© ELSEVIERParis 1987

Ann. Inst. PasteurlImmunol.1987, 138, 561-569

GENERATION OF LEISHMANIA-SPECIFIC SHORT-TERM T-CELL

LINES DERIVED FROM PERIPHERAL BWOD OF CURED

AMERICAN CUTANEOUS LEISHMANIASIS PATIENTS

J. Hendriks (*), S.G. Coutinho, L. Cysne and R.C.C. Dorea

Department of Protozoology and WHO Collaborating Centrefor Research and Training in Immunology of Parasitic Diseases,

Instituto Oswaldo Cruz, Rio de Janeiro (Brazil)

SUMMARY

The putative protective immune response in localized cutaneousleishmaniasis caused by Leishmania braziliensis braziliensis (Lbb) was analysedby generation of short term T-cell lines from peripheral blood lymphocytes(PBL) of three patients. All had had active skin lesions but were clinicallycured at the time of study. PBL were stimulated in vitro with sonicated Lbbpromastigotes; blasts were isolated after 5 days and propagated for 9 to 16 daysbefore being tested for proliferative capacity. The majority of propagatedcells were T4-positive and reacted with Leishmania antigens. An apparent cross­reactivity with Trypanosoma cruzi (T. cruzi) soluble antigen was recorded.Although preliminary, these data indicate that immune T cells derived fromcured LCL patients can recognize antigens common to Lbb and T. cruzi.

KEY-WORDS: Leishmania braziliensis, Trypanosoma cruzi, Antigenicity,T lymphocyte; Short-term human T-cell lines, Phenotype, Localized cutaneousleishmaniasis.

INTRODUCTION

Clinical and immunological observations over the past decades have at­tributed an important role to T-cell-mediated immune mechanisms in recovery

Submitted January 25, 1987, accepted May 29, 1987.

(*) PAHO/WHO associate expert in immunology.

Correspondence: Dr S.G. Coutinho, Protozoology Department, Instituto Oswaldo Cruz,Av. Brasil, 4365, 21040 Rio de Janeiro, Brazil.

562 J. HENDRIKS AND COLL.

from American cutaneous leishmaniasis (ACL) [3,4,6,12]. Yet, experimen­tal data on the exact nature of the T cells involved in the development of animmune response to Leishmania species in humans are scarce. Animal studieswith L. major have suggested the existence of two Leishmania-specific T-cellpopulations with apparent antagonistic effects upon the development of pro­tective immunity in the host [10, 14, 16, 17], but whether similar mechanismsalso occur in human leishmaniasis in unknown. ACL caused by L. brazilien­sis braziliensis (Lbb) can occur clinically in two different forms. The locali­zed cutaneous form (LCL) presents with one or several lesions that may healspontaneously or after treatment; antimony therapy usually results in increasedcell-mediated immune response, as measured by in vitro proliferation (LPR)of blood lymphocytes to Lbb antigen [12]. Patients with the other clinicalform (mucocutaneous leishmaniasis) usually respond poorly to therapy, andcompared to LCL this rare variant is frequently accompanied by increaseddelayed-type hypersensitivity reaction and LPR [2, 4]. As no suitable animalmodel is currently available to study Lbb infection, we analysed the T-cellpopulations involved by in vitro stimulation and expansion of lymphocytesderived from cured LCL patients having positive LPR to Lbb antigen. Thisreport describes the conditions under which Lbb-specific short-term T-celllines can be generated and presents data on their phenotype and antigenspecificity.

MATERIALS AND METHODS

Patients.

Patients were from the Jacarepagmi district in Rio de Janeiro, an area whereLbb-induced LCL is endemic [7]. The criteria for patient diagnosis have been described[12]. Only patients who responded well to therapy and who had positive in vitro lym­phoproliferative responses to Lbb antigen after cure were selected to generate andexpand short-term T-cell lines from their peripheral blood (table I).

The 3 patients presented with cutaneous lesions which contained Leishmaniaparasites upon microscopic examination of biopsy imprint smears. All lesions healedafter 2 to 3 series of glucantime.

Antigens.

Bacillus Calmette-Guerin (BCG) - Lyophilized BCG (Ataulpho de Paiva Foun­dation, Rio de Janeiro, Brazil) was reconstituted with sterile distilled water and usedat 50 Ilg/ml final concentration.

ACLBCGLCLLbbLdchLIT

American cutaneous leishmaniasis.bacillus Calmette-Guerin.localized cutaneous form of ACL.Leishmania braziliensis brazi/iensis.Leishmania donovani chagasi.liver infusion tryptose.

LmaLmmLPRNNNPBLPHA

Leishmania mexicana amazonensis.Leishmania mexicana mexicana.lymphoproliferative response.Novy, McNeal and Nicolle.peripheral blood lymphocyte.phytohaemagglutinin.

LEISHMANIA-SPECIFIC T-CELL LINES

TABLE I. - Summary of clinical data of 3 LCL patients studiedand in vitro LPR to Lbb antigen (Lbb-tot) after cure.

563

LCL Montenegro Time interval LPRpatient Age/sex skin test (*) cure-LPR test Lbb-tot Medium

(months) (dps) (dps)

1 28/F + 7.0 1059± 22 65 ± 12 53/F 22.0 693 ±297 82±463 48/M + 4.5 695 ± 175 69±26

LCL = localized cutaneous leishmaniasis.(*) Before treatment; a skin test was considered positive when the induration was 5 mm or more.LPR: represents 3H-TdR uptake expressed as disintegrations per second (dps) ± s.d. of triplicate

cultures.

Toxoplasma gondii (T. gondii) - Tachyzoites, passaged in mice, were harvestedfrom the peritoneal cavity and separated from the cells. Antigen was prepared, asdescribed below for total Leishmania-promastigote antigen. Leishmania antigens wereprepared from promastigotes of: L. braziliensis braziliensis (Lbb), L. mexicanamexicana (Lmm), L. mexicana amazonensis (Lma) and L. donovani chagasi (Ldch).Leishmania promastigotes were routinely grown in NNN supplement medium andTrypanosoma cruzi epimastigotes in LIT medium [5]. Antigens used to stimulate lym­phocytes were prepared as follows: total (tot) parasites were harvested, washed 3 timeswith phosphate-buffered saline (PBS pH 7.2), counted and disrupted by repeatedfreeze/thawing followed by ultrasonication (B15 sonifier, Branson, CT, USA). An­tigen concentration is expressed as the equivalent of the number of parasites/ml beforedisruption.

Soluble fraction (sol.) - The supernatant of tot-antigen was spun at 10,000 gfor 15 min. Protein concentrations were determined according to Lowry.

Antigens were sterilized by UV-irradiation overnight at 4°C, aliquoted and storedat - 20°C until use.

Generation and expansion of homogeneous Lbb-tot-stimulated IL-2-propagated T-cellpopulations (propagated T cells).

Mononuclear cells were isolated from heparinized blood samples by centrifuga­tion over Ficoll-Hypaque solution (Sigma, Chemical Co., St. Louis, USA) andresuspended in culture medium. The culture medium consisted of HEPES bufferedRPMI-1640 (Flow Lab., Irvine, Scotland), supplemented with penicillin (200 IU/ml),streptomycin (200 fLg/ml), 2-mercaptoethanol (0.04 mM/ml), 2 mM L-glutamine and10 070 heat-inactivated human AB serum. Cells (3 x 106/ml) were added in 1 mlvolumes (bulk cultures) to 24-well culture plates (Linbro, Flow Lab.). At the onsetof culture, 50 fLl of 1x 108/ml stock Lbb-tot antigen solution was added to eachwell. After 5-day culture in a humidified CO2 incubator at 37°C, the proliferatingblasts were isolated on a discontinous Percoll gradient as described by Kurnick etal. [9]. These blasts were expanded in 24-well culture plates, 1 ml volumes at3 x 105/ml in culture medium supplemented with 10 070 interleukin-2 (IL-2). Thesource of IL-2 was culture supernatant of phytohaemagglutinin (PHA)-stimulatedhuman tonsil lymphocytes, partially purified from PHA by passage over a Con­canavalin Sepharose 4B column [8]. Every 2 to 3 days, the growing cell cultures weredivided in half with fresh culture medium supplemented with IL-2.

564 J. HENDRIKS AND COLL.

The cells likewise expanded for 9 to 16 days following Percoll isolation beforefinal testing for antigen specificity and surface phenotype.

Proliferative assays of fresh peripheral blood lymphocytes (PBL) and propagatedT cells.

PEL.

Hypaque-isolated mononuclear cells were added to round bottom microtitre plates(Falcon, Oxnard, CA, USA), 3 X 105 cells/well in culture medium. Various antigenconcentrations (see «Results») were added in lO-fJ.I volumes and the cells culturedfor 5 days at 37°C in a humidified CO2 incubator.

Propagated T cells.

Expanded cells were harvested from bulk cultures, washed and cultured in round­bottom microtitre plates (3 x 104 cells/well) in culture medium for 4 days with an­tigen in the absence of IL-2. At the start of culture 1 X 105 freshly isolated gamma­irradiated (30 gy) autologous peripheral blood mononuclear cells were added to eachwell as a source of antigen-presenting cells.

Assessment ojproliferation oj PEL and propagated T cells.

Tritium-labelled thymidine (3H-TdR) (Amersham Int., Amersham, UK, spec. ac­tivity 5Cilmmol) was added (l fJ.Ci/well) 16 h before harvesting on a cell harvester(Titertek TM, Flow Lab.); radioactivity uptake was measured by a scintillationcounter.

Determination of cell surface phenotypes.

Surface phenotypes of propagated T cells were determined by standard indirectimmunofluorescence staining with Coulter clone monoclonal antibodies TIl, T4 andT8 (Coulter Immunology, Hialeah, FL, USA).

Briefly, 0.5 to 1 X 106 washed propagated T cells were incubated with 5 fJ.I ofmonoclonal antibody for 30 min at 4°C. After 2 washings, 100 fJ.I of a 1150 dilutedFITC-Iabelled goat anti-mouse immunoglobulin (GAM-FITC, Coulter Immunology)were added for a further 30 min at 4°C.

Finally, cells were washed twice, mounted on microscope slides and counted undera fluorescence microscope equipped with incident light illumination.

RESULTS

Proliferative response of propagated T cells to parasite antigens.

Figure IB illustrates the selective outgrowth of Lbb-specificpropagatedT cells after 5-day in vitrobulk cultivation with Lbb-totantigen, followedby expansion in IL-2-containing medium. While the subjects' initial PBL alsoproliferated well in the presence of other antigens (T. gondiiand BeG)(fig. lA), the propagated T cells only responded to Lbbantigen. Table II sum-

LEISHMANIA-SPECIFIC T-CELL LINES

TABLE II. - Antigen-induced response of Lbb·tot·stimulatedIL-2-propagated T cells derived from 3 cured LCL patients.

565

Stimulus

MediumIL-2 (10 070)BCG (50 fLg/ml)T. gondii (1 x 107/ml)Lbb-tot (5 x 106/ml)Lbb-sol (15 fLg/ml)Lmm-tot (5 x 106/ml)Lma-tot (5 x 106/ml)Ldch-tot(15 fLg/ml)T. cruzi-tot (5 x 106/ml)T. cruzi-sol (10 fLg/ml)

144245

100nd72ndndnd

246

LCL patients2

221002632

100697691ndnd

146

3

12591518

10082ndnd4759nd

Table represents percent of responses to homologous Lbb-tot antigens; dps values(100 070) for Lbb-tot antigens of patients 1,2 and 3 were respectively, 425, 400 and170; nd = not determined

marizes data on the antigen reactivity of three Lbb-tot-stimulatedIL-2-propagated T-cell lines derived from cured LCL patients. All cellsresponded in varying degrees to IL-2; no significant reaction was observedwith BCG or T. gondii, but there was apparent cross-reaction with L. m. mex­ieana, L. m. amazonensis and L. d. ehagasi. Surprisingly, the response toT. cruzi soluble antigen was higher than the Lbb-tot response in two cases.T. eruzi infection in mice has been shown to induce polyclonal T-cell activa­tion [13], but it is unlikely that this increased heterologous response representsa mitogenic effect, since normal PBL did not proliferate (data not shown).

tVo Of MAl RESPONSE

0 20 40 60 80 100 0 20 40 60 80 100

.EDIU. Jlll·TOT +UMII '-+--<

Ie, ~

·.m 8. PaoP-T CELLS

FIG. 1. - Proliferative responses of PBL (A) and (Lbb)-selected IL-2-propagated T cells(prop-T-cells) (B) from cured LCL patient nO 1 to Lbb-tot, BCG and T. gondii antigen.

Indicated are the percentages of maximal response (max. response was 1905 disintegrationsper second (dps) for PBL and 425 dps for prop-T-cells. Prior to this antigen restimulationtest, the propagated T cells were cultured for 16 days following Percoll gradient separation.

566 J. HENDRIKS AND COLL.

Cell phenotype of Lbb-selected IL-2-propagated cells.

Table III shows that the majority of expanded T cells belonged to the T4-celllineage in all three individuals. In contrast, PHA-stimulated IL-2-expandedlymphocytes from controls and cured patients exhibited an average of 55 %T4- and 45 070 T8-positivity (data not shown).

TABLE III. - Surface phenotype of propagated T cells.

LCLpatient

123

Tll+(%)

85 (*)9387

878179

T8+(010)

447

Table shows percent positive cells out of 200 cells counted.(*) = determined with sheep red blood cell rosetting method.

DISCUSSION

The study of specific T-cell lines and clones in murine leishmaniasis hasprovided a better insight into the cell-mediated immune mechanisms deter­mining the outcome of leishmania infection [11, 15, 16]. This report describesthe first isolation of short-term T-cell lines from patients with Americancutaneous leishmaniasis caused by L.b. braziliensis.

T-cell populations were derived from patients clinically cured at the timeof study with positive LPR (table I). Previous data from our laboratory havesuggested that antimony treatment may indirectly enhance a Lbb-specific T-cellresponse leading to cure and long-lasting immunity [12]. It is conceivable thatin vivo cells, similar to the Lbb-selected and propagated cells described here,are to a large extent responsible for immunity in the individual. Surfacephenotyping showed that cured LCL patients develop predominantly helper/in­ducer cells when stimulated with Lbb (table III). Extensive cross-reactivitywas observed with other Leishmania species and subspecies as well as withT. cruzi. Similar results of cross-reactivity between Leishmania and T. cruzihave been described in humoral responses [1] and cellular reactivity of bloodlymphocytes [4], but a higher heterologous response to T. cruzi has not, toour knowledge, been previously reported. The observed cross-reactivity couldreflect the occurrence of dual infection in the patient resulting in mixed T-cellclones of different specificities in our lines. We consider this explanation unlike­ly because (1) the technique applied readily selects Lbb-specific T cells evenif precursors to other antigens were present in the original peripheral blood

LEISHMANIA-SPECIFIC T-CELL LINES 567

(fig. 1), and (2) T. cruzi transmission has never been recorded in the area wherethe patients live. Thus, although preliminary, these data indicate the existenceof highly conserved antigenic determinants between these two species, whichare recognized by T cells,

Interestingly, Sheppard et al. [15] reported that cloned murine T cells exhibitsimilar cross-reactivity between Leishmania species and African procyclictrypanosomes. An intriguing question arising from these data is whether awell-mounted immune response to Lbb antigen could protect against T. cruziinfection. This possibility is currently under study in our laboratory on a mousemodel.

In conclusion, we have described conditions for developing human Lbb­specific T-cell lines from PBL of cured LCL patients. We postulate that similarcells may, in vivo, playa dominant role in conferring long-lasting immunityupon the individual. Preliminary data indicate that these cells derived fromcured LCL patients recognize a common antigen between T. cruzi and Lbb.

Further studies with cloned T cells and highly purified Lbb-antigen frac­tions are required to define the nature of these immunodominant and cross­reacting antigenic determinants (in preparation).

RESUME

PRODUCTION DE LIGNEES DE CELLULES T DE COURTE DUREESPECIFIQUES POUR LEISHMANIA A PARTIR DU SANG PERIPHERIQUE

DE PATIENTS GUERIS DE LEISHMANIOSE CUTANEE AMERICAINE

La n~ponse immunitaire probablement protectrice contre la leishmaniosecutanee localisee (LCL) due aLeishmania braziliensis braziliensis (Lbb) a eteanalysee apres production de lignees de cellules T de courte duree a partirde lymphocytes du sang peripherique (PBL) de trois patients. Tous avaienteu des lesions cutanees actives mais etaient gueris au moment de cette etude.Les PBL ont ete stimules in vitro avec des promastigotes de Lbb traites pardes ultrasons; apres 5 jours de culture, on a isole les cellules blastiques puison les a cultivees pendant 9 a16 jours avant de tester leur capacite aprolife­rer: les cellules sont en majorite T4 + et sensibles aux antigenes de Leishma­nia. Vne reaction croisee a ete observee avec des antigenes solubles deTrypanosoma cruzi. Bien que preliminaires, ces resultats indiquent que lescellules T immunes obtenues apartir de patients gueris de la LCL, sont capa­bles de reconnaltre des antigenes communs a Lbb et T. cruzi.

MOTS-CLES: Leishmania braziliensis, Trypanosoma cruzi, Antigenicite,Lymphocyte T; Lignees de cellules T de courte duree, Phenotype, Leishma­niose cutanee localisee.

568 J. HENDRIKS AND COLL.

ACKNOWLEDGMENTS

Partially supported by the UNDP/World Bank/WHO Special Program forResearch in Tropical Diseases (TDR), the Brazilian National Council for ScientificDevelopment (CNPq) and the Financing Agency for Studies and Projects (FINEP).

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