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4+ T cells and suggests HLA-based differences in epitope recognition
1 Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA 98101, USA
2 Department of Immunology, University of Washington, Seattle WA 98195, USA
Correspondence to: W. W. Kwok; E-mail: bkwok{at}benaroyaresearch.org
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Abstract |
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Tetanus toxoid is a routine positive control antigen for cellular assays. Previous studies identified multiple tetanus toxin (TT) epitopes, including some ‘universal’ epitopes. However, rigorous HLA-restricted study of tetanus toxoid responses is still lacking. In this study, the tetramer-guided epitope mapping approach was used to identify CD4+ T-cell epitopes within the TT heavy chain restricted by 10 different class II alleles. Of 106 peptides tested, 52 contained epitopes. Response frequencies toward specific epitopes varied, indicating prevalent and rare specificities. Most antigenic peptides (85%) were presented by one or two class II alleles. For peptides presented by three or more alleles, truncation studies revealed that some contained multiple epitopes. These results contrast with the perceived notion that tetanus toxoid responses are dominated by universal CD4+ T-cell epitopes. Rather these results illustrate heterogeneous T-cell responses for different class II alleles and individual-specific variation of the T-cell repertoire.
Keywords: class II MHC, epitope, tetanus toxoid, tetanus toxin
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Introduction |
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The US Advisory Committee on Immunization Practices recommends tetanus toxoid vaccination in all age groups. As such, almost all healthy subjects within the US exhibit both humoral and cellular responses against tetanus toxoid. Studies of tetanus toxin (TT)-specific CD4+ T-cell epitopes over the past 30 years suggest that particular regions of TT are highly immunogenic and that most individuals exhibit cellular immune responses to a few identical TT epitopes (1–5). Collectively, these studies give the impression that tetanus toxoid responses are primarily directed against a few promiscuous or universal CD4+ TT epitopes regardless of the HLA haplotype. However, the concept of universal epitopes seems opposed to the fact that highly polymorphic HLA proteins exhibit allele-specific peptide-binding preferences (6–8).
TT830–844, the most widely described universal TT epitope, is reported to be presented by at least 10 different class II alleles, including DRA1/DRB1*0401 (DR0401), DRA1/DRB1*1101 (DR1101) and DRA1/DRB1*1501 (DR1501) (1, 3). In developing a protocol and reagents for cellular immunoassays, we assayed for the presence of DR0401-, DR1101- and DR1501-restricted TT830–844 specific cells using class II tetramers. Although our assay could detect DR1101-restricted TT830–844 specific T cells, the assay failed to detect DR0401- and DR1501-restricted TT830–844 specific T cells in multiple subjects. Given the fact that these tetramer reagents have been extensively validated, these mixed results led us to question the universal recognition of the TT830–844 epitope and motivated a more comprehensive study of TT epitopes restricted by DR0401, DR1101, DR1501 and other class II alleles.
To rigorously address this question, the tetramer-guided epitope mapping (TGEM) approach was applied to identify TT-specific epitopes and their class II MHC restriction as described in our previous studies (9, 10). CD4+ T cells were stimulated with pooled mixtures of peptide and stained with class II tetramers loaded with the corresponding peptide mixture. For positive pools, staining was repeated with individual peptide-loaded tetramers to identify the antigenic peptide (or peptides) within the pool. We report here the detection of multiple epitopes within the TT heavy chain (residues 458–1315) for 10 different class II alleles. Although multiple epitopes were observed for each allele tested, no ‘universal’ high-avidity TT epitopes were detected. Rather, the majority of the identified epitopes was shared by one or two alleles.
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Methods |
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Human subjects
A total of 30 healthy immunized subjects (tetanus booster within the past 10 years) were recruited with informed consent. A wide interval of time after Tetanus booster was chosen to allow the timely recruitment of a reasonable number of donors for each class II allele of interest. The maximum interval of 10 years was chosen based on the US Advisory Committee on Immunization Practices recommendations. A two-step protocol was used to determine the class II HLA genotype for each subject. Low-resolution class II typing was carried out using an SSP UniTray typing kit (Invitrogen). High-resolution class II typing was accomplished by DNA sequencing with allele-specific primers based on low-resolution typing results.
Production of class II tetramers
The following class II monomers and multimers were produced for this study: DRA1/DRB1*0101 (DR0101), DRA1/DRB1*0301 (DR0301), DR0401, DRA1/DRB1*0404 (DR0404), DRA1/DRB1*0701 (DR0701), DR1101, DR1501, DRA1/DRB5*0101 (DRB5), DQA1*0102/DRB1*0602 (DQ0602) and DPA1*0103/DPB1*0401 (DP0401). Production of these molecules has been previously described (11–15). Briefly, soluble class II molecules were purified from the supernatants of insect cell cultures and biotinylated using biotin ligase enzyme (Avidity, Denver, CO, USA). Biotinylated class II molecules were dialyzed into phosphate storage buffer (pH 6.0) and loaded with pooled peptide mixtures or individual peptides by combining 0.5 mg ml–1 class II, 10 mg ml–1 of peptide (2 mg ml–1 of each individual peptide for pools) and 0.2% 
-octyl-D-glucopyranoside. After incubating at 37°C for 48 h, tetramers were cross-linked using PE–streptavidin (Biosource, Camarillo, CA, USA).
Tetanus peptides
A panel of 106 overlapping TT heavy-chain peptides was purchased from Mimotopes (Clayton, Victoria, Australia). Each peptide was 20 amino acid (aa) long with a 12 aa overlap between adjacent peptides. These peptides were divided into 21 pools of five consecutive peptides (pool 21 had six peptides).
Tetramer-guided epitope mapping
The TGEM procedure was as previously described (9, 10). PMBCs were freshly isolated from whole blood by Ficoll underlay for all experiments. Briefly, CD4+ T cells were isolated from PBMC using the Miltenyi ‘no-touch’ CD4+ T-cell isolation kit. CD4– cells recovered from the magnetic column were incubated in 48-well plates (3 x 106 cells per well) for 1 h and then washed, leaving adherent cells for use as APCs. After adding two million CD4+ T cells per well, each well was stimulated with pooled TT peptides. After 14 days incubation, 100 µl of re-suspended cells were stained with pooled peptide PE-conjugated tetramers for 60 min at 37°C. Subsequently, cells were stained with CD4–PerCP, CD3–APC and CD25–FITC mAbs (eBioscience) and analyzed by flow cytometry. Cells from pools that gave positive staining were analyzed again with individual peptide tetramers. Whenever the reagents were available, subjects were tested with tetramers corresponding to more than one DR molecule. In such cases, separate aliquots of cells were stained with tetramers for each allele of interest.
ELISPOT assay
Freshly isolated PBMCs were plated (0.5 x 105 cells per well) onto polyvinylidene difluoride membrane 96-well plates (Millipore, Bedford, MA, USA) pre-coated with 0.5 µg ml–1 (100 µl per well) anti-IFN-
mAb (Endogen, Cambridge, MA, USA) in the presence of TT peptides at 10 µg ml–1 and whole tetanus Toxoid (1% v/v of vaccine dialyzed against PBS). After 24-h incubation, cells were removed by washing with PBS and PBS–Tween. IFN- spots were labeled using biotinylated IFN- mAb, detected using Avidin–HRP (BD PharMingen, San Diego, CA, USA) and spots developed using Vectastain AEC substrate (Vector Laboratories, Burlingame, CA, USA).
CFSE assay
PBMCs were incubated in 24-well plates (5 x 106 cells per well) for 1 h. Adherent cells were used as APCs. CD4+ T cells were isolated from the non-adherent fraction by negative selection using the Miltenyi CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and labeled with 0.3 µM CFSE in PBS for 10 min at 37°C as described (11). Three million CFSE-labeled cells were plated over adherent APC and stimulated using 10 µg ml–1 of the TT peptide of interest. After 7 days, cells were stained with tetramer and surface antibodies and analyzed by flow cytometry to estimate the precursor frequency as described (11). Typically, the cells underwent eight to 10 divisions within the 7-day period.
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Results |
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Tetramer assay for TT830–844 specific cells
TT830–844 has been reported as a universal epitope presented by multiple alleles, including DR1101, DR0401 and DR1501 (1, 3). To detect TT830–844 specific T cells, PBMCs from DR0401, DR1101 and DR1501 subjects were stimulated with TT830–844 peptide and stained with the corresponding tetramers after 2 weeks of in vitro expansion. DR1101-restricted TT830–844 CD4+ T cells were readily detected (Fig. 1, panel 1), but DR0401- and DR1501-restricted TT830–844 T cells were not detectable (Fig. 1, panels 2 and 3). Similar results were observed for multiple subjects (data not shown). It is still possible that TT830–844 is presented by DR0401 and DR1501 and recognized by low-avidity T cells that could be detected using other methods. However, these results indicate that TT830–844 was not a high-avidity DR0401- or DR1501-restricted epitope for the individuals tested.
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DR0401-restricted TT epitopes
Motivated by this negative data, the TGEM approach was applied to identify DR0401-restricted epitopes within the 858 aa TT heavy chain. First, a panel of 106 peptides was designed (20 aa long with a 12 residue overlap) spanning the TT heavy chain. These peptides were divided into 21 pools of five (or six in pool 21) consecutive peptides. Enriched CD4+ T cells from DR0401 subjects were stimulated with peptide pools and analyzed using the corresponding pooled peptide tetramers after 2 weeks in culture. Peptide pools that were tetramer positive were analyzed a second time using individual peptide tetramers to reveal the antigenic peptides within each pool. Representative results from a single subject are shown in Figs 2 and 3. Positive staining was observed for nine peptide pools (Fig. 2) and these corresponded to 15 individual peptides (Fig. 3). Based on Tepitope-binding prediction (16, 17), p2 and p3 appear to contain an identical epitope (FIAEKNSFS). Similarly, consecutive peptides p20 and p21, p27 and p28, p58 and p59 and p86 and p87 all appear to contain a single epitope. TGEM was repeated for two additional DR0401 subjects with similar results (Table 1). Overall, at least 10 distinct DR0401-restricted epitopes were identified.
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Mapping of DR1501 TT epitopes
Using the peptide panel and TGEM approach described above, experiments were carried out for DR1501 subjects. Representative results from a single subject are shown in Figs 4 and 5. Positive staining was observed for four peptide pools (Fig. 4) and these corresponded to five individual peptides (Fig. 5). Based on binding prediction (16, 17), p23 and p24 appear to contain an identical epitope (IVPYIGPAL). TGEM was repeated for three additional DR1501 subjects, revealing additional epitopes within p97 and p99 (Table 1). Overall, at least six distinct DR1501-restricted epitopes were identified.
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Frequency of TT-specific T cells
ELISPOT assays or CFSE dilution experiments were used to estimate the precursor frequency for selected DR0401- and DR1501-restricted epitopes. These results are shown in Table 2. Generally, observed frequencies were <1 in 9000 CD4+ T cells. Apparently, there is no predominant DR0401 TT epitope, although TT610–629 does appear at a higher frequency compared with the other DR0401-restricted epitopes tested. Similarly, the frequencies of TT634–653 specific T cells were higher in all subjects compared with other DR1501-restricted epitopes.
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TT epitopes presented by additional class II alleles
As described above, the TGEM approach was used to identify TT epitopes presented by eight additional class II alleles: DR0101, DR0301, DR0404, DR0701, DR1101, DRB5, DQ0602 and DP0401 for multiple immunized subjects. These data are summarized in Tables 1 and 3
. For experiments on subjects with identical haplotypes (Table 3), we observed that some TT epitopes were shared by all subjects studied, while other epitopes were present in a smaller subset of the subjects. In total, we observed 14 antigenic peptides for DR0101 (containing 
10 distinct epitopes), five for DR0301 (4 epitopes), seven for DR0404 (5 epitopes), nine for DR0701 (8 epitopes), 14 for DR1101 (8 epitopes), eight for DRB5 (6 epitopes), seven for DQ0602 (6 epitopes) and one for DP0401.
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Unique and promiscuous TT epitopes
As summarized in Table 3, most of the TT-derived antigenic peptides were presented by 1 or 2 of the 10 alleles studied. However, there were some exceptions. For example, TT506–525, TT826–845 and TT1226–1245 were presented by three alleles; TT586–605, TT666–685 and TT1234–1253 by four alleles and TT674–693 by six alleles. Because each of these peptides is 20 residues in length, it is possible that some may contain multiple epitopes while others contain a single epitope that is truly promiscuous. To address this issue, we examined TT506–525, TT586–605 and TT674–693 using epitope prediction software and synthesized truncated peptides to discriminate between possible antigenic regions. TT506–525 is presented by DR0301, DR0401 and DR0404. Based on binding prediction, the truncated peptide TT511–521 should bind to these three alleles. This peptide was synthesized and used to make tetramers. As shown in Fig. 6(A), TT511–521 tetramer-positive responses were observed for TT506–525 stimulated T cells from DR0301, DR0401 and DR0404 subjects, indicating a single promiscuous epitope presented by DR0301, DR0401 and DR0404. TT586–605 is presented by DR0101, DR0401, DR0701 and DR1101. Three truncated peptides, TT586–598, TT591–603 and TT593–605, were synthesized and used to make tetramers. As shown in Fig. 6(B), following stimulation with TT586–605, TT591–603 tetramer-positive cells were observed for all four alleles while TT593–605 tetramer-positive cells were only observed for DR0101 and DR0401. No TT586–598 tetramer-positive responses were observed. These results suggest that DR0701 and DR1101 recognize an epitope that is found only within TT591–603 (most likely IYSYFPSVI), while DR0101 and DR0401 recognize an epitope that is found within both peptides (most likely YSYFPSVIS). It could be argued that DR0101, DR0401, DR0701 and DR1101 all recognize the same core epitope and that the observed differences in recognition are due to ‘end effects’ caused by different N- or C-terminal extensions of the peptide as bound by the MHC groove (18, 19). This would imply that the N-terminal KI extension is required for recognition by DR0701 and DR1101, but not by DR0101 and DR0401. However, the most straightforward interpretation is that these results indicate that TT586–605 does not contain a single promiscuous epitope. TT674–693 is presented by six different alleles, including DR0101, DR0401, DR0404 and DQ0602. Three truncated peptides, TT674–686, TT678–690 and TT680–692 were synthesized and used to make tetramers. As shown in Fig. 6(C), following stimulation with TT674–693, TT674–686 tetramer-positive cells were observed for DR0101 and DR0401 while TT680–692 tetramer-positive cells were observed for DR0404 and DQ0602. No tetramer-positive cells were observed for TT678–690. These results indicate that DR0101 and DR0401 recognize an epitope within TT674–686 (most likely YIPEITLPV), while DR0404 and DQ0602 recognize an epitope within TT680–692 (most likely VIAALSIAE). These results indicate that TT674–693 contains multiple epitopes rather than a single promiscuous epitope. Taken together, these results demonstrate that an apparently promiscuous peptide can contain either a single promiscuous epitope or a cluster of epitopes.
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Discussion |
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The idea that TT830–844 is a universal TT epitopes is widely held. However, difficulty in detecting both DR0401- and DR1501-restricted TT830–844 specific T cells prompted us to rigorously examine the TT CD4+ T-cell epitopes for several prevalent class II alleles. Using a panel of 106 overlapping 20mer peptides derived from the TT heavy chain, we applied the method of TGEM to identify TT-specific epitopes and their class II MHC restriction for 10 different class II alleles. In total, 52 different antigenic peptides (containing
36 distinct epitopes) were identified, including many previously reported epitopes such as TT593–599, TT616–631, TT632–651, TT652–663, TT830–844, TT916–932, TT947–969 (1–5, 20) and a substantial number of novel epitopes (see Table 3). The observation that nearly half of the TT peptides were antigenic illustrates the wide diversity of epitopes presented by various class II alleles. Overall, the vast majority (44 of 52) of the observed antigenic peptides contained epitopes for only one or two class II alleles. Although TT632–651, TT830–844 and TT946–968 have been reported as highly promiscuous epitopes, responses to these epitopes were only observed in two, three and one alleles, respectively, of the 10 alleles tested. TT632–651 is presented by DP0401, an allele that is estimated to be present in 20–40% of the Caucasian population (21). The prevalence of DP0401 may confer a degree of promiscuity, as observed in the previous reports. It is also possible that the discrepancy between the observation of this study and the previous results is due to the differences in experimental methodology since tetramers detect primarily high-avidity T cells capable of expansion in culture while other methods may be more sensitive to low-avidity T cells. As such, tetramers may fail to detect some low-avidity epitopes that would be seen using other methods. However, epitopes that elicit high-avidity responses and robust T-cell expansion are likely to be the most functionally relevant. Taken at face value, the diversity of epitopes observed for the 10 class II alleles studied demonstrates that the selection of peptides for antigen presentation is HLA dependent. This dependence can be illustrated by comparing epitopes restricted by DR0401 and DR0404. These closely related alleles differ only at residues 71 and 86 of their ß1 domains (lysine and glycine, respectively, for DR0401 and arginine and valine, respectively, for DR0404). Residue 86 influences the size of binding pocket 1. As such, DR0401 can be expected to accommodate bulkier residues in the pocket 1 position than DR0404. Of the 15 DR0401-restricted TT peptides identified in this study, only three contained epitopes shared by DR0404: TT506–525, TT914–933 and TT922–941 (TT674–693 was antigenic for both alleles, but contains two distinct epitopes—Fig. 6C). These shared epitopes contain smaller hydrophobic residues such as isoleucine at position 1 (based on motif prediction). In contrast, the non-shared epitopes, such as TT586–605, TT618–637, TT666–685, TT1122–1141 and TT1138–1157, contain bulkier hydrophobic residues such as tyrosine and phenylalanine at position 1 (based on motif prediction), which are presented by DR0401 and not by DR0404.
While most (85%) of the antigenic peptides observed in this study were presented by only one or two alleles, a few were presented by 3, 4 or even 6 of the 10 alleles tested. Further study (Fig. 6) revealed that these apparently promiscuous peptides fall into two categories. Some peptides, such as TT506–525, contain a single minimal epitope (TT511–521) that can be presented by multiple alleles. Other peptides, such as TT674–693 and TT586–605, contain more than one distinct epitope in close proximity. Clearly, the former represents a truly promiscuous (although not universal) epitope while the latter merely represents a densely antigenic region. It has been previously observed that DR0101, DR0401 and DR0701 exhibit similar peptide-binding preferences (22). In this study, DR0101 and DR0701 had three common epitopes, DR0101 and DR0401 had four common epitopes and DR0401 and DR0701 had two common epitopes while the remaining epitopes were not shared. As such, there are important MHC-based differences in epitope preference even between alleles with similar binding preferences.
Class II haplotype plays an important role in determining the TT-specific T-cell repertoire. However, examining the frequency of response to TT-specific epitopes among subjects with identical HLA haplotypes (Table 1), some epitopes appear to be more prevalent than others. For example, TT634–653 and TT1186–1205 were observed for all DR1501 subjects studied while TT1242–1261 was unique to a single individual. Although the number of subjects examined was limited, these differences suggest individualized intrinsic and extrinsic factors that influence the T-cell repertoire. The intrinsic factors that shape the immune repertoire may include genetic polymorphisms of antigen-processing genes and other MHC genes. Exposure to other micro-organisms and propinquity of vaccination should be the major extrinsic factors modulating the T-cell repertoire.
Most likely, the large number of antigenic peptides observed accounts for the almost universal immunogenicity of TT. Because most individuals are heterozygous in their MHC alleles, a given individual can be expected to recognize an extensive set of TT epitopes. For example, a DR0101/DR0401 subject could recognize epitopes within up to 22 of the 106 TT peptides. Taking into account non-DRB1 alleles, a DR0101/DR1501–DRB5–DQ0602 subject could recognize epitopes within up to 28 of the 106 TT peptides. Although tested for only a small subset of epitopes, the observed frequency of TT-specific CD4+ T cells for a specific epitope was <1 in 10 000. Thus, the robust CD4+ T-cell responses toward TT as observed are not due to any particular epitope, but rather are the summation of responses toward the multiple antigenic epitopes within the TT. Taken together, these findings suggest that class II-restricted tetanus toxoid responses are diverse and polyclonal.
As these epitope-mapping studies were carried using peptides rather than protein, it is possible that some of the epitopes identified are not naturally processed. This phenomenon has been seen in other studies. For example, Raju et al. (23) compared the TT-specific T-cell responses of PBMC stimulated using peptide pools with PBMC stimulated using whole tetanus toxoid. The majority of the epitopes they identified by peptide stimulation was also identified by protein stimulation, confirming that these were naturally processed. However, 7 of the 19 peptides were not recognized by T-cell lines propagated by tetanus toxoid protein. It should be noted that the T-cell lines used in this study were assayed following multiple cycles of peptide stimulation. As such, some of the original specificities may have been lost during expansion, leading to a low estimate of the reliability of these epitopes. In contrast, the TGEM approach monitors responses after a single cycle of peptide stimulation. It is also notable that T-cell responses toward some of the TGEM epitopes were detected ex vivo by ELISPOT. Epitopes detected in this manner are more likely to be bona fide epitopes and imply a protective response. Although we cannot rule out the possibility that some of the epitopes identified in this study are cryptic epitopes, it seems likely the majority is genuine TT CD4+ epitopes.
In conclusion, the results from this study indicated that the TT heavy chain contains numerous (at least 36) immunogenic epitopes within 52 of the 106 peptides tested. To date, there have been several comprehensive studies of complex viral antigens (24–26). However, to our knowledge, this work represents one of the most extensive and thorough investigations of T-cell epitopes and their MHC class II restriction within a complex antigen for multiple alleles in humans. The observed epitopes occur across the entire TT sequence and are recognized by high-avidity T cells in an MHC-specific fashion, as 85% of the antigenic peptides were presented by only one or two class II alleles. Surprisingly, even closely related class II alleles (such as DR0401 and DR0404) and alleles with demonstrated similarities in binding preference (such as DR0101, DR0401 and DR0701) exhibited clear differences in their recognition of TT epitopes. Although several of the TT peptides in this study were presented by multiple class II alleles, some of these contained a cluster of distinct epitopes rather than one truly promiscuous epitope. For individuals with identical class II haplotypes, response frequencies toward a specific epitope varied, implying the existence of both rare and prevalent epitopes. Together, these results contrast with the perceived notion that tetanus toxoid responses are dominated by a few universal T-cell epitopes. Indeed, these results clearly demonstrate that the heterogeneous-binding preference of highly polymorphic MHC proteins imposes a strenuous constraint limiting the likelihood that any peptide could be presented as a truly universal epitope. These results also imply significant obstacles for the design of peptide vaccines and for the rational modification of protein therapeutics to reduce their immunogenicity.
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Acknowledgements |
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The administrative support of Diana Sorus is appreciated. This work was in part supported by NIH contract HHSN266200400028C.
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Abbreviations |
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| ELISPOT, enzyme-linked immunosorbent spot |
| aa, amino acid |
| TGEM, tetramer-guided epitope mapping |
| TT, tetanus toxoid |
| APCs, antigen presenting cells |
| CFSE, carboxyfluoroscein succinimydal ester |
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Notes |
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Transmitting editor: T. F. Tedder
Received 24 April 2007, accepted 31 August 2007.
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