Recombinant Human Interleukin-2 Reverses In Vitro-Deficient Cell-

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1 INFECTION AND IMMUNITY, Sept. 1987, p /87/ $2./ Copyright 1987, Americn Society for Microbiology Vol. 55, No. 9 Recombinnt Humn Interleukin-2 Reverses In Vitro-Deficient Cell- Medited Immune Responses to Tuberculin Purified Protein Derivtive by Lymphocytes of Tuberculous Ptients HIROE SHIRATSUCHI,* YASUHISA OKUDA, AND IZUO TSUYUGUCHI Osk Prefecturl Hbikino Hospitl, Hbikino-shi, Osk 583, Jpn Received 4 Februry 1987/Accepted 9 June 1987 In vitro lymphocyte prolifertive response to purified protein derivtive of tuberculin (PPD) ws investigted in ptients with tuberculosis. Peripherl blood lymphocytes (PBL) from ptients with dvnced, refrctory tuberculosis showed significntly depressed response compred with the response of PBL from ptients with newly dignosed tuberculosis (P <.1). A further chrcteriztion of this low responsiveness to PPD reveled tht PBL from these dvnced tuberculous ptients filed to generte interleukin-2 (IL-2) in response to PPD stimultion. IL-2 receptor (Tc ntigen) expression on the surfce of T cells fter PPD stimultion ws lso impired, lthough to lesser extent, in the ptients with dvnced, refrctory tuberculosis. We ttempted to overcome the depressed in vitro response observed in PBL from ptients with dvnced, refrctory tuberculosis nd found tht the ddition of exogenous, recombinnt IL-2 returned the depressed PPD-induced PBL prolifertion in these ptients to the level of response observed in PBL from ptients with newly dignosed tuberculosis. The ddition of recombinnt IL-2 lso hd restortive effect (up regultion) in vitro on the prtly impired PPD-induced IL.2 receptor expression by PBL from the ptients with dvnced, refrctory tuberculosis. Our results suggest tht recombinnt IL-2 my offer novel pproch to the therpy of dvnced, drug-resistnt tuberculosis. In tuberculosis, the T-cell-medited immune response plys n importnt role in the pthogenesis of the disese nd lso in the protective immunity ginst the bcillus. This T-cell-medited immune response cn be detected in vitro by lymphocyte trnsformtion in response to purified protein derivtive of tuberculin (PPD). It is widely ccepted tht ntigen-induced T-cell prolifertion requires both the expression of receptors for nd the secretion of interleukin-2 (IL-2). Subsequent interction of IL-2 with its receptors leds to DNA synthesis nd expnsion of pproprite clones of ntigen-rective T cells (2, 7). The concentrtion of IL-2 nd the expression of IL-2 receptors on T cells determine the extent of T-cell prolifertion (3). Tuberculosis is chronic infectious disese nd exhibits brod clinicl s well s immunologicl spectrum (1) s hs been seen in leprosy. Leprosy is ssocited with ltertions of immune responses which vry long the spectrum of the disese (28). Ptients with lepromtous leprosy show n impired cell-medited immunity to Mycobcterium lepre ntigen. A number of studies bout this nergy in lepromtous leprosy hve been described, including impired IL-2 production by lymphocytes fter lepromin stimultion (13-16, 2, 24). Hregewoin et l. (13) reported tht peripherl blood lymphocytes (PBL) of ptients with lepromtous leprosy fil to proliferte fter in vitro stimultion with M. Iepre ntigen but tht the cells re mde fully rective by the ddition of exogenous IL-2. Ptients with fr-dvnced or miliry tuberculosis re often nergic to tuberculin PPD, s judged by in vivo skin testing nd in vitro lymphocyte trnsformtion (18, 23). However, little is known bout the mechnisms underlying the nergy in tuberculosis. In both humns nd mice (36), it hs been shown tht ntigen-specific unresponsiveness is ccompnied by filure of T cells to relese IL-2. Some * Corresponding uthor possible resons for tuberculin nergy include reltive loss of IL-2-producing cells, refrctoriness of IL-2-producing cells to the puttive stimultor (i.e., IL-1), nd unresponsiveness of effector T cells to IL-2 stimultion. In experimentl systems, T suppressor cells (12, 19, 21, 25) nd T-cell-derived suppressor fctors (17) hve been described. Previously, we reported n increse in the percentge of OKT8-positive (suppressor/cytotoxic) T cells in dvnced refrctory tuberculosis fter in vitro PPD stimultion (31). In the present study, ttempts were mde to chrcterize further the low PPD-induced PBL trnsformtion in dvnced tuberculosis. The results obtined show tht the low responsiveness to PPD ws mostly due to the impired IL-2 production. PPD-induced cquisition of IL-2 responsiveness ws, however, not impired. Therefore, the ddition of exogenous IL-2 substntilly restored the PPD-induced prolifertive responses in PBL from ptients with dvnced, refrctory tuberculosis, up to the high level of responsiveness seen in PBL from newly dignosed tuberculosis ptients. MATERIALS AND METHODS Ptients. A totl of 46 ptients with ctive pulmonry tuberculosis, ll of whom were inptients in our hospitl, were studied. These ptients were divided into two groups: 23 ptients (17 mles nd 6 femles, 1 to 73 yers old; men ge, 41 yers) with newly dignosed tuberculosis nd 23 ptients (18 mles nd 5 femles, 26 to 7 yers old; men ge, 51 yers) with dvnced, refrctory tuberculosis. Ptients clssified s hving newly dignosed tuberculosis were those in whom pulmonry tuberculosis ws dignosed for the first time by chest X ry nd by the demonstrtion of cid-fst bcilli in the sputum. Ptients considered to hve dvnced, refrctory tuberculosis were those who hd been hospitlized for severl yers without improvement. Cultures of sputum specimens from ptients with dvnced,

2 VOL. 55, 1987 ril-2 REVERSES PBL RESPONSE TO PPD 2127 Group TABLE 1. In vitro prolifertive response to PPD or lectin stimultion [3H]thymidine incorported (cpm, 13) + SD with: PPD ConA PHA PWM Pulinonry tuberculosis Advnced (n = 23) 2. ± ± ± ± 9.4 Newly dignosed (n = 23) b ± ± 7.3 Helthy controls (n = 32) 22.6 ± ± ± ± 9.9c PBL (16/ml) were cultured in vitro for 3 dys with ConA (1,ug/ml) or PHA (1,ug/ml) or for 6 dys with PPD (1,ug/ml) or PWM (2.5,ug/ml). b Significntly different from results with dvnced tuberculosis (P <.1) nd from results with helthy controls (P <.5). c Significntly different from results with dvnced tuberculosis (.1 < P <.2) nd from results with newly dignosed tuberculosis (P <.1). refrctory tuberculosis were continuously positive for cidfst bcilli resistnt to lmost ll ntituberculosis drugs. Most ptients with newly dignosed tuberculosis showed positive delyed-type skin rection (erythem, indurtion, nd in some cses, blister formtion) to the stndrd dose (5 TU) of tuberculin injection, wheres most of the ptients with dvnced, refrctory tuberculosis showed wek (erythem only) or no rection t ll to the stndrd dose of tuberculin. Helthy volunteers (16 mles nd 16 femles, 2 to 69 yers old; men ge, 37 yers), ll of whom were positive for the tuberculin skin test, served s control subjects. PBL preprtion. PBL were seprted from heprinized venous blood by the Ficoll-Hypque sedimenttion method. Antigen nd lectins. PPD ws kindly donted by the Institute for Microbil Diseses, Osk University, Jpn. Concnvlin A (ConA) ws purchsed from Miles-Yed Ltd., Rehovot, Isrel, phytohemgglutinin (PHA) ws obtined from Difco Lbortories, Detroit, Mich., nd pokeweed mitogen (PWM) ws obtined from GIBCO Lbortories, Grnd Islnd, N.Y. Preprtion of ril-2. Highly purified humn recombinnt IL-2 (ril-2), obtined from Escherichi coli crrying the cloned humn IL-2 gene, ws gift from Tked Chemicl Industries, Ltd., Osk, Jpn, nd ws used s source of exogenous IL-2 t finl concentrtion of 1 ng of protein per ml. This mteril yields only single bnd t 15, dltons by sodium dodecyl sulfte-polycrylmide gel electrophoresis (22). A 1-mg mount of this ril-2 contined 4 x 16 units in our ssy system s described below. Genertion of IL-2-contlining superntnts nd ssy for IL-2. For PPD-driven IL-2 production, PBL t 2 x 16/ml were ctivted with 1,ug of PPD or ConA per ml. After 24 h, the superntnts were collected, sterile filtered, nd ssyed for IL-2 ctivity. The IL-2 ctivity of culture superntnts ws determined on the bsis of incorportion of [3H]thymidine by the IL-2-dependent mnurine cytotoxic T- cell line NRB (22). NRB ws kindly provided by Eiichi Nkym of the Center for Adult Disese, Osk, Jpn. NRB ws mintined in the presence of rt spleenconditioned medium. Units of ctivity were defined s the reciprocl of the dilution necessry to obtin hlf-mxitnl incorportion of [3H]thymidine by the NRB cells. In the bsence of dded IL-2, the bckground incorportion ws only 2 to 4 cpm; pek incorportion (t 8 U of IL-2 per ml) ws pproximtely 4 x 14 cpm. PBL ctivtion with PPD nd nlysis for IL-2 receptors. PBL (16/tnl) were cultured in 24-well culture plte (no. 347; Becton Dickinson Lbwre, Oxnrd, Clif.) t 37 C nd 7.5% CO2 in ir for 6 dys with 1,ug of PPD per ml. The culture medium used ws RPMI 164 supplemented with 1% fetl clf serum. At the end of the culture period, the cells were hrvested, wshed twice, suspended in RPMI 164 medium contining 1% fetl clf serum, nd ssyed for IL-2 receptors. IL-2 receptors were determined by using monoclonl nti-tc ntibody (35), which ws kindly donted by T. Uchiym nd J. Yodoi, Kyoto University, Jpn. Cell suspension (1,ul) ws incubted with nti-tc ntibody (1/5, dilution in scites fluid) for 3 min t 4 C nd wshed, nd 1,ul of fluorescein-conjugted rbbit nti-mouse immunoglobulin G ntibody (1/2) (Miles-Yed) ws then dded. After further 3-min incubtion on ice, the cells were wshed nd suspended in 1 drop of fetl clf serum, nd the number of positive cells ws ssessed by using fluorescence microscope. Tc-positive cells were blstogenic nd belonged to E-rosetting T cells. In vitro ssy of prolifertive responses. The in vitro prolifertive responses were mesured by modifiction of the method reported previously (3, 33). In brief, 5 x 14 cells in.2 ml of RPMI 164 medium contining 1% het-inctivted pooled humn ser were plced in fltbottom microplte (no. 372; Becton Dickinson) with PPD or lectins nd were cultured for 3 dys with 1 jig of ConA or 1,ug of PHA per ml or for 6 dys with 1,g of PPD or 2.5,ug of PWM per ml in humidified 7.5% CO2 in ir t 37 C. [3H]thymidine (.2,uCi) ws dded to ech well 18 h before the cells were hrvested, nd the incorported rdioctivity ws counted in scintilltion counter (Tricrb; Pckrd Instrument Co., Inc., Rockville, Md.). Ech determintion ws performed in triplicte, nd the dt were expressed s couhts per minute ± the stndrd error of the men. To ssess the effects of exogenous IL-2 on ntigen-specific prolifertion, 2 RI of ril-2 (1 ng/ml) ws dded to the test culture t dy 3 of the culture period. Approprite control cultures were set up contining the sme dose of ril-2 but no ntigen. Results of dose-response experiments with ril-2 indicted tht dose of 1 ng of ril-2 per ml gve the highest enhncing effect on PPD culture, while giving lowest vlues in control cultures. Sttisticl nlysis. The significnce of the difference between groups ws clculted by Student's t test. Computerssisted evlutions of the results were used to clculte the P vlue in the dt. A P vlue of.5 ws used s the limit of sttisticl significnce. RESULTS Prolifertive responses to PPD nd lectins. PBL from ptients with pulmonry tuberculosis nd from tuberculin skin test-positive helthy donors were cultured with PPD or lectins (ConA, PHA, nd PWM), nd the prolifertive responses were evluted (Tble 1). There ws significnt difference between the two groups of tuberculous ptients in responsiveness of PBL to PPD. The extent of prolifertion of

3 2128 SHIRATSUCHI ET AL. TABLE 2. Group IL-2 ctivity in the superntnt of PPD- or ConA-stimulted cultures IL-2 units (no. of subjects) with: PPD ConA Pulmonry tuberculosis Advnced Newly dignosed 4.7 ± 1.6 (n = 11)b 15.2 ± 13.1 (n = 12) (n = 9) (n = 5) Helthy controls 9. ± 7.2 (n = 13) (n = 7) PBL (2 x 16/ml) were cultured for 24 h in the presence of PPD (1,ug/ml) or ConA (1,ug/ml). Culture superntnts were ssyed for IL-2 ctivity. For definition of units, see Mterils nd Methods. I.1 < P <.5 compred with results from newly dignosed tuberculosis. PBL from dvnced tuberculous ptients ws considerbly lower thn tht of PBL from ptients with newly dignosed tuberculosis (P <.1) but comprble with tht of PBL from helthy controls. PBL from ptients with newly dignosed tuberculosis showed higher prolifertion in response to PPD stimultion thn PBL from helthy controls (.1 < P <.5). In the responses to ConA or PHA, no significnt differences were seen mong the groups exmined. On the other hnd, the responses to PWM were significntly higher in PBL from tuberculous ptients thn in PBL from helthy controls (.1 < P <.2). IL-2 production. PBL (2 x 16) were stimulted with PPD or ConA, nd the culture superntnts were hrvested fter centrifugtion for 1 min t 2, rpm, membrne filtered (Millipore Corp., Bedford, Mss.), nd subjected to the IL-2 ssy s described bove. PPD-stimulted cultures of PBL from ptients with newly dignosed tuberculosis gve higher IL-2 ctivity thn did PPD-stimulted PBL from ptients with dvnced tuberculosis (.1 < P <.5), wheres no significnt differences were observed in IL-2 ctivity in ConA-stimulted culture superntnts mong the groups exmined (Tble 2). PPD-induced IL-2 receptor (Tc ntigen) expression. T-cell prolifertion is dependent on the interction of IL-2 with its cellulr receptor. We exmined the effect t PPD-induced IL-2 receptor expression on PBL fter 6 dys of in vitro culture. IL-2 receptor-bering lymphocytes were ssessed by using monoclonl nti-tc ntibody s described in Mterils nd Methods. The percentge, s well s the totl number, of Tc+ cells recovered in ech lymphocyte group ws significntly incresed fter in vitro stimultion with PPD (P <.1 compred with the control culture without PPD) (Tble 3). It is noteworthy tht fter PPD stimultion, the Tc+ cells were substntilly incresed in PBL frotn ptients with dvnced, refrctory tuberculosis, lthough the extent of the response ws not so lrge s tht of PBL from ptients with newly dignosed tuberculosis or helthy controls. These results suggest tht the low prolifertive response to PPD of PBL from the ptients with dvnced tuberculosis is due to the PPD-induced depressed IL-2 production nd not to the depressed expression of IL-2 receptors. The ddition of exogenous IL-2 ws, therefore, expected to restore the prolifertive response or to increse IL-2 receptor expression or both in PPD-treted cultures of PBL from such ptients. Effects of exogenous IL-2 (ril-2) on the prolifertive respotses to PPIl. Next, we exmined whether the defective responses of PBL from dvnced tuberculous ptients were INFECT. IMMUN. restored by the ddition of exogenous IL-2 (Fig. 1 nd 2). PBL were cultured in the presence of PPD, nd ril-2 ws dded t dy 3 of the culture period. ril-2 significntly incresed the PPD-induced prolifertion of PBL from dvnced tuberculous ptients (P <.1) (Fig. 1). The prolifertive responses of their PBL were restored by the ddition of ril-2 to the level of PPD-induced prolifertion of PBL from ptients with newly dignosed tuberculosis. The ugmenting effect of the ddition of ril-2 ws seen in PBL of ptients with dvnced, refrctory tuberculosis, wheres no significnt increse ws seen in responses to PPD of PBL from ptients with newly dignosed tuberculbsis or from helthy controls (Vig. 2). Effect of ril-2 on PPD-induced Tc expression. As described bove, ril-2 ugmented the PPD-induced prolifertion of PBL from ptients with dvnced, refrctory tuberculosis. We further investigted whether ril-2 lso incresed the PPD-induced IL-2 receptor (Tc) expression. Tests were performed on PBL from 7 dvnced tuberculosis ptients, 7 newly dignosed tuberculosis ptients, nd 11 helthy donors (Tble 4). The ddition of ril-2 to the PPDstimulted PBL culture cused significnt ugmenttion of the PPD-induced increse in Tc-positive cells in cultures of PBL from ptients with dvnced tuberculosis (36.1 ± 12.% versus 22.7 ± 12.5%, P <.1) nd in cultures from helthy controls (34.3 ± 11.4% versus %, P <.1), wheres no significnt ugmenttion by ril-2 ddition ws observed in cultures from ptients with newly dignosed tuberculosis ( % versus 35.3 ± 13.%,.2 < P). DISCUSSION The present study demonstrtes tht PBL from ptients with dvnced, refrctory tuberculosis hd defective PPDinduced in vitro prolifertive responses. The responses of these PBL to the nonspecific mitogens ConA, PHA, nd PWM, however, were comprble to those of ptients with newly dignosed tuberculosis or those of helthy donors. PPD-induced T-cell prolifertion consists of two prts: IL-2 production nd IL-2 receptor expression by sensitized T cells. In ptients with dvnced, refrctory tuberculosis, PPD-induced IL-2 production, but not IL-2 responsiveness, ws impired s compred with production nd responsiveness in PBL from newly dignosed tuberculosis ptients or helthy individuls, lthough the PPD-induced increse of Tc-positive cells ws lso not s high in dvnced, refrctory tuberculosis ptients. The ddition of ril-2 to the culture, however, restored the impired PPD-induced prolifertive responses of PBL from dvnced, refrctory tuberculosis ptients by the stimultion with PPD. Vismr et l. (36) reported tht the prolifertive unresponsiveness observed in humn PBL to ntigenic extrct from Cndid lbicns or to PPD of the spleen cells of mice infected with high dose of Mycobcterium bovis BCG ws due to primry lck of IL-2 production. In the present study, Tc ntigen-positive, IL-2 receptor-bering T cells which incresed fter in vitro PPD stimultion were further incresed in PBL of dvnced tuberculosis ptients by the ddition of exogenous IL-2. In ntigen-specific T-cell ctivtion, the IL-2 tht ws produced up regulted its own receptors on ctivted T cells nd triggered the prolifertion of T cells (27, 37). Thus, in dvnced, refrctory tuberculosis n impired IL-2 production gve n impired IL-2 expression nd led to n impired prolifertion of PPD-rective T cells. One plusible explntion for the impired PPD-induced prolifertion of PBL in dvnced, refrctory tuberculosis is

4 VOL. 55, 1987 ril-2 REVERSES PBL RESPONSE TO PPD 2129 TABLE 3. Increse in Tc-positive cells upon PPD stimultion No. of Tc-positive cells + SD with or without PPD Group % of totl No. of Tc-positive cells (1-4)/culture No PPD PPD No PPD PPD Pulmonry tuberculosis Advnced (n = 23) 4.7 ± b 1.7 ± ± 9.gb Newly dignosed (n = 23) 5.3 ± ± ± ± 2.8 Helthy controls (n = 32) 5.1 ± ± ± ± 16.5 PBL (16/ml) were cultured in vitro in the presence or bsence of PPD for 6 dys. Tc-positive lymphocytes were ssessed by the indirect-fluorescence technique. b.1 < P <.5 compred with helthy controls. tht suppressor cells might be generted nd inhibit PPDinduced IL-2 production. We reported previously (34) tht in dvnced, refrctory tuberculosis, immunoglobulin G Fc receptor-bering T cells or OKT8-positive T cells (31) were incresed in vitro fter stimultion of PBL with PPD. The Fc receptor-bering T cells tht were isolted suppressed the PPD-induced prolifertive response of utologous PBL. In dvnced tuberculosis, PBL depleted of OKT8-positive cells gve higher prolifertive response to stimultion with PPD thn unfrctionted PBL (31) did. In experimentl systems, mice injected intrvenously with high dose of BCG filed to develop delyed-type hypersensitivity to BCG nd were described s nergic (6). Colizzi et l. (5) demonstrted tht spleen cells from these nergic mice produce inhibitory fctors which block the IL-2 production. This stte of unresponsiveness cn be reversed both in vitro (14) nd in vivo (4) by the dministrtion of n IL-2-contining preprtion. Together with these observtions, our results suggest tht in dvnced, refrctory tuberculosis, the prolifertive T-cell defect in response to PPD stimultion is not due to the bsence of or generlized lck of triggering of immune T cells but my be relted to deficiency in events leding to IL-2 production. Addition of ril-2 lso ugmented the PPD-induced Tc-ntigen-positive (IL-2 receptor-bering) T cells in PBL from dvnced, refrctory tuberculosis ptients. Recently, Toossi et l. (32) described similr in vitro nlysis of lymphocytes of ptients with pulmonry tuberculosis. They reported tht ptients with newly dignosed pulmonry tuberculosis hve tuberculin-specific defect in IL-2 production. However, purified IL-2 fils to correct Downloded from 5 E 4 C.3-3. c 2 I- I 1 I~~~~I I Ē 1* S Q U. I- z C' on June 29, 218 by guest PPD PPD+IL 2 Advnced Tuberculosis PPD Newly dignosed Tuberculosis FIG. 1. Effect of exogenous IL-2 ddition on PPD-induced in vitro prolifertion of PBL from ptients with dvnced tuberculosis. PBL (5 x 14 per well) were cultured in vitro in the presence of PPD (1 p.g/ml) for 6 dys. ril-2 (1 ng/ml) ws dded to the culture on dy 3. The results re expressed s counts per minute of [3H]thymidine incorported during the lst 18 h of the culture period. The points connected by line represent dt from the sme donor. IL _ + + PPD Advnced Newly dignosed Helthy Tuberculosis Tuberculosis controls (n-23) (n-23) (n-32) FIG. 2. Summrized dt of the effects of exogenous ril-2 ddition on PPD-induced PBL prolifertion. The results re expressed s men counts per minute of [3H]thymidine incorportion ± the stndrd devition (br).

5 213 SHIRATSUCHI ET AL. INFECT. IMMUN. TABLE 4. Effects of ril-2 ddition on PPD-induced increse of Tc-positive cells % Tc-positive cells with or without PPD nd ril-2 Group nd cse no. No PPD PPD No ril-2 ril-2 % Increvse No ril-2 ril-2 % Increse Pulmonry tuberculosis Advnced Men 3.5 ± ± ± ± ± ± 4. Newly dignosed Men 4.7 ± ± ± ± ± ± 7. Helthy controls (n = 11) 4.1 ± ± ± ± ± ± 6.5 PBL (16/ml) were cultured in vitro for 6 dys in the presence or bsence of PPD nd ril-2 (1 ng/ml). Tc ntigen-positive cells were ssessed by the indirect-immunofluorescence method. PPD-induced blstogenesis in these ptients. In Jpn, most ptients with newly dignosed tuberculosis revel positive tuberculin skin rections nd their PBL re highly responsive in vitro to PPD stimultion, s hs been shown, with few exceptions, for the ptients in the present study. In our study, PBL from ptients with newly dignosed tuberculosis showed the highest response to PPD stimultion (Tble 1). In contrst, PBL from ptients with dvnced, refrctory tuberculosis with disese history of greter thn 3 yers showed significntly lower PPD responses thn PBL from newly dignosed tuberculous ptients did, lthough sputum specimens from the former group were continuously positive for cid-fst bcilli resistnt to ntituberculosis drugs. In ddition, in the present study, Tc ntigen-positive, IL-2 receptor-bering cells did pper in vitro fter stimultion with PPD of PBL from dvnced, refrctory tuberculosis ptients; the cells were further incresed upon introduction of ril-2 (Tbles 3 nd 4). Our results indicte tht even in PBL from ptients with dvnced, refrctory tuberculosis, PPD-sensitized T cells did exist nd becme responsive to exogenous IL-2 fter PPD stimultion. A lck of IL-2 production hs lso been reported to be responsible for T-cell unresponsiveness in severl other diseses. T-cell unresponsiveness in Hodgkin's disese (1) nd rheumtoid rthritis (8) is reversed in vitro by the ddition of IL-2. Clinicl trils using IL-2 dministrtion in vivo hve been conducted in children with Nezelof's syndrome (9) nd in cncer ptients (29). Preliminry results of these trils hve suggested tht IL-2 is ble to produce high degree of reconstitution of T-cell ctivity. In the present study, the restortion by ril-2 of depressed PPD-induced T-cell prolifertion in dvnced tuberculosis ws not complete (Fig. 2). Monocyte-medited suppressor bnormlities hve been reported in Hodgkin's disese (11) nd diffuse cutneous leishmnisis (26). In these diseses, the ddition of indomethcin, n inhibitor of prostglndin synthesis, to cultures restores the lymphocyte responses. In ddition to the deficiency of IL-2 production, prostglndin-medited suppression of IL-2 receptor expression might be prtly responsible for the impired prolifertion of PBL from ptients with dvnced tuberculosis in the present study. To dte, we hve no fesible mens of controlling frdvnced, drug-resistnt tuberculosis. From number of studies, evidence hs ccumulted tht immunity to intrcellulr prsites such s Mycobcterium tuberculosis or M. lepre is effected by T cells. This immunity is medited by the relese of lymphokines which my ctivte mcrophges to kill prsites. Although we re not t ll certin tht the in vitro studies presented here prllel the in vivo regultion of cellulr immunity in dvnced, refrctory tuberculosis, future studies of the effect of IL-2 dministrtion in vivo on cellulr immunity in these ptients pper wrrnted. If the effects in vitro presented here cn be reproduced in vivo, in vivo dministrtion of IL-2 might be beneficil for ptients with chronic refrctory infections. ACKNOWLEDGMENTS We thnk S. Kishimoto, Osk University, Jpn, for his vluble dvice throughout this study nd H. Tkhshi, the president of our hospitl, for his criticl review of the mnuscript. We lso thnk T. Uchiym nd J. Yodoi, Kyoto University, Jpn, for supplying us with the monoclonl ntibody nti-tc; E. Nkym, Center for Adult Diseses, Osk, Jpn, for supplying us with the murine cytotoxic T-cell line NRB; nd Centrl Reserch Division, Tked Chemicl Industries, Ltd., Osk, Jpn, for supplying us with ril-2.

6 VOL. 55, 1987 LITERATURE CITED 1. Bhtngr, R., A. N. Mlviy, S. Nrynn, P. Rjgopln, R. Kumr, nd. P. Bhrdwj Spectrum of immune response bnormlities in different clinicl forms of tuberculosis. Am. Rev. Respir. Dis. 115: Cntrell, D. A., nd K. A. Smith Trnsient expression of interleukin 2 receptor consequence for T cell growth. J. Exp. Med. 158: Cntrell, D. A., nd K. A. Smith The interleukin 2 T cell system: new cell growth model. Science 224: Colizzi, V In vivo nd in vitro dministrtion of interleukin 2-contining preprtion reverses T-cell unresponsiveness in Mycobcterium bovis BCG-infected mice. Infect. Immun. 45: Colizzi, V., J. Ferlug, F. Grreu, M. Mlkousky, nd G. L. Asherson Suppressor cells induced by BCG relese nonspecific fctors in vitro which inhibit DNA synthesis nd interleukin-2 production. Immunology 51: Collins, F. M., nd S. R. Wtson Suppressor T-cells in BCG-infected mice. Infect. Immun. 25: Cotner, T., J. M. Willims, L. Christenson, H. M. Shpiro, T. B. Strom, nd J. S. Stomenger Simultneous flow cytometric nlysis of humn T cell ctivtion ntigen expression nd DNA content. J. Exp. Med. 157: Emery, P., G. S. Pnyi, nd A. M. E. Nouri Interleukin-2 reverses deficient cell-medited immune responses in rheumtoid rthritis. Clin. Exp. Immunol. 57: Flomenberg, N., K. Welte, R. Mertelsmnn, N. Kernn, N. Ciobnu, S. Venut, S. Feldmn, G. Kruger, D. Kirkptrick, B. Dupont, nd R. O'Reilly Immunologic effects of interleukin 2 in primry immunodeficiency. J. Immunol. 13: Ford, R. J., J. Tso, N. M. Kouttb, C. G. Shsrbuddhe, nd S. R. Meht Assocition of n interleukin bnormlity with the T cell defect in Hodgkin's disese. Blood 64: Goodwin, J. S., R. P. Messner, A. D. Bnkhurst, G. T. Peke, J. H. Siki, nd R. C. Willims Prostglndin-producing suppressor cells in Hodgkin's disese. N. Engl. J. Med. 297: Gullberg, M., nd E. L. Lrsson Studies on induction nd effector functions of concnvlin A-induced suppressor cells tht limit TCGF production. J. Immunol. 128: Hregewoin, A., T. Godl, A. S. Mustf, A. Belehu, nd T. Yemneberhn T-cell conditioned medi reverse T-cell unresponsiveness in lepromtous leprosy. Nture (London) 33: Hoffenbch, A., P. H. Lgrnge, nd M.-A. Bch Deficit of interleukin 2 production ssocited with impired T-cell prolifertive responses in Mycobcterium lepremurium infection. Infect. Immun. 39: Horwitz, M. A., W. R. Levis, nd Z. A. Cohn Defective production of monocyte-ctivting cytokines in lepromtous leprosy. J. Exp. Med. 159: Kpln, G., D. E. Weinstein, R. M. Steinmn, W. R. Levis, U. Elvers, M. E. Potrroyo, nd Z. A. Cohn An nlysis of in vitro T cell responsiveness in lepromtous leprosy. J. Exp. Med. 162: Krmer, M., nd U. Koszinowski T cell-specific suppressor fctor(s) with regultory influence on interleukin 2 production nd function. J. Immunol. 128: McMurry, D. N., nd A. Echeverri Cell-medited immunity in nergic ptients with pulmonry tuberculosis. Am. Rev. Respir. Dis. 118: Mehr, V., J. Convit, A. Rubinstein, nd B. R. Bloom Activted suppressor T cells in leprosy. J. Immunol. 129: Mohgheghpour, N., R. H. Gelber, J. W. Lrrick, D. T. Sski, P. J. Brennn, nd E. G. Englemn Defective cellmedited immunity in leprosy: filure of T cells from leprom- ril-2 REVERSES PBL RESPONSE TO PPD 2131 tous leprosy ptients to respond to Microbcterium lepre is not reconstituted by interleukin 2. J. Immunol. 135: Mustf, A. S., nd T. Godl BCG-induced suppressor T cells optiml conditions for in vitro induction nd mode of ction. Clin. Exp. Immunol. 62: Nruo, K., S. Hinum, K. Kto, M. Koym, H. Td,. Shiho, nd K. Tsukmoto Comprison of the biologicl properties of purified nturl nd recombinnt humn interleukin-2. Biochem. Biophys. Res. Commun. 128: Nsh, D. R., nd J. E. Douglss Anergy in ctive tuberculosis: comprison between positive nd negtive rectors nd n evlution of 5 TU nd 25 TU skin test doses. Chest 77: Nth, I., M. Sthish, T. Jyrmn, L. K. Bhutni, nd A. K. Shrm Evidence for the presence of M. lepre rective T lymphocytes in ptients with lepromtous leprosy. Clin. Exp. Immunol. 58: Oht, N., M. Mini, nd T. Sszuki Antigen specific suppressor T lymphocytes (Leu2+ 3-) in humn schistosomisis jponic. J. Immunol. 131: Petersen, E. A., F. A. Nev, C. N. Oster, nd H. B. Diz Specific inhibition of lymphocyte-prolifertion response by dherent suppressor cells in diffuse cutneous leishmnisis. N. Engl. J. Med. 36: Reen, G. H., nd N. N. Yeh Interleukin 2 regultes expression of its receptor nd synthesis of gmm interferon by humn T lymphocytes. Science 224: Ridley, D. S., nd W. H. Jopling Clssifiction of leprosy ccording to immunity: five group system. Int. J. Lepr. 34: Rosenberg, S. A., M. T. Lotze, L. D. Muul, S. Leitmn, A. E. Chng, S. E. Ettinghusen, Y. L. Mtory, J. M. Skibber, E. Shiloni, J. T. Vetto, C. A. Seipp, C. Simpson, nd C. M. Reichert Observtions on the systemic dministrtion of utologous lymphokine-ctivted killer cells nd recombinnt interleukin-2 to ptients with metsttic cncer. N. Engl. J. Med. 313: Shirtsuchi, H., nd I. Tsuyuguchi Tuberculin purified protein derivtive-rective T cells in cord blood lymphocytes. Infect. Immun. 33: Shirtsuchi, H., nd I. Tsuyuguchi Anlysis of T cell subsets by monoclonl ntibodies in ptients with tuberculosis fter in vitro stimultion with purified protein derivtive of tuberculin. Clin. Exp. Immunol. 57: Toossi, Z., M. E. Kleinhenz, nd J. J. Elner Defective interleukin 2 production nd responsiveness in humn pulmonry tuberculosis. J. Exp. Med. 163: Tsuyuguchi, I., H. Shirtsuchi, H. Fujiwr, nd. Terok Nonspecific recruitment of lymphocytes in purified protein derivtive-induced lymphocyte prolifertive response of ptients with tuberculosis. Infect. Immun. 37: Tsuyuguchi, I., H. Shirtsuchi,. Terok, nd T. Hirno Increse in T cells bering IgG Fc receptors in peripherl blood of ptients with tuberculosis by in vitro stimultion with purified protein derivtive. Am. Rev. Respir. Dis. 121: Uchiym, T., S. Broder, nd T. A. Wldmnn A monoclonl ntibody (nti-tc) rective with ctivted nd functionlly mture humn T cells. 1. Production of nti-tc monoclonl ntibody nd distribution of Tc(+) cells. J. Immunol. 126: Vismr, D., G. Lombrdi, E. Piccolell, nd V. Colizzi Dissocition between interleukin-1 nd interleukin-2 production in prolifertive response to microbil ntigens: restortive effect of exogenous interleukin-2. Infect. Immun. 49: Welte, K., M. Andreeff, E. Pltzer, K. Holowy, B. Y. Rubin, M. A. S. Moore, nd R. Mertelmnn Interleukin 2 regultes the expression of Tc ntigen on peripherl blood T lymphocytes. J. Exp. Med. 16: