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The Journal of Immunology, 2006, 177: 1825-1832.
Copyright © 2006 by The American Association of Immunologists

{gamma}{delta} T Cells Facilitate Adaptive Immunity against West Nile Virus Infection in Mice1

Tian Wang2,*, Yunfei Gao{dagger}, Eileen Scully{dagger},{ddagger}, C. Todd Davis§, John F. Anderson, Thomas Welte||, Michel Ledizet#, Raymond Koski#, Joseph A. Madri||, Alan Barrett§, Zhinan Yin{dagger}, Joseph Craft{dagger},{ddagger} and Erol Fikrig2,{dagger}

* Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80521; {dagger} Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; {ddagger} Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; § Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, and Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77550; Department of Entomology, Connecticut Agricultural Experiment Station, New Haven, CT 06504; || Department of Pathology, Yale University School of Medicine, New Haven, CT 06520; and # L2 Diagnostics, New Haven, CT 06530


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and {gamma}{delta} T cells are involved in the protective immune response against viral challenge. We have now examined whether {gamma}{delta} T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCR{delta}–/– mice survived primary infection with WN virus compared with 80–85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of {gamma}{delta} T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, {gamma}{delta} T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8+ T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8+ T cells from TCR{delta}–/– mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that {gamma}{delta} T cells directly link innate and adaptive immunity during WN virus infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
West Nile (WN)3 virus, a mosquito-borne flavivirus, has caused annual epidemics of viral encephalitis in North America since 1999 (1). The virus is maintained in an enzootic cycle that primarily involves mosquitoes and birds, with humans and horses as incidental hosts (2). Although human infection is usually asymptomatic, life-threatening neurological disease including encephalitis can ensue, particularly in the elderly and immunocompromised (2). Thus, understanding the factors that contribute to pathogenesis and immunity is critical for combating this emerging viral infection.

The murine model of WN virus infection mimics human disease and has been used to address questions of viral pathogenesis and immunity (3, 4, 5). After the initial infection, the virus spreads systemically and eventually invades the CNS. Mice die rapidly when encephalitis develops, usually within 1–2 wk following infection (5). Components of the innate and adaptive murine immune system have been shown to facilitate the control of WN virus infection (6). Type 1 IFNs (7, 8, 9, 10), complement (11), {gamma}{delta} T cells (12), and early neutralizing IgM (13) help limit viremia and dissemination into the CNS. Macrophages, B cells, and dendritic cells as APCs play an important role in T cell activation and proliferation during WN virus infection (14, 15). Cellular immunity, including both CD4+ (15) and CD8+ {alpha}beta T cells, participate in the recovery of the host from WN virus infection (16, 17, 18). Although secondary immune responses provide long-lasting protection against WN virus, the mechanisms that link innate and adaptive immunity upon infection are not fully understood.

We recently demonstrated that {gamma}{delta} T cells provide a rapid response following WN virus infection in mice, limiting the viral load and invasion of the CNS within the first few days following infection, thereby protecting the host from lethal encephalitis (12). {gamma}{delta} T cells comprise a minority of the CD3+ T cells in lymphoid tissue, yet they are well represented in the peripheral blood and are abundant at epithelial and mucosal sites (19). Like {alpha}beta T cells, {gamma}{delta} T cells bear a TCR and can be polarized to produce Th1- or Th2-type cytokines (20, 21, 22), although they are more limited in TCR diversity (19). {gamma}{delta} T cells can rapidly produce cytokines in response to microbial Ags (23) and have unique features, including a lack of MHC restriction and the capacity to react with Ag without the requirement for undergoing conventional Ag processing (24, 25, 26). These features suggest a role for {gamma}{delta} T cells in pathogen control early after infection. Increased numbers of {gamma}{delta} T cells in the peripheral blood and/or localization to sites of infection have been documented in both human (27, 28, 29) and murine viral infections (30, 31).

{gamma}{delta} T cells are thought to bridge the innate and adaptive immune responses (32, 33). For example, human V{delta}2+ T cells have been recently shown to act as professional APCs and induce proliferation and differentiation of naive {alpha}beta T cells, forming a unique link between innate and adaptive immune responses (34). In primates, {gamma}{delta} T cells have characteristics of adaptive immunity upon mycobacteria infection, including the generation of a memory-type response (35). In this study, we have explored the possibility of whether {gamma}{delta} T cells are also involved in the development of protective adaptive immune responses during murine WN virus infection.


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

C57BL/6 (B6) and {gamma}{delta} T cell-deficient (TCR{delta}–/–) mice were purchased from The Jackson Laboratory and were maintained under specific pathogen-free conditions at the animal facilities at Yale University, School of Medicine. TCR{delta}–/– mice were bred on the B6 background and had been fully backcrossed. Experiments for primary and secondary infection were performed in 6- to 10-wk-old and 11- to 15-wk-old mice, respectively. Groups were age and sex matched for each experiment and were housed under identical conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee at Yale.

Infection in mice

WN virus isolate 2741 was initially cultivated by J. Anderson (Connecticut Agricultural Experiment Station, New Haven, CT) (36). For primary infection, mice were inoculated i.p. with 1/10 of an LD50 of WN virus CT 2741. For secondary infection, all mice were inoculated i.p. with an LD100 of WN virus isolate 2741. A total of 102 PFU corresponds to the LD50, and 103 PFU represents the LD100 for WN virus isolate CT 2741. For some experiments, mice were inoculated with a nonlethal dose (100 PFU) of WN virus isolate bird 1153, a naturally attenuated strain of WN virus (37). Infected mice were monitored twice daily for morbidity, including lethargy, anorexia, and difficulty in walking.

Quantitative PCR (Q-PCR)

At days 2 and 6 after WN virus challenge, RNA was extracted from the blood of B6 mice and TCR{delta}–/– mice using RNeasy extraction (Qiagen). The extracted RNA was eluted in a total volume of 60 µl of RNase-free water. Two hundred fifty nanograms of each extracted RNA sample was used to synthesize cDNA using the ProSTAR first-strand RT-PCR kit (Stratagene). A total of 40 ng of cDNA was then used for real-time PCR. The sequences of the primer-probe sets for WN virus were described earlier (38). The probe contained a 5' reporter, FAM, and a 3' quencher, TAMRA (Applied Biosystems). The reaction mixture contained a total volume of 50 µl, including each primer pair at a concentration of 1 µM and a probe at a concentration of 0.2 µM. The assay was performed on an iCycler (Bio-Rad). The thermal cycling consisted of 95°C for 3.5 min and 48 cycles of 95°C for 30 s and 60°C for 1 min. To prepare the DNA standard for real-time PCR, the 1.5-kb WN virus envelope protein Ag (WNV-E) gene region was cloned into pBADTOPO, as described earlier (5). To normalize the samples, the same amount of cDNA was used in the beta-actin Q-PCR. The quantity of each sample was determined using the standard curve of each Q-PCR. The ratio of the amount of amplified WNV-E DNA compared with the amount of beta-actin DNA represented the relative infection level of each sample.

In vivo depletion of {gamma}{delta} T cells

Before secondary infection, B6 mice surviving primary viral challenge were depleted of {gamma}{delta} T cells, as described previously (39). Briefly, mice were i.p. injected with 250 µg of hamster anti-TCR{gamma}{delta} mAb (UC-7; American Type Culture Collection) on three consecutive days. Depletion was confirmed via flow cytometry by staining with Abs specific for TCR{alpha}beta (clone H57-597) and TCR{gamma}{delta} (clone GL3) (BD Pharmingen).

ELISA

Microtiter plates were coated with rWNV-E expressed in Drosophila melanogaster S2 cells (40) overnight at 4°C at 100 ng/well in coating buffer (0.015 M Na2CO3, 0.03 M NaHCO3, and 0.003 M NaN3 (pH 9.6)). Sera from infected mice were diluted from 1/40 to 1/1000 in PBS with 2% BSA, added to the duplicate wells, and incubated for 1 h at room temperature. Plates were washed three times with PBS-T. Alkaline phosphatase-conjugated goat anti-mouse IgG or IgM (Sigma-Aldrich) at a dilution of 1/1000 in PBS-T was added for 1 h at room temperature. After washing three times with PBS-T, color was developed with p-nitrophenyl phosphate for 10 min and read at an absorbance of 405 nm using a spectrophotometer.

Passive immunization

Six-week-old B6 mice were intradermally injected with 150 µl of antiserum (diluted 1/3 in PBS) pooled either from day 28 infected wild-type or TCR{delta}–/– mice, or naive B6 mice. The animals were challenged with either one LD100 or 100 x LD100 of WN virus isolate 2741 24 h after the serum transfer. Infected mice were monitored twice daily, as described above.

Flow cytometry

Freshly isolated splenocytes were stained with Abs specific for TCR{alpha}beta and TCR{gamma}{delta}, or CD8{alpha} (clone 53-6.7) and CD44 (clone IM7) (BD Pharmingen). After staining, the cells were fixed in PBS with 2% paraformaldehyde and examined using a FACSCalibur flow cytometer (BD Biosciences). Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed using CellQuest software.

Intracellular cytokine staining

To measure cytokine production, splenocytes from WN virus-infected mice were isolated and were stimulated at 3 x 106 cells/tube with 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 h at 37°C. Golgi-plug (BD Pharmingen) was added during the final 2 h. Murine endothelial cells were isolated from brains of B6 mice and immortalized, as previously described (41). These cells were seeded on 12-well plates (1 x 105 cells/well), incubated for 24 h at 37°C, and infected with WN virus. A total of 8 x 105 splenocytes from infected wild-type or TCR{delta}–/– mice was incubated with 3 x 105 to 4 x 105 of WN virus-infected endothelial cells. All stimulations were performed for 5 h at 37°C in the presence of Golgi-plug. The cells were then harvested, stained with Abs (BD Pharmingen) for TCR{alpha}beta and CD8{alpha}, and fixed in 2% paraformaldehyde. The cells were then permeabilized with 0.5% saponin before adding PE-conjugated anti-IFN-{gamma} mAb (clone XMG 1.2) or control PE-conjugated rat IgG1 (BD Pharmingen). Cells were examined using a FACSCalibur flow cytometer, as described above.

Adoptive transfer of CD8+ T cells

Single-cell suspensions of CD8+ T cells were made from spleens of WN virus-infected wild-type or TCR{delta}–/– mice by positive selection method using anti-CD8{alpha} magnetic beads (Miltenyi Biotec), as described previously (42). A total of 8 x 106 cells was injected i.v. into naive 6-wk-old B6 mice 24 h before infection with one LD100 of WN virus 2741. Following challenge, infected mice were monitored twice daily, as described above.

In vitro cytotoxicity assays

The in vitro analysis of CTL activity was performed, as described (43). Splenocytes were isolated from naive B6 mice and were depleted of CD8+ T cells by negative selection MACS with anti-CD8{alpha} magnetic beads (Miltenyi Biotec) (42). These CD8-negative splenocytes were used as target cells. Briefly, they were pulsed with purified rWNV-E at 1 µg/106 cells at 37°C for 30 min. The pulsed cells were then washed and labeled with the fluorescent dye CFSE (Molecular Probes) by incubation of 5 x 106 cells/ml in PBS containing 50 µM CFSE for 10 min at 37°C, followed by one wash in 5 vol of ice-cold PBS and two washes in DMEM (Invitrogen Life Technologies). CD8-depleted splenocytes not pulsed with rWNV-E were labeled with 10-fold dilutions of CFSE. These two cell populations were then mixed equally and incubated with different ratios of purified CD8+ T cells from wild-type and TCR{delta}–/– mice at 37°C for 5 h. Cells were harvested and stained with anti-CD8{alpha} mAb. After staining, cells were fixed with 2% paraformaldehyde and examined by flow cytometry. Dead cells were excluded on the basis of forward and side light scatter. Cells were gated on the CD8-negative population and analyzed using CellQuest software. Percent specific lysis was determined using the following calculation: (1 – (mean CFSEhigh percentage of test sample x mean CFSElow percentage of test sample)/(mean CFSEhigh percentage of spontaneous release x mean CFSElow percentage of spontaneous release)) x 100.

Statistical analysis

Survival curve comparisons were performed using Prism software (GraphPad) statistical analysis, which uses the log rank test (equivalent to the Mantel-Haenszel test). Values of p for viral burden, Ab titer, and memory T cell number experiments were calculated with a nonpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Deficiency of {gamma}{delta} T cells affects host susceptibility during reinfection

We previously demonstrated that {gamma}{delta} T cells produced IFN-{gamma} within the first few days following WN virus infection in mice, reduced viral dissemination, and partially protected mice from lethality (12). In the current work, to determine whether {gamma}{delta} T cells also play a role in promotion of adaptive immunity, we more carefully examined the subset of mice that survived the primary infection with WN virus. Wild-type mice and TCR{delta}–/– mice were first infected with 1/10 of the LD50 of WN virus strain CT 2741 and monitored twice daily for morbidity. The low dose of virus was used to maximize the number of mice that survived following infection, particularly in the immunodeficient animals. As expected based upon our earlier work (12), only 15–20% of the TCR{delta}–/– mice survived viral challenge, while ~80–85% of the wild-type mice survived. We have not been able to detect WN virus in surviving mice from either group, when examining selected mice by PCR after 21 days (data not shown), suggesting that the virus does not persist in these animals.

We then assessed whether the TCR{delta}–/– and wild-type mice that were able to withstand primary infection with a low dose of WN virus were equally susceptible to challenge with a secondary infection by WN virus. For these studies, a higher dose of WN virus was used for the secondary challenge studies to generate a group of animals that readily succumbed so that the influence on protection could be more clearly discerned. Surviving mice were therefore challenged with an LD100 of WN virus at day 30 after primary infection. Age- and sex-matched naive mice (both wild-type and TCR{delta}–/– mice) were infected with the same dose of WN virus and used as experimental controls in the secondary challenge experiments. Wild-type mice that survived following primary infection were much more resistant to the secondary viral challenge (p < 0.01) than the age- and sex-matched naive wild-type mice, demonstrating development of a protective memory response (Fig. 1a). Likewise, TCR{delta}–/– mice surviving primary challenge were more resistant to WN virus infection than the age- and sex-matched naive TCR{delta}–/– mice (p < 0.01), suggesting the development of protective memory responses in the absence of {gamma}{delta} T cells. As expected (12), the naive TCR{delta}–/– mice were more susceptible to WN virus infection than naive wild-type mice (p < 0.01).


Figure 1
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  		FIGURE 1. {gamma}{delta} T cells are required for host resistance to secondary infection with WN virus. a, TCR{delta}–/– mice were more susceptible to the secondary infection with WN virus. Wild-type and TCR{delta}–/– mice were infected with 1/10 of an LD50 of WN virus and monitored twice daily for mortality. At day 30 postinfection, surviving mice were rechallenged with a dose close to an LD100 of WN virus. As controls, age- and sex-matched naive wild-type and TCR{delta}–/– mice were used. Infected mice were monitored twice daily for mortality. Data shown are pooled from five independent experiments. n = 30 and n = 27 for naive and secondary challenged (SC) wild-type mice, and n = 49 and n = 21 for naive and secondary challenged TCR{delta}–/– mice; **, p < 0.01 compared with wild-type naive mice; {dagger}, p < 0.01 compared with TCR{delta}–/– naive mice; §, p < 0.01 compared with secondary challenged wild-type mice; {ddagger}, p < 0.05 compared with wild-type naive mice. b, TCR{delta}–/– mice have a higher viral load during secondary challenge with WN virus. Viral load was determined from blood of secondary challenged wild-type and TCR{delta}–/– mice at the indicated days using Q-PCR. The y-axis depicts the ratio of the amplified WNV-E cDNA to beta-actin cDNA of each sample (unitless ratio ± 1 SEM). *, p < 0.05 compared with wild-type secondary challenged mice.

 
Surprisingly, the difference in susceptibility to WN virus infection was also sustained between the two experimental groups during secondary infection. The TCR{delta}–/– mice that survived primary infection with WN virus were more susceptible to secondary infection with WN virus than the wild-type mice that had endured the initial infection with virus. We therefore analyzed the viral burden in the blood from both groups of mice during secondary challenge. At day 2 after reinfection, viremia was significantly higher in TCR{delta}–/– mice than the wild-type mice (Fig. 1b). Moreover, at day 6, viral levels in the blood of wild-type mice were no longer detectable, while the viral burden remained elevated in TCR{delta}–/– mice (Fig. 1b). We next extended these observations by performing additional primary challenge studies using WN virus bird 1153 strain, which is a naturally attenuated WN virus that lost its neuroinvasiveness (37). Infection with the attenuated WN virus enabled us to generate a larger group of animals that survive the primary infection, and thereby perform the secondary challenges expeditiously. In addition, challenge with the attenuated strain in mice produces a nonlethal infection in the majority of the wild-type animals, which more closely resembles human cases (37). As above, mice lacking {gamma}{delta} T cells that survived primary infection with the attenuated isolate of WN virus were more susceptible (75% survival) to secondary infection with WN virus than the wild-type mice (100% survival) following inoculation of similar doses of WN virus. Together, these data suggest that {gamma}{delta} T cells contribute to the protective immune response upon recurrent exposure to WN virus. Although protective immunity induced by primary infection does appear to develop in the absence of {gamma}{delta} T cells, it is incomplete.

Depletion of {gamma}{delta} T cells before secondary infection does not affect susceptibility to WN virus in wild-type mice

The observation that TCR{delta}–/– mice had a higher level of viremia and lethality than wild-type mice upon secondary challenge with WN virus suggested that {gamma}{delta} T cells could be contributing directly to the antiviral response, in the absence of an effect upon the adaptive immune response, or alternatively, they might contribute to the development of protection mediated by the adaptive immune response. To test the former possibility, we depleted {gamma}{delta} T cells from wild-type mice that survived primary infection with WN virus, and then subjected these animals to a secondary challenge. To remove {gamma}{delta} T cells, mice were treated with an i.p. injection of 250 µg of hamster anti-TCR{gamma}{delta} mAb daily for 3 days, starting at day 28 after the primary WN virus infection. The efficiency of depletion was close to 90% at 24 h after treatment as analyzed by flow cytometry, as the percentage of splenic TCR{gamma}{delta}+TCR{alpha}beta– T cells dropped from 2.22% in untreated mice to 0.15% in treated mice (Fig. 2a). At 5 days posttreatment, 60% of {gamma}{delta} T cells remained absent (data not shown). Twenty-four hours after the depletion of {gamma}{delta} T cells, mice were challenged with one LD100 dose of WN virus. Mice treated with PBS were used as controls. Wild-type mice that had survived a primary infection with WN virus CT 2741 and then were depleted of {gamma}{delta} T cells were as resistant to secondary challenge with WN virus CT 2741 as control mice (Fig. 2b). These studies demonstrate that depletion of {gamma}{delta} T cells before secondary challenge does not alter the course of disease and suggests that {gamma}{delta} T cells do not act as memory T cells per se during secondary infection with WN virus.


Figure 2
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  		FIGURE 2. Depletion of {gamma}{delta} T cells after a primary infection does not affect host susceptibility during secondary infection of WN virus. a, Mice were given an i.p. injection of 250 µg of anti-TCR{gamma}{delta} Abs on three consecutive days. Following depletion, splenocytes were isolated and stained with Abs to TCR{alpha}beta and TCR{gamma}{delta} to confirm depletion via flow cytometry. b, Wild-type mice that survived WN virus infection were depleted of {gamma}{delta} T cells at day 28 for three consecutive days. Twenty-four hours posttreatment, mice were challenged with WN virus. n = 11 for the control group, and n = 10 for the depleted group; p = 0.29.

 
IgM and IgG production in TCR{delta}–/– mice during WN virus infection

{gamma}{delta} T cells were previously reported to be involved in regulating either Ab production or Ab isotype switching (44, 45). Moreover, B cell-mediated humoral immune responses are critical for host defenses against disseminated infection by WN virus (13, 46, 47). To determine whether {gamma}{delta} T cells influence the development of Abs to WN virus during infection, we measured WN virus-specific IgM (Fig. 3a, left panel) and IgG levels (Fig. 3a, right panel) in the sera from TCR{delta}–/– and wild-type mice that survived primary infection. Ab responses in both groups of mice were similar at 4 days, an early interval postinfection, and at later time points (days 21 and 28, p > 0.05, compared with sera in wild-type mice).


Figure 3
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  		FIGURE 3. IgM and IgG production during primary and secondary infection in TCR{delta}–/– mice. a, Development of specific Abs to WN virus in wild-type and TCR{delta}–/– mice during primary infection. Sera were collected from wild-type or TCR{delta}–/– mice at the indicated days during the primary infection. The development of specific IgM (left panel) or IgG (right panel) Abs to WN virus was determined after incubating sera with adsorbed purified rWNV-E protein. Data shown are representative of three similar experiments (n = 3) and are the averages of four to five mice in each experiment per time point, performed in duplicate. b, Mice were passively administered with sera from surviving mice and challenged with an LD100 dose (left panel; n = 5) or 100 x LD100 (right panel; n = 4) of WN virus. Control mice were given naive mouse sera in an identical fashion. **, p < 0.05 compared with control mice group. c, Development of specific Abs to WN virus in wild-type and TCR{delta}–/– mice during the secondary infection. Sera were collected from wild-type or TCR{delta}–/– mice at the indicated days postsecondary challenge. The development of specific IgM (left panel) or IgG (right panel) Abs to WN virus was determined after incubating sera with adsorbed purified rWNV-E protein. Data are the averages of four to five mice in each experiment per time point, performed in duplicate. Data shown are representative of three similar experiments (n = 3).

 
To determine whether neutralizing Abs to WN virus developed in both the wild-type and TCR{delta}–/– mice, sera from both groups at day 28 postinfection were used for passive immunization. Naive 6-wk-old B6 mice were administered sera from either group of animals 24 h before challenge with one LD100 of WN virus CT 2741. Sera from both the wild-type and TCR{delta}–/–-infected animals protected mice from lethal infection (Fig. 3b, left panel; p > 0.05, compared with naive mice that received wild-type sera), while mice given sera from uninfected mice all succumbed to infection (p < 0.05, compared with mice transferred with noninfected sera). In a separate experiment, mice were also challenged with a higher dose of WN virus (100 LD100) to determine whether this quantity of virus challenge could influence the outcome of the study. Sera from both groups partially, but equally (25% survival vs 25% survival; Fig. 3b, right panel; p > 0.05, compared with naive mice that received wild-type sera) protected naive mice from infection when the viral dose was increased, suggesting Ab production was not significantly different between these two groups during primary infection.

Finally, we measured WN virus-specific IgM and IgG during secondary challenge. At days 2 and 6 after reinfection, we did not detect significant differences between the two groups in either the IgM or IgG response (Fig. 3c, left and right panels; p > 0.05, compared with wild-type mice). However, at day 21 after reinfection, IgM response of wild-type mice was significantly higher than that in TCR{delta}–/– mice (Fig. 3c, left panel; p < 0.05, compared with wild-type mice). Overall, these data suggest that {gamma}{delta} T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus.

Mice lacking {gamma}{delta} T cells have impaired CD8+ T cell memory responses to WN virus

CD8+ effector T cells have been shown to have important functions in clearing WN virus infection from tissues and preventing viral persistence (10, 17, 18, 48). To understand the role of {gamma}{delta} T cells in regulation of adaptive immunity, we next focused on CD8+ T cells. We adoptively transferred CD8+ T cells (90–95% purity; Fig. 4a) from wild-type and TCR{delta}–/– mice at day 30 after WN virus infection to naive recipients 24 h before challenge with an LD100 of WN virus. Control groups, including mice that did not receive cells via adoptive transfer and mice given CD8+ T cells from naive mice, succumbed to infection within 2 wk. In contrast, mice that received CD8+ T cells from previously infected wild-type mice had a significantly higher resistance to lethal infection than the groups of control mice (Fig. 4b; 50% survival; p < 0.05 compared with control groups). Furthermore, mice administered CD8+ T cells from previously infected TCR{delta}–/– mice were much more susceptible to lethal infection than mice provided with CD8+ T cells from the infected wild-type mice, and succumbed to virus challenge at the same rate as control mice (Fig. 4b; 12.5% survival vs 0%; p > 0.05 compared with control mice). These studies clearly indicate that CD8+ T cell responses were significantly impaired in the absence of {gamma}{delta} T cells during the course of WN virus infection.


Figure 4
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  		FIGURE 4. Adoptive transfer of CD8 T cells from TCR{delta}–/– mice that survive a primary infection with WN virus does not protect naive mice from lethal infection. a, Splenocytes from WN virus-infected wild-type and TCR{delta}–/– mice were isolated at day 30 postinfection. CD8+ T cells were purified with microbeads coated with anti-CD8{alpha}, followed by positive selection with an autoMACS Separator (Miltenyi Biotec). Open area, Splenocytes stained with anti-CD8{alpha} mAb; gray area, purified CD8 T cells stained with anti-CD8{alpha} mAb. b, Adoptive transfer of CD8 T cells from surviving TCR{delta}–/– mice does not protect mice from lethal infection. Six-week-old B6 mice were adoptively transferred with CD8 T cells from surviving wild-type mice, TCR{delta}–/– mice, naive wild-type mice, or PBS. Following adoptive transfer, mice were infected with an LD100 of WN virus. n = 8 for each group. Data shown are representative of three similar experiments. *, A value of p < 0.05 compared with mice transferred with PBS or CD8 T cells from naive wild-type mice. {ddagger}, A value of p < 0.05 compared with mice transferred with CD8 T cells from surviving wild-type mice.

 
To further understand the role of {gamma}{delta} T cells in regulating CD8+ T cell responses, we assessed the function of these cells following primary infection with WN virus. Splenocytes from both wild-type and TCR{delta}–/– mice were isolated 30 days after primary infection and examined for the percentage of CD8 memory T cells. Wild-type mice had 36% of CD44highCD8+ memory T cells compared with 30% in TCR{delta}–/– mice (Fig. 5a). The total number of CD44highCD8+ memory T cells is 2.78 ± 0.07 x 106 in wild-type mice, compared with 2.5 ± 0.1 x 106 in TCR{delta}–/– mice (Fig. 5b; p < 0.05 compared with wild-type mice). The functional capacity of the total CD8+ T cell response in these mice was determined using a CTL assay. At day 30 following infection, purified CD8+ T cells from both wild-type and TCR{delta}–/– mice were incubated with various ratios of target cells labeled with rWNV-E. Flavivirus Ags that elicit CD8 T responses have been examined (49, 50, 51, 52, 53), and the E protein has been demonstrated to be a target (51, 54, 55). Moreover, WN virus E protein has been demonstrated in our previous studies as an antigenic target. Therefore, we focused on examining CD8 T cell responses to the E protein, recognizing that other proteins may elicit responses as well (5). The CTL activity of CD8+ T cells in TCR{delta}–/– mice was significantly reduced compared with wild-type animals (Fig. 5c). Finally, the functional CD8+ T cell response at day 30 postinfection was also determined by using an ex vivo intracellular cytokine staining assay for IFN-{gamma} after treatment with PMA and ionomycin. A total of 3.2% of the splenic CD8+ T cells in wild-type mice produced IFN-{gamma} compared with 2.7% of CD8+ T cells from TCR{delta}–/– mice (Fig. 5d, top panel). Moreover, incubation with WN virus-infected murine endothelial cells revealed that 18% of splenic CD8 T cells in wild-type mice produced IFN-{gamma} in comparison with 12% of CD8 T cells from TCR{delta}–/– mice (Fig. 5e). These data were consistent with an earlier time point during primary infection (day 12), in which 1.6% of splenic CD8+ T cells produced IFN-{gamma} compared with 0.9% of CD8+ T cells from TCR{delta}–/– mice (Fig. 5d, bottom panel). Overall, these studies suggest that {gamma}{delta} T cells facilitate the expansion and effector function of CD8+ memory T cells during WN virus infection.


Figure 5
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  		FIGURE 5. Numeric and functional reduction of CD8 memory T cells in TCR{delta}–/– mice. a, Numeric reduction of CD8 memory T cells in TCR{delta}–/– mice. Splenocytes from both groups were isolated at day 30 postinfection and stained with Abs for CD8 and CD44. Cells were gated on CD8+ populations for analysis of CD44 expression. The percentage of CD44highCD8+ is shown. Data presented from one study are representative of three similar experiments. b, Total number of CD8+CD44+ splenic T cells in wild-type or TCR{delta}–/– mice. Three mice per group were analyzed. *, A value of p < 0.05 compared with CD8+CD44+ T cells in wild-type mice. c, CD8+ T cell-mediated killing. Splenic CD8+ T cells were purified from wild-type mice or TCR{delta}–/– mice that survived from primary infection with either WN virus isolates 2741 or bird 1153 at day 30 postinfection. CD8-negative splenocytes from naive mice were purified by negative selection on anti-CD8 and were labeled with rWNV-E and CFSE, as described in Materials and Methods, and used as target cells. Purified CD8 T cells were incubated with target cells at various E:T ratios. *, A value of p < 0.05 compared with wild-type group. Data shown are average of three similar experiments. Splenocytes were also isolated from WN virus-infected wild-type mice and TCR{delta}–/– mice at day 30 (d, top panel) or at day 12 (d, bottom panel) postinfection and were cultured ex vivo with PMA plus ionomycin, as described in Materials and Methods, and stained for IFN-{gamma}, CD3, and CD8. The percentage of IFN-{gamma}+ CD8+ is shown. Data presented are representative of three similar experiments. e, Splenocytes were also isolated from WN virus-infected wild-type mice and TCR{delta}–/– mice at day 30 and incubated with WN virus-infected mouse endothelial cells, as described in Materials and Methods, and stained for IFN-{gamma}, CD3, and CD8. Histograms of IFN-{gamma} production of the gated CD3+CD8+ cells are shown. Three mice per group were analyzed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
{gamma}{delta} T cells have features that are characteristic of innate immune cells: they respond and expand quickly upon exposure to a pathogen, produce inflammatory cytokines, and are not Ag specific (19, 56). Our previous work has shown a role for {gamma}{delta} T cells in the early response during WN virus infection (12). {gamma}{delta} T cells expanded significantly early during WN virus infection, produced IFN-{gamma}, and enhanced perforin expression by splenic T cells, thereby enabling the host to clear the virus.

In the current work, we have further demonstrated an important role for {gamma}{delta} T cells in the development of a protective CD8+ T cell response against WN virus. We initially found that TCR{delta}–/– mice were more susceptible than wild-type mice to secondary challenge with WN virus, suggesting possible involvement of {gamma}{delta} T cells in adaptive immunity. {gamma}{delta} T cells were not directly acting as memory T cells in these studies, because depleting these cells in wild-type mice that survived primary infection with WN virus had no effect on host susceptibility to secondary viral challenge. Moreover, {gamma}{delta} T cells did not contribute to any alteration in WN virus-specific Ab production, because the TCR{delta}–/– and wild-type mice that survived primary infection with WN virus had similar levels of WN virus-specific IgG and IgM, and passive immunization with sera from both groups of mice could protect or partially protect naive mice from lethal WN virus infection. CD8+ memory T cell responses were significantly impaired in TCR{delta}–/– mice that survived primary infection. Both the total number of CD8+ memory T cells and the functional capacity (both CTL activity and ex vivo cytokine production) of the cells were reduced in TCR{delta}–/– mice that survived primary infection, compared with wild-type mice. Moreover, adoptive transfer of CD8+ {alpha}beta T cells from wild-type surviving mice into naive mice helped to enhance host resistance to lethal WN virus infection, while those transferred from surviving TCR{delta}–/– mice did not have a similar effect. These data demonstrate that {gamma}{delta} T cells are required for effective CD8+ T cell memory responses during WN virus infection.

Depletion of {gamma}{delta} T cells before secondary challenge did not affect susceptibility to WN virus in wild-type mice, suggesting that {gamma}{delta} T cells do not act as memory T cells per se during secondary challenge with WN virus. Although the mechanisms through which {gamma}{delta} T cells regulate CD8+ T cell memory responses are still under investigation, these depletion studies also exclude the possibility that {gamma}{delta} T cells are involved in the maintenance of CD8+ memory T cells. For an acute infection such as WN encephalitis, the CD8+ T cell response has three characteristic phases: a period of initial activation and expansion, a contraction or death phase, and the establishment and maintenance of memory (57). We found a significant decrease in the CD8+ T cell response in TCR{delta}–/– mice 12 days postinfection. WN virus is usually cleared from peripheral organs such as the spleen as early as day 8 (4). These data suggest that the impairment of CD8+ T cell response led by the deficiency of {gamma}{delta} T cells already exists during/before the contraction stage, possibly at the early T cell priming stage through cross talk with APCs, such as dendritic cells (58, 59, 60).

Increasing evidence from both human and primate studies has suggested that {gamma}{delta} T cells are involved in the genesis of adaptive immunity. Upon microbial activation, human V{delta}2+ T cells display some of the principal characteristics of professional APCs: they efficiently process and display Ags and provide costimulatory signals sufficient for strong induction of naive {alpha}beta T cell proliferation and differentiation (34). Moreover, the killer cell lectin-like receptor G1 is expressed in a significant proportion of human V{gamma}9/V{delta}2 T cells, which also have the phenotype for effector memory T cells (lack of CD27, CD45RA, CD62L, and CCR7 (61). In primates studies, the recall expansion of V{gamma}2/V{delta}2+ T cells developed during Mycobacterium tuberculosis infection of bacillus Calmette-Guerin-vaccinated macaques (35). Although the systemic Th1 response was impaired in {gamma}{delta} T cell-depleted mice following an intravaginal infection with HSV type 2, it was not clear whether this contributed directly to the higher susceptibility and lethal viral infection (62). Our findings in this study show that {gamma}{delta} T cells play an important role in regulating CD8 T cell memory responses in the murine model. Several groups have recently demonstrated that CD8+ effector T cells are critical in clearing WN virus infection from tissues and preventing viral persistence (10, 17, 18, 48). However, the development of CD8 T cell memory response to WN virus infection is not fully understood. Our studies have provided important insights into understanding the immune response to WN virus infection, and they will aid identification of the linkage between {gamma}{delta} T cells and adaptive immunity during WN virus infection. Overall, {gamma}{delta} T cells not only provide early defense against WN virus infection, but also contribute to the memory response, suggesting a potential role in facilitating future vaccine development.


   Acknowledgments
 
We thank Debbie Beck and Ping Zhu for technical assistance. We also thank Drs. Susan Kaech, Tim Quan, and Jin-Young Choi for helpful discussions.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (to E.F., J.C., Z.Y., and R.K.), the Burroughs Wellcome Fund (to E.F.), the Jeane Kempner Postdoctoral Fellowship (to T.W.), and the Medical-Scientist Training Program at the Yale School of Medicine (to E.S.). J.C. and Z.Y. are supported by grants from the Arthritis Foundation. T.W. is supported by a grant from American Federation for Aging Research. Back

2 Address correspondence and reprint requests to Dr. Erol Fikrig, Department of Internal Medicine, Section of Rheumatology, Yale University School of Medicine, S525A, 300 Cedar Street, P.O. Box 208031, New Haven, CT 06520-8031; E-mail address: erol.fikrig{at}yale.edu or Dr. Tian Wang, Department of Microbiology, Immunology and Pathology, Colorado State University, Campus Delivery 1692, Fort Collins, CO 80521; E-mail address: tian.wang{at}colostate.edu Back

3 Abbreviations used in this paper: WN, West Nile; Q-PCR, quantitative PCR; WNV-E, WN virus envelope protein. Back

Received for publication March 3, 2006. Accepted for publication May 2, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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