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The Journal of Immunology, 1999, 162: 3782-3789.
Copyright © 1999 by The American Association of Immunologists

Activation of CD4 T Cells by Somatic Transgenesis Induces Generalized Immunity of Uncommitted T Cells and Immunologic Memory1

Mara Gerloni*, Kent T. Miner{dagger}, Sidong Xiong*, Michael Croft{dagger} and Maurizio Zanetti2,*

* Department of Medicine and Cancer Center, University of California-San Diego, La Jolla, CA 92093; and {dagger} Division of Immunochemistry, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cellular immune responses were analyzed in vivo after a single intraspleen inoculation of DNA coding for a 12-residue Th cell determinant associated with a 12-residue B cell epitope, a process termed somatic transgene immunization. We show that CD4 T cells are readily activated and produce IL-2, IFN-{gamma} and IL-4, characteristics of an uncommitted phenotype. Linked recognition of the two epitopes coded in the same transgene promoted IgM-IgG1 switch and enhanced the total Ab response but had no effect on IgG2a Abs. Although originating in the spleen, T cell responsiveness was found to spread immediately and with similar characteristics to all lymph nodes in the body. A single inoculation was also effective in establishing long term immunologic memory as determined by limiting dilution analysis, with memory T cells displaying a cytokine profile different from that of primary effector T cells. These studies provide evidence that by initiating immunity directly in secondary lymphoid organs, an immune response is generated with characteristics that differ from those using vaccines of conventional DNA or protein in adjuvant administered in peripheral sites. Somatic transgene immunization can therefore be used to probe T cell responsiveness in vivo and represents a tool to further understanding of the nature of the adaptive immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The quantitative and qualitative aspects of T cell activation are thought to be controlled by at least three factors: the route of Ag entry, the physical form of the Ag, and the association of Ag with immunologic adjuvant. It is commonly accepted that Ag administered s.c. in immunologic adjuvant induces immunity, whereas Ag injected i.p. or i.v. in soluble form induces tolerance (1). However, model systems that permit visualization of the initiation of an adaptive immune response against endogenously produced Ag are not available, hence the possibility that current views on Ag-specific immune responses in vivo may not necessarily represent all the natural processes of immunity.

The impact of the physical form of Ag on the immune response is well known. Particulate Ags such as bacteria, Ags administered in aggregated form (2, 3), and Ags complexed with Abs in slight excess of Ag (4, 5) are immunogenic because of greater uptake by macrophages and dendritic cells (6). Deaggregated Ags, on the other hand, are by and large tolerogenic (7), an effect reflecting the extent to which different types of APC participate in the process (8). Soluble Ags injected i.v. induce rapid accumulation of T cells in secondary lymphoid organs. However, these T cells are several orders of magnitude less sensitive to Ag than T cells activated by Ag injected s.c. in immunologic adjuvant, are transient, and do not migrate into follicles (1). Consequently, soluble Ags injected i.v. induce tolerance rather than immunity, and immunologic memory is not established.

Nucleic acids in the form of plasmid DNA represent a new powerful way to induce immunity in vivo (9, 10). Conventional DNA vaccines are based on i.m. or intradermal administration and are designed to deliver transgenes under the control of viral promoters for ubiquitous gene transcription and expression. The resulting type of immunity resembles natural infection by exogenous viruses, includes both Ab and T cell responses (11, 12, 13, 14, 15), and is independent of classical immunologic adjuvants. For these reasons DNA vaccination is a powerful tool to analyze the initiation and further development of cellular immune responsiveness in vivo. However, immunity induced by conventional DNA vaccines is only in part understood. With few exceptions the transgene product is not detected in bodily fluids (16, 17), direct information about which cell(s) account for Ag presentation is limited (18, 19, 20, 21, 22), and immunity is in most instances the result of multiple injections (23).

Somatic transgene immunization (STI)3 is an alternative approach to DNA-based vaccination (24) developed to better understand the process in vivo. STI is induced with transgenes under the control of lymphoid tissue-specific regulatory elements. A single intraspleen inoculation of an Ig heavy (H) chain DNA under the control of a B lymphocyte promoter leads to 1) persistence in vivo of the transgene for up to 3–4 mo (25), 2) secretion of 15–30 ng/ml transgenic Igs (transgenic Ig) (24), 3) induction of a specific primary Ab response, and 4) establishment of durable immunologic memory (26) in 100% of instances. Since the variable domain of an Ig gene can be engineered to code for foreign peptides of discrete size (27), STI lends itself for studies on the nature and specificity of the adaptive response in vivo in which amounts of Ag (at least 1000-fold less than usual immunization regimens), origin of the Ag (endogenous synthesis), and conditions of immunization (no immunologic adjuvant) are all defined, controllable, and different from those commonly used (28). In the present work we used STI to analyze the in vivo activation of CD4 T cells specific for a well-characterized Th cell determinant.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Eight- to ten-week-old C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were kept in the animal facility of the University of California-San Diego.

Plasmid DNAs

Plasmids {gamma}1NV2NA3 (29) and {gamma}1NANP (30) were engineered as described previously. The pSV2Neo is the original plasmid forming the backbone of the pNeo{gamma}1 vector without the human {gamma}1 C region gene (31). This plasmid was used as a control in the immunization experiments. Plasmid DNAs were purified using a Qiagen Megaprep kit (Qiagen, Chatsworth, CA). The purity of the DNA was monitored using the following equation: %N = (11.1R - 6.32)/(2.16 -R), where R is 260 nm/280 nm, and %N is the percentage of nucleic acid (32). Purified plasmids were stored at -20°C until use.

Proteins and synthetic peptides

Recombinant antigenized Abs {gamma}1NV2NA3 and {gamma}1NANP were produced in transfectoma cells and purified as previously described (29, 30). Synthetic peptides NANPNANPNANP and NANPNVDPNANP were synthesized in the Peptide Chemistry Core Facility of the University of California-San Diego (29).

Immunizations

DNA inoculation. Mice were inoculated intraspleen with 100 µg of plasmid DNA in 50 µl of sterile saline solution as previously described (24).

Booster injections. Booster injections were administered on days 90, 110, 120, and 150 after priming by a single s.c. injection (50 µg/mouse) of affinity-purified {gamma}1NV2NA3 Ab emulsified in IFA.

[3H]Thymidine incorporation assay

Animals were inoculated as previously described. At the time of harvest, mice were sacrificed, and the lymph nodes and spleens were removed and crushed in a tissue shredder to remove excess tissues and release cells. Single cell suspensions were treated with RBC lysis buffer (Sigma, St. Louis, MO) and cultured (106 cells/ml) in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with HEPES buffer, glutamine, 7.5% FCS, and 50 µM 2-ME in the presence or the absence of synthetic peptides NANPNVDPNANP or NANPNANPNANP (50 µg/ml) in triplicate. The cells were incubated at 37°C in 10% CO2 for 3 days. [3H]thymidine was added at 1 µCi/well, and the cells were incubated for 16–18 h at 37°C. Cells were harvested onto glass-fiber filter mats using a Tomtec cell harvester (Orange, CT), and the radioactivity was measured in a liquid scintillation counter (Betaplate, Wallac, Turku, Finland). Results are expressed as the stimulation index, calculated as the ratio of counts per minute of cells cultured in the presence of synthetic peptide/counts per minute of cells cultured in the absence of peptide. Con A stimulation was used as a polyclonal activator and positive control.

Separation of CD4+ and CD8+ T cells

CD4+ and CD8+ T cells were isolated by Ab plus complement-mediated depletion from splenocytes of mice immunized 7 days earlier by DNA inoculation. Briefly, cell suspensions (30 x 106 cells/ml) were treated with mAb to CD8 (3.155) or CD4 (RL172) for 30 min on ice. After washing, anti-T cell Abs were cross-linked with a mouse anti-rat (MAR 18.5) mAb for 30 min on ice, and rabbit complement was added twice for 30 min each time at 37°C. The cell suspension was then washed twice and resuspended at the concentration of 5 x 106 cells/ml in RPMI (Irvine Scientific, Santa Ana, CA). The purity of the separated cell fractions was assessed by analysis on a FACScan with CellQuest software (Becton Dickinson, Mountain View, CA) at the flow cytometry facility of The La Jolla Institute for Allergy and Immunology (La Jolla, CA), using phycoerythrin-conjugated anti-CD4 and FITC-conjugated anti-CD8 mAbs (PharMingen, San Diego, CA).

Detection of cytokines

IL-2 assay. Culture supernatants were harvested 40 h after initial seeding and were stored at -20°C. The supernatants from three separate triplicate cultures were pooled for each mouse. IL-2 activity was determined in a bioassay using the IL-2- and IL-4-dependent NK.3 cells in the presence of anti-IL-4 (purified from the 11B11 cell line, American Type Culture Collection, Manassas, VA) (33). Briefly, 100 µl (1/2 dilution in medium) of 40-h culture supernatants were added in duplicate to 100 µl of NK.3 cells (106/ml) and incubated for 36 h. [3H]thymidine was added at 1 µCi/well during the last 12 h. Cells were harvested as specified above. Results are expressed as counts per minute.

IL-4, IL-5, and IFN-{gamma}. IL-4, IL-5, and IFN-{gamma} were measured in the same 40-h culture supernatants by ELISA as described previously (33), using the Abs 11B11 and biotinylated anti-IL-4 (BVD6, PharMingen), TRFK5 and biotinylated TRFK4, and R46A-2 and biotin-XMG1.2 (PharMingen), respectively. Standard curves were constructed with purified IL-2, IL-4, IL-5, and IFN-{gamma} (supernatants from the respective X63.Ag. cell lines). Tests were performed in duplicate.

Detection of transgenic Ig

The presence of transgenic Ig in the serum of mice was detected using a capture ELISA (34). Briefly, 1/10 dilutions of individual mouse sera in PBS containing 1% BSA and 1% Tween 20 (PBSA) were incubated on 96-well plates coated with a goat Ab to human IgG1 (10 µg/ml). The concentration of the transgenic Ig was calculated by plotting OD values against a standard curve constructed with a known amount of human IgG1 diluted in PBSA containing 10% normal mouse serum. The bound Abs were revealed using a horseradish peroxidase-conjugated goat Ab to human {gamma}-globulin (H chain specific) absorbed with murine Ig (Sigma). The bound peroxidase activity was revealed by adding o-phenylenediamine dihydrochloride and H2O2. Plates were read after 30 min in a microplate reader (Vmax, Molecular Devices, Menlo Park, CA) at 492 nm. Tests were performed in duplicate.

Detection of Abs and isotype determination

Abs to transgenic Ig were detected by ELISA on plates coated (2.5 µg/ml) with affinity-purified, transfectoma-derived {gamma}1NANP protein (35) by drying at 37°C. Pooled mouse sera were incubated at different dilutions overnight at 4°C in PBSA. The wells were then incubated for 1 h at room temperature with a goat Ab to mouse {gamma}-globulin (1/10,000 dilution) absorbed with human {gamma}-globulin and conjugated with horseradish peroxidase (Sigma). The bound peroxidase activity was revealed by adding o-phenylenediamine dihydrochloride and H2O2. The isotype of Abs was determined by ELISA. Serum samples diluted in PBSA were incubated overnight at 4°C on coated plates. After washing, the plates were incubated with horseradish peroxidase-conjugated goat anti-mouse IgM, IgG1 (Caltag, San Francisco, CA), and IgG2a (Southern Biotechnology Associates, Birmingham, AL) for 2 h at room temperature. The assay was developed as indicated above. Ab titers were determined on the basis of the last dilution with an absorbance (A492) >=0.200. Tests were performed in duplicate.

Limiting dilution analysis (LDA)

As a source of APCs we used spleen cells from unprimed mice cultured with LPS/dextran (25 µg/ml) for 24 h and treated for 30 min at 37 °C with 25 µg/ml mitomycin C (Sigma). Before use, spleen cells from naive, primed, or primed and boosted, mice were mixed with 2 x 106/ml APC in 96-well flat-bottom plates in the presence of 50 µg/ml synthetic peptide (-NVDP-). Each dilution of cells was plated in replicates of 48. Supernatants were harvested after 36 h, and 20 µl from each culture was tested for IL-2 activity using the NK.3 cell line. Single cultures supernatants were considered positive when the value of [3H]thymidine incorporation was greater than the mean of the replicate control cultures with no Ag plus 2 SDs. Frequencies of cytokine-producing cells were calculated using the program described by Waldman (36) and were calculated using maximum likelihood analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of STI on activation of T lymphocytes

T cell responses were assessed using DNA coding for 12 amino acid determinants of the circumsporozoite protein of Plasmodium falciparum malaria parasite (37). The plasmid {gamma}1NV2NA3 DNA contains an Ig H chain gene in which the V domain is engineered to code for a Th cell determinant (NANPNVDPNANP) in CDR2 and a B cell epitope (NANPNANPNANP) in CDR3 (antigenized Ab) (29). The Th cell determinant (-NVDP-) and the B cell epitope differ by only two amino acid residues (A->V and N->D) in positions 5 and 6, respectively. Previously, we have shown that an antigenized Ab product of the same gene, when injected in CFA, induces specific T cell proliferation and IL-2 secretion (29).

Spleen cells harvested 7 days after a single intraspleen inoculation of 100 µg of {gamma}1NV2NA3 DNA proliferated in culture after restimulation with the antigenized Ab expressing the Th cell determinant or the corresponding 12 mer Th cell determinant peptide (Fig. 1GoA). Proliferation occurred when cells were cultured with the T, but not the B, cell peptide, demonstrating specific activation by the heterologous peptide in CDR2. Proliferation after culture with the antigenized Ab expressing -NVDP- also suggests that the CDR2 peptide within the Ab molecule is processed and presented by APC. When compared with the proliferative response of cells from mice immunized with the antigenized Ab in CFA, STI induced a response of similar or greater magnitude (not shown). Specific activation of T cells was accompanied by marked production of IL-2 (Fig. 1GoB). The lower amounts of IL-2 measured in cultures restimulated in vitro with the NVDP peptide most likely reflect a higher consumption, as cells in these cultures were proliferating to a greater extent.



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  		FIGURE 1. Ag-specific activation of T lymphocytes by STI. A, The proliferative response of spleen cells from C57BL/6 mice inoculated with plasmid DNA {gamma}1NANP coding for the B cell epitope (four mice), {gamma}1NV2NA3 coding for the B and T cell epitopes (four mice), or control plasmid pSV2neo (two mice), and harvested on day 7. Cells were cultured in the presence of the Ags indicated along the abscissa. Results refer to the stimulation index, expressed as the mean ± SD. Results correspond to two independent experiments. AgAb, Antigenized Ab. Antigenized Ab expressing the B cell epitope and that expressing the B and T cell epitopes were previously described (29). Tests were run in triplicate. B, IL-2 production in spleen cell cultures from the same C57BL/6 mice shown in A. Results are expressed as counts per minute of the proliferative response of indicator NK.3 cells and are expressed as the mean ± SD. Tests were run in duplicate.

 
The kinetics of T cell activation in vivo was monitored in another experiment in which spleen cells were harvested 3, 7, 14, and 21 days after DNA inoculation (Fig. 2Go). Proliferation was absent on day 3, peaked on day 7, and was minimal by day 21. The response was specific, i.e., measured only when cells were restimulated in vitro with the Th cell determinant peptide. The production of IL-2 paralleled the proliferative response (not shown).



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  		FIGURE 2. Kinetics of T cell activation in vivo. The proliferative response of spleen cells from C57BL/6 mice inoculated with plasmid DNA as described in Fig. 1Go. Spleen cells were harvested on days 3, 7, 14, and 21. Cells were cultured in the presence of the synthetic peptide corresponding to the Th cell determinant (upper panel) or the B cell epitope (lower panel), as a control. Results refer to the stimulation index. At each time point groups consisted of two mice for the DNA coding for heterologous epitopes and one inoculated with plasmid pSV2neo control. Tests were run in triplicate.

 
Splenocytes harvested on days 7 and 14 were also assayed for production of IFN-{gamma}, IL-4, and IL-5 to assess whether any polarization to type 1 and type 2 phenotypes had occurred (Fig. 3Go). Both IFN-{gamma} and IL-4 were detected, albeit in different amounts, and IL-5 was absent (data not shown). Since IFN-{gamma} sp. act. is, on the average, 100-fold lower than that of IL-4, and IL-4 is typically secreted in much lower quantities than IFN-{gamma}, we conclude that both cytokines are produced proportionally and that cells activated through STI remain, by and large, uncommitted.



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  		FIGURE 3. Levels of IFN-{gamma} and IL-4 during the primary response. Spleen cells harvested 7 and 14 days after immunization were incubated with synthetic peptide corresponding to the Th cell determinant (50 µg/ml) for 40 h. Supernatants from triplicate cultures were harvested and tested in capture ELISA specific for IFN-{gamma} or IL-4 as detailed in Materials and Methods.

 
Activated cells are CD4+ T lymphocytes

CD4+ T cells were formally identified as the cell population proliferating and making cytokines. Spleen cells from mice immunized 7 days earlier were depleted of CD4+ and CD8+ cells by treatment in vitro with mAbs specific for CD8 or CD4 plus complement. By flow cytometry the purities of the two populations were 94% (CD4) and 99% (CD8), respectively (Fig. 4Go, C and D). The two cell populations were then cultured in vitro with the addition of fresh APC from naive mice and synthetic peptide (-NVDP-). Proliferation occurred in the CD4+, but not in the CD8+, T cell population (Fig. 4GoE). Similarly, IL-2 production was detected only in the CD4+ T cell population (Fig. 4GoF). The results demonstrate that under these experimental conditions STI selectively activates CD4+ T lymphocytes.



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  		FIGURE 4. Activated cells are CD4+ T cells. Seven days after DNA inoculation spleen cell populations were prepared and depleted of CD8+ (C) or CD4+ (D) cells by Ab plus complement. Unseparated CD8+ cells (A) and unseparated CD4+ cells (B) are shown as a reference. The proliferative response (E) and IL-2 production (F) of unfractionated (total), separated CD4 and CD8, and reconstituted (CD4+CD8) T cell populations are shown. Stimulation indexes and IL-2 production are expressed as described in Fig. 1Go.

 
T cell immunity spreads to other secondary lymphoid organs

Germane to the present studies was to determine the extent to which priming induces generalized T cell activation. In a first set of experiments we monitored spreading of immunity to other secondary lymphoid organs measuring cell proliferation and IL-2 production in a pool of inguinal, mesenteric, and cervical lymph node cells. Seven days after DNA inoculation cells of the lymph node pool proliferated specifically upon restimulation in vitro with the -NVDP- but not with the B cell epitope peptide (Fig. 5GoA). When compared with spleen cells, proliferation in lymph nodes was of a lesser magnitude. On day 14 the magnitude of the response in lymph node cells increased markedly, reaching values comparable to those in spleen cells. On day 21 only residual proliferative activity existed in both lymph node and spleen cells. The magnitude and specificity of the proliferative responses were reflected by the levels of IL-2 in the corresponding culture supernatants (Fig. 5GoB). These kinetic analyses reveal, therefore, that T cell activation in lymph nodes parallels that in the organ in which the process of immunity was initiated.



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  		FIGURE 5. T cell immunity induced by intraspleen DNA inoculation spreads to lymph nodes. Cell proliferation (A) and IL-2 production (B) in a pool of inguinal, mesenteric, and cervical lymph node and spleen cells harvested 7, 14, or 21 days after {gamma}1NV2NA3 DNA inoculation. Lymph nodes were isolated from four mice per experiment. Serum transgenic Ig (nanograms per milliliter) in the serum is expressed as the mean ± SD of six different mice at each time point (C). Cell proliferation (D) and IL-2 production (E) of lymph nodes collected from 1) axillary, brachial, deep, and superficial cervical (upper); 2) mesenteric, renal, and epigastric (middle); and 3) popliteal, caudal, sciatic, and lumbar (lower) lymph nodes 14 days after DNA inoculation are shown. Lymph nodes were isolated from six mice.

 
Analysis of the tempo of these responses in relation to other parameters of STI revealed something interesting. When the ratio between the stimulation indexes in lymph nodes and spleen was calculated, it became evident that by day 14 T cell responsiveness in lymph nodes was prevalent. Moreover, the peak of the proliferative response in lymph nodes appeared to correlate with the peak values of transgenic Ig in the serum (Fig. 5GoC). The results indicate that a pattern of proportionality exists between secretion of transgenic Ig and spreading of T cell immunity.

Whether pooled lymph node cells were a true representation of a generalized response was further analyzed in lymph nodes collected according to precise anatomical distribution, i.e., lower (popliteal, caudal, sciatic, and lumbar), middle (mesenteric, renal, and epigastric), and upper (axillary, brachial, deep, and superficial cervical) lymph nodes. T cell proliferation and IL-2 production were measured 14 days after DNA inoculation (Fig. 5Go, D and E). As shown, both parameters were comparably elevated in all three lymphoid districts.

Effects of linked recognition of Th and B cell epitopes on the Ab response

Expression of B and Th cell epitopes in linked association in transgenic Ig is expected to produce quantitative and qualitative effects on the B cell response. First, we determined Ab titers during priming. Mice given the transgene coding for both the Th cell determinant and the B cell epitope produced consistently higher Ab titers than mice immunized with the B cell epitope-containing gene (Fig. 6GoA), a result in agreement with our previous data (29). Second, we determined that specific activation of Th cells by the -NVDP- determinant was sufficient to promote the IgM->IgG1 switch. Previously we had reported that during STI mice produce mainly IgM and low level IgG2a, but no IgG1 (24) unless the transgene is appropriately manipulated to increase the activation of dendritic cells (38). The results obtained in mice immunized with the transgene coding for the B cell epitope (Fig. 6GoB) are in agreement with earlier results. In contrast, mice given the Th/B double-epitope transgene developed IgM and IgG1 Abs (Fig. 6GoC). The presence of the Th cell determinant in the transgene did not affect the IgG2a response, which was minimal in both groups. This suggests that the concomitant local activation of CD4+ T cells and B lymphocytes drove secretion of downstream cytokines required for isotype switch in B cells. We conclude that T cell immunity triggered by the Th cell determinant in linked association with a B cell epitope optimizes the B cell response by heightening the Ab titer and promoting isotype switch. Since similar levels of IgM and IgG2a were observed regardless of the presence of the Th cell determinant, it can also be argued that the increased amount of specific Abs is due presumably to IgG1 Abs.



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  		FIGURE 6. Effect of linked recognition of Th and B cell epitopes on the Ab response. The titer (log) of B cell epitope-reactive Abs in mice inoculated with plasmid DNA coding for T and B epitopes (triangle), B cell epitope (square), or control plasmid (circle; A). Plasmid DNA and modalities of serum Ab testing are defined in Materials and Methods. The titer (log) of IgG1, IgM, and IgG2a Abs determined in ELISA in the sera of mice inoculated with plasmid DNA coding for the B cell epitope only (B) or with plasmid DNA coding for the B and T cell epitopes (C). Every symbol refer to a single mouse. All mice were tested on day 14. Tests were performed in duplicate.

 
Response to secondary exposure to Ag in vivo

A central feature of the programmed adaptive response is the establishment of immunologic memory as the ability of the immune or vaccinated host to mount, upon re-encounter with Ag, a faster and greater specific response than a naive host (39). Although some data exist regarding the enhanced capacity of memory cells to respond to Ag (40), primarily the magnitude and kinetics of secondary responses reflect the response of a larger number of Ag-specific cells (41). The existence of immunologic memory was therefore assessed by analyzing the frequency of peptide-reactive CD4+ T cell precursors using LDA (36). Frequencies were determined in mice given a booster immunization with antigenized Ab {gamma}1NV2NA3 (50 µg) in IFA 90–110 days after DNA priming. In light of the fact that memory T cells are greatest in number 4 days after booster immunization (42), LDA was performed in spleen cells harvested at this time.

The frequency of Ag-responsive T cells was much higher after booster immunization. The effect was not merely due to expansion of specific T cells by immunization with protein Ag in IFA because in the absence of DNA priming the frequency was about 3 times lower. For comparative purposes LDA studies were also performed 4 and 7 days after single DNA inoculation (Table IGo). On days 4 and 7 the frequencies were 1/90,200 (group II) and 1/50,500 (group III), respectively. Four days after priming with protein Ag in IFA the frequency was 1/60,000 (group VII). The average frequency during the memory response was 1/21,900, i.e., 2.5–4 times higher. Table IGo also shows that early after DNA priming Ag-responsive T cells were enriched 75-fold over naive precursors but dropped to 1/424,500 (group V) by day 110. Collectively, the results indicate that priming by STI establishes T cell memory. Re-encounter with Ag induced a faster and higher specific response.


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  		Table I. Frequency of CD4+ T cells specific for the Th determinant

 
The levels of cytokines produced by reactivated memory T are illustrated in Table IIGo. IFN-{gamma} was detected in half (two of four) of the animals, IL-4 was produced in all four instances, and IL-5 was detected in two cases only.


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  		Table II. Cytokines in the supernatant of memory cell cultures

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Foreign Ags reach the immune system and incite immunity via two general routes. Some Ags localize in the organ or tissue they first encounter (e.g., skin, airways, intestinal, and genital mucosae) and are sequestered by DC, which then migrate to secondary lymphoid organs (lymph nodes and spleen) (43). Other Ags gain access to the general circulation through the vascular system and are trapped in the white pulp of the spleen where they are captured by DC in the follicular zone and are presented to B and T lymphocytes (44). In either case the adaptive immune response effectively develops when Ags reach secondary lymphoid organs where similarities in the architecture and physiology of the microenvironment ensure that lymphocyte function follows general patterns (45). By directing the synthesis and expression of a transgene in secondary lymphoid organs we obtained the simultaneous activation of B and T lymphocytes in a way that mimics a feature central to all routes of immunization with exogenous Ags. Together with the fact that STI also limits the extent to which the Ag interacts with diverse tissue environments, it is easy to see how targeting transgenes to the site of immune induction offers advantages to immunogenicity in vivo (24, 46).

Although low amounts of soluble Ag are notoriously tolerogenic (8), our experiments clearly show that nanomolar amounts of soluble Ag are immunogenic if synthesis and expression of Ag occur in B lymphocytes resident in the microenvironment. Under these conditions both Ab responses and effector T cells are readily induced. Thus, STI represents an exception to this paradigm and is a new approach for qualitative and quantitative studies on adaptive immunity with characteristics at least in part different from those resulting from immunization with protein Ag or conventional plasmid DNA.

Encounter with Ag and sufficient costimulation transform naive T cells into effector cells, accompanied by clonal expansion and expression of cytokines other than IL-2. Early effector Th cells (Th0 cells) produce both Th1 (IFN-{gamma}) and Th2 (IL-4, IL-5) cytokines (33). As differentiation progresses through pressure by environmental cytokines and Ag, T cells can be polarized toward the Th1 or Th2 phenotype (47, 48), but in the absence of such selective pressure they remain uncommitted and retain production of all cytokines (49). In our study effector T cells produced IL-2, IFN-{gamma}, and IL-4, suggesting that priming via STI expands T cells that maintain an uncommitted phenotype, a surprising result in view of the fact that DNA vaccines are thought to polarize the response toward Th1 (50, 51). Clearly, IFN-{gamma} was produced at 200-fold higher levels than IL-4, but even fully polarized Th2 cells produce much lower levels of IL-4 than the levels of IFN-{gamma} produced by Th1 cells (49, 52). Additional considerations favor lack of polarization. Firstly, IgG2a and IgG1 were both produced during priming, albeit at different levels (Fig. 3Go). However, when specific Ab responses were compared and relative rates of increase after booster were measured, it appears that IgG2a and IgG1 (4.1- and 4.2-fold increases) underwent comparable increases. IgM Abs only increased by a factor of 2, in agreement with our previous results (24). Secondly, we found no evidence of Th1 polarization in memory T cells. Since polarized primary effector T cells can retain their cytokine profile when reverting to memory cells (53), we conclude that in all likelihood STI does not bias either the primary or the secondary T cell response to secretion of a particular pattern of cytokines.

The spreading of T cell responsiveness from the spleen to lymph nodes throughout the body is the second original feature of our findings. As a rule, immunization with Ag in adjuvant activates specific T cells only in the lymph nodes proximal to the site of injection. Recent studies using adoptive transfer of TCR-transgenic T cells clearly showed that s.c. immunization with Ag in adjuvant attracts specific T cells only in the draining, not in the nondraining, lymph nodes (1). Thus, under conventional immunization procedures migrating T cells are sequestered in the draining lymph nodes by Ag transported by dendritic cells or macrophages via the lymphatics. In the present study T cell responsiveness in distal lymph nodes began approximately at the same time (day 7) as in the spleen and peaked on day 14 (Fig. 5GoA). Interestingly, maximal T cell responsiveness occurred when transgenic Ig were most abundant in the serum. The results suggest a model in which transgenic Ig are released into the circulation, undergo localization in the cortex of distal lymph nodes, and serve as an anchor for T cells. Whether T cells activated in lymph nodes derive from recirculating effector T cells or from naive CD4+ T lymphocytes undergoing de novo activation is not known. The first possibility is plausible, since the observed kinetics are consistent with the idea that effector T cells leave the site where they encountered Ag within 48 h and recirculate through the body in 24 h (54). The second possibility, de novo activation, is consistent with the fact that antigenized Ig clearly supported T cell activation in vitro (Fig. 1Go).

Overall the studies establish that STI, and DNA vaccination in general, are effective ways to activate CD4 T cells and establish durable T cell memory. The frequency of Ag-reactive T cells increased 3- to 4-fold in a long term primed animal and again severalfold after booster immunization. In addition, the response was faster that the primary response, consistent with a functional definition of immunologic memory (39). In all likelihood, early effector T cells gave rise to resting memory cells, which are known to recirculate as a pool through spleen and lymph nodes until they are sequestered again by Ag 24–48 h later (55). Surprisingly, the cytokines produced by reactivated memory T cells did not follow the pattern observed during priming. IFN-{gamma} was detected in half (two of four) of the animals, IL-4 was produced in all four instances, and IL-5 was detected in two cases only (Table IIGo). This suggests that the characteristics of priming are not maintained during the memory response unlike in the response against complex protein Ags during which IL-4 was found to increase up to 90-fold without an actual increase in the number of IL-4-producing cells (56). After booster immunization we observed a marked increase in IgG2a and IgG1 Abs. The apparent dichotomy between Ig isotypes and the cytokines produced may simply reflect the fact that in vivo the effects of IFN-{gamma} on immune regulation are short lived, and other factors may determine the outcome of a memory response as observed by others (57).

The importance of these findings is far reaching, since STI, as a working principle, is possibly one of the few methods, and certainly the simplest one, to incite an adaptive response closely mimicking immunity triggered by pathogens, tumor cells, or self Ag, (e.g., the rate of synthesis of the Ag and its endogenous origin). The possibility to induce effector Th cells reproducibly and without immune deviation offers advantages vis-à-vis its general applicability and the possibility to imprint the phenotypic characteristic of the developing immune response via ad hoc modifications of the transgene.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI36467 (to M.Z.) and AI36259 (to M.C.). Back

2 Address correspondence and reprint requests to Dr. Maurizio Zanetti, Department of Medicine and Cancer Center, University of California-San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0368. E-mail address: Back

3 Abbreviations used in this paper: STI, somatic transgene immunization; H chain, heavy chain; PBSA, PBS containing 1% BSA and 1% Tween 20; LDA, limiting dilution analysis. Back

Received for publication October 19, 1998. Accepted for publication December 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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