Characterization of Primary T Helper Cell Activation and T Helper Cell Lines Stimulated by Hapten-Modified, Cultured Langerhans Cells

Characterization of Primary T Helper Cell Activation and T Helper Cell Lines Stimulated by Hapten-Modified, Cultured Langerhans Cells

characterization of Primary T Helper Cell Activation and T Helper Cell Lines Stimulated by Hapten-modified, Cultured Langerhans Cells Conrad H auser, M .D.! Wayne M . Yokoyama, M.D., and Stephen 1. Katz, M .D ., Ph.D. Derm.atolog-y Branch. National Cancer Insritute (CH. SIK). and ubor2tory of Immunology. National Institute of Allergic and Infectious Dise.ases (WY), Nation.allnstitutes of Health, Bethesda. Maryl.and . U .S.A. It has recentl y been shown that hapten-modified cultured ation induced by cultured Langerhans cells. Restimulation of Langerhans cells are "ble to activate SInaB resting syngeneic in vitro primed T helper cells with hapten-modified cultured L3T4+ T helper cells from nonsensitized animals. Repeated Langerhans cells revealed the prese nce. within the primed T stimulation of these T cells with hapten-modified cultured helper ce ll population. of activated cells with specificiry to an Langerhans ce lls leads to the establishment of L3T4+ hap­ unrelated hapten, suggesting that, in hapten-dependent T ten-specific interleukin-4-producing T-cell Jjnes. Here we helper cell activation, hapten-nonspecific cells are activated report on further characteristics oflrimary hapten-depen­ along with those that are hapten specilic. Restimulation of a dent activ",ion of L3T4+ T cells an ofT-ceil lines derived hapten-specifi c long-term T helper cell subline using differ­ from rhem. Dendritic cell-enriched spleen cells were as able ent antigen-presenting cell types demonstrates that factors as Langerhans cells to activate nonsensitized T helper cells other than major histocompatibil ity complex class II density after hapten modification. However, M12e, a major hisco­ or ti ssue derivation of (he antigen-presenting cell playa role compatibility complex class U-positive B-ce11 line that was in tbe acti va tion of T cells in vitro. Finally, we demonstrate able to activate small , resting, allogeneic L3T4+ T cells was that in vi tro generated bapten-specifieT he1per ceJllines may not able co stimulate syngeneic T helper cells after hapten nor show strict major histocompatibility complex restriction. modification. Thyl + dendritic epidermal ceJls did not signif­ J !til/esc Dermaro/93:649-655, 1989 icantly affect the ma gnitude of primary T he1percdl prolifer- e previou~ly reported that c.ultured epidermal to major histocompatibility complex (MHC) class U antigem. L3T4 Langerhans' cells (cLC), after hapten modifi­ and LFA- l . In a subsequent series of experiments. we demonstrated cation, ue capabl~ of stimulating the prolifcr­ that L3T4+ Tb lines generared by repeated srimulation with hap­ ar.ion of sm"ll. resting, L3T4+ T helper cells ren-modifled cLC product' inrcrleukin 4 bur no detectable interleu­ (Th) from nonsensjtized animals! I]. Primary kin 2 (1L-2) [2]. Furthermore., these Th lines were able to stimulate Whapten-induced Tit proliferation was de pendenr on rhe presence of IgE produccion in smaJl, resting. nonprimed B cells after cognate specific Th within the un primed Th population: in addition. upon inter:tction [2J. resrimuiation with hapten-modified spleen cells. rhe in vitro In rhe present repon. we compare cLC to other antigen-present­ primed Th population responded in a h.apten-specific manner. Pri­ ing cells (APC) in the primary hapten-dependent pcolifcr4tion mary napten·dependenr Th proliferation was blocked by amibodies assay. as weJl as in resrimuiation assays using previously activated T h. Furthermore. we describe rhe effect of T hy 1+ dendritic epider­ mal cells on primary Th prolifeution. W e demonstr'l[e the exis· tence of " nonspecific" Tn activation that accompanies primary Ml-nuscripl received December 22. 1988: ~ccepted for publication June specific hapten-dependen[ T h activation. We ~so show [har hap­ 19. 1989. ten-specific Th lines generated in virro with cLC lack classical Reprint requests to: Dr. Stephen 1. Katz, Buildi ng 10. Room 12N238, MHC resrriction. Finally. we present a Th subline that responds to National Instirutes of Health, Bethesda. Maryland 20892. Presc:nted in pan 2.t the Annu2.1 Meeting of the Society of Investigative cLC and dendritic cell-enriched spleen cdls but not to spleen cells Dermatology. San Diego, May 4 -6. 1987. and the Annu al Meeting of the or M 12c ceJls. European Sociery for Demlatological Research, M.unich,June 20-22, 1988. MATERIALS AND METHODS ' Perm:ment addrcs~: Clinique de Dermarologie. Hopiul Cantonal Uni­ ven;it2ire, 1211 Geneva 4. Switzerland. Preparation ofTb Depleted of Autoreactivity As previously Supported in part by a grant (rom the Schweizerische Stifrung fur medi· described II J. nylon wool nonadherenr lymph node cells treated ziniKh-Biologische St.ipendien. wirh anti-MHC class n (M5/114.15.2); American T ype Culture Abbreviations: Coll.ecion [ATCe]. Rockville, MDJ. anri-Lyt-2 (53-6.72; ATCq, APC: antigen.presenring cell mouse anri-rat kappa chain (MAR 18.5; AT CC) antibodies. and C' cLC: cultured epidermal langerhans' cells 1L.-2: interleukin-2 (Cedarlane. Hornby. Ontario) wer~ cultured a.r 6 x 1()6 cells(well 6 FITe: fluorescei n isothiocyarute with either 0.3 x 10 synge neic eLC prepared :as described 1J or MHC: nujor histocompatibility c.omplex 0.5 X t ()6 miromycin-trea.ted syngeneic M 12c, a B-cdlline derived Th: T helper cells from rhe la-bearing M 12 B e<lI lymrhoma [3J (kindly provided by TNP: trinirrophcnyl Dr. Ronald Germain, Laboratory 0 lmmunology, NlAID, NTH. 0022.202X/ 89/S03.50 Copyright CO 1989 by The Society for lnvcstig:nive Dennatology. Inc. 649 650 HA USER IT At THE JOURNAL OF IN VEST IGATIV E DERMATOLOGY Bethesda, MO) in 2 ml culture medium, Culture medium consisted incorporation of radioacti vity into ce ll s. Results are given as means of RPM! 1640 (Biofluids , Rockville, MD or Gibco. G.-and Is land, oftriplicate cultures ± 1 SEM , NY) containing 10% feral calf serum (Biofluids). 100 U / ml peni­ Preparation of cLC and Thy 1 + Dendritic Epidermal T Cells cillin (Gibco), 100 pg streptomycin (Gibco), 2 p g/ml fu ngi.one cLC werc prepared as described [1]. Epidermal cells prepared from (Gibco); 5 X 10-' M 2-mercaproerhanol (Sigma, St. Loui., MO), 50 trunk skin of C3H mice by trypsinization for 40 min ar 37"C [4] I'g/ml gontamycin (Gibco), 2 mM glutamine (Gibco). and 1 pg/ ml were spun over a Ficoll gradient (3 parts 9% Fico1l 400 [Pharmacia] , indomcrhacin (Sigma). After 4 d of culture. acrivated Th cells were 2 partS Hypaque 90% [Winthrop, N ew York, NY]). The interface el iminared using bromodroxiuridi ne, bisbenzimide, light irradia­ cells were washed and incubated with anri-class I (36.7.5) and anti­ tion, anriIL-2 receptor antibody, and complement. described [1 ]. as class II (10.2. 16; both kindly provided by Dr. D. Sachs, Immunol­ T he remaining nonacriv3red Th were used for primary Th acriva­ ogy Branch, N C t, N IH) monoclonal antibodies and then trea ted rion experimencs. with low rox M rabbit complement (Cedarlane) J : 10 for 90 min. T hereafter. the cell s were washed and spun over a Ficoll gradient (as In Preparation of Vivo Primed Tb Animals were painted with above). The interface cells were washed and used for proliferation 100.ul 7% rrinit['ochlorobenzene (4 : 1. acerone:olive oil) on the assays. T he ce ll s were 30%-60% Thy1+ as assessed by indirect im­ abdominal skin. Five to seven days later. the animals were killed. munofluon::sce nce with an FIT e -conj ugated ami-T hyl antibody Lymphocytes from inguinal, axillary, and subscapular lymph nodes (Becton Dicki nson. Mountain View. CA). were prepared. After nylon-wool purification. the cells were treated with anti-MH C class 11 (M5/114.15.2) and anti-Lyt-2 (53-6.72) Flow Cytom etric Analysis of C lass n Intensity M1 2c, cLe, mouse anti-rat kappa chain (MAR 18.5) monoclonal antibodies and and dendritic ce ll -enriched seleen cdls wert" incubated with FIT C­ C'. The resulting ceUs were referred to as in vivo primed Th. conjugated MKD6 (anti-I-ACI) or 10.2.16 (anti-I-Ak) or with uncon­ j ugared anribodies and an FITC-conjuga red goat F(ab)2 anti-mouse Preparation of D endriric Cell-enriched Spleen C ells Spleen IgG (T ago. Burlingame. CA). The washed cel ls were then .analyzed ce ll s were incubated on plastic petri dishes (3 spleen/t O-em diame­ in a fl ow cytometer (Becton Dickinson or Coul ter. Hialeah, FL). ter plate: Costar. Cambridge, MA) in to m! culture medium. After Dead cel ls were excluded by propidium iod ide staining. T en rhou­ 90 min~ nonadherent cell s were removed. and the plate was rinsed sand ce ll s were scored for each sample. Results are given as number two times with fresh culture medium. The plates concaining fresh of events on the y axis and relative flu orescence intensity or flu ores­ culture medium were then incubated overnight at 37"C and 5% cence channel on the x axis. CO , The following day nonadherem cells were removed and spu n 2 RESU LT S over a 50% PercoII gradient (Phannacia. U ppsa.l a, Sweden) at 2000 g fo r 15 min at 4 "c. The interface cells were recovered and washed Comparison of cLC , M 12, and Spleen Cells Enriched fo r three rimes. D end ritic Cells in Primary Lymphocyte Proliferation and Lym phocyte R estimulation Assays Th depleted of autoreac­ Proliferatio n Assays Spleen ceJls. dendritic cell-enriched spleen tivity as describcd [tJ using M 12c ce lls were testcd in a primary ce lls. mi[Q m yci n~ r r eated M l 2c.

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