{gamma}{delta} T cells increase with Mycobacterium avium complex infection but not with tuberculosis in AIDS patients

J.-L. Pellegrin, J.-L. Taupin1, M. Dupon, J.-M. Ragnaud, J. Maugein2, M. Bonneville3 and J.-F. Moreau1

Services de Médecine Interne et Maladies Infectieuses,
1 Laboratoire d'Immunologie and
2 Laboratoire de Bactériologie, Centre Hospitalier Régional et Universitaire de Bordeaux, 33076 Bordeaux Cedex, France
3 INSERM U463, Hôtel-Dieu, Centre Hospitalier Régional et Universitaire de Nantes, 44035 Nantes, France

Correspondence to: J.-F. Moreau


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The aim of the present study was to better oinvokedcharacterize the expansion of double-negative (DN) T cells in vivo in AIDS patients and to ascertain the discrepant response of an immunodepressed immune system towards two distinct mycobacterial infections. In a large cohort of HIV-1 seropositive patients with low CD4+ T cell counts (<100/mm3), we have recently reported on an expansion of DN T cells which was observed only in patients with disseminated Mycobacterium avium infection, toxoplasmosis and Kaposi sarcoma, but not in patients with tuberculosis. The potential differential {gamma}{delta} T cells response observed in vivo in AIDS patients with tuberculosis or disseminated M. avium complex infection was investigated by collecting the concomitant or the closest T lymphocyte counts performed within 2 weeks of bacterial diagnosis of 112 disseminated M. avium infection and 41 tuberculosis patients. The DN and {gamma}{delta} T cell percentages were different between the two groups (P < 10–4) and the expansion of this compartment was found only with disseminated M. avium infections. An analysis of the variable {delta}2 segment versus pan-{delta} bearing T cells ratio disclosed a predominance of non-V{delta}2 T cells in these patients whose average values were identical in both groups. It is therefore concluded that the difference seen between these two types of mycobacterial infections concerning the DN T cells only involved the {gamma}{delta} T cells although the mechanism of their preferential expansion in disseminated M. avium infections remains a matter of speculation.

Keywords: {gamma}{delta} TCR, AIDS, immunosuppression, mycobacterial infections, opportunistic infections, T lymphocytes, tuberculosis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The recognition of antigen by T lymphocytes is accomplished through highly variable surface receptors, called the TCR, comprising either the {alpha}ß or the {gamma}{delta} heterodimers. The majority of the TCR {gamma}{delta}-bearing T cells found in the blood uses the V{gamma}9 together with the V{delta}2 gene variable segments to form a functional receptor. These cells belong to a subclass of non-CD8 and non-CD4 T cells called double negative (DN), which also includes other cells bearing the {alpha}ß TCR.

The functions of the {gamma}{delta} T cells remain largely unknown (1). Several lines of evidence suggest a particular role for them in immunity against mycobacteria and parasites, since they accumulate in the skin lesions of patients with leprosy or cutaneous leishmaniasis as well as in the blood of patients with leishmania, malaria, Epstein–Barr virus and HIV infections (1). In addition, V{gamma}9V{delta}2 cells proliferate in vitro when exposed to various mycobacterial extracts and parasites (2), whereas murine models strongly suggest a {gamma}{delta} T cell involvement in the host response against microbial pathogens (3). The most recent evidence demonstrates that V{gamma}9V{delta}2 cells are induced to proliferate, acquire broad cytotoxicity and produce tumor necrosis factor upon exposure to small phosphate non-peptidic ligands (previously called TUBag) in a manner similar to superantigenic stimulation of {alpha}ß T cells (4,5).

In contrast to the V{delta}2+ cells, those expressing the V{delta}1 gene segment are a minority at the periphery in normal subjects, but are increased in the blood of HIV-1-infected patients (6). In this case, no information is presently available on the function of these cells as well as their antigenic ligand leading to such expansion.

In a large cohort of HIV-1 seropositive patients with low CD4+ T cell counts (<100/mm3) we have recently reported on an expansion of CD3+CD4CD8 (DN) T cells observed in patients with disseminated Mycobacterium avium complex (D.MAC) infection, toxoplasmosis and Kaposi sarcoma, but not in patients with tuberculosis (7). The increase in DN T cells was found to be an independent predictor of D.MAC infection in patients with <100 CD4+ T cells/mm3.

In the present study, we compared the DN T cell and also the {gamma}{delta} T cell response in M. tuberculosis or D.MAC infections in AIDS patients from the same cohort of patients.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients
Patients were followed-up in the Bordeaux University Hospital by the `Groupe d'Epidémiologie Clinique du SIDA en Aquitaine' [GECSA, a surveillance system of HIV infection in Aquitaine established since 1987 (8)]. The diagnosis of tuberculosis or D.MAC infection was always microbiologically documented according to CDC criteria. Patients from the same cohort but free from any diseases for at least 1 year prior the blood determination were included as controls.

Flow cytometry
The immunological parameters were serially determined in the same laboratory using standard three-color flow cytometry carried out on EDTA-anticoagulated blood samples. Two successive immunophenotyping protocols were used.

Study 1.
From February 1994 to June 1995, patients samples were immunophenotyped by three-color flow cytometry (CD3, CD4 and CD8). The TCR subtype expressed by DN T cells was determined by flow cytometry using TCR {delta} (pan-{delta}) and ß (pan-ß) chain-specific mAb (Immunotech, Marseille, France) on randomly chosen patients from the study group. In these patients the DN T cells mainly expressed the {gamma}{delta} TCR heterodimer as already reported (7).

Study 2.
From July 1995 to May 1996, patients were prospectively followed-up and immunophenotyped using a four-color protocol (CD45, CD3, CD4 and CD8) (Tetrachrome; Coulter, Hialeah, Fl) before analysis with an Epics XL4 flow cytometer (Coulter). In addition {gamma}{delta} T cells were phenotyped using three-color staining (pan-{delta}, V{delta}2 and CD8{alpha}) (Immunotech) and read on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Normal blood donors (n = 54) were randomly taken as controls and their blood samples processed the same way.

Statistical analysis
CD4+, CD8+, DN T cell and {gamma}{delta} T cell percentages, and absolute counts for tuberculosis and D.MAC infection groups were compared using the non-parametric Mann–Whitney U-test, with a level of significance of 0.05. (Statistica; Statsoft, Tulsa, OK).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The potential differential {gamma}{delta} T cell response observed in vivo in AIDS patients with tuberculosis or D.MAC infection was investigated by collecting the concomitant or the closest T lymphocyte counts performed within 2 weeks of bacterial diagnosis of 112 D.MAC infections and 41 tuberculosis patients in total (n = 153). Although both groups of HIV+ patients were immunocompromised as estimated based on their percentage of peripheral CD4+ T cells when compared with normal blood donors, onset of tuberculosis was not dependent on the number of immunocompetent T cells. In agreement with this fact, CD4+ T cell percentages were significantly different between the two groups of illnesses (P < 10–5) and patients with opportunistic D.MAC infections exhibited the lowest level of CD4+ T cells in both studies (Table 1Go).


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Table 1. Comparison of percentages of CD4+, DN, {gamma}{delta} T cells and subclasses in patients with D.MAC versus patients with tuberculosis
 
The DN T cell percentages were also significantly different between the two groups (P < 10–4) and only patients with a D.MAC infection exhibited an expansion of this compartment in both studies (Table 1Go and Fig. 1AGo for the first study). In the second study where {gamma}{delta} T cells were directly enumerated, the same observation could be made (Fig. 1BGo), although not to the same extent as noticed in the first study on DN T cells, likely to be due to the smaller number of patients we could include in this second study.



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Fig. 1. Expansion of DN {gamma}{delta} T cells in HIV+ patients with D.MAC infection or tuberculosis. The percentages of DN T cells (A), of {gamma}{delta} T cells (B) among total blood lymphocytes and the ratio of V{delta}2 versus total {gamma}{delta}-bearing T cells (C) from either tuberculosis (BK) or D.MAC infection (MAC) infections are represented as box-plots (using Statistica). The open squares represent the median, the filled rectangles the values between the 75 and 25 percentiles, the limit bars the maximum and minimum limits of non-aberrant values, and the open circles the outliers.

 
Most blood {gamma}{delta} T cells in healthy adults use a particular combination of V region gene products (V{gamma}9 and V{delta}2) to form their TCR for antigen (1,2). We therefore carried out an analysis of the V{delta}2 gene usage in these patients and expressed the results as the ratio of the number of V{delta}2+ lymphocytes as identified with a specific mAb over the total number of {gamma}{delta} T cells as enumerated with a pan-{delta} mAb. The V{delta}2:pan-{delta} ratio showed a predominance of non-V{delta}2 T cells in these patients and their average values were identical in both groups (P = 0.96) (Fig. 1CGo).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
On a large number (n = 153) of HIV-1 seropositive patients infected with mycobacteria we confirm that an expansion of DN T cells is indeed associated with D.MAC infection but not with tuberculosis. In addition, we also established that these cells all belonged to the TCR {gamma}{delta} T cells and not to the {alpha}ß ones. Based on studies describing in vitro activation of {gamma}{delta} cells by mycobacteria and several parasites (35,9), an expansion of {gamma}{delta} T cells was expected to happen in this context. However, the finding that the percentage of {gamma}{delta} T cells remained in the normal range in patients infected with M. tuberculosis whereas it is increased in D.MAC infection is puzzling.

Because the occurrence of tuberculosis is not linked to immunosuppression as it occurs in non-immunosupressed individuals, we observed a higher percentage of CD4+ T lymphocytes in the tuberculosis group than in the D.MAC infection group of patients. Since the expansion of {gamma}{delta} T cells has been described to be helper dependent (10,11), the most severely CD4+ T cell-depleted patients would find it impossible to increase their {gamma}{delta} T cells. This is in total contrast to our data, suggesting that at least in HIV+ patients, the {gamma}{delta} T cell expansion is independent of the number of {alpha}ß CD4+ T cells. The differential response noted between tuberculosis and D.MAC infection cannot therefore be due to the immunodepression stage. This conclusion is further strengthened by the observation that some patients had a low CD4+ count, yet they did not display an elevated percentage or absolute number of {gamma}{delta} T cells when infected with M. tuberculosis.

The analysis of the V{delta}2:pan-{delta} ratio revealed a predominance of non-V{delta}2 T cells in agreement with previous reports showing that, in general, HIV+ patients have a strong reduction of their V{delta}2 subpopulation and a predominance of the V{delta}1 T cells subset (6). However, the mean V{delta}2:pan-{delta} ratio turned out to be identical within both groups of diseases, suggesting that if the V{delta}2 T cells are triggered to proliferate by mycobacterial compounds, they are made so only when challenged with M. avium complex and not with M. tuberculosis, and that both cell subsets are expanded. It is, however, noteworthy that there was no in vitro differences in the V{delta}2/V{gamma}9 cells responses when they were challenged in vitro with various mycobacteria (12).

It has already been reported that the size of the mycobacteria-reactive V{delta}2 T cells subset in both the blood and the lung was rather reduced in tuberculosis, and that these cells were refractory to in vitro mycobacterial antigenic challenge for unknown reasons (13). However, this report did not deal with HIV+ patients, in whom the V{delta}2-bearing subpopulation was reported by others to be anergic to antigenic challenge (6).

The discrepancy observed in the immune reaction following infections by these two infectious agents is new and questions the role of these particular cells in an immune response. The TCR {alpha}ß T cells perform well-characterized effector functions while the {gamma}{delta} T cell functions remain a matter of debate. Because these T cells are commonly abundant within epithelia they may be important in epithelial or mucosal infections. Mice lacking {gamma}{delta} T cells show enhanced intestinal damage in response to intra-intestinal protozoa infections likely to be due to a failure to regulate {alpha}ß T cell responses (14). Based on these data obtained from mouse models demonstrating a local role for these cells, it can be hypothesized that the expansion of both non-V{delta}2 and V{delta}2 T cells only seen in the case of D.MAC infection and not tuberculosis in HIV+ patients may be indicative of the route of infection. Indeed M. avium complex infection preferentially originates from the digestive tract, whereas M. tuberculosis preferentially disseminates from the lung, as all the cases collected in this study were pulmonary tuberculosis. Also related to this aspect is the far higher bacterial loads generally observed in D.MAC infection versus tuberculosis which may alter loco-regional T cell activation by inducing a strong recirculation of the antigen-activated cells. This may be of special importance in the case of HIV-infected patients since the virus profoundly and specifically depletes the CD4+ T cell compartment leaving the CD4 {gamma}{delta} T cells able to repopulate freed ancient TCR {alpha}ß T cell territories, susceptible to lead to a relative imbalance between both compartments.

In conclusion, this observation, in addition to its value for the diagnosis of D.MAC infection in HIV-1+ patients, (i) may help to address the controversial involvement of the {gamma}{delta} T cells in an immune response against tuberculosis and (ii) suggests that this functionally ill-defined cell subset may be important in order for immunocompromised patients to cope with disseminated mycobacterial opportunistic infections whose entry is thought to be intestinal.


    Acknowledgments
 
This work was made possible by a grant from SIDACTION 1995 and the `Ligue Régionale de Lutte contre le Cancer d'Aquitaine'. We are indebted to Ms M. Garcie, Ms N. Berrié and Mr J. C. Carron for excellent technical assistance.


    Abbreviations
 
DNdouble negative
D.MACdisseminated M. avium complex

    Notes
 
Transmitting editor: A. Fischer Back

Received 25 December 1998, accepted 21 May 1999.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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