with Tuberculous Pleurisy

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1 INFECTION AND IMMUNITY, Feb. 98, p /8/-8$./ Vol. 5, No. In Vitro Tuberculin Rectivity of Lymphocytes from Ptients with Tuberculous Pleurisy HIROSHI FUJIWARA,* YASUHISA OKUDA, TAKASHI FUKUKAWA, AND IZUO TSUYUGUCHI Osk Prefecturl Hbikino Hospitl, Hbikino City, Osk 58, Jpn Received June 98/Accepted October 98 Mononucler cells in pleurl fluid from ptients with tuberculous pleurisy were predominntly T cells. Responsiveness of pleurl fluid T cells to purified protein derivtive of tuberculin were studied by the ssy of cell prolifertion nd production of lymphocyte mitogenic fctor by the stimultion with purified protein derivtive. Peripherl blood lymphocytes were lso studied from ptients nd tuberculin-positive helthy controls. The order of responsiveness ws s follows: pleurl fluid lymphocytes > peripherl blood lymphocytes of ptients without effusion = peripherl blood lymphocytes of helthy controls > peripherl blood lymphocytes of ptients with effusion. The poor response of peripherl blood lymphocytes from pleurisy ptients were recovered by the elimintion of dherent cells in peripherl blood lymphocytes to the level of the response of peripherl blood lymphocytes from helthy controls. T cells purified from pleurl fluid mononucler cells responded more thn those from peripherl blood. These results suggested tht in the pleurisy ptients purified protein derivtive-rective T cells in peripherl blood did not decrese in ctivity, but were depressed by suppressor cells, nd further suggested tht highly purified protein derivtiverective T cells were ccumulted in the pleurl fluid. Tuberculous pleurisy with effusion is one of the diseses in which cell-medited immunity might ply n importnt role in pthogenesis. Although there re mny immunologicl investigtions from vrious points of view on pulmonry tuberculosis without effusion (, 8,, 7, 8,, ), there re few investigtions, especilly t the cellulr level, on tuberculous pleurisy (6). Tuberculous pleurl fluids contin lymphocytes, most of which belong to T cells (5, 6), in high concentrtion. In this regrd it is intriguing to investigte nd clrify the immunologicl chrcteristics of lymphocytes in pleurl fluid. In the present study, we exmined the immunologicl functions of lymphocytes from ptients with tuberculous pleurisy nd pulmonry tuberculosis ptients without pleurisy by using two in vitro ssy systems: purified protein derivtive of tuberculin (PPD)-induced prolifertive response nd PPD-induced production of lymphocyte mitogenic fctor (LMF), both of which re reported to be ssocited with T-cell functions (, 7, 9, ). MATERIALS AND METHODS Subjects. Twenty-seven ptients (7 to 69 yers old, men yers) with tuberculous pleurisy with effusion nd ptients (8 to 6 yers old, men 7 yers) with ctive pulmonry tuberculosis without effusion hospitlized t Osk Prefecturl Hbikino Hospitl were exmined before the initition of chemotherpy. All ptients with tuberculous pleurisy were newly detected nd hd tuberculous lesions in their lungs. Dignosis ws bsed on the histologicl findings of biopsied specimen of pleur. All ptients with pulmonry tuberculosis were lso newly detected nd dignosed by the demonstrtion of cid-fst bcilli in sput. No ptients hd miliry tuberculosis or underlying diseses. All ptients were positive for the tuberculin skin test with 5 tuberculin units (TU) of PPD, except for two ptients with pleurisy. Forty-one helthy hospitl employees nd lbortory workers (9 to 9 yers old, men 9 yers) tht were ll positive for the tuberculin skin test served s control subjects. The ptients nd controls were not immunized with BCG t lest within yer before study. Age-mtched controls were not necessry for the comprison with the ptients becuse the ptients over 5 yers old hd in vitro PPD responses like those of the younger ptients; ge did not pper to ffect the responses. Isoltion nd chrcteriztion of MNC. Mononucler cells (MNC) in peripherl blood were isolted from heprinized peripherl blood by Ficoll-Hypque (specific grvity,.77) density centrifugtion (). Pleurl fluid MNC were obtined from heprinized pleurl fluid by the sme method s for peripherl blood. Blood nd pleurl fluid smples were collected on the sme dy. Rosette-forming cells were tested for thein bility to form rosettes with sheep erythrocytes (SRBC) by the method of Jondl et l. (), except for the initil incubtion period of min of MNC with SRBC t 7 C. To stbilize the rosettes, MNC nd SRBC were suspended in % fetl bovine serum (Microbiologicl Assocites, Wlkersville, Md.).

2 VOL. 5, 98 T-CELL FUNCTIONS IN TUBERCULOUS PLEURISY More thn MNC were counted; smples with MNC binding more thn three SRBC were considered positive. Surfce immunoglobulin-bering cells were detected by stining with fluorescein isothiocynteconjugted got nti-humn immunoglobulin serum (polyvlent nti-immunoglobulin G, M, nd A; Behringwerke AG, Mrburg, Germny). MNC (5 x 5) in ml of RPMI 6 medium (Nissui Seiyku Co., Ltd., Tokyo, Jpn) were incubted t 7 C, in 5% CO in ir. After h, they were wshed twice nd then incubted with fluorescein isothiocynte-conjugted nti-humn immunoglobulin serum for 6 min t C, followed by three wshings in phosphte-buffered sline. The pellet ws resuspended in one drop of glycerol-phosphte-buffered sline (:). Over MNC were counted by using n Olympus model BH- RFL fluorescence microscope, nd cells displying more thn three shrply stined spots were considered positive. Nonspecific esterse stining of MNC ws performed by the method of Koski et l. (), with - nphthyl butyrte s substrte. Over 5 cells were counted, nd smples of MNC with multiple intensely red-stined grnules in the cytoplsm were considered positive. Seprtion of dherent nd nondherent cells. MNC from peripherl blood or pleurl fluid were suspended to x 6 cells per ml in RPMI 6 contining % het-inctivted fetl bovine serum; 5 ml ws trnsferred to plstic dishes (no. ; Flcon Plstics, Oxnrd, Clif.) nd incubted in humidified 5% CO in ir for h t 7 C. After incubtion, the dishes were shken slowly, nd nondherent (NA) cells were removed. The plstic dishes were then wshed five times with RPMI 6, nd dherent cells were obtined by dislodging the cells dhering to the dishes with rubber policemn. Approximtely 7% of dherent cells nd less thn 5% of NA cells stined positive by esterse stining. Purifiction of T cells. T cells were purified by the combined method of SRBC rosette formtion nd nylon wool column filtrtion. Erythrocyte-rosetting MNC s described bove were gently suspended, lyered over Ficoll-Hypque, nd centrifuged for min t x g. The rosette-rich frction ws collected from the bottom of the tube, hemolyzed in.8% NHCl, nd then filtered through nylon wool column by the method of Greves nd Brown (). Cells pssing through the column were designted s T cells. These T-cell frctions contined over 95% SRBC rosette-forming cells, less thn.% surfce immunoglobulin-bering cells, nd less thn.% esterse stin-positive cells. PPD nd culture medium. PPD ws kindly donted by H. Fujii, Institute for Microbil Disese, Osk University. Culture medium consisted of RPMI 6 supplemented with 9o pooled humn AB serum, plg of streptmycin per ml, nd U of penicillin per ml. The sme lot of PPD nd humn serum ws used throughout the study. PPD-induced prolifertive response. Peripherl blood or pleurl fluid MNC were cultured t x cells per. ml of culture medium in microculture plte (no. ; Flcon). The optiml culture period nd finl PPD concentrtion were 6 dys nd 5 p.g/ml, respectively. Cultures were performed in humidified tmosphere t 7 C in 5% CO in ir nd.,uci of [H]thymidine (specific ctivity, 5 Ci/mmol; Rdiochemicl Centre, Amershm, Englnd) ws dded to ech well 8 h before hrvesting. All cultures were performed in triplicte. Cells were collected onto glss filter disks nd wshed with semiutomted microhrvesting device (Lbo Msh; Lbo Science Co., Ltd., Tokyo, Jpn). H content ws determined by liquid scintilltion spectrometer (Tri-Crb; Pckrd Instrument Co., Inc., Downers Grove, Ill.). Results were expressed s counts per minute (cpm) of [H]thymidine incorported per culture nd s Acpm where Acpm = cpm of [H]thymidine incorported per stimulted culture - cpm of [H]thymidine incorported per unstimulted culture. PPD-induced production of LMF. MNC were suspended t concentrtion of 5 x 6 cells per ml in culture medium nd were cultured in tissue culture tubes (no., Flcon) for h t 7 C in humidified tmosphere of 5% CO in ir. Cultures used for LMF production received 5,ug of PPD per ml t the onset of the incubtion period nd were designted s P cultures. Control cultures received PPD (5,ug/ml) t the end of the incubtion period nd were designted s R cultures. Incubtion ws stopped by pelleting the cells for min t, x g. Culture superntnts were decnted from the cells, filtered through.5- plm Millipore filter (Millipore Corp., Bedford, Mss.) nd stored t - C until tested. According to the preliminry investigtions, the culture conditions described bove were optiml for the production of LMF by MNC, nd LMF ctivity in those culture superntnts remined constnt for t lest 6 months. Moreover, we confirmed tht LMF production ws ntigen specific nd LMF ws produced by T cells. Assy of LMF ctivity. Humn peripherl blood B lymphocytes obtined from single helthy donor were used s the indictor cells for the ssy of LMF ctivity. B lymphocytes were obtined s frction of SRBC rosette-forming cell-depleted MNC. They contined less thn 5% SRBC rosette-forming cells, nd they were unble to proliferte by the stimultion with PPD (from. to,ug/ml). These B lymphocytes were suspended t concentrtion of x 6 cells per ml in culture medium, nd.-ml liquots were plced in ech triplicte microculture plte well. Superntnts (. ml) of P or R cultures were dded to the cell suspensions. The optiml culture period ws 6 dys. [H]thymidine (. p.ci) ws dded to ech well 8 h before hrvesting, nd rdioctivity incorported to cells ws counted by the method described in the prolifertive response. LMF ctivity ws clculted ccording to the following formul: LMF ctivity = cpm incorported by B lymphocytes stimulted by P superntnts - cpm incorported by B lymphocytes stimulted by R superntnts. The cpm incorported by R superntnts were lwys less thn,. Irrdition. Adherent cells suspended in culture medium were irrdited in vitro by exposure to X rys from X-ry genertor (STABILIPAN /; Siemens-Reiniger-Werke AG, Erlgen, Germny). Sttistcl nlysis. The significnce of the difference between groups ws clculted by Student's t test. RESULTS Cell chrcteriztion of MNC in peripherl blood nd pleurl fluid. MNC in pleurl fluid nd

3 FUJIWARA ET AL. TABLE. INFECT. IMMUN. Chrcteriztion of MNC from the peripherl blood nd pleurl fluid in ptients with tuberculous pleurisy Source of MNC subjects No. of % forming SRBC rosettecells % Surfce bering immunoglobulincells % stin-positive Nonspecific esterse cells Peripherl blood Pleurl fluid ±. 88. ±.7b. ±.6.8 ±.b.7 ±.8. ±.7b Men + stndrd error of the men. b Significntly different from peripherl blood; P <.. MNC in peripherl blood of ptients were obtined on the sme dy. Bsed on morphologicl studies, pleurl fluid contined greter thn 9% lymphocytes. These MNC were employed for rosette formtion with SRBC, detection of surfce immunoglobulin determinnts, nd nonspecific esterse stining. The dt in Tble show tht the percentge of SRBC rosette-forming cells (T cells) ws significntly higher in pleurl fluid MNC thn in peripherl blood MNC, wheres surfce immunoglobulin-bering cells (B cells) nd esterse stin-positive cells (mcrophges) in pleurl fluid MNC were less thn in peripherl blood MNC (P <.). The percentges of T cells, B cells, nd mcrophges in peripherl blood MNC from these ptients were similr to those in peripherl blood MNC from helthy controls. PPD-induced prolifertive response nd LMF production. The PPD dose-response curves nd kinetic studies reveled the highest prolifertion in cultivtion of both pleurl fluid nd peripherl blood MNC for 6 dys with PPD dose of 5,ug/ ml. Peripherl blood MNC from ptients with tuberculous pleurisy responded significntly less thn those from pulmonry tuberculosis ptients without effusion nd helthy controls in the prolifertive response. These decresed responses in pleurisy ptients were not due to incresed "bselines," becuse the men vlues of [H]thymidine incorportion per unstimulted culture were 9 cpm in the pleurisy group nd Subjects TABLE. 8 cpm in the control group. On the contrry, pleurl fluid MNC from ptients with tuberculous pleurisy were significntly more responsive thn peripherl blood MNC from the pleurisy ptients, pulmonry tuberculosis ptients, nd helthy controls (Tble ). Responses of peripherl blood nd pleurl fluid MNC obtined from ech ptient on the sme dy nd ssyed simultneously re shown in Fig.. Pleurl fluid MNC from 7 of ptients hd greter responsiveness thn peripherl blood MNC in the prolifertive response (Fig. ). The results of the LMF production prlleled those of the prolifertive response. Peripherl blood MNC from ptients with tuberculous pleurisy showed less LMF production thn those from other groups, nd pleurl fluid MNC produced LMF more thn peripherl blood MNC from other groups (Tble, Fig. ). Response of NA cells. The effect of depletion of dherent cells from MNC ws exmined in the PPD-induced prolifertive response (Fig. ). Cells were cultured with 5 jig of PPD per ml for 6 dys. NA cells responded more thn unfrctionted MNC (UF cells) in the peripherl blood MNC from /6 (8%) ptients with tuberculous pleurisy. Moreover, the men vlue of [H]thymidine incorportion by these NA cells of (5. ±.) x cpm ws significntly greter thn tht by UF cells of (9.9 ±.) x cpm (P <.). NA cells responded over UF cells in the peripherl blood MNC from 5 of PPD-induced prolifertive response of MNC from tuberculous ptients [H]thymidine Source of MNC No. of incorported subjects ( Acpm) men + SE Tuberculous pleurisy Peripherl blood 7 9. ±.5" Pleurl fluid 6. ±.8c Plumonry tuberculosis Peripherl blood.5 ±.9 Helthy control' Peripherl blood.7 ±.7 MNC from peripherl blood or pleurl fluid were cultured with 5,u g of PPD per ml for 6 dys. [H]thymidine incorported during the lst 8 h ws counted. b Significntly different from helthy control nd pulmonry tuberculosis, P <.5. c Significntly different from peripherl blood of tuberculous pleurisy, P <.. Significntly different from helthy control nd pulmonry tuberculosis, P <.5. d Positive for tuberculin skin test.

4 VOL. 5, 98 T-CELL FUNCTIONS IN TUBERCULOUS PLEURISY 5 C. MNC from MNC from peripherl blood pleurl fluid FIG.. PPD-induced prolifertive response of MNC from peripherl blood nd pleurl fluid. Ech point connected by line indictes the response from the sme individul. Peripherl blood ws collected on the dy of collecting pleurl fluid. MNC were cultured with 5 Lg of PPD per ml for 6 dys. [H]thymidine incorported during the lst 8 h ws counted. The verticl brs represent the men ± stndrd error of the men. (%) ptients with pulmonry tuberculosis nd 7 of 7 (%) helthy controls; the men response of NA cells ws not significntly different from tht of UF cells in those subjects, nor ws the response of NA cells significntly different from tht of UF cells in pleurl fluid MNC. Figure shows the kinetics of the prolifertive TABLE. response of UF nd NA cells stimulted with 5,ug of PPD per ml nd Fig. 5 shows doseresponse curves of those cells stimulted with PPD in the 6-dy culture. The incresed response of prolifertion fter depletion of dherent cells ws observed only in peripherl blood MNC from ptients with tuberculous pleurisy. Tble shows PPD-induced LMF production s well s PPD-induced prolifertive response of both UF nd NA cells from peripherl blood MNC from three ptients with tuberculous pleurisy. NA cells showed greter LMF production thn UF cells; these results were in prllel with those of the prolifertive response. Response of T cells. To compre the rectivity of T cells in pleurl fluid with tht of T cells in peripherl blood, x 5 T cells purified from pleurl fluid MNC from pleurisy ptients or those from peripherl blood MNC from the sme ptient were reconstituted with vrious numbers of utologous dherent cells which hd been obtined from peripherl blood MNC nd irrdited with, rd. These T cells were cultured with 5 p.g of PPD per ml for 6 dys, nd [H]thymidine incorported during the lst 8 h ws counted. Representtive results re shown in Fig. 6. Pleurl fluid T cells showed greter response thn peripherl blood T cells from the sme ptient irrespective of the numbers of dherent cells dded (Fig. 6). The dt in Fig. 6 further indicte tht the ddition of incresing numbers of dherent cells suppressed the PPDinduced prolifertion of T cells from both pleurl fluid nd peripherl blood. DISCUSSION Our study showed tht pleurl fluid MNC were predominntly T cells nd highly responsive to in vitro stimultion with PPD. In contrst, peripherl blood MNC of these ptients were less responsive even thn those of tuberculin-positive helthy individuls. We showed tht responsiveness of ptients' peripherl blood LMF production by MNC from tuberculous ptients Subjects Source of MNC subjects Noȯf ~~~LMF (ctivity' subjects CpM) ~men ±- SE Tuberculous pleurisy Peripherl blood c Pleurl fluid 9..d Pulmonry tuberculosis Peripherl blood 5.7 ±. Helthy controle Peripherl blood ±.7 b MNC from peripherl blood or pleurl fluid were cultured with 5,ug of PPD per ml for h. Humn peripherl blood B lymphocytes obtined from single helthy donor were used s the indictor cells for the ssy of LMF ctivity. c Significntly different from pulmonry tuberculosis, P <.. Significntly different from helthy control, P <.. d e Significntly different from other three groups, P <.. Positive for tuberculin skin test.

5 6 FUJIWARA ET AL. I U xi MNC from MNC from peripherl blood pleurl fluld FIG.. LMF production by MNC from peripherl blood nd pleurl fluid. Ech point connected by line indictes the response from the sme individul. Peripherl blood ws collected on the dy of collecting pleurl fluid. MNC were cultured with 5,ug of PPD per ml for h to produce LMF. Humn peripherl blood B lymphocytes obtined from single helthy donor were used s the indictor cells for the ssy of LMF ctivity. The verticl brs represent the men + stndrd error of the men. MNC to PPD stimultion ws restored by the elimintion of dherent cells in MNC. These results suggest tht the hyporesponsiveness of peripherl blood MNC from pleurisy ptients ws not due to decline in the function of T-cell themselves, but to the effect of suppressor cells. These suppressor cells my be ctivted mcrophges, since they hve the bility to dhere rigidly, nd pleurl fluid MNC, which contin few mcrophges, were highly responsive. It is known tht ctivted mcrophges hve the cpcity to inhibit the T-cell function (9,,, ). On the other hnd, s mcrophges were required to ctivte T cells by soluble ntigen such s PPD () nd, s shown in our study, the number of mcrophges ffected the mgnitude of T-cell response (Fig. 6), it is conceivble tht the hyporesponsiveness of peripherl blood MNC from pleurisy ptients nd the recovery of the response by the elimintion of dherent cells were simply due to the percentge of mcrophges involved. This is, however, unlikely becuse the percentge of esterse-positive cells in peripherl blood MNC from pleurisy ptients ws similr to tht in peripherl blood MNC from helthy controls (Tble ). Ellner (5) showed the existence of suppressor mcrophges in the peripherl blood from tuberculous ptients with low tuberculin responses. Our dt suggested tht suppressor mcrophges were present in the peripherl blood from pleurisy ptients who responded to PPD within norml rnge s well s in low responders, lthough the differences in nondherent cell responses might reflect different efficiencies of removl of dherent cells on plstic dishes or different degrees of ccessory function of residul mcrophges or both (Fig. ). Suppressor mcrophges my be responsible for the depressed mnifesttion of delyed hypersensitivity observed in ptients with pleurisy. In regrd to the ntigen specificity of the depressed response of peripherl blood MNC in the pleurisy ptients, we did not exmine other ntigens in this study. However, peripherl - P INFECT. IMMUN. UF MA UF NA UF NA UF NA pw blood pdr fkd PO P&Ow bbod mnc mm UNC WC Tuberculous pleurisy Pko Helthy bbeesdoiscontrol FIG.. PPD-induced prolifertive response of UF MNC nd NA cells from peripherl blood or pleurl fluid. Ech point connected by line indictes the response from the sme individul. UF nd NA cells were cultured with 5,ug of PPD per ml for 6 dys. H]thymidine incorported during the lst 8 h ws counted. The verticl brs represent the men+ stndrd error of the men.

6 VOL. 5, 98 T-CELL FUNCTIONS IN TUBERCULOUS PLEURISY 7 IL b - ct b. C E. Tuberulous pleurisy perwl bbod MC pleurl fluiid MNC (nm) (n=6) T.....~ A- 8 Puhxw tuerulss perh.rlhwood MNC (n7), T Helthy control perh l bbod MNC (n.)..... I.... -L Culture period (dys) FIG.. Kinetics of the prolifertive response of UF MNC nd NA cells stimulted with PPD. UF () nd NA () cells from peripherl blood or pleurl fluid were cultured with 5 tig of PPD per ml for vrious periods. [H]thymidine incorported during the lst 8 h ws counted. Verticl brs represent the stndrd error of the men. in the pleurisy ptients showed greter LMF production thn unfrctionted blood MNC (Tble ). These observtions suggest tht suppressor mcrophges re responsible for the suppression of the lymphokine production of T cells s well s DNA synthesis. The dt in Fig. 6 indicte tht pleurl fluid T cells responded to PPD stimultion more thn peripherl blood T cells from the sme ptient t ny point of ddition of dherent cells, which is suggesting tht more PPD-rective T cells were IL C) o I - U. S E.C I-' perherl blood MNC (nx6),,, I'I 5 tubercuis d bood MNC (nz7).pero Tuberculous [ A- 5 5 Helthy control p hsrd bbod MC (n-) - J. pleurisy plwl fkid MNC (n-6) blood MNC from some pleurisy ptients which responded poorly to PPD stimultion gve prolifertion to phytohemgglutinin, polyclonl T cell ctivtor, s high s those from helthy controls (dt not shown). Results of PPD-induced LMF production of peripherl blood nd pleurl fluid MNC from pleurisy ptients were in prllel with those of the prolifertive response (Tble, Fig. ); NA cells from peripherl blood 55 Concentrtion 5 of PPD (tg/ml) FIG. 5. Dose-response curves of UF MNC nd NA cells stimulted with PPD. UF () nd NA () cells from peripherl blood or pleurl fluid were cultured for 6 dys in the presence of vrious concentrtions of PPD. [H]thymidine incorported during the lst 8 h ws counted. Verticl brs represent the stndrd error of the men.

7 8 FUJIWARA ET AL. TABLE. Effect of dherent cell-depletion on the PPD-induced LMF production nd prolifertive response of peripherl blood MNC from ptients with tuberculous pleurisy Ptient LMF ctivityb (cpm) Prolifertive rcsponse UF NA UF NA,9 9,79 9,999 55, 6,7 8,686,668 6,78,6,8,885,58 MNC were cultured with 5,ug of PPD per ml for h to produce LMF. b Humn peripherl blood B lymphocytes obtined from single helthy donor were used s the indictor cells for the ssy of LMF ctivity. c MNC were cultured with 5 jig of PPD per ml for 6 dys. [H]thymidine incorported during the lst 8 h ws counted. ccumulted in pleurl fluid thn in peripherl blood. Moreover, the ddition of incresing numbers of dherent cells suppressed the response of T cells from both pleurl fluid nd I '- i._ E Numbet of dherent 6 8 cells dded (" cells/well) FIG. 6. PPD-induced prolifertive response of T cells from ptient with tuberculous pleurisy. T cells from pleurl fluid () nd peripherl blood (O) mononucler cells from the sme ptient were obtined nd reconstituted with vrious numbers of utologous dherent cells. T cells ( x '/well) were cultured with 5,ug of PPD per ml for 6 dys. [H]thymidine incorported during the lst 8 h ws counted. Adherent cells were obtined from peripherl blood MNC nd irrdited (, rd). INFECT. IMMUN. peripherl blood. Tken together those observtions, hyperresponsiveness of pleurl fluid MNC to PPD stimultion my be cused by more PPDsensitive T cells or fewer dherent cells contining the suppressor mcrophges (or both) in pleurl fluid s compred with peripherl blood. It is conceivble tht lrge numbers of highly PPD-rective T cells were recruited to the pleurl fluid from peripherl blood to proliferte by the stimultion of tubercle bcilli or relted ntigens. ACKNOWLEDGMENTS We thnk Susumu Kishimoto, Osk University, nd Director Kzuo Ymmoto, Osk Prefecturl Hbikino Hospitl, for the preprtion of the mnuscript nd suggestions. LITERATURE CITED. Al-Twil, N. G., nd A. J. Thewini Study of the immunologicl sttus of ptients with pulmonry tuberculosis. Scnd. J. Immunol. 8:-8.. Bergholtz, B. O., nd E. Thorsby Mcrophgedependent response of immune humn T lymphocytes to PPD in vitro. Influence of HLA-D histocomptibility. Scnd. J. Immunol. 6: Boyum, A Isoltion of mononucler cells nd grnulocytes from humn blood. Isoltion of mononucler cells by one centrifugtion, nd of grnulocytes by combining centrifugtion nd sedimenttion t g. Scnd. J. Clin. Lb. Invest. (Suppl. 97): Chess, L., R. P. McDermott, nd S. F. Schlossmn. 97. Immunologic functions of isolted humn lymphocyte subpopultions. II. Antigen triggering of T nd B cells in vitro. J. Immunol. : Elner, J. J Suppressor dherent cells in humn tuberculosis. J. Immunol. : Eller, J. J Pleurl fluid nd peripherl blood lymphocyte function in tuberculosis. Ann. Intern. Med. 89: Evns, R. L., J. M. Brerd, H. Lzrus, S. F. Schlossmn, nd L. Chess Detection, isoltion, nd functionl chrcteriztion of two humn T-cell subclsses bering unique differentition ntigens. J. Exp. Med. 5:-. 8. Gtner, E. M. S., nd R. Anderson. 98. An in vitro ssessment of cellulr nd humorl immune function in pulmonry tuberculosis: correction of defective neutrophil motility by scorbte, levmisole, metoprolol nd proprnolol. Clin. Exp. Immunol. : Geh, R. S., nd E. Merler. 97. Humn lymphocyte mitogenic fctor: synthesis by sensitized thymus-derived lymphocytes, dependence of expression on the presence of ntigen. Cell. Immunol. :86-.. Greves, M. F., nd G. Brown. 97. Purifiction of humn T nd B lymphocytes. J. Immunol. :-.. Jondl, M., G. Holm, nd H. Wigzell. 97. Surfce mrkers on humn T nd B lymphocytes. I. A lrge popultion of lymphocytes forming nonimmune rosettes with sheep red blood cells. J. Exp. Med. 6:7-5.. Ktz, P., R. A. Goldstein, nd A. S. Fuci Immunoregultion in infection cused by Mycobcterium tuberculosis: the presence of suppressor monocytes nd the ltertion of subpopultions of T lymphocytes. J. Infect. Dis. :-.. Koskl, I. R., D. G. Poplck, nd R. M. Blese A nonspecific esterse strin for the identifiction of monocytes nd mcrophges, p In B. R. Bloom nd J. R. Dvid (ed.), In vitro methods in cell-medited nd tumor immunity. Acdemic Press, Inc., New York.. Littmn, B. H., nd J. R. Dvid An ssy for humn lymphocyte mitogenic fctor: B-lymphocyte stimultion by T-lymphocyte product. Cell. Immunol. 9:7-87.

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