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Infection and Immunity, May 2003, p. 2945-2949, Vol. 71, No. 5
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.5.2945-2949.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

V{gamma}9V{delta}2 T-Cell Anergy and Complementarity-Determining Region 3-Specific Depletion during Paroxysm of Nonendemic Malaria Infection

Federico Martini,1* Maria Grazia Paglia,1 Carla Montesano,1 Patrick J. Enders,2 Marco Gentile,3 C. David Pauza,2 Cristiana Gioia,1 Vittorio Colizzi,4 Pasquale Narciso,3 Leopoldo Paolo Pucillo,1 and Fabrizio Poccia1

Laboratory of Clinical Pathology,1 4th Clinical Division,3 ICAERI, National Institute for Infectious Diseases "Lazzaro Spallanzani" I.R.C.C.S., Rome, Italy,4 Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, Maryland 210212

Received 9 September 2002/ Returned for modification 5 November 2002/ Accepted 20 January 2003


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ABSTRACT
 
V{gamma}9V{delta}2 T lymphocytes strongly respond to phosphoantigens from Plasmodium parasites. Thus, we analyzed the changes in V{gamma}9V{delta}2 T-cell function and repertoire during the paroxysm phase of nonendemic malaria infection. During malaria paroxysm, V{gamma}9V{delta}2 T cells were early activated but rapidly became anergic and finally loose J{gamma}1.2 V{gamma}9 complementarity-determining region 3 transcripts.


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TEXT
 
Malaria affects hundreds of millions of persons worldwide, and Plasmodium falciparum, the most common species responsible, causes a severe disease resulting in millions of deaths. The degree of malaria pathology depends on both parasite and host factors (27). T cells bearing the {gamma}{delta} T-cell receptor (TCR) represent a variable fraction (1 to 10%) of peripheral blood lymphocytes. The main subset expresses a skewed V{gamma}9V{delta}2 TCR rearrangement. These cells are activated during malaria infection and recognize nonpeptidic phosphoantigens present in P. falciparum (1, 2) and in red blood cells that could be released by erythrocyte rupture (4). After stimulation, V{gamma}9V{delta}2 T lymphocytes can produce significant amounts of gamma interferon and tumor necrosis factor alpha (TNF-{alpha}) (8, 14, 22) that can mediate killing of blood stage malaria parasites (11). However, the role of {gamma}{delta} T cells in malaria infection is still unclear.

In this study, we analyzed the longitudinal pattern of {gamma}{delta} T-cell activation during the early events of nonendemic malaria infection, both during the paroxysm stage and during recovery after successful therapy. Four patients with nonendemic P. falciparum malaria from the "L. Spallanzani" Institute were enrolled (mean age, 44 years; age range, 32 to 53 years; 3 males, 1 female). Informed consent was obtained from all persons prior to enrollment, and only residual blood samples from the diagnostic routine were used. Blood samples were collected before antimalarial treatment, when patients showed high fever, chills, and rigors (paroxysm), and after recovery from clinical symptoms (after 24 to 36 h of antimalarial treatment). Malaria diagnosis was made by microscopic examination of thick blood film and confirmed by species-specific PCR (26). The clinical state was evaluated by using a clinical score as previously described (12). Table 1 shows clinical data obtained during paroxysm and after successful antimalarial treatment. Six healthy donors were used as controls. Surface staining was performed by using anti-CD3 (immunoglobulin G1 [IgG1], clone UCHT1), anti-HLA-DR (IgG2a, clone I2), and anti-TCR V{delta}2 (IgG1, clone IMMU389) monoclonal antibodies directly coupled to fluorochromes (fluorescein isothiocyanate, phycoerythrin, and Cy5; ImmunoTech-Coulter, Miami, Fla.). In vitro V{gamma}9V{delta}2 expansion after stimulation with 120 µM isopentenyl pyrophosphate (IPP) was determined as previously described (20, 21). Supernatants from cultures stimulated for 24 h with IPP were analyzed for TNF-{alpha} production by enzyme-linked immunosorbent assay kits used in accordance with the manufacturer's (Endogen, Woburn, Mass.) instructions. V{gamma}9 TCR complementarity-determining region 3 (CDR3) length heterogeneity was determined as previously described (6, 7). Group comparison was performed with the Mann-Whitney U test, and values of P that were <=0.05 were considered significant.


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  		TABLE 1. Clinical parameters of patients with nonendemic P. falciparum malariaa

In Fig. 1A, a significant increase in the absolute V{delta}2 T-cell subset number was found in malaria patients only after recovery. However, an increased percentage of activated HLA-DR+ V{delta}2 T cells (Fig. 1B) was observed during paroxysm (1). Peripheral V{gamma}9V{delta}2 T cells were stimulated in vitro with IPP ligand (20, 21), and 24-h supernatants were collected and studied for TNF-{alpha} production by enzyme-linked immunosorbent assay (Fig. 1C). Interestingly, V{gamma}9V{delta}2 T cells were unable to produce TNF-{alpha} upon phosphoantigen stimulation during paroxysm. After 10 days, the CD3/V{delta}2/HLA-DR phenotype was assessed by flow cytometry and a relative expansion index was determined as previously described (20, 21) (Fig. 1D). During paroxysm, the IPP reactivity was significantly reduced but it reverted after successful chemotherapy. This anergy to IPP stimulation was specific for V{gamma}9V{delta}2 T cells, since the {alpha}ß T-cell response to phytohemagglutinin was unaffected (data not shown). Figure 2 shows flow cytometry panels from a representative patient. An increase in the V{delta}2 T-cell number and an increased frequency of V{delta}2+ HLA-DR+ activated cells were observed after recovery (Fig. 2A and B). When stimulated in vitro for 10 days with IPP, more than half of the lymphocytes were V{delta}2+ HLA-DR+ after recovery (Fig. 2F), compared with less than 1% in the same culture performed during paroxysm (Fig. 2E). Since it has recently been shown that V{gamma}9V{delta}2 T cells stimulated with nonpeptidic antigens mostly exhibit J{gamma}1.2 in the V{gamma}9 TCR{gamma} chains (17), we used spectratyping analysis to assess V{gamma}9 TCR heterogeneity (18). In Fig. 3, V{gamma}9 CDR3 length profiles from a representative patient with P. falciparum infection are shown both during paroxysm and after recovery (panel A). Two normal controls are shown in Fig. 3B. In healthy donors, the V{gamma}9 CDR3 lengths were biased to favor V{gamma}9 mRNA open reading frames 990 to 996 bases in length (Fig. 3B), corresponding to J{gamma}1.2 fragment usage (6, 7). In contrast, a shift from longer to shorter V{gamma}9 lengths was shown during paroxysm in malaria patients (Fig. 3A). Indeed, V{gamma}9 transcripts of 990 to 996 bases (J{gamma}1.2) accounted for 75% ± 13% (standard deviation) in healthy controls, compared to only 11% ± 16% (P < 0.005) and 42% ± 14% (P < 0.05) in malaria patients during paroxysm and recovery, respectively. Interestingly, there were increases in the proportions of normally minor 972-, 975-, and 978-base V{gamma}9 transcripts for both the paroxysm samples and the recovery samples compared to uninfected controls (Fig. 3A and B).



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  		FIG. 1. Malaria-induced changes in ex vivo V{gamma}9V{delta}2 T-cell subset absolute counts (A), ex vivo activation phenotype (HLA-DR+ percentage in the V{gamma}9V{delta}2 subset of T cells) (B), in vitro phosphoantigen-driven TNF-{alpha} synthesis (C), and V{delta}2 expansion index (D). Results are expressed as medians plus the upper quartiles. Four P. falciparum patients were studied in the paroxysm phase and after successful antimalarial treatment (24 to 36 h after paroxysm). As controls, six uninfected persons were studied. Differences between groups were evaluated with the Mann-Whitney U test. *, P <= 0.05; **, P <= 0.01.



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  		FIG. 2. Representative cytometric panel from a P. falciparum patient (120299) shown as V{delta}2/HLA-DR plots during paroxysm and after successful antimalarial treatment (24 h after paroxysm). In panels A and B, ex vivo distributions are shown. In panels C and D, in vitro cultures stimulated with phosphoantigen (PPD) plus interleukin-2 for 10 days are shown. In panels E and F, in vitro cultures stimulated with phosphoantigen (interleukin-2 plus IPP) for 10 days are shown. Percentages refer to the indicated quadrant among total T cells. FITC, fluorescein isothiocyanate; PE, phycoerythrin; CTRL, control.



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  		FIG. 3. V{gamma}9 CDR3 length profile frequencies of a representative P. falciparum patient (120299) studied during the paroxysm and recovery phases (A) and those of two normal controls (B). CDR3 lengths 990 to 996 refer to phosphoantigen-reactive V{gamma}9/J{gamma}1.2 clones.

The possible role of the host immune response in determining the clinical outcome of malaria infection is controversial (27), with data supporting both a protective (9) and an immunopathogenetic (5, 13) role mediated by cytokines. In this context, V{gamma}9V{delta}2 T lymphocytes have been shown to undergo deep changes during acute malaria infection: V{gamma}9V{delta}2 expansion at onset was described by some authors (19, 23), while others found a delayed but persistent expansion occurring only after recovery (25). In this study, we observed a paroxysm-linked decrease in response to phosphoantigens, as both in vitro TNF-{alpha} synthesis and expansion, presumably as a consequence of specific hyperactivation. Moreover, {gamma}{delta} T-cell function recovered after successful antimalarial treatment, confirming that {gamma}{delta} T-cell anergy during infectious diseases is reversible (16). Interestingly, a study of experimental infection with P. falciparum (24) showed fast activation of V{gamma}9V{delta}2 T cells, associated with a steep decline, linked to the erythrocyte cycle during which both Plasmodium phosphometabolites and red cell components are released. The overall outcome of V{gamma}9V{delta}2 T-cell stimulation strongly depends on the activation level. At the early phases of infection, a massive antigenic exposure makes possible the induction of an abortive response (10, 15), as activated V{gamma}9V{delta}2 T cells are highly susceptible to activation-induced cell death (4) triggered by Fas-FasL interaction (15). Moreover, the phosphoantigen 2,3-diphosphoglycerate, contained in red blood cells at millimolar concentrations, could be released by erythrocyte rupture and act as an antagonist (4), thus contributing to {gamma}{delta} T-cell anergy. Response to phosphoantigens is dependent on the V{gamma}9V{delta}2 receptor (3). V{gamma}9V{delta}2 T-cell populations maintain diversity in the CDR3s of V{gamma}9 mRNA after phosphoantigen stimulation, indicating that the response is polyclonal or oligoclonal, and are enriched for V{gamma}9 TCR chains containing the J{gamma}1.2 segment (7). During paroxysm, the changes in the V{gamma}9 repertoire indicated a preferential depletion of J{gamma}1.2 V{gamma}9 transcripts with higher affinity to phosphoantigens. Furthermore, human immunodeficiency virus-infected individuals were shown to have significant reductions in the frequency of V{gamma}9 containing the J{gamma}1.2 segment (6). Thus, the decrease in V{gamma}9V{delta}2 response during active infectious diseases may reflect a specific depletion of V{gamma}9/J{gamma}1.2+ phosphoantigen-responding clones by a mechanism of hyperactivation-apoptosis. The recovery phase associated with successful chemotherapy is followed by fast reconstitution of the V{gamma}9V{delta}2 T-cell number and response capability (16). This pattern of rapid activation, anergy depletion, and recovery after resolution of the infection may represent a common behavior of V{gamma}9V{delta}2 T cells in response to acute-phase infections.


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ACKNOWLEDGMENTS
 
We thank Gennaro De Libero, Enrico Girardi, and Massimo Amicosante for critical reading of the manuscript.

This work was supported by a current-research grant from the Italian Ministry of Health. P.J.E. was supported by a Public Health Service viral oncology training grant (5T32 CA09075).


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FOOTNOTES
 
* Corresponding author. Mailing address: National Institute for Infectious Diseases "Lazzaro Spallanzani" I.R.C.C.S., Via Portuense 292, 00149 Rome, Italy. Phone: 39 06 55170 907. Fax: 39 06 55170 904. E-mail: martini{at}inmi.it. Back

Editor: S. H. E. Kaufmann


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Infection and Immunity, May 2003, p. 2945-2949, Vol. 71, No. 5
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.5.2945-2949.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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