ADONIS 030094759 !00024B
Scand. J. Immunol. 33, 203-209. 1991
Flow Cytometric Analysis of CD2 Modulation on Human Peripheral Blood T Lymphocytes by Dharmendra Preparation of Mycobacterium leprae R. SHEELA, S. I L A N G U M A R A N & V. R. M U T H U K K A R U P P A N Department of immunology. School of Biological Sciences. Madurai Kamaraj University, Madtirai. India
Sheela. R., Ilangumaran. S. & Muthukkaruppan, V.R, KIow Cytometric Analysis or CD2 Modulation on Human Peripheral Blood T Lymphocytes by Dharmendra preparation of Mycobacterium teprae. Scand. J. Immunol. 33, 20.1-209, 1991 It has been reported previously that Mymhaclerium leprue modulated CD2 on human peripheral blood T lymphocytes and that this modulation was accompanied by a marked reduction in the proliferauve response of these cells to mitogens and antigens. In this study, we report that treatment of peripheral blood mononuclear cells from healthy individuals with Dharmedra preparation of M. leprue inhibited their ability to form rosettes with sheep red blood ceils. Flow cytomotric analysis of Dharmendra lepromin-lrcated eells showed that, in addition to CD2. CD4 and CD8 were modulated while the surface expression of CD.l was not alTecied. The specifieiiy of CD2 modulation was confirmed by similar effects of Dharmedra lepromin on ihymocytes and lymph node cells from human CD2 transgenic mice. The modulatory elTeci of Dharmendra lepromin was not observed at lower temperatures. Dharmendra lepromin treatment of activated T cells resulted in reduced binding of monoclonal antibodies to IL-2Rand Dfifi epitope of CD2. The modulalory effects were not observed with Dharmendra preparation of BCG or other preparations of M. leprae. Our results indicate that certain M. leprae factor(s) specifically modulate{s) CD2, CD4, CDS and IL-2R but not CD3 on T lymphocytes. The suppressive elTeet of Dharmendra lepromin on the T-cell proliferative response reporled earlier may be explained by its modulatory effect on a number of T-cell surface molecules. Profcs.\or V. R. .Mi/ihukktirtippan, Department nJ Immunology. School of Biological Seierices, Madurai Kamaraj University. Madurai 625 021, India
Leprosy is a c!ironic infectious disease catiscd by Mycohacierium leprae. The clinical spcctrutn of the disease correlates we!t with the immune statu.s o'f the patients. The lepromatous form of the disease is associated with impaired cell-mediated immunity (CMI) and dissctninaied infection with M. leprae [4]. Earlier studies had shown that the proportion of cells forming rosettes with sheep red blood cell.s (SRBC) was reduced in the peripheral blood of lepromatous leprosy patients [16]. Previously, we have shown by immunotiuorcsccnce microscopy that treatment of peripheral blood fnononuclear cells from healthy individuals with Dharmendra preparation of M. teprae significantly modulated the expression of CD2 [14]. This modulation was accompanied by marked suppression of the T-cell proliferative response to mitogens and antigens [12, 14, 15],
In this study, we investigated the effect of Dhannendra lepromin treatment on a number of CD molecules on peripheral blood mononuclear cells from healthy individuals by (low cytometric analysis. We present evidence that, in addition to CD2. the expression of CD4, CDS and 1L-2R were reduced, while that of CD3 was not affected. Further, these effects were brought about specifically by Dharmendra lepromin, but not by Dharmendra preparation of Bacillus Calmette-Guerin (BCG) or by other preparations of M. teprae.
MATERIALS AND METHODS Chemicals. Metrizoate sodium. Hank's balanced salt solution (HBSS). powdered culture medium RPMI 1640. penicillin-streptomycin mixture, propidium iodide, Tween-80, 2-aminoethvl isothiouronium bro203
204
R. Sheela. S. Ilangumanui & V. R. Muthukkaruppan
mide(AET).cnnc;tn;iViilin A (Con A)iiiid sndiuin a^ide were purchased from Sigma Chemical Co.. USA. Ficoll 400 was obtained from Pharmacia, Sweden, Mycnhavterial pn-iMniiions. Dharmcndra prcparalion of .HyctihcicWriiini h-pnic (Dhitrnicndra lepromin) was eilher ohiained from Dr U. Scngtipia (Central JALMA [nstitulcfor Leprosy, Agra, India for prepared rrom armadillo-derived, freett-driod A/, Icpnic obtained from WHO-IMMLF.P St. U-pnw Bank through Dr R, J. W. Rees (National Institute for Medical Research. Mill Hill. London. UK). BCG, Danish strain 1331, was kindly provided by The Director, BCG Vaccine laboratories, Madras. India. Dharmendra lepromin was prepared from freezcdried M. leprav following the standard procedure J5|. Briefly, the bacteria were suspended in ehloroforni by grinding in a glass mortar. The chloroform was then allowed lo evaporate complelely in a hot water bath. The residual substance was suspended in solvent elher and cenlrifuged at 5900 j,' for I h al 4 C. The peilet was resuspended in ether and centrifuged again. The linal pellet was dried under vacuum for 4S h al room temperature. The dried malerJal was ground in 2 ml phosphate-bulTcrcd saline (PBS) containing 0.05"AI Twccn SO to suspend ihe bacilli. After counting by acidfast staining the bacterial count was adjusted to I x lO" bacilli per ml in PBS and autoelaved. Aliquots were stored frozen al - 2 0 C until use. BCG. subjected to Dharmendra preparation, was used as a eontrot for Dharmendra lepromin, Mitsuda lepromin and soluble M. k-pnu- antigen (leprosin A. batch CDI22) were obtained from Dr R. J. W. Rees. Live M. leprav and delipidified cell wall preparation of M. leprae (DCW) were generous gifls from Dr P. R. Mahadevart (Foundation for Medical Research. Bombay. India). MomiclotUil Anlihiitlici l.Mti.'ihI. MoAb Leu 4 (anti-CD3), Leu 5b (anti-CD:). Leu 2a (anti-CD8) and anti-lL-2R (anti-CD25) conjugated to fluoreseetn isoIhiocyanate (FITC) or phycoerythrin (PE) and FITCeonjugated goal anti-mouse antibody (GAM) were purchased from Bccton Dickinson (Mountain View, Calif, USA), Rhodamine-conjugated anli-T4 (antiCD4) was obtained from Coulter Electronics, UK, MoAh D66 directed against a conformational epitope on CD2 [1] was a kind gift from Dr A. Bernard. Paris, France. Mice. Human CD2 (hCD2) transgenic mice (CBAxC57 BL/IO) carrying a single eopy of hCD: gene [11] were a kind gift from Dr M. ,1. Crumpton through the Imperial Caneer Researeh Fund, UK. Balb. c mice served as controls. Four- to six-wcek-oid mice were used I'or experiments. Prcfiiiratlon «/ ('f'//,v. Peripheral blood mononuelear eells (PBMC) were isolated from hepariiiized venous blood obtained from healthy normal volunteers over Fieoll Metrizoate gradient [10], After washing thrice in HBSS. cells were suspended in RPMI 1640 supplemented with penicillin (100 U,ml) and streptomycin (100 ;ig,,m!) containing O.!".. bovine serum albumin (BSA). Con A blasts were generated by stimulating PBMC with 10 fi^iml Con A for 24 h in mediutii containing 10''^ FCS. Single-cell suspensions were prepared from thymus and lymph nodes of mice. Treatment of veils with DIuirmendra lepromin. One
million cells were incubated with 20 //i or indicated concentrations of Dharmendra iepromin, BCG or other A/./c/jmc preparations in lOO/ilofRPMI 1640. supplemented with penicillin (MM) U ml), streptomycin (100 //g/mDandO-l':,. BSA for !2hat.l7 Clunless otherwise indicated). At the end of the incubation period, treated cells were washed three times in PBS before they were subjected to ro.setting with SRBC or immunofluore.seence staining. Cell viability as assessed by How cytometric analysis afler propidium iodide staining was more than W\, up to 72 h after Dharmendra lepromin treatment. E-rosetiing assays. Sheep erythroeytes were treated wiih 0.14 M 2-aminoeth\l isothiouronium bromide (AFT) [IS] and resuspended as a !"i. suspension in RPMI 1640 eontaining 10".. FCS. One million PBMC in a lOU tA volume were mixed with an equal \olume of AET-treated I'!.. SRBC in a test lube and cenlrifuged at 2(H) ,e for 1 min at room temperature. The cells were incubated at .'17 C for 30 min and then at 4 C overnight. The pellet was gently tapped and rosettes were counted in a haemocytometer under phase-contrast microscope. Two-hundred lymphocytes were seanned and the n umber of rosetting and non-rosetting lymphocytes was counted. Any cell binding to four or more SRBC was classified as a rosette. Imnninofittiire.scvnie siuinin^ and flow cviomerruunttly.-ii.^ IFCMI. Cells were incubated with appropriate MoAb diluted in PBS containing 0.1".. BSA and 0,1% sodium azide at 4 C for 30 min. Unconjiigated MoAb D66 was followed by the secondary antibody reagent GAM-FITC for 30 min at 4 C. Labelled cells were washed twice in PBS eontaining U.T'r. sodium azide and subjected to flow cytometric analysis in FACScan (Beeton Dickinson) using ihe Consorl 30 software program with log amplification gains for fluorescence. Fach experiment was repeated three times and one set of representative data is presented.
RESULTS Inhibition of E-roselting by Dharmendra lepromin The ability of normal human PBMC to form roselles with AET-irealed SRBC was studied after treatment with Dharmendra lepromin for 12 h at 37 C. A concentration dependent redtiction was observed in the proportion of rosette forming eeils (Table I). This effect ol" Dharmendra lepromin acted directly on E-rosctte-forming cells, since pretreatment of SRBC with Dharmendra lepromin did not affect their ability to form rosettes with T cells. Further, the inhibition of E rosetting was specific to M. leprae, as no such effect was observed after treatment with Dharmendra preparation of BCG, Modulation of CD2 by Dharmendra lepromin Since it has been shown that CD2 is the T-cell
C'D2 Modulation hy M. leprae TABLE I, Reduction in E-rosette forming ability of peripheral blood monontjciear cells after treatment with Dharmendra lepromin Volume added (Ml)
Treatment
% E-rosetting cells
None
74 + 4
Dharmendra lepromin 2+2 12 + 5 44 + 5 74 + 5 7.3+4
20
to
5. 2.5 1.25
Dharmendra BCG 20
72 + 6
One million PBMC treated wilh Ihe indicated volutne of Dharmendra lepromin or Dhiirniendra preparation ofBCGd y 10^hacilli/ml)forl2hat37 Cwerewashed and an E-rosetting assay was performed with AETtreated SRBC as described in Materials and Methods. Two-hundred eells were scanned and any cell with more than four SRBC hound was eounted as rosette positive. The results are meiin + SE ai three experitiienls.
• HAKKLNOliA
205
surface molecule involved in rosetting with SRBC. the accessibility orCD2 as well as other CD molecules on the T-cell surface to the corresponding MoAb was studied after trcatnietit of PBMC with Dharmendra lepromin. It is evident from Fig. I that MoAb binding to CD2 was complelely abrogated by treatment with Dharmendra lepromin but not with Dharmendra preparation of BCG. In addition, binding of antiCD4 was lost cotnpletely. while that of anti-CD8 was partially reduced. However. Dharmendra lepromin treatment did not inhibit the binding of anti-CD3 MoAb. indicating that the modulation of CD2, CD4 and CD8 by Dharmendra lepromin, but nol by Dharmendra preparation of BCG, is specific and is not due to a generahzed effect of mycobacteria on lymphocyte cell-surface molecules.
Modulation of human CD2 molecules on cells from hCD2 transgenic mouse Thai the CD2 modulation by Dharmendra lepromin is specific, was confirmed using hCD2 transgenic mouse. Dharmendra lepromin treatment of thymocytes and lymph node cells from these mice showed a marked reduction in the surface expression orCD2 (Fig. 2).
DHAKMENURA
LEPROHIN
BCG
Kincrics of CD2 modulation hv Dharmendra k'promin
I;.
Modulation of CD2 was found to be dependent on the concentration of Dharmendra lepromin used as well as the duration of incubation. The minimal concentration of Dharmendra lepromin THYMOCYTES
LOG FLUORESCENCE
INTENSITY
FIG. I. Effect of Dharmendra lepromin treatment on CD2. CD3. CD4 and CD8 on PBMC. 1 x 10" PBMC were treated with 20 ^I (2 x 10' bacilli) of Dharmendra lepromin or Dharmendra preparation of BCG for 12 h at 37 Cin 100/d of RPMI 1640 contaming 0.1% BSA. Treated cells were analysed for the surface expression of the indicated CD molecules.
LYMPH
NODE
CELLS
LOG FLUORESCENCE INTENSITY FIG. 2. Modulation of CD2 on eells from hCD2 transgenic mice. Thymocytes and lymphnode eells from hCD2 transgenic mice were subjected to Dharmendra lepromin treatment as described in Fig. I and analysed for the expression of hCD2. ( } Background fluoreseenee; ( ) untreated cells; ( ) Dharmendra lepromin treated cells.
206
/?. Sheela. S. Ilangumaran & V. R. Muthukkaruppan B THtflTED
1 h
5
3h
s: zi
6 h
9 h
?4 h
A A A
.UNTREATED 1 CONTROL 20 pi
i
•I-k Oh
A ^
i
37"
if
.•UNTREATED CONTROL
I
LOG FLUORESCENCE INrENSlTY
FIG. 4. Effecl of Dharmendra lepromin on aclivaied T cells. 24 h Con A blasts were incubaled with Dharmendra lepromin for 12 hal 37 C. Treated cells and controls were analysed for the expression of CD2, D66 epitope of CD2 and IL-2R. ( ) Background fluorescence: ( ) unactivaled. untreated cells; ( ) activated. untreated cells; ( ) activated, treated eells.
C /I ''CONTROL U'c
LOG FLUDHESCENCE INTENSITY
FIG. 3. Kinelies of CD2 modulation. 1 x 10" PBMC were treated wilh (A) indicated concent rai ions of Dharmendra lepromin for 12 h al 37 C; (B) 20 ^1 of Dharmendra lepromin at 37 C for indicated time intervals; (C) 20 n\ of Dharmendra lepromin at 37 C for 12 h. washed ihree times and ineubated in RPMI 1640 eontaining 10'^., FCS for 72 h: and (D) 20 /il of Dharmendra lepromin for 12 h :it 37 C or 4 C. and analysed for the expression orCD2 by Row cytometry. In all experiments. Dharmendra iepromin treatment wasdonein lOO/ilorRPMI I640containing0.l'I'l, BSA. (
k
) Indicates background fluorescence in A, C and
D,
required to cause maximal CD2 modulation after 12 h was 20 /d (2 x 10^ bacilli) in 1(K) /A final volume (Fig. 3A). At this concentration of Dharmendra lepromin. maximal reduction in CD2 expression was observed after incubation for al least 9 h (Fig. .IB). When ecIls treated with Dharmendra lepromin were washed and incubaled in medium containing IO"n FCS. the reeovery of CD2 expression was observed by 72 h (Fig. 3C). Modulation of CD2 by Dharmendra lepromin was influenced by ihe temperature of incubation. When PBMC were treated with Dharmendra lepromin at 4 C. instead of at 37 C, levels of CD2 expression were not affected signitieantly. even after 24 h of incubation (Fig. 3D). Effect of Dharmendra tcpnmiin on actirateil T cells To determine whether CD2 modulation by Dhar-
mcndra lepromin was influenced by the activation state o[" T cells, 24-h Con A blasts were thoroughly washed and treated with Dharmendra lepromin. In addition to the modulation of CD2. MoAb binding lo IL-2R was inhibited on T lymphoblasls(Fig. 4). The binding of MoAb D66 to a conformational epilope on CD2. whieh is expressed more after activation, was also reduced on Con A blasts after treatment with Dharmendra lepromin. indicating ihat Ihe whole CD2 molecule was modulated (Fig. 4). Treatment with Dharmendra lepromin also inhibited the binding of anti-CD2 and [D66 MoAb on Jurkat cells indicaling thai the modulatory effect of Dharmendra lepromin acted directly on T cells (F"ig. 5). Specificity oJ CD2 modulation bv Dharmendra preparation of M. leprae Different preparations of M. teprae. live M. k'prae. Mitsuda iepromin, soluble A/, leprae
LOG FLUOBFSCENCE INTENSITY
FIG, 5-ModulalionofCD2onJurkal Cells. Jurkat cells were treated with Dhurmendra lepromin for 12 h at 37 Cin RPMI 164()a)ntainingO. I".. BSA. Treated cells were labelled with MoAb to CD2 itnd D66 epitope of CD2. ( ) Background fluorescence: ( ) untreated cells: ( — ) Dharmendra iepromin-lreated cells.
HBER
CD2 Modulation hy M. leprae
Z -J UI
o
.A
\ D
A
E
A
\ /I
F
A
LOG FLUORESCENCE INTENSITY FIG. 6. Effect ofdifrercnt pa-piiru lions of ,V/. /c/j/t/c on CD2. I X 10'' PBMC in ;i I(K) //I voliinic were ireitled with {A), none (B) 20 /il Dharmendra lepromin. (C) 20 ft] {2x \0^ biidlli) MilKLida lepromin. (D) 2 x 10' live M. leprae. (E) 40 /jg.ml leprosin A. or (F) 40 /ig-ml DCW for 24 h al 37 C and analysetJ Tor CD2 e.\pression.
aniigcn (leprosin A) and delipidified cell wall (DCW) preparation were lesled for their ability lo modulate CD2. Il is evident from Fig. 6 thai even aRer 24-h treatment, other M. leprae preparations tested were not able to modulale CD2, indiealing ihat certain M. /f/jrc/c-specific factors, exposed and/or retained especially in Dharmendra preparation, is responsible for the observed CD2 modulation in normal PBMC.
DISCUSSION Earlier studies by Huoreseent microscopy have shown thai treatment of normal human PBMC with Dharmendra preparation of M. tcprac results in the modulation of CD2 on the T-ccll surface [14]. In this report, we confirm that Dharmendra lepromin indeed modulates CD2. by E-rosetting assays and flow cylometric analysis. Treatment of normal human PBMC with Dharmendra lepromin resulted in a marked reduction in the proportion of E-rosette-forming cells (Table 1). It has been well established that CD2 is the molecule involved in the E rosetting of T cells with SRBC [9]. Flow cytometric analysis of treated cells showed that CD2 was modulated significantly; in addition. MoAb binding to CD4 and CD8 were reduced (Fig. i). This modulation was nol due to a generalized effect of Dharmendra lepromin on T cell surface, since ihe expression of CD3 was not affected. Additionally.
207
no modulalory effect was observed with Dharmendra preparation oi' BCG (Table I; Fig. I). indicaling that the factor is M. leprae specific. Further. Dharmendra lepromin modulated CD2 on thymocytes and lymph node eells from hCD2 triinsgenic mice. Kinetic studies showed gradual regeneration of CD2 after modulation by Dharmendra lepromin. Similar regeneration kinetics was reported for CD2 after modulation with antiCD2 MoAb [13]. This suggests thai the observed modulation of CD2 could be due to its loss from the cell surface. This view is supported by the temperature dependenee of the modulation, since lower temperatures would affect ihe membrane fluidity and membrane protein mobility, and thus [he modulation of cell surface molecules. Dhamiendra lepromin treatment of Con A blasts resulted in a marked reduction in the binding of MoAb D66 to a conformational epitope of CD2 on activated T cells and that of Leu 5b, indicating that the whole CD2 molecule was affected. In addition, IL-2R was modulated on these cells (Fig. 4). Since CD2, CD4, CDS and IL-2R arc involved in signal transduction during T-eell activation [2, 8]. it is logical to suggest that the modulation of these molecules might have contributed to the suppressive effect of Dharmendra lepromin on ihe proliferalive response of PBMC to mitogens and antigens reported earlier (12. 14, 15]. Various preparations of M. leprae were tested for iheir ability to modulate CD2 (Fig. 6). The cell-wail structure of M. leprae is highly complex with extensive extracellular lipid coats [7]. Live M. leprae and Mitsuda lepromin [3] are integral M. leprae preparations with their thick lipid coats inlact. Soluble M- leprae antigen (leprosin A) [6] is devoid of eell wall and most of the wallassociated components. In delipidified cell-wall preparation [19], lipids and myeoiates were almost completely removed. In Dharmendra preparation, treatment of the bacilli wilh chloroform and ether removes much of the lipid covering leaving the cell-wall structure with associated carbohydrates and proteins exposed. The observation that the Dharmendra preparation of M. /('/irac alone modulated CD2 suggests that ihe M. leprae factor responsible for the modulatory effects could be a carbohydrate moieiy. Recently, it has been shown that CD2 and CD4 could be modulated by dexlran sulphate, a polymeric carbohydrate [17, 20]. The view that Ihe factor responsible could be a polysacchaHde is further
208
R. Sheela. S. Ilangumaran & V. R. Muthukkaruppan
supported by the fact that autoclaving did not affect the modulatory effects of Dharmendra lepromin. Our results establish clearly that a certain factor(s) from M. leprae has the ability to modulate CD2, CD4, CD8 and II.-2R on the T-cell surface, which would possibly influence the T-cell functions. Though we can speculate that this may happen in the micro-environment of highly bacilliferous lesions of lepromatous leprosy patients. reduction in the expression of CD2, CD4 and CDS was observed neither in peripheral blood T lymphocytes [21, and our unpublished observation] nor in ihe lesional lymphocytes of these patients [21]. Yet. Ihe proportion of E-rosetting cells is decreased while thai of T cells (as seen by MoAb binding to CD3) is normal in PBMC from these patients (our unpublished observation). We cannot exclude the possibility that the breakdown products of M. leprae in the bacillifcrous lesions of these patients may affect CD2 to the extent of inhibiting ihe E-rosetting ability of T cells without causing a discernible effect on anti-CD2 binding. This probably has a role in the immunological anergy observed in lepromatous leprosy patients.
ACKNOWLEDGMENTS This work was supported by grants from Department o^ Science and Technology. India and Indo-Swiss cooperations in Biotechnology. R.S. and S.I. are recipients of Junior Research Fellowships from the Council for Scientific and Industrial Research. Government of India. The authors wish to thank Dr V. D. Ramanathan (Tuberculosis Research Centre, Madras) for determining the bacterial counts of Dharmendra preparations of M. leprae and BCG. The excellent technical assistance of P. Nataraja Sundaram and R. Meenakshi Sundaram is gratefully acknowledged.
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2 Biercr, B.E.. Sleckmim. B.P,. Ratnotsky. S.E. & BiirakolT. S.J. The biologic roles of CD2. CD4. CD8 inTcellactivitlion. .Ann. Rev. Immunol. 1,519. l*;89. 3 Bloom, B.R.. Convit, J.. Godal. T.. Noordeen, S.K.. Perkins. F T . . Rees. R.J.W.. Sansarricq. H.. Shepard. C.C. Torrigiani. G. & Walter, J. Recommended safety requiremenls for the preparations of lepromirt: a WHO memorandum. Bull. WHO 57. 921, 1979. 4 Bloom, B.R, & Godal, T. Selective primary health care: strategies for control of distance in the developing world. V. Leprosy. Rev htfr. Di.s. 5, 765. 1983. 5 Dharmendra. Notes on leprosy. Ministry of Health, Govcrnmeni of India, 1967. 6 Draper. P- Protocol 1/79: purification of W. leprae PP. 4 in Anne.\- I to the Report of the Enlarged Steering Committee for Research on the Immunohgv of Leprosy (IMMLEP) Meeting of 7 H February. 197^. Geneva. World Health Organization. 1979.' 7 Draper, P. Leprosy review: the bacteriology of Mycohatteriunj leprae. Tubercle 64, 4}. I9H3. 8 Halvorseii. R.. Leivestad. T.. Gandernack, G. & Thorsby. E. Accessory cell-dependent T-ccll activation via Ti-CD3. InvoKement of CD2-LFA-3 interactions. Sictiul. J. Immtmol. 28. 277. 198S. 9 Howard. F.D.. Lcdbciter, J.A., Wong. J.. Biebcr. C.P.-Stinson, E.B.& Herzenberg, L.A. A human Tlymphocyte dilTerenliation marker defined hy monoclonal antibodies thai block E-rosette formation. J Immunol. 126, 2117, I9SI. 10 Hunt. S,V, Preparative immunoselcciion of lymphocyte populations. Pp. 55.! 1 in Weir. D.M. (ed.) Hmuthiiok of E.xpcrimental Imiiuiiu'liigy. 2. Cellular Immunology. 4th edn. Scientific Publications, London. 1986. i 1 Lang, G., WoUon, D.. Owen. M.J.. Sewell. W.A.. Brown. M.H.. Mason. D.Y.. Crumpton. M.J. & Kioussi.s. D. The structure of the human CD2 gene and its expression in transgenic mice. EMBO J. 7, 1675. 1988. 12 Maiarkannan. S., Chakkalath. H R . & Muthukkaruppar, V.R. Impairment of alternate pathway (CD2) of T cell activation in Leprosy. J. Bio.sci. 14, 29, 1989. 13 Morelta. A., Olive. D.. Poggi. A., Pantaleo, G., Mawas, C, & Moretta. L. Modulation of surface T i l molecules induced by monoclonal antibodies: analysis oi the functional relationship between antigen dependent and antigen independent pathways of human T cell activation. Ettr. J. Immunol. 16, 1427, 1986. 14 Muthukkaruppan. V.R., Chakkalath, H.R. & James. M.M. Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor. ImmunoL Lett. 15, 199. 1987, 15 Muthukkaruppan. V.R., Chakkalath. H.R. & Maiarkannan. S. The classical and alternate pathways of T cell activation are impaired in leprosy, Immunol. Lett. 19, 55. 1988. 16 Nath. r . Curtis, J.. Sharma. A.K. & Talwar. G.P. Circulating T-cell numbers and their niitogenic potential in leprosy correlations with mycobacterial load. Chn. Exp. Immunol. 29. 393. 1977. 17 Parish, C.R., McPhtm, V. & Warren. H.S. Is a
CD2 Modulalhm by M. leprae natural ligand ofthcT lymphocyte CD2 molecule as a sulfated Carbohydrale'.' J. Immwwl. 141. 349S, 1988. 18 Pellegrino. M.A., Ferrone, S., Dietrick. M.P. & Reisfeld, R.A, Enhancement of sheep red blood cell human lymphocyte rosette formation by sulphydry! compound Z-amino-ethyl isothiouronium. Clin. Inununol. Pathol. 3, 324. 1975. 19 Robinson, P. & Mahadevan, P.R. A cotnponent of Mvcohacterium leprae as immunomodulating agent Tor immune deficient cells of leprosy patients. ./. Clin. Liih. Immunol. 24, 171. 1987. 20 Thiele. B., Braig, H.R., Ehm. I., Kuiiz.e. R. & Ruf.
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B. Influence of sui fated carbohydrates on the accessibility of CD4 and other CD molecules on the coll surface and implications for human immunodeficiency virus infection. Eitr../. Immunol. 19. I IM, 1989. 21 Wong. L.. Salgame. P,. Torigian, V.K.. Fu, T,H., Rea. T.H. & Modlin, R.L, CD2 expression and function in lepromatous leprosy. Infect. Immun. 57, ^SL';, 1489.
Received 26 June 1990 Accepted in revised form 3 September 1990
Scand. J. Immunol. 33, 203-209. 1991
Flow Cytometric Analysis of CD2 Modulation on Human Peripheral Blood T Lymphocytes by Dharmendra Preparation of Mycobacterium leprae R. SHEELA, S. I L A N G U M A R A N & V. R. M U T H U K K A R U P P A N Department of immunology. School of Biological Sciences. Madurai Kamaraj University, Madtirai. India
Sheela. R., Ilangumaran. S. & Muthukkaruppan, V.R, KIow Cytometric Analysis or CD2 Modulation on Human Peripheral Blood T Lymphocytes by Dharmendra preparation of Mycobacterium teprae. Scand. J. Immunol. 33, 20.1-209, 1991 It has been reported previously that Mymhaclerium leprue modulated CD2 on human peripheral blood T lymphocytes and that this modulation was accompanied by a marked reduction in the proliferauve response of these cells to mitogens and antigens. In this study, we report that treatment of peripheral blood mononuclear cells from healthy individuals with Dharmedra preparation of M. leprue inhibited their ability to form rosettes with sheep red blood ceils. Flow cytomotric analysis of Dharmendra lepromin-lrcated eells showed that, in addition to CD2. CD4 and CD8 were modulated while the surface expression of CD.l was not alTecied. The specifieiiy of CD2 modulation was confirmed by similar effects of Dharmedra lepromin on ihymocytes and lymph node cells from human CD2 transgenic mice. The modulatory elTeci of Dharmendra lepromin was not observed at lower temperatures. Dharmendra lepromin treatment of activated T cells resulted in reduced binding of monoclonal antibodies to IL-2Rand Dfifi epitope of CD2. The modulalory effects were not observed with Dharmendra preparation of BCG or other preparations of M. leprae. Our results indicate that certain M. leprae factor(s) specifically modulate{s) CD2, CD4, CDS and IL-2R but not CD3 on T lymphocytes. The suppressive elTeet of Dharmendra lepromin on the T-cell proliferative response reporled earlier may be explained by its modulatory effect on a number of T-cell surface molecules. Profcs.\or V. R. .Mi/ihukktirtippan, Department nJ Immunology. School of Biological Seierices, Madurai Kamaraj University. Madurai 625 021, India
Leprosy is a c!ironic infectious disease catiscd by Mycohacierium leprae. The clinical spcctrutn of the disease correlates we!t with the immune statu.s o'f the patients. The lepromatous form of the disease is associated with impaired cell-mediated immunity (CMI) and dissctninaied infection with M. leprae [4]. Earlier studies had shown that the proportion of cells forming rosettes with sheep red blood cell.s (SRBC) was reduced in the peripheral blood of lepromatous leprosy patients [16]. Previously, we have shown by immunotiuorcsccnce microscopy that treatment of peripheral blood fnononuclear cells from healthy individuals with Dharmendra preparation of M. teprae significantly modulated the expression of CD2 [14]. This modulation was accompanied by marked suppression of the T-cell proliferative response to mitogens and antigens [12, 14, 15],
In this study, we investigated the effect of Dhannendra lepromin treatment on a number of CD molecules on peripheral blood mononuclear cells from healthy individuals by (low cytometric analysis. We present evidence that, in addition to CD2. the expression of CD4, CDS and 1L-2R were reduced, while that of CD3 was not affected. Further, these effects were brought about specifically by Dharmendra lepromin, but not by Dharmendra preparation of Bacillus Calmette-Guerin (BCG) or by other preparations of M. teprae.
MATERIALS AND METHODS Chemicals. Metrizoate sodium. Hank's balanced salt solution (HBSS). powdered culture medium RPMI 1640. penicillin-streptomycin mixture, propidium iodide, Tween-80, 2-aminoethvl isothiouronium bro203
204
R. Sheela. S. Ilangumanui & V. R. Muthukkaruppan
mide(AET).cnnc;tn;iViilin A (Con A)iiiid sndiuin a^ide were purchased from Sigma Chemical Co.. USA. Ficoll 400 was obtained from Pharmacia, Sweden, Mycnhavterial pn-iMniiions. Dharmcndra prcparalion of .HyctihcicWriiini h-pnic (Dhitrnicndra lepromin) was eilher ohiained from Dr U. Scngtipia (Central JALMA [nstitulcfor Leprosy, Agra, India for prepared rrom armadillo-derived, freett-driod A/, Icpnic obtained from WHO-IMMLF.P St. U-pnw Bank through Dr R, J. W. Rees (National Institute for Medical Research. Mill Hill. London. UK). BCG, Danish strain 1331, was kindly provided by The Director, BCG Vaccine laboratories, Madras. India. Dharmendra lepromin was prepared from freezcdried M. leprav following the standard procedure J5|. Briefly, the bacteria were suspended in ehloroforni by grinding in a glass mortar. The chloroform was then allowed lo evaporate complelely in a hot water bath. The residual substance was suspended in solvent elher and cenlrifuged at 5900 j,' for I h al 4 C. The peilet was resuspended in ether and centrifuged again. The linal pellet was dried under vacuum for 4S h al room temperature. The dried malerJal was ground in 2 ml phosphate-bulTcrcd saline (PBS) containing 0.05"AI Twccn SO to suspend ihe bacilli. After counting by acidfast staining the bacterial count was adjusted to I x lO" bacilli per ml in PBS and autoelaved. Aliquots were stored frozen al - 2 0 C until use. BCG. subjected to Dharmendra preparation, was used as a eontrot for Dharmendra lepromin, Mitsuda lepromin and soluble M. k-pnu- antigen (leprosin A. batch CDI22) were obtained from Dr R. J. W. Rees. Live M. leprav and delipidified cell wall preparation of M. leprae (DCW) were generous gifls from Dr P. R. Mahadevart (Foundation for Medical Research. Bombay. India). MomiclotUil Anlihiitlici l.Mti.'ihI. MoAb Leu 4 (anti-CD3), Leu 5b (anti-CD:). Leu 2a (anti-CD8) and anti-lL-2R (anti-CD25) conjugated to fluoreseetn isoIhiocyanate (FITC) or phycoerythrin (PE) and FITCeonjugated goal anti-mouse antibody (GAM) were purchased from Bccton Dickinson (Mountain View, Calif, USA), Rhodamine-conjugated anli-T4 (antiCD4) was obtained from Coulter Electronics, UK, MoAh D66 directed against a conformational epitope on CD2 [1] was a kind gift from Dr A. Bernard. Paris, France. Mice. Human CD2 (hCD2) transgenic mice (CBAxC57 BL/IO) carrying a single eopy of hCD: gene [11] were a kind gift from Dr M. ,1. Crumpton through the Imperial Caneer Researeh Fund, UK. Balb. c mice served as controls. Four- to six-wcek-oid mice were used I'or experiments. Prcfiiiratlon «/ ('f'//,v. Peripheral blood mononuelear eells (PBMC) were isolated from hepariiiized venous blood obtained from healthy normal volunteers over Fieoll Metrizoate gradient [10], After washing thrice in HBSS. cells were suspended in RPMI 1640 supplemented with penicillin (100 U,ml) and streptomycin (100 ;ig,,m!) containing O.!".. bovine serum albumin (BSA). Con A blasts were generated by stimulating PBMC with 10 fi^iml Con A for 24 h in mediutii containing 10''^ FCS. Single-cell suspensions were prepared from thymus and lymph nodes of mice. Treatment of veils with DIuirmendra lepromin. One
million cells were incubated with 20 //i or indicated concentrations of Dharmendra iepromin, BCG or other A/./c/jmc preparations in lOO/ilofRPMI 1640. supplemented with penicillin (MM) U ml), streptomycin (100 //g/mDandO-l':,. BSA for !2hat.l7 Clunless otherwise indicated). At the end of the incubation period, treated cells were washed three times in PBS before they were subjected to ro.setting with SRBC or immunofluore.seence staining. Cell viability as assessed by How cytometric analysis afler propidium iodide staining was more than W\, up to 72 h after Dharmendra lepromin treatment. E-rosetiing assays. Sheep erythroeytes were treated wiih 0.14 M 2-aminoeth\l isothiouronium bromide (AFT) [IS] and resuspended as a !"i. suspension in RPMI 1640 eontaining 10".. FCS. One million PBMC in a lOU tA volume were mixed with an equal \olume of AET-treated I'!.. SRBC in a test lube and cenlrifuged at 2(H) ,e for 1 min at room temperature. The cells were incubated at .'17 C for 30 min and then at 4 C overnight. The pellet was gently tapped and rosettes were counted in a haemocytometer under phase-contrast microscope. Two-hundred lymphocytes were seanned and the n umber of rosetting and non-rosetting lymphocytes was counted. Any cell binding to four or more SRBC was classified as a rosette. Imnninofittiire.scvnie siuinin^ and flow cviomerruunttly.-ii.^ IFCMI. Cells were incubated with appropriate MoAb diluted in PBS containing 0.1".. BSA and 0,1% sodium azide at 4 C for 30 min. Unconjiigated MoAb D66 was followed by the secondary antibody reagent GAM-FITC for 30 min at 4 C. Labelled cells were washed twice in PBS eontaining U.T'r. sodium azide and subjected to flow cytometric analysis in FACScan (Beeton Dickinson) using ihe Consorl 30 software program with log amplification gains for fluorescence. Fach experiment was repeated three times and one set of representative data is presented.
RESULTS Inhibition of E-roselting by Dharmendra lepromin The ability of normal human PBMC to form roselles with AET-irealed SRBC was studied after treatment with Dharmendra lepromin for 12 h at 37 C. A concentration dependent redtiction was observed in the proportion of rosette forming eeils (Table I). This effect ol" Dharmendra lepromin acted directly on E-rosctte-forming cells, since pretreatment of SRBC with Dharmendra lepromin did not affect their ability to form rosettes with T cells. Further, the inhibition of E rosetting was specific to M. leprae, as no such effect was observed after treatment with Dharmendra preparation of BCG, Modulation of CD2 by Dharmendra lepromin Since it has been shown that CD2 is the T-cell
C'D2 Modulation hy M. leprae TABLE I, Reduction in E-rosette forming ability of peripheral blood monontjciear cells after treatment with Dharmendra lepromin Volume added (Ml)
Treatment
% E-rosetting cells
None
74 + 4
Dharmendra lepromin 2+2 12 + 5 44 + 5 74 + 5 7.3+4
20
to
5. 2.5 1.25
Dharmendra BCG 20
72 + 6
One million PBMC treated wilh Ihe indicated volutne of Dharmendra lepromin or Dhiirniendra preparation ofBCGd y 10^hacilli/ml)forl2hat37 Cwerewashed and an E-rosetting assay was performed with AETtreated SRBC as described in Materials and Methods. Two-hundred eells were scanned and any cell with more than four SRBC hound was eounted as rosette positive. The results are meiin + SE ai three experitiienls.
• HAKKLNOliA
205
surface molecule involved in rosetting with SRBC. the accessibility orCD2 as well as other CD molecules on the T-cell surface to the corresponding MoAb was studied after trcatnietit of PBMC with Dharmendra lepromin. It is evident from Fig. I that MoAb binding to CD2 was complelely abrogated by treatment with Dharmendra lepromin but not with Dharmendra preparation of BCG. In addition, binding of antiCD4 was lost cotnpletely. while that of anti-CD8 was partially reduced. However. Dharmendra lepromin treatment did not inhibit the binding of anti-CD3 MoAb. indicating that the modulation of CD2, CD4 and CD8 by Dharmendra lepromin, but nol by Dharmendra preparation of BCG, is specific and is not due to a generahzed effect of mycobacteria on lymphocyte cell-surface molecules.
Modulation of human CD2 molecules on cells from hCD2 transgenic mouse Thai the CD2 modulation by Dharmendra lepromin is specific, was confirmed using hCD2 transgenic mouse. Dharmendra lepromin treatment of thymocytes and lymph node cells from these mice showed a marked reduction in the surface expression orCD2 (Fig. 2).
DHAKMENURA
LEPROHIN
BCG
Kincrics of CD2 modulation hv Dharmendra k'promin
I;.
Modulation of CD2 was found to be dependent on the concentration of Dharmendra lepromin used as well as the duration of incubation. The minimal concentration of Dharmendra lepromin THYMOCYTES
LOG FLUORESCENCE
INTENSITY
FIG. I. Effect of Dharmendra lepromin treatment on CD2. CD3. CD4 and CD8 on PBMC. 1 x 10" PBMC were treated with 20 ^I (2 x 10' bacilli) of Dharmendra lepromin or Dharmendra preparation of BCG for 12 h at 37 Cin 100/d of RPMI 1640 contaming 0.1% BSA. Treated cells were analysed for the surface expression of the indicated CD molecules.
LYMPH
NODE
CELLS
LOG FLUORESCENCE INTENSITY FIG. 2. Modulation of CD2 on eells from hCD2 transgenic mice. Thymocytes and lymphnode eells from hCD2 transgenic mice were subjected to Dharmendra lepromin treatment as described in Fig. I and analysed for the expression of hCD2. ( } Background fluoreseenee; ( ) untreated cells; ( ) Dharmendra lepromin treated cells.
206
/?. Sheela. S. Ilangumaran & V. R. Muthukkaruppan B THtflTED
1 h
5
3h
s: zi
6 h
9 h
?4 h
A A A
.UNTREATED 1 CONTROL 20 pi
i
•I-k Oh
A ^
i
37"
if
.•UNTREATED CONTROL
I
LOG FLUORESCENCE INrENSlTY
FIG. 4. Effecl of Dharmendra lepromin on aclivaied T cells. 24 h Con A blasts were incubaled with Dharmendra lepromin for 12 hal 37 C. Treated cells and controls were analysed for the expression of CD2, D66 epitope of CD2 and IL-2R. ( ) Background fluorescence: ( ) unactivaled. untreated cells; ( ) activated. untreated cells; ( ) activated, treated eells.
C /I ''CONTROL U'c
LOG FLUDHESCENCE INTENSITY
FIG. 3. Kinelies of CD2 modulation. 1 x 10" PBMC were treated wilh (A) indicated concent rai ions of Dharmendra lepromin for 12 h al 37 C; (B) 20 ^1 of Dharmendra lepromin at 37 C for indicated time intervals; (C) 20 n\ of Dharmendra lepromin at 37 C for 12 h. washed ihree times and ineubated in RPMI 1640 eontaining 10'^., FCS for 72 h: and (D) 20 /il of Dharmendra lepromin for 12 h :it 37 C or 4 C. and analysed for the expression orCD2 by Row cytometry. In all experiments. Dharmendra iepromin treatment wasdonein lOO/ilorRPMI I640containing0.l'I'l, BSA. (
k
) Indicates background fluorescence in A, C and
D,
required to cause maximal CD2 modulation after 12 h was 20 /d (2 x 10^ bacilli) in 1(K) /A final volume (Fig. 3A). At this concentration of Dharmendra lepromin. maximal reduction in CD2 expression was observed after incubation for al least 9 h (Fig. .IB). When ecIls treated with Dharmendra lepromin were washed and incubaled in medium containing IO"n FCS. the reeovery of CD2 expression was observed by 72 h (Fig. 3C). Modulation of CD2 by Dharmendra lepromin was influenced by ihe temperature of incubation. When PBMC were treated with Dharmendra lepromin at 4 C. instead of at 37 C, levels of CD2 expression were not affected signitieantly. even after 24 h of incubation (Fig. 3D). Effect of Dharmendra tcpnmiin on actirateil T cells To determine whether CD2 modulation by Dhar-
mcndra lepromin was influenced by the activation state o[" T cells, 24-h Con A blasts were thoroughly washed and treated with Dharmendra lepromin. In addition to the modulation of CD2. MoAb binding lo IL-2R was inhibited on T lymphoblasls(Fig. 4). The binding of MoAb D66 to a conformational epilope on CD2. whieh is expressed more after activation, was also reduced on Con A blasts after treatment with Dharmendra lepromin. indicating ihat Ihe whole CD2 molecule was modulated (Fig. 4). Treatment with Dharmendra lepromin also inhibited the binding of anti-CD2 and [D66 MoAb on Jurkat cells indicaling thai the modulatory effect of Dharmendra lepromin acted directly on T cells (F"ig. 5). Specificity oJ CD2 modulation bv Dharmendra preparation of M. leprae Different preparations of M. teprae. live M. k'prae. Mitsuda iepromin, soluble A/, leprae
LOG FLUOBFSCENCE INTENSITY
FIG, 5-ModulalionofCD2onJurkal Cells. Jurkat cells were treated with Dhurmendra lepromin for 12 h at 37 Cin RPMI 164()a)ntainingO. I".. BSA. Treated cells were labelled with MoAb to CD2 itnd D66 epitope of CD2. ( ) Background fluorescence: ( ) untreated cells: ( — ) Dharmendra iepromin-lreated cells.
HBER
CD2 Modulation hy M. leprae
Z -J UI
o
.A
\ D
A
E
A
\ /I
F
A
LOG FLUORESCENCE INTENSITY FIG. 6. Effect ofdifrercnt pa-piiru lions of ,V/. /c/j/t/c on CD2. I X 10'' PBMC in ;i I(K) //I voliinic were ireitled with {A), none (B) 20 /il Dharmendra lepromin. (C) 20 ft] {2x \0^ biidlli) MilKLida lepromin. (D) 2 x 10' live M. leprae. (E) 40 /jg.ml leprosin A. or (F) 40 /ig-ml DCW for 24 h al 37 C and analysetJ Tor CD2 e.\pression.
aniigcn (leprosin A) and delipidified cell wall (DCW) preparation were lesled for their ability lo modulate CD2. Il is evident from Fig. 6 thai even aRer 24-h treatment, other M. leprae preparations tested were not able to modulale CD2, indiealing ihat certain M. /f/jrc/c-specific factors, exposed and/or retained especially in Dharmendra preparation, is responsible for the observed CD2 modulation in normal PBMC.
DISCUSSION Earlier studies by Huoreseent microscopy have shown thai treatment of normal human PBMC with Dharmendra preparation of M. tcprac results in the modulation of CD2 on the T-ccll surface [14]. In this report, we confirm that Dharmendra lepromin indeed modulates CD2. by E-rosetting assays and flow cylometric analysis. Treatment of normal human PBMC with Dharmendra lepromin resulted in a marked reduction in the proportion of E-rosette-forming cells (Table 1). It has been well established that CD2 is the molecule involved in the E rosetting of T cells with SRBC [9]. Flow cytometric analysis of treated cells showed that CD2 was modulated significantly; in addition. MoAb binding to CD4 and CD8 were reduced (Fig. i). This modulation was nol due to a generalized effect of Dharmendra lepromin on T cell surface, since ihe expression of CD3 was not affected. Additionally.
207
no modulalory effect was observed with Dharmendra preparation oi' BCG (Table I; Fig. I). indicaling that the factor is M. leprae specific. Further. Dharmendra lepromin modulated CD2 on thymocytes and lymph node eells from hCD2 triinsgenic mice. Kinetic studies showed gradual regeneration of CD2 after modulation by Dharmendra lepromin. Similar regeneration kinetics was reported for CD2 after modulation with antiCD2 MoAb [13]. This suggests thai the observed modulation of CD2 could be due to its loss from the cell surface. This view is supported by the temperature dependenee of the modulation, since lower temperatures would affect ihe membrane fluidity and membrane protein mobility, and thus [he modulation of cell surface molecules. Dhamiendra lepromin treatment of Con A blasts resulted in a marked reduction in the binding of MoAb D66 to a conformational epitope of CD2 on activated T cells and that of Leu 5b, indicating that the whole CD2 molecule was affected. In addition, IL-2R was modulated on these cells (Fig. 4). Since CD2, CD4, CDS and IL-2R arc involved in signal transduction during T-eell activation [2, 8]. it is logical to suggest that the modulation of these molecules might have contributed to the suppressive effect of Dharmendra lepromin on ihe proliferalive response of PBMC to mitogens and antigens reported earlier (12. 14, 15]. Various preparations of M. leprae were tested for iheir ability to modulate CD2 (Fig. 6). The cell-wail structure of M. leprae is highly complex with extensive extracellular lipid coats [7]. Live M. leprae and Mitsuda lepromin [3] are integral M. leprae preparations with their thick lipid coats inlact. Soluble M- leprae antigen (leprosin A) [6] is devoid of eell wall and most of the wallassociated components. In delipidified cell-wall preparation [19], lipids and myeoiates were almost completely removed. In Dharmendra preparation, treatment of the bacilli wilh chloroform and ether removes much of the lipid covering leaving the cell-wall structure with associated carbohydrates and proteins exposed. The observation that the Dharmendra preparation of M. /('/irac alone modulated CD2 suggests that ihe M. leprae factor responsible for the modulatory effects could be a carbohydrate moieiy. Recently, it has been shown that CD2 and CD4 could be modulated by dexlran sulphate, a polymeric carbohydrate [17, 20]. The view that Ihe factor responsible could be a polysacchaHde is further
208
R. Sheela. S. Ilangumaran & V. R. Muthukkaruppan
supported by the fact that autoclaving did not affect the modulatory effects of Dharmendra lepromin. Our results establish clearly that a certain factor(s) from M. leprae has the ability to modulate CD2, CD4, CD8 and II.-2R on the T-cell surface, which would possibly influence the T-cell functions. Though we can speculate that this may happen in the micro-environment of highly bacilliferous lesions of lepromatous leprosy patients. reduction in the expression of CD2, CD4 and CDS was observed neither in peripheral blood T lymphocytes [21, and our unpublished observation] nor in ihe lesional lymphocytes of these patients [21]. Yet. Ihe proportion of E-rosetting cells is decreased while thai of T cells (as seen by MoAb binding to CD3) is normal in PBMC from these patients (our unpublished observation). We cannot exclude the possibility that the breakdown products of M. leprae in the bacillifcrous lesions of these patients may affect CD2 to the extent of inhibiting ihe E-rosetting ability of T cells without causing a discernible effect on anti-CD2 binding. This probably has a role in the immunological anergy observed in lepromatous leprosy patients.
ACKNOWLEDGMENTS This work was supported by grants from Department o^ Science and Technology. India and Indo-Swiss cooperations in Biotechnology. R.S. and S.I. are recipients of Junior Research Fellowships from the Council for Scientific and Industrial Research. Government of India. The authors wish to thank Dr V. D. Ramanathan (Tuberculosis Research Centre, Madras) for determining the bacterial counts of Dharmendra preparations of M. leprae and BCG. The excellent technical assistance of P. Nataraja Sundaram and R. Meenakshi Sundaram is gratefully acknowledged.
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2 Biercr, B.E.. Sleckmim. B.P,. Ratnotsky. S.E. & BiirakolT. S.J. The biologic roles of CD2. CD4. CD8 inTcellactivitlion. .Ann. Rev. Immunol. 1,519. l*;89. 3 Bloom, B.R.. Convit, J.. Godal. T.. Noordeen, S.K.. Perkins. F T . . Rees. R.J.W.. Sansarricq. H.. Shepard. C.C. Torrigiani. G. & Walter, J. Recommended safety requiremenls for the preparations of lepromirt: a WHO memorandum. Bull. WHO 57. 921, 1979. 4 Bloom, B.R, & Godal, T. Selective primary health care: strategies for control of distance in the developing world. V. Leprosy. Rev htfr. Di.s. 5, 765. 1983. 5 Dharmendra. Notes on leprosy. Ministry of Health, Govcrnmeni of India, 1967. 6 Draper. P- Protocol 1/79: purification of W. leprae PP. 4 in Anne.\- I to the Report of the Enlarged Steering Committee for Research on the Immunohgv of Leprosy (IMMLEP) Meeting of 7 H February. 197^. Geneva. World Health Organization. 1979.' 7 Draper, P. Leprosy review: the bacteriology of Mycohatteriunj leprae. Tubercle 64, 4}. I9H3. 8 Halvorseii. R.. Leivestad. T.. Gandernack, G. & Thorsby. E. Accessory cell-dependent T-ccll activation via Ti-CD3. InvoKement of CD2-LFA-3 interactions. Sictiul. J. Immtmol. 28. 277. 198S. 9 Howard. F.D.. Lcdbciter, J.A., Wong. J.. Biebcr. C.P.-Stinson, E.B.& Herzenberg, L.A. A human Tlymphocyte dilTerenliation marker defined hy monoclonal antibodies thai block E-rosette formation. J Immunol. 126, 2117, I9SI. 10 Hunt. S,V, Preparative immunoselcciion of lymphocyte populations. Pp. 55.! 1 in Weir. D.M. (ed.) Hmuthiiok of E.xpcrimental Imiiuiiu'liigy. 2. Cellular Immunology. 4th edn. Scientific Publications, London. 1986. i 1 Lang, G., WoUon, D.. Owen. M.J.. Sewell. W.A.. Brown. M.H.. Mason. D.Y.. Crumpton. M.J. & Kioussi.s. D. The structure of the human CD2 gene and its expression in transgenic mice. EMBO J. 7, 1675. 1988. 12 Maiarkannan. S., Chakkalath. H R . & Muthukkaruppar, V.R. Impairment of alternate pathway (CD2) of T cell activation in Leprosy. J. Bio.sci. 14, 29, 1989. 13 Morelta. A., Olive. D.. Poggi. A., Pantaleo, G., Mawas, C, & Moretta. L. Modulation of surface T i l molecules induced by monoclonal antibodies: analysis oi the functional relationship between antigen dependent and antigen independent pathways of human T cell activation. Ettr. J. Immunol. 16, 1427, 1986. 14 Muthukkaruppan. V.R., Chakkalath, H.R. & James. M.M. Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor. ImmunoL Lett. 15, 199. 1987, 15 Muthukkaruppan. V.R., Chakkalath. H.R. & Maiarkannan. S. The classical and alternate pathways of T cell activation are impaired in leprosy, Immunol. Lett. 19, 55. 1988. 16 Nath. r . Curtis, J.. Sharma. A.K. & Talwar. G.P. Circulating T-cell numbers and their niitogenic potential in leprosy correlations with mycobacterial load. Chn. Exp. Immunol. 29. 393. 1977. 17 Parish, C.R., McPhtm, V. & Warren. H.S. Is a
CD2 Modulalhm by M. leprae natural ligand ofthcT lymphocyte CD2 molecule as a sulfated Carbohydrale'.' J. Immwwl. 141. 349S, 1988. 18 Pellegrino. M.A., Ferrone, S., Dietrick. M.P. & Reisfeld, R.A, Enhancement of sheep red blood cell human lymphocyte rosette formation by sulphydry! compound Z-amino-ethyl isothiouronium. Clin. Inununol. Pathol. 3, 324. 1975. 19 Robinson, P. & Mahadevan, P.R. A cotnponent of Mvcohacterium leprae as immunomodulating agent Tor immune deficient cells of leprosy patients. ./. Clin. Liih. Immunol. 24, 171. 1987. 20 Thiele. B., Braig, H.R., Ehm. I., Kuiiz.e. R. & Ruf.
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B. Influence of sui fated carbohydrates on the accessibility of CD4 and other CD molecules on the coll surface and implications for human immunodeficiency virus infection. Eitr../. Immunol. 19. I IM, 1989. 21 Wong. L.. Salgame. P,. Torigian, V.K.. Fu, T,H., Rea. T.H. & Modlin, R.L, CD2 expression and function in lepromatous leprosy. Infect. Immun. 57, ^SL';, 1489.
Received 26 June 1990 Accepted in revised form 3 September 1990
