Direct Ex Vivo Kinetic and Phenotypic Analyses of CD8⁺T-Cell Responses Induced by DNA Immunization

Abstract

In many natural infections and experimental models, virusspecific CD8 ϩ T cells are critical for clearance of primary virus infection and for subsequent protective immunity. In addition, these cells are an important component of the immunity conferred by attenuated and recombinant viruses and by plasmid DNA vaccines. Although the kinetics of the antiviral CD8 ϩ T-cell response during the inductive and memory phases of certain systemic viral infections have been well characterized (8,16,19), there is little detailed knowledge about the kinetics of cellular immunity induced by conventional vaccines or by DNA immunization. Of particular interest is how rapidly CD8 ϩ T-cell responses develop after vaccination, as these cells play a critical role in limiting virus replication.Although a number of groups have independently demonstrated DNA vaccine-induced antigen-specific CD8 ϩ T cells in a variety of animal models (1,6,12,14,18,24,26,31) as well as in humans (29), the kinetics and functional attributes of these responses have not been fully characterized. In particular, most DNA vaccine studies to date have employed in vitro cytotoxicity assays to detect CD8 ϩ T-cell responses, but these assays are not sufficiently sensitive to permit the direct ex vivo detection of DNA-induced cytotoxic T lymphocytes (CTLs); consequently, DNA-induced CTLs have been extensively restimulated in vivo or in vitro to expand their numbers to a detectable level. It has therefore been difficult to confidently enumerate the CD8 ϩ T cells in a DNA-vaccinated host. Here we employ a more sensitive technique, intracellular cytokine staining (ICCS), to detect CD8 ϩ T-cell responses, thereby avoiding the need for lengthy restimulation. In this way, we have been able to measure DNA-induced CD8 ϩ T-cell numbers directly ex vivo at various times after a single inoculation of plasmid DNA. Using a well-defined murine model of viral pathogenesis and immunity, infection with the arenavirus lymphocytic choriomeningitis virus (LCMV), we have examined the temporal kinetics and functional attributes of CD8 ϩ T cells induced by DNA vaccination and compared them to the responses induced by LCMV infection and by immunization with a recombinant vaccinia virus vaccine. We have also determined how rapidly a DNA vaccine can induce protective antiviral immunity. MATERIALS AND METHODSMice. BALB/c mice were purchased from the Scripps Research Institute animal facility and housed in specific-pathogen-free conditions. Mice were used between 4 and 16 weeks of age.Viruses and viral infections. Stocks of the Armstrong strain of LCMV were grown on BHK cells in RPMI containing 10% fetal bovine serum (FBS), penicillin G (50 U/liter), streptomycin (50 g/liter), and 20 mM L-glutamine (all from Gibco-BRL, Rockville, Md.). LCMV titers were determined by plaque assay on Vero cells grown in medium 199 (Gibco-BRL) containing 5% FBS, penicillin G (50 U/liter), streptomycin (50 g/liter), 20 mM L-glutamine, and 0.5% agarose as previously described (11). Mice were infected by the intrap...

The Immunodominant CD8 T Cell Response to the Human Cytomegalovirus Tegument Phosphoprotein pp65495–503 Epitope Critically Depends on CD4 T Cell Help in Vaccinated HLA-A*0201 Transgenic Mice

“…We did not detect differences in the kinetics of e6-, e3-, and e8-specific CD8 T cell responses (data not shown). The DNA-primed CD8 T cell responses peaked at 12-14 d postvaccination and declined to low levels in the following 20-30 d as previously reported for other Ags (49,50). We therefore assume that CD4 T cell help facilitates priming of e6-specific CD8 T cells in pp65-immune mice, which limit e3 and e8 CD8 T cell responses by a yet unknown mechanism.…”

“…A2-HHD-II mice were immunized with pCI/st-pp65 or control pCI DNA. Splenocytes were harvested at day 12 postpriming (i.e., a time point when clonal expansion of DNA-primed CD8 T cells reaches maximal levels) (49,50) and restimulated ex vivo for 4 h with the respective pp65 peptides. The numbers of CD8 T cells showing specifically inducible IFN-g expression were determined by FCM (Fig.…”

“…We complexed immunostimulatory ODNs to short, synthetic peptides in which the cationic HIV-tat [49][50][51][52][53][54][55][56][57] RKKRRQRRR domain (tat) was fused NH 2 terminally to the antigenic e6, e3, and e8 epitopes (47). This generated the cationic tat/e3 (tat-LMNGQQIFL), tat/e6 (tat-NLVPMVATV), and tat/e8 (tat-RIFAELGEV) peptides.…”

Robust Recall and Long-Term Memory T-Cell Responses Induced by Prime-Boost Regimens with Heterologous Live Viral Vectors Expressing Human Immunodeficiency Virus Type 1 Gag and Env Proteins

“…About half of these cells produced TNF-␣, which is likely a reflection of the fact that activated T cells are divided between IFN-␥ ϩ TNF-␣ ϩ and IFN-␥ ϩ TNF-␣ Ϫ populations (18,19,47). Mock boosting of VSV-Envvaccinated mice with vTF7-3, an rVV expressing the T7 RNA polymerase, revealed that a total of ϳ3.7% of CD8 ϩ splenocytes were Env tetramer positive, and these cells produced IFN-␥ upon stimulation with p18-I10 peptide (Fig.…”

HER2 peptide-specific CD8+ T cells are proportionally detectable long after multiple DNA vaccinations

“…While DNA vaccines induce CD8 + cytotoxic T lymphocytes (CTL) specific for peptides derived from plasmid-encoded molecules, detailed analysis of the kinetics of CD8 + T cells induced by vaccinations is still limited. 7,8 We have recently demonstrated in a murine syngeneic tumor system that 9mer peptide, murine HER2p63 (TYLPANASL), can induce CD8 + CTLs with MHC class I, K d restriction and more importantly, can be a target for in vivo tumor-rejection immune responses. [9][10][11] HLA-A2402, which is expressed by more than 60% of the Japanese population and also by a significant proportion of individuals in other countries, shares a similar peptidebinding motif with murine MHC class I, K d .…”