TY - JOUR
AU - Burastero, S E
AB - SUMMARY Individuals with a negative intradermal reaction to tuberculin PPD have long been described in the Mycobacterium tuberculosis exposed, immune-competent population. Here, we studied PPD-specific blood T lymphocytes from these subjects for phenotypic markers relevant to skin migration, including the expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen (CLA). Out of 82 patients with active tuberculosis we identified four subjects who were repeatedly PPD skin test-negative. CD4 T lymphocytes specific to mycobacterial antigens were derived from these individuals, which (i) proliferated in vitro to M. tuberculosis antigens comparably to those from PPD+ patients; (ii) secreted comparable amounts of IL-2 but lower amounts of IFN-γ; (iii) were confined within the CLA-negative T cell subset. We conclude that the negative tuberculin reaction in a small subset of patients exposed to mycobacteria is associated with impaired production of IFN-γ by circulating PPD-specific T cells that are lacking CLA expression. On this basis in vitro proliferation to PPD can discriminate bona fide non-responders from infected patients with a deficit in the margination of M. tuberculosis-specific T lymphocytes. tuberculosis, skin test, cutaneous lymphocyte antigen INTRODUCTION PPD skin test-negative individuals (i.e. individuals who do not develop an indurative reaction in the 48–72 h following the intradermal injection of a standardized amount of PPD) have long been described among immune-competent subjects previously vaccinated with bacille Calmette–Guérin (BCG) or infected with Mycobacterium tuberculosis [1]. In a proportion of these individuals the skin anergy to PPD is transient since, in the weeks following the presumed exposure to M. tuberculosis, the reaction turns positive [2, 3]; however, some remain persistently PPD skin test-negative at subsequent controls [4]. These latter patients do not have any distinct clinical peculiarities, but may escape a first-line diagnostic evaluation. It has been suggested that a relatively low sensitivity of skin reaction may explain a negative PPD test in patients with a lower level of immunological reactivity, but there is general agreement about the fact that qualitative differences underly persistent PPD negativity in some patients with tuberculosis (TB) [1]. PPD-specific peripheral blood CD4 T cells can be identified in M. tuberculosis-exposed individuals using an in vitro lymphocyte proliferation assay [5]. It has been reported that T lymphocytes taken from M. tuberculosis-exposed (or BCG-vaccinated) individuals with a negative skin reaction to PPD undergo blast cell transformation when incubated in vitro with PPD [6]. In this study we compared blood lymphocytes taken from PPD skin test-negative and PPD+ patients with TB, focusing on the expression of the cutaneous lymphocyte-associated antigen (CLA) and on the pattern of lymphokines secreted by PPD-reactive T cells. Our results suggest a correlation between reduced IFN-γ production and impaired CLA expression. PATIENTS AND METHODS Patients We studied 82 patients with newly diagnosed pulmonary (n = 76) or extrapulmonary (n = 6) TB. The diagnosis was made by means of positive acid-fast smears and/or by the microbiological isolation of M. tuberculosis from biological specimens. They were all treated with conventional therapy and they all responded to the primary chemotherapeutic regimen. All of the patients underwent a PPD reaction test performed by means of an intradermal injection of 20 U of PPD (Biocine, Siena, Italy). The infiltrative reaction was evaluated 72 h after injection and scored as positive when the average of the maximum and minimum diameters was > 5 mm. When a negative PPD reaction was observed, the test was repeated twice (1 month and 6–8 months later). A subgroup of 18 patients with pulmonary TB and positive skin reaction to PPD was randomly selected and subjected to further in vitro studies, as specified below. Intradermal reactivity to a panel of common environmental antigens (tetanus toxoid (TT), Diphtheria, Streptolysin, PPD, Candida, Trycophiton, Proteus) was also assessed in selected patients using a commercial assay (Multitest, Merieaux, Paris, France). Antibodies and antigens The purified MoAb HECA-452 against human CLA was a generous gift from L. J. Picker (Dallas, TX) [7]. FITC-conjugated HECA-452 and the corresponding isotype-matched FITC-conjugated MoAb with irrelevant specificity were purchased from PharMingen (San Diego, CA). The purified unconjugated anti-CD14, anti-CD16, anti-CD19 and anti-CD8 MoAbs and the (Fab)′2 goat anti-rat (H + L) affinity-purified antibody used for the cell separation experiments came from Sigma (St Louis, MO). The mycobacterial antigens used for the proliferation assays included: PPD from M. tuberculosis for in vitro use (Statens Serum Institute, Copenhagen, Denmark); and heat-killed whole cells from M. tuberculosis, strains H37Rv and H37Ra (WC-H37Ra and WC-H37Rv) or crude culture filtrate from M. tuberculosis, strains H37Rv and H37Ra, without lipoarabinomannan (CF-H37Ra and CF-H37Rv), generously provided by P. Brennan and J. Belisle (Fort Collins, Colorado State University, CO). Proliferation assays Five-day proliferation assays of peripheral blood mononuclear cells (PBMC) were performed in order to evaluate the in vitro proliferation of specific T cells from M. tuberculosis-infected patients following stimulation with mycobacterial antigens. The PBMC were separated from whole blood using a Ficoll density gradient (Pharmacia, Uppsala, Sweden), washed in PBS (Gibco, Milan, Italy) and re suspended in RPMI 1640 medium (Gibco). They were then seeded (2 × 105/well) in 96-well flat-bottomed microtitre plates (Costar, Milan, Italy). The cells were cultured for 5 days in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine and 10% fetal bovine serum (FBS; Gibco) (complete medium). Either complete medium (in control wells) or antigens were added (each at 20 μg/ml) to quadruplicate microcultures. Tritiated thymidine was added to each microculture well (0.5 μCi/well) for the last 8 h of culture. The cells were harvested using a cell harvester (Skatron, Lier, Norway), and thymidine incorporation was measured as ct/min using a β-counter (LKB). Proliferation was expressed as a proliferation index, calculated as the ratio between the thymidine incorporation in the test wells (containing PBMC stimulated with antigens) and that in the control wells (containing unstimulated PBMC). The proliferation assays were considered positive on the basis of restricted criteria: i.e. when the proliferation index was > 3 and the thymidine incorporation was > 2000 ct/min greater than that in the unstimulated control wells [8]. All of the patients included in this study were positive for more than one of the mycobacterial antigen preparations used to stimulate the PBMC. Preparation of CLA-enriched and CLA-depleted CD4 T cells Experiments were performed to assess if the in vitro proliferation to PPD of CD4 T lymphocytes could be positively or negatively segregated on the basis of CLA expression. These were designed as previously described by Santamaria Babi et al. with modifications [9]. Briefly, the CD4 T lymphocytes were prepared by means of negative immunobead separation. The PBMC were reacted simultaneously with MoAbs to human CD14, CD16, CD19 and CD8 in ice, and then co-incubated with anti-mouse IgG antibody-conjugated magnetic beads, according to the manufacturer’s instructions (Dynal, Oslo, Norway). The bead-rosetted cells were separated from the others using a magnetic support containing the cell suspension in a tube. The CD4 T lymphocyte-enriched T cell population consisted at cytofluorometric analysis of 92–97% of CD4+ cells in preliminary experiments. Subsequently, the CD4 T lymphocytes were enriched and depleted of CLA+ cells by means of panning. The cells were reacted with HECA-452 MoAb (1 μg/106 cells) and plated on anti-rat immunoglobulin (H + L)-coated, non-tissue culture-treated plates (coating conditions: 4°C overnight with an antibody concentration of 20 μg/ml in PBS). The non-HECA-452-reacting cells (CLA-depleted) were gently harvested using a pipette; the HECA-452-reacting cells (CLA-enriched) were detached by means of cold PBS. The HECA-452-enriched and -depleted lymphocytes were counted and plated (5 × 105/well) with 15 Gy irradiated autologous PBMC as a source of antigen-presenting cells (APC; 104/well), with or without antigens. Typically, > 80% and < 2% of CD4 T cells were HECA-452+ at cytofluorometric analysis in the CLA-enriched and in the CLA-depleted preparations, respectively. Establishment of (secondary) M. tuberculosis-specific T cell lines for the analysis of cytokine secretion Aliquots of 2 × 106 PBMC were seeded in 24-well plates (Costar) in 2 ml of complete medium with PPD (20 μg/ml). On day 7 the cells were washed and re-stimulated with autologous, 15 Gy irradiated PBMC (as a source of APC) pulsed with antigen (PPD, 20 μg/ml). On day 15, the supernatants were removed and the cells were washed and stimulated for 18 h with phorbol myristate acetate (PMA; 50 ng/ml). Before adding brefeldin A for intracellular staining (see below), the supernatants were removed and stored for analysis of their IL-4 and IFN-γ content, which was done using an in-house ELISA (Roche Milano Ricerche, Milano, Italy). IFN-γ and IL-4 production by polyclonally activated PBMC As a measure of the overall capability to produce IFN-γ, microcultures of 2 × 105 CD4 T cells per well were established in complete medium in the presence of irradiated autologous PBMC as a source of APC 10 μg/ml phytohaemagglutinin (PHA; Sigma). After 72 h supernatants were collected for IFN-γ and IL-4 measurement. Analysis of p70 IL-12 production by monocytes Monocyte-enriched preparations were obtained by incubating PBMC for 1 h in tissue culture flasks in complete medium; the adherent cells were subsequently detached by washing with cold PBS and scraping and used for IL-12 production assays. In order to optimize IL-12 secretion, we basically used the protocol described by Ma et al. [10]. Briefly, monocyte-enriched, adherent cell preparations (1 × 106/ml in complete medium) were primed for 18 h with 1000 U/ml of IFN-γ and then cultured for 24 h with 1 μg/ml of lipopolysaccharide (LPS; Sigma) or heat-killed whole cells from M. tuberculosis, strain H37Rv (20 μg/ml). IL-12 was measured in the supernatants using an in-house ELISA system (Roche Milano Ricerche). Single-cell analysis of cytokine production on secondary PPD-specific T cell lines Brefeldin A was added to the PMA-stimulated T cell lines during the last 2 h of culture, and then the cells were fixed with 4% paraformaldehyde and permeabilized with saponin. The fixed, permeabilized cells were stained with FITC–anti-IFN-γ and PE–anti-IL-4 MoAbs, according to manufacturer’s instructions (PharMingen). FACS analysis Cells were stained with the indicated MoAb and analysed using the cytometer (FACScan cytometer in a four-parameter acquisition setting; Becton Dickinson, Mountain View, CA). The results were analysed by means of Cellquest software (Becton Dickinson). Statistical analysis Unpaired t-tests were used for between-group comparisons. P < 0.05 was considered statistically significant. The data are expressed as mean values ± s.d. RESULTS Identification of PPD skin test-negative individuals among patients exposed to M. tuberculosis The PBMC taken from 82 individuals with active TB were tested for proliferation to a panel of five different preparations containing mycobacterial antigens, i.e. PPD, culture fluid from the H37-Ra or Rv strains, and heat-killed whole cells from the same strains. All patients were tested for skin reactivity to PPD. Seventy-eight patients were positive to both the in vivo PPD reaction and in vitro proliferation to PPD; four were PPD skin test-negative and maintained this negativity at two subsequent controls 6 months apart. The proliferation to all of the mycobacterial antigens, expressed as a proliferation index, was similar in the PPD skin test-negative and PPD skin test-positive subjects, indicating that the same pool of antigens was recognized by T cells (Table 1). The clinical profile of the four PPD skin test-negative patients was similar to that of most PPD+ patients with pulmonary TB (extensive unilateral lesions, comparable age range, no antibiotic resistance) and they all responded (according to clinical and radiological criteria) to a primary chemotherapeutic standard regimen. The four PPD− patients had positive acid-fast smears and multiple positive cultures for M. tuberculosis. Microbiological processing of the samples was performed in a laboratory belonging to a national network of clinical reference laboratories, yearly subjected to quality control procedures. The isolation of M. tuberculosis from different aliquots of the four original specimens was confirmed by an independent laboratory, to exclude false-positive results due to cross-contamination. Table 1 Proliferation of peripheral blood mononuclear cells (PBMC) from patients with active lung tuberculosis to different Mycobacterial antigens Numbers indicate number of patients.Positivity was determined according to the criteria described in Patients and Methods.H37Ra and H37Rv are the two mycobacterial strains originating from the raw antigenic preparations of either WC (heat-killed whole cells) or CF (lipoarabinomannan-depleted protein from culture fluid). Open in new tab Table 1 Proliferation of peripheral blood mononuclear cells (PBMC) from patients with active lung tuberculosis to different Mycobacterial antigens Numbers indicate number of patients.Positivity was determined according to the criteria described in Patients and Methods.H37Ra and H37Rv are the two mycobacterial strains originating from the raw antigenic preparations of either WC (heat-killed whole cells) or CF (lipoarabinomannan-depleted protein from culture fluid). Open in new tab Cellular immunity to other common antigens was tested in vivo in the PPD skin test-negative patients using a commercial intradermal assay. They had individual patterns of skin reactivity which excluded generalized skin anergy (Table 2) and were comparable to those observed in the general population and in PPD+ TB patients. Table 2 Intradermal skin reaction to standard antigens (Multitest) Numbers indicate the mean diameter (maximum + minimum diameter, averaged) of the infiltrative reaction to each antigen measured at 72 h, in millimetres. Open in new tab Table 2 Intradermal skin reaction to standard antigens (Multitest) Numbers indicate the mean diameter (maximum + minimum diameter, averaged) of the infiltrative reaction to each antigen measured at 72 h, in millimetres. Open in new tab M. tuberculosis-specific T cells from PPD skin test-negative patients with active TB segregate in the CLA− CD4 T cell subset The cutaneous lymphocyte antigen is the candidate homing receptor for routing CD4 T cells to the skin endothelium. CLA+ and CLA− CD4 T lymphocytes were isolated from PBMC and separately assayed in cell proliferation assays, in the presence of autologous irradiated PBMC as a source of APC. The proliferation to PPD was prevalently restricted to the CLA− CD4 T cell subset in the four PPD skin test-negative individuals. In contrast, the CLA+ but not the CLA− CD4 T cells proliferated to PPD in a panel (n = 18) of PPD+ patients with TB, as expected (Fig. 1). Fig. 1 Open in new tabDownload slide In vitro proliferation to PPD of cutaneous lymphocyte-associated antigen (CLA)-enriched (CLA+) and CLA-depleted (CLA−) CD4 T lymphocytes from PPD skin test-negative patients ((a), individual values) and PPD skin test-positive patients ((b), grouped results) with tuberculosis. Fig. 1 Open in new tabDownload slide In vitro proliferation to PPD of cutaneous lymphocyte-associated antigen (CLA)-enriched (CLA+) and CLA-depleted (CLA−) CD4 T lymphocytes from PPD skin test-negative patients ((a), individual values) and PPD skin test-positive patients ((b), grouped results) with tuberculosis. In a parallel and independent set of experiments, we further controlled this observation by using a different read out. The proportion of CLA+ CD4 cells was measured before and after culturing PBMC from different individuals with PPD. The expansion of the CLA subset upon addition of the antigen will indicate that CLA+ CD4 T cells contain PPD-specific T lymphocytes. We found that the addition of PPD induced an expansion of CD4 CLA+ cells in a panel of PPD skin test-positive patients (n = 18), but in none of the PPD skin test-negative patients. Fig. 2 shows CLA expression by CD4 T cells from one PPD skin test-negative and one PPD skin test-positive TB patients following in vitro incubation with PPD. Individual results are shown as percent variation of CLA+ CD4 T cells induced by PPD in the four PPD skin test-negative patients (Fig. 3). Percent variations obtained from 18 PPD+ patients are indicated as grouped results for comparison. Fig. 2 Open in new tabDownload slide Cytofluorometric analysis of cutaneous lymphocyte-associated antigen (CLA) expression at baseline (left panels) and after 8 days of in vitro PPD stimulation (+ PPD) on CD4 T cell lines from one representative PPD skin-test positive (PPD+) and one representative PPD skin test-negative (PPD-neg-1) patient with tuberculosis. Fig. 2 Open in new tabDownload slide Cytofluorometric analysis of cutaneous lymphocyte-associated antigen (CLA) expression at baseline (left panels) and after 8 days of in vitro PPD stimulation (+ PPD) on CD4 T cell lines from one representative PPD skin-test positive (PPD+) and one representative PPD skin test-negative (PPD-neg-1) patient with tuberculosis. Fig. 3 Open in new tabDownload slide Percent variation of cutaneous lymphocyte-associated antigen (CLA)+ CD4 T cells from PPD skin test-negative (individual values) and PPD+ patients (grouped values) with tuberculosis, following 8-day in vitro stimulation with PPD. Fig. 3 Open in new tabDownload slide Percent variation of cutaneous lymphocyte-associated antigen (CLA)+ CD4 T cells from PPD skin test-negative (individual values) and PPD+ patients (grouped values) with tuberculosis, following 8-day in vitro stimulation with PPD. As an internal antigen control for the correlation between CLA expression and margination to the skin, as detected with this assay, we used tetanus toxoid (TT). In fact, patients PPD-neg-1 and PPD-neg-4, but not PPD-neg-2 and PPD-neg-3, had a positive skin reaction following the intradermal injection of this antigen (Table 2). Indeed, we found that TT induced an in vitro expansion of CLA+ CD4 T cells in PPD-neg-1 and PPD-neg-4, but not in PPD-neg-2 and PPD-neg-3 (Fig. 4). Fig. 4 Open in new tabDownload slide Percent variation of cutaneous lymphocyte-associated antigen (CLA)+ CD4 T cells from PPD skin test-negative patients with tuberculosis, following 8-day in vitro stimulation with tetanus toxoid (TT). Subjects PPD-neg-1 and -4 scored positive and patients PPD-neg-2 and -3 scored negative in the in vivo intradermal reaction to TT. Fig. 4 Open in new tabDownload slide Percent variation of cutaneous lymphocyte-associated antigen (CLA)+ CD4 T cells from PPD skin test-negative patients with tuberculosis, following 8-day in vitro stimulation with tetanus toxoid (TT). Subjects PPD-neg-1 and -4 scored positive and patients PPD-neg-2 and -3 scored negative in the in vivo intradermal reaction to TT. In order to evaluate whether the proportion of CLA-expressing T cells as a whole was comparable in the four PPD skin test-negative patients with TB and in the PPD skin test-positive controls, FACS analysis was performed. The percentage of T cells (identified by a CD3 monoclonal) expressing the CLA marker was comparable in the single PPD skin test-negative patients PPD-neg-1 to PPD-neg-4 (8.2, 9.0, 3.8, 4.1%, respectively) versus the PPD skin test-positive controls (n = 18, 6.7 ± 4.9%, mean ± s.d. of percent values). Lack of IFN-γ production by M. tuberculosis-specific T cell lines The lymphokine profile of M. tuberculosis antigen-stimulated T cells is expected to be largely of the Th1 type, i.e. with a prevalent production of IL-2 and IFN-γ. In addition to its other activities, IFN-γ up-regulates CLA expression by inducing the secretion of IL-12 by monocytes. We therefore measured IFN-γ production in the supernatants of PPD-stimulated CD4 T cells from the four PPD skin test-negative patients, and compared the results with those obtained from 18 PPD skin test-positive patients. We found a profound deficiency in the IFN-γ production of the PPD lines taken from the peripheral blood of the four PPD skin test-negative versus a panel of 18 PPD skin test-positive patients (Fig. 5); in contrast, these T cell lines produced comparable levels of IL-2 (as expected from the proliferative response) and comparable (low) levels of IL-4 (not shown). These results were confirmed by the intracellular staining of PPD-stimulated CD4 T cell lines with anti-IFN-γ and anti-IL-4 antibodies (Fig. 6). Stimulation with polyclonal activators (either PHA or an insolubilized anti-CD3 antibody) induced IFN-γ and IL-4 production by the PBMC from the four PPD skin test-negative patients with TB to levels comparable to those observed in the PPD skin test-positive patients (not shown), thus suggesting an antigen-specific, not generalized deficiency. Fig. 5 Open in new tabDownload slide In vitro production of IFN-γ and IL-4 from PPD-stimulated T cell lines established from PPD skin test-negative (individual values) and from PPD+ (grouped values) patients with tuberculosis. Fig. 5 Open in new tabDownload slide In vitro production of IFN-γ and IL-4 from PPD-stimulated T cell lines established from PPD skin test-negative (individual values) and from PPD+ (grouped values) patients with tuberculosis. Fig. 6 Open in new tabDownload slide Cytofluorometric analysis of intracellular IFN-γ (FITC-stained, on the abscissa) and IL-4 (PE-stained, on the abscissa) by PPD-stimulated T cell lines from one representative PPD+ and one representative PPD skin test-negative patient with tuberculosis. Fig. 6 Open in new tabDownload slide Cytofluorometric analysis of intracellular IFN-γ (FITC-stained, on the abscissa) and IL-4 (PE-stained, on the abscissa) by PPD-stimulated T cell lines from one representative PPD+ and one representative PPD skin test-negative patient with tuberculosis. IL-12 production by blood monocytes from PPD skin test-negative patients with active TB is preserved Since IL-12 is a critical lymphokine in Th1 responses and can up-regulate CLA expression, we designed experiments to check whether the monocytes from the PPD skin test-negative patients were intrinsically defective in producing IL-12 under optimal activation conditions (IFN-γ priming plus LPS stimulation) and under specific activation with a pathogenic, heat-killed strain of Mycobacterium (H37-Rv). In all conditions tested, no differences were found between the IL-12 production of these patients and those of 18 PPD+ patients (Table 4). Table 4 IFN-γ-primed IL-12 production (as pg/ml of the p70 heterodimer) by monocytes from PPD skin test-negative individuals with tuberculosis (TB), stimulated with Staphylococcus aureus Cowan (SAC) strain, lipopolysaccharide (LPS) or heat-inactivated Mycobacterial tuberculosis, strain H37Rv *,**,***Not significantly different.Controls are PPD+ TB patients. Healthy reactors and PPD skin test-negative patients gave similar results (not shown). Open in new tab Table 4 IFN-γ-primed IL-12 production (as pg/ml of the p70 heterodimer) by monocytes from PPD skin test-negative individuals with tuberculosis (TB), stimulated with Staphylococcus aureus Cowan (SAC) strain, lipopolysaccharide (LPS) or heat-inactivated Mycobacterial tuberculosis, strain H37Rv *,**,***Not significantly different.Controls are PPD+ TB patients. Healthy reactors and PPD skin test-negative patients gave similar results (not shown). Open in new tab DISCUSSION The main findings of this study are that (i): PPD skin test-negative M. tuberculosis-infected patients have PPD-specific peripheral blood T lymphocytes that efficiently proliferate upon antigen stimulation but produce lower amounts of IFN-γ than controls; (ii) the expression of the skin-selective homing receptor (CLA) is defective on these (antigen-specific) T cells; (iii) the impaired IFN-γ production is limited to the response of blood T cells to PPD, and is not associated with either a worse prognosis or a reduced monocyte production of IL-12. We have previously reported that the proliferation of PPD-specific lymphocytes in the pleural space of infected patients with tubercular pleuritis can be detected 2–4 weeks before the PPD reaction turns positive [2]. This suggests that the compartmentalization of the immune response can temporarily prevent the migration of PPD-specific lymphocytes from the organ targeted by the infection to the skin. It is possible that the subsequent acquisition of homing receptor allowing the recirculation of T cells to the skin parallels the acquisition of a positive PPD reaction. In this context, the lack of proliferation to PPD in blood correlates with PPD negativity. In contrast, we here describe M. tuberculosis-infected individuals with a persistently negative PPD reaction and a positive in vitro proliferative response to mycobacterial antigens. The in vitro proliferation to mycobacterial antigens in the four PPD skin test-negative subjects we identified was measured using a wide range of antigen doses and different preparations, including proteins from culture filtrate and inactivated whole cells. Our results indicate that PPD negativity was not related to the greater sensitivity of the in vitro proliferation assay [11], nor to a skewing towards peculiar antigenic specificities. Moreover, the four PPD skin test-negative patients did not have progressive, disseminated disease. Instead, they successfully responded to the primary chemotherapeutic regimen and the blastogenic response of their lymphocytes to polyclonal activators (e.g. PHA) was comparable to that of the lymphocytes from PPD+ patients (not shown). In conclusion, neither a specific nor an aspecific quantitative deficiency of the immune response offers a reasonable explanation for the observed skin anergy to PPD in these individuals. We therefore focused on possible qualitative differences and studied the expression of the CLA, the major T cell ligand for the vascular adhesion molecule E-selectin [12, 13]. Indeed, the interaction between circulating CD4 T cells and microvascular endothelial cells is critical to the homing process, and CLA has been proposed as a receptor for tissue-selective T cell extravasation to the skin [7–14]. CLA is expressed by a subset of memory T cells in peripheral blood, by allergen-reactive T cells in patients with atopic dermatitis [9], and by malignant T cells of cutaneous T cell lymphomas [7]. CLA expression is up-regulated by IL-12 secreted by the cells of the monocyte/macrophage lineages and IFN-γ has a powerful priming effect on IL-12 production [15]. The pattern of CLA expression by antigen-reactive T cells is probably influenced by a complex array of antigen-related factors, e.g. the type, the dosage and the route of exposure. It was reported that whereas CLA+ T cells from atopic dermatitis patients preferentially responded to house dust mite and CLA+ T cells from nickel contact dermatitis patients showed an increased response to nickel, CLA− T cells showed very little response in either case [16]. Similarly, we found here that only CLA+ T cells proliferated to PPD in PPD skin test-positive individuals and CLA− T cells did so in PPD skin test-negative individuals. In contrast, TT, a systemically acting antigen, induced in some vaccinated individuals a proliferative response in both CLA+ and CLA− cells ([16] and S. E. Burastero, personal observation). The proportion of CLA-expressing T cells as a whole was comparable in the four PPD skin test-negative patients with TB and in the PPD+ controls. Moreover, IL-12 production by IFN-γ-primed, Staphylococcus aureus Cowan I (SAC)-stimulated monocytes was similar to that observed in the PPD+ patients. Taken together, these findings clearly indicate the presence of a functional IL-12 receptor and of an overall intact Th1 cell subset [17]. Moreover, the PPD− TB patients we describe here do not have any impairment in the immune response to mycobacteria due to IFN-γ receptor [18] or IL-12 receptor deficiency, which are severely symptomatic immunodeficiencies to intracellular pathogens [19, 20]. However, in these same patients CLA expression was selectively defective on PPD-specific T cells. This could explain the negative skin reaction, and a similar correlation was found for the intradermal reaction to another antigen. Notably, CLA−, PPD-specific T cells from the four PPD skin test-negative patients were largely of the CD45RO memory phenotype (not shown). Moreover, we found that individuals who were not exposed to M. tuberculosis and who were not BCG vaccinated were PPD skin test-negative (not shown), in agreement with previous reports [5, 6]. Taken together, these results indicate that PPD-specific T lymphocytes are proliferating to a recall antigen, not through a superantigen type of stimulation [21]. The available data offer no indications concerning the mechanisms inducing the selective defect in IFN-γ production by M. tuberculosis-specific T cells found in these patients. We can speculate that local production of this cytokine is preserved in the infected target organ(s). Indeed, in the case of sarcoidosis it has been previously reported that IFN-γ is spontaneously released by T lymphocytes from the lung, but not from peripheral blood [22]. The development of the DTH reaction to PPD requires the IFN-γ-primed production of IL-12 by M. tuberculosis-infected macrophages, which in turn induces CLA expression on antigen-specific T cells. However, it has been reported that RANTES produced by macrophages and endothelial cells is also a key chemokine for attracting T cells to DTH sites [23]. Similarly, the macrophage migration inhibitory factor (MIF) produced by macrophages and T lymphocytes has been reported to play an essential role in the tuberculin reaction [24]. However, it is interesting to note that IFN-γ can up-regulate the production of both RANTES and MIF. Thus, the impaired production of this single cytokine could negatively affect DTH by mechanisms other than the mere control of CLA expression. 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TI - Circulating, Mycobacterium tuberculosis-specific lymphocytes from PPD skin test-negative patients with tuberculosis do not secrete interferon-gamma (IFN-γ) and lack the cutaneous lymphocyte antigen skin-selective homing receptor
JO - Clinical & Experimental Immunology
DO - 10.1046/j.1365-2249.2000.01128.x
DA - 2002-04-05
UR - https://www.deepdyve.com/lp/oxford-university-press/circulating-mycobacterium-tuberculosis-specific-lymphocytes-from-ppd-3U3tHVBAQS
SP - 99
EP - 106
VL - 119
IS - 1
DP - DeepDyve
ER -