Induction of a type 1 regulatory CD4 T cell response following Vß8.2 DNA vaccination results in immune deviation and protection from experimental autoimmune encephalomyelitis
Vipin Kumar1,,
Jeannie Maglione,
Jayant Thatte2,,
Brian Pederson,
Eli Sercarz and
E. Sally Ward2,
Department of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
1 Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA
2 Center for Immunology and Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Correspondence to:
V. Kumar
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Abstract
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DNA vaccination has been used to generate effective cellular as well as humoral immunity against target antigens. Here we have investigated the induction and involvement of regulatory T cell (Treg) responses in mediating prevention of experimental autoimmune encephalomyelitis (EAE), following vaccination with plasmid DNA encoding the TCR Vß8.2 chain predominantly displayed on disease-causing lymphocytes. Vaccination with DNA encoding the wild-type TCR results in priming of type 1 CD4 Treg and skewing of the global response to myelin basic protein in a Th2 direction, leading to significant protection from disease. In contrast, vaccination with mutant DNA encoding altered residues critically involved in recognition by the Treg results in priming of a type 2 regulatory response which fails to mediate immune deviation or protection from EAE. Control mice immunized with DNA, encoding TCR with changes at an irrelevant site, were protected from antigen-induced disease. Furthermore, protection can be transferred into naive recipients with CD4 Treg from wild-type DNA-immunized mice but not from animals vaccinated with the mutant DNA. These data suggest that vaccination with plasmid DNA encoding one or multiple Vß genes can be exploited to enhance natural regulatory responses for intervention in autoimmune conditions.
Keywords: DNA vaccine, experimental autoimmune encephalomyelitis, regulatory T cells, TCR, Th1/Th2
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Introduction
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Immunization with plasmid DNA encoding a protein antigen results in effective and long-lasting cellular and humoral immunity in several antigenic systems (1,2). Such DNA immunization can evoke both CD8 and CD4 T cell responses mediated by presentation of antigenic determinants in the context of class I and class II MHC molecules respectively (35). The intramuscular delivery of DNA generally induces an antigen-reactive, proinflammatory type 1 CD4 T cell response, through the activation and processing and presentation of antigen by bone marrow-derived professional antigen-presenting cells (APC) (68). The plasmid vector also contains immunostimulatory nucleotide sequencesunmethylated CpG motifs that activate APC, e.g. dendritic cells (DC), resulting in secretion of IL-12. The bone marrow-derived DC could potentially get transfected with the DNA, and directly process and present the encoded antigen (911). Alternatively, DC could cross-prime T cells after acquiring antigenic protein from other cell types; data supporting both routes of antigen presentation by DC have been reported. In rodents, DNA vaccines have been shown to induce protective, cell-mediated immunity against organisms such as Leishmaniasis major, Mycobacterium tuberculosis, and conditions such as malaria, tumors and allergen-induced anaphylaxis (1,2). A recent report of the induction of cellular immune responses to a peptide of Plasmodium falciparum in humans by a DNA vaccine raises hopes for the clinical applicability of this method of immunization (12).
Experimental autoimmune encephalomyelitis (EAE) is a prototypic CD4 T cell-mediated autoimmune disease model for the human demyelinating disease multiple sclerosis (13). It is characterized by inflammation and demyelination in the central nervous system accompanied by paralysis following immunization with myelin antigens, e.g. myelin basic protein (MBP). A majority of the MBP-primed effector CD4 T cells which mediate EAE in H-2u mice recognize the N-terminal peptide MBPAc19 or Ac120 and predominantly use the TCR Vß8.2 gene segment (14,15). Although the regulation and function of individual cytokines is complex, most experimental observations are consistent with the idea that myelin antigen-specific Th1 cells are encephalitogenic, whereas a Th2 response is protective (16,17).
Recently, vaccination with TCR Vß8.2 plasmid DNA has been shown to result in significant protection from subsequent induction of antigen-induced EAE (18). However, the mechanism or the involvement of a regulatory T cell (Treg) response in the prevention of disease has not yet been defined. Furthermore, it has not been described how intramuscular Vß8.2 DNA immunization that predominantly induces CD4 Th1 cells results in deviating the response to MBP in a protective Th2 direction. Here we have examined whether DNA vaccination can prime an appropriate Treg response that controls EAE (19). Using mutant Vß8 DNA molecules and cell transfer strategies, we demonstrate that Treg reactive to the TCR peptide B5 (amino acids 76101) containing the framework region (FR) 3 determinant are involved in mediating skewing of the anti-MBP response in a protective type 2 direction and prevention of disease. These findings have important implications for preventive or therapeutic vaccine approaches for T cell-mediated pathological conditions.
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Methods
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Construction of plasmid DNA
The plasmids for genetic vaccination were made as follows: for the Vß8.2 construct, a plasmid containing the Vß domain gene (VßDßJß) with a C-terminal c-myc epitope has been described previously (36). A 5' EcoRI site followed by an in-frame methionine codon were appended to the 5' end of the gene encoding the mature Vß domain using the PCR and the following oligonucleotide primer: 5'-ATC AGA ATT CAT GGA GGC TGC AGT CAC CCA A-3'. The 3' end of the gene (i.e. the c-myc epitope tag) was tailored with a BamHI site using the PCR and the primer: 5'-TGA TGG ATC CTT ATT AGA GAA CAG TCA GTC TGG T-3'. The gene was initially cloned into the vector pCMV5 as an EcoRIBamHI fragment and then subsequently ligated into pCMV8 (a derivative of pCMV5 with an additional leader intron which expresses higher levels of protein) as a SalIBamHI fragment. Mutated variants of the Vß8.2 gene were generated by site-directed mutagenesis (37) (Q85A, V88A and F90A, where Q85A indicates mutation of Gln85 to alanine, etc.) or splicing by overlap extension (38) (V10A, V12S and L107A). These mutated Vß genes were then used to replace the wild-type region of the Vß8.2 gene as PstI (site overlapping codons 24 of the mature gene)BstEII (site 5' to c-myc tag fragments). pCMV8 has a PstI site in the polylinker proximal to the 5' end of the Vß gene and this cloning therefore resulted in the loss of ~25 bases of coding sequence. Following PstI digestion, this sequence was ligated into the construct as an oligonucleotide duplex to reconstitute the complete coding sequence. The Vß3 construct, containing the Vß domain gene derived from the 2B4 hybridoma (a generous gift of Dr Mark Davis, Stanford University), was made in an analogous way to the wild-type Vß8.2 construct. The inserts of all constructs were sequenced prior to use. All constructs were transiently transfected into COS cells and a similar level of expression of Vß-myc protein was verified by analysis of cell lysates on immunoblots using the anti-c-myc antibody 9E10 as described previously (36).
Plasmid DNA vaccination
The purified plasmid DNA samples were dissolved in PBS. DNA (100 µg) was injected 23 times intramuscularly at weekly intervals. One week following the last injection of DNA, mice were s.c. immunized with MBPAc19/complete Freund's adjuvant (CFA) for the induction of EAE.
Mice
B10.PL and SJL/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). (SJLxB10.PL)F1 mice were bred under specific pathogen-free conditions in our colony. Female B10.PL or (SJLxB10.PL)F1 mice, as indicated in the text or in legends for the figures and tables, were generally used at 1014 weeks of age, and were maintained on standard laboratory diet and water ad libitum in specific pathogen-free animal facilities at UCLA and LIAI.
TCR peptides
The TCR Vß8.2 peptides used were the same as reported previously (19): B1, amino acids 130L (TCR FR1 region peptide); B4, amino acids 6190; B5, amino acids 76101 (TCR FR3 region peptide).
Measurement of antigen-specific proliferative responses
Proliferative responses to MBPAc19 or Ac120 and TCR peptides were determined in lymph nodes or spleens essentially as described earlier (19). To monitor priming of CD4 Treg, spleens were removed 710 days after the last plasmid DNA injection. Lymph node cells (4x10 5 cells/well) and splenocytes (8x105 cells/well) were cultured in 96-well microtiter plates in 200 µl of serum-free medium (HL-1; Ventrex, Portland, ME/X-vivo 10; BioWhittaker, Walkersville, MD) supplemented with 2 mM glutamine; peptides were added at concentrations ranging from 0.1 to 7 µM final concentration. Proliferation was assayed by addition of 1 µCi [3H]thymidine (ICN, Irvine, CA) for the last 18 h of a 5-day culture and incorporation of label was measured by liquid scintillation counting.
Induction and clinical evaluation of EAE
Mice were immunized s.c. with 100 µg of guinea pig MBP or Ac19 or its high-affinity variant (9.4Met) emulsified in CFA; 0.15 µg pertussis toxin (PT) (List Biological, Campbell, CA) was injected in 200 µl saline i.v. 48 h later. Mice were observed daily for signs of EAE until 4060 days after immunization. The average disease score for each group was calculated by averaging the maximum severity for all of the affected animals in the group. Disease severity was scored on a five-point scale, as described earlier (19): 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and front limb or whole body paralysis; 5, moribund or death.
Measurement of lymphokine secretion
The frequency of antigen-induced IFN-
- or IL-4-producing T cells was determined using the sensitive, single-cell ELISA-spot assay or ELISA, as described earlier (39). For ELISA-spot assays, after culture of lymph node or splenic cells with antigen for 48 h, live cells were recovered, washed and transferred by serial dilution (from 104 to 5x105 cells/well) to 96-well microtiter plates (Millipore, Bedford, MA) that had been precoated with the capturing mAb (anti-IFN-
or anti-IL-4) at 2 mg/ml. After 24 h, cells were removed, and spots were visualized using biotinylated detecting mAb and avidin D-peroxidase in conjunction with 3-amino-9-ethylcarbazole (Sigma, St Louis, MO) substrate. Spots were counted under a dissecting microscope, and the frequency of antigen-specific cells was determined from the difference between the number of spots seen with and without antigen. All capturing and detecting antibody pairs were purchased from PharMingen (San Diego, CA).
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Results
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Vß 8.2wt DNA vaccination protects B10.PL mice from antigen-induced EAE
B10.PL mice were immunized 23 times at weekly intervals intramuscularly with plasmid DNA encoding the Vß8.2wt gene in PBS. In parallel, mice were also vaccinated with an irrelevant plasmid DNA encoding the Vß3 gene or with PBS. One week following the last DNA injection, animals were challenged with MBPAc19/CFA/PT for the induction of EAE and monitored daily for clinical symptoms. As shown in Fig. 1
, mice vaccinated with the Vß8.2 DNA were significantly protected from disease. In the Vß8-vaccinated group only three out of 12 mice developed mild paralysis and recovered quickly (duration of disease ranged from 4 to 8 days). In contrast, all animals in the PBS or the Vß3 DNA vaccinated group contracted severe EAE (duration of disease ranged from 9 to 20 days). Data from two different experiments are summarized in Table 1
. The levels of expression of different DNA constructs were found to be similar (see Methods).

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Fig. 1. Mice vaccinated with the Vß8.2wt DNA but not with Vß8.2mut-rel DNA are protected from antigen-induced EAE. Groups of mice (four or five each) were immunized intramuscularly with 100 µg of plasmid DNAs (Vß8.2wt, Vß8.2mut-rel, Vß3 or PBS) 3 times at weekly intervals. One week after the last DNA injection, animals were injected with MBPAc19/CFA/PT for the induction of EAE. The clinical symptoms of EAE were monitored and scored daily, as described in Methods.
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Table 1. Mice vaccinated with the Vß8.2wt DNA but not with Vß8.2mut-rel DNA encoding altered residues in the critical FR3 region are protected from antigen-induced EAE
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Vaccination with mutant Vß8.2 DNA encoding altered residues in the FR3 region recognized by Treg does not prevent EAE
We have shown earlier that CD4 Treg reactive to the dominant FR3 region of the Vß8.2 chain (peptide `B5', amino acids 76101/Au, also referred to as the TCR FR3 peptide) are spontaneously primed in B10.PL mice following MBP injection and mediate recovery from EAE (19,20). We asked whether TCR peptide B5-reactive Treg expanded following DNA vaccination. Ten days following the last DNA challenge, proliferative recall responses to B5 peptide in draining lymph node cells isolated from PBS-, Vß3- or Vß8.2 DNA-immunized mice were examined. Stimulation indices (SI) in the Vß8.2 group ranged from 6.1 to 17.4 in comparison to 1.6 to 2.7 in the two control groups. There was no proliferative response to another Vß8.2-derived peptide, B1 (amino acids 130L), in any of the vaccinated animals. To determine the cytokine phenotype of Treg, secretion of IL-4 or IFN-
was determined in the culture supernatants. IFN-
production in response to B5 was ~4-fold higher (average 912 pg/ml) in the Vß8.2 DNA-immunized group versus Vß3- or PBS-treated controls (230 pg/ml). There was no detectable level of IL-4 in cultures from any of the groups. These data suggest that processing and presentation of the FR3 region determinant of the Vß8.2 chain following DNA vaccination results in expansion of B5-reactive type 1 Treg in vivo.
Having established the expansion of Treg, we next examined whether these cells directly participate in the DNA-induced protection from disease. To test this notion, mutated Vß8.2 plasmid DNA was created and used for vaccination. In the first instance, mutations were introduced in a relevant region, Vß8.2mut-rel, to change three critical residues (Q85, V88 and F90) involved in the recognition of FR3 peptide by Treg (21). In parallel, another mutant DNA (Vß8.2mut-irr) was made encoding three altered residues (A10, S12 and L107) in an irrelevant portion of the Vß8.2 chain that is not involved in recognition by Treg but juxtaposed on the TCR. As shown in Fig. 1
and Table 1
, mice vaccinated with the Vß8.2mut-rel DNA encoding changes in the FR3 determinant region are not protected from EAE. In contrast, animals vaccinated with the Vß8.2mut-irr DNA are significantly protected from disease (see Table 1
). Interestingly, mice vaccinated with Vß8.2-mut-rel DNA contracted more severe disease in comparison to mice in the other control groups: seven out of 10 animals died following severe paralysis in this group. These data suggest that the FR3 region determinant represents a crucial target for the induction of regulation of EAE following DNA vaccination.
CD4 Treg reactive to the FR3 region of the Vß8.2 chain mediate protection from EAE
Although data presented above suggest that FR3 peptide-reactive Treg are involved in regulation, it was still possible that mutation in the FR3 region of the Vß8.2 chain altered both the Treg response as well as an anti-TCR humoral response. To examine this possibility, we determined whether sera collected from DNA vaccinated mice from any of the groups (PBS, Vß3 or Vß8.2 immunized) contained anti-Vß8.2 antibodies. Contrary to the earlier suggestion (18), using flow cytometry analysis, we did not detect significant staining of Vß8.2+ T cells with any of the sera tested (data not shown). To further examine the involvement of FR3 peptide-reactive Treg, CD4 T cells were isolated from mice vaccinated with Vß8.2wt, Vß8.2mut-rel, Vß8.2mut-irr or Vß3 DNA molecules. Following in vitro stimulation with the TCR peptide B5, purified CD4 T cell populations were transferred into naive B10.PL mice. Recipients were challenged with MBPAc19/CFA/PT for the induction of EAE. As shown in Table 2
, recipient animals which received T cells from mice vaccinated with Vß8.2wt or Vß8.2mut-irr DNA molecules were significantly protected from disease. In contrast, recipients of T cells from control mice vaccinated with either the Vß8.2mut-rel or Vß3 DNA are not protected from EAE. These data clearly establish that CD4 Treg are crucially involved in Vß8.2 DNA-mediated prevention of EAE in B10.PL mice.
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Table 2. Adoptive transfer of CD4 Treg, isolated from animals vaccinated with the Vß8.2 DNA, but not from mice immunized with the Vß8.2mut-rel DNA prevents EAE in recipient B10.PL mice
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Type 1 CD4 Treg are involved in the Vß8.2 DNA-mediated immune deviation of the MBPAc120 response
What is the effect of TCR DNA immunization on the cytokine pattern of the anti-MBP response? One week following the last DNA challenge, mice (three in each group) were s.c. immunized with the dominant encephalitogenic determinant of MBP, Ac120. Ten days later, lymph node or splenic cells were isolated and used for in vitro recall assays for proliferation and cytokine ELISA-spot analysis (IFN-
and IL-4) in response to MBPAc120 or TCR peptide B5 respectively. In parallel, responses to another Vß8.2-derived peptide, B1 (amino acids 130L), and to the purified protein derivative of Mycobacterium (PPD) were determined and served as controls. There was no significant proliferative response (SI < 2) to TCR peptide B1 in any of the groups of mice. A similar proliferative response (SI = 2.65.3) to peptide B5 was detected in all three groups of mice immunized with the Vß8.2wt, Vß8.2mut-rel or Vß8.2mut-irr DNA plasmids (Fig. 2
). In contrast, there was no significant response (SI < 2) to B5 in the PBS- or the Vß3-immunized animals. Interestingly, while the Treg response was Th1-like in animals immunized with either the Vß8.2wt or the Vß8.2mut-irr, mice vaccinated with the Vß8.2mut-rel DNA showed a predominantly Th2-like response (Fig. 2
). In all groups proliferative responses to Ac120 were similar, with SI = 6.3 to 9.1. However, as shown in Fig. 2
, the frequencies of IL-4- or IFN-
-secreting Ac120-reactive T cells were quite different: a Th1-predominant response correlated with susceptibility to EAE, whereas Th2-like responses were dominant in protected mice. Thus, following Vß8.2wt or Vß8.2mut-irr, the response to Ac120 was deviated in a Th2 direction. In contrast, in other groups the frequency of Ac120-reactive T cells secreting IFN-
was much higher than the cells secreting IL-4, representing a Th1-like response. The frequency of IFN-
- or IL-4-secreting cells in response to PPD did not vary significantly in any of the groups (data not shown). These data indicate that type 1 Treg are required to deviate the MBP-reactive response in a Th2 direction and are consistent with our recent experiments using mucosal priming with TCR peptide B5 (17).

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Fig. 2. Immune deviation of the MBPAc120 response following Vß 8.2 plasmid DNA vaccination. Groups of B10.PL mice (three each) were given 100 µg of plasmid DNAs intramuscularly 3 times at weekly intervals. One week after the last injection, these mice were s.c. challenged with 100 µg of Ac120. Nine days later lymph node or spleen cells were harvested, pooled and subjected to proliferation and ELISA-spot analysis in response to Ac120 or TCR peptide B5. The frequencies of IL-4- or IFN- -producing T cells are given as spot forming cells/million. The background in proliferation assay ranged from 1731 ± 243 to 3340 ± 510 c.p.m. These data are from one representative of three independent experiments.
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Discussion
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The data presented here clearly demonstrate that vaccination of B10.PL mice with plasmid DNA encoding the TCR Vß8.2 gene segment, predominantly expressed on MBP-reactive encephalitogenic T cells, results in significant protection from antigen-induced EAE. The protection is specific in that vaccination with DNA encoding the TCR Vß3 gene segment, not displayed on disease-causing T cells, does not influence the course of disease. Furthermore, the Vß8.2 DNA-mediated protection involves type 1 CD4 Treg reactive with the dominant determinant from the FR3 region of the Vß8.2 chain. Thus, vaccination with Vß8.2mut-rel DNA encoding point mutations in the FR3 region, critical for recognition by Treg, does not prevent EAE, whereas vaccination with the Vß8.2mut-irr DNA encoding alterations in an irrelevant region of the TCR chain is protective. Prevention of EAE is accompanied by deviation of the anti-MBPAc120 response in a Th2 direction.
It is interesting that the in vivo processing and presentation of the TCR Vß8.2 chain following DNA vaccination leads to priming/expansion of type 1 CD4 T cells reactive to the naturally processed, dominant determinant from the FR3 region. As proposed earlier (2,911), it is likely that professional APC, e.g. DC, process and present the TCR peptide to CD4 Treg resulting in their expansion. This is consistent with the finding that Treg, primed following DNA vaccination, predominantly secrete inflammatory cytokines, such as IFN-
. Indeed, we have recently demonstrated that type 1 Treg are required to deviate the anti-MBPAc120 response in a Th2 direction (17,22). It is noteworthy that vaccination with the Vß8.2mut-rel DNA encoding relevant changes in the FR3 region results in activation of type 2 T cells specific for TCR peptide B5 and exacerbation of disease. Thus the relevant mutant DNA apparently encodes an altered peptide ligand for the CD4 Treg resulting in their priming in a Th2 direction (23,24). In our preliminary experiments, in contrast to the wild-type FR3 region peptide, a mutated peptide containing such alterations does not prevent MBP-induced EAE. This is consistent with recent findings (17), which demonstrated that priming of type 2 Treg results in a state of dysregulation leading to exacerbation of disease and the death of most animals following paralysis. Thus, the encephalitogenic potential of the MBP-reactive effector population is crucially and dominantly influenced by the cytokine secretion phenotype of CD4 Treg.
Although, a precise molecular mechanism of the eventual deviation of the anti-MBP response following the action of type 1 Treg is not yet clear, our data suggest that Treg indirectly influence cytokine predominance in the MBP-reactive T cell population (25). The secretion of proinflammatory cytokines by CD4 Treg is required for efficient recruitment/activation of CD8 Treg reactive to another determinant, from the FR2/CDR2 region of the Vß8.2 chain. For example, secretion of these cytokines may result in up-regulation of co-stimulatory or adhesion molecules on APC for an efficient induction of the CD8 population. Consistent with the involvement of a distinct CD8 Treg in this regulation, in preliminary experiments we found that mice vaccinated with the mutant Vß8.2 DNA encoding alterations in the FR2/CDR2 region were not protected from EAE. Recently, two different groups have demonstrated that CD4 T cell help via a class II MHC-dependent pathway is required for the efficient generation of an effective CTL response following DNA immunization (5,26). CD8 Treg cells may induce apoptosis or anergy (27,28) of the initially rapidly expanding, high-avidity, MBP-reactive Vß8.2 Th1 cell population. Since Th2 cells are less susceptible to apoptosis (29), this would enable a relatively slower reacting compartment of low-avidity, MBP-specific type 2 cells (which may or may not express Vß8.2) to expand in the absence of cross-regulatory IFN-
secreting cells, resulting in an apparent shift of the population as a whole in a Th2 direction. Our recent preliminary data (V. Kumar, unpublished data) using mice lacking a functional IFN-
gene suggest that this cytokine is critically involved in TCR-based regulation.
Immune deviation of antigen-specific T cells at the population level may explain how TCR-based regulation directed to a single Vß chain is able to also control disease-inducing, MBPAc19-specific T cells that use other TCR Vß chains, e.g. Vß13 or Vß4 (14). Such modulation of T cell responsiveness to one target antigenic determinant may suppress bystander responses to other antigenic determinants (30,31), from the same protein, as well as from other myelin components that may arise as a result of determinant spreading during chronic demyelination (32). Furthermore, all newly primed T cell responses in the B10.PL mouse model of EAE resulting from determinant spreading are not necessarily pathogenic; some of them could rather be protective (33). Consistent with this, it has been shown that deviation of a dominant disease-causing T cell population using an altered peptide ligand or DNA vaccination can prevent EAE or diabetes respectively (23,31). Overall these findings suggest that vaccination with plasmid DNA encoding one or multiple Vß genes could represent a powerful approach for intervention in T cell-mediated pathological conditions. In disease conditions where T cells using multiple V genes are involved, it is likely that plasmid DNA encoding multiple TCR V genes may be used to intervene. In a recent report, DNA vaccination using two diverse Vß TCR chains of cardiac myosin-restricted T cells regulated autoimmune myocarditis demonstrating that T cell-centered regulation can be achieved when more than a single Vß repertoire is involved in pathogenesis (34). Furthermore, Vß DNA along with DNA encoding appropriate co-stimulatory, cytokine or chemokine molecules could be used to render regulatory responses more effective (35).
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Acknowledgments
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This work was supported by grants from the National Multiple Sclerosis Society (V. K. and E. S. W.), NIH (E. S. and E. S. W.) and the Yellow Rose Foundation (E. S. W.). This is publication no. 375 from the La Jolla Institute for Allergy and Immunology. We would like to thank Dr Sergei Popov for help in making DNA constructs, Drs Randle Ware and Susanne Schneider for critically reading the manuscript, and Alex Jahng for help in producing the figures.
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Abbreviations
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APC antigen-presenting cell |
B5 TCR peptide containing FR3 region (amino acid 76101) from the Vß8.2 chain |
CFA complete Freund's adjuvant |
EAE experimental autoimmune encephalomyelitis |
DC dendritic cell |
FR3 framework 3 region |
MBP myelin basic protein |
PPD purified protein derivative |
PT pertussis toxin |
SI stimulation index |
Treg regulatory T cell |
Vß8.2wt DNA TCR DNA encoding the wild-type Vß8.2 domain |
Vß8.2mut-rel mutant TCR DNA encoding changes in three residues critical for the recognition of FR3 peptide by Treg, also referred to as Vß8.2CD4 Mut. |
Vß8.2mut-irr mutant TCR DNA encoding three altered residues in an irrelevant portion of the Vß8.2 domain |
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Notes
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Transmitting editor: L. Steinman
Received 28 December 2000,
accepted 9 March 2001.
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References
|
---|
-
Donnelly, J. J., Ulmer, J. B., Shiver, J. W. and Liu, M. A. 1997. DNA vaccines. Annu. Rev. Immunol. 15:617.[ISI][Medline]
-
Gurunathan, S., Klinman, D. M. and Seder, R. A. 2000. DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol. 18:927.[ISI][Medline]
-
Manickan, E., Rouse, R. J., Yu, Z., Wire, W. S. and Rouse, B. T. 1995. Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes. J. Immunol. 155:259.[Abstract]
-
Ulmer, J. B., Fu, T. M., Deck, R. R., Friedman, A., Guan, L., DeWitt, C., Liu, X., Wang, S., Liu, M. A., Donnelly, J. J. and Caulfield, M. J. 1998. Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA. J. Virol. 72:5648.[Abstract/Free Full Text]
-
Maecker, H. T., Umetsu, D. T., DeKruyff, R. H. and Levy, S. 1998. Cytotoxic T cell responses to DNA vaccination: dependence on antigen presentation via class II MHC. J. Immunol. 161:6532.[Abstract/Free Full Text]
-
Carson, D. A. and Raz, E. 1997. Oligonucleotide adjuvants for T helper 1 (Th1)-specific vaccination. J. Exp. Med. 186:1621.[Free Full Text]
-
Chu, R. S., Targoni, O. S., Krieg, A. M., Lehmann, P. V. and Harding, C. V. 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 186:1623.[Abstract/Free Full Text]
-
Krieg, A. M., Hartmann, G. and Yi, A. K. 2000. Mechanism of action of CpG DNA. Curr. Topics Microbiol. Immunol. 247:1.[ISI][Medline]
-
Akbari, O., Panjwani, N., Garcia, S., Tascon, R., Lowrie, D. and Stockinger, B. 1999. DNA vaccination: transfection and activation of dendritic cells as key events for immunity. J. Exp. Med. 189:169.[Abstract/Free Full Text]
-
Casares, S., Inaba, K., Brumeanu, T. D., Steinman, R. M. and Bona, C. A. 1997. Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope. J. Exp. Med. 186:1481.[Abstract/Free Full Text]
-
Porgador, A., Irvine, K. R., Iwasaki, A., Barber, B. H., Restifo, N. P. and Germain, R. N. 1998. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization. J. Exp. Med. 188:1075.[Abstract/Free Full Text]
-
Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charoenvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedegah, M., de Taisne, C., Norman, J. A. and Hoffman, S. L. 1998. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282:476.[Abstract/Free Full Text]
-
Paterson, P. Y. 1980. Autoimmune diseases of myelin. Prog. Clin. Biol. Res. 49:19.[Medline]
-
Urban, J. L., Kumar, V., Kono, D. H., Gomez, C., Horvath, S. J., Clayton, J., Ando, D. G., Sercarz, E. E. and Hood, L. 1988. Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy. Cell 54:577.[ISI][Medline]
-
Acha-Orbea, H., Mitchell, D. J., Timmermann, L., Wraith, D. C., Tausch, G. S., Waldor, M. K., Zamvil, S. S., McDevitt, H. O. and Steinman, L. 1988. Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell 54:263.[ISI][Medline]
-
Zamvil, S. S. and Steinman, L. 1990. The T lymphocyte in experimental allergic encephalomyelitis. Annu. Rev. Immunol. 8:579.[ISI][Medline]
-
Kumar, V. and Sercarz, E. 1998. Induction or protection from experimental autoimmune encephalomyelitis depends on the cytokine secretion profile of TCR peptide-specific regulatory CD4 T cells. J. Immunol. 161:6585.[Abstract/Free Full Text]
-
Waisman, A., Ruiz, P. J., Hirschberg, D. L., Gelman, A., Oksenberg, J. R., Brocke, S., Mor, F., Cohen, I. R. and Steinman, L. 1996. Suppressive vaccination with DNA encoding a variable region gene of the T-cell receptor prevents autoimmune encephalomyelitis and activates Th2 immunity. Nat. Med. 2:899.[ISI][Medline]
-
Kumar, V. and Sercarz, E. E. 1993. The involvement of T cell receptor peptide-specific regulatory CD4+ T cells in recovery from antigen-induced autoimmune disease. J. Exp. Med. 178:909.[Abstract]
-
Kumar, V., Stellrecht, K. and Sercarz, E. 1996. Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE). J. Exp. Med. 184:1609.[Abstract]
-
Kumar, V., Tabibiazar, R., Geysen, H. M. and Sercarz, E. 1995. Immunodominant framework region 3 peptide from TCR V beta 8.2 chain controls murine experimental autoimmune encephalomyelitis. J. Immunol. 154:1941.[Abstract/Free Full Text]
-
Kumar, V. 1998. TCR peptide-reactive T cells and peripheral tolerance to myelin basic protein. Res. Immunol. 149:827.[ISI][Medline]
-
Nicholson, L. B., Greer, J. M., Sobel, R. A., Lees, M. B. and Kuchroo, V. K. 1995. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity 3:397.[ISI][Medline]
-
Brocke, S., Gijbels, K., Allegretta, M., Ferber, I., Piercy, C., Blankenstein, T., Martin, R., Utz, U., Karin, N., Mitchell, D., et al. 1996. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature 379:343.[ISI][Medline]
-
Kumar, V. and Sercarz, E. 1996. Genetic vaccination: the advantages of going naked. Nat. Med. 2:857.[ISI][Medline]
-
Wild, J., Grusby, M. J., Schirmbeck, R. and Reimann, J. 1999. Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent. J. Immunol. 163:1880.[Abstract/Free Full Text]
-
Gaur, A., Ruberti, G., Haspel, R., Mayer, J. P. and Fathman, C. G. 1993. Requirement for CD8+ cells in T cell receptor peptide-induced clonal unresponsiveness. Science 259:91.[ISI][Medline]
-
Jiang, H., Zhang, S. I. and Pernis, B. 1992. Role of CD8+ T cells in murine experimental allergic encephalomyelitis. Science 256:1213.[ISI][Medline]
-
Zhang, X., Brunner, T., Carter, L., Dutton, R. W., Rogers, P., Bradley, L., Sato, T., Reed, J. C., Green, D. and Swain, S. L. 1997. Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837.[Abstract/Free Full Text]
-
Weiner, H. L., Friedman, A., Miller, A., Khoury, S. J., al-Sabbagh, A., Santos, L., Sayegh, M., Nussenblatt, R. B., Trentham, D. E. and Hafler, D. A. 1994. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12:809.[ISI][Medline]
-
Coon, B., An, L. L., Whitton, J. L. and von Herrath, M. G. 1999. DNA immunization to prevent autoimmune diabetes. J. Clin. Invest. 104:189.[Abstract/Free Full Text]
-
Vanderlugt, C. J. and Miller, S. D. 1996. Epitope spreading. Curr. Opin. Immunol. 8:831.[ISI][Medline]
-
Kumar, V. 1998. Determinant spreading during experimental autoimmune encephalomyelitis: is it potentiating, protecting or participating in the disease? Immunol. Rev. 164:73.[ISI][Medline]
-
Matsumoto, Y., Jee, Y. and Sugisaki, M. 2000. Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR. J. Immunol. 164:2248.[Abstract/Free Full Text]
-
Cohen, A. D., Boyer, J. D. and Weiner, D. B. 1998. Modulating the immune response to genetic immunization. FASEB J. 12:1611.[Abstract/Free Full Text]
-
Ward, E. S. 1991. Expression and secretion of T-cell receptor V alpha and V beta domains using Escherichia coli as a host. Scand J. Immunol. 34:215.[ISI][Medline]
-
Kunkel, T. A., Roberts, J. D. and Zakour, R. A. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367.[ISI][Medline]
-
Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61.[ISI][Medline]
-
Kumar, V., Bhardwaj, V., Soares, L., Alexander, J., Sette, A. and Sercarz, E. 1995. Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon gamma by T cells. Proc. Natl Acad. Sci. USA 92:9510.[Abstract]