lymphocyte - thoraxainslie, solomon,bateman pneumoconiosis.'7 a composite profusion score...

Thorax 1992;47:513-518

Lymphocyte and lymphocyte subset numbers inblood and in bronchoalveolar lavage and pleuralfluid in various forms ofhuman pulmonarytuberculosis at presentation and during recovery

GM Ainslie, J A Solomon, E D Bateman

AbstractBackground Lymphocytes have a cen-tral role in human defences againstmycobacteria. A study was designed toassess the relation between lymphocyteresponses and clinical pattern of disease,nutrition and recovery during treatmentin patients with tuberculosis.Methods Lymphocyte numbers andsubsets (on the basis of CD3, CD4, andCD8 monoclonal antibodies) weremeasured in peripheral blood and,where appropriate, bronchoalveolarlavage or pleural fluid of patients withdifferent forms of pulmonary tuber-culosis. Eleven had localised pulmonarytuberculosis, 18 miliary tuberculosis andseven a tuberculous pleural effusion.Results CD4 lymphocytes were foundin greatly increased numbers in pleuralfluid and were relatively depleted in theblood. Lymphocyte numbers inbronchoalveolar lavage fluid variedwidely in localised pulmonary andmiliary tuberculosis but were highest inlavage fluid from patients with miliarytuberculosis. This was due to an increasein CD8 lymphocytes, which were alsoincreased in the blood. Lymphocytenumbers bore no relation to nutrition,symptom duration, or radiographicprofusion scores. In miliary tuberculosisthe time taken for the chest radiographto clear (mean (SD) 17-6 (7 8) weeks)correlated with lymphocyte numbers inlavage fluid, especially CD8 cells(r = 074), but not with the patients' ageor nutrition. After 8 weeks' treatment,total and CD4 lymphocyte numbers inlavage fluid showed a substantialincrease.Conclusion The association of CD8cells with delayed recovery is compatiblewith suppression of the antimycobac-terial action of macrophages. The switchto predominance of CD4 cells in lavagefluid during successful treatment sup-ports the view that they may have a rolein eliminating mycobacteria.

The variable manifestations and natural his-tory of pulmonary tuberculosis are deter-mined by host factors, including cell mediated

immunity, rather than differences in virulenceof different strains of Mycobacterium tuber-culosis.' The immune response to M tuber-culosis involves subpopulations of specificallysensitised CD4 helper/inducer or cytolytic Tlymphocytes.`5 Most reports of human dis-ease are based on findings in blood and pleuralfluid.5'0 In advanced or disseminated tuber-culosis circulating T cells, especially CD4cells, are reduced in number and CD8 T cellsare increased relatively.58101' In tuberculouspleural effusions CD4 functionally helper Tcells are increased in the pleural fluid com-pared with blood.61213 There have been fewreports of lymphocytes in infected lungs.Options for studying these include examina-tion of pathology specimens and cellsrecovered by bronchoalveolar lavage. Previousstudies of lymphocytes in lavage fluid in tuber-culosis have shown increased numbers oflymphocytes in local'4 and miliary tuber-culosis.'5We have compared lymphocyte numbers

and subsets in the lungs, blood and, wherepossible, the pleura of patients with differentforms of pulmonary tuberculosis (local,miliary and pleural) and assessed the serialchanges that occurred during successful treat-ment.

MethodsSTUDY POPULATIONApproval for the study was obtained from theMedical Faculty Research and Ethics Commit-tee. All patients gave informed written consentfor participation in the study. The studygroups comprised seven patients with tuber-culosis pleurisy, 11 with localised pulmonarytuberculosis (tuberculosis confined to one lobeor segment) and 18 with miliary tuberculosis.All patients were black or of mixed race. Ineach case direct sputum examination failed toshow acid and alcohol fast bacilli and broncho-scopy and/or thoracocentesis were required toconfirm active tuberculosis. The clinical assess-ment included measurement of body mass,height, triceps skinfold thickness,'6 midarmcircumference,'6 and serum albumin; chestradiography; a full blood count; and a Mantouxskin test employing 5 TU purified proteinderivative (PPD). Chest radiographs were re-viewed and the size, profusion and distributionof lung changes assessed according to theUICC/ILO standard radiographs for

Respiratory Clinic,Department ofMedicine, Universityof Cape Town andGroote SchuurHospital, Cape Town,South AfricaGM AinslieJ A SolomonE D BatemanRequests for reprints to:Dr GM Ainslie,E16 Respiratory Clinic,Groote Schuur Hospital,Observatory 7925,Cape Town,Republic of South AfricaAccepted 10 January 1992

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Ainslie, Solomon, Bateman

pneumoconiosis.'7 A composite profusionscore combining these three features (with amaximal possible score of 18 points) was cal-culated for each plate. Human immuno-deficiency virus status was not tested as therewere no cases of AIDS (other than in whitehomosexuals) in the Cape Town region duringthe study period (1982-86), and retrospectivepopulationwide serum testing identified only 20positive cases in persons of mixed race, andnone in blacks. No patients received cortico-steroid treatment.

COLLECTION OF LYMPHOCYTESLymphocytes were obtained before or within3 days of commencement of antituberculosisdrugs from peripheral blood in all patients,from bronchoalveolar lavage fluid in patientswith localised pulmonary or miliary tuber-culosis and from pleural fluid in patients with apleural effusion. Bronchoalveolar lavage wasperformed during fibreoptic bronchoscopyunder topical anaesthesia with 4% lignocaine,following premedication with 06 mg atropineand 5-10 mg diazepam intravenously. Sterile0-9% saline was instilled in 20 ml aliquots to atotal of 220 ml. The first aliquot was presumedto be bronchially contaminated and was discar-ded. In miliary tuberculosis the lavage wasperformed in a lateral subsegment of the rightmiddle lobe or the lingula. In localised pul-monary tuberculosis an affected (usually upper)and an unaffected lobe (usually the middle orlingula) were lavaged separately. Bronchialbrushings and transbronchial lung biopsyspecimens were also obtained for diagnosticpurposes. The percentage yield of lavage fluidranged from 50% to 84%. There was nosignificant difference in the yields from the threegroups (mean yield in miliary tuberculosis64%, localised pulmonary tuberculosis, affec-ted lobe 58% and unaffected lobe 61%). Pleuralfluid, obtained by thoracocentesis, was heparin-ised and subjected to cytological and bac-teriological examination. Pleural biopsysamples obtained with an Abram's needle wereexamined histologically and cultured. Peri-pheral blood was obtained by venepunctureimmediately after bronchoscopy orthoracocentesis and heparinised.

PREPARATION AND ANALYSIS OF CELLSLavage and pleural fluid were kept on ice,strained through gauze, spun down and theresultant cell pellet washed three times withRPMI-1640 and resuspended in RPMI con-taining 10% fetal calf serum. Total cell countswere performed in a haemocytometer and dif-ferential counts from May-Grunewald-Giemsastained cytocentrifuge preparations of I05 cells.Mononuclear cell preparations were madefrom lavage fluid, pleural fluid or peripheralblood by Ficoll-Hypaque centrifugation,"8 andlymphocytes separated from monocytes!macrophages by adherence on to plastic petridishes for 1-2 hours followed by passagethrough nylon wool columns.'9 Identification ofT lymphocyte subsets was performed withOKT murine monoclonal human T cellantibodies (Ortho Pharmaceuticals, Raritan,

New Jersey, USA) where OKT3 (CD3) stainsfor all mature T cells, OKT4 (CD4) for helper/inducer and OKT8 (CD8) for suppressor/cytotoxic subsets. A volume of 5 pl of each ofOKT3, OKT4 and OKT8 antibodies wasadded to 200 ,il aliquots of a 2-5 x 106 mlpreparation of lymphocytes at 4°C for 30minutes. Following two washes with medium,the cells were stained with 100 ,ul of a 40 xdilution of fluorescein conjugated goat antimouse immunoglobulin (Cappel Laboratories,Cochranville, Pennsylvania, USA) for 20 min-utes at 4 °C, then washed three times andcounted in a fluorescence activated cell sorter(Coulter model TPS-1, Coulter ElectronicsInc, Hialeah, Florida, USA). An identicalaliquot of cells, treated in the same way butwithout OKT antibody, served as control.Channel by channel subtraction of controlfluorescence from test specimen counts wasperformed by a specially prepared computerprogram. Studies of T cell subsets were per-formed on peripheral blood and pleural fluidfrom all patients with pleural tuberculosis but,for logistical reasons, on blood and lavage fluidfrom only eight patients with localised pul-monary tuberculosis and 12 patients withmiliary disease.

ASSESSMENT DURING RECOVERYClinical assessment was repeated at monthlyintervals for 6 months after initiation ofantituberculous treatment. Studies of lavage Tcell numbers and subsets were repeated on fivepatients with miliary tuberculosis after eightweeks of antituberculous treatment onceradiographic clearing had begun. Repeatstudies of blood T cell numbers and subsetswere performed at four, eight, and twelveweeks on these five subjects, and in a further fivepatients with miliary tuberculosis, five withlocalised pulmonary tuberculosis and five withpleural disease. These subgroups were similarto and representative of the original groups.

NORMAL VALUESNormal values for lavage results were obtainedfrom seven normal control subjects and thosefor T cell subsets in peripheral blood from12 normal control subjects with a mean age of33-4 (8 9) years. Seven were males, five (42%)were smokers, ten were of mixed race and twowere black. There was no significant differencebetween the controls and-patients with respectto any of the above. The normal values quotedfor other tests are those validated and acceptedin our hospital and medical school laboratory.

ANALYSISAll data unless stated to the contrary arepresented as mean (SD). Statistical compar-isons are made with the two-tailed Student's ttest, Wilcoxon sign test or Mann-Whitney Utest.

ResultsSTUDY POPULATION AND NUTRITION (table 1)There were more females in the group withmiliary tuberculosis than in the other twogroups, but ages, numbers of smokers and

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Lymphocyte and lymphocyte subset numbers in blood and in bronchoalveolar lavage and pleuralfluid in tuberculosis

Table 1 Mean (SD) demographic and nutritional data at presentation ofpatientswith pulmonary and pleural tuberculosis

Pleural Local Miliarytuberculosis tuberculosis tuberculosis(n = 7) (n = 11) (n = 18)

Age (years) 31 9 (15-1) 37-4 (17-9) 36-2 (17-5)Sex (M: F) 6: 1 7: 4 7: 11Smokers 3 8 10Duration of symptoms (weeks) 1-9 (2-2) 2-8 (3-4) 2-2 (2 03)Body mass (% ideal)t 91-7 (22 6) 96-3 (8 8) 95-1 (17-8)Midarm circumference 19-5 (27 9) 22-8 (22-2) 15-1 (21-5)(% of predicted)$

Triceps skinfold thickness 31 (18-8) 24 (26 2) 16-8 (26 2)(% of predicted)t

Serum albumin (g/l)§ 36-7 (5 9) 37-7 (6-1) 30 3 (4.8)*Positive Mantoux reactivity 5 11 8

*p values (Mann-Whitney U test) versus local tuberculosis, p < 0-05. t1969Metropolitan Life Insurance Co standards. $Frisancho, 1974.16 §Normal values: albumin35-50 g/l.

duration of symptoms were similar. Fourpatients with miliary tuberculosis and two eachwith localised pulmonary disease and tuber-culous pleural effusions had a regular heavyintake ofalcohol. Body mass was above the 90thcentile in all groups but midarm circumferenceand triceps skin fold thickness were markedlyreduced (below 24th centile). Serum albuminwas significantly reduced in patients withmiliary tuberculosis compared with those withlocalised pulmonary tuberculosis. Morepatients with miliary disease displayed anergyto PPD.

CELL COUNTS IN BLOOD AND LAVAGE ANDPLEURAL FLUID (table 2)Peripheral blood lymphopenia (less than 1-5 x109/1) was present in six of seven patients withpleural disease, nine of 18 with miliary diseaseand seven of 11 with localised pulmonarytuberculosis. In the groups as a whole bloodlymphocyte numbers were only reducedsignificantly in those with pleural disease(1-06 (0A46) x 109/1, p < 005). The total cellcount and lymphocyte yield in lavage fluid,whether expressed as total lymphocytes, per-centage of lymphocytes or lymphocytes/mllavage fluid, were significantly increased inmiliary tuberculosis. Lymphocyte numbers

expressed per ml lavage fluid were elevated inboth affected and unaffected lobes in localisedpulmonary tuberculosis with a trend towardsmore lymphocytes in the affected than theunaffected lobe. A modest increase in the per-centage of neutrophils was seen in the affectedlobe of localised pulmonary tuberculosis butthe proportion of eosinophils was normal in allcategories of disease. More than 90% of cells inpleural fluid were lymphocytes (lymphocytenumber 1234 (1196) x 103/ml).Lymphocyte numbers in lavage fluid of

patients with miliary tuberculosis bore a weakpositive relationship to patient's age (r =

0-544; p < 0 05) but were unrelated to alcoholintake, duration of symptoms, chest radiogra-phic profusion, measures of nutrition, serumalbumin or peripheral blood lymphocytenumbers.

T LYMPHOCYTE SUBSETS (figure 1)Peripheral blood CD3 cells (T lymphocytes)were present in similar proportions in controland disease categories (mean (SD) percentagesof CD3 lymphocytes in peripheral blood were68-6 (10-6) in localised pulmonary tuber-culosis, 73 (9-1) in miliary tuberculosis and71-8 (11) in pleural tuberculosis versus 68-4(9-8) in control subjects). The ratio of CD4 toCD8 cells, however, was significantly lower inperipheral blood from patients (mean (SD) ofCD4: 8 was 1 6 (0 58) in miliary disease, 1 76(0-88) in localised pulmonary tuberculosis and1-21 (043) in pleural disease) than in bloodfrom normal subjects (2-2 (0-8), p < 0 05). Asimilar but more pronounced trend was presentin lavage fluid, especially in miliary disease(mean (SD) of CD4: 8 lymphocytes in lavagefluid was 0-86 (0-43) in miliary tuberculosis,1-23 (0-28) in localised pulmonary tuberculosisaffected lobe, and 1-02 (0-23) in the unaffectedlobe versus 1-98 (0-36) in normal subjects, p <0-01). The low CD4: 8 ratio in blood was dueto an absolute decrease of CD4 cells (means ofabsolute numbers of CD4/CD8 lymphocytesx 1o6 in blood: 629/451 in miliary tuberculosis,692/454 in localised pulmonary tuberculosisand 406/355 in tuberculous effusions versus771/453 in control subjects). In the lavage fluid

Table 2 Mean (SD) numbers and proportions of cells in peripheral blood and bronchoalveolar lavagefluid

Bronchoalveolar lavagefluid

MacrophagesPeripheral Totalblood Total Lympho- lympho- Lympho- Inorganic Clear Giantlymphocytes cell yield cytes cytes cytes Neutrophils Eosinophils inclusions foamy cells( x 109/1) ( x 106) (Go) ( X 103) ( x 103/ml) (%) (G) (G) (%) (Go)

Control 1 79 10 9 5 4 601 5 4 2-3 0-6 29 0 55 4 7-4(n = 8) (0-57) (5-4) (3-6) (469) (2-7) (1.7) (0-6) (28-7) (27-5) (3-8)

Local tuberculosis 1-67 20 6 14 2 2118t 415t 5-3t 2-3 34-4 39-7 5 2affected lobe (0 66) (13 4) (12.2) (1891) (61-7) (4 5) (2 9) (26 9) (22 2) (3-3)(n = 11)

Local tuberculosis 19 9 7-1 1633t 13 4t 0-8 0 9 35 8 49 8 6-5unaffected lobe (129) (50) (730) (64) (08) (1 1) (177) (142) (1.2)(n = 7)

Miliary tuberculosis 1 48 33-3t 19.1* 6916* 58.9t 2-3 0-3 34.5 32.7* 8 8(n = 18) (0-76) (20 9) (15 4) (7795) (69) (1-7) (0-5) (25-1) (21 1) (6 8)

p values (versus controls): *p < 0-05, tp < 0-01.

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the low CD4: 8 ratios were due to a greaterincrease in CD8 than CD4 lymphocytes (meansofabsolute numbers ofCD4/CD8 lymphocytesx 103 in lavage fluid: 1916/2469 in miliarytuberculosis and 534/446 in localised pulmon-ary tuberculosis versus 235/138 in normalsubjects). In the pleural fluid the majority ofcells were CD4 T lymphocytes. The CD4: 8ratio was high in pleural fluid (3-92 (1-3) ) andlow in blood (1 21 (0 43) ).

LYMPHOCYTE NUMBERS AND SUBSETS DURING

RECOVERY

Total lymphocyte numbers in peripheral blood

in miliary tuberculosis did not change sig-nificantly during the first 12 weeks oftreatment,remaining in the low normal range, althoughthe proportion ofCD4 cells increased (table 3).By contrast, in localised pulmonary tuber-culosis and pleural disease, lymphocyte num-

bers increased from 1 14 (0 44) to 1 66 (061) x

109/l (p < O01) in the first four weeks, without a

significant change in subsets.The proportion and absolute numbers of

lymphocytes in bronchoalveolar lavage rose

strikingly by eight weeks owing to an increasein CD4 cells.

RADIOGRAPHIC RESOLUTIONRadiographic resolution occurred most rapidlyin patients with tuberculous pleural effusions(by 8 7 (6) weeks) and was similar for localisedpulmonary (17-8 (5 8) weeks) and miliarytuberculosis (17-6 (7 8) weeks). In patients withmiliary tuberculosis there was a significantlinear correlation between radiographicrecovery time and total lymphocyte numbers(r = 0 55; p < 0-05), percentage of lym-phocytes (r = 0-66; p < 0 01) and, mostnotably, the CD8 lymphocyte number (r =

0 74; p < 0-01) in lavage fluid. The higher theinitial CD8 lymphocyte numbers in lavagefluid, the longer it took for the chest radiographto improve.

DiscussionAn increased number of lymphocytes is afeature of the histopathology of pulmonarytuberculosis, and was therefore an expectedfinding in the bronchoalveolar lavage fluid ofsuch patients. Contrary to expectations,however, they were elevated in only 55% ofpatients with local pulmonary tuberculosis andin 60% of those with miliary disease. Thevariable yield of lymphocytes probably reflectsthe individual variation in inflammatory res-

ponse to mycobacterial infection. A secondobservation was that there was a greaterincrease in CD8 T lymphocytes than in theCD4 lymphocytes usually considered central toantimycobacterial defences.`5 This change wasalso in the peripheral blood in radiologicallynormal lobes in patients with non-cavitatorylocalised disease. Differences in the lung lym-phocyte subpopulation in different clinicalforms of tuberculosis were quantitative only.

Table 3 Mean (SD) changes in lymphocyte numbers and T lymphocyte subsets during treatment of miliarytuberculosis

Weeks of treatment

Before treatment 4 weeks 8 weeks 12 weeks

Peripheral blood (n = 10)Lymphocytes ( x 0I/l) 1-56 (0-27) 1 33 (0-22) 1-38 (0-23) 1-63 (0-41)CD4 (% ofT cells) 57-8 (4-7) 61-8 (4 7) 62-1 (4-2) 63.6 (8.3)*CD4: CD8 1 59 (0 26) 2-08 (0 45) 1 91 (0 43) 2 54 (0 83)*

Bronchoalveolar lavage (n 5)Lymphocytes (%) 23-6 (8-8) 40-0 (9-9)*Lymphocytes ( x 106) 41 3 (20 5) 75-2 (25-8)*CD4 (% of T cells) 47 9 (4-7) 72-3 (1-6)tCD4: CD8 0-99 (0 19) 2-66 (0-2)t

p values versus before treatment: *p < 0 05; tP < 0-01

T lymphocyte OKT4:8(CD4:8) ratios inperipheral blood,bronchoalveolar lavagefluid and pleuralfluid inpatients with differentforms ofpulmonarytuberculosis. Bar valuesrepresent mean (standarddeviation).

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The changes in pleural tuberculosis, namelymild peripheral blood lymphopenia and a lowCD4: 8 ratio, in association with a markedpleural lymphocytosis, have been describedpreviously.7 An interesting finding in thecurrent study is the differing responses in thepleura and lung. In one patient with bothmiliary tuberculosis and a pleural effusion, thepleural fluid lymphocyte CD4: 8 ratio was 4-3(representing the expected excess ofCD4 cells)while a marked CD8 lymphocyte excess(CD4: 8 = 1 1) was noted in the lavage fluid.The reason for this difference in T cell subsetsin the different compartments is not clear. It ispossible that there are differences in the acces-sory cell function of pleural macrophages20 orthat a brief and spontaneously remitting CD8phase occurs during the early stage of pleuralinfection, before the CD4 cell proliferation thatis associated with marked pleural exudates, andin the lung in miliary disease during successfultreatment.

It is relevant to ask what factors determinethe intensity of lymphocyte response in localand miliary tuberculosis. In the latter, norelationship with profusion of chest radiogra-phic abnormality, duration of symptoms andmeasures of nutrition was observed. Dhandet all5 have shown a relative increase in totalcell and lymphocyte counts in poorly nourishedpatients with local disease, especially whenextensive. Increased T suppressor cells and alower T helper to suppressor ratio have beenobserved in malnutrition2 22and with advancedage.23 This shift, however, is usually associatedwith reduced T lymphocyte numbers. Aheavier load of infecting organisms is an alter-native explanation and, as this was notmeasured in our study, remains a possibility. Aswitch to functionally suppressive T cellproliferation with increased dose of mycobac-teria has been observed in experimentalanimals, but total cell numbers did not increasegreatly.24

Previous studies looking at the pattern oflymphocyte responses during treatment havebeen confined to peripheral blood. Our patientswith sputum negative localised pulmonary andpleural disease showed a lymphopenia atpresentation, which resolved on treatment, aswas seen with Onwubalili and Scott withsputum positive localised pulmonary tuber-culosis.25 The CD4: 8 ratio did not rise sig-nificantly in our patients, unlike the sputumpositive and sputum negative patients ofSinghal et al." Our patients with miliary tuber-culosis showed a rise in the blood CD4: 8 ratioduring treatment, as did the patients ofBhatnagar et al.8 Their lymphopenia, however,did not resolve within the first 12 weeks.This is the first study to assess the changes in

cellular response in the lungs of patients withmiliary tuberculosis during treatment, and torelate them to radiographic resolution. Therewas an inverse relationship between rate ofrecovery of miliary tuberculosis and total lym-phocyte numbers and CD8 cells in lavage fluidat presentation. The differing rates of recoverywere not accounted for by different profusion ofabnormality on chest radiography or the

patients' nutrition. It is not clear whether theincreased CD8 cells form an essential part ofthe delayed recovery or whether they merelyreflect more severe infection.

It is important to establish the functional roleofthe CD8 cells observed in our study. Effectivehost defence against mycobacteria involvescollaboration between mononuclear phago-cytes and lymphocytes, the final pathway beinginhibition of mycobacterial replication withinmacrophages.26 Lymphokines from specificallysensitised clones of CD4 helper lymphocyteshave been thought to be central to thisprocess.327 The CD4 lymphocyte response ispotentially tissue damaging as lymphokines areproduced which prime macrophages for mas-sive release of tumour necrosis factor whenmycobacteria are ingested.2 If the CD8 cellsshown in this study have suppressor function,they may serve to downregulate the prolifera-tion ofCD4 cells and potentially damaging hostresponses. This might be done at the expense ofdelaying clearance of the organism. Function-ally suppressive lymphocytes have been iden-tified in human tuberculous infections29 3' andexperimental models.3132 As yet there is noevidence for CD8 cytotoxic T cells in humantuberculosis.33 3There was a substantial increase in lym-

phocyte numbers in lavage fluid after eightweeks of treatment despite an improvement inthe chest radiographs. This increasing lym-phocytosis was associated with an impressiveswitch from CD8 to CD4 dominance. It is notclear what factors determine this change. Itmight follow as a consequence of diminishingbacterial load following antimycobacterialdrug treatment. These observations in humanpulmonary tuberculosis provide support for invitro data emphasising the central role of CD4cells in enhancing antimycobacterial responsesmediated by macrophages. 52735 The CD4lymphocytes found in tuberculous pleuraleffusions have previously been shown to havehelper function.7 36 Further studies of thefunctional characteristics of both early CD8and later CD4 cells in human pulmonarytuberculosis, particularly those associated withhigh intensity lung lymphocyte responses, arenecessary.

In summary, we have established that theintensity of the lymphocyte responses in lunginfected with M tuberculosis varies widely inindividual patients.

The authors wish to thank Professor Eugene Dowdle of theDepartment of Clinical Science and Immunology for advice anduse of laboratory facilities, Sister Cecelia Wilson for herexcellent assistance during bronchoscopies and MrsA Singer fortyping this manuscript. Financial support from the SouthAfrican Medical Research Council and Guy Elliot PostgraduateResearch Fellowship of the University of Cape Town isgratefully acknowledged.

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2 Edwards D, Kirkpatrick CH. The immunology of myco-bacterial diseases. Am Rev Respir Dis 1986;134:1062-71.

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4 OttenhoffTH, Ab BK, van Embden JD, Thole JE, KiesslingR. The recombinant 65-kD heat shock protein ofMycobacterium bovis Bacillus Calmette-Guerrin/M tuber-

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