| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
Departments of Immunology [C. S. G., E. R., P. L. A.] and Haematology [L. F., J. A. M.], Royal Free and University College Medical School, London NW3 2PF, United Kingdom
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
secretion greater than that seen with non-self peptides, whereas none of five patients with progressive disease responded to their own lymphoma Id. We have shown that mutated regions of lymphoma Id contain MHC class I binding peptides that are potential targets for cytotoxic T cells. Immunotherapy using the tumor-specific mutated regions from lymphoma Id avoids the need to break innate tolerance toward the germ-line protein sequences present on normal and malignant B cells. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immunization with idiotypic protein and adjuvant induces anti-idiotypic antibodies in both mice (8) and humans in the absence of clinically detectable lymphoma (9) , indicating the immunogenic potential of Ids. The production of anti-idiotypic antibodies has been shown to mediate protection against tumor challenge in mouse models (10) . Alternatively, immunization with idiotypic proteins or DNA vaccines without adjuvant produced low levels of idiotypic antibody and poor protective immunity in mice and humans (10 , 11) . Addition of an adjuvant, for example tetanus toxin fragment C, to DNA encoding the idiotype induced higher levels of idiotypic antibodies in mice (12) , and this approach is currently being tested in humans. Although the vast majority of studies have used anti-idiotype antibody as an indicator of immune response, more recent work has focused on the generation of specific cellular immunity. Immunization of follicular lymphoma patients with idiotypic protein plus granulocyte/macrophage-colony stimulating factor, induced secretion of tumor necrosis factor from postvaccine PBMCs, shown to be mediated by CD4+ and CD8+ T cells (13) . Specific lysis of autologous tumor cells was also observed and was blocked by antibodies against MHC class I, indicating the role of CD8+ cytotoxic T cells. Immunization of lymphoma patients with idiotype protein-pulsed autologous dendritic cells induced cellular proliferative responses rather than humoral immunity (14) . A large body of evidence favors cell-mediated rather than antibody control of tumors, and this is supported by studies in melanoma (15 , 16) and virally induced tumors (17) .
The increased knowledge of epitopes within tumor-associated proteins recognized by CTL and bioinformatics enabling prediction of peptide binding to HLA class I have provided tools with which to search for the existence of CTLs and immunogenic peptides within tumors from patients with malignancy (16 , 18 , 19) . This approach has also been used to monitor CTL responses in patients infected with viruses such as cytomegalovirus and EBV (20 , 21) . A recent study by Trojan et al. (22) described HLA-A2-restricted peptide epitopes from the tumor-derived immunoglobulin variable region from patients mainly with CLL. Some of the peptides were shared among patients, indicating their unmutated, germ-line nature. The authors described the in vitro generation of donor and some patient CTLs against the peptides and reported low-level killing of CLL tumor cells.
The aims of our study were to define peptide sequences from somatically mutated regions in lymphoma immunoglobulin Ids capable of binding to the patient’s own HLA class I molecules and hypothetically with a greater immunogenicity, because the mutated regions should not be regarded as self. The mutant cell line, T2, provided a means of testing peptide binding to the HLA-A2 allele by the ability to up-regulate and stabilize cell surface expression of HLA-A2 peptide complexes (23)
. In addition, peptides of lymphoma Ids from patients with HLA-A3 were tested on a T2 cell line transfected with HLA A3 (24)
. Selected peptides restricted by A1, A2, A3, and A11 were also tested for binding in a competitive inhibition assay using either LCLs derived from the patients themselves or LCLs homozygous for the HLA type of interest. Finally, stimulation of patient lymphocytes by their own idiotypic peptides was examined in ELISPOT assays. The demonstration of frequent self-HLA binding lymphoma idiotypic peptides and lymphocytes from lymphoma patients in long-term remission capable of reacting to idiotypic peptides by IFN- secretion suggests that mutated idiotypic peptides provide a target for control of lymphomas by cell-mediated immune responses.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Genomic DNA Isolation.
Diagnostic biopsies from 45 patients with B-cell malignant lymphomas were processed for DNA extraction using Proteinase K and a Puregene kit (Flowgen, Lichfield, Staffordshire, United Kingdom). Patients were assessed for IgH clonality by Southern blotting and hybridization to a JH probe, as described previously (26
, 27)
. Patients with clonal JH pattern were processed for amplification of the rearranged IgH genes as detailed below.
PCR.
PCR was performed using the manufacturer’s instruction (Promega, Southampton, United Kingdom) in a 30-µl volume containing 1 µg of genomic DNA and 100 pM of each primer. The forward primers were the family-specific VH1–6 leader primers (28
, 29)
, and the reverse primer was a JH consensus (30)
. Restriction sites were added to facilitate cloning. Conditions of amplification were as follows: 1 cycle for 5 min at 94°C, followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 66°C, and 2 min extension at 72°C, with a final cycle of 10 min extension at 72°C. The amplified PCR products were purified using a Hybaid recovery kit (Hybaid Ltd., Teddington, Middlesex, United Kingdom), cloned into Bluescript KS+ vector (Promega) and DNA, obtained using Qiagen miniprep kit (Qiagen, Crawley, West Sussex, United Kingdom), and sequenced using an ABI 377 Sequencer.
RT-PCR.
RNA was prepared using the guanidine isothiocyanate method (31)
and reverse transcribed as described previously (32)
, and IgH rearrangements were amplified using antisense primers from IgM, IgG, and IgA constant regions (33)
and VH leader primers (as described above). All immunoglobulin sequences were submitted to the IMGT/DNAPLOT website (34)
for DNA and protein comparison to germ-line sequences.
Peptides.
VDJ protein sequences were searched for nonamer or decamer peptides that conformed to the binding motif of the patients’ HLA type by submitting the protein sequence to online databases, which predict binding peptides and score them accordingly (19
, 18) . We were also influenced by published literature regarding anchor and secondary residues (35, 36, 37, 38)
. The majority of peptides selected covered an area of somatic hypermutation or spanned a VD or DJ junction. Peptides were synthesized to 95% purity (MWG-Biotech UK Ltd., Peartree Bridge, Milton Keynes, United Kingdom). They were supplied lyophilized and then redissolved in DMSO and stored at -70°C at a concentration of 25 mM.
T2 Assay.
T2 cells were cultured in RPMI1640 + 10% FCS and grown in 5% CO2 at 37°C. Peptides from HLA-A2-positive patients were assayed for their ability to bind to and stabilize A2 molecules on T2 cells (39)
. Cells (1 x 105) in a volume of 100 µl of RPMI 1640 (serum free) were aliquoted into 96-well, U-bottomed plates and incubated with peptide at a final concentration of 100 µM plus 5 nM ß2 microglobulin (Cymbus Biotechnology Ltd., Chandlers Ford, Hampshire, United Kingdom) for 18 h at 37°C in 5% CO2. The level of stabilized HLA-A2 on the surface of the T2 cells was detected using the pan HLA class I monoclonal antibody W6/32 (European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury) that recognizes stabilized HLA-A2 complexes (40)
. This was detected using a goat antimouse, FITC-conjugated IgG (Cambridge Biosciences, Cambridge, United Kingdom) as the second layer. Samples were fixed in 1% paraformaldehyde in PBS prior to analysis and analyzed on a Becton Dickinson FACScan. The fluorescence ratio = MFI of the test peptide/MFI of DMSO control.
The T2 cell line transfected with HLA-A3 (24) allowed analysis of peptides from HLA-A3-positive patients. Cells (1 x 105) in a volume of 100 µl of RPMI 1640 (serum free) were incubated with 100 µM peptide for 18 h at 26°C in 5% CO2. Binding of peptides was detected as described above, except that a monoclonal anti-HLA-A3 antibody, GAP-A3, was used (ECACC).
EBV Transformation.
PBMCs were isolated from patients’ blood using Lymphoprep (Nycomed Pharma AS, Oslo, Norway). EBV transformed B lymphoblastoid lines (B-LCL) were created as described previously (41)
.
Competition Assay.
The competition assay was carried out as described by van der Burg et al. (42)
. In brief, LCLs that were homozygous for either A1, A2, A3, or A11 (cell lines JY, HOM-1, VAVY, and FB) or the patient’s own B-LCL were washed twice in PBS and left to rest as a pellet on ice for 5 min. The cells were then acid stripped for 90 s using ice-cold Na2HPO4 buffer, pH 3.1 for A1, pH 3.2 for A2, and pH 2.9 for A3 and A11. The cells were buffered with ice-cold Iscove’s modified Dulbecco’s medium (ICN Flow, Basingstoke, Hampshire, United Kingdom), and then washed and resuspended at 1 x 106/ml in Iscove’s modified Dulbecco’s medium plus 7.5 µg/ml ß2-microglobulin (Cymbus Biotechnology Ltd.). Twenty-five µl of fluorescent-labeled reference peptide [HLA-A1, YLEPACFITCAKY; HLA-A2, FLPSDCFITCFPSV; HLA-A3/11, KVFPCFITCALINK (Department of Immunohematology and Blood Bank, Leiden University Medical Center)] at a final concentration of 133 nM were incubated with 25 µl of Id competitor peptide (MWG-Biotech) at final concentrations of 0.5–33.3 µM in a 96-well, U-bottomed plate with 100 µl (1 x 105) of cells for 24 h at 4°C (42)
. The percentage inhibition demonstrated by immunoglobulin peptides competing with the FITC-labeled reference peptide was calculated as described (42)
.
ELISPOT Assay for IFN-.
PBMCs (1 x 105) were plated in triplicate in round-bottomed, 96-well plates and stimulated for 18 h with 50 µg/ml peptide at 37°C in 5% CO2. Culture medium was replaced following the 18-h stimulation, and cells were mixed, aliquoted onto the ELISPOT capture plate (Immunodiagnostic Systems Ltd., Bolden, Tyne and Wear, United Kingdom), and incubated for an additional 20 h. IFN- spots were detected according to the manufacturer’s instructions.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), indicating a monoclonal population with a biallelic rearrangement, which is commonly associated with an unproductive rearrangement on one allele. In 3 of 45 patients, more than two rearranged bands or two rearranged bands of unequal intensity (referred to as R and r in Table 1
) were identified, which is most likely explained by the presence of more than one clonal population, referred to as oligoclonality, a frequent finding in B-cell acute leukemias (43)
but infrequent in lymphomas (44)
. In the remaining 3 patients, Southern blotting analysis was not carried out, but evidence of a clonal B-cell population was provided by immunophenotyping (data not shown).
|
With the exception of 3 cases, somatic mutations within the VH sequence resulted in between 1 and 37 amino acid replacements. The highest replacement frequency was seen in DLBL and FCL (Table 1)
.
Peptide Searches.
Immunoglobulin sequences from those patients who were HLA-A1, HLA-A2, HLA-A3, or HLA-A11 positive were searched for nonameric and/or decameric peptides that carried the appropriate binding motif (18
, 19
, 36, 37, 38
, 45, 46, 47, 48, 49)
. The peptides were analyzed for anchor, preferred, and deleterious residues. Forty-two peptides restricted by HLA-A1, HLA-A2, HLA-A3, or HLA-A11 were selected, 39 of which either contained at least one amino acid change attributable to somatic hypermutation or covered a VD or DJ junction, making them tumor specific (Tables 2
and 3
). The remaining 3 peptides (MP1, WF1, and GW3) were germ line in nature. Tables 2
and 3
show that 67% of peptides with the potential to bind to A1, A2, A3, and A11 were located in the FR, and only 33% were in overlapping FR and CDR regions.
|
|
and 2
show the shift in mean fluorescence ratios of peptide binding to T2-A2 or T2-A3 cells. The influenza matrix peptide is known to bind with high affinity to HLA-A2 (38)
and gave a MFI ratio of 2.31 in our experiments. Peptides with a MFI ratio >1.5 defines high affinity binding to HLA-A2. Altogether, 45% of the peptides selected for HLA-A2 binding motifs demonstrated binding in the T2 assay, of which 24% (7 of 29) were high affinity and 21% (6 of 29) bound with intermediate affinity (MFI ratio of 1.25–1.5). Addition of 5 nM ß2-microglobulin to the cells in serum-free medium when incubated with peptide increased the sensitivity and reproducibility of the T2-A2 assay. We also found that because of the hydrophobic motif preferred by HLA-A2, many of the peptides were insoluble in water and had to be dissolved in DMSO. We observed precipitation of peptides when incubated with cells in RPMI 1640, which may be a limitation in this assay because insolubility may impair binding.
|
|
The positive control for the HLA-A3 assay was also an influenza derived peptide, shown previously to bind well to A3 (45) . Various conditions were tested when optimizing this assay. Although it was found to work under the conditions used for T2-A2, in contrast to T2-A2 the MFI ratios increased considerably when incubated at 26°C in serum-free medium without the addition of ß2-microglobulin. Seventy-five % (3 of 4) of immunoglobulin peptides were found to increase the MFI ratio, and two of them bound with equivalent affinity to the positive influenza control.
Competition Assay.
Fig. 3
provides a representative example of inhibition of the FluFITC peptide by Id peptides in the competition assay. The IC50s (concentration of Id peptide at which the FL-labeled peptide is inhibited by 50%) for A2 and A3 peptides were calculated and are shown in Fig. 4
. Sixteen peptides that bound in the T2 assay (A2 and A3) were tested in the competition assay. Eight of 16 (50%) had IC50 <5 µM, comparable with known CTL epitopes; 1 of 16 (6%) had IC50s between 5 and 15 µM and would be considered a candidate CTL epitope (42)
; 3 of 16 (19%) competed with IC50 >15 µM, and 4 of 16 (25%; RH3, FA2, GS1, and WF1) did not compete at all. We also selected A1- and A11-restricted Id peptides based on the same selection procedure as for A2 and A3 peptides and tested them in the competition assay (data not shown). Similar results were gained with 2 of 5 (40%) A1 peptides and 2 of 4 (50%) A11 peptides competing with IC50 <5 µM. The IC50s of peptides with high (>1.5) T2 binding affinities were significantly lower than those with low (<1.5) binding affinity (P = 0.02 by Mann-Whitney U test), indicating agreement between these two assays. Because of the highly polymorphic nature of the HLA genes, the patient’s own HLA proteins may have slight differences to those HLA proteins presented by the unrelated homozygous LCLs (JY, HOM-1, VAVY, and FB). To investigate this, we created EBV-transformed B-cell lines from patients’ peripheral blood and used these in the competition assay. Nine peptides (HLA-A1, HLA-A2, HLA-A3, and HLA-A11) were tested on both unrelated and patients’ own cell lines. IC50 data were similar for both LCLs and are shown in Fig. 5
. Peptides MM1 and TL5 did not show binding to the unrelated LCLs but showed low affinity binding (IC50 = 20.01 and 27.35, respectively) to the patient’s own cell line (not shown in Fig. 5
).
|
|
|
.
and 5
show the number of IFN--producing cells/105 PBMCs in response to different peptides. PBMCs were stimulated with peptides derived from the patient’s own lymphoma Id, HLA-matched Id peptides derived from other patients’ lymphomas, and influenza or EBV peptides. PBMCs from 3 patients (JD, SJ, and MM) responded to stimulation with their own lymphoma Id peptides (JD2, SJ1, and MM1) above any response toward non-self Id peptides. Both JD and SJ have been in complete remission from follicle center lymphoma for 3 and 10 years, respectively. Patient MM currently has stable follicle center lymphoma with a few small neck nodes (
1 cm). PBMCs from the other 7 patients did not respond to self-Id above responses made to non-self-Id peptides, and this included 1 in complete remission of 10 years duration (SP), 1 with stable disease (3-cm neck node), and 5 with progressive disease at the time of testing. Clinical details of the patients tested in this assay are shown in Table 4
.
|
|
-producing cells ranged from 5 to 65 per 105 PBMCs, with some of the Id peptides producing higher responses than those seen with viral peptides. However, the HLA-A2-negative donors did not respond to the A2 peptides, supporting the HLA-restricted nature of the responses. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Peptides were chosen on the basis of scores predicted by two online databases and published literature. Peptides without anchor residues at positions 2 and 9 had low scores and did not bind in the T2 assay. The use of different peptide scoring algorithms, which are based on natural ligands (18 , 19 , 36) or the dissociation kinetics of HLA complexes (19) , provides independent evaluation of peptide binding. The database predictions for any one peptide do not always agree, and even when they do correlate, they do not guarantee binding for in vitro assays. Prediction of peptide binding is complex because it depends on both the presence of anchor residues and the influence of secondary residues at other sites (52) , and it is unlikely that peptide binding databases are able to predict all potential epitopes. However, detection of all binding peptides from the Id sequences would require analysis of a panel of overlapping peptides, which would be very labor intensive. Peptide prediction databases reduce the time taken to analyze protein sequences for the presence of potential epitopes, and the accuracy of prediction will increase as our knowledge of binding peptides expands. The results from this study have shown that high binding scores with databases using different peptide binding algorithms increased the accuracy of prediction. Using only a single MHC class I allele, HLA-A2, about half of the selected peptides were capable of binding, and a quarter of them had binding affinities in the range known to make good viral CTL epitopes (42) . The same is true for other class I alleles, A1, A3, or A11, suggesting that an effective cytotoxic target is achievable from almost any lymphoma Id sequence.
The range of MFI ratios obtained using T2-A3 cells were of a much higher magnitude than those gained using T2-A2 cells. HLA-A2 molecules on the surface of T2 cells are reported to be at least partially occupied with signal peptides derived from normal cellular proteins, such as IP-30 and calreticulin (53, 54, 55) . The resulting high background fluorescence accounts for the differences in MFI ratios.
There was a statistical correlation between peptide binding in the T2 assay and IC50 data from the competition assay, although there were some discrepancies in the results. For example, ME2 showed a higher IC50 in the competition assay but proved to be a high affinity binder in the T2 assay. This may be because of minor differences in the HLA-A2 molecules presented by the LCL and T2 cell lines. The T2 assay relies on stabilization and up-regulation of HLA on the cell surface, and although this does not yield quantitative data, it provides an indication of the ability of a peptide to bind. The competition assay allows the semiquantitative determination of peptide binding, the kinetics of which are comparable with that of soluble HLA class I molecules (42)
. Another advantage of the competition assay is that the patients’ own EBV-transformed B cells can be used instead of a cell line. The HLA locus is highly polymorphic, and although peptides from a patient may bind to the HLA on a cell line, single amino acid differences in their own HLA molecules may prevent binding. We demonstrated similar binding characteristics for all of the peptides tested with HLA class I homozygous lines and the patients own EBV-transformed cell line (Fig. 5)
.
Virtually all (39 of 42) lymphoma Id peptides analyzed by in vitro HLA-binding assays in this study carried amino acid changes because of somatic hypermutation or covered a junctional region (Tables 2
and 3
) and were therefore tumor specific. We were interested to establish whether the numbers of binding peptides differ as a result of mutation. The germ-line counterparts of the variable genes used in the immunoglobulins studied were scanned for potential binding peptides using the scoring systems and databases described earlier. Similar numbers of HLA-binding motifs were found in both mutated and germ-line immunoglobulin variable segments. In some cases, peptide binding motifs were lost because of a mutation that introduced an amino acid into a position affecting binding adversely, and in other cases existing peptides had their scores increased when a mutation inserted a favorable amino acid.
Finally, three patients had blood lymphocytes that made low magnitude responses to their lymphoma Id peptides by secretion of IFN-, suggesting previous sensitization to these determinants. Patients SJ, JD, and MM made responses to peptides SJ1, JD2, and MM1, respectively. Peptides SJ1 and MM1 showed binding in the peptide binding assays; however, peptide JD2 showed no binding on either unrelated or autologous LCLs. HLA class I molecules are known to present peptides of fewer than 9 amino acids (56
, 57) ; therefore this peptide may have been further processed before presentation. Two of three of these patients were in complete remission from their disease, and the third had detectable but low-volume stable disease. This is consistent with murine models of immunotherapy where immunization was successful in mice free of lymphoma or with minimal disease but not where the lymphoma was of large volume or progressing (58)
. All patients in this study with progressive lymphoma made no or very low responses to their lymphoma Ids, whereas they were often capable of reacting to viral or unrelated lymphoma Id peptides, indicating their immunocompetence in this respect. Patients’ PBMCs were also stimulated with a range of non-self peptides, some of which showed binding in the peptide assays and some of which did not. Six patients made responses to non-self-Id peptides, including 12 of 19 responses to peptides that did not bind in the peptide binding assays. The reasons for this are unclear but may be attributable to binding to other HLA class I molecules expressed by the patients’ cells or further processing of the peptides. Reactivity with non-self, HLA-matched Id peptides may indicate cross-reactive T-cell responses to similar epitopes encountered previously by the immune system, as illustrated by the recognition of a myeloma-specific peptide by CTLs specific for an influenza epitope (59)
. These responses are similar to the generation of CTLs from HLA-matched donors against lymphoma immunoglobulin peptides, described recently (22)
. It could be argued that the autologous responses made to self-Id peptides are from cross-reacting T cells, but if these T cells could be amplified by immunotherapy, they may provide useful responses in vivo. The absence of an immune response to self-Id peptides in those patients with progressive disease is likely to indicate the induction of anergy in responding T cells, as shown in metastatic melanoma (16)
.
In a recent study by Trojan et al. (22) , HLA-A2-restricted, unmutated germ-line peptides from the immunoglobulin V regions of patients with B-cell malignancies could bind in the T2 assay, also demonstrated by peptide WF1 in this study. They showed that a small proportion of these peptides could stimulate the expansion of CTLs from healthy donors and a few patients after repeated rounds of in vitro stimulation. Many of the peptides were shared among patients, indicating their germ-line nature. Unmutated peptide epitopes of this nature will be present not only in the lymphoma immunoglobulin but also within the V regions of normal B cells, making it a tumor-associated antigen rather than tumor-specific antigen. Immunotherapeutic targeting of these epitopes has the potential to destroy normal B cells but to do so would need to break tolerance to self-antigens in vivo. Id peptides covering mutated regions are tumor specific and more likely to be immunogenic because there is no need to break self-tolerance. Support for this position was demonstrated recently where unmutated immunoglobulin in CLL had a more aggressive course than those in which the immunoglobulin had undergone somatic mutation (60) .
On the basis of murine studies, lymphoma immunotherapy for over a decade has concentrated on antibody-mediated immunity (9 , 61) , and attempts at peptide therapy were ineffective in mice (62) . However, the experiments performed at that time were relatively crude, and the peptide binding motifs for murine MHC were unknown. Recently, more promising immunotherapy of lymphoma and other malignancies has been achieved using dendritic cells to immunize patients (14) . The efficacy of dendritic cells to prime CTLs may be the means by which immunotherapy has improved in this disease. The identification of CTL epitopes in melanoma and graft versus host disease has enabled MHC:peptide tetramer staining of specific CTL populations during disease progression (16 , 63) , providing invaluable data on the immune response. Our current research is involved with synthesizing lymphoma Id peptide tetramers to determine whether we can detect specific CTLs in peripheral blood or lymph node biopsies of these patients to substantiate the ELISPOT data.
We have shown that the somatically mutated regions of lymphoma Id from patients with non-Hodgkin’s lymphoma have the potential to be immunogenic, based on the presence of peptides with the ability to bind to the patients’ own HLA type and that patients in complete remission from their disease have memory T cells capable of being stimulated by their lymphoma Id peptides. These are important steps in defining immunotherapeutic targets for lymphoma.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by the Leukaemia Research Fund (to E. R.) and by the Marc Fisher Trust (to C. S. G.). 
2 To whom requests for reprints should be addressed, at Department of Immunology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF. Phone: 44-20-7472-6266; Fax: 44-20-7431-3416; E-mail: p.amlot{at}rfc.ucl.ac.uk
3 The abbreviations used are: Id, idiotype; PBMC, peripheral blood mononuclear cell; CLL, chronic lymphocytic leukemia; LCL, lymphoblastoid cell line; ELISPOT, enzyme-linked immunospot; RT-PCR, reverse transcription-PCR; MFI, mean fluorescence intensity; FR, framework region; CDR, complementarity-determining region; DLBL, diffuse large B-cell lymphoma; FCL, follicle center lymphoma.
Received 1/21/01. Accepted 5/ 1/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. M. Zirlik, D. Zahrieh, D. Neuberg, and J. G. Gribben Cytotoxic T cells generated against heteroclitic peptides kill primary tumor cells independent of the binding affinity of the native tumor antigen peptide Blood, December 1, 2006; 108(12): 3865 - 3870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhou, D. Xu, X. Li, H. Li, M. Shan, J. Tang, M. Wang, F.-S. Wang, X. Zhu, H. Tao, et al. Screening and Identification of Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific CTL Epitopes J. Immunol., August 15, 2006; 177(4): 2138 - 2145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-D. Wang, W.-Y. F. Sin, G.-B. Xu, H.-H. Yang, T.-y. Wong, X.-W. Pang, X.-Y. He, H.-G. Zhang, J. N. L. Ng, C.-S. S. Cheng, et al. T-Cell Epitopes in Severe Acute Respiratory Syndrome (SARS) Coronavirus Spike Protein Elicit a Specific T-Cell Immune Response in Patients Who Recover from SARS J. Virol., June 1, 2004; 78(11): 5612 - 5618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. von Bergwelt-Baildon, B. Maecker, J. Schultze, and J. G. Gribben CD40 activation: potential for specific immunotherapy in B-CLL Ann. Onc., June 1, 2004; 15(6): 853 - 857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Gallez-Hawkins, M. C. Villacres, X. Li, M. C. Sanborn, N. A. Lomeli, and J. A. Zaia Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response J. Virol., April 1, 2003; 77(7): 4457 - 4462. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, MA2; 
, ME1; *, SJ1; 
, SP2; 
, A2 Flu).
) Id peptides was calculated from the percentage inhibition of the labeled peptide demonstrated in the competition assay.