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Molecular Medicine

, Volume 5, Issue 7, pp 471–476 | Cite as

In vivo γδ T Cell Priming to Mycobacterial Antigens by Primary Mycobacterium tuberculosis Infection and Exposure to Nonpeptidic Ligands

  • Fabrizio Poccia
  • Miroslav MalkovskyEmail author
  • Aaron Pollak
  • Vittorio Colizzi
  • Guido Sireci
  • Alfredo Salerno
  • Francesco Dieli
Open Access
Original Articles

Abstract

Background

The recognition of phosphorylated nonpeptidic microbial metabolites by Vγ9Vδ2 T cells does not appear to require the presence of MHC molecules or antigen processing, permitting rapid responses against microbial pathogens. These may constitute an important area of natural anti-infectious immunity. To provide evidence of their involvement in immune reactivities against mycobacteria, we measured the responsiveness of peripheral blood Vγ9Vδ2 T cells in children with primary Mycobacterium tuberculosis (MTB) infections.

Materials and Methods

Peripheral blood mononuclear cells from 22 children with MTB infections and 16 positivity of tuberculin (PPD)-negative healthy children were exposed to nonpeptidic antigens in vitro and the reactivity of the Vγ9Vδ2 T cell subset with these antigens was determined using proliferation and cytokine assays. Also, responses of γδ T cells from rhesus monkeys stimulated with phosphoantigens in vivo were measured.

Results

The Vγ9Vδ2 T cell responses were highly increased in infected children in comparison with age-matched controls. This augmented Vγ9Vδ2 T cell reactivity subsided after successful antibiotic chemotherapy, suggesting that persistent exposure to mycobacterial antigens is required for the maintenance of γδ T cell activation in vivo. The in vivo reactivity of Vγ9Vδ2 T cells to phosphoantigens was also analyzed in a rhesus monkey model system. Intravenous injections of phosphoantigens induced an activated state of simian Vγ9Vδ2 T cells which decreased after 2 months, i.e., with a time course similar to that seen in MTB-infected children.

Conclusions

The increased reactivity of Vγ9Vδ2 T cells to phosphoantigens appears to be dependent on constant antigenic exposure. Consequently, the assessment of Vγ9Vδ2 responses may be useful for monitoring the efficacy of antimycobacterial therapies.

Introduction

Tuberculosis in children is usually due to a primary infection (1). Anti-Mycobacterium tuberculosis (MTB) immunity depends on the interaction of antigen-specific CD4+ αβ+ T lymphocytes with macrophages (2,3). However, several studies indicate that γδ T lymphocytes also play an important role in MTB immunosurveillance (4,5). In Homo sapiens, most γδ T cells express the Vγ9Vδ2 rearrangements (6,7). Vγ9Vδ2 T cells from healthy donors recognize nonpeptidic, tumor-associated or microbial phosphoantigens (8, 9, 10, 11) and, similar to natural killer (NK) cells, display the inhibitory receptors for major histocompatibility (MHC) class I molecules (12, 13, 14, 15, 16, 17). These inhibitory receptors may control their reactivities toward conserved self-antigens and exogenous mycobacterial ligands (12). The phosphoantigenic recognition requires neither antigen uptake/processing nor classical polymorphic or nonpolymorphic MHC molecules, allowing for a rapid response to microbial immune challenge (18). This recognition is severely impaired in some patients with chronic viral (19,20) or bacterial (21) infections. Here we report our analyses of γδ T cell reactivities to phosphoantigens ex vivo in primary MTB-infected children and in vivo in rhesus monkeys (Macaca mulatta).

Materials and Methods

Cell Preparation and Stimulation

Peripheral blood mononuclear cells (PBMC) were isolated from 22 children with MTB infections (13 males, 9 females; 5.2 ± 3.3 years of age, range 1–12 years). Fourteen patients suffered from pulmonary MTB, four had MTB meningitis, three had lymphatic MTB, and one had renal MTB. The diagnoses of MTB infections were established by the presence of clinical symptoms, by the positivity of tuberculin (PPD) skin test, and by chest radiography. In some cases (i.e., MTB meningitis and renal MTB), positive cultures of microorganisms and/or MTB detection by polymerase chain reaction (PCR) further supported the clinical diagnosis. PPD-negative healthy children (9 males, 7 females; 6.2 ± 2.5 years of age, range 3–12 years) served as controls. Informed consent was obtained for each patient and control subject. In addition, PBMC were isolated from 12 healthy rhesus monkeys (5–13 years old). Mononuclear cells were cultured at 106 cells/ml in a complete culture medium [RPMI-1640, 10% v/v heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin]. Long-term cultures (10–14 days) were supplemented with 100 U/ml of recombinant interleukin-2 (IL-2) (Boehringer Mannheim, Mannheim, Germany), whereas short-term cultures (4 days) were performed in the presence of 5 U/ml of IL-2. Vγ9Vδ2 T cells were stimulated with the following: 0.5 mM ribose-1-phosphate (Rib-l-P, Sigma, St. Louis, MO), 0.5 mM xylose-1-phosphate (Xyl-l-P, Sigma), 0.5 mM dimethylallyl-pyrophosphate (DMAPP, Sigma), 50 µM monoethyl-pyrophopsphate [MEP, kindly provided by Drs. Y. Tanaka and B.R. Bloom (9)], 100 µM diphosphoglyceric acid (DPG, Sigma), 100 µM isopentenyl-pyrophosphate (IPP, Sigma), and the mycobacterial TUB Agl sample diluted 1/1000 v/v [approximately at a final concentration of 1 nM, kindly provided by Dr. J.J. Fournié (8)]. After 1 week of culture, 50% of culture medium was replaced by fresh medium. The expansion of Vγ9Vδ2 T cells was followed by cytometric analysis as previously described (14,19), following double staining of the stimulated cells with anti-CD3 or anti-CD2 [phycoerythrin (PE)] and Vδ2 [fluorescein isothiocyanate (FITC)] M Ab. The absolute number of Vδ2 T cell in each culture was calculated as follows: (% of Vδ2 T cells among total cells) X (total cell count)/100. The Vδ2 expansion index was then calculated by dividing the absolute number of Vδ2 T cells in specifically stimulated cultures by the absolute number of Vδ2 T cells before the initiation of culture (14,19). Thus, an expansion index higher than 1 represents a specific expansion of the Vδ2 T cell population. The Mann-Whitney U test was used and p values of <0.05 were considered significant.

Monoclonal Antibodies and FACS Analysis

The anti-CD3 (IgG1, clone SK7, Becton-Dickinson. Mountain View, CA) was coupled with PE. The B6.1 MAb (IgG1, Pharmingen, San Diego, CA), which recognizes the Vδ2 region of the γδTCR, was unlabeled or coupled with FITC. The following anti-human MAbs cross-reactive with rhesus monkey antigens were used: anti-CD2-PE (Antigenix America, New York) and anti-Vδ2-FITC (T Cell Diagnostic, Wabum, MA). The antiinterferon γ (IFN-γ) antibodies (clone 4S.B3, IgG1) were purchased from Pharmingen. Control MAb (IgGl or IgG2a) for cell surface labeling were purchased from Becton Dickinson. Analysis of surface and intracellular antigen expression was done as described previously (14). For each sample, 2 × 104 double-stained viable lymphocytes were gated following size (FSC) and granularity (SSC) criteria and analyzed with the Lysis II Software Program (Becton-Dickinson).

Intravenous Injection of DPG in Rhesus Monkeys

All monkeys received a single injection of 100 mg/kg DPG (the maximum dose tolerated by rodents). A slight increase in body temperature was observed in all injected monkeys for 2 hr and two monkeys out of eight developed fever, but recovered within a few hours. (The experimental protocol was approved by the Animal Research Committee of the University of Wisconsin.)

T Cell Proliferation Assay

PBMC from healthy donors (107/ml) were resuspended in complete medium and stimulated with 100 µM IPP and 5 U/ml IL-2. Cells were cultured for 4 days at 37°C in flat-bottomed microtiter wells, and T cell proliferation was measured following a 6-hr pulse with [3H]-thymidine [0.5 µCi/well, Amersham, Bucks, U.K. (22)]. Cultures were harvested (Skatron Instruments, Lier, Norway) and the number of cpm was determined using a β-counter (Packard Gamma 5500).

TNF-α and IFN-γ Detection

Tumor necrosis factor α (TNF-α) release in the supernatants of IPP-stimulated PBMC was measured by ELISA (Amersham) after 24 hr. The intracellular staining of Vδ2 T cells producing IFN-γ was analyzed after 6 hr of culture with IPP as previously described (14). Monensin (10 µM, Sigma) was added during the last 4 hr of culture to block intracellular transport and allow cytokine accumulation. The stimulated cells were washed in phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.1% sodium azide and stained with the anti-Vδ2-FITC MAb for 15 min at 4°C. The cells were fixed and permeabilized in PBS 1% paraformaldehyde for 10 min at 4°C, and incubated for 30 min at room temperature in the dark with the anti-cytokine MAb diluted in PBS with 1% BS A and 0.05% saponin. Finally, they were washed twice in PBS (1% BSA, 0.01% saponin) and analyzed using a FACScan (Becton Dickinson). The controls for nonspecific staining included cells stained with isotype-matched monoclonal antibodies. The proportions of cytokine-producing Vδ2 T cells were determined by FACScan analyses.

Results

To determine whether primary MTB-infected children possess Vγ9Vδ2 T cells exhibiting their constitutive reactivities (8, 9, 10, 11), PBMC from 22 patients and 16 age-matched controls were stimulated with different phosphoantigens. These studies assessed the ability of Vγ9Vδ2 cells to expand in 14-day cultures with different synthetic [such as ribose-1-phosphate (Rib-l-P), xylose-1-phosphate (Xyl-l-P), dimethylallyl-pyrophosphate (DMAPP), monoethyl-pyrophosphate (MEP), diphosphoglyceric acid (DPG), and isopentenyl-pyrophosphate (IPP)] (9,10), or natural [TUBAg-1 (8)] phosphoantigens. The Vγ9Vδ2 T cell responses were highly increased in MTB-infected children in comparison to age-matched controls (Table 1). The Vγ9Vδ2 T cell subset in tuberculin-positive MTB-infected children responded well to all antigens used. The strongest responses were detected using IPP, with the mean expansion index of 61. In contrast, the IPP-induced expansion of Vγ9Vδ2 T cells from healthy, tuberculin-negative children was approximately 10 times lower, with the mean expansion index of 6. The absolute and relative numbers of γδ T cells, Vγ9Vδ2 T cells, and Vδ2/CD45RO cells measured in these children as well as in some additional young TB patients and controls were comparable (Table 2). The γδ T cell response was further studied in 15 tuberculin-positive children 3 to 9 months after chemotherapy (23). The increased responsiveness of Vγ9Vδ2 cells sharply declined close to the levels detected in healthy tuberculin-negative children (Fig. 1). This suggests that persistent mycobacterial exposure was required for the presence of hyperactivity against phosphoantigens. In contrast, responses of αβ T lymphocytes to tuberculin (PPD) and to the immunodominant 38 kDa protein of MTB (24) were increased after chemotherapy (data not shown).
Table 1

Vγ9/Vδ2 responses to different phosphoantigens in PPD skin test-positive children, age-matched PPD-negative controls, and healthy rhesus monkeys

  

Vγ9/Vδ2 expansion index after in vitro culture with

PBMC

(n)

Rib-1-P

Xyl-1-P

DMAPP

TUBAg

IPP

DPG

MEP

Healthy PPDchildren

16

7 ± 6

2 ± 1

5 ± 2

6 ± 2

6 ± 1

n.d.

n.d.

MTB-infected PPD+ childrena

22

18 ± 7

30 ± 14

54 ± 24

50 ± 14

61 ± 20

n.d.

n.d.

Healthy rhesus monkeys

12

n.d.

n.d.

n.d.

8 ± 2

8 ± 3

6 ± 2

9 ± 3

PBMC were stimulated for 10–14 days with nonpeptidic antigens as described in Materials and Methods. The expansion index was calculated as the absolute number of Vδ2 T cells in stimulated cultures divided by the absolute number of Vδ2 T cells in the initial cultures prior to the antigenic exposure, n.d., not determined.

ap < 0.01 when compared to the healthy PPD group.

Table 2

Frequency of γδ T lymphocytes in the peripheral blood of healthy PPD skin test-positive children, healthy PPD skin test-negative children, and PPD skin test-positive children with primary tuberculosis

Children Category

Proportion of CD3+ Cells (Mean ± Standard Error)a

γδ +

Vγ9Vδ2+

Vδ2/CD45RO+

Healthy PPD+ (n = 46)

5.6 ± 0.5%

4.3 ± 0.4%

3.0 ± 0.4%

Healthy PPD (n = 17)

5.3 ± 0.6%

4.0 ± 0.5%

3.2 ± 0.3%

TB patients PPD+ (n = 27)

5.2 ± 0.6%

4.1 ± 0.4%

3.2 ± 0.4%

aThe absolute numbers of γδ T cells (mean ± standard error) in healthy PPD+: 84 ± 36 γδ+ cells/µl (n = 46); healthy PPD: 79 ± 33 + cells/µl (n = 17); TB patients PPD+: 78 ± 39 γδ+ cells/µl (n = 27).

Fig. 1

Successful chemotherapies of M. tuberculosis infections are accompanied by reduced γδ T cell reactivities to nonpeptidic antigens. (A) Vδ2 expansion index to Rib-IP, (B) Vδ2 expansion index to TUBAg, (C) Vδ2 expansion index to IPP. Each symbol corresponds to one individual.

To investigate the possibility of phosphoantigen-priming of the Vγ9Vδ2 T cell subset in vivo, rhesus monkeys whose Vγ9Vδ2 T lymphocytes react in vitro to IPP, MEP, and DPG (Table 1) received intravenous injections of DPG. Peripheral blood samples were collected at different time intervals and analyzed for functional reactivities in vitro. The DPG treatment resulted in a substantial up-regulation of both proliferative and cytokine (IFN-γ) responses to isopentenyl pyrophosphate in immunized animals (Fig. 2). IFN-γ-producing Vδ2 T cells increased from 2.2% prior to the in vivo treatment to 48.9% 1 month after the treatment. TNF-α-producing Vδ2 T cells were not detectable before, but were clearly present after, the treatment (Fig. 2).
Fig. 2

Intravenous injection of DPG induces an increased reactivity of γδ T cells. The Vγ9Vδ2 T cell expansion was monitored by measuring DNA synthesis after 5-day stimulation (A) or by flow cytometry after 10 days (B). Previous experiments (data not shown) have indicated that among the peripheral blood leukocytes proliferating in the IPP response, the Vγ9Vδ2 T cell subset forms a dominant population, whereas other proliferating leukocytes (which respond by DNA synthesis to Vγ9Vδ2 T cell-produced cytokines) constitute a minority. Cytokine production (C) was monitored by ELISA (TNF-α) or by flow cytometry (IFN-γ) before and 1 month after the injection of DPG in vivo. The production of TNF-α was detectable by the ELISA assay only after the administration of DPG.

Discussion

Historically, most vaccination strategies have utilized protein antigens. However, there are many lymphocytes that do not seem to recognize primarily peptide antigens bound to MHC molecules (8, 9, 10, 11, 12). Our data indicate that in vivo exposures to nonpeptidic antigens substantially augment the immunological reactivity of genetically unrestricted γδ T cells. When activated, these cells exert a powerful antibacterial activity (12, 13, 14) and release IFN-γ and TNF-α (14). These properties may be useful in the design and development of novel vaccines and/or adjuvants. The long-lasting memory response of αβ T lymphocytes is clinically applicable for evaluating previous exposures to MTB, but provides unsatisfactory information about the presence or absence of productive infection. In contrast, the γδ T cell hyperactivity against phosphoantigens requires persistent antigenic exposure. Thus, the assessment of Vγ9Vδ2 T cell responses may be a useful tool to monitor active MTB infections and potential failures of antimycobacterial therapies.

The recent elegant studies of Hoft et al. (25) provide evidence that the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine augments human γδ T cell responsiveness to mycobacteria. This study is fully compatible with our observations of human and simian γδ T cells primed in vivo by either TB disease or intravenously administered phosphoantigen. Therefore, both studies strongly suggest that γδ T cells in vivo may (a) allow for rapid and potent responses against the invading pathogen and (b) serve as targets for new TB vaccines.

Notes

Acknowledgments

This work was supported by grants from the NIH (AI42712 and RR00167) and AIDS/Tuberculosis Projects of the Italian Institute of Health “Istituto Superiore delia Sanità.” The authors are indebted to Jacque Mitchen for his expert help with animal experiments. This work is publication no. 39–017 of the Wisconsin Regional Primate Research Center.

References

  1. 1.
    Miller JWF. (1981) Tuberculosis in Children. Churchill Livingstone, Edinburgh.Google Scholar
  2. 2.
    North RJ. (1974) T cell dependence of macrophage activation and mobilization during infection with Mycobacterium tuberculosis. Infect. Immun. 10: 66–71.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Kaufmann SHE. (1989) In vitro analysis of the cellular mechanisms involved in immunity to tuberculosis. Rev. Infect. Dis. 11: 48–53.CrossRefGoogle Scholar
  4. 4.
    Ladel CH, Blum C, Dreher A, Reifenberg K, Kaufmann SHE. (1995) Protective role of γ/δ and α/β T cells in tuberculosis. Eur. J. Immunol. 25: 2877–2881.CrossRefGoogle Scholar
  5. 5.
    Kabelitz D, Bender A, Shondelmaier S, Schoel B, Kaufmann SHE. (1991) The primary response of human γ/δ T cells to Mycobacterium tuberculosis is restricted to Vγ9-bearing cells. J. Exp. Med. 173: 1331–1339.CrossRefGoogle Scholar
  6. 6.
    Parker CM, Groh V, Band H, et al. (1990) Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire. J. Exp. Med. 171: 1597–1612.CrossRefGoogle Scholar
  7. 7.
    Brenner MB, McLean J, Scheft H, et al. (1987) Two forms of the T-cell receptor gamma protein found on peripheral blood cytotoxic T lymphocytes. Nature 325: 689–694.CrossRefGoogle Scholar
  8. 8.
    Constant P, Davodeau F, Peyrat MA, et al. (1994) Stimulation of γδ T cells by nonpeptidic mycobacterial ligands. Science 264: 267–270.CrossRefGoogle Scholar
  9. 9.
    Tanaka I, Morita CT, Tanaka Y, Nieves E, Brenner MB, Bloom BR. (1995) Natural and synthetic non-peptide antigens recognized by human γδ T cells. Nature 375: 155–158.CrossRefGoogle Scholar
  10. 10.
    Burk MR, Mori L, De Libero G. (1995) Human Vγ9-Vδ2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites. Eur. J. Immunol. 25: 2052–2058.CrossRefGoogle Scholar
  11. 11.
    Fisch P, Malkovsky M, Kovats S, et al. (1990) Recognition by human Vγ9/Vδ2 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma cells. Science 250: 1269–1273.CrossRefGoogle Scholar
  12. 12.
    Poccia F, Gougeon ML, Bonneville M, et al. (1998) Innate T-cell immunity to nonpeptidic antigens. Immunol. Today 19: 253–256.CrossRefGoogle Scholar
  13. 13.
    Poccia F, Malkovsky M, Gougeon ML, et al. (1997) γδ T cell activation or anergy during infections: the role of nonpeptidic TCR ligands and HLA class I molecules. J. Leukoc. Biol. 62: 1–5.CrossRefGoogle Scholar
  14. 14.
    Poccia F, Cipriani B, Vendetti S, et al. (1997) CD94/NKG2 inhibitory receptor complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive Vγ9Vδ2 T lymphocytes. J. Immunol. 159: 6009–6017.PubMedGoogle Scholar
  15. 15.
    Battistini L, Borsellino G, Sawicki G, et al. (1997) Phenotypic and cytokine analysis of human peripheral blood γδ T cells expressing natural killer receptors. J. Immunol. 159: 3723–3730.PubMedGoogle Scholar
  16. 16.
    Halary F, Peyrat M-A, Champagne E, et al. (1997) Control of self-reactive cytotoxic T lymphocytes expressing γδ T cell receptors by natural killer inhibitory receptors. Eur. J. Immunol. 27: 2812–2821.CrossRefGoogle Scholar
  17. 17.
    Carena I, Shamshiev A, Donda A, Colonna M, De Libero G. (1997) Major histocompatibility complex class I molecules modulate activation threshold and early signalling of T cell antigen receptor-γ/δ stimulated by nonpeptidic ligands. J. Exp. Med. 186: 1769–1774.CrossRefGoogle Scholar
  18. 18.
    Lang F, Peyrat MA, Constant P, et al. (1995) Early activation of human Vγ9Vδ2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands. J. Immunol. 154: 5986–5994.PubMedGoogle Scholar
  19. 19.
    Poccia F, Boullier S, Lecoeur H, et al. (1996) Peripheral Vγ9/Vδ2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-1 infected persons. J. Immunol. 157: 4449–4461.Google Scholar
  20. 20.
    Wallace M, Scharko AM, Pauza CD, et al. (1997) Functional γδ T-lymphocyte defect associated with human immunodeficiency virus infections. Mol. Med. 3: 60–71.CrossRefGoogle Scholar
  21. 21.
    Li B, Rossman MD, Imir T, et al. (1996) Disease-specific changes in γδ T cell repertoire and function in patients with pulmonary tuberculosis. J. Immunol. 157: 4222–4229.PubMedGoogle Scholar
  22. 22.
    Malkovsky M, Asherson GL, Stockinger B, Watkins MC. (1982) Nonspecific inhibitor released by T acceptor cells reduces the production of interleukin-2. Nature 300: 652–655.CrossRefGoogle Scholar
  23. 23.
    Bothamley GH. (1995) Serological diagnosis of tuberculosis. Eur. Respir. J. 8: 676–688.Google Scholar
  24. 24.
    Kadival GV, Chaparas SD, Hussong D. (1987) Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis. J. Immunol. 139: 2447–2451.PubMedGoogle Scholar
  25. 25.
    Hoft DF, Brown RM, Roodman ST. (1998) Bacille Calmette-Guérin vaccination enhances human γδ T cell responsiveness to mycobacteria suggestive of a memory-like phenotype. J. Immunol. 161: 1045–1054.PubMedGoogle Scholar

Copyright information

© Picower Institute Press 1999

Authors and Affiliations

  • Fabrizio Poccia
    • 1
    • 2
  • Miroslav Malkovsky
    • 1
  • Aaron Pollak
    • 1
  • Vittorio Colizzi
    • 2
  • Guido Sireci
    • 3
  • Alfredo Salerno
    • 3
  • Francesco Dieli
    • 3
  1. 1.Department of Medical Microbiology and ImmunologyUniversity of Wisconsin Medical School, University of Wisconsin Comprehensive Cancer Center, and Wisconsin Regional Primate Research CenterMadisonUSA
  2. 2.International Center for AIDS and Other Emerging Infections, L. Spallanzani Institute and Department of BiologyUniversity of Rome “Tor Vergata,”RomeItaly
  3. 3.Institute of General PathologyUniversity of PalermoPalermoItaly

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