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Experimental Therapeutics, Preclinical Pharmacology |
1 Department of Pathology, Asahikawa Medical College, Asahikawa, Japan; and 2 Department of Immunology, Mayo Clinic, Rochester Minnesota
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Synthetic peptides prepared from several regions of the HTLV-I envelope (Env) sequence that were predicted to serve as helper T-cell epitopes were prepared with use of computer-based algorithms and tested for their capacity to trigger in vitro helper T-cell responses using lymphocytes from normal volunteers.
Results: The results show that the HTLV-I–Env317–331, and HTLV-I–Env384–398-reactive helper T lymphocytes restricted by HLA-DQw6 and HLA-DR15, respectively, could recognize intact HTLV-I+ T-cell lymphoma cells and, as a consequence, secrete lymphokines. In addition, HTLV-I Env196–210-reactive helper T lymphocytes restricted by HLA-DR9 were able to directly kill HTLV-I+ lymphoma cells and recognize naturally processed antigen derived from killed HTLV-I+ lymphoma cells, which was presented to the helper T cells by autologous antigen-presenting cells.
Conclusions: The present findings hold relevance for the design and optimization of T-cell epitope-based immunotherapy against HTLV-I–induced diseases such as ATLL.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although being an asymptomatic virus carrier is usually not a life-threatening situation, aggressive ATLL is difficult to treat and is usually fatal. Thus far, current prophylactic and therapeutic approaches for ATLL are far from optimal. Because of the limitations in the standard treatment of ATLL, much focus has been placed in the development of vaccine-based immunotherapy, but unfortunately, vaccines to induce HTLV-I–specific T-cell responses are not yet available.
It is known that both antibody and CTL responses can play a role in protection from viral infection and in eradicating viral-infected cells (9 , 10) . An attractive and relatively expedient approach to develop vaccines that are intended to elicit antigen-specific T-cell responses is the use of synthetic peptides representing CTL and helper T lymphocyte epitopes. This strategy has been explored for various viral and malignant diseases (11, 12, 13, 14) . A large number of T-cell epitopes derived from HTLV-I Tax protein have been identified, and most of these efforts have focused on MHC class I-restricted peptide epitopes to induce CD8+ CTL responses to HTLV-I because these cells are considered to be the prime effector cells that will presumably annihilate the virus-infected and -transformed cells (4 , 5 , 15, 16, 17) .
Recent experiments by a number of groups have demonstrated that CD4+ helper T lymphocytes play an important role in potentiating antiviral and antitumor cellular and humoral immune responses (18 , 19) . Moreover, in some instances, helper T lymphocytes can exhibit a direct effector function by directly recognizing and killing MHC class II+ virally infected or tumor cells that present helper T lymphocyte epitopes on their surface (20, 21, 22) . Because helper T lymphocytes play an important role both in the induction and maintenance of CTL responses, vaccines that activate both CTLs and helper T lymphocytes should be more effective than vaccines that only target CTL responses. However, there is limited information available regarding MHC class II-restricted T-cell responses against HTLV-I and the existence of the corresponding helper T lymphocyte peptide epitopes.
In the present study, we report the identification of MHC class II epitopes from the HTLV-I envelope (Env) glycoprotein that are capable of stimulating CD4+ T-cell responses from HTLV-I–naive individuals. These peptides, Env196–210, Env317–331, and Env384–398, induced HTLV-I–specific responses restricted by the HLA-DR9, HLA-DQw6, and HLA-DR15 alleles, respectively. Most importantly, some of the peptide-generated helper T lymphocytes were also capable of directly recognizing HTLV-I–infected, MHC class II+ T-cell lymphomas and naturally processed antigen in the form of cell lysates or apoptotic cells prepared from HTLV-I+ T cell lymphomas, which were indirectly presented by autologous antigen presenting cells (APCs). The present findings should hold some value for the development of epitope-based vaccines designed to elicit CTL and helper T lymphocyte responses for the treatment/prevention of ATLL and other diseases mediated by HTLV-I.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthetic Peptides.
Potential HLA-DR–restricted CD4+ T-cell epitopes were selected from the amino acid sequence of the HTLV-I–Env protein using the algorithm tables for three HLA-DR alleles (DRB1*0101, DRB1*0401, and DRB1*0701) described by Southwood et al. (24)
. The predicted peptide epitopes were synthesized by solid phase organic chemistry and purified by high-performance liquid chromatography. The purity (>80%) and identity of peptides were assessed by high-performance liquid chromatography and mass spectrometry, respectively.
In vitro Induction of Antigen-Specific Helper T Lymphocytes Lines with Synthetic Peptides.
The procedure selected for the generation of HTLV-I–Env-reactive helper T lymphocyte lines using peptide-stimulated PBMCs has been described in detail (25
, 26)
. Briefly, dendritic cells were produced in tissue culture from purified CD14+ monocytes (using antibody-coated magnetic microbeads from Miltenyi Biotech, Auburn CA) that were cultured for 7 days at 37°C in a humidified CO2 (5%) incubator in the presence of 50 ng/mL granulocyte macrophage colony-stimulating factor and 1000 IU/mL interleukin 4. Peptide-pulsed dendritic cells (3 µg/mL for 2 hours at room temperature) were irradiated (4200 rad) and cocultured with autologous-purified CD4+ T cells (Miltenyi Biotech) in 96 round-bottomed well culture plates. One week later, the CD4+ T cells were restimulated with peptide-pulsed irradiated autologous PBMCs, and 2 days later, human recombinant interleukin 2 was added at a final concentration of 10 IU/mL. One week later, the T cells were tested for antigen reactivity using a cytokine release assay as described below. Those cultures exhibiting a significant response of cytokine release to peptide (at least 2.5-fold over background) were expanded in 24- or 48-well plates by weekly restimulation with peptides and irradiated autologous PBMCs. Complete culture medium for all procedures consisted of AIM-V medium (Life Technologies, Inc., Rockville, MD) supplemented with 3% human male antibody serum. All blood samples were obtained after the appropriate informed consent.
Measurement of Antigen-Specific Responses with Helper T Lymphocytes.
CD4+ T cells (3 x 104/well) were mixed with irradiated APCs in the presence of various concentrations of antigen (peptides, tumor lysates, and apoptotic cells) in 96-well culture plates. APCs consisted of either autologous PBMCs (1 x 105/well), HLA-DR–expressing L cells (3 x 104/well), MHC-typed EBV-LCLs (3 x 104/well), T-cell lymphoma cell lines (3 x 104/well), or autologous dendritic cells (5 x 103/well). Tumor cell lysates were prepared by three freeze-thaw cycles of 1 x 108 tumor cells and resuspended in 1 mL of serum-free RPMI 1640. Lysates were used as a source of antigen at 5 x 105 cell equivalents per mL. Culture supernatants were collected after 48 hours for measuring antigen-induced lymphokine (granulocyte macrophage colony-stimulating factor or IFN-
) production by the helper T cells using commercially available ELISA kits (PharMingen, San Diego, CA). To demonstrate antigen specificity and MHC restriction, blocking of the antigen-induced proliferative response was assessed by adding anti-HLA-DR monoclonal antibody L243 (IgG2a, prepared from supernatants of the hybridoma HB-55 obtained from the American Type Culture Collection), anti-HLA-DQ monoclonal antibody SPV-L3 (IgG2a; Beckman Coulter, Inc., Fullerton, CA) or anti-HLA-A, anti-HLA-B, and anti-HLA-C monoclonal antibody W6/32 (IgG2a; American Type Culture Collection). All antibodies were used at a final concentration of 10 µg/mL throughout the 48-hour incubation period. All assessments of ELISA were carried out at least in triplicate, and results correspond to the mean values with the SD of the mean.
Cell-Mediated Cytotoxicity Assays.
Cytotoxic activity of CD4+ T cells was determined in a colorimetric CytoTox 96 assay (Promega, Madison WI) for measuring the release of lactate dehydrogenase (LDH) from target cells. Targets were prepared by incubating T-cell lymphoma cells or EBV-LCLs with or without 10 µg/mL peptides and tumor cell lysates at 37°C overnight. T Cells were mixed with 2 x 104 targets at different E:T ratios in 96 round-bottomed well plates. After 6 to 9 hours of incubation at 37°C, 50 µL of supernatant were collected from each well to measure LDH content. To correct for spontaneous LDH release from effector cells, LDH levels were measured for each individual effector cell concentration used in the experimental set up (effector spontaneous). All measured values were assayed in triplicate and corrected for the culture medium LDH background. The percentage of specific LDH release was determined as percentage of cytotoxicity = [(experimental – effector spontaneous – target spontaneous)/(target maximum – target spontaneous)] x 100.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of T-Cell Responses to Peptide Epitopes from HTLV-I–Env Protein.
Peptides representing the three sequences derived from HTLV-I Env described above were synthesized and tested for their ability to stimulate T-cell responses using PBMCs obtained from four MHC class II-typed, healthy individuals (HLA-DR1/15, HLA-DR4/15, HLA-DR4/9, and HLA-DR8/9). Purified CD4+ T cells were stimulated in primary cultures using peptide-pulsed autologous dendritic cells as APCs as described in Materials and Methods. After three to four cycles of peptide restimulation using autologous irradiated PBMCs as APCs, the lymphocyte cultures were tested for their capacity to respond to the peptide presented by autologous PBMCs as APCs using cytokine release assays. Those cultures that exhibited at least 2.5-fold increase of cytokine release to peptide over background were selected and expanded for additional analysis. Interestingly, peptide-reactive helper T lymphocyte lines were obtained from all four individuals, and after expansion, these were analyzed for their antigen specificity and MHC restriction pattern. In the case of the DR4/DR9 individual, the CD4+ T-cell line that was successfully isolated responded to peptide HTLV-I–Env196–210 when either autologous PBMCs or dendritic cells were used as APCs (Fig. 1A)
. Furthermore, these responses were inhibited to a great extent by anti-HLA-DR antibodies but not by anti-MHC class I antibodies (Fig. 1A)
. These T cells did not respond significantly to an irrelevant control peptide (data not shown). When mouse fibroblasts (L cells) transfected with HLA-DR4, HLA-DR9, or HLA-DR53 were used as APCs, it became evident that the helper T lymphocytes from this individual recognized peptide HTLV-I–Env196–210 in the context of HLA-DR9 (Fig. 1B)
. To evaluate the overall affinity (avidity) of the HLA-DR9–restricted helper T lymphocytes for its ligand, peptide titration curves were performed using various types of autologous APCs (dendritic cells, PBMCs, CD14+ monocytes, and EBV-LCLs). These results show that the helper T lymphocyte line displayed high avidity for peptide HTLV–I-Env196–210 because <0.1 µg/mL peptide was required to attain 50% of maximal response, regardless of the type of APCs used in these assays (Fig. 1C)
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. In both cases, the T cells responded to peptide presented by autologous PBMCs in a dose-dependent manner, and the affinity of T-cell responses derived from the DQ6/9 donor appeared to be slightly higher than the response derived from the DQ5/6 donor, as determined by the amount of peptide required to achieve 50% maximal cytokine release (Fig. 2, C and D)
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. In addition, as shown in Fig. 3B
, the dose-response curve to peptide HTLV-I–Env384–398 indicated that the affinity of this cell for antigen seemed to be high, requiring 
0.1 µg/mL peptide to achieve 50% of the maximal response.
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, the HLA-DR9–restricted HTLV-I-Env196–210-specific CD4+ T-cell line was very effective in directly recognizing DR9+ HTLV-I–infected T-cell lymphoma cell lines (Kan and Su) but did not react with the DR9-negative HTLV-I–infected T-cell lymphoma cell line MT2 or autologous lymphoblastoid B cells that do not express HTLV-I viral antigens. Moreover, the recognition of HTLV-I+ T-cell lymphoma cells by the helper T lymphocyte was inhibited by anti-HLA-DR antibodies (Fig. 4B)
, confirming that this interaction requires the presentation of peptide by MHC class II molecules.
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demonstrate that the DR9-restricted HTLV-I–Env196–210-specific T-cell line was able to respond in a dose-dependent manner to APCs presenting cell lysates (Fig. 5A)
or apoptotic/dead tumor cells (Fig. 5C)
from the HTLV-I+ T-cell lymphoma MT2 but not with APCs presenting a lysate or apoptotic/dead cell from the HTLV-I–negative Jurkat T-cell lymphoma. In addition, these results also indicate that dendritic cells and EBV-LCLs were the most potent types of APCs and that were able to capture and process the cell lysates but not the apoptotic/dead tumor cells (Fig. 5, A and C)
. The capacity of the helper T lymphocytes to recognize naturally processed HTLV-I Env antigen in the form of tumor lysates (MAT, Kan, Su, and MT2) or apoptotic/dead tumor cells (MT2) presented by dendritic cells was effectively blocked by anti-HLA-DR antibodies but not by anti-HLA class I antibodies (Fig. 5, B and D)
, confirming that the epitopes are presented via MHC class II molecules. In summary, the overall results indicate that the T-cell epitope represented by peptide HTLV-I–Env196–210 can be effectively recognized by helper T lymphocytes, indirectly by APCs that process cell lysates or apoptotic/dead cells or directly by MHC class II+ HTLV-I–transformed T-cell lymphoma cells.
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. Most importantly, the recognition of HTLV-I+ T-cell lymphomas by these helper T lymphocytes was inhibited by anti-HLA-DQ antibodies (Fig. 6)
, indicating that the T-cell epitopes are also expressed on MHC class II molecules on HTLV-I–infected T-cell lymphoma cell lines.
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. Because this helper T lymphocyte line ceased to continuously grow in tissue culture, we were not able to assess whether autologous dendritic cells fed with tumor lysates or apoptotic cells would be able to function as APCs. Nevertheless, these results indicate that the HTLV-I–Env384–398 peptide epitope can be presented to helper T lymphocytes in the context of HLA-DR15 directly on the HTLV-I+ T-cell lymphoma cells.
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, this helper T lymphocyte was very efficient in killing the DR9+ HTLV-I+ T-cell lymphoma cells (Su and Kan) but was unable to kill the DR9-negative HTLV-I+ MT2 T-cell lymphoma and autologous EBV-LCLs. In addition, the HTLV-I–Env196–210-reactive T-cell line was not only able to kill the autologous EBV-LCLs pulsed with synthetic peptide, but also the autologous EBV-LCLs that were pulsed with the cell lysate from HTLV-I+ T-cell lymphoma MT2 but not the EBV-LCLs pulsed with Jurkat lymphoma (HTLV-I negative) lysates (Fig. 8B)
. These results demonstrate that lysis by this helper T lymphocyte is MHC restricted and requires the presence of the specific MHC peptide complexes on the target cells. With respect to the other HTLV-I–Env peptide-reactive helper T lymphocyte lines, although low levels of cytotoxicity were observed, these did not appear to be statistically significant (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our original goal was to identify epitopes of HTLV-I that elicit strong MHC class II-restricted CD4+ T-cell responses, which could be used as components of immunotherapeutic vaccines against ATLL. Here, we have described three potent MHC class II-binding CD4+ helper T lymphocyte epitopes represented by sequences HTLV-I–Env196–210, HTLV-I–Env317–331, and HTLV-I–Env384–398. To our knowledge, this is the first description of peptide epitopes capable of stimulating MHC class II-restricted helper T lymphocyte responses that represent true T-cell epitopes because these perform as naturally processed antigenic peptides. One of the most important functions of CD4+ helper T lymphocytes is related to their helper activity for B cells, which is necessary for IgG class switching and antibody affinity maturation. Interestingly, two of the described peptide sequences described here (HTLV-I–Env196–210 and HTLV-I–Env384–398) overlap or lie proximal to previously described humoral B-cell epitopes for the HTLV-I envelope protein (9 , 30, 31, 32) . It has been reported that peptide HTLV-I–Env190–209 frequently binds antibodies in samples from HTLV-I+ individuals (9) . Thus, these regions of HTLV-I Env glycoprotein seem to be highly immunogenic and should be considered as important targets for inducing both for T-cell and the B-cell immune responses (T-B vaccine). Thus, the two newly identified HTLV-I–Env helper T lymphocyte peptide epitopes containing both T-helper epitopes and humoral B-cell epitope should offer significant therapeutic benefits for the development of immunotherapy against HTLV-I–mediated lymphoproliferative disorders.
In addition to the two newly described helper T lymphocyte epitopes, we present data demonstrating that peptide HTLV-I–Env196–210 was able to induce helper T lymphocyte responses to naturally processed epitope, which were restricted by HLA-DR9. The same peptide was previously described to function as a cytotoxic helper T lymphocyte epitope restricted by the HLA-DQ5 and HLA-DR16 (33) . In other studies, it was reported that peptides HTLV-I–Env382–403 and HTLV-I–Env381–395 could elicit T-cell responses restricted by HLA-DR1 and HLA-DR15, respectively (29 , 34) . Thus, peptides represented by sequences HTLV-I–Env196–210 and HTLV-I–Env384–398 should be categorized as promiscuous MHC class II epitopes because they are capable of being presented to CD4+ T cells by multiple HLA-DR (and HLA-DQ) alleles and therefore are attractive candidates for vaccine development because they would offer expanded population coverage. Additional studies are warranted to determine whether these peptides can elicit helper T lymphocyte responses to additional MHC class II alleles.
An interesting finding that deserves additional consideration was the observation that some of the peptide-induced helper T lymphocyte lines were capable of recognizing antigen directly on HTLV-I+ lymphoma cells but not indirectly on APCs that were fed with dead tumor cells. It is generally thought that MHC class II-restricted responses are directed against exogenous antigens that are captured by APCs via phagocytosis/endocytosis and are processed in the endosomal compartments. However, it should be noted that the majority of the peptides (>85%) that are isolated from cell surface MHC class II molecules are derived from endogenous cell components, which predominantly are membrane proteins (35) . Thus, it should not be all that surprising that the HTLV-I+ lymphoma cells are capable of effectively producing helper T lymphocyte epitopes from the endogenous envelope membrane protein. The inability of APCs to present some of the HTLV-I Env epitopes to helper T lymphocytes via the exogenous pathway could be due to the presence of the large number of other proteins/peptides present in the tumor lysates/apoptotic cells, which would compete for binding to MHC class II. This type of competition with exogenous materials would not be as severe in the T-cell lymphoma cells because these are not typically phagocytic as with dendritic cells and other professional APCs. Alternatively, the possibility exists that protease composition in the antigen-processing compartments of T-cell lymphoma and professional APCs (e.g., dendritic cells) may differ. If this was the case, some helper T lymphocyte epitopes may not be generated or could be destroyed (by cleavage) in the dendritic cells, whereas they could be effectively produced by the T-cell lymphomas. Regardless of the mechanism involved in the generation of these MHC class II epitopes, it is clear that direct recognition of HTLV-I–infected T cells or HTLV-I+ T-cell lymphomas by helper T lymphocytes can aid in the prevention of viral or tumor spread.
The importance of CD4+ helper T lymphocytes for the induction and maintenance of antitumor responses is generally accepted. Recently, it was reported that HTLV-I–specific CD4+ helper T lymphocytes, as well as CD8+ CTLs, collaborated to eradicate HTLV-I+ T-cell lymphoma cells in an animal tumor model (36)
. The therapeutic value of helper T lymphocyte should not only be seen as mere helpers for CTLs and antibody production but also as potent effectors capable of directly inhibiting viral/tumor spread. It has been widely reported that CD4+ helper T lymphocytes can exhibit high levels of cytotoxicity via either the perforin or Fas/Fas ligand pathway against MHC class II+ tumors or viral-infected cells (21
, 37)
. In addition, CD4+ helper T lymphocytes may inhibit EBV or HTLV-I–induced lymphoproliferation by producing lymphokines such as IFN- or tumor necrosis factor 
. Thus, it could be conceived that peptide-based vaccines solely containing helper T lymphocyte epitopes could prove to be effective against tumors or viral infections affecting MHC class II-expressing tissues. Nevertheless, vaccination strategies involving both helper T lymphocyte and CTL epitopes would most likely offer better results than vaccines that focus in only one of the two cell types. Interestingly, three potent CD8+ CTL epitopes restricted by HLA-A2 (one of the most common MHC class I alleles) represented by peptides Env175–183, Env182–190, and Env395–403 lie closely to the Env196–210 and Env384–398 helper T lymphocyte epitopes (38)
. One attractive strategy for the selection of peptides to be used as vaccines is the possibility of using a single peptide of relatively small size (<2530 residues) that contains epitopes for both CTLs and helper T lymphocytes. Thus, a synthetic peptide of 29 residues (HTLV-I–Env182–210) or 1 of 20 residues (HTLV-I-Env384–403) could be capable of eliciting antivirus CTL and helper T lymphocyte responses in patients expressing HLA-A2 and one of the MHC class II allele that restricts the corresponding HTL response.
In conclusion, we present here experimental evidence that the HTLV-I–Env196–210, HTLV-I–Env317–331, and HTLV-I–Env384–398 helper T lymphocyte epitopes are effective at eliciting potent anti-HTLV-I CD4+ T-cell responses capable of recognizing directly the HTLV-I+ T-cell lymphoma cells. Two of these epitopes (HTLV-I–Env196–210 and HTLV-I–Env384–398) lie proximal to or overlap with previously characterized antibody epitopes and HLA-A2–restricted CTL epitopes found in the HTLV-I envelope glycoprotein. The present findings advocate that some synthetic peptides derived from the HTLV-I–Env glycoprotein could be used as prophylactic/therapeutic vaccines to effectively and simultaneously stimulate CTLs, helper T lymphocytes, and antibody responses against ATLL and other diseases mediated by HTLV-I.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Hiroya Kobayashi, Department of Pathology, Asahikawa Medical College, Midorigaoka-Higashi 2-1-1, Asahikawa, 078-8510, Japan. Phone: 81-166-68-2381; Fax: 81-166-68-2389; E-mail: hiroya{at}asahikawa-med.ac.jp
Received 5/ 6/04; revised 6/21/04; accepted 6/23/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kan(DR9/4); 
, Su(DR9/15); 
, MT2(DR4/15); 
, auto.LCL(DR9/4). B. The peptide-reactive helper T lymphocyte line produced granulocyte macrophage colony-stimulating factor (GM-CSF) as the result of recognizing antigen on DR9+ HTLV-I+ T-cell leukemia cell lines presenting naturally processed epitope. Production of GM-CSF was inhibited by anti-HLA-DR mAb L243 and could not be detected using DR9 negative HTLV-I+ MT2 cell and HTLV-I negative autologous EBV-LCLs. Values shown are triplicate determinations; error bars, SD. Columns and symbols without error bars had SD <10% the value of the mean.
