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The Journal of Immunology, 1999, 162: 560-567.
Copyright © 1999 by The American Association of Immunologists

Comparison of Primary Sensitization of Naive Human T Cells to Varicella-Zoster Virus Peptides by Dendritic Cells In Vitro with Responses Elicited In Vivo by Varicella Vaccination1

Darlene E. Jenkins, Linda L. Yasukawa, Randy Bergen, Claudia Benike, Edgar G. Engleman and Ann M. Arvin2

Departments of Pediatrics and Pathology, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71–100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; {chi}2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection with varicella-zoster virus (VZV)3, a human alpha herpesvirus, induces cellular immunity that protects against reinfection and reactivation of the virus from neuronal sites of latency (1, 2). As is true of other human viral pathogens, current understanding of adaptive immunity to VZV is based largely upon the analysis of secondary T cell responses in immune individuals (3). Memory T cell recognition of VZV proteins has been characterized using in vitro methods to assess CD4+ and CD8+ T cell activation and the induction of cytokine and CTL responses (4, 5, 6, 7, 8, 9). Although the initial sensitization of CD4+ T cells is required in antiviral immunity, it has been difficult to generate a primary adaptive response by naive T cells in vitro. The capacity of dendritic cells (DC) to mediate the sensitization of unprimed T cells was demonstrated first in murine systems in which epidermal Langerhans cells and splenic DC were found to elicit Ag-specific helper and cytotoxic T cells in vitro whereas macrophages did not (10, 11, 12, 13, 14, 15, 16). It is now possible to isolate human DC from peripheral blood even though DC represent fewer than 0.1–1.0% of circulating PBMC. The functional capacity of DC to sensitize CD4+ and CD8+ T cells against HIV, influenza A, and other nonviral Ag has been documented (17, 18, 19, 20, 21, 22).

The pathogenesis of primary VZV infection begins with the inoculation of mucosal epithelial cells. After inoculation, the virus is presumed to be transported to regional lymph nodes where interactions of viral Ags with host immune cells may initiate the virus-specific T cell response. The mechanism of VZV transport from mucosa to lymph nodes is not known, but involvement of DCs is a possibility because VZV envelope glycoproteins are known to bind mannose receptors that are expressed in abundance by immature or nonlymphoid DC present in peripheral tissues (10, 19, 23, 24). Mature DC, which localize to the secondary lymphoid tissues in vivo, act as potent APC and express high levels of MHC class I and II, CD40, costimulatory molecules, CD80 and CD86, and adhesion molecules LFA-1, LFA-3, and ICAM-1 (10, 19). Abs against CD86, HLA-DR, and other surface molecules expressed on mature DC inhibit the sensitization of naive human CD4+ T cells in vitro, confirming their role in the induction of adaptive immunity (20, 25). Although mature DC may be less efficient at capturing and processing whole proteins than immature DC found at mucosal and skin sites, they are highly effective in presenting peptides to naive T cells (26, 27, 28).

The first objective of this study was to determine whether naive CD4+ T cells could respond to VZV peptides presented by autologous DC in vitro. Synthetic VZV peptides that represent amino acid residues of the immediate early 62 (IE62) protein were used because this protein is recognized by memory T cells induced by natural infection or after varicella immunization (7, 8). The IE62 protein is the major immediate early transactivating protein of VZV; it initiates the transcription of viral genes required to produce infectious virus progeny and is the predominant component of the virion tegument (29). Because it is present in the tegument, the IE62 protein is released into the cytoplasm of the infected cell immediately after virion uncoating and can be processed for Ag presentation before any viral replication has occurred. Memory CD4+ T cells from individuals with natural or vaccine-induced immunity to VZV recognize IE62 protein as demonstrated by proliferation and production of IL-2 and IFN-{gamma} (8, 30). The IE62 protein is also a target of VZV-specific CTL (30). The IE62 peptides that we used to investigate primary in vitro sensitization of naive T cells by DC in this study were analyzed previously for recognition by memory T cells from donors with naturally acquired immunity to VZV (31).

Assuming that primary sensitization could be demonstrated, our second objective was to assess how the pattern of VZV peptide recognition that was observed using T cells from nonimmune individuals related to the responses of the same donors after VZV immunity was induced in vivo by immunization with the varicella vaccine. The live attenuated varicella vaccine is known to elicit protective immunity to VZV in susceptible children and adults (32). Evaluation before and after varicella vaccination provided an opportunity to assess the relevance of immunogenic T cell epitopes, defined by primary DC presentation in vitro, to IE62 peptide recognition detected after replication of the virus in the host. Naive and vaccine-induced responses were also compared with the patterns of T cell recognition of the IE62 peptides in individuals with naturally acquired immunity to VZV.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
VZV IE62 peptides and other antigens

Synthetic IE62 peptides were made to correspond to amino acid sequences predicted from the nucleotide sequence of VZV ORF62 using an automated-peptide synthesizer (31). Eight peptides of 11–16 amino acid residues were designed to include sequences consistent with algorithms for potential T cell epitopes (33, 34) (Table IGo). After synthesis, the crude peptides were purified by HPLC, and purity was confirmed by analytical reverse-phase HPLC and amino acid analysis. VZV IE62 shares some functions with related proteins of herpes simplex virus and simian varicella virus, but no homologies with the IE62 peptides were identified in these proteins or in general searches of DNA and protein databases (35, 36). The peptides were designated P1–P8, based upon their order in the IE62 protein sequence. Whole VZV Ag was made from detergent-solubilized VZV-infected melanoma cells; an uninfected cell lysate was used as control Ag (30). The whole VZV Ag preparation contains IE62 protein along with viral glycoproteins, enzymes, and other regulatory/structural proteins. Other antigens included keyhole limpit hemocyanin (KLH) (Sigma, St. Louis, MO), purified tetanus toxoid (TT; Michigan Public Health Department, Ann Arbor, MI) and phytohemagglutinin (Difco, Detroit, MI).


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  		Table I. Synthetic peptides corresponding to VZV IE62 sequences

 
Identification of VZV-susceptible donors and immunization protocol

Donors who were not immune to VZV were identified from a cohort of adults with no history of varicella by demonstrating absence of IgG Abs to VZV in serum, as tested by ELISA, and lack of proliferative responses when PBMC cultures were stimulated with whole VZV Ag (30). The stimulation index (SI) is calculated as the ratio of cpm in whole VZV Ag-stimulated wells (3.0 x 105 cells/well) pulsed with [3H]thymidine, compared with wells incubated with an uninfected-cell control. An SI of <3.0 correlates with susceptiblity to VZV. Responses of 12 donors with naturally acquired immunity to VZV, defined by a history of varicella IgG Abs to VZV and SI > 3.0, were also analyzed (31).

The live, attenuated varicella vaccine used in this study was produced from the Oka-Merck strain by Merck (West Point, PA). Lyophilized vaccine (lots 0410B, 0773D, 1104D, 1355D) was stored at -70°C before use. The vaccine was administered s.c. in two doses, containing >3000 plaque-forming units of infectious virus per dose, given at an interval of 6–8 wk. All donors had received TT vaccine. Immunologic assays were done before varicella vaccination and 1–8 mo after the second dose of vaccine.

Isolation of DC, monocytes, and CD4+ T cells

DC were isolated from human peripheral blood by a series of density gradient centrifugation steps as described previously (25). Briefly, PBMCs were isolated from buffy coats by Ficoll-Hypaque gradient and then fractionated over a four-step discontinuous Percoll gradient composed of 30, 40, 50, and 75% layers. The fraction located as the interface of the 40 and 50% layers was enriched further by adherence to a culture flask for 3 h at 37°C and served as a source of monocytes for experiments with naive T cells. PBLs were obtained from the interface betweeen the 50 and 75% layers and were cultured for ~36 h at 37°C in complete media consisting of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 10% heat-inactivated pooled human serum. Cultured PBLs were separated over a 15% hypertonic metrizamide (Sigma) to enrich for DC in the the lower density fraction; the high density pellet was used for isolation of CD4+ T cells. The DC fraction was incubated overnight at 37°C and a second metrizamide gradient (14%) was used to enrich DC further. DC isolations resulted in 50–80% purity based on FACS analysis (high MHC class II DR, CD14-negative) and microscopic evalution of cell morphology. DC and monocytes were irradiated (2400 rad) before incubation with CD4+ T cells and peptides to decrease background. CD4+ T cells were isolated from the first high density metrizamide pellet with CD4+ magnetic microbeads (Miltenyi Biotec, Auburn, CA) using a positive selection column; purity of 92–99% was confirmed by FACS analysis. Monocytes were isolated from PBMC preparations from vaccinated donors by adherance; these preparations contained >=78% monocytes by FACS analysis. CD4+ T cells were recovered from nonadherant populations using CD4+ magnetic beads.

CD4+ T cell proliferation

Proliferation assays were conducted in U-bottom 96-well plates using 50 µl of irradiated DC or monocytes (8.0–10 x 103 cells/well), 50 µl of individual IE62 peptides (final concentrations of 40, 60, or 80 µg/ml), and 50 µl of CD4+ T cells (3.0 x 104–1.0 x 105 cells/well), brought to a total volume of 200 µl/well with complete media. Control wells consisted of DC or monocytes with CD4+ T cells and 50 µl KLH (25 µg/ml) or 50 µl TT (5 Lfu/µl final concentration); DC or monocytes with CD4+ T cells (no peptide or Ag); CD4+ T cells alone; or CD4+ T cells stimulated with phytohemagglutinin. KLH and TT were used as control Ag for naive and memory immune responses, respectively. This procedure was also used to test DC as APC after immunization of the donors. In addition, the immunized donors were also tested for T proliferation to IE62 peptides using monocytes as APC. CD4+ T cells (2.4 x 104/well) and monocytes (8 x 103/well) were incubated with peptides at two concentrations (60 and 80 µg/ml) and 6–10 replicates per concentration. Naturally immune or susceptible subjects were also tested as described previously, using 3.0 x 104 PBMC/well, with 22 replicates per peptide (60 µg/ml), with monocytes acting as APC (31). Proliferation was measured by incubating T cell/DC, T cell/monocyte, or PBMC cultures for 6 days, pulsing with [3H]thymidine, and harvesting after 18–24 h.

Proliferation was determined by calculating the mean cpm ± SE from 3 to 5 replicates for wells with peptide or Ag and from 6 to 10 replicates for wells measuring background cpm of DC or monocytes with CD4+ T cells alone. In experiments using DC as APC, the numbers of DC isolated from each donor restricted the number of peptides that could be tested to fewer than 8 in some cases. [3H]thymidine incorporation measured in peptide or Ag-stimulated wells and control wells was analyzed by statistical comparison using the Student’s t test for two means. The donor response was considered to be positive to the peptide or Ag if the p value was <0.05 using a two-tail analysis. When monocytes were used as APC in the assays done after immunization, positive wells were identified as those with cpm > mean cpm for all wells; the donor response was defined as significant when the mean cpm to the peptide was <0.05 compared with the mean for all wells using the Student’s t test for two means. The positive predictive value was calculated using the formula, a/(a+b), in which "a" is the number of donors who had positive responses before and after immunization and "b" is the number of donors with a positive response before and negative response after immunization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Optimal conditions for detection of naive T cell proliferative responses to VZV IE62 peptides presented by DC

Preliminary titrations of DC showed that a minimum of 8–10 x 103 DC/well was necessary to detect proliferation of naive T cells in response to IE62 peptides (data not shown). This observation is similar to the optimal DC concentration identified in experiments using HIV protein or peptides (17, 18). Because the number of DC isolated from peripheral blood was limited, 8.0 x 103 DC/well was chosen as the standard concentration for these experiments. Titrations of peptides (20–80 µg/ml) showed that T cell proliferative responses varied depending on the concentration of peptide (Fig. 1GoA). Because responses were consistently low or undetectable to most peptides tested at 20 and 40 µg/ml, assays were done using peptide concentrations of 60 and 80 µg/ml; P5 was also tested at 40 µg/ml because responses to this peptide were detected at the lower concentration. Incubation of DC with autologous CD4+ T cells and no Ag resulted in nonspecific background proliferation, as was shown previously (18). Background cpm with IE62 peptides varied with the number of CD4+ T cells/well; a range of 3.0 x 104 to 1.0 x 105 CD4+ autologous T cells/well was optimal for the detection of responses to the IE62 peptides.



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  		FIGURE 1. In vitro proliferation of naive CD4+ T cells to VZV IE62 peptides presented by autologous DC or monocytes. The proliferation of T cells from a VZV-susceptible donor in response to VZV IE62 peptides is shown for a representative experiment comparing DC (A) and monocytes (B) as APC. Each bar shows the mean [3H]thymidine uptake (cpm x 103 + SE) from replicate wells after the incubation of T cells with IE62 peptides, P1, P2, P4 or P7, and APC. The solid bars indicate responses using IE62 peptides at a concentration of 60 µg/ml, and the shaded bars show responses with 40 µg/ml per well. KLH and TT were used as controls for naive and memory responses, respectively. Background mean cpm is indicated by the horizontal line and represents [3H]thymidine incorporation when CD4+ T cells and APC were incubated without peptide or Ag. The asterisks indicate a positive response in peptide-stimulated wells compared with control wells, defined as p < 0.05 by Student’s t test for 2 means.

 
Proliferation of naive T cells to VZV IE62 peptides is elicited using DC, but not monocytes, as APC

The primary in vitro proliferative response to IE62 peptides (Table IGo) by CD4+ T cells obtained from seven VZV-susceptible donors was evaluated using autologous DC or monocytes as APC. Only DC were effective as APC in these naive responses. In a representative experiment, three of the four IE62 peptides that were tested induced proliferation of T cells from a nonimmune donor, relative to background cpm, when autologous CD4+ T cells were cultured with DC and peptide (Fig. 1GoA). Proliferative responses also resulted from stimulation with TT, the control Ag for recall responses, and with KLH, the control Ag for a naive response. In contrast, no in vitro CD4+ T cell response to IE62 peptides or KLH was detected in wells when monocytes were used as APC (Fig. 1GoB). Proliferation of memory T cells was elicited by TT in the presence of monocytes as expected, because all donors had been immunized with tetanus vaccine.

The in vitro proliferative responses of seven VZV-susceptible donors to the panel of IE62 peptides are summarized in Table IIGo. Presentation by DC elicited T cell proliferation to a minumum of three peptides in the naive donors, and one donor responded to all of the eight peptides. P4 was the most immunogenic peptide, inducing a positive response in all of the donors who were tested. Other peptides that were highly immunogenic included P1, P5, and P7, which elicited T cell proliferation in 67–83% of the naive donors. P3, P6, and P8 induced responses in fewer donors (43–60%) whereas P2 presented by DC elicited proliferation of CD4+ T cells from only two of six (33%) nonimmune subjects.


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  		Table II. Summary of proliferative responses to VZV IE62 peptides by CD4+ T cells obtained from naive donors before varicella immunization

 
None of the naive donors had T cell responses to whole VZV Ag in a standard proliferation assay using PBMC in which monocytes act as APC (Fig. 2Go). Nonimmune donors had a mean stimulation index of 1.6 ± 0.40 SE to whole VZV Ag.



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  		FIGURE 2. Confirmation of cellular immunity to VZV after immunization with the varicella vaccine. Five VZV susceptible donors were tested for proliferation responses to whole VZV Ag in PBMC cultures. Donors were tested before immunization (prevaccine), 21 days after the first dose (V1D21), just before the second dose (V2D0), 21 days after the second dose (V2D21) and 6 mo (6 M) after immunization. The SI was calculated as the ratio of mean cpm ([3H]thymidine incorporation) in wells incubated with whole VZV Ag compared with wells containing uninfected-cell control.

 
Comparison of T cell recognition of VZV IE62 peptides after varicella immunization with naive responses elicited by DC presentation

The induction of cellular immunity to VZV after immunization of VZV-susceptible donors with varicella vaccine was confirmed by demonstrating T cell proliferation in PBMC cultures stimulated with whole VZV Ag (Fig. 2Go). The mean SI for vaccine recipients tested 3–4 wk after the first vaccine dose was 21.2 ± 3.70 SE and all donors had SI > 3.0. The responses were maintained or increased when evaluated 3–4 wk after the second dose and persisted at 6 mo (mean SI: 18.1 ± 5.13 SE). VZV IgG Abs were detected by ELISA after immunization of all donors (data not shown).

CD4+ T cell proliferative responses to IE62 peptides were evaluated in five donors after varicella immunization and compared with the responses of their naive T cells to these peptides in vitro. Proliferation to IE62 peptides by T cells from donors who were tested after immunization was assessed in cultures using either DC or monocytes as APC. Examples of the responses of two donors who were evaluated for T cell proliferation to IE62 peptides before and after vaccination are illustrated in Fig. 3Go. Donor 1 responded to all peptides that were tested before and after immunization using DC as APC (Fig. 3Go, panels 1A and 1B) and to seven of eight peptides using monocytes as APC after immunization (Fig. 3Go, panel 1C). Donor 3 had naive and memory T cells that responded to P4 and P7 using DC as APC (Fig. 3Go, panels 3A and 3B). Responses to P2, P3, P5, P6, and P8, as well as to P4 and P7, were detected after vaccination using monocytes as APC (Fig. 3Go, panel 3C).



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  		FIGURE 3. In vitro CD4+ T cell proliferation responses to VZV IE62 peptides before and after varicella vaccination. T cell-proliferative responses to the IE62 peptides, P1–P8, are shown for donors 1 and 3; panels 1A and 3A show responses before immunization with varicella vaccine. Panels 1B and 1C, and 3B and 3C, show postvaccination responses. Panels 1A and 3A show the mean [3H]thymidine uptake (cpm ± SE) from replicate wells after the incubation of naive CD4+ T cells with autologous DC alone or with IE62 peptides. Panels 1B and 3B show mean proliferative responses from replicate wells of CD4+ T cells from donors 1 and 3 after immunization using DC as APC. Panels 1C and 3C show mean proliferation in replicate wells when CD4+ T cells obtained after immunization were incubated with autologous monocytes alone or IE62 peptides and monocytes. Data with peptides indicate the maximum response observed when CD4+ T cells were incubated with peptide concentrations of 40, 60, or 80 µg/ml using DC as APC, or 60 or 80 µg/ml using monocytes as APC. Results are indicated only if at least 2 concentrations of peptide were tested. The asterisks indicate a positive response in peptide-stimulated wells compared with control wells, defined as p < 0.05 by Student’s t test for two means.

 
A summary of the responses of all five donors who were tested before and after vaccination is given in Table IIIGo. Postvaccination, all donors had memory T cell proliferation to a minimum of three peptides. As was observed in experiments using naive T cells, P4 was the most immunogenic IE62 peptide after varicella immunization using monocytes or DC as APC; positive responses were detected in all five donors. When monocytes were used as APC to test IE62 peptide recognition after immunization, P1, P4, and P6 induced proliferation of T cells from 80 to 100% of donors whereas P2 and P8 were less immunogenic (40–60%). Using DC as APC, P1 and P7 were immunogenic for most donors before and after immunization whereas P2 and P8 were poor stimulators of T cells from both naive and immune donors; P6 was recognized by memory T cells from 80% donors after vaccination when peptides were presented by DC, compared with 60% in susceptible donors (data not shown).


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  		Table III. Summary of proliferative responses to VZV IE62 peptides by CD4+ T cells obtained from donors before and after varicella immunization

 
The pattern of IE62 peptides recognized by naive and memory T cells from each donor was compared by statistical analysis (Table IIIGo). Using monocytes as APC, T cell recognition of specific peptides was concordant between naive DC-mediated responses and postvaccine responses in 69% of assays (p = 0.05; {chi}2). In 19 of 36 comparisons, naive and memory responses were both positive and in 6 comparisons, the peptide was not immunogenic either before or after varicella immunization. Discordant responses that were negative in naive T cell cultures and positive after in vivo sensitization occurred in 7 of 36 comparisons (19%), and in 4 cases (11%) naive T cells responded but memory T cells stimulated by the IE62 peptide did not. The positive predictive value of peptide recognition by naive T cells detected before vaccination relative to responses to the same peptide after in vivo sensitization by varicella vaccine was 82% using monocytes as APC for memory T cells. When DC were used as APC both before and after vaccination, a concordance of the proliferative responses to specific peptides was observed in 25 of 36 comparisons (p = 0.03; {chi}2).

Comparison of VZV IE62 peptide responses in donors with natural immunity, VZV-susceptible donors, and donors immunized with varicella vaccine

A comparison of the percentage of naive, immunized, and naturally immune donors who had proliferative responses to each of the eight IE62 peptides is shown in Fig. 4Go. More than 70% of donors in all groups had T cell proliferation to P1 and P4. A memory T cell proliferation response to the IE62 peptides, P1, P3, P4, P5, and P6, was detected previously in 75–87% of 12 donors with naturally acquired immunity to VZV using unfractionated PBMC cultures (31). Fewer donors responded to P2 (42%), P7 (50%), and P8 (67%). The mean cpm in proliferation assays was highest with P1 (2.6 x 103 ± 430 SE) and P4 (2.3 x 103 ± 491 SE) and lowest with P8 (1.6 x 103 ± 718 SE) among donors who responded to the peptide.



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  		FIGURE 4. Comparison of T cell proliferation responses to VZV IE62 peptides in naturally immune donors, naive donors, and donors immunized with varicella vaccine. The percentage of naturally immune (NI) subjects (black bars) who had detectable T cell proliferation to IE62 peptides is compared with those of naive donors (gray bars); the percentage of donors immunized with varicella vaccine who had responses postvaccination in cultures with DC as APC is shown with open bars; the percentage with responses using monocytes as APC is shown with hatched bars. The horizontal axis indicates the peptide tested, P1-P8.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These experiments demonstrate that a naive T cell response can be induced in vitro by peptides corresponding to residues of the IE62 protein, an immunogenic protein of VZV, when Ag presentation is mediated by autologous DC. Our findings are consistent with the observations that human DC are required to stimulate primary immune responses in vitro to HIV gp120 epitopes, TT, or superantigen, and that macrophages or monocytes can present Ag effectively only to T cells from sensitized donors (17, 18, 19, 23, 26, 37). As was found using HIV gp120 peptides, the generation of responses to VZV IE62 peptides showed that DC with the capacity to present viral peptides to naive human T cells are present among circulating PBMC populations (18, 38). When presented by DC, T cells from each of seven donors responded to at least three of the eight IE62 peptides and 70–100% of the donors responded to P1, P4, and P7, suggesting that the algorithms for probable T cell epitopes are useful to predict amino acid sequences that can be recognized by naive T cells (33, 34). When presented by monocytes, P1 and P4 were also recognized by T cells from 80 and 100% of donors after vaccination. The observation that peptides selected with MHC class I algorithms were immunogenic in CD4+ T cell/MHC class II interactions has also been noted with other microbial proteins and probably reflects the relative promiscuity of MHC class II peptide binding (39, 40).

The isolation of DC from PBMC initially yields immature DC (37). The DC enrichment protocol used in these experiments included a 2-day incubation in the presence of autologous lymphocytes to allow maturation of DC to a strong Ag-presentation phenotype (25). By the second day in culture, DC had the morphological characteristics of mature cells, including long cytoplasmic processses, irregularly shaped nuclei, and few cytoplasmic organelles. Furthermore, DC were observed in conjunction with clusters of lymphocytes and exhibited the functional capacity to elicit proliferation of naive CD4+ T cells to IE62 peptides at this time point. Ag presentation by DC has been shown to involve binding of CD40 to CD40 ligand on T cells (41, 42). This interaction is associated with a marked increase in IL-12 production and favors a predominantly Th1 response, which is the cytokine pattern that we have observed during the acquisition of primary immunity to VZV (43).

According to current models of DC development, uncultured DC correspond to less mature cells in vivo; these cells express low levels of MHC and costimulatory molecules and high levels of FcR and mannose receptors and function primarily to take up and process Ag (10, 37). Uncultured, immature DC process foreign proteins and present peptides to memory T cells efficiently but do not sensitize naive T cells. The subset of DC that process and present Ag to naive T cells has been shown to be derived from CD2+ precursors (20). The fact that we detected memory responses to whole Ag, such as TT, as well as proliferation of naive T cells to IE62 peptides, suggests that both immature and mature forms of DC were present under the culture conditions that we used. Immature as well as mature DC are also detected when human DC are derived in vitro in the presence of granulocyte-macrophage CSF and TNF-{alpha}, as defined by their Ag-processing and -presentation capacities (26).

In addition to confirming that cultured DC are required for priming naive T cell responses to viral peptides in vitro, our experiments extended previous observations with VZV Ag by analyzing the relationship between naive responses generated in vitro with those elicited in the same donors after in vivo sensitization to IE62 peptides. The specific patterns of IE62 peptide recognition observed when the primary response of VZV-susceptible donors was evaluated in vitro correlated with responses documented after VZV immunity was induced in vivo by immunization with live-attenuated varicella vaccine. A match between pre- and postvaccination responses to IE62 peptides occurred in 69% of comparisons using monocytes as APC; the positive-predictive value was 82%. These data indicate that the assessment of naive T cell responses in vitro has potential value for predicting the immunogencity of viral peptides in vivo. Because failure to demonstrate a naive T cell response to a peptide had less predictive value than a positive response, some peptides that are immunogenic in vivo would be excluded by this criterion. However, in the case of large viral proteins like IE62 that have many potential T cell epitopes, the observation that a positive response to a particular peptide in vitro suggests that it will also be immunogenic in vivo and should help to limit the options to be considered in subunit vaccine design. The lack of a complete match between responses before and after vaccination is consistent with an evaluation of HIV immunity after initial infection that showed a progressive increase in the number of HIV epitopes recognized by T cells over time, as well as a loss of reactivity to some epitopes (45).

Similarities in IE62 peptide recognition were also observed when the responses of VZV-susceptible donors were compared with those of donors who had naturally acquired immunity to VZV. For example, the IE62 peptides, P1 and P4, that stimulated naive CD4+ T cells effectively in vitro, were also recognized by T cells from most donors with natural immunity; conversely, recognition of P2 and P8 was limited in both groups. A somewhat broader recognition of IE62 peptides was noted in naturally immune individuals compared with vaccine recipients, which could reflect an expanding T cell repertoire induced by re-exposures to varicella or by subclinical reactivations of the endogenous latent virus in these donors (2, 3).

The donors in these experiments were selected for their susceptiblity to VZV and represented an "outbred" population expected to have diverse MHC class II alleles. Nevertheless, relatively broad recognition of VZV peptides by naive T cells and by T cells obtained after in vivo sensitization of the donors was demonstrated. Naive donors had T cells that proliferated in response to a mean of 65% of the IE62 peptides and T cells from immunized donors responded to a mean of 88% of the peptides using monocytes as APC. When memory responses to the VZV IE62 peptides were evaluated in naturally immune individuals who had widely varying MHC haplotypes, all donors showed broad recognition of VZV peptides (31). The sensitization of T cells from all, or almost all, donors to a single peptide such as P4, suggests that these more immunogenic peptides represent epitopes that can mediate MHC class II/TCR interactions despite genetic variations. The value of using primary in vitro sensitization to identify peptides that are immunogenic across diverse class II MHC types will require confirmation in larger populations when DC separations can be done more efficiently and with smaller volumes of blood. However, CD4+ T cell recognition of conserved influenza A hemagglutinin epitopes, defined using synthetic peptides, has been described after immunization of donors with diverse MHC haplotypes using a subunit influenza vaccine (46). Other studies of in vitro sensitization using HIV peptides or the hapten, TNP, have shown that the same peptides induced proliferation of naive T cell proliferation responses despite differences in MHC alleles (12, 47). An analysis of memory responses to measles peptides in vaccinated and naturally immune donors also identified several peptides that were commonly recognized by T cells from an HLA-diverse donor population (48).

In addition to its value for understanding the cellular interactions and epitope characteristics that are required to generate adaptive immunity, the analysis of primary CD4 and CD8 T cell recognition of viral peptides in vitro using DC-based assays may be useful for designing subunit vaccines against human pathogens, especially those for which no animal model is available (49, 50).


   Acknowledgments
 
We thank Julia Martinsen for assistance. The live attenuated varicella vaccine was provided by Merck. Informed consent was obtained from participants according to the U.S. Department of Health and Human Services and Stanford University guidelines for research involving human subjects.


   Footnotes
 
1 This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI20459) and by a postdoctoral fellowship award to D.E.J. from the Varicella-Zoster Virus Research Foundation. This work was presented in part at the Third International Conference on Varicella Zoster Virus, Palm Beach, FL, March 9–11, 1997. Back

2 Address correspondence and reprint requests to Dr. Ann M. Arvin, G312, Stanford University School of Medicine, Stanford, CA 94305. E-mail address: Back

3 Abbreviations used in this paper: VZV, varicella zoster virus; DC, dendritic cell; IE62, immediate early 62; KLH, keyhole limpit hemocyanin; TT, tetanus toxoid; SI, stimulation index. Back

Received for publication June 16, 1998. Accepted for publication September 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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