cei0119-0099.pdf

Circulating, Mycobacterium tuberculosis-specific lymphocytes fromPPD skin test-negative patients with tuberculosis do not secrete

interferon-gamma (IFN-g) and lack the cutaneous lymphocyte antigenskin-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 theMycobacterium tuberculosis exposed, immune-competent population. Here, we studied PPD-specificblood T lymphocytes from these subjects for phenotypic markers relevant to skin migration, includingthe 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 PPDskin test-negative. CD4 T lymphocytes specific to mycobacterial antigens were derived from theseindividuals, which (i) proliferated in vitro to M. tuberculosis antigens comparably to those from PPDpatients; (ii) secreted comparable amounts of IL-2 but lower amounts of IFN-g; (iii) were confinedwithin the CLA-negative T cell subset. We conclude that the negative tuberculin reaction in a smallsubset of patients exposed to mycobacteria is associated with impaired production of IFN-g bycirculating PPD-specific T cells that are lacking CLA expression. On this basis in vitro proliferationto PPD can discriminate bona fide non-responders from infected patients with a deficit in themargination of M. tuberculosis-specific T lymphocytes.

Keywords tuberculosis skin test cutaneous lymphocyte antigen

INTRODUCTION

PPD skin test-negative individuals (i.e. individuals who do notdevelop an indurative reaction in the 4872 h following theintradermal injection of a standardized amount of PPD) havelong been described among immune-competent subjects pre-viously vaccinated with bacille CalmetteGuerin (BCG) orinfected with Mycobacterium tuberculosis [1]. In a proportion ofthese individuals the skin anergy to PPD is transient since, in theweeks following the presumed exposure to M. tuberculosis, thereaction turns positive [23]; however, some remain persistentlyPPD skin test-negative at subsequent controls [4]. These latterpatients do not have any distinct clinical peculiarities, but mayescape a first-line diagnostic evaluation.

It has been suggested that a relatively low sensitivity of skinreaction may explain a negative PPD test in patients with a lowerlevel of immunological reactivity, but there is general agreement

about the fact that qualitative differences underly persistent PPDnegativity in some patients with tuberculosis (TB) [1].

PPD-specific peripheral blood CD4 T cells can be identified inM. tuberculosis-exposed individuals using an in vitro lymphocyteproliferation assay [5]. It has been reported that T lymphocytestaken from M. tuberculosis-exposed (or BCG-vaccinated) indivi-duals with a negative skin reaction to PPD undergo blast celltransformation when incubated in vitro with PPD [6].

In this study we compared blood lymphocytes taken from PPDskin test-negative and PPD patients with TB, focusing on theexpression of the cutaneous lymphocyte-associated antigen (CLA)and on the pattern of lymphokines secreted by PPD-reactive Tcells. Our results suggest a correlation between reduced IFN-gproduction and impaired CLA expression.

PATIENTS AND METHODSPatientsWe studied 82 patients with newly diagnosed pulmonary (n 76)or extrapulmonary (n 6) TB. The diagnosis was made by means

Clin Exp Immunol 2000; 119:99106

99q 2000 Blackwell Science

Correspondence: Samuele E. Burastero MD, San Raffaele ScientificInstitute, Milan, Italy 20132.

E-mail: [email protected]

of positive acid-fast smears and/or by the microbiological isolationof M. tuberculosis from biological specimens. They were alltreated with conventional therapy and they all responded to theprimary chemotherapeutic regimen. All of the patients underwent aPPD reaction test performed by means of an intradermal injectionof 20 U of PPD (Biocine, Siena, Italy). The infiltrative reaction wasevaluated 72 h after injection and scored as positive when theaverage of the maximum and minimum diameters was > 5 mm.When a negative PPD reaction was observed, the test was repeatedtwice (1 month and 68 months later).

A subgroup of 18 patients with pulmonary TB and positive skinreaction to PPD was randomly selected and subjected to furtherin vitro studies, as specified below.

Intradermal reactivity to a panel of common environmentalantigens (tetanus toxoid (TT), Diphtheria, Streptolysin, PPD,Candida, Trycophiton, Proteus) was also assessed in selectedpatients using a commercial assay (Multitest, Merieaux, Paris,France).

Antibodies and antigensThe purified MoAb HECA-452 against human CLA was a generousgift from L. J. Picker (Dallas, TX) [7]. FITC-conjugated HECA-452and the corresponding isotype-matched FITC-conjugated MoAbwith irrelevant specificity were purchased from PharMingen (SanDiego, CA). The purified unconjugated anti-CD14, anti-CD16, anti-CD19 and anti-CD8 MoAbs and the (Fab)02 goat anti-rat (H L)affinity-purified antibody used for the cell separation experimentscame from Sigma (St Louis, MO).

The mycobacterial antigens used for the proliferation assaysincluded: PPD from M. tuberculosis for in vitro use (Statens SerumInstitute, Copenhagen, Denmark); and heat-killed whole cellsfrom M. tuberculosis, strains H37Rv and H37Ra (WC-H37Raand WC-H37Rv) or crude culture filtrate from M. tuberculosis, strainsH37Rv and H37Ra, without lipoarabinomannan (CF-H37Ra andCF-H37Rv), generously provided by P. Brennan and J. Belisle(Fort Collins, Colorado State University, CO).

Proliferation assaysFive-day proliferation assays of peripheral blood mononuclearcells (PBMC) were performed in order to evaluate the in vitroproliferation of specific T cells from M. tuberculosis-infectedpatients following stimulation with mycobacterial antigens.

The PBMC were separated from whole blood using a Ficolldensity 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 werecultured for 5 days in RPMI 1640 supplemented with 100 U/mlpenicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 10% fetalbovine serum (FBS; GIBCO) (complete medium). Either completemedium (in control wells) or antigens were added (each at 20 mg/ml)to quadruplicate microcultures. Tritiated thymidine was added toeach microculture well (05 mCi/well) for the last 8 h of culture. Thecells were harvested using a cell harvester (Skatron, Lier, Norway),and thymidine incorporation was measured as ct/min using ab-counter (LKB). Proliferation was expressed as a proliferationindex, calculated as the ratio between the thymidine incorporationin the test wells (containing PBMC stimulated with antigens) andthat in the control wells (containing unstimulated PBMC). Theproliferation assays were considered positive on the basis ofrestricted criteria: i.e. when the proliferation index was > 3 and

the thymidine incorporation was > 2000 ct/min greater than that inthe unstimulated control wells [8]. All of the patients included inthis study were positive for more than one of the mycobacterialantigen preparations used to stimulate the PBMC.

Preparation of CLA-enriched and CLA-depleted CD4 T cellsExperiments were performed to assess if the in vitro proliferationto PPD of CD4 T lymphocytes could be positively or negativelysegregated on the basis of CLA expression. These were designed aspreviously described by Santamaria Babi et al. with modifications[9]. Briefly, the CD4 T lymphocytes were prepared by means ofnegative immunobead separation. The PBMC were reacted simul-taneously with MoAbs to human CD14, CD16, CD19 and CD8in ice, and then co-incubated with anti-mouse IgG antibody-conjugated magnetic beads, according to the manufacturersinstructions (Dynal, Oslo, Norway). The bead-rosetted cells wereseparated from the others using a magnetic support containing thecell suspension in a tube. The CD4 T lymphocyte-enriched T cellpopulation consisted at cytofluorometric analysis of 9297% ofCD4 cells in preliminary experiments. Subsequently, the CD4 Tlymphocytes were enriched and depleted of CLA cells by means ofpanning. The cells were reacted with HECA-452 MoAb (1 mg/106cells) and plated on anti-rat immunoglobulin (H L)-coated, non-tissue culture-treated plates (coating conditions: 48C overnightwith an antibody concentration of 20 mg/ml in PBS). The non-HECA-452-reacting cells (CLA-depleted) were gently harvestedusing a pipette; the HECA-452-reacting cells (CLA-enriched)were detached by means of cold PBS. The HECA-452-enrichedand -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 cytofluoro-metric analysis in the CLA-enriched and in the CLA-depletedpreparations, respectively.

Establishment of (secondary) M. tuberculosis-specific T cell linesfor the analysis of cytokine secretionAliquots of 2 106 PBMC were seeded in 24-well plates (Costar)in 2 ml of complete medium with PPD (20 mg/ml). On day 7 thecells were washed and re-stimulated with autologous, 15 Gyirradiated PBMC (as a source of APC) pulsed with antigen(PPD, 20 mg/ml). On day 15, the supernatants were removed andthe cells were washed and stimulated for 18 h with phorbolmyristate acetate (PMA; 50 ng/ml). Before adding brefeldin Afor intracellular staining (see below), the supernatants wereremoved and stored for analysis of their IL-4 and IFN-g content,which was done using an in-house ELISA (Roche MilanoRicerche, Milano, Italy).

IFN-g and IL-4 production by polyclonally activated PBMCAs a measure of the overall capability to produce IFN-g, micro-cultures of 2 105 CD4 T cells per well were established in completemedium in the presence of irradiated autologous PBMC as a sourceof APC 10 mg/ml phytohaemagglutinin (PHA; Sigma). After 72 hsupernatants were collected for IFN-g and IL-4 measurement.

Analysis of p70 IL-12 production by monocytesMonocyte-enriched preparations were obtained by incubatingPBMC for 1 h in tissue culture flasks in complete medium; theadherent cells were subsequently detached by washing with coldPBS and scraping and used for IL-12 production assays. In order to

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optimize IL-12 secretion, we basically used the protocol describedby Ma et al. [10]. Briefly, monocyte-enriched, adherent cellpreparations (1 106/ml in complete medium) were primed for18 h with 1000 U/ml of IFN-g and then cultured for 24 h with 1 mg/mlof lipopolysaccharide (LPS; Sigma) or heat-killed whole cells fromM. tuberculosis, strain H37Rv (20 mg/ml). IL-12 was measured inthe supernatants using an in-house ELISA system (Roche MilanoRicerche).

Single-cell analysis of cytokine production on secondaryPPD-specific T cell linesBrefeldin A was added to the PMA-stimulated T cell lines during thelast 2 h of culture, and then the cells were fixed with 4% parafor-maldehyde and permeabilized with saponin. The fixed, permeabi-lized cells were stained with FITCanti-IFN-g and PEanti-IL-4MoAbs, according to manufacturers instructions (PharMingen).

FACS analysisCells were stained with the indicated MoAb and analysed using thecytometer (FACScan cytometer in a four-parameter acquisitionsetting; Becton Dickinson, Mountain View, CA). The results wereanalysed by means of Cellquest software (Becton Dickinson).

Statistical analysisUnpaired t-tests were used for between-group comparisons.P < 005 was considered statistically significant. The data areexpressed as mean values 6 s.d.

RESULTSIdentification of PPD skin test-negative individuals amongpatients exposed to M. tuberculosisThe PBMC taken from 82 individuals with active TB were testedfor proliferation to a panel of five different preparations containingmycobacterial antigens, i.e. PPD, culture fluid from the H37-Ra orRv strains, and heat-killed whole cells from the same strains. All

patients were tested for skin reactivity to PPD. Seventy-eightpatients were positive to both the in vivo PPD reaction and in vitroproliferation to PPD; four were PPD skin test-negative and main-tained this negativity at two subsequent controls 6 months apart.The proliferation to all of the mycobacterial antigens, expressed asa proliferation index, was similar in the PPD skin test-negative andPPD skin test-positive subjects, indicating that the same pool ofantigens was recognized by T cells (Table 1). The clinical profile ofthe four PPD skin test-negative patients was similar to that of mostPPD patients with pulmonary TB (extensive unilateral lesions,comparable age range, no antibiotic resistance) and they allresponded (according to clinical and radiological criteria) to aprimary chemotherapeutic standard regimen. The four PPDpatients had positive acid-fast smears and multiple positive cul-tures for M. tuberculosis. Microbiological processing of the sam-ples was performed in a laboratory belonging to a national networkof clinical reference laboratories, yearly subjected to qualitycontrol procedures. The isolation of M. tuberculosis from differentaliquots of the four original specimens was confirmed by anindependent laboratory, to exclude false-positive results due tocross-contamination.

Cellular immunity to other common antigens was tested in vivoin the PPD skin test-negative patients using a commercial intra-dermal assay. They had individual patterns of skin reactivity whichexcluded generalized skin anergy (Table 2) and were comparableto those observed in the general population and in PPD TBpatients.

M. tuberculosis-specific T cells from PPD skin test-negativepatients with active TB segregate in the CLA CD4 T cell subsetThe cutaneous lymphocyte antigen is the candidate homing recep-tor for routing CD4 T cells to the skin endothelium. CLA andCLA CD4 T lymphocytes were isolated from PBMC and sepa-rately assayed in cell proliferation assays, in the presence ofautologous irradiated PBMC as a source of APC. The proliferationto PPD was prevalently restricted to the CLA CD4 T cell subset in

CD4 T cells in PPD skin test-negative TB 101


Table 1. Proliferation of peripheral blood mononuclear cells (PBMC) from patients with active lung tuberculosis to different Mycobacterial antigens

Age, meanPatients No. M/F (range) Nil PPD CF-H37Ra CF-H37Rv WC-H37Ra WC-H37Rv PHA

All 82 40/42 409 (2081) 314 6 381 5723 6 413 5441 6 5899 5085 6 3842 5193 6 4022 5449 6 4069 11 312 6 7654PPD 78 38/40 408 (2081) 343 6 401 5634 6 454 5346 6 5999 4788 6 3544 4998 6 3988 5399 6 4002 12 432 6 10 987PPD 4 2/2 317 (2345) 166 6 43 4171 6 4584 4927 6 3416 9298 6 5298 8930 6 5456 7765 6 3788 11 287 6 12 376

PHA, Phytohaemagglutinin; M/F, male/female numbers.Numbers indicate mean 6 s.d. of ct/min.

Patients with tuberculosis positive for proliferation to each single Mycobacterial antigen preparation

No. PPD CF-H37Ra CF-H37Rv WC-H37Ra WC-H37Rv

All patients 82 79 74 71 73 73PPD patients 78 75 70 70 69 69PPD patients 4 4 4 3 4 4

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).

the four PPD skin test-negative individuals. In contrast, the CLAbut not the CLA CD4 T cells proliferated to PPD in a panel(n 18) of PPD patients with TB, as expected (Fig. 1).

In a parallel and independent set of experiments, we furthercontrolled this observation by using a different read out. Theproportion of CLA CD4 cells was measured before and afterculturing PBMC from different individuals with PPD. The expan-sion of the CLA subset upon addition of the antigen will indicatethat CLA CD4 T cells contain PPD-specific T lymphocytes. Wefound that the addition of PPD induced an expansion of CD4CLA cells in a panel of PPD skin test-positive patients (n 18),but in none of the PPD skin test-negative patients. Figure 2 showsCLA expression by CD4 T cells from one PPD skin test-negativeand one PPD skin test-positive TB patients following in vitroincubation with PPD. Individual results are shown as percentvariation of CLA CD4 T cells induced by PPD in the four PPDskin test-negative patients (Fig. 3). Percent variations obtained from18 PPD patients are indicated as grouped results for comparison.

As an internal antigen control for the correlation between CLAexpression and margination to the skin, as detected with this assay,we used tetanus toxoid (TT). In fact, patients PPD-neg-1 andPPD-neg-4, but not PPD-neg-2 and PPD-neg-3, had a positiveskin reaction following the intradermal injection of this antigen(Table 2). Indeed, we found that TT induced an in vitro expansionof CLA CD4 T cells in PPD-neg-1 and PPD-neg-4, but not inPPD-neg-2 and PPD-neg-3 (Fig. 4).

In order to evaluate whether the proportion of CLA-expressingT cells as a whole was comparable in the four PPD skin test-negative patients with TB and in the PPD skin test-positivecontrols, FACS analysis was performed. The percentage of T cells(identified by a CD3 monoclonal) expressing the CLA marker wascomparable in the single PPD skin test-negative patients PPD-neg-1to PPD-neg-4 (82, 90, 38, 41%, respectively) versus the PPDskin test-positive controls (n 18, 67 6 49%, mean 6 s.d. ofpercent values).

Lack of IFN-g production by M. tuberculosis-specific T cell linesThe lymphokine profile of M. tuberculosis antigen-stimulated Tcells is expected to be largely of the Th1 type, i.e. with a prevalentproduction of IL-2 and IFN-g. In addition to its other activities,IFN-g up-regulates CLA expression by inducing the secretion ofIL-12 by monocytes. We therefore measured IFN-g production inthe supernatants of PPD-stimulated CD4 T cells from the four PPDskin test-negative patients, and compared the results with thoseobtained from 18 PPD skin test-positive patients. We found aprofound deficiency in the IFN-g production of the PPD lines takenfrom 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 expectedfrom the proliferative response) and comparable (low) levels ofIL-4 (not shown). These results were confirmed by the intra-cellular staining of PPD-stimulated CD4 T cell lines with anti-IFN-g and anti-IL-4 antibodies (two representative results areshown in Fig. 6). Stimulation with polyclonal activators (eitherPHA or an insolubilized anti-CD3 antibody) induced IFN-g andIL-4 production by the PBMC from the four PPD skin test-negativepatients with TB to levels comparable to those observed in thePPD skin test-positive patients (not shown), thus suggesting anantigen-specific, not generalized deficiency.



Fig. 1. In vitro proliferation to PPD of cutaneous lymphocyte-associatedantigen (CLA)-enriched (CLA) and CLA-depleted (CLA) CD4 Tlymphocytes from PPD skin test-negative patients ((a), individual values)and PPD skin test-positive patients ((b), grouped results) with tuberculosis.

Table 2. Intradermal skin reaction to standard antigens (Multitest)

TetanusPatient Glycerol toxoid Diphtheria Streptolysin PPD Candida Trycophiton Proteus

PPD-neg-1 0 5 6 0 0 6 0 0PPD-neg-2 0 0 0 13 0 0 0 10PPD-neg-3 0 0 0 0 0 0 12 0PPD-neg-4 0 11 3 14 2 0 0 0

Numbers indicate the mean diameter (maximum minimum diameter, averaged) of the infiltrative reaction to each antigen measured at 72 h, inmillimetres.

PPD-neg-4

PPD-neg-3

PPD-neg-2

PPD-neg-1

0 10 000 20 000 30 000 40 000

PPD_/CLA

_

PPD_/CLA

+

(a)

PPD+

(n = 18)

ct/min

0 10 000 20 000 30 000 40 000

(b)

IL-12 production by blood monocytes from PPD skintest-negative patients with active TB is preservedSince IL-12 is a critical lymphokine in Th1 responses and can up-regulate CLA expression, we designed experiments to checkwhether the monocytes from the PPD skin test-negative patientswere intrinsically defective in producing IL-12 under optimalactivation conditions (IFN-g priming plus LPS stimulation) andunder specific activation with a pathogenic, heat-killed strain ofMycobacterium (H37-Rv). In all conditions tested, no differenceswere found between the IL-12 production of these patients andthose of 18 PPD patients (Table 4).

DISCUSSIONThe main findings of this study are that (i): PPD skin test-negativeM. tuberculosis-infected patients have PPD-specific peripheralblood T lymphocytes that efficiently proliferate upon antigenstimulation but produce lower amounts of IFN-g than controls;(ii) the expression of the skin-selective homing receptor (CLA)is defective on these (antigen-specific) T cells; (iii) the impairedIFN-g production is limited to the response of blood T cells toPPD, and is not associated with either a worse prognosis or areduced monocyte production of IL-12.

We have previously reported that the proliferation of PPD-specific lymphocytes in the pleural space of infected patients withtubercular pleuritis can be detected 24 weeks before the PPDreaction turns positive [2]. This suggests that the compartmentaliz-ation of the immune response can temporarily prevent the migra-tion of PPD-specific lymphocytes from the organ targeted by the

infection to the skin. It is possible that the subsequent acquisitionof homing receptor allowing the recirculation of T cells to the skinparallels the acquisition of a positive PPD reaction. In this context,the lack of proliferation to PPD in blood correlates with PPDnegativity. In contrast, we here describe M. tuberculosis-infected



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Fig. 2. Cytofluorometric analysis of cutaneous lymphocyte-associatedantigen (CLA) expression at baseline (left panels) and after 8 days ofin vitro PPD stimulation ( PPD) on CD4 T cell lines from one represen-tative PPD skin-test positive (PPD) and one representative PPD skin test-negative (PPD-neg-1) patient with tuberculosis.

Table 3. IFN-g and IL-4 production (pg/ml) by peripheralblood mononuclear cells stimulated with phytohaem-agglutinin from PPD and PPD tuberculosis patients

IFN-g, IL-4,Subjects pg/ml pg/ml

PPD-neg-1 156 342PPD-neg-2 231 423PPD-neg-3 345 234PPD-neg-4 235 341

241 6 78* 335 6 77**PPD (n 18)Mean 6 s.d. 236 6 99* 250 6 61**

*,**Not significantly different.

PPD-neg-4

(a)

PPD-neg-3

PPD-neg-2

PPD-neg-1

Percent variation of CLA+ CD4 T cellsin vitro stimulated with PPD(individual PPD skin-negative patients)

_10 10 30 50 70 90

(b)

PPD+ (n = 18)

Mean % variation of CLA+ CD4 T cellsin vitro stimulated with PPD(n = 18 PPD skin-positive patients)

_10 10 30 50 70 90

Fig. 3. Percent variation of cutaneous lymphocyte-associated antigen(CLA) CD4 T cells from PPD skin test-negative (individual values) andPPD patients (grouped values) with tuberculosis, following 8-day in vitrostimulation with PPD.

individuals with a persistently negative PPD reaction and a positivein vitro proliferative response to mycobacterial antigens.

The in vitro proliferation to mycobacterial antigens in the fourPPD skin test-negative subjects we identified was measured using awide range of antigen doses and different preparations, includingproteins from culture filtrate and inactivated whole cells. Ourresults indicate that PPD negativity was not related to the greatersensitivity of the in vitro proliferation assay [11], nor to a skewing

towards peculiar antigenic specificities. Moreover, the four PPDskin test-negative patients did not have progressive, disseminateddisease. Instead, they successfully responded to the primarychemotherapeutic regimen and the blastogenic response of theirlymphocytes to polyclonal activators (e.g. PHA) was comparableto that of the lymphocytes from PPD patients (not shown). Inconclusion, neither a specific nor an aspecific quantitative defi-ciency of the immune response offers a reasonable explanation forthe observed skin anergy to PPD in these individuals.

We therefore focused on possible qualitative differences andstudied the expression of the CLA, the major T cell ligand for the



PPD-neg-4

PPD-neg-3

PPD-neg-2

PPD-neg-1

(TT+)

(TT_)

(TT_)

(TT+)

Percent variation of CLA+ CD4 T cellsin vitro stimulated with TT(individual PPD skin-negative patients)

_20 0 20 40 60 80

Fig. 4. Percent variation of cutaneous lymphocyte-associated antigen(CLA) CD4 T cells from PPD skin test-negative patients with tubercu-losis, 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.

PPD-neg-4

(a)

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PPD-neg-1

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Fig. 5. In vitro production of IFN-g and IL-4 from PPD-stimulated T celllines established from PPD skin test-negative (individual values) and fromPPD (grouped values) patients with tuberculosis.

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Fig. 6. Cytofluorometric analysis of intracellular IFN-g (FITC-stained, onthe abscissa) and IL-4 (PE-stained, on the abscissa) by PPD-stimulated Tcell lines from one representative PPD and one representative PPD skintest-negative patient with tuberculosis.

Table 4. IFN-g-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, lipopo-lysaccharide (LPS) or heat-inactivated Mycobacterial tuberculosis, strain

H37Rv

Patient SAC LPS H37Rv-WC

Mneg-1 874 1254 251Mneg-2 574 1234 321Mneg-3 1221 2514 451Mneg-4 898 1874 401

891 6 264* 1719 6 607** 356 6 88***Controls (n 18)Mean 6 s.d. 649 6 181* 2402 6 809** 364 6 146***

*,**,***Not significantly different.Controls are PPD TB patients. Healthy reactors and PPD skin test-

negative patients gave similar results (not shown).

0 1000 2000

(b)

PPD+

(n = 18)

IFN-g (pg/ml)

vascular adhesion molecule E-selectin [12,13]. Indeed, the inter-action between circulating CD4 T cells and microvascularendothelial cells is critical to the homing process, and CLA hasbeen proposed as a receptor for tissue-selective T cell extravasationto the skin [714]. CLA is expressed by a subset of memory T cellsin peripheral blood, by allergen-reactive T cells in patients withatopic dermatitis [9], and by malignant T cells of cutaneous T celllymphomas [7]. CLA expression is up-regulated by IL-12 secretedby the cells of the monocyte/macrophage lineages and IFN-g has apowerful priming effect on IL-12 production [15].

The pattern of CLA expression by antigen-reactive T cells isprobably influenced by a complex array of antigen-related factors,e.g. the type, the dosage and the route of exposure. It was reportedthat whereas CLA T cells from atopic dermatitis patients pre-ferentially responded to house dust mite and CLA T cells fromnickel contact dermatitis patients showed an increased response tonickel, CLA T cells showed very little response in either case[16]. Similarly, we found here that only CLA T cells proliferatedto PPD in PPD skin test-positive individuals and CLA T cells didso in PPD skin test-negative individuals. In contrast, TT, asystemically acting antigen, induced in some vaccinated indivi-duals 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 wascomparable in the four PPD skin test-negative patients with TB andin the PPD controls. Moreover, IL-12 production by IFN-g-primed, Staphylococcus aureus Cowan I (SAC)-stimulated mono-cytes was similar to that observed in the PPD patients. Takentogether, these findings clearly indicate the presence of a functionalIL-12 receptor and of an overall intact Th1 cell subset [17].Moreover, the PPD TB patients we describe here do not haveany impairment in the immune response to mycobacteria dueto IFN-g receptor [18] or IL-12 receptor deficiency, which areseverely symptomatic immunodeficiencies to intracellular patho-gens [19,20]. However, in these same patients CLA expression wasselectively defective on PPD-specific T cells. This could explainthe negative skin reaction, and a similar correlation was foundfor the intradermal reaction to another antigen. Notably, CLA,PPD-specific T cells from the four PPD skin test-negative patientswere largely of the CD45RO memory phenotype (not shown).Moreover, we found that individuals who were not exposed toM. tuberculosis and who were not BCG vaccinated were PPD skintest-negative (not shown), in agreement with previous reports[5,6]. Taken together, these results indicate that PPD-specific Tlymphocytes are proliferating to a recall antigen, not through asuperantigen type of stimulation [21].

The available data offer no indications concerning themechanisms inducing the selective defect in IFN-g productionby M. tuberculosis-specific T cells found in these patients. We canspeculate that local production of this cytokine is preserved in theinfected target organ(s). Indeed, in the case of sarcoidosis it hasbeen previously reported that IFN-g is spontaneously released byT lymphocytes from the lung, but not from peripheral blood [22].

The development of the DTH reaction to PPD requires the IFN-g-primed production of IL-12 by M. tuberculosis-infected macro-phages, which in turn induces CLA expression on antigen-specificT cells. However, it has been reported that RANTES produced bymacrophages and endothelial cells is also a key chemokine forattracting T cells to DTH sites [23]. Similarly, the macrophagemigration inhibitory factor (MIF) produced by macrophages and Tlymphocytes has been reported to play an essential role in the

tuberculin reaction [24]. However, it is interesting to note that IFN-g can up-regulate the production of both RANTES and MIF. Thus,the impaired production of this single cytokine could negativelyaffect DTH by mechanisms other than the mere control of CLAexpression.

In conclusion, the impaired production of IFN-g and the lack ofCLA expression by circulating M. tuberculosis-specific T cells isassociated with a negative tuberculin reaction in a portion ofimmune-competent patients with clinically undistinguished tuber-culosis. The characterization of the molecular mechanism of thislocally abnormal Th1 response awaits further studies.

ACKNOWLEDGMENTSThis work was supported by grants from Istituto Superiore di Sanita` to R.P.and S.E.B.

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