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The Journal of Immunology, 2007, 179, 8287-8296
Copyright © 2007 by The American Association of Immunologists, Inc.
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Prolonged (E)-4-Hydroxy-3-Methyl-But-2-Enyl Pyrophosphate-Driven Antimicrobial and Cytotoxic Responses of Pulmonary and Systemic V{gamma}2V{delta}2 T Cells in Macaques1

Zahida Ali*, Lingyun Shao*,{dagger}, Lisa Halliday{ddagger}, Armin Reichenberg§, Martin Hintz§, Hassan Jomaa§ and Zheng W. Chen2,*

* Department of Microbiology and Immunology, Center for Primate Biomedical Research, University of Illinois, College of Medicine, Chicago, IL 60612; {dagger} Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China; {ddagger} Biological Resources Laboratory, University of Illinois, Chicago, IL 60612; and § Institut für Klinische Chemie und Pathobiochemie, Justus-Liebig-Universität Giessen, Giessen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although phosphoantigen-specific V{gamma}2V{delta}2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these {gamma}{delta} T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating V{gamma}2V{delta}2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated V{gamma}2V{delta}2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3–4 mo, although circulating V{gamma}2V{delta}2 T cells had returned to baseline levels weeks prior. The V{gamma}2V{delta}2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7–CD45RA–CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-{gamma} up to 15 wk after treatment. Furthermore, the capacity of massively expanded V{gamma}2V{delta}2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ {alpha}β T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+V{gamma}2– T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic V{gamma}2V{delta}2 T cells may confer immunotherapeutics against infectious diseases and cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Accumulating evidence suggests that human V{gamma}2V{delta}2 (also called V{gamma}9V{delta}2) T cells play a role in mediating immunity against microbial pathogens (1, 2, 3, 4, 5, 6, 7, 8) and tumors (9, 10). V{gamma}2V{delta}2 T cells are a major circulating {gamma}{delta} T cell subset that normally constitutes 2–5% of peripheral blood T cells, and are unique in their ability to massively expand during various bacterial and protozoal infections (11) and notably increase in patients with certain cancers (10, 12). V{gamma}2V{delta}2 T cell expansion appears to be specifically mediated by certain low m.w. foreign- and self-nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis (e.g., (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP),3 isopentenyl pyrophosphate (IPP), and its isomer dimethylallyl pyrophosphate) (13, 14, 15) commonly referred to as phosphoantigens. HMBPP is produced in the newly discovered 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis of most eubacteria, apicomplexan protozoa, plant chloroplasts, and algae, but not in vertebrates and thus not in the human host (16). Although IPP is also produced in humans, its bioactivity is ~104 lower than that of HMBPP, which is the most potent V{gamma}2V{delta}2 T cell activator known, with an EC50 of 0.1 nM (13, 17, 18). Thus, much higher levels of endogenous IPP (e.g., those produced during cellular stress or transformation) (10, 19, 20) are most likely needed to trigger IPP-specific V{gamma}2V{delta}2 T cell responses. Although the mechanism of phosphoantigen recognition by V{gamma}2V{delta}2 T cells is not well defined, it appears to be mediated by the V{gamma}2V{delta}2 TCR (21) and requires cell-cell contact (22).

We and others have recently demonstrated that nonhuman primates can serve as a useful animal model to study the immune biology of human V{gamma}2V{delta}2 T cells in response to phosphoantigen-producing pathogens or synthetic phospholigands (1, 23, 24). The remarkable expansion of V{gamma}2V{delta}2 T cells after infection or treatment with synthetic V{gamma}2V{delta}2 TCR ligands has indeed raised an exciting possibility to explore the potential of activated V{gamma}2V{delta}2 T cells as immunotherapeutics against cancers and infectious diseases. Because the majority of pathogenic infections occur as a result of airborne, oral, or sexual transmission, it is crucial to characterize phosphoantigen-specific V{gamma}2V{delta}2 T cell immune responses at these mucosal sites. However, many immunological questions regarding mucosal V{gamma}2V{delta}2 T cell responses after infection or phospholigand treatment have not been addressed. Particularly, the ability of massively expanded V{gamma}2V{delta}2 T cells to undergo mucosal migration and accumulate at these sites upon phospholigand treatment or infection remains largely unknown. In addition, the ability of activated V{gamma}2V{delta}2 T cells to recognize naturally occurring phosphoantigen and exert effector functions in mucosae or the circulation has not been well characterized. Because the microbial phosphoantigen HMBPP is the most potent V{gamma}2V{delta}2 T cell-activating ligand known, we have used HMBPP plus IL-2 treatment regimens to address a series of open questions as to whether HMBPP-activated V{gamma}2V{delta}2 T cells are able to undergo prolonged expansion as seen in various infections, migrate to or accumulate in pulmonary and other mucosae in the context of phenotypic changes in homing or memory markers, re-recognize phosphoantigen and exert effector functions, and impact {alpha}β T cell responses.


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

Four- to 8-year-old, 3- to 4-kg, cynomolgus macaques (Macaca fascicularis) were used in this study. All animals were maintained and used in accordance with guidelines of the institutional animal care and use committee. Animals were anesthetized with 10 mg/kg ketamine HCl (Fort Dodge Animal Health) i.m. for all blood sampling and treatments. EDTA-anticoagulated blood was collected at various time points before and after treatment. Day 0 blood was drawn immediately before treatment.

HMBPP and IL-2 administration

HMBPP was synthesized using a previously described procedure (25), which was scaled up to a final yield of 10 g. The purity determined by ion chromatography was >98%. Immediately before injection, HMBPP was reconstituted with saline and sterile filtered. Human rIL-2 (Proleukin; Chiron) was reconstituted with sterile ddH2O immediately before injection. Based on the in vitro bioactivity of HMBPP and with the objective of having a high in vivo exposure to HMBPP with a single injection, two groups of cynomolgus macaques received a 1-ml i.m. injection of either 10 mg/kg (n = 4) or 50 mg/kg (n = 4) HMBPP. These animals also received 0.5-ml s.c. injections of 1 million IU of IL-2 once daily for 5 consecutive days beginning on the day of HMBPP treatment. One macaque receiving 10 mg/kg HMBPP was only given 0.6 million IU of IL-2/day. As a control group, HMBPP alone was given at the following concentrations: 50 mg/kg (n = 4), 10 mg/kg (n = 2), and 5 mg/kg HMBPP (n = 1). Another control group was given 1 million IU of IL-2/day for 5 consecutive days (n = 2). No acute or long-term adverse effects were exhibited by any animal, including fever or chills, after HMBPP or IL-2 treatment. The general behavior and health of the animals remained normal throughout the study and after its conclusion.

Bronchoalveolar lavage (BAL), gingival, and rectal mucosae sampling

The procedures were modified from our previously described protocols (1). Before BAL and rectal biopsy sampling, animals were subjected to overnight or 24-h fasting, respectively, and were tranquilized i.m. with 1–2 mg/kg xylazine (Ben Venue Laboratories) and 10 mg/kg ketamine HCl. For BAL, animals also received 0.05 mg/kg atropine (Phoenix Scientific) i.m. as an anticholinergic and were restrained in an upright position. A pediatric feeding tube was inserted down the larynx, into the trachea through direct visualization with a laryngoscope to the level of the carina. Saline (10 ml) was instilled into the trachea and immediately withdrawn and repeated a maximum of three times until a total of 12–15 ml of BAL fluid was retrieved. For rectal biopsies, animals were restrained in ventral recumbency with the pelvic area supported and elevated ~4–5 above remainder of body. With the aid of a speculum standard, 3 x 5-mm biopsy forceps were used to collect two to three tissue pieces at each time point. Gingival mucosa samples were performed on anesthetized animals by atraumatically brushing their gum line with small sterile soft-bristled toothbrushes and rinsing the area with ~50 ml of saline and collecting the oral rinse.

Isolation of lymphocytes from blood, BAL fluid, gingival, and rectal mucosae

PBL were isolated from freshly collected EDTA blood by Ficoll-PaquePlus (Amersham) density gradient centrifugation before analysis. Lymphocyte isolation from freshly collected rectal mucosal biopsies was done according to previously described procedures (26) with minor changes. Briefly, biopsies were collected in RPMI 1640 containing 5% FBS (Invitrogen Life Technologies), washed, incubated for 30 min (37°C, 300 rpm) in 5% FBS-HBSS plus 5 mM EDTA, and, upon washing, incubated in 5% FBS-RPMI 1640 plus 90 U/ml collagenase (Sigma-Aldrich) for 1 h (37°C, 300 rpm). Samples were repeatedly aspirated with a 16-gauge needle to disrupt tissue and filtered through 70-µm cell strainers before layering on Ficoll-PaquePlus and centrifuging at 3000 rpm for 20 min, after which the mononuclear cell layers were collected and washed with 10% FBS-RPMI 1640 before analysis. Freshly collected BAL and gingival mucosa samples were washed with 5% FBS-PBS and filtered through 40-µm cell strainers (BD Biosciences) before analysis.

Immunofluorescent staining and flow cytometric analysis

For cell surface staining, 100 µl of EDTA blood was treated with RBC lysing buffer (Sigma-Aldrich) and washed twice with 5% FBS-PBS before staining. PBMC, BAL, rectal, and gingival mucosa cells were stained with up to five Abs (conjugated to FITC, PE, allophycocyanin, Pacific Blue, and PE-Cy5 or allophycocyanin-Cy7) for at least 15 min. After staining, cells were fixed with 2% formaldehyde-PBS (Protocol Formalin) before analysis on a CyAn ADP flow cytometer (DakoCytomation). Lymphocytes were gated based on forward and side scatters, and pulse-width and at least 40,000 gated events were generally analyzed using Summit Data Acquisition and Analysis Software (DakoCytomation). Absolute cell numbers were calculated based on flow cytometry data and complete blood counts performed on a hematology system (Advia 120; Siemens).

The following mouse mAbs were used: V{gamma}9 (7A5), V{delta}2 (15D), V{delta}1 (TS8.2), and Pan{gamma}{delta} (5A6.E9) (Pierce); V{delta}3 (P11.5B) (Beckman Coulter); CD3 (SP34, SP34-2), CD4 (L200), CD8 (RPA-T8), CD27 (M-T271), CD28 (CD28.2), CD45RA (5H9), CD49d (9F10), CCR5 (3A9), and IFN-{gamma} (4S.B3) (BD Pharmingen); CD4 (OKT4) and CD27 (O323) (eBioscience); CD8 (DK25) (DakoCytomation); CCR7 (150503) (R&D Systems); perforin-biotin (Pf-344) (Mabtech); and rat mAb to integrin β7 (FIB504) (BD Pharmingen). The following secondary Abs were used for indirect staining: Pacific Blue-conjugated streptavidin (Invitrogen Life Technologies) and PE-conjugated goat F(ab')2 anti-mouse IgG (Fc{gamma}) (Beckman Coulter). Staining panels were as follows: CD3/CD4/CD8/V{gamma}2/V{delta}2; CD3/V{gamma}2/CD27/CD45RA; CD3/pan{gamma}{delta}/V{delta}1 or V{delta}3; CD3/V{gamma}2/CCR5/CCR7; CD3/V{gamma}2/{alpha}4/β7.

Intracellular cytokine staining

For intracellular cytokine staining, 105-106 BAL cells or 106 PBL plus costimulatory mAbs CD28 (1 µg/ml) and CD49d (1 µg/ml) were incubated with HMBPP (40 ng/ml) or medium alone in 200 µl final volume for 1 h at 37°C, 5% CO2, followed by an additional 5-h incubation in the presence of brefeldin A (GolgiPlug; BD Biosciences). After staining cell surface CD3 and V{gamma}2 for at least 15 min, cells were permeabilized for 45 min (Cytofix/Cytoperm; BD Biosciences) and stained another 45 min for IFN-{gamma} and perforin before resuspending in 2% formaldehyde-PBS.

Statistical analysis

Statistical analysis was done using ANOVA and Student’s t test, as previously described (1).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HMBPP/IL-2 cotreatment induced prolonged expansion of V{gamma}2V{delta}2 T cells and a transient increase in CD4+ and CD8+ {alpha}β T cells in the circulation

Because nonmicrobial phospholigands tested to date appear to stimulate a shorter-term expansion of V{gamma}2V{delta}2 T cells than those observed during human and macaque infections with phosphoantigen-producing microbes (23, 24, 27), we sought to determine whether the microbial phosphoantigen HMBPP can induce a prolonged expansion of V{gamma}2V{delta}2 T cells in monkeys. HMBPP (n = 7) or IL-2 (n = 2) treatment alone did not induce detectable V{gamma}2V{delta}2 T cell expansion (Fig. 1a). However, after a single 10 mg/kg i.m. HMBPP injection plus low-dose IL-2 treatment, circulating V{gamma}2V{delta}2 T cells specifically expanded to 41–62% of total CD3+ T cells (41- to 57-fold above baseline) and 1250–9900 cells/µl (102- to 442-fold above baseline) at day 4 after the treatment and remained 4- to 7-fold above baseline in percentage of total T cells at day 14 before returning to baseline at day 56 (n = 3) (Fig. 1, b–d). Upon increasing the HMBPP dose to 50 mg/kg plus IL-2, the duration of the V{gamma}2V{delta}2 T cell expansion was significantly increased in half of the animals tested (animals 7235 and 7318) as compared with those treated with 10 mg of HMBPP/kg (Fig. 1e). V{gamma}2V{delta}2 T cell levels remained 4- and 2.3-fold above baseline at day 56 in relative percentage of CD3+ T cells in animals 7235 and 7318, respectively, and did not return to baseline until day 105 (Fig. 1e). Such a prolonged expansion of V{gamma}2V{delta}2 T cells appeared to result from greater increases in their absolute numbers during peak expansion, as follows: 12,200 and 41,700 cells/µl (127- and 914-fold above baseline) in the two animals, respectively (Fig. 1f). V{gamma}2V{delta}2 T cells in the other two animals that received the 50 mg/kg HMBPP dose (7311 and 7317) expanded at day 4 and returned to baseline by day 21. Interestingly, majority of expanded V{gamma}2V{delta}2 T cells in HMBPP/IL-2-cotreated animals expressed CD8 on their cell surface (Fig. 2, a–c). Furthermore, in these cotreated animals, absolute numbers of circulating CD4+ and CD8+ {alpha}β T cells increased 3.5- and 3.7-fold on average, respectively, at day 7 after the HMBPP/IL-2 cotreatment and then returned near baseline levels on day 11, whereas HMBPP or IL-2 alone resulted in no or only subtle increases in CD4+ and CD8+ {alpha}β T cell numbers (Fig. 2, d and e). Also, there was a marked lymphopenia of all examined T cells at day 1 after HMBPP treatment before the massive increases in V{gamma}2V{delta}2 and {alpha}β CD4+ and CD8+ T cells in cotreated animals (Figs. 1 and 2). These results indicate that the microbial phosphoantigen HMBPP is unique compared with other previously described phospholigands in its ability to stimulate a prolonged, major expansion of circulating V{gamma}2V{delta}2 T cells, and also a transient increase in circulating CD4+ and CD8+ {alpha}β T cells when coadministered with IL-2.


Figure 1
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  		FIGURE 1. HMBPP/IL-2 cotreatment induced a prolonged and massive expansion of circulating V{gamma}2V{delta}2 T cells in vivo. Relative percentages of CD3+ T cells that express V{gamma}2V{delta}2 after treatment with HMBPP or IL-2 alone are shown over time as the SEM for the number of indicated animals (a). V{gamma}2V{delta}2 T cell expansion from pretreatment to day 4 in one representative cotreated animal is shown (b). Various {gamma}{delta} TCR-expressing lymphocytes in cotreated animals are shown over time as a relative percentage of total CD3+ T cells (c and e) and absolute numbers/µl blood (d and f). V{delta}1 levels are shown as the SEM for the indicated animals.

 

Figure 2
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  		FIGURE 2. HMBPP/IL-2 cotreatment induced an expansion of circulating CD8- and CD4-expressing T cells in vivo. Cotreated animals were treated with 50 mg/kg (open symbols) or 10 mg/kg (filled symbols) HMBPP plus IL-2. As controls, four animals received 50 mg/kg HMBPP only and two received IL-2 only. All animals were examined for CD8- and CD4-expressing lymphocytes. CD3-gated CD8+V{gamma}2+ cells are shown for one representative cotreated animal at day 7 (a) and over time for all examined animals (b). Absolute numbers of CD8+V{gamma}2+ (c), {alpha}β CD8+V{gamma}2– (d), and {alpha}β CD4+ (e) cells/µl blood are shown over time. Relative percentages of CD3+ T cells that are CD4+ or CD8+V{gamma}2– decreased in cotreated animals due to V{gamma}2V{delta}2 T cell expansion (data not shown). Results for HMBPP- and IL-2 alone-treated animals are shown over time as the SEM for the number of indicated animals (b–e). Asterisks indicate statistically different T cell levels between cotreated and control groups at those time points (p < 0.05) (b–e).

 
HMBPP/IL-2 cotreatment induced a prolonged accumulation of V{gamma}2V{delta}2 T cells in pulmonary mucosa and their short-term increase in gingival and rectal mucosae

Given the possibility that HMBPP/V{gamma}2V{delta}2 T cell-based immunotherapeutics against infections might rely on the ability of these cells to migrate to the mucosal interface after their systemic expansion, we sought to examine whether massively expanded V{gamma}2V{delta}2 T cells can accumulate in pulmonary and other mucosae after HMBPP/IL-2 cotreatment. HMBPP-activated V{gamma}2V{delta}2 T cells appeared to readily undergo pulmonary migration during their massive expansion after HMBPP/IL-2 cotreatment, accounting for up to 80% of total CD3+ T cells and up to 4 million in absolute number in the BAL fluid at the peak accumulation time, days 4–11 (Figs. 3, a–c). Importantly, V{gamma}2V{delta}2 T cell levels remained up to 46% of total CD3+ T cells (~10.4-fold above baseline) in the BAL fluid of all examined animals at day 56, whereas at this time blood V{gamma}2V{delta}2 T cells were <5% of CD3+ T cells (Figs. 3b and 1, c and e). Sixteen weeks after the single HMBPP/IL-2 cotreatment, V{gamma}2V{delta}2 T cells were as high as 13% of total BAL fluid CD3+ T cells in the five monkeys, whereas blood V{gamma}2V{delta}2 T cells had returned to baseline several weeks prior. Also, the majority of pulmonary accumulated V{gamma}2V{delta}2 T cells expressed CD8 (data not shown). On average, HMBPP/IL-2 cotreatment also led to 6- and 2-fold increases in pulmonary CD4+ and CD8+V{gamma}2– {alpha}β T cell numbers, respectively, at day 4 (Fig. 3, d and e). Interestingly, although HMBPP treatment alone did not result in detectable increases in peripheral {alpha}β T cell numbers (Fig. 2, d and e), we did detect 2- and 4-fold increases in pulmonary CD4+ and CD8+V{gamma}2– T cell numbers, respectively, at day 4, whereas the V{gamma}2V{delta}2 T cell levels remained the same posttreatment (Fig. 3, c–e). Thus, although HMBPP alone did not result in marked expansion of peripheral or pulmonary V{gamma}2V{delta}2 T cells, it may have been sufficient to activate these cells and affect pulmonary {alpha}β T cell levels.


Figure 3
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  		FIGURE 3. HMBPP/IL-2 cotreatment induced a prolonged accumulation of V{gamma}2V{delta}2 T cells and a transient increase in {alpha}β T cells in pulmonary mucosa. Freshly collected cells from BAL fluid of animals treated with 50 mg/kg (open symbols) or 10 mg/kg (filled symbols) HMBPP plus IL-2, or HMBPP (50 mg/kg) or IL-2 alone, were stained over time for V{gamma}2V{delta}2 (b and c)-, {alpha}β CD8+V{gamma}2– (d)-, and {alpha}β CD4+ (e)-expressing T cells. V{gamma}2V{delta}2 T cell accumulation in pulmonary mucosa from pretreatment to day 11 in one representative cotreated animal (a) and over time for all examined animals (b) is shown from CD3-gated cells. Absolute numbers of V{gamma}2V{delta}2 (c), {alpha}β CD8+V{gamma}2– (d), and {alpha}β CD4+ (e) cells are shown over time. Relative percentages of CD3+ T cells that are CD4+ or CD8+V{gamma}2– decreased in cotreated animals due to V{gamma}2V{delta}2 T cell expansion (data not shown). T cell levels for HMBPP- and IL-2 alone-treated animals are shown as the SEM. Asterisks indicate statistically different T cell levels in cotreated animals from pretreatment (day 0) (p < 0.05) (b).

 
We then examined whether the massively expanded V{gamma}2V{delta}2 T cells could similarly accumulate in rectal and gingival mucosae after the HMBPP/IL-2 cotreatment. Baseline V{gamma}2V{delta}2 T cell levels in the rectal and gingival mucosae were on average 0.4 and 3.4% of the total CD3+ T cells at these sites, respectively (Fig. 4, b and d). On day 4, V{gamma}2V{delta}2 T cells accumulated to make up 12–70% of total CD3+ T cells in the gingival mucosa of all tested animals except animal number 7317, whose V{gamma}2V{delta}2 T cell expansion level in the blood and lung was also the lowest among the animals examined (Fig. 4, a and b). In the rectal mucosa, an increase in V{gamma}2V{delta}2 T cells was prominently apparent in animals 7318 and 7319 on day 4 (Fig. 4, c and d). V{gamma}2V{delta}2 T cells returned to baseline levels at day 21 or 28 at both of these sites. Also, HMBPP alone did not result in detectable changes in gingival or rectal V{gamma}2V{delta}2 T cell levels (Fig. 4, b and d). Altogether, these results demonstrate that HMBPP/IL-2 cotreatment induced a prolonged accumulation of V{gamma}2V{delta}2 T cells in pulmonary mucosa and also their short-term increase in gingival and rectal mucosae.


Figure 4
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  		FIGURE 4. HMBPP/IL-2 cotreatment induced a short-term increase in V{gamma}2V{delta}2 T cells in gingival and rectal mucosae. Freshly collected cells from gingival mucosa (a and b) and rectal mucosal biopsies (c and d) from animals cotreated with 50 mg/kg (open symbols) or 10 mg/kg (filled symbols) HMBPP plus IL-2, or with HMBPP alone (50 mg/kg) were stained over time for V{gamma}2V{delta}2-expressing CD3+ T cells. V{gamma}2V{delta}2 T cell levels in gingival (a and b) and rectal (c and d) mucosae from pretreatment to day 4 in one representative cotreated animal (a and c) and over time for all examined animals (b and d) are shown from CD3-gated cells. Gingival and rectal V{gamma}2V{delta}2 T cell levels for HMBPP alone-treated animals are shown as the SEM (b and d).

 
Effector memory V{gamma}2V{delta}2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment were CCR5+CCR7–CD45RA–CD27+

The high levels of V{gamma}2V{delta}2 T cells that accumulated at the pulmonary mucosa upon HMBPP/IL-2 activation most likely underwent transendothelial migration from the circulation by specific interactions with the endothelium (27). Given that CCR7 and integrins (28) play an important role in driving lymphoid or mucosal migration of {alpha}β T cells, and that CCR5 is implicated in endothelial migration of human {gamma}{delta} T cells in vitro (29), we focused on evaluation of CCR7, integrins {alpha}4/β7, and CCR5 expression by pulmonary and circulating V{gamma}2V{delta}2 T cells. Interestingly, whereas integrin {alpha}4 was expressed by virtually all pulmonary and circulating V{gamma}2V{delta}2 T cells before and after treatment, integrin β7 expression increased from ~46 to ~68% on circulating V{gamma}2V{delta}2 T cells and decreased from ~63 to ~23% on pulmonary V{gamma}2V{delta}2 T cells (Fig. 5, a and b). Before treatment, circulating V{gamma}2V{delta}2 T cells expressed over 3-fold higher levels of CCR5 on their cell surface as compared with those V{gamma}2V{delta}2 T cells in the lung, whereas pulmonary V{gamma}2V{delta}2 T cells expressed over 4-fold higher levels posttreatment (Fig. 5, a and b). In contrast, whereas <10% of pulmonary V{gamma}2V{delta}2 T cells expressed CCR7 before and after treatment, blood V{gamma}2V{delta}2 T cells expressed significantly higher levels of CCR7 posttreatment (Fig. 5, a and b). These results suggest that pulmonary V{gamma}2V{delta}2 T cells that accumulated after HMBPP/IL-2 cotreatment possess a proinflammatory phenotype. Conversely, blood V{gamma}2V{delta}2 T cells may maintain the ability to migrate to lymph nodes or other lymphoid tissues due to the predominant expression of CCR7 during HMBPP/IL-2-mediated massive expansion, which potentially explains the V{gamma}2V{delta}2 lymphopenia observed on day 1 post-HMBPP treatment (Fig. 1, c and e).


Figure 5
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  		FIGURE 5. Migration and memory marker expression by pulmonary and circulating V{gamma}2V{delta}2 T cells. BAL fluid and whole blood from the following cotreated animals: 7311, 7315, 7317, 7318, and 7319 were examined. CD3- and V{gamma}2-gated BAL cells or PBL were examined for {alpha}4 integrin, β7 integrin, CCR5, and CCR7 expression before (a) and at 16 wk post-HMBPP treatment (b) and are shown as the SEM. V{gamma}2-gated BAL cells (c and d) or PBL (e) from five cotreated animals were examined for CD27 and CD45RA expression over time, and the relative percentages (c and e) or absolute numbers (d) of V{gamma}2 cells are shown as the SEM. All four subpopulations in the blood were statistically different from pretreatment (day 0) to day 4 (p < 0.05) (e). The expression of the following markers was statistically different between lung and blood V{gamma}2 T cells, as follows: CD45RA+CD27+ at days 0 and 56, CD45RA–CD27+ at days 0 and 4, and CD45RA–CD27– at day 4 (c and e).

 
We then compared the potential memory status of pulmonary and circulating V{gamma}2V{delta}2 T cells based on their expression of the surrogate memory markers CD27 and CD45RA at various time points post-HMBPP treatment. On average, ~82–95% of the V{gamma}2V{delta}2 T cells in the lung displayed a memory phenotype (CD27+CD45RA–) before and after treatment (Fig. 5c). In addition, on average, pulmonary CD27+CD45RA– and CD27–CD45RA– V{gamma}2V{delta}2 T cells increased from 7.9 x 105 to 2.1 x 106 (2.7-fold) and from 1.4 x 104 to 5.9 x 105 (40-fold) in absolute number on day 4, respectively (Fig. 5d). V{gamma}2V{delta}2 T cells in the blood also predominantly exhibited the CD27+CD45RA– memory phenotype at most time points except at peak expansion, whereby the effector memory phenotype (CD27–CD45RA–) significantly increased (Fig. 5e). However, majority of these circulating {gamma}{delta} T cells, not those in the pulmonary compartment, maintained the expression of the lymph node homing receptor CCR7 (Fig. 5b). Thus, although V{gamma}2V{delta}2 T cells in both pulmonary and blood compartments predominantly displayed the CD27+CD45RA– memory phenotype, an expression of CCR5 or CCR7 potentially distinguished effector memory from central memory phenotypes of these cells. In this regard, V{gamma}2V{delta}2 T cells that migrated to the lungs and remained there for months after HMBPP/IL-2 cotreatment appeared to be CCR5+CCR7–CD27+CD45RA– effector memory cells, as majority of them were able to exert effector function (see Fig. 8 below).


Figure 8
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  		FIGURE 8. Massive antimicrobial responses of long-lived pulmonary V{gamma}2V{delta}2 T cells. Freshly collected BAL cells from cotreated animals at 12 and 15 wk post-in vivi HMBPP treatment were incubated for 6 h with HMBPP plus anti-CD28 and anti-CD49d mAbs before staining. IFN-{gamma} expression by CD3-gated V{gamma}2+ and V{gamma}2– T cells is shown for each examined animal on week 12 (a) or as the SEM for four examined animals on week 15 (b). Perforin expression by CD3-gated V{gamma}2+ and V{gamma}2– T cells is shown as the SEM for the number of indicated animals on weeks 12 and 15 (c). The percentages of V{gamma}2 T cells that express IFN-{gamma} or perforin are indicated within parentheses in the dot plots.

 
HMBPP-activated V{gamma}2V{delta}2 T cells in the lung and circulation were able to re-recognize phosphoantigen and exert effector functions of IFN-{gamma} and perforin production

To determine whether the V{gamma}2V{delta}2 T cells that profoundly increased in the blood and pulmonary mucosa after HMBPP/IL-2 cotreatment could potentially re-recognize phosphoantigen produced by pathogens and mount antimicrobial immune responses, V{gamma}2V{delta}2 T cells in blood or BAL fluid were assessed for their ability to produce cytokines in vivo as well as to re-recognize phosphoantigen and exert those effector functions ex vivo. Up to 35% of circulating V{gamma}2 T cells that expanded on day 4 after HMBPP/IL-2 cotreatment were able to produce the cytotoxic molecule perforin even without ex vivo HMBPP restimulation (Fig. 6, a and b), whereas 0.5–3.8% of expanded V{gamma}2 T cells produced the antimicrobial cytokine IFN-{gamma} without further HMBPP exposure (Fig. 7, a and b). The in vivo production of these effector cytokines by V{gamma}2V{delta}2 T cells at their peak expansion coincided with the apparent increases in numbers of CD4+ and CD8+ {alpha}β T cells at day 7 (Fig. 2, d and e). Interestingly, the entire circulating V{gamma}2 T cell subpopulation that expanded in vivo at day 4 after HMBPP/IL-2 cotreatment produced perforin following ex vivo HMBPP restimulation (Fig. 6, c–e), whereas 13–61% of blood V{gamma}2 T cells (4.9–10.6% of total CD3+) produced IFN-{gamma} upon HMBPP restimulation at this time point (Fig. 7, c–e). Perforin expression by 4–15 and 2–6% of circulating CD3+V{gamma}2– T cells was also detected upon HMBPP restimulation on days 4 and 28, respectively (Fig. 6c). However, perforin-producing V{gamma}2 T cells were not detected at time points after day 4, suggesting the cytotoxic function of HMBPP-specific V{gamma}2V{delta}2 T cells may be a transient feature. The percentage of circulating V{gamma}2 T cells that produced IFN-{gamma} upon HMBPP restimulation varied among the animals tested, but were detectable at significant levels at all time points (Fig. 7e), suggesting the antimicrobial function of these cells is a lasting feature.


Figure 6
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  		FIGURE 6. Cytotoxic responses of circulating V{gamma}2V{delta}2 T cells. Freshly collected PBL from cotreated animals were stimulated for 6 h with HMBPP (c–e) or medium alone (a and b) plus anti-CD28 and anti-CD49d mAbs before staining. Perforin expression by CD3-gated V{gamma}2+ and V{gamma}2– T cells is shown as the SEM for five examined animals after incubation with medium alone (a) or HMBPP restimulation (c). The percentages of V{gamma}2 T cells that express perforin are indicated within parentheses in the dot plots. Percentages of CD3+ cells that are V{gamma}2+perforin+ after incubation with medium alone are shown for each individual animal examined over time (b). Percentages of CD3+ (d) or V{gamma}2+ (e) cells that are V{gamma}2+perforin+ after HMBPP restimulation for each animal are also shown. Asterisks indicate statistically different T cell levels from pretreatment (day 0) (p < 0.05) (b, d, and e). V{gamma}2+Perforin+ cell levels were statistically different between medium-alone and HMBPP-restimulated groups at day 4 (p < 0.05) (b and d).

 

Figure 7
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  		FIGURE 7. Antimicrobial responses of circulating V{gamma}2V{delta}2 T cells. Freshly collected PBL from cotreated animals were stimulated for 6 h with HMBPP (c–e) or medium alone (a and b) plus anti-CD28 and anti-CD49d mAbs before staining. IFN-{gamma} expression by CD3-gated V{gamma}2+ and V{gamma}2– T cells is shown as the SEM for five examined animals after incubation with medium alone (a) or HMBPP restimulation (c). The percentages of V{gamma}2 T cells that express IFN-{gamma} are indicated within parentheses in the dot plots. The percentages of CD3+ cells that are V{gamma}2+IFN-{gamma}+ after incubation with medium alone are shown for each individual animal examined over time(b). The percentages of CD3+ (d) or V{gamma}2+ (e) cells that are V{gamma}2+IFN-{gamma}+ after HMBPP restimulation in each animal are also shown. Asterisks indicate statistically different T cell levels from pretreatment (day 0) (p < 0.05) (b and d). V{gamma}2+IFN-{gamma}+ cell levels were statistically different between medium-alone and HMBPP-restimulated groups at day 4 (p < 0.05) (b and d).

 
Surprisingly, even at 12 and 15 wk post-HMBPP treatment, majority of the V{gamma}2 T cells that accumulated at high levels in the pulmonary mucosa were able to re-recognize the phosphoantigen HMBPP and exert effector function of cytokine production. Although up to 20% of BAL CD3+ T cells were V{gamma}2V{delta}2 T cells at weeks 12 and 15, on average 84.9 and 82.8% of these BAL V{gamma}2+ T cells produced massive amounts of IFN-{gamma} upon HMBPP restimulation at these time points, respectively (Fig. 8, a and b). This was in contrast to the low percentage of IFN-{gamma}-producing V{gamma}2V{delta}2 T cells in the blood even at their peak expansion after HMBPP/IL-2 cotreatment. Also, ~1.5 and 3.1% of pulmonary V{gamma}2+ T cells were able to produce perforin upon HMBPP restimulation at weeks 12 and 15, respectively (Fig. 8c). Furthermore, cytokine production by CD3+V{gamma}2– T cells was detected in the lung (Fig. 8). These results indicate that the long-lived HMBPP-specific V{gamma}2 T cells in the blood and lung are not anergic, but rather, are capable of responding to further antigenic stimulation to become IFN-{gamma}- and/or perforin-producing effectors and may regulate {alpha}β T cell responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This is the first study using the microbial phosphoantigen HMBPP for potential V{gamma}2V{delta}2 T cell-based immunotherapeutics in macaques. In comparison with recent studies using a nonmicrobial phospholigand (23, 24), several new observations have been made in our extensive study using HMBPP, as follows: 1) HMBPP/IL-2 cotreatment can induce a longer massive expansion of V{gamma}2V{delta}2 T cells, perhaps due to the fact that HMBPP is the most potent V{gamma}2V{delta}2-TCR ligand tested to date (13). 2) HMBPP-activated V{gamma}2V{delta}2 T cells can undergo an extraordinary pulmonary accumulation or migration, which lasts for months even after the expansion of these cells becomes undetectable in the blood. 3) These pulmonary V{gamma}2V{delta}2 T cells displayed an effector memory phenotype, as follows: CCR5+CCR7–CD45RA–CD27+. 4) Although the peak peripheral expansion of V{gamma}2V{delta}2 T cells confers upon these cells the ability to produce cytotoxic perforin in response to HMBPP, most V{gamma}2V{delta}2 T cells that migrated to the lung after HMBPP/IL-2 cotreatment are able to re-recognize microbial phosphoantigen and mount effector function via IFN-{gamma} production. 5) The capacity of massively expanded V{gamma}2V{delta}2 T cells to autonomously produce cytokines without ex vivo restimulation coincides with an increase in numbers of CD4+ and CD8+ {alpha}β T cells in the blood and lung after HMBPP/IL-2 cotreatment.

The prolonged accumulation of V{gamma}2V{delta}2 T cells in the lung airspaces implies that the pulmonary mucosa is the favorable migration site for phosphoantigen-specific V{gamma}2V{delta}2 T cells. In general, the magnitude and duration of V{gamma}2V{delta}2 T cell expansion in the lung are much greater than in the blood and gingival or rectal mucosae after the HMBPP/IL-2 cotreatment. This is consistent with our earlier studies demonstrating that a major expansion of V{gamma}2V{delta}2 T cells after pulmonary Mycobacterium tuberculosis infection occurs in the pulmonary compartment, but not in the blood (1). Importantly, the current study indicates that the pulmonary mucosa can selectively recruit a large number of V{gamma}2V{delta}2 T cells that predominantly display an effector memory phenotype and produce large quantities of IFN-{gamma} after HMBPP restimulation. The preferential expression of CCR5 by pulmonary V{gamma}2V{delta}2 T cells suggests that CCR5 may contribute in recruiting HMBPP-activated V{gamma}2V{delta}2 T cells to the lung. This scenario is indeed supported by the in vitro migration study describing a role of CCR5 and its ligands (MIP-1{alpha}, MIP-1β, and RANTES) in the transendothelial migration of human V{gamma}2 T cells (29). Although the CCR5-driven migration may account for the prolonged accumulation of V{gamma}2V{delta}2 T cells in the lung after the single HMBPP/IL-2 cotreatment, we cannot exclude the possibility that proliferation of these {gamma}{delta} T cells in pulmonary/bronchial mucosal lymphoid follicles contributes to their long-lasting accumulation. It is worth mentioning that the timing of the drop in peripheral V{gamma}2V{delta}2 and {alpha}β T cell numbers after expansion does not directly correspond to the increases of these cells in the lung. Thus, the decline of these cells in the circulation after their expansion may be due to activation-induced cell death.

Another extraordinary observation found in the present study is that over 80% of V{gamma}2V{delta}2 T cells that migrated to the lung can re-recognize phosphoantigen and produce copious amounts of the antimicrobial cytokine IFN-{gamma} even at 12–15 wk after the single HMBPP/IL-2 cotreatment. This observation suggests that the HMBPP/IL-2 regimen can confer V{gamma}2V{delta}2 T cell-based immunotherapeutics against a variety of pulmonary infections induced by phosphoantigen-producing pathogens. Presumably, these HMBPP-activated V{gamma}2V{delta}2 T cells can readily sense or recognize phosphoantigen-producing pathogens and mount IFN-{gamma}-mediated antimicrobial immune responses in the mucosa and lymphoid-tissue interface. Because HMBPP-activated V{gamma}2V{delta}2 T cells can express granulysin (L. Shao and Z. W. Chen, unpublished study), they may directly mediate bactericidal effects. Furthermore, HMBPP-activated V{gamma}2V{delta}2 T cells may readily attack target cells infected with phosphoantigen-producing pathogens, because some of these {gamma}{delta} T cells can produce the cytotoxic molecule perforin.

The long-lived pulmonary V{gamma}2V{delta}2 T cell response induced by a single HMBPP/IL-2 cotreatment may be sufficient to provide some degree of effect against various pathogens. However, multiple treatments may be needed to obtain optimal effects. Although an exhaustive response of V{gamma}2V{delta}2 T cells after repeated phospholigand/human IL-2 treatments has been previously reported (23), it is yet to be determined whether this is a true exhaustive phenomenon or simply attributed to the development of tolerance to human IL-2 by the monkeys instead of V{gamma}2V{delta}2 T cell exhaustion after repeated phospholigand/human IL-2 cotreatments.

We also found that the majority of HMBPP-activated V{gamma}2V{delta}2 T cells express CD8. Although under conditions in which these cells are not undergoing expansion, they constitute a minor proportion of total CD8+ T cells, our study demonstrates that these cells expand to make up >50% of the total CD8+ T cell population when V{gamma}2V{delta}2 T cell phosphoantigen is present along with IL-2. Thus, during situations in which V{gamma}2V{delta}2 T cells may be induced (e.g., during most bacterial or protozoal infections), it is important to distinguish CD8+ T cells based on their TCR to accurately determine potentially distinct responses of these two T cell populations. Furthermore, HMBPP-activated V{gamma}2V{delta}2 T cells appear to impact {alpha}β T cell responses in vivo. In animals whose V{gamma}2V{delta}2 T cells underwent massive expansion and produced detectable levels of cytokines in vivo, circulating {alpha}β T cells increased almost 4-fold. The increase in {alpha}β T cells may have resulted from a non-TCR-mediated expansion of total {alpha}β T cells (e.g., via IL-2R-, CD27/28-, {alpha}4/β7-, or other adhesion molecule-mediated signaling). The increase that we observed was transient most likely due to the absence of cognate {alpha}β TCR Ags, yet circulating and pulmonary CD3+V{gamma}2– T cells produced considerable levels of perforin and IFN-{gamma}. Thus, massively expanded V{gamma}2V{delta}2 T cells after the HMBPP/IL-2 cotreatment may confer an adjuvant effect on the development of adaptive {alpha}β T cell responses in the setting of an infection or immunization with a vector-based vaccine (1, 30, 31).


   Acknowledgments
 
We thank B. Paige, J. Graves, and Dr. K. Hagen for technical assistance with flow cytometry, and the University of Illinois Biological Resources Laboratory staff for animal care and technical assistance with complete blood counts.


   Disclosures
Top
 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 National Institutes of Health R01 Grants HL64560 and RR13601 (both to Z.W.C.) and Deutsche Forschungsgemeinschaft Grant JO565/1-1 (to H. J.). Back

2 Address correspondence and reprint requests to Dr. Zheng W. Chen, 835 South Wolcott Avenue, MC790, Chicago, IL 60612. E-mail address: zchen{at}uic.edu Back

3 Abbreviations used in this paper: HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; BAL, bronchoalveolar lavage; IPP, isopentenyl pyrophosphate. Back

Received for publication June 27, 2007. Accepted for publication October 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Shen, Y., D. Zhou, L. Qiu, X. Lai, M. Simon, L. Shen, Z. Kou, Q. Wang, L. Jiang, J. Estep, et al 2002. Adaptive immune response of V{gamma}2V{delta}2+ T cells during mycobacterial infections. Science 295: 2255-2258. [Abstract/Free Full Text]
  2. Wang, L., A. Kamath, H. Das, L. Li, J. F. Bukowski. 2001. Antibacterial effect of human V{gamma}2V{delta}2 T cells in vivo. J. Clin. Invest. 108: 1349-1357. [Medline]
  3. Poccia, F., L. Battistini, B. Cipriani, G. Mancino, F. Martini, M. L. Gougeon, V. Colizzi. 1999. Phosphoantigen-reactive V{gamma}9V{delta}2 T lymphocytes suppress in vitro human immunodeficiency virus type 1 replication by cell-released antiviral factors including CC chemokines. J. Infect. Dis. 180: 858-861. [Medline]
  4. Dieli, F., M. Troye-Blomberg, J. Ivanyi, J. J. Fournie, M. Bonneville, M. A. Peyrat, G. Sireci, A. Salerno. 2000. V{gamma}9/V{delta}2 T lymphocytes reduce the viability of intracellular Mycobacterium tuberculosis. Eur. J. Immunol. 30: 1512-1519. [Medline]
  5. Ottones, F., J. Dornand, A. Naroeni, J. P. Liautard, J. Favero. 2000. V{gamma}9V{delta}2 T cells impair intracellular multiplication of Brucella suis in autologous monocytes through soluble factor release and contact-dependent cytotoxic effect. J. Immunol. 165: 7133-7139. [Abstract/Free Full Text]
  6. Poccia, F., C. Agrati, F. Martini, M. R. Capobianchi, M. Wallace, M. Malkovsky. 2005. Antiviral reactivities of {gamma}{delta} T cells. Microbes Infect. 7: 518-528. [Medline]
  7. Farouk, S. E., L. Mincheva-Nilsson, A. M. Krensky, F. Dieli, M. Troye-Blomberg. 2004. {gamma}{delta} T cells inhibit in vitro growth of the asexual blood stages of Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that requires granulysin. Eur. J. Immunol. 34: 2248-2256. [Medline]
  8. Troye-Blomberg, M., S. Worku, P. Tangteerawatana, R. Jamshaid, K. Soderstrom, G. Elghazali, L. Moretta, M. Hammarstrom, L. Mincheva-Nilsson. 1999. Human {gamma}{delta} T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules. Scand. J. Immunol. 50: 642-650. [Medline]
  9. Liu, Z., B. L. Guo, B. C. Gehrs, L. Nan, R. D. Lopez. 2005. Ex vivo expanded human V{gamma}9V{delta}2+ {gamma}{delta}-T cells mediate innate antitumor activity against human prostate cancer cells in vitro. J. Urol. 173: 1552-1556. [Medline]
  10. Corvaisier, M., A. Moreau-Aubry, E. Diez, J. Bennouna, J. F. Mosnier, E. Scotet, M. Bonneville, F. Jotereau. 2005. V {gamma}9V{delta}2 T cell response to colon carcinoma cells. J. Immunol. 175: 5481-5488. [Abstract/Free Full Text]
  11. Chen, Z. W., N. L. Letvin. 2003. V{gamma}2V{delta}2+ T cells and anti-microbial immune responses. Microbes Infect. 5: 491-498. [Medline]
  12. McClanahan, J., P. I. Fukushima, M. Stetler-Stevenson. 1999. Increased peripheral blood {gamma}{delta} T-cells in patients with lymphoid neoplasia: a diagnostic dilemma in flow cytometry. Cytometry 38: 280-285. [Medline]
  13. Hintz, M., A. Reichenberg, B. Altincicek, U. Bahr, R. M. Gschwind, A. K. Kollas, E. Beck, J. Wiesner, M. Eberl, H. Jomaa. 2001. Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human {gamma}{delta} T cells in Escherichia coli. FEBS Lett. 509: 317-322. [Medline]
  14. Tanaka, Y., C. T. Morita, Y. Tanaka, E. Nieves, M. B. Brenner, B. R. Bloom. 1995. Natural and synthetic nonpeptide antigens recognized by human {gamma}{delta} T cells. Nature 375: 155-158. [Medline]
  15. Altincicek, B., J. Moll, N. Campos, G. Foerster, E. Beck, J. F. Hoeffler, C. Grosdemange-Billiard, M. Rodriguez-Concepcion, M. Rohmer, A. Boronat, et al 2001. Cutting edge: human {gamma}{delta} T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis. J. Immunol. 166: 3655-3658. [Abstract/Free Full Text]
  16. Eberl, M., M. Hintz, A. Reichenberg, A. K. Kollas, J. Wiesner, H. Jomaa. 2003. Microbial isoprenoid biosynthesis and human {gamma}{delta} T cell activation. FEBS Lett. 544: 4-10. [Medline]
  17. Eberl, M., M. Hintz, Z. Jamba, E. Beck, H. Jomaa, G. Christiansen. 2004. Mycoplasma penetrans is capable of activating V{gamma}9/V{delta}2 T cells while other human pathogenic mycoplasmas fail to do so. Infect. Immun. 72: 4881-4883. [Abstract/Free Full Text]
  18. Zhang, Y., Y. Song, F. Yin, E. Broderick, K. Siegel, A. Goddard, E. Nieves, L. Pasa-Tolic, Y. Tanaka, H. Wang, et al 2006. Structural studies of V{gamma}2V{delta}2 T cell phosphoantigens. Chem. Biol. 13: 985-992. [Medline]
  19. Gober, H. J., M. Kistowska, L. Angman, P. Jeno, L. Mori, G. De Libero. 2003. Human T cell receptor {gamma}{delta} cells recognize endogenous mevalonate metabolites in tumor cells. J. Exp. Med. 197: 163-168. [Abstract/Free Full Text]
  20. Bonneville, M., J. J. Fournie. 2005. Sensing cell stress and transformation through V{gamma}9V{delta}2 T cell-mediated recognition of the isoprenoid pathway metabolites. Microbes Infect. 7: 503-509. [Medline]
  21. Bukowski, J. F., C. T. Morita, Y. Tanaka, B. R. Bloom, M. B. Brenner, H. Band. 1995. V{gamma}2V{delta}2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer. J. Immunol. 154: 998-1006. [Abstract]
  22. Morita, C. T., E. M. Beckman, J. F. Bukowski, Y. Tanaka, H. Band, B. R. Bloom, D. E. Golan, M. B. Brenner. 1995. Direct presentation of nonpeptide prenyl pyrophosphate antigens to human {gamma}{delta} T cells. Immunity 3: 495-507. [Medline]
  23. Sicard, H., S. Ingoure, B. Luciani, C. Serraz, J. J. Fournie, M. Bonneville, J. Tiollier, F. Romagne. 2005. In vivo immunomanipulation of V{gamma}9V{delta}2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model. J. Immunol. 175: 5471-5480. [Abstract/Free Full Text]
  24. Casetti, R., G. Perretta, A. Taglioni, M. Mattei, V. Colizzi, F. Dieli, G. D’Offizi, M. Malkovsky, F. Poccia. 2005. Drug-induced expansion and differentiation of V{gamma}9V{delta}2 T cells in vivo: the role of exogenous IL-2. J. Immunol. 175: 1593-1598. [Abstract/Free Full Text]
  25. Hecht, S., S. Amslinger, J. Jauch, K. Kis, V. Trentinaglia, P. Adam, W. Eisenreich, A. Bacher, F. F. Rohdich. 2002. Studies on the non-mevalonate isoprenoid biosynthetic pathway: simple methods for preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate. Tetrahedron Lett. 43: 8929-8933.
  26. Veazey, R. S., M. DeMaria, L. V. Chalifoux, D. E. Shvetz, D. R. Pauley, H. L. Knight, M. Rosenzweig, R. P. Johnson, R. C. Desrosiers, A. A. Lackner. 1998. Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280: 427-431. [Abstract/Free Full Text]
  27. Chen, Z. W., N. L. Letvin. 2003. Adaptive immune response of V{gamma}2V{delta}2 T cells: a new paradigm. Trends Immunol. 24: 213-219. [Medline]
  28. Lefrancois, L., L. Puddington. 2006. Intestinal and pulmonary mucosal T cells: local heroes fight to maintain the status quo. Annu. Rev. Immunol. 24: 681-704. [Medline]
  29. Roth, S. J., T. G. Diacovo, M. B. Brenner, J. P. Rosat, J. Buccola, C. T. Morita, T. A. Springer. 1998. Transendothelial chemotaxis of human {alpha}/β and {gamma}/{delta} T lymphocytes to chemokines. Eur. J. Immunol. 28: 104-113. [Medline]
  30. Cendron, D., S. Ingoure, A. Martino, R. Casetti, F. Horand, F. Romagne, H. Sicard, J. J. Fournie, F. Poccia. 2007. A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct {gamma}{delta} and {alpha}β T cell responses in primates. Eur. J. Immunol. 37: 549-565. [Medline]
  31. Martino, A., R. Casetti, F. Poccia. 2007. Enhancement of BCG-induced Th1 immune response through V{gamma}9V{delta}2 T cell activation with non-peptidic drugs. Vaccine 25: 1023-1029. [Medline]



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