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Clin Exp Immunol 2000; 119:99–106

Circulating, Mycobacterium tuberculosis-specific lymphocytes from
PPD skin test-negative patients with tuberculosis do not secrete
interferon-gamma (IFN-g) and lack the cutaneous lymphocyte antigen
skin-selective homing receptor
Z. I. MAGNANI, C. CONFETTI, G. BESOZZI*, L. R. CODECASA*, P. PANINA-BORDIGNON†, R. LANG†,
G. A. ROSSI‡, R. PARDI & S. E. BURASTERO San Raffaele Scientific Institute, the *Institute Villa Marelli for Lung
Diseases, †Roche Milano Ricerche (Roche), Milan, and the ‡G. Gaslini Institute, Genoa, Italy

(Accepted for publication 20 October 1999)

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 PPDþ
patients; (ii) secreted comparable amounts of IL-2 but lower amounts of IFN-g; (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-g 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.
Keywords

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–Gue´rin (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
Correspondence: Samuele E. Burastero MD, San Raffaele Scientific
Institute, Milan, Italy 20132.
E-mail: BURASTERO.SAMUELE@HSR.IT
q 2000 Blackwell Science

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 PPDþ 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-g
production and impaired CLA expression.
PATIENTS AND METHODS
Patients
We studied 82 patients with newly diagnosed pulmonary (n ¼ 76)
or extrapulmonary (n ¼ 6) TB. The diagnosis was made by means

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according to the manufacturer’s instructions (Dynal. strains H37Rv and H37Ra (WC-H37Ra and WC-H37Rv) or crude culture filtrate from M. Copenhagen. Brennan and J. The mycobacterial antigens used for the proliferation assays included: PPD from M. The nonHECA-452-reacting cells (CLA-depleted) were gently harvested using a pipette. On day 15. 119:99–106 . and thymidine incorporation was measured as ct/min using a b-counter (LKB). Typically. J. generously provided by P. The PBMC were separated from whole blood using a Ficoll density gradient (Pharmacia. anti-CD16. the HECA-452-reacting cells (CLA-enriched) were detached by means of cold PBS. 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 mg/ml). Norway). All of the patients underwent a PPD reaction test performed by means of an intradermal injection of 20 U of PPD (Biocine. A subgroup of 18 patients with pulmonary TB and positive skin reaction to PPD was randomly selected and subjected to further in vitro studies. strains H37Rv and H37Ra. tuberculosis-infected patients following stimulation with mycobacterial antigens. 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 mg/ml phytohaemagglutinin (PHA. > 80% and < 2% of CD4 T cells were HECA-452þ at cytofluorometric analysis in the CLA-enriched and in the CLA-depleted preparations. nontissue culture-treated plates (coating conditions: 48C overnight with an antibody concentration of 20 mg/ml in PBS). washed in PBS (GIBCO. Italy). Magnani et al. 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). Milan. Paris. Milano.100 Z. when the proliferation index was > 3 and the thymidine incorporation was > 2000 ct/min greater than that in the unstimulated control wells [8]. Norway). France). Intradermal reactivity to a panel of common environmental antigens (tetanus toxoid (TT). 2 mM L-glutamine and 10% fetal bovine serum (FBS. The proliferation assays were considered positive on the basis of restricted criteria: i. the supernatants were removed and the cells were washed and stimulated for 18 h with phorbol myristate acetate (PMA. antiCD19 and anti-CD8 MoAbs and the (Fab)02 goat anti-rat (H þ L) affinity-purified antibody used for the cell separation experiments came from Sigma (St Louis. The PBMC were reacted simultaneously with MoAbs to human CD14. tuberculosis for in vitro use (Statens Serum Institute. as specified below. Milan. which was done using an in-house ELISA (Roche Milano Ricerche. The bead-rosetted cells were separated from the others using a magnetic support containing the cell suspension in a tube. On day 7 the cells were washed and re-stimulated with autologous. Clinical and Experimental Immunology. of positive acid-fast smears and/or by the microbiological isolation of M. respectively. 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. 20 mg/ml). 50 ng/ml). Denmark). Picker (Dallas. GIBCO) (complete medium). 104/well). and heat-killed whole cells from M. the test was repeated twice (1 month and 6–8 months later). the CD4 T lymphocytes were enriched and depleted of CLAþ cells by means of panning. The CD4 T lymphocyte-enriched T cell population consisted at cytofluorometric analysis of 92–97% of CD4þ cells in preliminary experiments. Diphtheria. tuberculosis. Sigma). 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. without lipoarabinomannan (CF-H37Ra and CF-H37Rv). Italy) and re suspended in RPMI 1640 medium (GIBCO). Italy). The HECA-452-enriched and -depleted lymphocytes were counted and plated (5 × 105/well) with 15 Gy irradiated autologous PBMC as a source of antigenpresenting cells (APC. CD16. Either complete medium (in control wells) or antigens were added (each at 20 mg/ml) to quadruplicate microcultures. Streptolysin. Sweden). When a negative PPD reaction was observed. They were all treated with conventional therapy and they all responded to the primary chemotherapeutic regimen. and then co-incubated with anti-mouse IgG antibodyconjugated magnetic beads. Merieaux. I. Trycophiton. Tritiated thymidine was added to each microculture well (0·5 mCi/well) for the last 8 h of culture. 15 Gy irradiated PBMC (as a source of APC) pulsed with antigen (PPD. Proliferation was expressed as a proliferation index. Siena. Uppsala. with modifications [9]. The cells were harvested using a cell harvester (Skatron. Antibodies and antigens The purified MoAb HECA-452 against human CLA was a generous gift from L. CD19 and CD8 in ice. Belisle (Fort Collins. 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. CO). Establishment of (secondary) M. The cells were cultured for 5 days in RPMI 1640 supplemented with 100 U/ml penicillin. In order to q 2000 Blackwell Science Ltd. PPD. Before adding brefeldin A for intracellular staining (see below). Colorado State University. The cells were reacted with HECA-452 MoAb (1 mg/106 cells) and plated on anti-rat immunoglobulin (H þ L)-coated. the CD4 T lymphocytes were prepared by means of negative immunobead separation. All of the patients included in this study were positive for more than one of the mycobacterial antigen preparations used to stimulate the PBMC. TX) [7]. 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. Lier. Proteus) was also assessed in selected patients using a commercial assay (Multitest. FITC-conjugated HECA-452 and the corresponding isotype-matched FITC-conjugated MoAb with irrelevant specificity were purchased from PharMingen (San Diego. The purified unconjugated anti-CD14. Subsequently. 100 mg/ml streptomycin. the adherent cells were subsequently detached by washing with cold PBS and scraping and used for IL-12 production assays. Briefly.e. They were then seeded (2 × 105/well) in 96-well flatbottomed microtitre plates (Costar. Italy). After 72 h supernatants were collected for IFN-g and IL-4 measurement. CA). the supernatants were removed and stored for analysis of their IL-4 and IFN-g content. Candida. IFN-g and IL-4 production by polyclonally activated PBMC As a measure of the overall capability to produce IFN-g. These were designed as previously described by Santamaria Babi et al. with or without antigens. tuberculosis from biological specimens. Oslo. tuberculosis. MO).

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). M. four were PPD skin test-negative and maintained this negativity at two subsequent controls 6 months apart. in the presence of autologous irradiated PBMC as a source of APC.CD4 T cells in PPD skin test-negative TB optimize IL-12 secretion. of ct/min. 119:99–106 . no antibiotic resistance) and they all responded (according to clinical and radiological criteria) to a primary chemotherapeutic standard regimen. Microbiological processing of the samples was performed in a laboratory belonging to a national network of clinical reference laboratories. The four PPD¹ patients had positive acid-fast smears and multiple positive cultures for M. Statistical analysis Unpaired t-tests were used for between-group comparisons. FACS analysis Cells were stained with the indicated MoAb and analysed using the cytometer (FACScan cytometer in a four-parameter acquisition setting. comparable age range. indicating that the same pool of antigens was recognized by T cells (Table 1). expressed as a proliferation index.d. Briefly. Becton Dickinson. male/female numbers. according to manufacturer’s instructions (PharMingen).d. Clinical and Experimental Immunology. The results were analysed by means of Cellquest software (Becton Dickinson). 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 PPDþ TB patients. CA). Phytohaemagglutinin. IL-12 was measured in the supernatants using an in-house ELISA system (Roche Milano Ricerche). PPD. The fixed. tuberculosis The PBMC taken from 82 individuals with active TB were tested for proliferation to a panel of five different preparations containing mycobacterial antigens. Proliferation of peripheral blood mononuclear cells (PBMC) from patients with active lung tuberculosis to different Mycobacterial antigens Patients All PPDþ PPD¹ No. The proliferation to all of the mycobacterial antigens. Mountain View. M/F. Cellular immunity to other common antigens was tested in vivo in the PPD skin test-negative patients using a commercial intradermal assay. PPD CF-H37Ra CF-H37Rv WC-H37Ra WC-H37Rv 82 78 4 79 75 4 74 70 4 71 70 3 73 69 4 73 69 4 Numbers indicate number of patients. P < 0·05 was considered statistically significant. Patients with tuberculosis positive for proliferation to each single Mycobacterial antigen preparation All patients PPDþ patients PPD¹ patients No. tuberculosis. The proliferation to PPD was prevalently restricted to the CLA¹ CD4 T cell subset in Table 1. monocyte-enriched. and heat-killed whole cells from the same strains. culture fluid from the H37-Ra or Rv strains. 82 78 4 M/F Age. Positivity was determined according to the criteria described in Patients and Methods. [10]. Numbers indicate mean 6 s. strain H37Rv (20 mg/ml). The clinical profile of the four PPD skin test-negative patients was similar to that of most PPDþ patients with pulmonary TB (extensive unilateral lesions. to exclude false-positive results due to cross-contamination. RESULTS Identification of PPD skin test-negative individuals among patients exposed to M. All 101 patients were tested for skin reactivity to PPD. permeabilized cells were stained with FITC–anti-IFN-g and PE–anti-IL-4 MoAbs.e. 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. Sigma) or heat-killed whole cells from M. CLAþ and CLA¹ CD4 T lymphocytes were isolated from PBMC and separately assayed in cell proliferation assays. and then the cells were fixed with 4% paraformaldehyde and permeabilized with saponin. adherent cell preparations (1 × 106/ml in complete medium) were primed for 18 h with 1000 U/ml of IFN-g and then cultured for 24 h with 1 mg/ml of lipopolysaccharide (LPS. The isolation of M. Seventy-eight patients were positive to both the in vivo PPD reaction and in vitro proliferation to PPD. q 2000 Blackwell Science Ltd. i. tuberculosis. yearly subjected to quality control procedures. was similar in the PPD skin test-negative and PPD skin test-positive subjects. The data are expressed as mean values 6 s. tuberculosis-specific T cells from PPD skin test-negative patients with active TB segregate in the CLA¹ CD4 T cell subset The cutaneous lymphocyte antigen is the candidate homing receptor for routing CD4 T cells to the skin endothelium. mean (range) Nil PPD CF-H37Ra CF-H37Rv WC-H37Ra WC-H37Rv 40/42 38/40 2/2 40·9 (20–81) 40·8 (20–81) 31·7 (23–45) 314 6 381 343 6 401 166 6 43 5723 6 413 5634 6 454 4171 6 4584 5441 6 5899 5346 6 5999 4927 6 3416 5085 6 3842 4788 6 3544 9298 6 5298 5193 6 4022 4998 6 3988 8930 6 5456 5449 6 4069 5399 6 4002 7765 6 3788 PHA 11 312 6 7654 12 432 6 10 987 11 287 6 12 376 PHA. we basically used the protocol described by Ma et al. tuberculosis from different aliquots of the four original specimens was confirmed by an independent laboratory.

averaged) of the infiltrative reaction to each antigen measured at 72 h. We found that the addition of PPD induced an expansion of CD4 CLAþ cells in a panel of PPD skin test-positive patients (n ¼ 18). 9·0. We therefore measured IFN-g production in the supernatants of PPD-stimulated CD4 T cells from the four PPD skin test-negative patients. 3·8. grouped results) with tuberculosis. but in none of the PPD skin test-negative patients. not generalized deficiency. 119:99–106 . and compared the results with those obtained from 18 PPD skin test-positive patients. we found that TT induced an in vitro expansion of CLAþ CD4 T cells in PPD-neg-1 and PPD-neg-4. the CLAþ but not the CLA¹ CD4 T cells proliferated to PPD in a panel (n ¼ 18) of PPDþ patients with TB. as detected with this assay.d.e. thus suggesting an antigen-specific. as expected (Fig. 1. of percent values). In a parallel and independent set of experiments. 4·1%. but not in PPD-neg-2 and PPD-neg-3 (Fig. with a prevalent production of IL-2 and IFN-g. tuberculosis antigen-stimulated T cells is expected to be largely of the Th1 type. The proportion of CLAþ CD4 cells was measured before and after culturing PBMC from different individuals with PPD. Percent variations obtained from 18 PPDþ patients are indicated as grouped results for comparison. In fact. individual values) and PPD skin test-positive patients ((b). q 2000 Blackwell Science Ltd. Intradermal skin reaction to standard antigens (Multitest) Patient PPD-neg-1 PPD-neg-2 PPD-neg-3 PPD-neg-4 Glycerol Tetanus toxoid Diphtheria 0 0 0 0 5 0 0 11 6 0 0 3 Streptolysin PPD Candida Trycophiton Proteus 0 0 0 2 6 0 0 0 0 0 12 0 0 10 0 0 0 13 0 14 Numbers indicate the mean diameter (maximum þ minimum diameter. respectively) versus the PPD skin test-positive controls (n ¼ 18. we further controlled this observation by using a different read out. Indeed. (a) _ _ _ + PPD /CLA PPD /CLA PPD-neg-4 PPD-neg-3 PPD-neg-2 PPD-neg-1 0 10 000 20 000 30 000 40 000 30 000 40 000 (b) + PPD (n = 18) 0 10 000 20 000 ct/min Fig. In vitro proliferation to PPD of cutaneous lymphocyte-associated antigen (CLA)-enriched (CLAþ) and CLA-depleted (CLA¹) CD4 T lymphocytes from PPD skin test-negative patients ((a). In addition to its other activities. had a positive skin reaction following the intradermal injection of this antigen (Table 2). 1). in contrast. 3). 6). Figure 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. but not PPD-neg-2 and PPD-neg-3. we used tetanus toxoid (TT). Table 2. We found a profound deficiency in the IFN-g 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. The expansion of the CLA subset upon addition of the antigen will indicate that CLAþ CD4 T cells contain PPD-specific T lymphocytes. 4). Stimulation with polyclonal activators (either PHA or an insolubilized anti-CD3 antibody) induced IFN-g 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). Individual results are shown as percent variation of CLAþ CD4 T cells induced by PPD in the four PPD skin test-negative patients (Fig. I. Magnani et al. In order to evaluate whether the proportion of CLA-expressing T cells as a whole was comparable in the four PPD skin testnegative patients with TB and in the PPD skin test-positive controls. These results were confirmed by the intracellular staining of PPD-stimulated CD4 T cell lines with antiIFN-g and anti-IL-4 antibodies (two representative results are shown in Fig. IFN-g up-regulates CLA expression by inducing the secretion of IL-12 by monocytes. tuberculosis-specific T cell lines The lymphokine profile of M. 5). FACS analysis was performed. patients PPD-neg-1 and PPD-neg-4. i. the four PPD skin test-negative individuals. 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. 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). Clinical and Experimental Immunology. In contrast.102 Z. in millimetres. As an internal antigen control for the correlation between CLA expression and margination to the skin. 6·7 6 4·9%. mean 6 s. Lack of IFN-g production by M.

In all conditions tested. pg/ml IL-4.**Not significantly different. Percent variation of cutaneous lymphocyte-associated antigen (CLA)þ CD4 T cells from PPD skin test-negative (individual values) and PPDþ patients (grouped values) with tuberculosis. we here describe M. 119:99–106 Table 3. Clinical and Experimental Immunology. infection to the skin. 2. tuberculosis-infected DISCUSSION The main findings of this study are that (i): PPD skin test-negative M. 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 upregulate CLA expression. Cytofluorometric analysis of cutaneous lymphocyte-associated antigen (CLA) expression at baseline (left panels) and after 8 days of in vitro PPD stimulation (þ PPD) on CD4 T cell lines from one representative PPD skin-test positive (PPDþ) and one representative PPD skin testnegative (PPD-neg-1) patient with tuberculosis. following 8-day in vitro stimulation with PPD. This suggests that the compartmentalization of the immune response can temporarily prevent the migration of PPD-specific lymphocytes from the organ targeted by the q 2000 Blackwell Science Ltd. _10 10 30 50 70 90 Mean % variation of CLA+ CD4 T cells in vitro stimulated with PPD (n = 18 PPD skin-positive patients) Fig. heat-killed strain of Mycobacterium (H37-Rv). tuberculosis-infected patients have PPD-specific peripheral blood T lymphocytes that efficiently proliferate upon antigen stimulation but produce lower amounts of IFN-g than controls.7% 103 104 + PPD (n = 18) Fig.2% (b) M1 10 103 104 0 100 101 102 FL1-H 8. the lack of proliferation to PPD in blood correlates with PPD negativity. 3. IFN-g. 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-g priming plus LPS stimulation) and under specific activation with a pathogenic. In contrast. no differences were found between the IL-12 production of these patients and those of 18 PPDþ patients (Table 4). IFN-g and IL-4 production (pg/ml) by peripheral blood mononuclear cells stimulated with phytohaemagglutinin from PPD¹ and PPDþ tuberculosis patients Subjects PPD-neg-1 PPD-neg-2 PPD-neg-3 PPD-neg-4 PPDþ (n ¼ 18) Mean 6 s. and is not associated with either a worse prognosis or a reduced monocyte production of IL-12. 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.d. We have previously reported that the proliferation of PPDspecific lymphocytes in the pleural space of infected patients with tubercular pleuritis can be detected 2–4 weeks before the PPD reaction turns positive [2]. In this context.2% PPD-neg-2 103 104 PPD-neg-1 _10 10 30 50 70 90 _ PPD patient (PPD-neg-1) + Counts Baseline +PPD 40 40 30 30 20 20 M1 10 0 100 Percent variation of CLA CD4 T cells in vitro stimulated with PPD (individual PPD skin-negative patients) 50 50 101 102 FL1-H 8. . (ii) the expression of the skin-selective homing receptor (CLA) is defective on these (antigen-specific) T cells.2% 103 104 0 100 PPD-neg-4 PPD-neg-3 M1 10 M1 0 100 103 101 102 FL1-H 18. (iii) the impaired IFN-g production is limited to the response of blood T cells to PPD. pg/ml 156 231 345 235 241 6 78* 342 423 234 341 335 6 77** 236 6 99* 250 6 61** *.CD4 T cells in PPD skin test-negative TB (a) + PPD patient 50 50 Counts Baseline 40 40 30 30 20 20 10 101 +PPD 102 FL1-H 9.

the four PPD skin test-negative patients did not have progressive. Controls are PPDþ TB patients. strain H37Rv (TT +) PPD-neg-4 _ (TT ) PPD-neg-3 _ (TT ) PPD-neg-2 + (TT ) PPD-neg-1 _20 0 20 40 60 80 Percent variation of CLA+ CD4 T cells in vitro stimulated with TT (individual PPD skin-negative patients) Fig. In vitro production of IFN-g and IL-4 from PPD-stimulated T cell lines established from PPD skin test-negative (individual values) and from PPDþ (grouped values) patients with tuberculosis. towards peculiar antigenic specificities. lipopolysaccharide (LPS) or heat-inactivated Mycobacterial tuberculosis. including proteins from culture filtrate and inactivated whole cells.104 Z. In conclusion. 6. 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. We therefore focused on possible qualitative differences and studied the expression of the CLA. stimulated with Staphylococcus aureus Cowan (SAC) strain.***Not significantly different. Cytofluorometric analysis of intracellular IFN-g (FITC-stained. Clinical and Experimental Immunology. Moreover. Instead.**. on the abscissa) and IL-4 (PE-stained. Percent variation of cutaneous lymphocyte-associated antigen (CLA)þ CD4 T cells from PPD skin test-negative patients with tuberculosis. 5. PHA) was comparable to that of the lymphocytes from PPDþ patients (not shown). 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. on the abscissa) by PPD-stimulated T cell lines from one representative PPDþ and one representative PPD skin test-negative patient with tuberculosis. 4. 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. they successfully responded to the primary chemotherapeutic regimen and the blastogenic response of their lymphocytes to polyclonal activators (e. IFN-g-primed IL-12 production (as pg/ml of the p70 heterodimer) by monocytes from PPD skin test-negative individuals with tuberculosis (TB). individuals with a persistently negative PPD reaction and a positive in vitro proliferative response to mycobacterial antigens. nor to a skewing (a) Patient SAC LPS H37Rv-WC Mneg-1 Mneg-2 Mneg-3 Mneg-4 874 574 1221 898 891 6 264* 1254 1234 2514 1874 1719 6 607** 251 321 451 401 356 6 88*** 649 6 181* 2402 6 809** 364 6 146*** Controls (n ¼ 18) Mean 6 s. Magnani et al. q 2000 Blackwell Science Ltd. I. *. Our results indicate that PPD negativity was not related to the greater sensitivity of the in vitro proliferation assay [11]. disseminated disease.d.g. the major T cell ligand for the PPD-neg-4 + PPD patient 4 104 10 PPD-neg-3 Control IL-4-PE PPD-neg-2 PPD-neg-1 0 1000 103 102 102 101 101 100 100 2000 +PMA 103 101 102 103 IFN-γ FITC (b) 104 100 100 101 102 103 IFN-γ FITC 104 _ PPD patient (PPD-neg-1) 104 104 Control + IL-4-PE PPD (n = 18) 0 1000 2000 +PMA 103 103 102 102 101 101 100 100 101 102 103 IFN-γ FITC 104 100 100 101 102 103 IFN-γ FITC 104 IFN-γ (pg/ml) Fig. 119:99–106 . Table 4. Healthy reactors and PPD skin testnegative patients gave similar results (not shown). following 8-day in vitro stimulation with tetanus toxoid (TT). Fig.

Trumble A et al. Perez Soler MT et al. 6 Muller HK. which are severely symptomatic immunodeficiencies to intracellular pathogens [19. This could explain the negative skin reaction. Eur J Immunol 1994. Moser R. 66:133–48. 8 Jensen B. 136:1053–68. Kurpisz M. The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells. Antigen specific lymphocyte activity in vitro by peripheral blood leucocytes from Mantoux positive and negative human beings. However. Moreover. CLA¹ T cells showed very little response in either case [16]. CLA expression is up-regulated by IL-12 secreted by the cells of the monocyte/macrophage lineages and IFN-g has a powerful priming effect on IL-12 production [15]. 119:99–106 . J Immunol Methods 1984. The characterization of the molecular mechanism of this locally abnormal Th1 response awaits further studies. 70:65–70. tuberculosis-specific T cells found in these patients. Echeverri A. the macrophage migration inhibitory factor (MIF) produced by macrophages and T lymphocytes has been reported to play an essential role in the 105 tuberculin reaction [24]. The development of the DTH reaction to PPD requires the IFNg-primed production of IL-12 by M. induced in some vaccinated individuals a proliferative response in both CLAþ and CLA¹ cells ([16] and S. Statistical evaluation of the lymphocyte proliferation assay with non-stimulated cultures. Kishimoto TK. 89:932–3. However. Int Arch Allergy Appl Immunol 1983.B. Indeed. these results indicate that PPD-specific T lymphocytes are proliferating to a recall antigen. Clinical and Experimental Immunology. J Immunol Methods 1981. Thus. J Exp Med 1995. e. but not from peripheral blood [22]. 3 Ellner JJ. I. Taken together. Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor. Indeed. and by malignant T cells of cutaneous T cell lymphomas [7]. Moreover. we found that individuals who were not exposed to M. Preferential expression of the HECA-452 epitope by benign and malignant T cells at cutaneous sites. in agreement with previous reports [5. Perez Soler MT et al. 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]. Comparison of quantitative and qualitative differences in the PPD-specific lymphoproliferative response of lymphocytes from the two kinds of donors. Pleural fluid and peripheral blood lymphocyte function in tuberculosis. 7 Picker LJ. a systemically acting antigen. Am Rev Respir Dis 1978. 24:1269–77. personal observation). 5 Jensen B. Chow JM. Clin Exp Immunol 1977. Antigen specific in vitro activity of lymphocyte subpopulations from Mantoux positive and negative subjects assessed by lymphocyte proliferation assay. Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites. which in turn induces CLA expression on antigen-specific T cells. PPD-specific T cells from the four PPD skin test-negative patients were largely of the CD45RO memory phenotype (not shown). Ann Intern Med 1978. Nature 1991.P. ACKNOWLEDGMENTS This work was supported by grants from Istituto Superiore di Sanita` to R. Similarly. the PPD¹ TB patients we describe here do not have any impairment in the immune response to mycobacteria due to IFN-g receptor [18] or IL-12 receptor deficiency. the cutaneous lymphocyte-associated antigen. Gri G et al. 181:1935–40.CD4 T cells in PPD skin test-negative TB vascular adhesion molecule E-selectin [12. 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 PPDþ controls. Smith CW et al.6].g. the type. Picker LJ. Moller S et al. Staphylococcus aureus Cowan I (SAC)-stimulated monocytes was similar to that observed in the PPDþ patients. 4 Collins FM. it is interesting to note that IFNg can up-regulate the production of both RANTES and MIF. It was reported that whereas CLAþ T cells from atopic dermatitis patients preferentially responded to house dust mite and CLAþ T cells from nickel contact dermatitis patients showed an increased response to nickel. Lymphokine and lymphocyte transformation studies.20]. 349:796–9. Thorarinsdottir T. the dosage and the route of exposure. We can speculate that local production of this cytokine is preserved in the infected target organ(s). in these same patients CLA expression was selectively defective on PPD-specific T cells. by allergen-reactive T cells in patients with atopic dermatitis [9]. tuberculosis-specific T cells is associated with a negative tuberculin reaction in a portion of immune-competent patients with clinically undistinguished tuberculosis. tuberculosis and who were not BCG vaccinated were PPD skin test-negative (not shown). Tuberculin anergy in clinically normal individuals. CLA¹. Taken together. 10 Ma X. E. Tuberculous pleural effusions. Migration of skinhoming T cells across cytokine-activated human endothelial cell layers involves interaction of the cutaneous lymphocyte-associated antigen q 2000 Blackwell Science Ltd. The available data offer no indications concerning the mechanisms inducing the selective defect in IFN-g production by M. Rubin B. in the case of sarcoidosis it has been previously reported that IFN-g is spontaneously released by T lymphocytes from the lung. Am J Pathol 1990. The immunology of tuberculosis. In conclusion. CLA is expressed by a subset of memory T cells in peripheral blood. J Exp Med 1996. 11 Jensen B. 125:42–49. REFERENCES 1 McMurray DN. the impaired production of this single cytokine could negatively affect DTH by mechanisms other than the mere control of CLA expression. Moreover. the impaired production of IFN-g and the lack of CLA expression by circulating M. Martin CL et al. Burastero. 136:575–9. Am Rev Respir Dis 1987. However. and a similar correlation was found for the intradermal reaction to another antigen.13]. Pye DW. In contrast. Notably. 183:147–57. Cell-mediated immunity in anergic patients with pulmonary tuberculosis. tuberculosis-infected macrophages. I. 12 Picker LJ. 14 Santamaria Babi LF. 2 Rossi GA. IL-12 production by IFN-gprimed. Moller S. 27:303–12. not through a superantigen type of stimulation [21]. we found here that only CLAþ T cells proliferated to PPD in PPD skin test-positive individuals and CLA¹ T cells did so in PPD skin test-negative individuals. Manca F. Am Rev Respir Dis 1982. 13 Picker LJ. Evidence for selective presence of PPD-specific T-lymphocytes at site of inflammation in the early phase of the infection. The pattern of CLA expression by antigen-reactive T cells is probably influenced by a complex array of antigen-related factors. Rott LS et al. TT. 118:827–34. Similarly. Bentzon MW. Michie SA.E. Martin RJ. ELAM-1 is an adhesion molecule for skin-homing T cells. the interaction between circulating CD4 T cells and microvascular endothelial cells is critical to the homing process. 40:259–74. Balbi B. and CLA has been proposed as a receptor for tissue-selective T cell extravasation to the skin [7–14]. 9 Santamaria Babi LF. and S. these findings clearly indicate the presence of a functional IL-12 receptor and of an overall intact Th1 cell subset [17]. A unique phenotype of skinassociated lymphocytes in humans.

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