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Cytotoxic T Cell Responses to DNA Vaccination: Dependence on Antigen Presentation via Class II MHC¹

Holden T. Maecker^2,*, Dale T. Umetsu, Rosemarie H. DeKruyff and Shoshana Levy^*,4

Departments of ^* Medicine/Oncology and Pediatrics, Stanford University Medical Center, Stanford, CA 94305

	Abstract

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This study was designed to test whether cytotoxic T cell (CTL) responses to DNA vaccination are dependent upon MHC class II-restricted priming of CD4⁺ T cells. Because DNA vaccination may directly transfect dendritic cells, and dendritic cells may be capable of directly stimulating CD8⁺ T cell responses, such priming might be unnecessary. To test this hypothesis, C57BL/6 mice were immunized intramuscularly or intradermally with DNA encoding either whole OVA, a class I (K^b)-restricted peptide epitope of OVA (amino acids 257–264, SIINFEKL), or this class I-restricted epitope plus the adjacent class II (I-A^b)-restricted epitope of OVA (amino acids 265–280, TEWTSSNVMEERKIKV). Very low to negligible CTL responses were observed in mice vaccinated with the SIINFEKL construct, whereas mice vaccinated with the SIINFEKLTEWTSSNVMEERKIKV or with the complete OVA construct made equally robust CTL responses. These responses were sensitive to blocking by anti-CD8 mAb and were shown to be SIINFEKL-specific by using SIINFEKL peptide-pulsed EL-4 cells as targets. To ensure that the generation of these CTL responses was indeed dependent upon CD4⁺ T cell help, mice were depleted of either CD4⁺ or CD8⁺ cells before immunization. Depletion of CD4⁺ cells completely abrogated the CTL response to OVA DNA, as did depletion of CD8⁺ cells. Thus, we conclude that the CTL response to both intramuscular and intradermal DNA vaccination is highly dependent upon the generation of CD4⁺ T cell help via a class II MHC-dependent pathway. These results will be relevant for the construction of minimal-epitope vaccines for DNA immunization.

	Introduction

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The mechanism by which direct injection of DNA into tissues induces humoral and cellular immune responses to encoded Ags has been the subject of much recent investigation. Experiments have demonstrated that gene gun-mediated delivery of DNA to skin can result in the direct transfection of dendritic-like cells that migrate to local lymph nodes (1). The role of migratory cells is further reinforced by studies in which the site of DNA inoculation is excised shortly after immunization, with little or no effect upon the resulting immune response (2, 3). Furthermore, studies using bone marrow chimeras have demonstrated that cellular responses to the DNA-encoded Ags are dependent upon bone marrow-derived cells rather than on resident host tissue cells (4, 5).

The above studies do not exclude the possibility of "cross-priming" (6) after DNA vaccination. In this process, non-APCs in the local tissue secrete Ag or peptides that are taken up by professional APCs, such as dendritic cells. Normally, exogenous Ags preferentially enter the MHC class II loading pathway. However, dendritic cells have been shown to be unique among APC for their ability to "cross-prime," presenting exogenous Ags in association with class I (as well as class II) MHC molecules (7, 8).

To assess the role of cross-priming by secreted Ags during DNA vaccination, transfected myoblasts have been transplanted into host tissues and used to generate cellular responses (9, 10). These responses are most likely explained through a process of cross-priming via secreted peptides or proteins. Alternately, in the normal situation of DNA vaccination into muscle, dendritic cells may phagocytose apoptotic bodies derived from transfected tissue cells (as they do for virally infected cells (11)), resulting in cross-priming. However, there is no evidence that nonprofessional APC can directly prime immune responses after transfection via DNA vaccination.

Once Ag has been processed by APC such as dendritic cells, whether by direct transfection or cross-priming, T cell responses can be elicited. But what is the precise role of CD4⁺ T cells in contributing to CD8⁺ T cell responses? In general, CD8⁺ T cell responses are highly dependent upon the prior stimulation of CD4⁺ T cells (12, 13, 14). This stimulation can be circumvented by stimulation of CD40 on dendritic cells, among other means (15, 16, 17). In vitro priming of dendritic cells with class I MHC-binding peptides can also result in the production of CD8⁺, class I-restricted CTL responses (18, 19). In addition, i.v. injection of synthetic peptides can prime CTL responses, even in CD4-deficient mice (20). Thus, it has been postulated that dendritic cells can directly prime naive CD8⁺ T cells and induce CTL responses.

If dendritic cells are indeed transfected by DNA vaccination, and/or if they present MHC class I-associated peptides via cross-priming, one might assume that CD8⁺ T cell responses to DNA immunization could be induced in the absence of CD4⁺ T cell help. Indeed, some strategies for DNA vaccination have apparently been based upon this implicit assumption. For example, multiple CTL epitopes have been assembled into expression vectors and successfully used for DNA vaccination and induction of CTL responses (21, 22). On the surface, this would seem to validate the idea that class II-restricted T cell help is not required for generation of CTL responses by DNA vaccination. Alternately, this approach may succeed because of the inadvertent creation of MHC class II-binding helper epitopes via the head-to-tail assembly of CTL epitopes. Another group has used "single" T cell epitopes of up to 17 amino acids, with or without a leader sequence, as DNA vaccines to generate CTL responses to HIV gp120 and mutant p53 via gene gun immunization (23). Although these responses would be predicted to be CD4-independent, this was not directly tested. In fact, the possibility remains that MHC class II-binding epitopes were also present in their construct, potentially generating CD4-dependent T cell help.

In this study, we set out to directly test the hypothesis that CTL generation via DNA vaccination does not require class II-restricted T cell help. We did this by constructing vectors containing only MHC class I or MHC class I + class II-restricted epitopes of OVA. Our results demonstrate that, contrary to the above reasoning, class II-restricted T cell help via CD4⁺ T cells is absolutely required for efficient generation of CTL responses to OVA DNA immunization. These results have both theoretical and practical importance to the design of future minimal epitope constructs for DNA vaccination.

	Materials and Methods

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DNA constructs

A vector for DNA immunization (pOVA) containing the OVA gene linked to a human V leader sequence and driven by a CMV promoter has been described (24). This vector contains CpG motifs known to be important for immunogenicity of DNA vaccines (25) and generates strong CTL responses in vivo (24). The plasmid was modified by removal of the OVA gene by restriction digestion with DraIII and BamHI. The OVA gene was replaced with a sequence generated by two overlapping synthetic oligonucleotides encoding the MHC class I-restricted T cell epitope of OVA, SIINFEKL (amino acids 257–264), and resulting in restriction site overhangs to allow directional cloning into the vector backbone. A second vector was also created by insertion of overlapping synthetic oligonucleotides encoding the above SIINFEKL epitope followed by the MHC class II-restricted T cell epitope of OVA, TEWTSSNVMEERKIKV (amino acids 265–280). The correct construction of these vectors was confirmed by DNA sequencing.

Mice and injections

Female C57BL/6 mice, 6–8 wk of age, were purchased from Harlan BioScience (Indianapolis, IN). They were injected i.m. with 100 µg of one of the above vectors, or intradermally (i.d.)³ with 50 µg of one of the above vectors in normal saline. The i.m. injections were done either once or twice (4 wk apart), with CTL assays done 4 wk after a single injection or 2 wk after a second injection. All i.d. injections were done twice, 4 wk apart, with CTL assays done 2 wk after the second injection.

CTL assays

CTL assays were performed as described (24). Briefly, mouse spleens were aseptically harvested, single cell suspensions made, and the cells treated with a hypotonic buffer to remove RBC. They were then incubated at a 10:1 ratio with irradiated EG.7-OVA cells (a gift of M. Bevan, University of Washington, Seattle, WA) for 6 days, followed by analysis of lytic ability using a standard ⁵¹Cr release assay. Target cells were EG.7-OVA, EL-4 (obtained from the American Type Culture Collection, Manassas, VA), or EL-4 pulsed for 1.5 h with 1 µg of SIINFEKL peptide.

Depletion of CD4⁺ or CD8⁺ cells

Mice were given daily i.p. injections of 200 µg of mAb for 3 days, then rested for 4 days. Representative mice were then bled to quantitate depletions by flow cytometry. All mice were also immunized with DNA on this day, and received an additional 200 µg injection of mAb, which was repeated every 4–5 days for the duration of the experiment. The i.d.-immunized groups received a second DNA injection 4 wk after the first injection, with spleens being harvested 2 wk after this second injection. Representative mice were also bled at the time of splenic harvest to recheck depletions via flow cytometry. Depletions at all time points checked were always 95%. mAb used for depletions included: irrelevant mouse IgG1 (clone LC4, produced in our laboratory (26)); anti-CD4 (clone GK1.5); and anti-CD8 (clone 53-6.7). Anti-CD4 and anti-CD8 hybridomas were obtained from Irving Weissman (Stanford University). They were produced as ascites in Swiss nu/nu mice, and the ascites was clarified by high-speed centrifugation, sterile filtered, and titered for IgG1 concentration by ELISA.

	Results

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A single CTL epitope DNA vaccine induces very low CTL activity

To test whether a single CTL epitope could be used as a DNA vaccine to induce CTL responses in mice, we constructed a vector (referred to as pep DNA) expressing the SIINFEKL peptide of OVA (amino acids 257–264). This peptide was joined to a 5' Ig V leader, since this leader was used in our OVA DNA vaccination vector containing the complete OVA cDNA (referred to as OVA DNA). The OVA DNA construct was previously shown to induce strong CTL responses in mice (24). Also, we reasoned that the V leader may aid in directing translated products into the endoplasmic reticulum for loading onto class I MHC molecules.

The pep DNA vaccine was given i.m. to more than 20 C57BL/6 mice over several experiments and always induced a weak or negative CTL response, as assayed on the OVA-transfected EG.7-OVA cell line (Fig. 1). By contrast, the OVA DNA vaccine generated reproducibly high CTL responses. This was true after either one or two immunizations; i.e., the pep DNA vaccine did not generate significant CTL responses even after boosting.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. A single CTL epitope DNA vaccine is inefficient at generating CTL responses. Groups of five mice each were immunized twice i.m., 4 wk apart, with 100 µg of DNA vector encoding either the complete OVA sequence (OVA DNA) or a class I-restricted peptide epitope of OVA (pep DNA). Mice were individually analyzed for CTL responses 2 wk after the second immunization. Target cells were the OVA-transfected cell line EG.7-OVA, or its nontransfected parental cell line, EL-4. The data are representative of two similar experiments.

The pep DNA vaccine was modified by addition of a Th cell epitope from OVA, TEWTSSNVMEERKIKV (amino acids 265–280). This two-epitope DNA vaccine (called dbl pep DNA) was then compared with the pep DNA vaccine and the OVA DNA vaccine for the ability to induce CTL responses (Fig. 2). Whether assayed on EG.7-OVA targets, or SIINFEKL peptide-pulsed EL-4 targets, the pep DNA construct again induced low (Fig. 2A) or negative (Fig. 2B) CTL responses. However, the dbl pep DNA vaccine induced a strong CTL response. In one experiment (Fig. 2B), the dbl pep DNA induced superior lysis compared with OVA DNA; in another experiment, OVA DNA was slightly superior (Fig. 2A). The OVA DNA and dbl pep DNA responses were SIINFEKL peptide-specific as seen by the use of peptide-pulsed target cells. The responses were also CD8-dependent since addition of anti-CD8 mAb during the lysis portion of the assay abrogated cytotoxicity against EG.7-OVA (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Addition of an MHC class II-binding epitope rescues the ability to generate CTL responses to the single CTL epitope. Groups of two to three mice each were immunized once i.m. with 100 µg of a vector encoding an irrelevant protein (Irrel. DNA), OVA (OVA DNA), a single CTL epitope of OVA (pep DNA), or that CTL epitope plus an MHC class II-binding epitope from OVA (dbl pep DNA). CTL responses were measured 4 wk after a single immunization. Spleen cells from each group were pooled and tested on EG.7-OVA vs EL-4 cells (A) or EG.7-OVA vs SIINFEKL peptide-pulsed EL-4 cells (B). To ensure that lysis of EG.7-OVA was CD8-restricted, anti-CD8 mAb was included in some culture wells during the lysis portion of the assay (OVA DNA + aCD8, dbl pep DNA + aCD8).

It remained possible that the CTL response to the dbl pep DNA vaccine (as well as the OVA DNA vaccine) was not mediated via MHC class II-dependent help, as suggested by the above experiment, but rather was due to some other effect of the additional sequence(s) surrounding the SIINFEKL epitope. To test the dependence on T cell help directly, mice were depleted of either CD4⁺ or CD8⁺ cells before vaccination with the OVA DNA vaccine. As seen in Fig. 3, depletion of either CD4⁺ or CD8⁺ cells completely abrogated the CTL response against either EG.7-OVA or peptide-pulsed EL-4 cells. Similar results were obtained using the dbl pep DNA vaccine, in that the CTL response to this construct was also dependent upon CD4⁺ cells (data not shown). To demonstrate that the response against EG.7-OVA was CD8-dependent, anti-CD8 mAb was added to cultures during the lysis portion of the assay, abrogating cytotoxicity against EG.7-OVA. Thus, the CTL response to i.m. OVA DNA vaccination is dependent upon MHC class II-mediated, CD4⁺ T cell help.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. CTL responses to OVA DNA vaccination are CD4-dependent. Groups of two to three mice each were treated three times daily with 200 µg of anti-CD4 (GK1.5) or anti-CD8 (53–6.7) or irrelevant mAb, given i.p., then immunized i.m. 5 days later with 100 µg of OVA DNA. Depletion was maintained by repeated i.p. injections of 200 µg mAb of every 4 days, and spleens were harvested and pooled for CTL assays 3 wk after immunization. Depletion was checked by flow cytometry at immunization and harvest, and was >95% for both CD4 and CD8 at each time point. To ensure that the responses of irrelevant mAb-treated mice were CD8-restricted, anti-CD8 mAb was included in some culture wells during the lysis portion of the assay (Irrel Ab-treated + aCD8). The results shown are representative of two experiments.

The skin is thought to be an efficient site for the induction of immune responses to DNA vaccination because of the large number of Langerhans cells, which can be activated to become Ag-presenting dendritic cells. It thus seemed possible that the direct transfection of these cells via i.d. DNA vaccination might be more efficient and might lead to direct stimulation of CD8⁺ T cell responses. We therefore immunized mice i.d. with either the pep DNA vaccine or the OVA DNA vaccine, and tested their CTL responses after two immunizations (Fig. 4A). As with i.m. DNA vaccination, the pep DNA construct failed to elicit detectable CTL responses, while the OVA DNA construct generated robust CTL activity against EG.7-OVA, but not parental EL-4 cells. To test the effect of CD4⁺ cells directly in this system, depletion experiments were conducted as in Fig. 3, but mice were vaccinated twice by the i.d. route. As with i.m. DNA vaccination, the CTL response to i.d. OVA DNA vaccination was highly dependent upon the presence of CD4⁺ cells; depletion of either CD4⁺ or CD8⁺ cells before vaccination abrogated the response (Fig. 4B). Thus, despite the increased number of potential dendritic cell targets in the skin, i.d. DNA vaccination was also found to be dependent upon CD4-mediated, MHC class II-restricted T cell help.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Intradermal DNA vaccination is also dependent upon MHC class II-restricted, CD4-mediated T cell help. A, Groups of two to three mice each were immunized twice i.d., 4 wk apart, with 50 µg each of DNA encoding the entire OVA sequence (OVA DNA) or a class I-restricted peptide epitope of OVA (pep DNA). Spleen cells were pooled and analyzed for CTL responses 2 wk after the second immunization and analyzed on the indicated targets. B, Groups of two to three mice each were treated with depleting mAb as in Fig. 3, then immunized twice i.d., 4 wk apart, with 50 µg of OVA DNA. Spleen cells were harvested and pooled for CTL assays 2 wk after the last injection.

	Discussion

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Can dendritic cells directly stimulate CD8⁺ CTL responses? Two studies have shown that pulsing dendritic cells with a single MHC class I-restricted peptide can elicit strong CTL responses (18, 19). However, because these dendritic cells were pulsed ex vivo, they may have been subjected to stimulation with other foreign Ags from FCS-containing medium. Thus, they may present peptides via class II MHC in addition to the class I epitopes with which they were pulsed and may therefore engender CD4⁺ T cell help via these other peptides. In fact, Porgador and Gilboa (19) showed that the CTL response to peptide-pulsed dendritic cells was CD4-dependent by depleting CD4⁺ cells in vivo. This is a strong argument against the ability of dendritic cells to stimulate CD8⁺ responses in the absence of CD4⁺ help.

Another study demonstrated the ability to induce CTL by injection of synthetic peptides in vivo into mice (20). These authors found that CTL could be generated even after CD4⁺ cell depletion or in CD4-deficient mice. However, the peptides in these experiments were given in incomplete Freund’s adjuvant, which may somehow overcome the need for CD4⁺ cells. Also, the dose of peptides (100 µg given s.c.) may be significantly higher than that engendered by DNA vaccination. Thus, these results may be explained by the effect of Ag dose, and DNA vaccination may simply result in too little Ag presentation to stimulate CD4-independent CTL responses.

Other studies have pointed to the importance of CD4⁺ cells in maintaining CD8-dependent responses. Walter et al. (27) studied the CTL response to CMV in bone marrow transplant recipients receiving adoptive transfer of autologous CD8⁺ T cells. Those patients who maintained strong CTL responses over time had recovered endogenous CD4⁺ T cell help; those who did not recover sufficient CD4⁺ help failed to maintain their CTL responses due to apoptosis of the transferred CD8⁺ cells.

Some investigators have created constructs for DNA vaccination which consist entirely of CTL epitopes (21, 22). These "polytope" constructs use multiple CTL epitopes strung head-to-tail but do not include any helper epitopes. If our results can be generalized to other systems, such constructs would not be predicted to work. However, they may have been successful because the assembly of the polytope construct created neoepitopes that served to generate class II MHC-restricted help. In fact, the construct of Thomson et al. (21) contains a potential MHC class II-binding epitope, LSYIPSAEK, formed by the fusion of CTL epitopes 7 and 8. This sequence contains anchor residues L and K at positions 1 and 9, and the preferred residue I at position 4. This would be predicted to bind to I-E molecules of both H-2^b and H-2^d haplotypes, according to the data compiled by Rammensee et al. (28). The polytope construct was tested by Thomson et al. (21) in H-2^b and H-2^d mice. Thus, we would predict that this sequence, and/or others formed as part of the polytope construct, may contribute to the ability to generate CTL responses in mice of these haplotypes.

Perhaps most intriguing of previous investigations into DNA vaccination with CTL epitopes is the work of Ciernik et al. (23). These authors constructed "single epitope" DNA vaccines that induced CTL responses against HIV gp120 and mutant p53. The responses were improved by addition of an adenovirus E3 leader for targeting to the endoplasmic reticulum. How could these vaccines work without the inclusion of helper epitopes? Several possibilities include the following. 1) The 17-amino acid p53 peptide and/or the E3 leader may generate a helper epitope, and CTL responses without the leader or with a more minimal CTL epitope may be analogous to low-level responses occasionally seen with our single epitope vaccine (see Figs. 1 and 2). 2) The E3 leader may target the peptide to the ER more efficiently than our Ig V leader, and this efficient targeting is required for generating strong CTL responses without CD4⁺ T cell help. 3) The use of gold particle bombardment for delivery in their study may generate different results from i.m. and i.d. injection used in our study. In any event, the dependence of CTL generation on CD4⁺ cells by Ciernik et al. was not tested, making distinguishing among these possibilities difficult. Furthermore, our results argue that single epitope DNA vaccines will not always work well, despite the optimism generated by the above studies. Our results also argue that single epitope vaccines can be greatly improved by the addition of a helper epitope.

Because of these considerations, future DNA vaccines that target class I MHC molecules should include both MHC class I and class II-binding epitopes in multiple epitope DNA vaccines.

	Acknowledgments

 
We thank Vishal Doctor for technical assistance with DNA injections, and Ronald Levy for critical review of the work.

	Footnotes

 
¹ This project was supported by Grant R01 AI37219 from the National Institutes of Health.

² Current address: Becton Dickinson Immunocytometry Systems, 2350 Quine Drive, San Jose, CA 95131.

³ Address correspondence and reprint requests to Dr. Shoshana Levy, Department of Medicine/Oncology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305.

⁴ Abbreviations used in this paper: i.d., intradermally; dbl pep DNA, DNA vaccine expressing a CTL and a helper epitope of OVA; OVA DNA, DNA vaccine expressing the complete OVA sequence; pep DNA, DNA vaccine expressing a single CTL epitope of OVA.

Received for publication June 25, 1998. Accepted for publication August 17, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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				L. Steinaa, P. B. Rasmussen, A. M. Wegener, L. Sonderbye, D. R. Leach, J. Rygaard, S. Mouritsen, and A. M. Gautam Linked Foreign T-Cell Help Activates Self-Reactive CTL and Inhibits Tumor Growth J. Immunol., July 1, 2005; 175(1): 329 - 334. [Abstract] [Full Text] [PDF]

				R. Kennedy, A. H. Undale, W. C. Kieper, M. S. Block, L. R. Pease, and E. Celis Direct Cross-Priming by Th Lymphocytes Generates Memory Cytotoxic T Cell Responses J. Immunol., April 1, 2005; 174(7): 3967 - 3977. [Abstract] [Full Text] [PDF]

				H. Okada, T. Tsugawa, H. Sato, N. Kuwashima, A. Gambotto, K. Okada, J. E. Dusak, W. K. Fellows-Mayle, G. D. Papworth, S. C. Watkins, et al. Delivery of Interferon-{alpha} Transfected Dendritic Cells into Central Nervous System Tumors Enhances the Antitumor Efficacy of Peripheral Peptide-Based Vaccines Cancer Res., August 15, 2004; 64(16): 5830 - 5838. [Abstract] [Full Text] [PDF]

				K. Teramoto, K. Kontani, Y. Ozaki, S. Sawai, N. Tezuka, T. Nagata, S. Fujino, Y. Itoh, O. Taguchi, Y. Koide, et al. Deoxyribonucleic Acid (DNA) Encoding a Pan-Major Histocompatibility Complex Class II Peptide Analogue Augmented Antigen-specific Cellular Immunity and Suppressive Effects on Tumor Growth Elicited by DNA Vaccine Immunotherapy Cancer Res., November 15, 2003; 63(22): 7920 - 7925. [Abstract] [Full Text] [PDF]

				A. Kariyone, T. Tamura, H. Kano, Y. Iwakura, K. Takeda, S. Akira, and K. Takatsu Immunogenicity of Peptide-25 of Ag85B in Th1 development: role of IFN-{gamma} Int. Immunol., October 1, 2003; 15(10): 1183 - 1194. [Abstract] [Full Text] [PDF]

				K. Dayball, J. Millar, M. Miller, Y. H. Wan, and J. Bramson Electroporation Enables Plasmid Vaccines to Elicit CD8+ T Cell Responses in the Absence of CD4+ T Cells J. Immunol., October 1, 2003; 171(7): 3379 - 3384. [Abstract] [Full Text] [PDF]

				E. T. A. Marques Jr., P. Chikhlikar, L. B. de Arruda, I. C. Leao, Y. Lu, J. Wong, J.-S. Chen, B. Byrne, and J. T. August HIV-1 p55Gag Encoded in the Lysosome-associated Membrane Protein-1 as a DNA Plasmid Vaccine Chimera Is Highly Expressed, Traffics to the Major Histocompatibility Class II Compartment, and Elicits Enhanced Immune Responses J. Biol. Chem., September 26, 2003; 278(39): 37926 - 37936. [Abstract] [Full Text] [PDF]

				Y. Hashimoto, H.-S. Chen, C. Cunningham, T. H. Malik, and P. K. Lai Two Major Histocompatibility Complex Class I-Restricted Epitopes of the Borna Disease Virus p10 Protein Identified by Cytotoxic T Lymphocytes Induced by DNA-Based Immunization J. Virol., May 15, 2003; 77(10): 6076 - 6081. [Abstract] [Full Text] [PDF]

				A. Lobell, R. Weissert, S. Eltayeb, K. L. de Graaf, J. Wefer, M. K. Storch, H. Lassmann, H. Wigzell, and T. Olsson Suppressive DNA Vaccination in Myelin Oligodendrocyte Glycoprotein Peptide-Induced Experimental Autoimmune Encephalomyelitis Involves a T1-Biased Immune Response J. Immunol., February 15, 2003; 170(4): 1806 - 1813. [Abstract] [Full Text] [PDF]

				J. Rice, S. Buchan, and F. K. Stevenson Critical Components of a DNA Fusion Vaccine Able to Induce Protective Cytotoxic T Cells Against a Single Epitope of a Tumor Antigen J. Immunol., October 1, 2002; 169(7): 3908 - 3913. [Abstract] [Full Text] [PDF]

				A. Campos-Neto, J. R. Webb, K. Greeson, R. N. Coler, Y. A. W. Skeiky, and S. G. Reed Vaccination with Plasmid DNA Encoding TSA/LmSTI1 Leishmanial Fusion Proteins Confers Protection against Leishmania major Infection in Susceptible BALB/c Mice Infect. Immun., June 1, 2002; 70(6): 2828 - 2836. [Abstract] [Full Text] [PDF]

				M. C. Wolkers, M. Toebes, M. Okabe, J. B. A. G. Haanen, and T. N. M. Schumacher Optimizing the Efficacy of Epitope-Directed DNA Vaccination J. Immunol., May 15, 2002; 168(10): 4998 - 5004. [Abstract] [Full Text] [PDF]

				A. L. Marzo, V. Vezys, K. Williams, D. F. Tough, and L. Lefrancois Tissue-Level Regulation of Th1 and Th2 Primary and Memory CD4 T Cells in Response to Listeria Infection J. Immunol., May 1, 2002; 168(9): 4504 - 4510. [Abstract] [Full Text] [PDF]

				C. Alfonso, J.-O. Han, G. S. Williams, and L. Karlsson The Impact of H2-DM on Humoral Immune Responses J. Immunol., December 1, 2001; 167(11): 6348 - 6355. [Abstract] [Full Text] [PDF]

				J. H. Cho, J. W. Youn, and Y. C. Sung Cross-Priming as a Predominant Mechanism for Inducing CD8+ T Cell Responses in Gene Gun DNA Immunization J. Immunol., November 15, 2001; 167(10): 5549 - 5557. [Abstract] [Full Text] [PDF]

				F. Rodriguez, S. Harkins, J. M. Redwine, J. M. de Pereda, and J. L. Whitton CD4+ T Cells Induced by a DNA Vaccine: Immunological Consequences of Epitope-Specific Lysosomal Targeting J. Virol., November 1, 2001; 75(21): 10421 - 10430. [Abstract] [Full Text] [PDF]

				P. Parajuli, V. Pisarev, J. Sublet, A. Steffel, M. Varney, R. Singh, D. LaFace, and J. E. Talmadge Immunization with Wild-Type p53 Gene Sequences Coadministered with Flt3 Ligand Induces an Antigen-specific Type 1 T-Cell Response Cancer Res., November 1, 2001; 61(22): 8227 - 8234. [Abstract] [Full Text] [PDF]

				A. E. Fujimura, S. S. Kinoshita, V. L. Pereira-Chioccola, and M. M. Rodrigues DNA Sequences Encoding CD4+ and CD8+ T-Cell Epitopes Are Important for Efficient Protective Immunity Induced by DNA Vaccination with a Trypanosoma cruzi Gene Infect. Immun., September 1, 2001; 69(9): 5477 - 5486. [Abstract] [Full Text] [PDF]

				B. Wang, C. C. Norbury, R. Greenwood, J. R. Bennink, J. W. Yewdell, and J. A. Frelinger Multiple Paths for Activation of Naive CD8+ T Cells: CD4-Independent Help J. Immunol., August 1, 2001; 167(3): 1283 - 1289. [Abstract] [Full Text] [PDF]

				J. Rice, T. Elliott, S. Buchan, and F. K. Stevenson DNA Fusion Vaccine Designed to Induce Cytotoxic T Cell Responses Against Defined Peptide Motifs: Implications for Cancer Vaccines J. Immunol., August 1, 2001; 167(3): 1558 - 1565. [Abstract] [Full Text] [PDF]

				V. Kumar, J. Maglione, J. Thatte, B. Pederson, E. Sercarz, and E. S. Ward Induction of a type 1 regulatory CD4 T cell response following V{beta}8.2 DNA vaccination results in immune deviation and protection from experimental autoimmune encephalomyelitis Int. Immunol., June 1, 2001; 13(6): 835 - 841. [Abstract] [Full Text] [PDF]

				K. Chan, D. J. Lee, A. Schubert, C. M. Tang, B. Crain, S. P. Schoenberger, and M. Corr The Roles of MHC Class II, CD40, and B7 Costimulation in CTL Induction by Plasmid DNA J. Immunol., March 1, 2001; 166(5): 3061 - 3066. [Abstract] [Full Text] [PDF]

				U. Kumaraguru, I. A. Davis, S. Deshpande, S. S. Tevethia, and B. T. Rouse Lymphotoxin {{alpha}}-/- Mice Develop Functionally Impaired CD8+ T Cell Responses and Fail to Contain Virus Infection of the Central Nervous System J. Immunol., January 15, 2001; 166(2): 1066 - 1074. [Abstract] [Full Text] [PDF]

				L. Krishnan, S. Sad, G. B. Patel, and G. D. Sprott Archaeosomes Induce Long-Term CD8+ Cytotoxic T Cell Response to Entrapped Soluble Protein by the Exogenous Cytosolic Pathway, in the Absence of CD4+ T Cell Help J. Immunol., November 1, 2000; 165(9): 5177 - 5185. [Abstract] [Full Text] [PDF]

B. Cochlovius, M. Stassar, O. Christ, L. Raddrizzani, J. Hammer, I. Mytilineos, and M. Zoller In Vitro and In Vivo Induction of a Th Cell Response Toward Peptides of the Melanoma-Associated Glycoprotein 100 Protein Selected by the TEPITOPE Prog