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The Journal of Immunology, 2001, 167: 3592-3599.
Copyright © 2001 by The American Association of Immunologists

Plasmid DNA Encoding CCR7 Ligands Compensate for Dysfunctional CD8+ T Cell Responses by Effects on Dendritic Cells1

Seong Kug Eo2, Udayasankar Kumaraguru2 and Barry T. Rouse3

Laboratory of Viral Immunology, Department of Microbiology, University of Tennessee, Knoxville, TN 37996


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lymphotoxin {alpha}-deficient (LT{alpha}-/-) mice, which lack lymph nodes and possess a disorganized spleen, develop dysfunctional CD8+ T cells upon HSV infection and readily succumb to herpes encephalitis. Such mice do develop apparently normal peptide-specific CD8+ T cell responses, as measured by MHC class I tetramer staining, but the majority of cells fail to become cytotoxic or express peptide-induced IFN-{gamma} production. In the present study, we demonstrate that functional defects of CD8+ T cells in LT{alpha}-/- mice can be largely rectified by the administration of plasmid DNA encoding CCR7 ligands before HSV infection. Treated mutant mice developed increased peptide-specific cytotoxic responses, enhanced numbers of CD8+ T cells capable of producing IFN-{gamma}, as well as improved resistance to HSV challenge. The corrective effect of chemokine treatment appeared to result from improved dendritic cell-mediated Ag presentation. Thus, a major consequence of the treatment was an increase in splenic dendritic cell number in CCR7 ligand-treated LT{alpha}-/- mice with such splenocyte populations showing improved APC activity in vitro. Our results document that functional defects of CD8+ T cells can be corrected, and indicate the value of plasmid vector encoding appropriate chemokines to achieve such immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD8+ T cells act as important effectors in defense against several viruses and certain tumors. Recently, with the development of multiple assays to detect and quantify CD8+ T cells, it has become evident that such cells may be functionally heterogeneous. Several examples have now been described in which cells detectable by novel sensitive assays such as avidin-linked MHC tetramers show incomplete activation markers and lack one or more functions responsible for effector activity (1, 2, 3). Examples have been described for persistent virus infections, especially under circumstances in which animals have some form of immune suppression (4, 5, 6). One of the earliest reports came from the Ahmed (5) laboratory, which observed that the minor specificity of lymphocytic choriomeningitis virus-specific CD8+ T cells in persistently infected helper cell-deficient mice failed to express cytotoxicity or to produce IFN-{gamma}. Such cells were termed "Sisyphean" to describe their effector cell futility. Other examples of effector cell dysfunction were observed in SIV infection in macaques (6, 7) and in the brains of mice infected with coronavirus (8). In the cancer field, too, CD8+ T cells may be detectable by MHC class I tetramer-binding assays, yet such cells lack effector function (9). In all instances in which dysfunctional CD8+ T cell effectors were noted, no attempts were made to correct such defenses and to observe the outcome of such maneuvers.

Our laboratory recently reported that the CD8+ T cell response in lymphotoxin {alpha}-deficient (LT{alpha}-/-)4 mice may also fail to functionally mature upon infection with HSV or immunization with OVA protein (10). Such mice were highly susceptible to challenge with HSV as previously observed with some other pathogens (11, 12, 13). The reasons for their increased susceptibility to infection probably relate to the fact that LT{alpha}-/- mice have defective lymphoid tissue (14, 15, 16, 17, 18, 19). Accordingly, they lack lymph nodes and Peyer’s patches, and their splenic white pulp is disorganized in structure and contains greatly reduced numbers of mature dendritic cells (DCs) (20). LT{alpha}-/- spleens also lack normal expression of certain chemokines such as CCR7 ligands (21). Because these chemokines are known to mediate the interaction between mature DCs and naive T cells (22, 23, 24), it could be that their reduced expression in LT{alpha}-/- spleens accounts for the dysfunctional CD8+ T cell response observed in such animals. Consequently, we reasoned that if LT{alpha}-/- mice were given expression plasmids encoding CCR7 ligands, CD8+ T cell function could be restored. Our results support this notion, although functional restoration was not fully complete. Accordingly, systemic administration of plasmid DNA encoding either CCL21 (formerly secondary lymphoid tissue chemokine) or CCL19 (formerly EBV-induced molecules 1 ligand chemokine) to LT{alpha}-/- mice before infection with HSV resulted in enhanced CD8+ T cell responses. More importantly, such responses were functionally superior to those of control animals, as measured by cytotoxicity and immunodominant epitope peptide-induced intracellular IFN-{gamma} production. In addition, treated animals were more resistant to HSV-induced encephalitis. Our novel observations document that dysfunctional CD8+ T cell responses can be corrected, and indicate the value of expression plasmids encoding appropriate chemokines to achieve this objective. The mechanisms by which chemokine immunotherapy most likely functions to compensate for the immune defects in LT{alpha}-/- mice were also described.


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

Female 5- to 6-wk-old C57BL/6 (H-2b) mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed in the animal facilities at the University of Tennessee. LT{alpha}-/- mice were maintained on a pure C57BL/6 background, as described (15). All investigations follow guidelines of the Committee on the Care of Laboratory Animals Resources, Commission on Life Science, National Research Council. HSV-1 strain KOS was grown in Vero cells obtained from American Type Culture Collection (Manassas, VA). The viruses were concentrated, titrated, and stored in aliquots at -80°C until use. Titers were measured in Vero cells and expressed as PFU per milliliter.

Synthetic peptide

HSV gB498–505 peptide (SSIEFARL) specific for MHC class I (H-2b)-restricted CD8+ T lymphocytes (25, 26) was chemically synthesized, purified, and quantitated by Genemed Synthesis (South San Francisco, CA).

Plasmid DNA preparation

Plasmid DNA encoding CCL21 or CCL19 was kindly provided by A. Zlotnik (DNAX Research Institute, Palo Alto, CA) and J. G. Cyster (University of California, San Francisco, CA), respectively, and then inserted into the pCI-neo eukaryotic expression vector (Promega, Madison, WI). Plasmid DNA encoding gB (gB DNA) of HSV-1 KOS under the CMV promoter has been described in detail elsewhere (27). The plasmid DNAs were purified by polyethylene glycol precipitation, as described previously (28).

Immunization

Groups of wild-type (wt) and LT{alpha}-/- mice (5- to 6-wk-old female mice) previously given 200 µg of plasmid DNA encoding CCR7 ligand via the i.m. route were immunized with a sublethal dose (106 PFU) of live HSV-1 KOS in the hind footpad 5 days later. In some experiments, wt and LT{alpha}-/- mice were injected i.p. with plasmid DNA encoding CCR7 ligand to preferentially direct plasmid DNA into spleen, and then 5 days later immunized with live HSV-1 KOS in the hind footpad. The control mice were injected with 200 µg of empty pCI-neo control vector.

SSIEFARL-specific CD8+ T cell proliferation

CD8+ T cell proliferation was evaluated following in vitro restimulation of splenocytes with MHC class I (H-2b)-restricted SSIEFARL peptide. Briefly, the splenocytes collected from immunized mice were in vitro restimulated with irradiated SSIEFARL peptide (5 µg/ml)-pulsed syngeneic splenocytes for 3 days. [3H]Thymidine (1 µCi/well) was added to each well 18 h before harvest. Harvested cells were measured for radioactivity using a beta scintillation counter (Inotech, Lansing, MI). Con A (5 µg/ml) was used as a polyclonal stimulator, a positive control for lymphoproliferation assay.

CTL activity

CTL activity was assessed by a standard 5-h 51Cr release assay against labeled target cells, as previously described (29). Splenic T cells obtained from immunized mice were in vitro restimulated with SSIEFARL peptide (5 µg/ml)-pulsed syngeneic splenocytes for 5 days and then used as effector cells. The effector cells were then mixed at various ratios with 51Cr-labeled target cells for 5 h. Target cells included SSIEFARL-pulsed MHC-matched EL4 (H-2b), mismatched EMT6 (H-2d), and unpulsed EL4. Spontaneous release of 51Cr was determined by incubating the target cells with medium alone, and maximum release was determined by adding Triton X-100 to a final concentration of 5%. The percent specific lysis was calculated as follows: 100 x ((experimental release - spontaneous release)/(maximum release - spontaneous release)). Each experiment was performed twice using triplicate samples.

FACS analysis

The following mAbs obtained from BD PharMingen (San Diego, CA) were used for FACS analysis: FITC anti-CD8{alpha}; PE-labeled anti-CD4, CD62 ligand (CD62L), CD44, CD25, CD40 ligand (CD40L), and CD69; PE anti-CD11c; FITC anti-CD11b; FITC anti-MHC II (I-Ab {alpha}-chain); biotinylated anti-TCR V{beta}10; PE-labeled IgG2a; FITC- and PE-labeled IgG2b; and PerCP-labeled streptavidin. For staining, cells were resuspended with PBS containing 1% BSA and 0.05% NaN3 at a concentration of 106–107 cells/ml, followed by incubation at 4°C for 30 min with properly diluted mAb. After staining, the cells were washed twice by spinning at 1200 rpm, 4°C for 5 min. Following refixation, the cells were resuspended in PBS and analyzed on FACScan cytometer using CellQuest software (BD Biosciences, Mountain View, CA).

MHC class I tetramer staining and FACS sorting

MHC class I (H-2b) tetramers containing CTL immunodominant peptide (SSIEFARL) of HSV gB were made available to us by Dr. S. S. Tevethia (30). A total of 1 x 106 cells was suspended in FACS buffer and stained for surface marker with a mixture of Ab that included the tetramers. They were incubated for 45 min to 1 h and washed and analyzed using BD Biosciences hardware and software. To isolate SSIEFARL-specific CD8+ T cell binding to tetramer, cells stained with tetramers were sorted by FACS sorter (FACSVantage).

Intracellular cytokine staining

To enumerate the number of IFN-{gamma}-producing T cells, intracellular cytokine staining was performed as previously described (31). In brief, 106 freshly explanted splenocytes were cultured in flat-bottom 96-well plates. Cells were left untreated, stimulated with SSIEFARL peptide (1 µg/ml), or treated with PMA (10 ng/ml) and ionomycin (500 ng/ml), and incubated for 6 h at 37°C in 5% CO2. Brefeldin A (10 µg/ml) was added for the duration of the culture period to facilitate intracellular cytokine accumulation. After this period, cell surface staining was performed, followed by intracellular cytokine staining using a Cytofix/Cytoperm kit (BD PharMingen), in accordance with the manufacturer’s recommendations. For intracellular cytokine IFN-{gamma} staining, the Ab used was anti-IFN-{gamma} (clone XMG1.2). The fixed cells were resuspended in PBS and analyzed with CellQuest.

Immunohistochemistry and histology

Spleens were harvested, embedded in OCT compound (Miles, Elkhart, IN), and frozen at -70°C. Frozen sections (6–10 µm thick) were fixed in cold acetone. Endogenous peroxidase was quenched with 0.2% H2O2 in methanol. The sections were then blocked with 3% BSA and stained by first incubating with biotinylated anti-mouse CD11c. Avidin-biotin complex reagent (ABC kit; Vector Laboratories, Burlingame, CA) was added 1 h later, according to the manufacturer’s instructions. All of the sections were then treated with aminoethyl carbozole substrate (Zymed Laboratories, San Francisco, CA), counterstained with hematoxylin, coverslipped with aquamount (Lerner Laboratories, Pittsburgh, PA), and examined. As a negative control, irrelevant biotinylated rat Abs and normal rabbit serum were used. For identification of spleen architecture, spleens were fixed in 10% neutral buffered Formalin solution and embedded in paraffin. Sections were cut and stained with H&E.

Virus challenge

Five- to 6-wk-old wt and LT{alpha}-/- mice pretreated with control vector or CCR7 ligand were injected i.m. with a lethal dose (5 LD50) of HSV-1 KOS. They were examined daily starting from day 3 for signs of HSV infection that included mobility, wasting, limb paralysis, and encephalitis.

Statistics

Significant differences between groups were evaluated using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LT{alpha}-/- mice develop functionally inferior CD8+ T cell responses correctable by CCR7 ligands

As shown in Table IGo, following footpad infection of wt and LT{alpha}-/- mice with a sublethal dose (106 PFU) of HSV-1 KOS, splenocytes collected 14 days after infection possessed similar numbers of SSIEFARL-specific CD8+ T cells following in vitro stimulation with peptide (5.2% for wt and 4.3% for LT{alpha}-/-). However, when testing tetramer-positive sorted cells for intracellular IFN-{gamma} production after brief peptide stimulation, whereas 82.4% of tetramer-positive wt cells scored positive, only 10.6% of tetramer-positive LT{alpha}-/- cells produced IFN-{gamma} (Table IGo). Furthermore, when measuring peptide-specific CTL activity, the response of LT{alpha}-/- splenocytes (4 LU) was markedly less than was evident in wt splenocyte populations (81 LU) (Table IGo). Similar data were reported in a previous publication (10), in which it was suggested that the functional defect was the consequence of inappropriate Ag presentation by a disorganized spleen (14, 15, 16, 17, 18, 19). Moreover, LT{alpha}-/- spleens are known to have a deficiency in DC content (20).


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  		Table I. Tetramer-positive CD8+ T cells producing IFN-{gamma} and CTL activity of in vitro expanded splenocytes collected from wt and LT{alpha}-/- mice

 
Because chemokines such as CCR7 ligands are known to facilitate interaction between DCs and naive T cells in lymphoid tissue (22, 23, 24), the effects of pretreatment with such chemokines on the quantity and quality of the immune responses were evaluated. Both LT{alpha}-/- and wt mice were given plasmid DNA encoding either CCR7 ligands or controls (vector or the chemokine CCL3, formerly macrophage-inflammatory protein 1{alpha}) 5 days before infection with HSV. Fourteen days after infection, splenocytes collected from sacrificed animals were analyzed. Because the majority of SSIEFARL-specific CD8+ T cells in mice of the B6 background express V{beta}10 TCR (26, 32), the number of such cells in spleens, as well as their activation status, was measured in the various groups of mice. The data, shown in Table IIGo, indicate that mice pretreated with either of the CCR7 ligands had increased number of splenic V{beta}10+ CD8+ T cells. The increase was particularly evident in LT{alpha}-/--pretreated animals (Table IIGo). In such animals treated with CCL21 or CCL19, total splenic V{beta}10+ CD8+ T cell numbers were increased 8- to 9-fold compared with controls given empty vector DNA. Modest increases were also evident in wt mice given CCR7 ligand DNA (5- to 6-fold). The effect of the CCR7 ligand pretreatment had no effect on the ratio of CD4+ to CD8+ T cells (Table IIGo). The activation status of V{beta}10+ CD8+ T cells for CD62L, CD44, CD25, CD40L, and CD69 was also measured in untreated and treated LT{alpha}-/- mice. As is evident in Fig. 1Go, whereas the majority of LT{alpha}-/- V{beta}10+ CD8+ T cells in control vector-treated animals retained the nonactivation phenotype, following CCL21 or CCL19 pretreatment the percentage of such cells expressing activation markers increased, and appeared similar in number to those of wt animals.


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  		Table II. Summary of numbers of lymphocyte subtypes in splenocytes collected from wt and LT{alpha}-/- mice infected with HSV

 


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  		FIGURE 1. Expression of activation markers on T cells gated with double-positive V{beta}10+CD8+ in the spleen of CCR7 ligand-pretreated LT{alpha}-/- mice. Splenocytes of control vector or CCR7 ligand-pretreated wt and LT{alpha}-/- mice were collected at day 14 post HSV infection, and then stained with anti-mouse CD8 and anti-mouse V{beta}10 in combination with one of the activation markers, including anti-CD62L, CD44, CD25, CD40L, CD69, and its isotype control using three-color analysis. The gray lines represent activation phenotypes for control vector-treated wt mice, and the black area represents those for mutant mice. The isotype controls were not represented. The graphs are a representation of four different mice.

 
To measure SSIEFARL peptide-specific CD8+ T cell responses in the various groups of mice, equal aliquots of splenocytes were stimulated in vitro with peptide-pulsed syngeneic splenocytes from normal uninfected mice. After 5 days, cultured cells were harvested to measure SSIEFARL-specific CD8+ T cell responses. The results recorded in Table IIIGo indicate that CCR7 ligand preadministration acted as an adjuvant increasing the number of tetramer-positive CD8+ T cells detectable in cultures from treated LT{alpha}-/- mice. In such mice given the CCR7 ligands, the increased response was of the order of 3-fold (Table IIIGo). Interestingly, the CD8+ T cells from CCR7 ligand-pretreated LT{alpha}-/- mice also showed significant changes in function. Accordingly, pretreatment with CCL21 or CCL19 resulted in increased percentages of tetramer-positive cells that produced intracellular IFN-{gamma} upon brief peptide stimulation (Table IIIGo). Responses were increased from ~10% to ~30% (Table IIIGo). However, these values were still less than ~80% IFN-{gamma}+ tetramer-positive cells observed in both untreated or CCR7 ligand-treated wt mice (Table IIIGo).


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  		Table III. The numbers of tetramer-positive cells producing IFN-{gamma} and CTL activity recovered in in vitro expanded splenocytes of wt and LT{alpha}-/- mice pretreated with CCR7 ligands

 
Changes in function were also evident in CTL activity following CCR7 ligand pretreatment. As is evident in the experiment in Fig. 2Go, the CTL activity of CCR7 ligand-pretreated LT{alpha}-/- mice was almost restored to that observed in the wt population. In other experiments, the levels of restoration were of the order of ~60–70% (data not shown).



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  		FIGURE 2. The CTL activity of SSIEFARL-specific CD8+ T cells in in vitro expanded splenocytes of CCR7 ligand-pretreated wt and LT{alpha}-/- mice. Splenocytes collected from wt or LT{alpha}-/- mice pretreated as described in Materials and Methods were expanded in vitro with SSIEFARL-pulsed syngeneic splenocytes for 5 days and used as effectors in a 5-h 51Cr release CTL assay. The targets include SSIEFARL-pulsed MHC-matched EL4 (H-2b), mismatched EMT6 (H-2d), and unpulsed EL4. The data show 51Cr release for peptide-pulsed EL4 targets. The experiment was performed using six mice, and figures are the average of such experiments.

 
Consequence of CCR7 ligand DNA administration on splenic DCs

The splenic architecture in LT{alpha}-/- mice is disorganized with the content of APC, particularly DCs, less than in normal mice (14, 15, 16, 17, 18, 19, 20). Because receptors for the CCR7 ligand are present on mature DCs and naive T cells (22, 23, 24), the immunostimulatory effects of the chemokine pretreatment were anticipated to be explained by increases in cell numbers and interaction between such cells in lymphoid tissue, especially the spleen. Seven days after administration of either CCL21 or CCL19, the spleens of treated mice had increased in total cell numbers (~2- to 3-fold), as well as increases in both total T cells and especially DCs. Thus, as shown in Fig. 3GoA, the content of CD11chigh class IIhigh cells per spleen in control vector-treated LT{alpha}-/- mice was 0.7%. This contrasts with values of 2.8% and 3.1% in CCL21- and CCL19-treated LT{alpha}-/- mice, respectively (Fig. 3GoA). CCR7 ligand treatment also significantly increased the content of mature DCs in the spleen of wt mice (Fig. 3GoA).



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  		FIGURE 3. CCR7 ligands restore the reduced number of mature DCs in the secondary lymphoid tissue of LT{alpha}-/- mice. Splenocytes were collected from control vector or CCR7 ligand-treated wt and LT{alpha}-/- mice using collagenase digestion 7 days later and used for analysis. A, Determination of mature DC numbers in wt and LT{alpha}-/- mice following treatment of CCR7 ligand. Splenocytes were stained for the DC marker (CD11c) and class II marker (I-Ab {alpha}-chain), as indicated. B, Number of both lymphoid and myeloid DC subsets in CCR7 ligand-treated or untreated LT{alpha}-/- mice. The splenocytes were stained for DC subsets with CD11b/CD11c and CD11c/CD8{alpha}, as indicated.

 
To study whether pretreatment of CCR7 ligand preferentially regulates the migration of one or the other of DC subset, FACS analysis was used to compare the content of both myeloid (CD8{alpha}-CD11b+) and lymphoid (CD8{alpha}+CD11b-) DC subsets in untreated and treated LT{alpha}-/- mice. The ratio of myeloid to lymphoid DCs in LT{alpha}-/- spleen was unchanged by treatment, and in fact was approximately the same as observed in wt mice (Fig. 3GoB). Hence, the consequence of CCR7 ligand administration appeared to increase the quantity of DCs in spleen, but not to change the subset ratio.

In Fig. 4Go, the data demonstrate that the splenic cells from CCR7 ligand-treated mice had superior APC function to that of control vector-treated mice. To demonstrate this, spleens from both wt and LT{alpha}-/- mice were subjected to collagenase digestion to completely release DCs from the architecture of spleen. The collagenase-treated peptide-pulsed splenocytes from untreated or treated wt and LT{alpha}-/- mice were then used as stimulators for enriched gB DNA-primed T cells. In such experiments, the population of CCR7 ligand-treated splenocytes induced higher peptide-specific CD8+ T cell proliferation and CTL responses than that of control vector-treated LT{alpha}-/- splenocytes (Fig. 4Go, A and B). Interestingly, the increased APC activity of CCR7 ligand-treated LT{alpha}-/- splenocytes was ~3- to 4-fold, correlating closely with the content of extra-DCs in the treated mutant population (Fig. 4Go, A and B). These results indicate that CCR7 ligand administration increases APC activity, and that this effect may be dependent on the increased number of DCs induced by CCR7 ligand treatment.



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  		FIGURE 4. CCR7 ligand treatment restores the diminished Ag-presenting capacity of LT{alpha}-/- spleens. A, Proliferation of SSIEFARL-specific CD8+ T cells. Enriched T cells isolated from spleen of gB DNA-immunized mice were in vitro expanded with collagenase-digested splenocytes of CCR7 ligand-treated wt or LT{alpha}-/- mice following SSIEFARL peptide stimulation, and then incubated with [3H]thymidine for the final 18 h. B, CTL activity of SSIEFARL-specific CD8+ T cells. gB DNA-primed T cells were in vitro expanded for 5 days, as described above, and used as effectors in a 5-h 51Cr release CTL assay. The figures show one representative experiment of four experiments performed.

 
Histological consequence of CCR7 ligand administration on splenic architecture

With the observation that CCR7 ligand administration restored immunocompetence and in vitro APC activity, ectopic expression of CCR7 ligand appears sufficient to trigger lymphoid neogenesis (33). To assess the influence of CCR7 ligand administration on the disorganized lymphoid tissue, the architecture of the spleen was analyzed histologically. As is evident in Fig. 5Go, CCR7 ligand treatment of LT{alpha}-/- mice appeared to have no discernable effect on the disorganized splenic structure. However, when the distribution and number of DCs were visualized immunohistologically, the density of DCs distributed in spleens of CCR7 ligand-treated LT{alpha}-/- mice was clearly increased (Fig. 5Go). This result correlated with the FACS analysis indicating increased percentages of DCs in the spleen of CCR7 ligand-treated LT{alpha}-/- mice.



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  		FIGURE 5. The splenic architecture and the distribution of CD11c+ DCs in CCR7 ligand-treated wt and LT{alpha}-/- mice. The architecture of spleen was visualized by staining with H&E (magnification, x40). For determining the number and distribution of CD11c+ DCs in the spleens, the frozen spleen sections were stained for anti-CD11c Ab for DCs, as described in Materials and Methods (magnification, x100). Arrows point to brown color-stained CD11c+ DCs.

 
The change of resistance to HSV systemic challenge in CCR7 ligand-treated LT{alpha}-/- mice

Immunity to HSV involves aspects of both CD4+ and CD8+ T cell immunity (34, 35, 36, 37, 38). However, protection of the peripheral and CNS appears to be mainly a property of CD8+ T cells (37, 38). Because gB498-505 peptide-specific CD8+ T cell number and function were elevated following CCR7 ligand pretreatment of LT{alpha}-/- mice, such animals were expected to be more resistant to encephalitis following HSV systemic challenge. To evaluate the resistance of CCR7 ligand-pretreated LT{alpha}-/- mice against HSV systemic infection, LT{alpha}-/- mice previously given control vector or CCR7 ligand DNA were infected i.m. with lethal dose (5 LD50) of HSV-1 KOS. As shown in Table IVGo, whereas control vector-pretreated LT{alpha}-/- mice showed no protection, CCR7 ligand pretreatment of LT{alpha}-/- mice elicited resistance up to 67% for CCL21 and 83% for CCL19 at day 10 postchallenge. However, the resistance level of CCR7 ligand-pretreated LT{alpha}-/- mice was still less than that of control vector-pretreated wt mice (Table IVGo). These results indicate that CCR7 ligand pretreatment results in heightened resistance to HSV-induced encephalitis.


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  		Table IV. Resistance of CCR7 ligand-pretreated LT{alpha}-/- mice to HSV systemic challenge

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Functionally intact CD8+ effector T cells may mediate immunity in several ways. These include target cell killing following direct contact and immunity mediated by secreted cytokines such as IFN-{gamma} (39, 40). Under certain circumstances, such as during some persistent virus infections or limited T cell help, CD8+ T cells may be present that possess languid effector function (4, 5, 6, 7, 8). As noted previously, LT{alpha}-/- mice, which lack lymph nodes and possess a disorganized spleen, develop dysfunctional CD8+ T cells upon HSV infection and readily succumb to herpes encephalitis (10). Such mice do develop apparently normal peptide-specific CD8+ T cell responses, as measured by MHC class I tetramer staining, but the majority of cells fail to become cytotoxic or express peptide-induced IFN-{gamma} production. In the present study, we demonstrate that functional defects of CD8+ T cells in LT{alpha}-/- mice can be largely rectified by the administration of plasmid DNA encoding CCR7 ligands before HSV infection. Treated mutant mice developed increased peptide-specific cytotoxic responses, enhanced numbers of CD8+ T cells capable of producing IFN-{gamma}, as well as improved resistance to HSV challenge. The corrective effect of chemokine treatment appeared to result from improved DC-mediated Ag presentation. Thus, a major consequence of the treatment was an increase in splenic DC number in CCR7 ligand-treated LT{alpha}-/- mice with such splenocyte populations showing improved APC activity in vitro. Our results document that functional defects of CD8+ T cells can be corrected, and indicate the value of plasmid vector encoding appropriate chemokines to achieve such immunotherapy.

The development of multiple assays to identify specific T cell reactivity has revealed that such cells may be functionally heterogeneous. Several examples have now been described in which cells detectable by sensitive assays, such as avidin-linked MHC tetramers, may lack one or more functions, as measurable by cytotoxicity, cytokine production, or proliferation (1, 2, 3). An early example was described by the Ahmed group (5), which coined the term "Sisyphean" to describe their effector cell futility. Such cells were present in helper cell-deficient mice persistently infected with lymphocytic choriomeningitis virus. A similar circumstance was observed in macaques persistently infected with SIV and that had suppressed CD4+ Th cell function (7). In this instance, the functional defect was correctable in vitro by prolonged in vitro culture in IL-2 (7). These and other examples of functionally defective CD8+ T cells occur in conditions of extended Ag exposure and may represent examples in which cells differentiate normally, but are subsequently anergized (41). Such a situation may not be the case in LT{alpha}-/- mice used in the present study. These animals generate CD8+ T cell responses to HSV infection or to OVA protein that numerically, as detected by MHC class I tetramer staining, were similar to the responses of normal mice (10). However, most of the cells lack functions such as cytotoxicity and peptide-induced IFN-{gamma} expression. In this instance, the dysfunctional effector phenotype most likely indicates a failure of maturation rather than the induction of anergy in previously normal cells (42).

This failure of CD8+ T cells to functionally mature may reflect the fact that LT{alpha}-/- mice have abnormal secondary lymphoid tissue, lacking lymph nodes and possessing architecturally disorganized spleens (14, 15, 16, 17, 18, 19). In such tissue, immune induction appears inadequate, perhaps the consequence of LT{alpha}-/- spleens lacking normal numbers of DCs, the major cell type involved in Ag presentation in primary responses (20). Thus, as reported by others and confirmed in this study, LT{alpha}-/- spleens have markedly reduced number of DCs, although the DCs present appeared to represent a normal ratio of myeloid to lymphoid subsets. Others have suggested that DC recruitment to the spleen is driven by membrane-expressed LT{alpha}, this in turn perhaps driving chemokine expression by as yet unidentified producer cells (20). Furthermore, certain chemokines appear to be minimally expressed by LT{alpha}-/- spleens (21). These include the B cell-attracting chemokine CXCL13 (formerly called BCA-1), as well as the two CCR7 ligands, CCL21 and CCL19 (21). These latter chemokines may function to attract naive T cells, as well as mature DCs (22, 23, 24). Accordingly, any deficiency in CCR7 ligand production could explain the reduced accumulation of mature Ag-presenting DCs in LT{alpha}-/- spleen.

Our observations support such ideas and extend them by showing that injection of plasmid DNA encoding CCR7 ligand into LT{alpha}-/- mice results in a major increase (4- to 5-fold) in the numbers of mature DCs in LT{alpha}-/- spleens. The reconstituted DCs included both myeloid and lymphoid subtypes, with ratio between them approximately the same as observed in wt spleen. Thus, the corrective effect of CCR7 ligand administration on DC activity could be mainly quantitative, rather than qualitative. In support of this idea, a comparison of in vitro APC activity of splenocytes from LT{alpha}-/- mice treated with control vector or CCR7 ligand revealed that the greater efficacy of the latter population roughly correlated with the increased percentages of DCs.

Most importantly, CCR7 ligand-pretreated mice developed numerically and functionally improved CD8+ T cell responses against the HSV immunodominant epitope peptide SSIEFARL compared with control vector-treated mice. In CCR7 ligand-treated animals, the V{beta}10+ subset of CD8+ T cells, which contains the majority of SSIEFARL-specific CD8+ T cells (26, 32), was increased in number, and more cells showed activation phenotypes than was evident in control vector-treated mice. Upon expansion in vitro, a necessary procedure to demonstrate tetramer-positive cells as well as CTL specific for epitope peptide SSIEFARL (30), LT{alpha}-/- CD8+ T cells from treated mice were markedly more cytolytic, and significantly more cells produced IFN-{gamma} following brief peptide stimulation. Such responses were of practical consequence because the outcome of immune correction was that treated animals were significantly more resistant to viral challenge than were control vector-treated animals.

Accumulating numbers of observations document that T cell responses may be induced that lack one or more effector functions, explaining in some circumstances susceptibility to infection or neoplasia (4, 5, 6, 7, 8, 9). Few of any previous reports used measures to correct such functional defects. Our studies do achieve this objective and demonstrate significant immune reconstitution. Our study also emphasizes the value of systemic administration of expression plasmids encoding appropriate chemokines, as a valuable means of achieving immunotherapy. In our investigation, although significant immune correction was accomplished, perhaps of no surprise, the effect was incomplete and in need of improvement. It appears likely that improvement could come as a result of more appropriate chemokine expression in relevant tissues, or could be achieved by using additional means of corrective immunotherapy that optimize T cell maturation in LT{alpha}-/- mice. Regarding the former, it is not clear whether the plasmid DNA used in our study was optimally expressed in appropriate cells in the spleen. As shown in other studies using plasmids encoding marker proteins, i.m. administered plasmids result in significant and quite prolonged expression in the spleen (43). This includes expression in DCs themselves, perhaps an unnecessary event to achieve the function required in our investigations. Experiments with plasmids targeted to known cells in the spleen are required to resolve the issue of appropriate protein expression.

Preliminary experiments have also been done to address the issue of the need for additional approach to cause LT{alpha}-/- CD8+ T cell maturation. These experiments have included the use of mixture of expression plasmids such as the coadministration of both CCR7 ligands. Marginal, although not statistically significant, improvement was observed (data not shown). Further studies with this experimental approach are currently underway in our laboratory.


   Acknowledgments
 
We thank Dr. Albert Zlotnik (DNAX Research Institute) and Dr. Jason G. Cyster (University of California) for generously providing CCL21- and CCL19-encoding plasmid DNA, respectively. We are thankful for the technical assistance of Teresa Sobhani, and for the helpful advice of Christopher Pack in reviewing the manuscript. We thank Nancy Nielsen for FACS sorting of tetramer-positive cells. We also acknowledge the technical assistance and discussion of Dr. Sangjun Chun, who tragically passed away in December 1999.


   Footnotes
 
1 This work is supported by National Institutes of Health Grants AI 46462 and AI 14981. Back

2 S.K.E. and U.K. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Barry T. Rouse, Laboratory of Viral Immunology, Department of Microbiology, University of Tennessee, Knoxville, TN 37996. E-mail address: btr{at}utk.edu Back

4 Abbreviations used in this paper: LT{alpha}-/-, lymphotoxin {alpha}-deficient; DC, dendritic cell; wt, wild-type; CD62L, CD62 ligand; CD40L, CD40 ligand. Back

Received for publication June 14, 2001. Accepted for publication July 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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