MSU R_E]'URNING MATERIALS; P1ace in book drop to ”saunas remove this checkout from ”- your record. FINES will be charged if book is returned after the date stamped be10w. I filafifi W"), 97341;? ‘3 I u s v». Ammmohz SUPPRESSION'OF'HUMBN’T’IEMPHDCYTEIRESPONSES.BYIIIXQQQQ§QEQ_QIgzi By LisaArmBeltz A_DISSERTNTION Suhnitted to Michigan State University in partial fulfillment of the requirmmts for the degree of DOCTOR.OF'PHIICSOPH¥’ Department of Microbiology and mblic Health 1988 Am WIWOFWTWRESWBYW BY LisaAnnBeltz Wisaparasiticpmtozoanofmanmidicausesa debilitating and often fatal disease whose early stages are accarpanied bydecreased inmme reactivity, the extent andunderlying causes of midiweremflcncwn. Inordertoaddrssstboseissues,weutilizedan mmmwhidamimpmastigcteswemcc-wlunedwith mrmaltnmanperipnralblocdwmnlearcellsflflfl). Inthis systan, m1 reduced PHI: proliferation following stinulatim by severalmitogeniclectinscrantibcdiestoeithertbeTcellreceptor cmplexcrwz. Msreductimmsmtduetomadequatelevelsof nitrientsormitogensortoalossofPBCViabilityaftercc-mlm withtheparasite. Walsoirhibitedthegrwthofseveralhrt mtallinmortalizedcelllines. milemaocytesweremtrequiredfor decreased pm: responsiveness, parasite viability was necessary. Similarresultswereddtairedmmmsseparatedfmcellsby a Millipcre filter, demmstrating that amessim occurred via a factorsecretedbytheparasite. Maximalirhibitimwasmtedmly “immiwasaddedtowltureswitlfinuhrofstimlatim; therefore, early stages of activaticn were affected. Interleukin 1 and interlwkin2(n2)arepmductscfstimlatednnnocytesarchells, respectively, required early during '1' cell activation. Following optiml stinulatim of lumen PRC, production of theselynuaokinesardinterferurrwasmaffectedbymimfleume LisaAnnBeltz parasitedecreasedIIZandinterferm-rprcductimbymse splaaocytes. IL2 restoredproliferatim of amressednmsebut not mmnlymaocyts. 'meimbilityoftnman'rcellstorespaidto adogenwsorexogenmsflZconelatedwithirhibitedexpressimofIlz receptors. BoththenmberofcellsbearingIIZreceptorsardreceptor dersityweredecreasedbymiwithinnhr. Iowandhighaffinity receptorswerebcthaffected. 'Iheexpression of T113, anearly activatim narker, and the transferrin receptor, a growth factor receptor appearing late in activatim, were also irhibited bym whileEAl,theearliestrepcrtedactivatimmarkerochells,was unaffected. SuppressimofmmanTcellfimctimsbyLmiistms selective,withthekeyeventslyingmtinalteredlynfiiokine productimhxtratherindecreasedenqnessimofcmcialeactor receptors. COpyright by Lisa Ann Beltz 1988 'Ibmyfather, whoselifehasbeenmyinspiratm, arrimynnuaer,mhelpsdnetotransfommydreansintoreality IwufldliketothankDr.FelipeKierszenbarmforhisguidancearri patiencedm'ingtheyearsIspenttraininginhislaboratory. He believedinmeandgaveneencanagementandsmportwhenthe irevitableproblarsarose. Iwishtothankthemenbersofmyguidamecaunittee,nrs.Jeffrey Willians, Panela Fraker, Walter Esselman, andJchn Wang, fortheirtime ardeffortsmmybehalfandforhelpingmetodevelcpabroaderview ofscienoe. Additiaaally,Iwishtoexpressmygratiufletobdo researdxerswithwl’m I was fortunate to collaborate, Dr. Gerald Samenfeld of the University of Iouisville and Dr. Maroelo Sztein of GeorgeWashingthniversity. Aspecialwordoftharflcsnustbegiven toDr.Szteinformtonlyreadingthecamtlessnmberofsanples validilsattohim,h1talsoforhissenseoflnmorandhis stimlatingcmversatims. IwanttothankDrs.Ju1iaWirth,AlfredAyala,ardMarkm1el1y fornany interestingdiswssions and occasionally, for the use of their naterials,mtnostinpcrtantly,fortheircanaraderie. Imstalso expressmyamreciatim fortheexoellenttedmiml assistanceofur. James Kidder, Mr. William mgan, Mrs. Patricia Hoops, mums. Lisa Santangelo. lmeextentoftheircontributionhecclnesmoreapparent daily. Anothergroupofpeopletowtmlgivemyvarnesttlmficsaremy blood donors who literally gave of thanselves for this work. vi Mofmyasscciatesdeserveqaecialnentim. Drs.Fernando VillaltaarrlMariadeFatimLimwereinstnmentalinshapingneasa researcher. 'meir love and dedication to science were highly contagiom. 'mey,anitheirdaughterRadxel,havebeenasasecarl familytoneardthedebtwhidllowethanmnneverberepaid. Iamdeeplyirdebtedaswelltomyfamily-mymther,mybrother, arrinygrandparents-miosesqportneverwavered. Wiflnxttheirlcve andencmragatent,nygoalsvmldhavebeenattainablebuteupty. Fimlly,IgivemydeepsstttmflcstomyGod,mioistheultimate sanoeofalllomledgeandstrength. mummismrkwasdone, anitoflim,1mwdedicateit. vii MOFGNTENIS Page List of Fignes ...... xi Listof'l‘ables ........................................ .. ...... xii I. W: anoverviav ..... .. .................... 2 II. Immosuppressim causedl:yj[,__<,;r3,1z_;iI ..................... 4 III. Ancverviav of'rcell stimlation .................... 8 IV. Hmnanirrterlafldnz ................ .............. ........ 13 V.'Iheinterlaflcin2receptor(IIZR) ..... 15 VI. Interferm-‘r ......... . ....... ............ 20 VII. Rosearchgoals .. ..... 22 VIII. Reference . ........ ..... 25 omits-r1 Wm! ofmmnWWbyW 47 Abstract 48 Introduztion ................... . ...... ......... 49 Materialsanduethods ........ 50 Results ....... . ............ ....... 55 Disalssim .. ....... ......... ............ ........ 67 Refererms ........ 70 viii Chapter 2 Page Inhibits Interferm-r Productim by mmleenmllsbutmtmmanmrifiaeral Blood W0.0.0.0.....0.........OOIOOOOOOOOOOOO00...... ..... 72 Introductim .. .................................. . ..... . ...... 75 Materialsanduethods . .............. . ........................ 77 Results .... ..... .............. ....... . ...... ... .............. 80 Disamicn ...... 86 mfemxces ......................... . ...... ....... 90 Gupta-’3 NwellbdmimforWi-fiflmed alppression ofI-hnnanlynpxocyte: Inhibition of Interlaakin 2 Receptor Abstract ....... . .......................... . .......... 95 Materials andMethods 99 Results ........... ............. ........ ...... . ............ ... 102 Mm ................................................... 118 W4 Wimthemqnessimofaoflime InterlaflcinZard'I‘rarzsferrinWorsmtmtEAl, anEarlyMar-keroflyngzocytehctivatim 121 Abstract ..................................................... 122 Haterialsarduethods ....... 125 Ranks ...................................................... 128 Discussim ................................................... 134 References ........ 138 ix Chapters Wion of the (1)2 Pathway of Hunan T Cell Activation by W 0.000......0.0.00.0.........OOOOOOOOOOOOOOOO mterialsarriuethods........ ..... msultsuuunw ...... . ..... ......... Discussion....... ........ ............ ............... . ..... ... mfereaces ... ............ .. ...... AppendixI ' Mediates its alppressive Effect via a $1L1b1e mmr 0.00.00.00.00I.........OOOOOOOOOOOOOOOO0....... AppendixII Inhibits the Growth of Several but not all Wim @1111” 00.0.0000.........OOOOOOOOOOOOO... W m WWII“ ......O.......OOOOOOOOOOOOOOOOO.......... 142 143 144 146 149 152 156 159 168 175 LIST OF FIGJRES Page Effects ofmi on 112Reaq>rossimbytnman lynphocytes 105 Table IISTOF'EBIB Chapterl Suppressim of Con A-induced PBJIC resporses by blood fm Ofm I.0....OOO......OOOOOOOOOOOOOOOOOO... Suppression of PHI: responses irriuced with subcpt- inal, optimalarrisupraoptinalcmcentratims of OmA, PHAorPWbyblood forms ofmi Mitogenic capacity of Con A solutims before and after absorption with a suppressive concentration of m ....0.0......OOOOOOOOOO......OOOOOOOOOOOOOOOOO Ability of RHII+5%F$ medium to support Con A-induced Effects of addition of mi at different times ammmafimflmuAOOOOOOOOOOOOO ..... I... ReducedproductimofIIZbyPflcirnlbatedwith m1 ......OOOOOOOOOOOOOOOOOI......OOOOOOOOOOOOO... Failure of exogenous Imp}.l to restore Oan rm- Sim Of m ...00.000.000.000...0.00.00.00.00...O. Chapterz mi—iMJced idiibitim of IFN-r prodnctim by ”WM......OOOOOOOOOOOO......OOOOOOOOOOOO IackofrestoratimbyexogemusIFN-r oft-hemp- acity of BIA-stimulated use to proliferate after Lack of effect of m on IFN-r prochctim w m .0.0...O.........OOOOOOOOOOOOOOCO......OOOOOO. 56 57 59 60 62 63 65 66 81 83 85 Table Page (hapter3 1 mi-ixfliced suppressim of 112R expressim by 1M .......................O................... 103 2 Effectsofmmthecapacityofrnman lynfiiocytstoincorpcrate3H-thymidihearrisecrete minrospmsetostimlatimwithHIAoranti-Cm 106 3 Effects of W m 3IwI--tl'xym.i.dine incorporatim ammgqaressimbymmnlynphocytes stimlated withHiAoranti-CIB inthepreserceorabsenceof exogenusIIZ ......... ........ .. ........ ......... ..... 109 4 RestoratimbyexogeIanIIZOfthecapacityofmse butmtmmanlynphocyterespmsss (3H-thymidine imorporatim)a1;pressedby1,_qmzi ............... 111 5 mprodnactimbytnmnmonocytesWinthe presexneorabsenceofmi. ................... 112 Chapter4 1 'meeffectofmmtheemessimofthem wmamm..... ......... . OOOOOOOOOOOOOOOOOOOOO 129 2 'Iheeffect ofmionthebinding of 1251412 to the ILZRurrlerhigh affinity conditions 130 3 Iackofeffectofmmtheacpressimofm wstimawm......O............................. 132 4 'nneffectofmimfleapressimofflem wmwm...................................O 133 Chapters 1 mimusblastogenesisbybcmuxe'rcell receptorandanpatmays ......... 150 2 'meeffectofmimMprodwtimardImR expressimafterstimlatimbyeitheronr mm ........................ ........ ....... ....... 151 Appendixl 1 mamasesmmnmcproliferatiminflae absane of direct cartact with the cells 161 2 'Ihesuppressive affectofSSFisreversible........... 163 xiii Table WofPBCtotheSSFdidmtiflfibitm IIZRexpressimisdecreasedbytheLmiSSF...... WII . WideneasesfleMOfCIIL-Zarfimfl “1.1 11” ......OOOOOOOOOOOOOOOO......OOOOOOOOOOOOO0.0 mi does not effect the ability of 1171' 10282 cells to proliferate or express ILZR ..... Page 165 166 171 172 (hlA RHII + S ERIE-2.23173 RBII+5%F$ ABBREVIATIGIS Ocrxzanavalin A counts per minrte early activation antigen 1 fetal bovine serum interlalkin 1 interlaxkin 2 crude human interleukin 2 purified Imman interleukin 2 interlaflcin 2 receptor crude rat interleukin 2 interferm-r lynphocy‘te functim—associated antigen 2 lynphocyte fixation-associated antigen 3 lipopolysacdaaride mandemaelnmberofthelogarittmofthefluoresence intensitiesdeteminedbyflowcytanetry msespleencells lumen peripheral blood noncruclear cells {magnate-buffered saline cartaining 1% bovine serum albumin Wlutinin WNW RHEIMOmdimcartainiJgFBS malaOmedimcartainimz.S%m mul640nedimcartainimfirm XV RHE+10%F'BS RM 1640 medimn containing 10% FBS SSF secreted suppressive factor Tm uansferrin receptor mama: INIKJIIJCI‘IW I. W: an overview W is the henoflagellated protozoan which is the causative agent of Chagas' disease. Fifteen to twenty million people areestimatedtobeinfectedwiththisparasiteardanadditional forty to forty-five million are at risk of acquiring the infection (1) . milefllevastmajorityofulecaseshavebeencmfinedtothetrqaical andsubtropical regimeofSouthardOentralAnerica, severalreports havedanmstratedinstarcesofmmaninfectimacquiredinthevnited States (2-4), where ahighperoentage of intermediate invertarate and vertebmtemstsharborirqmihasbeenfandinsaegeogramical regions (5). Chagas' diseasecanbedividedintothreerhases: aarte, latent, andchronic. 'Iheearly, aartephasenaybeasynptaraticandocclrs nostfrequentlyindlildren (5). Diagnosisnaybeuadebythepresence ofan irduratedskinlesim (chagana) oranmlilateraledenaofthe eyelid, conjmlctivitis, and enlarged satellite 1m node (Ratana's sign) (5,6). Parasitemia may also be delrmstrated at this time and diminishes within two to three months. Other possible nanifestations include fever, hepatcsplaunegaly, lynfllaielnpathy, lynrhocytosis, m; alterations, heart failure, and nenirqoencefllalitis (5,6). Mortality duringtheacutestageof infectionisfivetotenperoent (5,7). 3 Afteralatentpericdofvariablelength,lastinguptouventyor thirtyyears,aperoentageofttnseinfectedpassintofllenoresevere, chrmicstageofthedisease. misstageischaracterizedbydamageto the cardiovascular systan (myocarditis, cardiac failmre) or the digestive system (megacolon or megaesqhagus), aswell as nervous tissue(5,8-10). 'Ienperoentofthedeathsanalgadultsmybedueto dlraiicChagas'diseaseinsateregiasofOentralaniSaJthAmerica (11). Most of the early descriptions of parasite norphology and life cyclearetheworkofCarlosclagasueviededinS). 'Ihelifecycleof thispamsiteimmlvestransmissimbetveenaninvertebratehostofthe family Rediviidae,‘ aibfamily Triataninae, and a vertebrate host. A widerangeofnamalsserveasalitablehosts: theseincludeman, danestic aninals, androdents, asmllassylvatic reservoirs. Anphibians andbirds are refractory to infection (12,13). Infectim of themalianhostbeginswtmtheelalgated, flagel- latednetacyolictrypmastigotesfranflleinsectfecesarembbedinto mlccsaorfllesiteofareduviidbite. 'metrypanastigotesinvade nearbyoells (especiallythoseofthemlartissuesorthereticllo- eniothelial systan)andt.ransfomintotheamastigote form. the latter mltiplybybinary fissioninthehostcell's cytoplasmandthen transform into nadividing bloodstream trypanstigotes which are releasedfrunthehlrstinghostoell. ‘Ihesetrypanastigotesmyeifller imradeothercellstocmtinlethenamaliancycleornaybeingested byareduviidbugduringabloodneal. Inthevector'sminbrt, trypcmastigotes transform into epimastigotes, the dividing form in the 4 insect. Afterpassagetothehinigut, theepimastigotostransforminto the infective metacyclic trypanastigotes. II. Inmmosuppressimcausedbym Boththecellulararrihmnoralarmsoftheimmesystanplay inportantmlasinmstdeferseduringthelatentarddmflcmasesof dagas'diseaseueviadedinSandM). 'Iheearly,acrtestageof infectiminhmnansandmice,however,isdlaracterizedbyastateof specific and norspecific immoalppression. 'Ihis condition is not unique tom infection, occurring in several otherparasitic diseasesaswell. Severalreportshavedemonstratedtheocwrrenceof amessedcell-nediatedrospmsssinmmarsduringtheawtemaseof thedisease(15,16). 'Ihisphelmetmisaccatpaniedbyanincreasein theabsolutenmberofaB+Tsupprossor/cytotmciccellsarriadecrease inthemnberof cm+Thelper lynphocytes (16). cellular inuunity,as measured by lynphocyte blastogenesis and delayed-type hypersensitivity reactions, remrnstonormal levelsdur'ingthedmlicstage (17-19). Shriiesofthemderlyirgnednnism(s)ofthisacltemaseimnnsq>- pressimhavemtbeenmriertakalinmmaninfectim,perhapsduein part to the difficulty in obtaining and/or diagnosing patients during thispaaseofthedisease. ‘ 'nle vast najority of studies of W-inmced immlcsuppression haveutilizedthenousenodelswten. Cell-mediatedimmnem areirhibitedinmiceduringtheaalterhaseof infectim. Splenocytes frunthesemiceeldiibitdecreasedblastogenicrospmsestotheTcell mitogensccncanavalinAwonMandmytdlatagglutinin (PHA)anitothe 5 B cell mitogens lipopclysaccharide (IPS) and dettran sulfate (20-27) . Partialtocmpleterecoveryoftheserespmsesocclrsduringthe chrmic phase (24-26) . Proliferative responses to trypanosanal antigelsarealsoirhibitedduringtheaartebltmtflledlrmicstage ofthedisease inthenoderatelysrsceptibleCBlVJarflresistantCS? BIL/6 mice (25,26), whereas in the more alsceptible (Bu/Hal mice, the suppressionexterls intothedlronicmase aswell (26). Inadditicn to decreased proliferative responses, '1' cells frun ELM-infected mice are also defective in providing helper activity to B lyuphocytes (28). When either epimastigotes (27,29) or bloodstream trypatastigotes (29,30) are added to clltm'es of splencytes fran uninfected mice, thereisasimificantreductimintheblastogeiicrospmsetoConA andLPS. ‘Ihisdecreaseisproducedmlywheitheparasiteispresert during the initial 24 hours of stinulation (29,30), suggesting that the summive event occurs at an early stage of lyn'prlocyte activation. W also inhibits the delayed—type hyperseisitivity reaction to skin selsitizing agerts (21,31) and trypanosanal antigen (21,32) duringtheacrtephaseofthedisease. 'nleinhibitimintherespaee tomiantigexspersistsintothedlronicfiiasewhile repmsiveness to an unrelated antigen is restored (33). Spleen cells franacutelybutnot frundarmically infectedmicearealsounableto produce migration infibitory factor 111113219 (32,34). 'memnnoralarmoftheimmsystenisalsoaffecbedbym infection. wlelocytes frun acutely infected mice display deficielt names of plan-forming cells (PFC) to both T cell-dependent 6 (heterologous erythrocytes, trinitrophenyl—bovine serum albumin) and -irdepe'dent (di- and trinitrqhenyl-Ficoll, W. IPS) antigeisinm (26,35-41). 'Ihedecrease in Ingutnot IgMPI-o persists well into the chrmic phase (40,41). Both primry and seccndarngGrsspmsesareaffected, milemlytheprimrylgu respmse is reduced (36,38). A restriction in the IgG isotype profile intheseraofdirmicallyinfectednicehasalsobeelmted: the predanilant isotype being 1962, with deficielt production of Ign and 1963 (42). Ammberofnedlanienshavebeelsuggestedtoplayacausative roleintheabovemtedimmosqprsssim. Severalreseardiershave reportedthepreselceoprpressorTcellswhidldecreasedTandB cell proliferation (22) , delayed-type hypersensitivity reactions (33) , ardIngroductim (40). otherworkers, however, haveshownthatthe rerovalofIytZJor'Ihylpositivecellsdoesnotleadtoadecrease in suppressive activity (20,25,43,44) . Another cell type whidl has been demonstrated to play a role in I‘M-1m immosurpressim isthesuppressormacromage, whidlhasbeelshowntodecreaseblasto- genie responses (20,26,39,45) and the amber of PFC (39,46). Indoueth- acinwasstmntoincreaseresponsiveiess (45), suggestingthe involvenentof PGEZ. Othernacrcphage fmctionswhidlarerequired for immereqaonses, Masantigentptakeardpreseltatim, epressicn of major histocmpatibility couple: MIC) antigels, and release of interleukin 1, are not altered by m infection (35,36,47). Decreased nmbers of splenic T cells (24,44) and polyolmal activatim of B (38,48,49) and '1' (49-51) cell responses, leading to clonal 7 depletim,havealsobeenalggastedtohaveapartincausingthe immcsuglressim. Interlefldn2(112)productimisalsodecreasedin stinulated splelocytesfraninfected mice (47,52). Sincethis lynphokineplaysavitalroleinboth'l‘arrchellrespmsesfiee Interlelkin 2),thedecreaseinIL21evelsmaybepartially responsible for the deficiency in lynphocyte rospcnsivenss. Various soluble factors have also been suggested to play a role in I‘m-m imn'lcdeficiercy. The first and: factor to be reportedwasfoundintheserumof acutely infectedaninals (37,50,53- 56): thismrkwasnotreproduciblearrlwaslaterretractedbythe authors(57). Anotherfactorwasreportedbythisgrwpofreseardlers tobepresertintheallblresupematantofsplencytesfrminfected mice (54,58) arriactscnlymsyngeneic splency‘tes. Recently, amt-her suppressivefactorfrunthesemltnempenxatantstasbeelrepcrted (59). this factor has a molecular weight of 14 to 15 Rd, a pI of 6.6, ismthaplotype-restrictedandisbelievedtobeofhcstcell origin. Finally, allmresofinfectedsplelocytesarereportedtoproducea suppressive factor when incubated with epinestigotes, trypmastigotes, crthe 104,000 xg supernatant fraction of epinastigotes (60). SeveralattetptshavebeeinadetooveromeW-ixduced immosugpressim. Since 112 producticn/secretim isdecreased in spleelcellsfraninfectedniceardthislynfixfldneisremiredforT cell proliferatim as well as for B cell differentatim (see Inter- leflan),severalgrwpsofreseardiershavetriedtoovercanethe suppressiveeffectoftheparasitebytheadditimofemgemsIIZto cultures of splencytes fran infected mice. '1‘ cell blastogenic 8 respmsestoCmAstimlatimwerenotrestoredun,whflearecovery ochellrespmseswaspromcedbyeithercrude(52,61)orpmified 112(62). IL2wasalsoabletorestoretheabilityochellsto providehelperactivitytoBcells (28). Whenadm'nisteredtoinfected mice either alone (63) orincatbination with parasite antigens (64), wrestoredtliemmmoralrospcnsesoftlesemicewitha subsequeitdecreaseinparasiteniaaniasligltircreaseinlelgevity. 'meacflitimofIIZandparasiteantigeisismsteffectiveinrestora- tie) (64). misgmlphasalsofomdthattheadministratimof parasite antigens alone is able to overcame the suppressive effect of Wifadministerednorethanelceandgiveiattheamropriate tineintewals(65). III. An Overview of T Cell Stinllatim lenphocytestinulatim,withtheaahseque1tsynuiesisara release of factors involved inmacrophage, Blynphocyte, ardnatural killercellMQactivatimaswellasinclalalexpansimofantigei- specific'rcells, playsacrucial role inthehost immerespmse. Severalpatlnlaysof'l‘cellactivatimrmrebeelreported. 'Ihenost cumulnearsofjnliygstiwlatimocclrsviaelgageertoftheTcell antigelreceprtorcalplec(w3-Ti). 'mefirsteverttooccurinthis patlmayisthephagocytosisandprocessirgoftheantigelbymcro- phage/unlocytes, Bcells, anddendritic cells, followedby itspresen- tatimtoTcellsinthecmtettofthecorrectlHCantigeluevieved in66). 'Ihe T4+subsetwhidlcmsistsofhelperaniamressor- irducercellsrecognizesprocessedantigeiinfllecmtettofmclass 9 II antigens (67). 'IheT8"’ subsettowhidnboth suppressor-effector arnd cytotondcTcellsbelengreqnndstoantigenplnmclassIulCantigens (67). 'nneprocessedantigenarriuiCnoleculearereccgnizedby the (In-Ti canplex on T cells (67). CD3 (T3) is a molecule which is famdmallmaunetnmanlenunocytesanicmsistsofamenbrane- bound heterotrimer (68) which is non-cmvalently linked to Ti (69). Ti is the clanotypically unique structure which allows specific antigen recognitian (70,71). It is a nnenbrane-bourri heterodinner belcnging to the immogldoulin superfamily (72) whose individual dnains each mflergoanticrearrangenenttoprovidethelargediversityof antigen-recognizing structures required by the host (73-76) . Recogni- tien of antigenplus MHC or the addition of antibodies to either Ti or CD3 leadstoarenovalofthecanplexfranthecell surface (70,77) and provides the first signal in T cell activation (70,78-80) . In addition to their role in antigen presentation, nacrofinages, B cells, and dendritic cells also synthesize and secrete interleukin 1 (I11) upon stimlation (81,82). This lynrholdm elicits a large variety of responses inamnnberofdifferent cell types (83). Oneof its actiens is toprovide a secand signal inTcell stimlation (81). Bnorbol myristyl acetate is able to mimick ILl activity (84,85), possibly throlgh the activation of protein kinase C. The canbination of signalsprovidedbym-Jriandm leadootheproductianof interlenkin 2 (11.2) and its surface receptor (ILZR) (83,86). I12 isalynpnokinesynunesizedandreleasedbyactivatedThelper cells (37). Uponbindixgtoitsreceptor, mtransnitsanintra- 10 cellular signal for cell progression fron the early to the late G1 stage of the cell cycle (88,89). Resting cells bear only very low rlnlber'sofalowaffinity formoftheIIIZR, hrtupo‘ncontactwith processedantigenandILl, Tcellsecpressgreatlyenharcedlevelsof nnnenbrane-bomdreceptors, includingsonnewithahighaffinity forIL2 (see Interlelkin 2 mceptors) . Receptor pnnospnmylatim (90), activation of a Nat/Hi- punnp (91), protein kinase C mobilization (92,93), activation of an unique protein kinase (94), increased levels of cytosolic (2+2 (95,96), inositol triphosphate generation (97) , and theinhibitionofdMPacomlation (98) havebeenreportedtobe involved in the signal transnnission. 'nnesynthesisofbothmandtheIIZRoconrsearlydurinchell activation and is transcriptionally regulated. 112 m is first seen at 9 hours after stinulation and peaks at 24 hours (99,100), while ILZR MisfirstdetectableatBMIrsaMismximlbeureenéard24 hours (100). Releaseofnzbythe'cellsoconrsbylzholrsof activation and ismaxilnal at 48 honrs (101). The expression of the IIBRonthesurfaceofthecellsbeginsamrodnately6honrsafter stimlationardpeaksat48holrs (102). 'nnesynthesisofbothIIZand its receptor are subsequently donmregulated (100,102,103) . Other-eventsoconrringdurinchell activation includethe synthesis of IFN-r (See Interferon-r), the expression of several growth factor receptors (104-108), oncogene transcription (103,109), [NA synthesis, and cell division. Most of these events are regulated at leastpartiallybytheinteractionofIIZwiththeIIZR. Theirduction of IFN-r transcription occurs apprrndmately 3 hours after stimulation, . 11 peaksat9t015hours,anribeginstodecreaseat24tnnrs(100). mileseveralreportsshowthatnzlnayupregulate IFN-r production, thesekineticstnriiessuggcstthatIFN-rsynthesisisatleast partiallyindeperientofll2regulation (see Interferon-r). Tramferrin is reqnired for lymhocyte proliferation, and anti- transferrin receptor (Tfli) antibodies block thymidine incorporation in '1' cells (106,110,111), indicating thevital role of this receptor in lynphccyteblastncgnesis. 'Ihisgroathfactorrecqtorisecpressed late during lynphocyte activation, with its m first being detectable at6tol4hoursardpealdngbetween14and481nns(100,103). EbcpressionoftheTfRonthecellsnlrfaceisdetectableat4BInnrsarnd ismximl72to96holrslater(112). 'nneexpressionofthisreceptor appearstobedeperientonthepresenceofnz,andantibodiestothe IIZRblockthearpearanceofflneTfRonthecellsurface (106), suggesting that the IL‘Z-ILZR interaction regulates the expression of theTflz. Othergrwthfactorreceptorsmidnareexpressedathigher levelsmactivatechellsinclnriethemreceptothhhighandlov affinityfonnns; 113),theinsulinreceptor(104)andthetype1ardII insulin-like growth factor receptors (107). 'nnetranscriptionofprotooncongenesalsooconrsdurinchell activation (103,109). Soneofoncogenem, snxinasc-nuycandc—fos, appearearly,priortotheiniuctionofthen2m,mileothers, suchasc-myb,N-ras,andp53, aretranscribed later. 'Iheetpression ofthelattergropiseinancedbytheadditionofnzuon, suggestingaregulatory effect of this lynphokine. 12 Ultimately, thestimlatedlynpnocytepassesunroughthesmcz phasesofthecellcycletotheurhasewhereitmriergoesdivision. Thus, activation of the T cells by the CD3-Ti pathway leads to lynpho- kine production and expansion of antigen-reactive cells. Mitogenic lectinsareabletomimickthisprocessbutproducepolyclonal lynpho- cyte activation. In addition to the above-motioned antigen-dependent pathway, several antigen-independent pathways of T lyndnocyte stimlation have been reported, involving c112 (114), np44 (115-117), and 'Ip90 (118). Of these, the 002 pathway has been best characterized (reviewed in 114). The first demonstration that (:02 [the sheep erythrocyte receptor, T11, lynphocyte function-associated antigen 2 (IFA-2), IeuS] my be involved in an alternative pathway of lymphocyte activation came fronn the finiingthatapairofantibcdiesdirectedagainsttwodistinct epitopes of (132, T112 and T113, were able to induce blastogenesis. 'nnis stinulation is nonocyte-iniependent (112). Upon stinulation with FHA, unenmberofGDZmoleculosonthecellsurfaceincreases, and T113beconesdetectablewithin 24 hours. 'Ihisepitope isnotexpressed onrestingcellsarriitseanessionisbelievedtoresultfronadname in molecular conformation (112) . An antibody directed against the T112 epitope, whidn is found on all T cells, also rapidly induces T113 expression, in as little as 30 minutes (112) . The ligand of (132 has recently been identified as Inn-3 (119,120) , a molecule expressed in endothelial, epithelial, and connective tissues, aswellasonnanyblocdcells (121). milealchellsmay bind to an LFA-like molecule on sheep erythrocytes via 002, only 13 activatechellsbirdtommanerythrocytes, midnecpressanudn lower level of IPA-3 than sheep erythrocytes (122,123). Two forns of m-3ImebeeIdnaracterized, oneformattadnedtothecellnabrane by a hydrophobic C-terminls ard the other via a phosphatidylinositol tail (124,125). Both fornns show significant honology to (:02 (124) . 'nnebixdirgofHA-3tocmallowsTcellstobeconerosponsiveto stimlation by anti-’rl13 (126) . It is possible, therefore, that the interaction of (1)2 with Inn-3 on accesory cells triggers T113 expres- sionardthattheelbsequentbirdilgofthisepitopetoits ligand irdnms antigen-irdendent proliferation. 'nnis pathway may be of particular inportance for immature thynnocytes which lack the cm-mi conplex (114) . The putative ligand of T113 has yet to be identified. OzardtheGJB-Ticonplexareseparateentitiasardarenot associated on the cell surface (127). Moreover, COB-Ti is not reqnired for CD2 activation since the latter pathway is operative in CD3‘ thynocytes (128). Nevertheless, the renoval of CD3-Ti fronthe cell surface inhibits (Dz-induced proliferation (112) , sungesting that the antigen-dependent pathway may regulate CD2 responsiveness. Like stimlation via CD3-Ti, triggering by C132 also involves the synthesis of 112 axd the expression of 112R (128). Furthermore, (:02 activation induces phosphorylation of CD3 (129). Taken together, these data irdicate that at least two pathways of T cell activation exist, either antigen-dependent or -i1depedent. These pathways involve separate signaling mlecules interacting with separate receptors tut may merge subsecpenttoreceptorbirding, witheadnpathwayregulatingthe activity of the other. 14 IV. Human Interleukin 2 112 isalyudnokinesecretedbyactivated'rcellsmidnallovs progressionfronntheearlytothelateclphaseofthecell cycle (see An Overview of T Cell Stimlation) . It has been well characterized, at theamimacidaswellasthelflilevel. 'lhislynphokinehasa molecular weight of 15 1:0 and consists of a 133 amino acid polypeptide containing one inntranolecular dimlfide bridge (87) . Altholgh one 0- linked glyccsylation site is present, carbohydrate is not neccessary for biologiml activity (130-132) . x-ray crystallography studies indicate a significant amount of a helical secondary structure (133) . 'IhereexistsonlyasinglecopyoftheIIdene, locatedon dnronncscnne4q (134). 'nnisgenecontainns4emnsseparatedbyinter- venin'g seqneces (135,136). The cm for 11.2 has alsobeenclonedard sequenced (137), and encodes apolypeptide of 153 amino acids, witha putative signal sequence of 20 N-terminal residues. IIZhasbeenfoundtohaveavarietyofactivitiesinseveral different cell types. In T lynnphocytes, ILZ triggers the production of other lynfinokines, the expansion of reactive clones and the generation of cytotoxic activity (101). In B lyuphocytes, 112 has been reported to play a role in both differentiation and division (138,139) , although thefornnerfunctionisstillamatterofcontroversy. IIZeinancesthe cytotoxicity of nnonocytes and NK cells (140,141), as well as stim- latinng the respiratory burst and degrarulation of neutrophils (142). Deficient IL’Z production is fond in several pathologiml codi- tions. These inclnde infection with W (47,62), W 15 m (143.144). W (145). W (145): and WM (147): and in lepmnatws leprosy (148). pulmonary tuberonlosis (149), certain types of cancer (150), systenic lupus erythennatcsus (151), Hodgkin's disease (152), and anus (153- 155). Inthefirstfouroftheseinfections, ecogenousILZhas restorativeeffectseitlnerigfloronmlynfinocyte functions (28,47,61-64,143-146). V. The Interlelkin 2 mar (ILZR) The biological activities of 112 are mediated through the 112R, which, afterbinding its ligand, is internalizedandtransportedtothe lyscsonalconpartlnentmereitisdegraded (156). 'lheILZRis expressedonactivatedTandBlynphocytes (156-158), withthefornner etpressingapproodmatelymiceasmanyreceptorsasthelatter (159). Inmatmre thymocytes (160) and IFN-r- or IFS-induced monocytes (161,162) alsobearthereceptor. 'nneetpressionofthemRonTcellsmaybe unregulated throlgh several agents: these include 11.1 (163), I12 (164- 166), Il-‘N-n (167,168), phorbol myristic acetate (169) and thymic lnomnes (170). The initial binding studies using radiolabeled-II2 detected 200- 11,000 receptors on activated '1' cells with a Kd of 10"11 to 10"12 (170). Studies with 3H-anti-Tac, an antibodytothe receptornmid'n blocks the binding of 112 (172,173), however, detected 30,000 to 60,000 ILZRpercell (169). ‘nnisdiscrepancyinreceptormnberwasresolved bystndiesmnidnusedabroaderrargeof radiolabeled-ILZ concentra- . tions. 'nnesestudiesdenostratedthepresenceofonoclassesof 16 receptors: tl'e first class bound ligand with the previonsly noted high affinity and = 10'11 to 10712) , while the second class had a 1a of approudmately 10"3 and was represented by 40,000 to 50,000 molecules per cell (171). Anti-Tao birds both classes of. the receptor (173), while physiological levels of 112 are believed to interact with only thehigh affinity form (172). In orderto furthercharacterize tie 112R, severalgrolpsofimrestigatorsmadeuseofvarionscell lines fronpatientswitholtaneonslenunonnas transformedbyfietuman'l‘ cell lynphotropic virus I (HI‘UV-l) (174). These cell lines include HUT 102, YT, ardmhl andconstitnntivelyproduce andexpressmenbrane-bonrd IlzRatSto10timeshigherlevelsthanactivatednomachells (169,175) . Additionally, several of these linnes spontaneously release I12 (175) . Since these cells have greatly elevated numbers of 112R, they were used to perform the initial purification and characteriza- tions of the receptor (176-178) . Subseqnent stndies have sham that fiegenesenccdingthisnoleonleasvellastleaminoacidsegnences are the sane in both nornnnal and the I-H'UI-l-infected lines (179,180), although differences in molecular weight have been reported (177,178) and are due to variations in post-translational processing (180). Information gleaned fron tie study of both I-rI‘IN-l-infected cell lines and normal lyupleblasts have revealed that the low affinity form ofthereceptorisconposedofasinglepolypeptidednainthatreacts with the anti-Tao antibody (HG-178,181) . It has a molecular weight of agnroxinnately 55 1d) on lynnphoblasts (50 kD on 1111102) with a pI of 5.6- 6.0, contains N- and O-linked glyccsylation, and is fincsphorylated and sulfated (176-178, 180). Proteolytic analysis of this molecnle suggests 17 theexisteraeofmdisulfide-linkeddonains, withtheII2birding site in the N-terminal region (182). ConplenentarymAfortteabove-nnentioedp55polypeptidehasbeen clonedardsequenced, andenccdesaproteinconsistingofzslamim acids with a N-terminal extracellular region, a 19 amino acid trans- menbrare sequent, and a predicted cytoplaennic region of 13 residues (183-185) . This last finding was nmelqlected sinnce most growth factor receptors have more extensive cytoplasmic tails, which frequently contain tyrosine kinase activity. 'nnis innfornnation suggested that the p55 polypeptide may be unable to geerate an intracellular signal itself and my be associated with a separate molecule whidn is able to do so. GemiclNAforthepSSpclypeptidehasalsobeenecamined. There existsonlyasirglecqnyofthegeeeeodingthismoleclleanditis located on chronosone 10 (179). The gee consists of 8 axons, of which two, eaonsZand4, arebelieredtotevearisenfronageednnplica- tion. Interestingly, alternatively spliced mRJAs which lack tie secod oftl'esesequemsdonotproducefumtionalreceptors (179). 'nneII2R genehastwotranscription initiationsitesinnornnachells (threein HI‘IN-I-infected lines) and three different polyadenylation sites (179) . NomajorsizegrolpsofnRIAhavebeenfond, of 1500ard3500base pairs, withthe 1500basepairnoietiesnakinngnnseofthe5'-most polyadenylation site (183) . The fornner grolp is believed to contain at leasttwokindsofnRJAardthelatter, atleast four, althoghthe actualmnberofspeciesineadngronpmaybegreaterdnetothe presenceofseveraltranscriptionstartsitesaswellastoalternative 18 RNA splicing (186). Both of these groups containmwhich give rise to functional receptors (183). mnentlecmenccdingtlernmanfomofttepSSpolypeptidewas transfected into a mouse T-lynfinocytic lire, both low and high affinity fornsoftheII2Rwereexpressedandtlecellswereabletorespodto tnmnanII2 (187). WnenmouseLcellsweretherecipients oftrecINA for either human (187,188) or miss (181,189) p55, low but not high affinity IL2Rwereproduced. Tress low affinity receptors conldbe converted to the high affinity form following the fusion of tie transfected L cells with membranes of l‘nmnan T cells (189) . Together, these results suggested that p55 is respos-ible for low affinity bindingandacts incocertwithaseco'dmolecule, fondintte meahranes of '1' cells, to produce high affinity binding. Evidence fortteexistenceofthepntativesecoddnainoftl'e I12Rwasprovidedbysmdies inwhich 12f’I-Imwascross-lirnloedto its receptor neing tl'e bifunctional agent disuccinimidyl suberate and analyzed on sue-polyacrylamids gels (190—194) . When the cross-linking wasperfornnedwitheithernormalleupnoblastsorfln‘loz cells, two bank of 55 and 70-75 kD were detected (190-192,194,195). Tie 55 kD ncleolle is precipitable by anti-Tao (190-192, 195) and is also deton- strable on cells transfected with p55 can (191) . This polypeptide thus appearstoccn'espodtotlepreviolslydnaracterizeddnainofthe recqator. The 70-75 kD polypeptide (tenceforth referred to as p75) , however, does not react with anti-Tao (l90-l92,194,195) and represents a novel 112-binding molecule. 19 Further clarification of tte roles of p55 and p75 in 112 binding werecbtainedbyperformingcross-linkingsudieswithflcells, aNK- like HITN-l—infected cell line (196) . Normally, these cells bind II2 with a Kd of 10'9 (intermediate affinity binding) and this binding is not innhibitable by anti-Tao. Cross-linking studies using vr cells revealed a single 112-binding band of 75 kD (192,195) . These cells can alsobeinducedtoexpressthehignaffinity fornnofthereceptor (196,197). Cross-linking of tie induced YT cells to 112 yields both the p75 and p55 chains (192,195). Taken together, these findings indicate that p55 aloe is capable of low affinity II2 binding, p75 aloe produces binding of an intermediate affinity, and together, p55 and p75 form the high affinity 11.212. since the mnber of low affinity receptorsfarecceedsthemderofthoseofhighaffinity, thelevels of p75 are believed to be the limiting factor in the formation of high affinity receptors. The respective contributions of p55 and p75 to high affinity binding were examined recently (198,199). The p55 chain allows rapid association (5 sec) and dissociation (6-10 sec), while both tl'e association (42-47 min) and dissociation (250-330 min) of 112 with p75 ismndnslower. Together, theyformareceptorwiththerapidassccia- tion (37 sec) characteristic of p55 and the slow dissociation (285 min) of p75. As previously rcted, p55 contains an extremely short cytoplaenic region which may be unable to function in signal transmission. 'ne p75 molecule, on the other hard, is able to internalize 112 (193) and transmit a signal for cell division (200,201) or immoglcbulin 20 synthesis (202,203) in the absence of p55. Additionally, low levels of p75butmtp55arepmesentonresting'rcells (194,200) andthusmay explainhcwhigh levels ch12 areabletcactivateunstinulatedcells (204). In addition to the marbrane—bonnd form of the ILZR, a soluble form alsoexists. 'nnsesolublereceptorsarereleasedfronactivated nominal T cells and W-l-infected lines (205) and this release is oinanced by :12 (206). line soluble form is approcinetely 10 kD less thanthemanbrane—associatedreceptorandmayttmsarisebyeither alternative Rm splicing or by proteolytic cleavage fron the cell surface (205,207) . Elbe serum levels of the soluble ILZR are enhanced incertaindiseasestates, inclndingfiodgldn'sdim, adult'Ihcell leukemia, chronic lynphocytic leukania, Sezaiy syndrone, and AIIB (208- 210). Since the soluble receptor is able to bind 11.2, it my act as a conpetitive inhibitor of the methane-bond form, decreasing the 11.2- responsiveness of T cells in these diseases (211). Decleasedexpressionofthembrane-associatedIIZRhasalscbeen danostrated in certain pathological coditions: pllmnary tuberonlosis (149), Hodgkin's disease (152), A105 (154,212), and infection withL m (144). Inthelattercase, thisdecreaseistheresultof summessorcell activityandmtdirectlyinflncedbytheparasite. VI. Interferon-n Inn-n isanotherlymnoldneptoduoedbyactivated'rcells. Its synthesisisregulated, atleastinpart, bylLZsinnceanti-Iac (164,213) and culture conditions which inhibit n2 modicum (164,214) 21 alsodecreaseIFN-r synthesis. 'nneadditioncfllztothesecnltures restores IFN-n production (214). 112 is also able to induce IFN-r synthesisinmnstimlatedlynphocytesandthiseffectisohancedby pnorbol myristate acetate (213) . mile these reports show the ability cfnztcnpregulateIFN-rproduction, thepresenoechLz isnnctan absoluterequiranentsincenonnnalIFN-r productioncccursian infections in the face of deficient levels of I12 (144). Taper-ally, IFN-rmisprodwedpriortoflnatofm (seeAnOverviavchOell Stimulation), againanggestingthatILZ isnnotthesole factor regulating IFN-‘r production. IFN—rhasawiderangeof fingtionsinnammbercfdifferentcell types (reviewed in 215). In addition to its antiviral effects, this lynfinokinne induces the expression of NBC and Inn-1 antigens (215,216) and the Fc receptor for 196 (215) , activates nentrophils (217), increases tunnoricidal activity in monocytes and NK cells (141,218), activates macrophages and monocytes for antimicrobial activity (219) and induces differentiation of myelo—monocytic and B cells (215) . In T cells, IPN-rnnayeitherinmeasecrdecneasegrcwth, dependingonthe dosage and time of administration (219,220). ‘Ihis enhancement nnnay be due, inpart, tctheabilitychFN-rtcinncreasem (221) andILZ (222,223) production as well as the expression of 112R on ‘1‘ cells (167,168) andmnocytes (161,224). IFN-r isreleasedfronthestinnulated'rcellsasaglycoprotein with a pI of 8.6 (225) which exists in three numeric forms with molecular weights of 15, 20, and 25 kD, in increasing order of occur- rence (226). 'lhese form have identical amino acid seqnonces, with the 22 25 kD molecule containing tnno N-linked glycosylation sites and the 20 kD formhaving only one (227). | IFN-thAhasbeenclonedandsequenced, andencodesa polypeptide of 146 amino acids, 20 of which are believed to function as a signnal sequence (228). 'Ihereexists onlya singlecopyofthegenne form-1, locatedondnronosonelz (229). 'nnisgenneisconposedof four axons and containns a repetitive element (230). ReceptorsforIFN—rhavebeendonuctratedonmoccytesand moccyte—liJce cell lines (231-234), fibroblasts (235), lymnoblastoid cells (236,237), and WISH amniotic cells (238). ‘Ihis receptor binds Il‘N-r with high affinity (m ranging from 10'9 to 10'”) (231-235,237, 238). Cross-linldng of radiolabeled IFN-r to the cell menbranes, followed by annalysis by SIB-PAGE, shows a receptor with a molecular weight of 100-150 kD (234,236-238) , whereas isolation of the receptor using anti-receptor antibodies produces two molecules of 50 and 90 kD, bothofwhichcanbind IFN-r (239). films, the receptorappears tobe conposed of two suhnnits. Deficient production of IFN-r is found in several disease states, including acute tuberculosis (240), Lemma infections (241,242), and lepronatcus but not tuberculoid leprosy (243). Since leprosy is a spectrnm of disease states with the lepr'onatcus form exhibiting greater pathogenicity than the tuberculoid form, increased pathology correlates with defective IFN-r production in this codition. Additionally, the decreaseinnIfN-r synthesis inlgigmgnia infectionsisnctedin acceptible on: not resistant strains of mice (241,242). 'Ihus, the 23 abilityofarcsttoproduceIFN-rmaydeterminnethesnnbseqnent severityofsonediseases. VII. mseardnGoals an-ingtheinitialphasecfcnagas'disease,thezeexistsastate ofsuppressedresponsivenessinboththemmcralardthecellular branchesoftheimmesystan. Alloftheprevionslyreportedsudies examiningtlcmderlyingnednanissofthisynenmenmhaveutilized flnemousennodelsysten. fiegoalsofthisneseardnweretoshdyl, mi-indlcedimucmppressionofmmanleuuccyterespocesandto examine at which stage of lynplccyte activation this suppression first isseen. Qnapteronedescribestheabilityofmtryponastigotesto inhibit the proliferative response of normal human T lynfinocytes stinulated by a variety of mitogenns. 'Ihe ability of activated human andmnselymccytestopredtceardrespodtoIFN-nisdescribedin dnapterunowhilethesynthesisofmandIIZandtheexpressionof theIIZRarethetopicsofdnapterthree. Severalmarkersochellactivationhavebeondescribedwhidn appearinadefinitetenporalorder. 'mesennerkersincludeearly activation antigen 1,theIL2R, andthetransferrin receptor. Chapter fonrexplorestheabilityofmitoaffecteadnofthesemarkers overtimeinordertosudythespecificityoftheimucsugnressionas wellastodeterminetheearlieststagesatwhidnlynfimytesare inhibited. 'nneexpression of boththehighandthe low affinity form oftheILZRareeuamined. 24 The C132 pathway of lynptccyte activation provides an alternative route to the better dnacterized CD3-Ti patlmay of stinnlation. 'nne ability of ml to inhibit T cell stimlation through this pathway isexaminedinncnapterfive. Appendixlexaminneswhethercell-to—parasitecontactisrequired fortheinductionofimncsuppressionandwhetherthiseventis reversible. lymccyteactivationinvolvesaseriesofstagesasthecell ncvesfrontherestingstageofcointothecellcyclentheparasite couldenaertits inhibitory effect atanyofthesestages. Innortalized celllines,however,area1readyinthecellcycleandthusbypass severeloftheeventswhidnoconrduringactivation,perhapsevennthe stageswhichareacteduponbymi. 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Immunel. 129:344. Sadick, M.D., Locksley, R.M., m, C., and Raff, H.V. 1986. Murine cutaneous leislmnaniasis correlates with tle capacity to geerate interferen-rinrespensetoleislmeniaantigensm m. J. Iummuuel. 136:655. Nogueira, N., laplan, G., Levy, 8., Sarne, S.N., hushner, P., Granelli-Piperne, A., Vieira, L., Gould, V.C., Levis, N., Steinmen, R., Yip, Y.K., and Grim, LA. 1983. Defective r interferenproductieninleprosy: reversalwithantigenand innter'leflcin 2. J. Exp. Med. 158:2165. 47 Ml SUPPRESSIQIOFHMWWBYW 48 Virtallynethingislmomabouttlebasisfortleimuesuppression associatedwithluumanmiinfection. Wehaveusedanjnlitrg systen to explore this effect. Incubation of luuman peripheral blood muclear cells (PRC) with blood forus of mi abrogated tleir responsestosuboptiuel,optiuuelardsupraoptina1dosesof0onA,PHA oerhetheror-notnenocytosveredqnleted. Killedparasiteswere notsuguressive. Maxinnel suppression (74%) occurredwlentleparasitos werepresentduringtheentireoultnreperiod (96-hr),although significant empression (33%) was seenwhen the organiens were added 24, 4Bor72hrafterinitiation,suggestingthattheearlystagesof lynfiecyteactivatienhadbeeninnpairedandtlatasecedgeerationof cellswasalsoaffected. 'nne4-daysupernatantuuediumofami auspennsionsugportedmnrospensostoOonAaswellasuediumalee, indicating tlat suppression did not result froun parasite renoval of essentialnutrients. nun-tlernnore, 96hraftermitogenic stimulation tleproportiensofviablepmcinoulturescontainingorlacldngtle parasiteswerecorparable. AlthoughmibindsconAandHiA,this absorptionmsnottlecauseofreducedresposiveesssirceoptimal coeentratiensofoonAandPHArenainedinsolutionmderour conditions. IevelsofIIZinPHA-stiumlatedmcoultureswere markedlyreducedinthepreseeeofmi. I-Iowever,exogeeusIL2 failedtorestorelynplecyterespensiveness. Wneitlerabsorbed rerinactivatedIL‘z. 'nnus,tlenotedsumressiona;pearedtoinvolve at least deficient production and utilization of IL2. 49 Experimental and luuman infections by W1 - tle cauusative agent of Clagas' disease - are accepanied (particularly duringtleacuteperiod) byseverealterationsoftlelumeraland cellular arms of tle iummune system (Breer, 1980: Kuhn, 1981: Clinton M, 1975: Teixeira fl, 1978: Maleckar & Kierszenbaum, 1983: Ramos, Sdnadtler-Siwon & Ortiz-Ortiz, 1979). his condition luas been regarded as a neans by which tle parasite eludes immunelogical defeces while it establishes itself in the lest (W, 1980: Kuhn, 1981) . Stdieswithmurinenedelsysteusofd'agas' diseasehaveproduuoed evideee suggesting several mechanisms of immunesurpression, including alteration of accessory cell function (Cunningham & Kuhn, 1980: Kierszenbaum, 1982) , reduced levels of T cells in the spleen (Hayes 8: Kierszenbaum, 1981) and altered lyuphokine-producing ability (Harel-Bellan M, 1983; Reed, Inverso & Rates, 1984, 1984a: Tarleton & Kuhn, 1983). In contrast, our hmledge of lumen lynnfiecyte alterations in lumen W infection is negligible and, given tle differences between murine and luuman Clagas' disease, extrapolations umldbeunwarranted. Inthiswork, anmmsystenwasusedto study tle effects of mi on lumen lynpleproliferative responses inducedbymitogens. Itwillbeshominthispapertlatco—culture with tle parasite omesses lumen lyuplecytes at a relatively early stageoftleactivationprocessandthatalteredlyuphocyte functions includeaneflcedlyreducedcapacitytobothprroduceandutilize interleukin 2 (IL2). WWW m '33 4-week-old Crl-CDl(ICR)m Swiss mice used to ueintain and produce blood forueofmiandthefeuelelewisratsueedasasouroeof spleencellstoprooucenz (mat) werepurdnasedfronndarlesRiver laboratory (Portage, MI). Parasites flemlalunenstrainofmiwasueedinthiswork. Trypouestigotes were purified froun tle blood of mice (infected intraperitoneally 2 weeks previously with 2 x 105 organisms) by density gradient centrifugation over a mixture of Ficoll-Hypaque of density 1. 077 (Bxizko & Kierszenbaum, 1974) followed by dnrolatcgrariny throgh a dieflnylaminoethyl-cellulose column (Villalta & 13m, 1979). The parasites were washed twice by centrifugation (800 x G, 20 min, 4'0) in RPMI 1640 medium containing L-glutamine (Gibco, Grand Island, NY), penicillin (100 units/ml) and streptomycin (100 ug/ml). parasite suspensioewerepreparedattledesiredcoeentration (seem) in tle same radium supplemented with 5% heat-inactivated (56'C, 20 min) fetal bovine serum (FBS, Gibco (REMI+5%F$). In sone experiuuants, trypanesonesgrown incultrr'es of ratleartmyoblasts (Line & Kierszenbaum, 1982) or epiuestigotes grown in Warren's medium (Warren, 1969) were used. When killed blood trypouastiguta were needed, the organisms were incubated with 0.025% glutaraldehyde in {regulate-buffered saline (20‘C, 2 min) , washed by centrifugation, 51 quenchedwith 0.1M lysineinplosrhate-hlffered salineandwashedtwioe wimlzmnsitm. ' Him CabanaVaIinA (Con A), phytd'xanagglutinin (FHA) andpokeweedmitogen (ma) werepmdlasedfranSigmacmiczlco. (St. louis, in). W 'nlem-omtainirgapermtmltlsedtomintainmzwls(seebelm) was prepared by stilmlating rat spleen cells (1 x 105 cells/m1) with 2 mealA/mlinthepresenoeofsxm'SMZ-mptoeuiarnLusingthe mediumdescribedabove. 'mesupematantswereoollectedafter inwbatimthesewlmresatW'CaniflCszormhrinanamsphere saturated with water vapor, andstoredat -20'Cuntilused. 'Ihis mterialwillbereferredtointhetextasnzrat. mritiedmman 112 (mph) was purchased from Collaborative Resend: (Imdngton, MA). mnepreparatimsofmnnanIIZ(I12ch)oonsistedofthe48-hr supernatants of peripheral blood mononuclear cell (mac) (5 x lo6 mac/ml) cilmres stimulated with 25 ug BIA/m1 (Tilden & Belch, 1932). Insaneoases,pmductionof112d,inthepresenoeorabsaneof1; mimompamd3theomoentratimofparasites,vmenpresart,wass x105organisne/mlaraallctlercaaitiasrenaimdtresame. Ella 'IhePHKZusedinthisworkwerefranhealthyvolmrteers. 'Iheir mifioatim was by oartrifugatim over Iyuphoprep (Nyegaard, Oslo) at 34OXGarfl20'Cfor4SminardtheywerewashedtM'eetimeswith senm-freeRHII 1640 medium priortome: oellviability, detenninedby trypan blue dye exclusion, was always >99%. 'n-le final alspensims of 52 flmeoellswrerepreparedinmn+5%m. 'meIIZ-depenientlflfl cell line (kindly provided by Dr. Phillippa Mar-rack firm the University of ColoradoHealthScienoescenter, Denver, (1)) wasusedtomeaalrenz activity in biological fluids. 'Ihese cells were maintained in REMI-I-lOfl‘E at 37°C by mixing equal volumes of cell culture and the mrat preparation (see above). Suspensions of mac (3.5 ml at 5 x 106 cells/ml) were incubated at 37'C (5%CD2 incubator) forlhrinaGO—mndiamebersterileplasticpetri dish. ‘memladhererrtoellswer'erawvedardmbjectedtothesame procedure once more, and then centrifuged (280 x G, 10 min, re). The adherent, nonspecific-esterase- positive cells were further depleted by dmtography over a quhadeu G-10 (Rumcia, Piscataway, NJ) column (Mid-ell, Mishell & Shigii, 1980) . 'me nonspecific esterase test has been described (Yam, Li & Crosby, 1971). W Cell cultures were set up in triplicate in 96-well microwlulre plates. Each culture contained 1.25 X 105 mac and the appropriate mitogen concentration (see W) in a total volume of 0.1 ml. When parasitesorotherreagentsweretobepresent, theywereoa'rtainedin 0.025 ml and albstiurted for the equivalent volume of RHII+5%FBS. All allun'eswereirnibatedat 37'Cand5%m2 for96hr (mlessortherwise stated) and pulsed with 1 Mai 38W (specific activity 2 mGi/mmle, Amersham, Arlington Heights, IL) during the last 24 hr. Ollmres were interrupted by harvesting (MASH II, ILA. Biqmoducts, 53 Walkersville, MD) and radioactivity was measured in a liquid scintillatim qaectruneter. Solutims of Con A (concentrations described under M) were inxbated with 5 x 105 blood forms of mi per milliliter at 37°C (002 irmbator) for24hr. meparasiteswerethenrewvedby filtration through sterile 0.22-um Millipore filters (Bedford, MA) . 'me filtratewasusedasthealluzrenediuminblastogelmis assaystoteet Parrespmsestotheresidualannmtofmitogen. W 'I'. cmzi After incubating metres medium with or without 5 x 105 blood forms of mi per milliliter at 37’C (m2 imbator) for 4 days and filtration (0.22 pm pore size), the filtrates were used in blastogenesis assays to test mac responses to various caicentraticns of Con A. W Cultures of m2 cells were set up in triplicate in microculture wells, eadicmtainirg4x103 cells. misfiralvolimecftheeecilnnasnas 0.2 ml, including 0.1 ml of two-fold dilutims of the biological materialtobetested. ‘mealltureswereirnibatedat 37'C for48hr (5%CD2) arriprlsedwithluciafl-thymidineduringulelast24 hr. Cell harvesting and measuranent of radioactivity incorporated into synthesized INA was as described above. '1'- cruzi Wd) Solutims of I1231 were ircubated with purified blood trypanastigota at final concentrations varying from 1.25 x 105 to 2 x 107 organisms/m1 54 at 37°C for 48 hr. After rencving the parasites by filtration through sterile 0.22-um-pore-size filters, the filtrates were tested for IL2 activity as described above. For cartrol purposes, aliquots of 112,131 berealbjectedtothesanecorditia'semceptthattheparasiteswere absent. WWW Eadisetofrewltspresentedintletablesistypicallyrepre- sentative oftwotofoureuperimentswithasimilarprotoool. 'Ihe results represent the mean of triplicate determinations 1- 1 sm. Differences between means were considered to be statistically significant if 150.05 by Student's °°t°° test. “mpreserrtinthealltures,plrified blood forms 015W amessedPflCrespaeostoCmACI‘ablel). 'IheconcentratimofOon Aproducingoptinalrespa'sosvariedamcngrepeatexperiments (datanot 81m),prdaab1yduetotheuseofm£frandifferentda\orsard different batdies of the mitogen. Boater, significant sugaression by Wmdaservedinalleaqaerimmts. Alflnmmmexperiments asignificantreductimofflflresponsoetothetestedmitogenswas producedwith 2.5x105 blood forns/ml,theminimalconoentratim of parasitescausihgsudleffectinmostedqaerinentswas5xlo5 organisns/mlandwasusedinallsubsequentexperinerrts. Ofinterest, tissueallmre-derivedtrypatestigotsardepimastigotesgrwninan axenic median also expressed Con A-induced lynrhcproliferative mac resposes(datamtstmn). 'mesugprossiveeffectofbloodtrypmastiqctesmsalsoseenm eitlerHiAoerereusedtostimlatetlePflflandocalrredcvera widerangeofmitogencaioentratiae,imltdingsuboptinel,optimlmd sipraoptinaldoeesaablez). ShueLmicanbfidchrAardRfl(Pereiram,1980),we caeideredthepossibilitythattheparasitemigrtlevereduoedthe cunentratimofthesemitogenstosuboptimallevels. 'Ibtestthis possibility, Pmc were stimlated with solutims of Om A or FHA which hadbeeneitherabscrbedwitthlOGorganisms/mlforuhrormock- absorbed without parasites. Absorption of Can A solutims with 56 ..ueomosa mou.aasoo cev os—ae .csuccu as» ou accuses new: .mo.qWe a .eoo¢«.& ozu sou». apouosuose_ ..o.. .ospu csmu as emcee use: moa.uascc new use cw amt. use m=_sse 0:.uesmsu-: .u: fl nu.) campus «to: es. s: mo so» noonaaus. crux mouse—cu we» m .2 ... so some .... 12 .3 n 2 ~.s .... 2 as s a; .2 . ....s .3 . 2: .3 . ..2 N... . a... a: x as so u 3: ..o u can as ... cam ~.o n 3 a2 x as nausea so“ the 93%.: no... a; c u— m e o Apsmem.ecuscv A—s\ma. «combusueouecu me.rc——ce on“ on < ecu saw: euc.ouoo Ania“ xv .s.c.u co_uasocmuecu ou_mctos .~=su .h to mssce coo—a so noncocmos uzmo noosecw-< ecu eo cosmmmtccsm .H open» 57 Table 2. Suppression ofmcresponses inducedwith suboptimal, optinelardaipraoptimalconcentratiomofconA, PHAorPWIby blood formsofmi Mitogen Mitogen concerrt. 3I-I-‘Ihymidine incorporation (cpn X 10'3) Ml) Parasfi' absent W Con A 0 1.4 1 0.2 1.6 1 0.4 0.4 14.4 1 1.0 1.4 1 0.11 4 45.2 1 2.9 2.0 1 0.11 8 40.5 1 1.0 2.9 1 0.01 16 1.9 1 0.6 1.1 1 0.1 FHA 0 0.7 1 0.1 2.2 1 0.2 6.3 27.6 1 1.0 1.3 1 0.11 12.5 18.7 1 0.2 0.3 1 0.11 25 42.9 1 1.6 5.7 1 0.21 50 26.8 1 0.3 2.3 1 0.31 mm 0 0.2 1 0.0 0.1 1 0.0 2.5 3.3 1 0.4 0.4 1 0.02 5 2.9 1 0.1 0.9 1 0.12 10 2.3 1 0.2 1.3 1 0.12 'meexpefimentswiflreachmitogenverecafiuctedseparately. 1'2 p50.001 and p50.05, respectively, for reductims in cpn with respecttotheconespondingcontrolvalue (parasites absent). 58 mishiftedpeakrespmsestwardstrehigherlevelsflableB), corrdaorating the ability of the parasite to bind thismitogen. However, enoughmitogm renained in the solutions Whidl initially cartainedGoraugcmA/mltoirmlceqvtimlmcrespmses. Significant PBX: stinulation was also produced by solutions of FHA Alsooonsideredheretheposeibilitioe a) thatmi cmsumed rutrients required for optimal lynphocyte proliferation and b) that reduced levels of 3H-thymidine incorporation reallted fran a greater lossofPBCviabilityduetothepresenceofmi. Acorriiticned medimmidihadbeminmbatedwifllasurpressivecaicentratimofz; mifor96hrwasaseffectiveinsumorting3H-fllymidjre incorporatimbyPBCasmck—absorbednedim (Table4). Imenthe prwortiaeoftrypan-blre—exchflingPBcweredetermiredinCmA- stinflated cultures attheerd'ofthe96-hr incubatim period,the valtesdrtairedintleabserneofmiinrepeatexperimentswere 77toa3%whereasinthepresenceoftheorganisnstreoonespmding valueswere72to74%. Wealsoinvestigatedmefllernniocytes/necrrpegemvtnseacoesory cellfmctionmyhavebeenalteredupmtheirinfectimbymi, verearequiruentforparasite-irdwedsuppressimtoocan‘. When mnpopllatiaemiosemncyte/nacroghagecmtentshadbemreduced frané-9.7%to<0.7%werestinflatedwithcrnAormAinthepresaloe of mi, their responses were still significantly sugpressed. ‘nms,thelynrhocyteresponsesinthepresenceofnedimalae,8ug ConA/mlandzsugawmlwere400211618,29,7681900and52,7431 59 Table 3 . Mitogenic capacity of solutiors before and after absorptim with a sunaressive concentration of mi Hitogen 3I-I-’Il°1ymidine incorporation .(cpn x 10'3) after Mock absorption mi absorptim Name 7.6 1 0.4 Con A 4 ug/ml 21.1 1 2.3 5.1 1 1.4 Om A 6 ug/ml 6.3 1 0.3 19.5 1 1.0 0:31 A 8 ug/ml 6.5 1 0.3 17.0 1 1.0 Nae 7.7 1 0.5 BIA 5 ug/ml 55.5 1 0.2 50.6 1 3.3 FHA 7.5 ug/ml 55.9 1 1.2 45.7 1 3.6 FHA 10 ug/ml 55.4 1 1.5 42.4 1 3.1 'mesolutionsofOonAamimAveremock-absorbed (sanerhysical treaunents, no parasites) or absorbed with 5 x 106 parasites/ml for 24 hr, filteredthrcugh 0.22-m-pore-size filters, andthenusedto stinllatemicin96-hrcdlmresintheabsenceofparasitos. 'lhe wluneswerepllsedwithluCi3H-thymidimdm'ingthelast24hr. 60 Table 4. Ability of RPMI+5%F$ medium to support Cm A-induced respmses after incubatim with W Con A 3H-thymidine incorporation (cpn X 10'3) in (pg/ml) Untreated medium Medium preincubated with parasites 0 3.5 1 0.1 3.3 1- 0.1 4 24.9 1- 0.6 21.9 1 0.3 6 31.5 i- 0.9 23.]. 1- 0.4 8 31.1 i 3.9 19.0 i 0.6 M96-hrPchfltureswereperfornedintheabsenoeofmi. 'me culture media consisted of filtered (0.22-um filter) Ram-sues whidihadbeenincubatedintheabsence ('Wmtreated") orpresenceofs X105parasitas/ml. ConAwasaddedatzerotime. 'Ihecultureswere pnsedwithluCi3n-thymidixedmirgtrelast24hr. 61 1443 qn, respectively, whereas in the presence of 5 x 106 parasites/ml flewmrrtedto38321ll33, 17,2481496arfi15,3671348 qm, respectively. No surprossion was seen when gluteraldehyde—killed blood trypanastigctesweresdbstitutedforlivingorganiaeinthem cultures (datanotshown). mperinaitswereflmdesigredtoestablishtheperiodoftine during which the suppressive effect of the parasite was exerted. In thesePBflanulres,trypanastigoteswereaddedatvarimstinesafter mitogenicstirulatim. llaximlampressimwasprodnedwnmthe organimewerepreeentintheollmreefranttebegimfihguemtine), although significant expression occurred when the parasite was irnorporatedintothemcailmreszmllaornhrlater (TableS). Wtcmzimmwmflm SiresIIZisproducedbystimzlatechellsardplaysakeyrelein lynrhocyte proliferation, we set out to establish whether W Wmeyaffectimmmim. Isvelsofm wereneasuredafter48hrofPBCiJn1batimwith25ug/m1HiAinthe presenceorabsenceof5x105bloodtrypmastigotesperml. Asshown in'Iable 6, thelevels ofIlZactivity foundinthefiltrates of BIA- stimlated mac cultures were significantly smaller when the parasites werepresent. Ifmisuppressedmitogax-irflicedreworsesbym nerelybyinpairirgflZproductim,exogerrmsII2shafldoorrectthe deficiercy,aswasseenbyinvestigatorswhostuiiedantibody productim to I‘M-unrelated antigens by lynphocytes frun infected mice (Reedetal, 1934,1934a;1arletm&mm, 1983). 62 Table 5. Effects of addition of 11.5.2311 at different times after PRC stimlation with Con A Time of addition 3H-thymidire incorporation (cpm x 10-3) 015W (hr) Malone M+Lmi(%R)1 O 43.4 1 1.2 11.2 1 0.9 (74.2) 24 45.0 1 2.2 30.0 1 0.7 (33.3) 48 47.4 1 1.3 31.1 1 0.7 (33.4) 72 41.5 1 0.4 27.7 1 1.7 (33.3) Ninety-six—hr cultures; stimlated with 6 ug Con A/ml and pulsed with 1 uCi 3H-thymidine during the last 24 hr. 1 tR, percentreductiminqmduetothepresenceof parasites. All %R values mmsent statistically significant reductions in cpm (1350.05). 63 .<:mou:ma gear eweeobac nape» or“ as «cosmos seer oucmsueeme .apossaoucuos .mo.an use ~co.qwm e .6 Oh: we so. a__60 seass_s assessors soaasse use .6 easse_oa . as: .aza. .ts «N seas use assess oesaseses-=n .u: n gu.r senses are: worsapau or» .s: me so» neosuspon omega ea.) essences. use: “escapee ——oc ~-h= or» ..sxnuuem-soc oo~ u m ea amuasu .»+c ac: scene .99%. m-mluneomditiors. Suspensiors ofnSCwere imlbatedinthe presence or absence of 5 ug/ml Lhytohenagglutinin (FHA-P: Sigma 78 Clerical oc., St. Louis, 10) with or without 2.5 x 106 W. unless cthmwise noted. The cultures were irnlbated at 37'C (5% (Dz) forthe desired periods of time (see Resnflts). Cultures ofhPflCwere treated similarly accept that the final parasite concentration was 5 x 165 organisms/ml. Clhese concentrations of mi were selected becausetheyrepresenttheminiml levelswhidnconsistentlyproduce immosugzressim under optimal stinulatcry conditions for use and hm (1: Beltz and Kierszenbaum, unpublished results). rhesuranentorfIFN-‘r. allmresofnSCothBKIwereincubatedin 24-wellplatesinavclumeof1ml for480r72hunderthecmditiots describedintheprecedingparagrapn. Cellsandparasiteswereranncved by passage through 0.22—um—pore—size filters and the filtrates were stored at -70'C until assayed for IFN-r. Murine IIN-r activity was determinedbyaplaquereduztionassayusingnmseIr929 cellsarndthe Indiana strain of bovine vesicular stanatitis vine (30). line titer wasexpressedasnmits/mlccrrespolflingtcthereciprocal ofthe highestdilutiontoreduceplaquesbysoik. Inthisassay, orneunitwas equivalent to 0.88 NIH G-002-904511 reference units. Identification of the antiviral activity as IFN-r was provided by its lability at pH 2 and inhibitian by anti-nurine IFN-r antibodies (a gift of Dr. E. Havell, Trudeau Institute, Saranac, NY). Human IFN-was assayed using a radioimunoassay kit (Centocor, Malvern, NY). 11115 systenn uses two antibodies directed at different epitopes of IFN—r and is designed to detect only biologically active IFN-r . AbscrpfimcfnlrineIFN-r. RecarbinantmmineIFN-r (specific activity - 2.3 x 107 units/mg protein: a gift frann Genentedn Inc., 79 Swth San Francisco, on) was incubated in 24-well plates at a cawrtratim of 500 units/m1 in RPMI+2.5%I-'BS in the presence or absenceofSorleloémi/mlat37’Cfor48h. Afterpassage of the supernatants through 0.22-um-pore-size filters, residual IFN-r activity was determined as described above. Ithocyteproliferatimassays. Cultures ofnSCothBCwere iranbatedinthepresaneorabserneofmi in96-wellplatesina volume of 0.1 ml in the ramer described under Cc-cllture coalitions. Exogenous reccnbinant nurine IFN-‘r , partially purified human IFN-r (Melcy laboratories, Springfield, VA), and/or recanbinant glycosylated Innnannz (Genzyme, Boston, MA) wereaddedtcsaneoftheallturesat the desired concentrations (see Results). The lynphckines, when added to the cultures, replaced an equivalent volume of medium. The cultures were pulsed with l uci 3H-thymidine (specific activity - 2.0 ci/nmole; New England Nuclear Biotechnology Systans, Wilmingtan, m) at 48 h (1160) or72h (hPBC) andterminated 24hlaterbyautaneted harvesting. Incorporated radioactivity was determined in a liquid scintillation counter. A11 determinations were performed in triplicate andtheresultswereexpressedasneancamtspermin(cpn)1-standard deviation. 80 lbs levels of IFN-r activity in the supernatants of PHA-stinulated mnse spleen cells containing M were fannd to be significantly lowerthanthoseinparasite-free cultures (Table 1). 'lhisreduction was denunstrable 48 h after the initiatim of the cultures (decrease of 47%) wt was more prunnnced at 72 h (decrease of 259%). Similar resultsverefcmdwhenZug/mlofcmcanavalinAwasusedasthe mitogen (data not shown). mi neither secreted an IFN-r-like activityrnordiditirmncemnstimllatednSCtodcsoCI‘ablel). 'IhencteddecreaseinthelevelsofIFN-‘rintheculmre supernatantsmighttavereslltedfrunareductimintreproductiav secretimofthislynfinkineorfrmitsrencvalbytheparasite. '1!) determine which of these possibilities explained our observatims, solutia'sofrecanbinantmlrireIFN-rmreimnbatedinthepresenweor absenceofLMforwhardtheammtofresidnnlantiviral activityvasthendetermined. 'nneammtsofIPN-rremaininginthe supenetantsofclltnnesaftertheabsorptimwithmdidmt differ significantly frun that in the mockdtreated cantrols (p50. 1) . thus, for exanple, inoneof theeuperinnents, the IFN-r activities of thesolnrtionsafterincubationwith5x105or1x107 trypanastigotes/ m1were357il40axfl303i51lmits/ml, respectively,finerea8284i 74mits/mlweredetectableincantmlcllturestovmidnparasiteshad nctbeenadded. Itshouldbemtedthattheconcentratimsof parasitesusedfortheseabsorptimsrepresenteduvoardfwrtims, respectively, the level which was sufficient to reduce IFN-r activity 81 ms 1. mi-induced Irhibitim of IFN-r Productim by FHA-stimulated msca Material tested IFN-r (W1) 48 h 72 11 ISO 530 530 W 530 530 ISO + mi 530 530 W + m 530 530 IISC + an 58 67 use + FHA + W 31 530 a‘Ihetestedmaterialsconsistedcftheculturesupernat- antscfnSC (2.5::106 cells/ml) and/arm (2.5x1060rganisns/ml) inthepresencecrabsenceofSug/ml FHA. 'nnesuperrnatantswerecollectedattheirriicatedtimes afterinitiatimoftheanlunres. 'missetofresultsis typically representative of two separate repeat experiments. 82 instimlatednSCalltures(seeTable1). 'nneadditimofmitcwl‘tJlresofstimlatednSCdecneases lynphoproliferatian (14). Because IEN-r is able tomodify the proliferatim of T cells, either reducing or enhancing it depending an thedoseardthetimeofadministration (30),thepossibilitythatthe deservedalppressiminlynfinocytegrwthmyhaveresultedfmthe inhibitian of IFN-r predictionbymmsexplored. Varying ammtsofenngenmsrecatbinantnurineIPN-rwereaddedtcthe cunnesandtheirertectsmh-thymidireimorporatimbym- mressednSCvneredetermimd. AsshcwninTablez,theadditianof IFN-ratcmcentratiorsrangingfranBto2SOnmits/mldidmtovercane the alppressive effect. Higher IFN-r ccncentratians were not tested in theseenqlerinentsbecauseprelimimryresults(datamtshcm)had indicated that levels greater than 188 units/ml exert an inhibitory effect an lynphoproliferation. 'lhis can also be seen in Table 2 for thecmtrol resultobtainedwith 250 units IFN-r/ml. muzhasbeenshcmtorestoretheproliferativerespmse cfHIA-stimlatednSCsuppressedmmbymim A. Beltz,M. B. Sztein and F. Kierszenbaum, J. Inmnncl., inpress). Since IFN-T has beenreportedtoaffecttheinteractionofIIZwithlynfinocytesby increasingtheexpressimoereceptors,wetestedunetherIPN-r would act synergistically with IL2 and enhance the restorative effect of the latter lymncldne. The results indicated that treatm‘nt with 16 orlZSmits/mlIFN—tdidmtcverccnemi-inmcedsumressim vhenaddedtogeunerwithsuhqntinalmlevels(50mits/ml)anddid mtinprwenSCrespmsivenessnmenarestorativecacentratianofm 83 M32. IackofRestcrationbyEbtogenousIFN-r oftheCapacitycfPl-IA- stimulated use to Proliferate after m—m Suppression'na IFN-r (mute/ml) o 15.2 1 1.0 0.8 1 o.o° 95 8 14.6 1 1.0 0.3 1 0.2‘3 95 16 15.9 1 0.8 0.7 1 o.2° 95 32 14.6 1 0.4 1.0 1 0.49 93 63 14.7 1 1.7 0.9 1 0.5c 94 125 13.9 1 0.5 0.9 1 0.13 94 250 4.6 1 o.7° 0.9 1 0.0c 94 a‘Reccnioil'ant nurine II-‘N-twasaddedattheindicated concentraticxs toculturesofmSC(2.5x106cells/ml) cantaininngspgpnwmlinthe presenceorabsenceonJxlO‘ILM/ml. 'lhecultlnreswere incnbatedror72handluci3H-thymidinevaspresentduringthelast 24 h. ‘Ihis set of results is typically representative of three separaterepeatexperiments. bpercentdecreasewithrespeottothecorrespondingcantml (nsc+ HIA,noIFN-r). cp_<_0.05, forflnereductia‘lsinqlnwithrespecttoeither m1, i.e., nSC+PHAwithorwithout IFN-r, asmlculatedbysuflent's "t" test. 84 (100 units/ml) wasused (datanctshown). WenexteaminedvmeflnermvmldinhibitHN-rproductim orsecretimbyhm. 'Ihepresenceof5x106parasites/mlin cultures of FHA-stimllated hm did not lead to a significant reductioninthelevelsofIFN-rinthempematants(decreaseof5%at 72h:'1‘able3). ‘nnisparasiteconcantratimvasulicethatfamdto casistentlydecreasemnrineIFN—rproductim (Tablel)andsuppress reproducibly mitogen-induced lynphoproliferatian of mar: ( 1) . 'Ihe additionofexogernusmmanIFN-rdidnctrestorethesugpressed pmliferativerespaseofhfltexposedtcmmwtherormtm waspresent(datanctshcwn). 85 Table3.1ackofEffectof1,_qyz_imIFN-7Productianbyhm Material testeda IFN-r (M11 48 h 72 h m 55 $5 ELM 55 55 hm + mi 55 55 W + FHA 55 55 mar: + BIA 120 125 m + BIA + m 96 119 a'Ihetestedmaterials mistedofthecdlunre supernatants of hm (1.25 x lo6 cells/ml) and/or W (5 x lo6 organisns/nnl) imnbatedinthepresenceorabsenceome/ml FHA. 'Ihe supermtants were collected at the indicated time of culture and IFN-r activity was assayed by radioimmcassay. this set of results is typically representative of two separate repeat experiments. 86 DISGBSICN 'mesezeantsstnvedthatthepresenceofmmanhnesof Hm-stimlated mouse spleen cells decreased the levels of IFN-r activityinthesupernatants (Tablel). misdecreasewasmtdueto absoutptim, cummptim, or inactivatim of the lynplnkine bythe parasite since incubation of recmbinant IFN-r with W did not leadtoalossinmltiviralactivityevmvtmimlbatedwithfm timesasmanyparasitesaswerenecessaxytocmsistentlysuppress lynphoproliferatim and reduce the levels of I‘m-r in our culture systan. More,thedecreaseinIFN-rlevelsmsduetoreduced produtimorsecretimofthelynfinkim. Wehavepreviaslyreported thattheixmbatimofPMwithmidoesmtleadtoloasesin mitecellnnnbexsorviabilityanithattheparasitedoesmtrawve simificantmmtsofnrtrientsormitogmfranthemltures (1). DecreasedproliferatimbyuSCfruninfectedmiceorbynonnalnsc humatedwithmimispamlleledhydecreasesinm (8.32:8e1tzardKierszenbam, mpublisbed results) andIFN-r productimcrablel). Inca1trast,1,_g3;ziismabletodeczeasen2 productimbyhPBCmfler optiml mltuze caditims (L. A. Beltz, M. B. SzteinaniF. Kierszemaum, J. Immnl.,inpress) and,asreported herein, also has no significant effect an IFN—r productimbyhmc ('I'able3). M.Mameartoadstmtabledifferermsinmume pansiteaffectsnSCanthflnrespmseston. Im-rarflIIZareelatentsofacmplexregulatozynetvnrkandaxe able to affect ead't other's synthesis ard utilizatim (5,11,12,24,27, 35), with IFN-r productim being upregulated by 11.2 (5,24,35). 87 Acoordingly,antibodiestothen2reoeptorm1ddexmnethasaie,adnig which blocksIIZsynthesis,decreaseIFN—r productimbymitogen- stimlatethflC(24). Furthermre,theadditimofexogernsn2to Wage-depletedmixedlynfiiocyte cultures (5) arximstimlated macawmnw-rmis. shamidecreasesthe pmductimofbothIIZandIPN-rbynscmilehavingmeffectmthe productimofeitherlynfinkinebyhm,itistlmspossiblefliatfl1e parasite's abilitytoinlfibit synthesis of the former lynfilddne is at least partially responsible for the decrease in the latter. It should bemted,rmaever,thatlyuphocyte£franmioeinfectedwifllm MWaninpairmmtinIIZhItmtIFN-rseczetimmm, dalaistratingthatmmlmIevelsmaynctbeanabsoluterequiranent for optimal IFN-r synthesis and opening the alternative possibility mtmimyemertseveralirdepmdentwppressiveeffectsmthe Tcells. AdecreasedcapacitytoproduoeIFN-risduaracteristicof lynphocytes fran patients with leprunatws, but not tuberwloid, leprosy (18). Simeleprosyisaspectrmnofdiseasestateswiththe leprunatmsandtmbermloid formbeingthemstardleastpathogenic, respectively, increased pathology in this condition appears to correlatewithareduoedcapacitytopmduoeIFN-r. Asimilardefect isseeninwsoeptible,butmtresistarrt,stxaimofmioeinfected wit-11W (15) armor (26). 'Ihus, theability of flielnsttopmduoeIFN-rmaydetemimfliesubsequztseverityofthe disease. 88 Inthecnseofmi, sevenlmnmandefirdings suggest a possible role of IFN-r in host defense. ‘Ihus, the addition ofIFN-r tocultures cfbothnxrine fibroblastsardmcrqhages increasestheirresistametomminfectimbymitiypo- nastigctes (21,37). Ruthermore, exogenous IFN-r acts synergistically with anti-trypanosanal antibodies to decrease parasitania and prclcng survival of infected mice (20). 'Ihe ability ofmi to reduce II-N-r prcdntimbynscmismightdecreasethehcst'scapacityto eliminate the parasite. W has been reported to decrease mitcgen-iniuced proliferatim of lynphocytes fran either infected mice (8-10,13) or tnmns (33). 'Ihisdefectisalsocbservedwtmtheparasiteisco- cultured withnsc orhm frunminfected dcnors (1,14). mile Im-r has been found to amplify lymphocyte responses to mitogenic stimlaticn under certain circumstances (7,12,27,31), the data presented in Table 2 stmtlnteamgeranIFN-r caildmtcvercanethesqapressiveeffectof mi. 'Iherefore, it seems unlikely that reduced IFN-r productim lead to the Wi-ixduced reduction in lynphcproliferatim. 'Ihempacityofnsctcprcducenz isdecreasedfollcwimeither Wamwtom (8,32: L. BeltzandF. Kierszerbaum, mpiblished basalts). 'me addition of IL2 to these expressed admires restores their ability to secrete immoglchilin (22,23,32) and to proliferate in respmse to mitogen stimlatim (L. A. Beltz, M. B. Sztein and F. Kierszenbaum, J. 1111113101., in press). BecauseIFN-rhasbeenreportedtoincreasetheexpressimofm receptors am both '1' cells (12,27) and monocytes (11) an: higher levels 89 offlerIZzeceptcrallcwcellstorespondtolwerconcentratimsof n2(4),mtestedmemerIFN-rvmldetmancetherestorativecapacity chL2. 'mis,however,wasnctthecase:Il=N-rdidmtactsynergist- icnllywithIIZcrlcwerthecmcentratimofIIquuizedtoadiieve userecoverymatamtshcwn). In conclusion, we have danaxstrated a deficient capacity of use to proaneorsecreteIFN-raftereaqaoanetom. Whilethis deficiexcydoesmtappeartobeinvolvedinflieaippressimof lynghocyte proliferatim, it nevertheless may decrease the resistance ofctherhostcells,sud1asmacrqhages,toparasiteinvasimard .4 growth. Furthermre,tlmems11tsdanastrateasalientdifferencein the suppressive activities of mi towardnsc anthflIC. Miether this difference stats from the use of different populations of lynfincytescrfrananacmaldiffezenceinnmseammmnlynghccyte respasestoLMrerainstoberesolved. 'misworkwasaipportedbyNASAgrantMGQ-lalardasimledical Researdi Support Grant fran the College of Osteopathic Medicine, Michigan State University. 1. 6. 10. 11. 12. 9O 'dfi' 0’43- ‘2». mltz, L. A., atd F. W. 1987. Suppressim of mman 1W W by Wm- Immlogy 60:309-315- Rrasdii, D., S. Oasini, atd A. Minnie. 1984. Interferon-r reduces nacrcgiage-suppressive activity by inhibiting prostaglardin F2 release ard ixducing interleikin 1 production. J. Immol. 133:764-768. ndzkc, D. D., and F. Kierszabain. 1974. Isolation of W M frcn blood. J. Parasitol. 60: 1037-1038. when, D. A., ard K. A. mid). 1984. ‘me interleikin-Z T-cell systan: A new cell growth model. Science 224:1312-1316. Farrar, W. L., H. II. Jdmscn, ard J. J. Farrar. 1981. Regulation of thepmductimofimmemterfermardcytctcxichynfiiccytesby interlamin 2. J. Imuol. 126:1120-1125. Itasca, D., L. Achrini, S. Ianblfc, aid G. mria. 1985. Enhancing effect of IFN-r on helper T cell activity and IL2 productim. J. Inuunol. 134:3907-3911. Prim, R. M., and S. N. Vogel. 1983. IFN's with special @2515 m the imme system Adv.Im1nol. 34:97-140. Karel-Baum, A., H. Joslmwicz, D. Fradelizi, ard H. Risen. 1983. Modification of T—cell proliferatim ard interleukin 2 production inmice infected with W1. Proc. Natl. Acad. Sci. ISA 803466-3469. Harel—Bellan, A., H. Jcskcwicz, D. Fradelizi, ard H. Risen. 1985. lenphccyte fmcticnduringexperinental Chagas' disease: production of ard resporse to interleukin 2. Eur. J. Inmmol. 15:438-442. byes, H. M., ard P. Kierszabaun. 1981. Ecperinental diagas' disease: kinetics of lynphccyte responses and immological control ofthetrarsition fmawtetodxrmicW infection. Infect. Inuun. 31:1117-1124. Harman, F., S. A. Carnistra, H. Isvine, ardJ. D. man. 1985. ”mica of interleukin 2 receptors ard birding of interan 2 bygamnainterferm-ixducedmmanlaflcenicaxdmrmalmonocytic @118. J. Btp. M. 162:1111-1116. Jdlmm, H. M., ardw. L. Farrar. 1983. lbs role of a ganna interfexm-like lynpholdne in the activation of T cells for expression of interleukin 2 receptors. Cell. Immol. 75:154-159. 14. 16. 17 . 20. 21. 22. 91 .Kierszebann, I". 1981. mevasimofWi franthe host imme respense. Iynphcproliferative responses to antigens during amte ard dnrcnic experimental dnagas' disease. Immlcgy. 44:641-648. mlednar, J. R., ad 1". Kim. 1983. Inhibitian of mitcgen- irduced proliferation of monise T and B lynphccytes by bloodstream forms of W. J. Inmmol. 130:908-911. Ian-ray, B. V., B. Hasur, ad J. S. Keithly. 1982. Cell-mediated immune W in experimental viseral leishneniasis. 1. Cbrrelaticn between resistance to W and Immune-generating capacity. J. Immol. 129: 344-350. m, B. V., B. Y. Min, ad c. D. muneml. 1983. Killing of intracellular W by lynfinddne-stimlated hunan Wear magccyte. Evidence that interferon-r is the activating lynpholdne. J. Clin. Invest. 72:1506-1510. Nathan, C. P., H. w. army, 1!. R. Wieae, ad B. Y. Rbin. 1983. Identifiaticn of interferun-r as the lynphokine that activates human nacncphage oxidative metabolisn ad anti-microbial activity. J. Exp. Med. 158:670-689. Nogueira, N.,G.Raplan, R.Ievy, R.N.Sanno, P.nishner,A. W-Pipenn,1.Vieira V.C.Gcnld, W.Ievis, R.Steinnan, Y. R. Yip, and z. A. aim.1983. Defective r-interferon productim in leprosy. Reversal with antigen ad interleikin 2. J. mp. Med. 158:2165-2170. Pfefferlmn, R. R., ad P. 14. Gym. 1983. Recanbinant hunan ganma interfermblccksthegrcwthofmmmmlummmn fibrdalasts. Fed. Proc. 42: 964-967. Plata, P., P. Garcia-Pens, adJ. Wietzerbin. 1987. mums resistance to W: synergy of specific antibodies and 138:397-415. Plata, P., J. Wietzerbin, F. Garcia—Pens, R. Falcoff, adR. Risen. 1984 . Synergistic protection by specific antibodies and interferon aqamstinfectimbyIWiinm- Euro-I- Immol- 14:930-935. Med, 8. G., J. A. Invere), ads. B. Enters. 1984. Heterolcgcus antibodyresponses inmicewithchrvmicf.['_,__<:,mz_;lI infectim: depressed T helper functien restored with mpernatants certaining interleukin 2. J. Immol. 133:1558-1563. 23.1hed, s. G., J. A.Inverso ads. B.lbters 1984.31ppressed antibodyresponsestosheeperythrocytesinmicewithdnreiic W infections are restored with interlankin 2. J. m1. 133:3333-3337. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 92 then, G. R., ad N.-B. Yeh. 1984. Interleikin 2 regulates expressienofitsreceptoradsynthesisofgamainterfermby tunan ‘1‘ lynphocytes. Science 225:429-430. msztcczy, 1., C. Siroki, ad I. madi. 1986. Effects of interferes-a, -fi, and -7 an human interleikin-Z productien. J. Salrick, n. D., R. n. chcsley, c. nuns, adH. v. Raff. 1986. nirine cutaneous leishmaniasis: resistance correlates with the capacitytogenerate interfere-rt inrespmsetonggmania antigens m. J. Immol. 136:655—661. Sdnenridn, P.,U. Der, M.Rillian, adLPfizenaier. 1985. Differentialeffects ofgama—interferunanlnmnan'rcellsduringam primary activatimin vim, p. 63-67. In C. Sorg, A. Sdninpl, 14. Lady (eds. ), Cellular ard molecular biology of lynphokinesfm ProceedingsofIntematicnalWorkshcpinw.Germny, Academic Press, Crlardo. Sileghen, 14., R. miners, ad P. De Baetselier. 1987. Bmerimental 1mm infections selectively suppress interleukin 2 production ad interleukin 2 receptor expressian. Eur. J. Immol. 17:1417-1421. mith, R. A. 1984. Interleukin 2. Ann. Rev. Innmol. 2:319-333. Serenfeld, G., A. D. Handel, ad '1'. C. lur'igan. 1977. line inmmosnmpressive effect of type II wise interferon preparaticns an antibody production. Cell. Immol. 34:193-206. Senenfeld, G., A. D. Hardel, ad'r. C. lurigan. 1978. Time ard preparaticns. Cell. Inmunol. 40:285-293. 'l‘arletan,R. L., adR. R. m. 1984. Restoratimcfjnm immerespmsesofspleencellsfrunmice infectedwith 'bysupematantscontaininginterlendnz. J. m1. 133:15‘70-1575. minim, A. R. L., G. W, V. m, “A. Prata. 1978. Acquired cell-mediated immunodepressicn in acute Chagas' disease. J. Clin. We 62:1132-11410 W, 6., ad B. Perussia. 1985. Immune interferon: a pleictrcpic lynpnokine with nultiple effects. Inumnol. Today. 6:131-136. Vilcek, J., D. Henriksen—Destefan, D. Sisal, A. Klim, R. J. Rib, ad J. Is. 1985. Regulation of IPN-r inductian in human peripheral bloodcellsbyeonogeunsadendogenonmlyprcduced interleikin 2. J. Imml. 135: 1851-1856. 93 36. Villalta, F., ad W. lean. 1979. Effect of purifiaticn by [EAR- cellulcse column on infectivity of W blood forns. J. Parasitol. 65:188-189. 37. Wirth, J. J., P. Rierszeimn, G. Sanenfeld, and A. Zlctnik. 1985. Wimeffectsofganmainterfermcnmagccyticcell associatim with and killing of W1. Infect. mum. 49:61-66. 94 M3 MWMW-MWIWOF WW: NHBI'I‘IQ‘IOFINIERIEJKINZ RECEPKR WEN 95 Co-culmre of blood forms of W - the causative agentofdnagas'disease-withtnmanperipheralblcodmmiclear cellsinpairedthecapacityoleynfinocytestoeanresssurface receptorsforinterleukin2(IL2R). 'Ihiseffectwasevidencedby mrkedrednnctiesinbotlnthepr'oportimcf'lac+cellsardttnedensity cflacantigenmthesnn'faceofthepositivecells,determinedbyflow cytanetry. 'nneextentoftheixhibitianincreasedwithincreasing parasite concentrations. Urderoptimalorsubqrtimalcorditions of stimlaticn with either finytchenagglutinin or mmoclonal anti-CD3 - specific for an epitope of the (133-Ti humn T cell antigen receptor canplex-thepresenceofmcurtailedthecapacityofT lynphccytestoproliferateadexpressIIZRbitdidnotaffectIIz production. Furthermore,theadiitimofemgenonsn2didnotrestore therespasivesssofsuppressedmmnlynrhccyteshxtdidwrsnmse theswereused irstead. Therefore, unlikemouse lynrphocytes, human lynphccyte suppression by L931 did not involve deficient IL2 prcdnntionandwasaccanpaniedbyinpairednzutilizatian. Co-culture of human mnocytes/macrcphages with suppressive concentraticrs of L miircreasedinterlenkinl(fl1)productimardtheparasitedidmt decrease In seceretion stimulated by a bacterial lipqaolysaccharide. 'Iherefore, the suppression of II2R expression and lynphoproliferaticn ismtlikelytohavebeenanixdirectcesequencecfinsufficientm productionduetoinfectimofmonocytesormacrofinages. Wehave previously shown that sippression of human lynphccyte proliferation by 96 W isnotcansedbymtrientconsunption, absorptionof II2, lymhccyte killing or mitogen removal by the parasite. 'Iherefcre, theseresultsmsoveranovelsuppressivenednanisminducedbyfi mi, involving inhibited expressien of II2R following lynphocyte activation and rendering T cells unable to receive the 112 signal required for continuation of their cell cycle ard monmting effective imme responses. 97 INIROIIJCI‘IQJ Chagas' disease-causedbyflneprotozoanWi-isa major health problem in South and Central America. Its acute phase, bothinlaboratoryanimalsardinmnnars, isaccanpaniedbyastateof suppressed immnity believed to facilitate the establishment ad dissemination of the etiologic agent in the host (1-15) . Several imnnolcgical abnnornnalities have been identified in mice infected with W, imlndinginxzreasedlevelscfsuppressorlynfinccytesard macrophages (1-4) ad diminished levels of '1‘ cells (5) in the spleen, inpaired lynpnocyte proliferation in response to mitcgens (2,4-9) or parasite antigens (10), suppressed antibody-forming capacity (11) and inpaired 112 production (4,7,12). How these abnormlities are induced isnotknownard, unfortunately, differences inhowmseardmnman 1W are affected by W nake it difficult to extrapolate these findings to human infection. 'Ihus, nnurine splenic lynphccytes - whether frann infected animals (12, 16, 17) or co-culmred with the parasite 11111211? (Beltz ard Kierszenbanmn, unpublished mite)- display reduced interlenkin 2 (Il2)-prcducirg capacity, the ceseqnerncesofwlnidnareovercanebytheadditimofenaogennsm (12, 16, 17): thisisnotthecaseforhnmanlynptnocytessnn'pressedby the parasite (15). '1b stndy the early alterations that m induces in hunen lynnphccyta ad to explore the mechanisn(s) involved, we used an jam systen in which lynphccytes ard monocytes/macrqinageswere inanbatedwiththeparasite inthepresence of lynphocyte—activating stimuli. We report that W inhibits the 98 capacityofhnman'rlynnfinocytestoexpresssurface interlenkinz receptors (112R) upon activatim. This effect my render ‘1' lynfinocytes unabletoreceivetheIl2 signalrequiredtoproceedwiththeircell division cycle and nount signnificant levels of immunity. 99 WNW m. Blood (trypanastigote) fornns of Tulahuen strain 1.. m were isolated fran the blood of Crl-CD-l (ICR) Swiss mice (Charles River Breeding laboratories) infected 2 weeks previously with 2 X 105 parasites intraperitoneally. 'lhe parasites were purified by centrifugation over a mixture of Ficoll-Iiypaqne of density 1.077 (18), followed by dnranatograpny through EAR-cellulose (19) . 'me eluted organisns (100% trypanastigotes) were concentrated by centrifugation (800 x g, 20 min, 4'C) ard mended at the desired concentrationn in RPMI 1640 medium enpplenented with 59. (vol/vol) heat-inactivated fetal bovinesermnardcentainingstreprtanycinat 100ug/mlardpenicillinat 100 units/ml (henceforth referred to as name) . healthy volunteers were purified by desity gradient centrifugation through a mixture of Ficoll-Hypaque of density 1.077 (350 x g, zo'c, 45 min). Afterthreewashingswith serum-freeRBdI 1640 medium, the cells wereresuspededatthedesiredconcentratieninRHfl+S. Cell viability, determined by trypan-blue-dye exclnsian, was >99%. W. Single cell suspesicrs of inbred CBIVJ mouse (Jackson laboratory) spleen cells were prepared in RHII+S as described (13) . W. Recanbinant, glycosylated hunan 112 was pirdnased fran Genzyme (Bosten, MA). WW. PRC were irwbated in RIMES (5% (Dz; 96-well plates) at 37'C for 96 hours with or without 0.6 or S 100 ug/mlHR(SignadnenicalCo.)cr6or251g/mlanti-w3(0rtho Diagnostics) [a menoclcnal antibody specific for an epitope of the T cellantigenreceptorcanpleccm-Ti (20)]inthepresenoeorabsence ofmi. '1heculturevolumewas0.1ml. All corditieswere testedintriplicate. Ehdnanlmrereceivedluci3H-thymidine 24 hours before termination by autanated harvesting. Radioactivity was determined in a liquid scintillation counter: the resultswere expressedasneancanntsperminrte(qn)1-lstadarddeviatim. 'nne cencentratiasofPBradparasitesatzerotime,andafter48ad96 hrof culturearegivennn'derm. Viabilitywasestablishedby trypanblueenclnsicn. www.mmvitywasdetemirsdinflne supenatantsof48-honmmncnlturessetupasdescribedabweewept thatthefinal volumewasl.5mlan'd24-well plateswereused. The anpernnatantswerepassedthmnghaO.22—m—pore—size filtertorenove parasitesifpresentandstoredat-ZO'Cmntiltested. '1heI12- depedentlflflcelllinewasusedtodeterminell2activity(15)ad theresultswereecpressedasmnits/mlinreferencetoastardardm preparatian (cemnavalin A-stinnulated rat spleen cell mlhnre super- ernatants) which was arbitarily assigned a value of 100 units/ml (21). WW- m<1.2SX105c=ells/m1)m inanbatedinRPMI+S (5% CD2: 24-well plates) at 37'C for 48hmrswith moranti-wammclmalantibodyinthepresenceorabsenceofL m1. After48hcurs,thecellswerewashedthreetineswith Wte-bnfferedsalin'spliLZcentainirgRbovinesenmalbnmin (Signa)adwerestairsdbytreatnsntwithanti-Tacnugclenalantibody 101 [which recognizesanepitcpe cfthehumanII2R (22) andwaskirdly provided by Dr. T. A. Waldmam, National Institutes of Health] followed, after washing, by flourescein-cenjugated F(ab')2 derived frann goatanti-mouselgGantibody('1'agoImnnnodiagnnostics). 'nnecellswere fbaedin1%fcrna1dehydeadwereanalyzedinaMIVflwcytmeter. TenthanardPBlC, gatedtoexclndemardcelldebris, were accnmlated for each histogram. The perwntage of IL2R+ cells [i.e., %Tac+ cells (22)] in each preparation was ealenlated after anbtracting the background of nonspecific labeling with mPC-Zl (a nespecific 1% derived fran a mouse myelana cell cnlture) and fluorescein-conjugated F(ab')2 anti-mouse IgG. ‘Ihe mean dnannel nunnber of the logarithm of thefluorescenceinte'sities (MFCn) wastheparameterusedtocanpare the relative desity of 'I'ac antigen on the different Tac+ cell populations. The logaritlmn of fluorescence intensities was distributed over 256 dnannels. CnemlofPBc suspension at 2.5 x 105 Pant/m1 was incabated at 37'C for 2 hours in 24-well plates; the adherent cells (>98% nanocytes/macrophages by both norphological criteria ard positive staining for with esterase) mreirnnbatedwithmedinnnaleneorcontainingSX1060r1X107 trypanastigotes/ml inthepresencecrabsenceonOug/mlbacterial lipopolysaccharide (LPS, Difco) at 37'C for 24 hours (5% (:32). Culture supernatantdilutimswereaddedtomsethymcytecllunres stimlated withasuboptimalmAcorgentration (lug/m1) asdescribedindetailby Heltzerandeenheim (23). 'nneresultswereeanessedasqn representing 3H-thymidine incorporation by proliferating thymcytes. presencecfblcodstreamforuschmi,PBCstimlatedwithHiA sinnedanarkedlydecreasedcapacitytoecpresssurfacean. This effect was parasite concentratian dependent (Table 1) ad was evidenced byadecreaseintheproportiancfllficellsaswellasinthe ldensityofTacantigencnthesurfaceofthepositivelynfinccytes unethercptimlormboptimalPHAcancentratieswereusedWig.1). 'lbestablishmnetherthiseffectwasalsoprcducedmder oddities lonown to mimick antigen-induced lynphccyte activatim (23), wecarriedartsimilarecperimentsnsirganti—dnasthestimllant. 'nnereenltsdemrstratedthatmalsoinpairedMeanessimin thiscasewhetherthelynfinccyteswerestimnlatedwithcptimalor subcptimlamountscfanti-Cm (Fig. 1). mididnotstain positively fa: 'Iac antigen [i.e., did not bind anti-Tao or the fluorescein-labeled F(ab')2 anti-mouse IgGthetherornotco-culunred withmcinthepresenceorabsenceofPHAcranti-ab,addidnot respa'dto recanbinant. I12 (20 to 250 units/ml) with altered levels of 3nn-thymidireimorporatimmatamtsnmn). WmmmmWWmWfimw PanstimnlatedwithsuboptinalorcptimlmAconcentraties (15). 'nnereenltspresentedinlableZirdicatedthatthiswasalsothecase mnenthelynpnocyteeweretriggeredwithsubcptimlcrqntiml cancentratimsofanti-cn. Urdertheanboptimalorcptimal stimlatoryceditia'susedintheecperimentsdescribedabove(o.6ad W FHA %Tac+cells (%V) m 0 Absent <2 0 Present 46.3 114 l x 105 Present 34.9 (-25) 112 5 x lo6 Present 26.5 (-43) 67 10 x lo6 Present 4.2 (-91) 86 3‘ alltures of Pmc (1.25 x lo6 cells/m1) with or without W wereincubatedwithorwithortSug/mlmAfor48 hr. 'Ihecellswere stained with anti-Tao and fluorescein-labeled goat F(ab')2 anti-mes IgG. 'neperoentageof'l‘ac+cellswascalonlatedaftersnnbtractingt1e background of nonspecific labeling (see Materials ad Methods). Peleentvariation (%V) withrespecttothevaluecbtainedwithm alone = [(value with parasites - value without parasites) / value without parasites x 100]. This set of resalts is typically representative of two separate repeat experiments. 104 Figure 1. Effects ofWonIlzRecpressionbymmnan lymnocytes. Pacnere inncubatedat 37'C fcr48hourswithPHAcr ami-w3moecloelantibodyinflepresereeorabseeeof5X105L cunt/m1. The cells were processed for flow cytonnetric analysis as describedunder'rable 1. (A) ResposestoancptimalHiAcoeentration (5 ug/ml): ma, 55.0% naci‘ cells, are: 120; mm, 40.3% Tac+ cells, MFCh 103. (B) Responses to a subcptiml HiA concentration (0.6 cells (lg/m1): an, 28.6% Tac" cells, m 126: mm, 19.6% nac”, men 109. ((2) Responses to an optimal anti-CD3 concentration (25 ng/ml): anti-CD3, 41.5% Tac+ cells, m 137; anti- mm, 27.0% Tac+ cells, m 121. (0) Responses to a suboptimal anti-cm concentration (6 ng/ml): anti-cm, 20.5% Tac+ cells, m 133; anti-mm, 16.0% Tac+ cells, MFCh 117. In control Parantures (no mAcranti-Cm), theproporticn of Tac+ cellsnevereaoeeded4%. Thesetsofdata forPHA-andanti—Cm- irducedresposesarerenresentativeoffiveadthreeseparaterepeat experiments, respectively. Wistlnenneandnamelmmberofthe logarithm of tie fluorescence intensity. neseeosuaml D . m 105 .m: ea. uh ”so-MM”. .333 H + ressionnorthe lerelof3H-thymidineuptakereumnedtorormlityafteradditianof 2501mits/mlIL2. Similarresaltswereobtainedinecperimentsin midndosesofIIZuptOGOOmits/mlwereusedard3H-thymidine 11)9 ._ epoch ucomo. mom .>u .maauaos as“ ea uspuoscomosaus appuowQAa use mou-muco use <:e so» seas to “can use ..ssas .5. n.5easssm .No.QWa. seaosc_ea_s appaomss_s.sa are: mas_a...a hearse; as» cum: coe_ooaa mus—as seasons mooeoeoee_n Fp< .apms_uooomoe .~e_ sup: sea «sores: snare .» newcmaucoo muesupsu roe 5.9 cc. O.“ can .apuspuoocmoe .~h_ gu.3 cc. “segue: mossy—so so» m.~ no. ~.o not»: nus—as or» mouemoeaa eo «oceans as“ c. mou-.ueo gu_r “cospcmaxu as“ so» .apussuuoamos .~b_ su_r cc. asses.) .Nsso .» ocmcsaaeoo “mesa—so so» v.m ecu q.u use “no“.mceaa «segue: <=a gas: aeoewemsxu «cu toe .a_o>_uuusmos .~b_ gs.) use users.) moeaupsu so» 9.“ as» n.o "consume eo nucomosa ecu c. accesses ounce eoee unacceuasm «so: concave “segue: noc_auao wasp.» use .eau ucamsogu ma commutes» oea_co.»aeootouc, ocwo.EAcu-=m ea mos—o» —_< .ncuumc, ss.cos eo «cases acupasssoo co easement noise—so posucou "coaches «ea eases apouo.uoes_ .psxmuwcs omm an. «ossupso «anecdotes» us“ ca gonna use ~e~ cuss; possessoua—u .ucoc_c50uo¢ .__ o—ao» ca ucomm— ugu c. uuawsummo ma noseoeeoa as»: muesupau nine or» a 2- 23.3 32...: :3 no u 3 as u as .. 39. a £8.35 an. esn\s.as -~\m.om .ss-. a.~ “.m.m ..H.u ..am - .asae mN .neo-seea en- as~\a.me an.\e.ma ln5-. a.o u.s.ms e.o_n e.am . .exaa m .aza .n- aofi\m.an a~_\a.am ims-. a.o.u ..a ~.s.u_o.~s - ,s\aa m .aea a__oo eudsx+oaea euax\+oahu +uae .Nsto .e.u=sa a=o_. ozma .su. c~3.o .e.usma oea_a urea ~s_ seasons.» toe >u cowmmoraxo mm._ comsoeooeooes ocwuweaga-zm eo copspuua combos: ame— msocomoxo eo success to coconuts or“ cm mou-sscm to «:a cue: newnesswum moswcoznss. case; an coemmoenxo “we” use ccmuaeoctooc, scrumsxcs-: mm _~seo .h eo nuooeeu m _._ whm

to, T.Uchiyala,R.R.Bardy, G.Yamh, ard T. Taniqxhi. 1985. Reconstitution of functional receptor for taxman interleukin-2 in mouse cells. W Brian), D. 8., ard 1". Kierezeltnn 1974. Isolation of W fmn blood- 1W Villalta, P., ard W. law. 1979. Effect of purifimtim by M- cellulose column on infectivity of W1 blood forms. 11W R11),R. J., W. C. @838, me. 1!. Basic. 1984. Imardhigh affinity cellular receptors for interleukin-2. ' inplicaticns for u‘elevelfimm MW Udliyana, T., S. Broder, ard T. A. Walmanl. 1981. A ncnoclcnal antibody (anti-Tao) reactive with activated and functimally mature lumen T cells. I. Production of anti-Tao monoclmal antibodyarridistrib.rtimof‘l‘ac(+) cells. W Green, W. C., anl W. J. Iemard. 1986. 1118 human interlalkin-z receptor. Wm Iemard, W. J., J. H. quer, G. R. (:rabtree, S. eriikoff, J. W, R. J. Mb, )1. mike, P. B. Svetlik, N. J. Raffer, T. A. Waldnanl, and w. C. Greene. 1984. Molecular cloning and expression of cINAs for the hunan interleukin-2 receptor. Nature 3.1% m, R. J. 1986. Calversicn of low-affinity interlelkin 2 receptors to a high-affinity state following fusion of cell membranes W Bar-a, T., L. K. 1.. July, J. H. Bjcrlrhhl, ms. ll. Pu. 1986. when T cell activatim. III. Rapid inductim of a filesphorylated 28 kD/32 kD disulfide-linked early activation antigen (EA 1) by 12-o-tetradecanoy1 phorbol-lB-acetate, mitogens, ard antigens. (L W 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 140 m, s. C., J'. c. mdgdm, R. a. messy, J. P. Protentis, s. F. Sdlloesmn, an! E. L. mirilerz. 1983. Antigua-like effects of monoclonal antibodiesdilectedatreoeptorsofmmanTcell cleric. W m, 14., R. W. Kczak, C. K. Golden, aniT. A. Helm. 1986. Danonstraticn of a non-Tac peptide that binds interleukin 2:a pctentialparticipantinamltidlain interleukinz receptor 00191835 WW aural,ll.,R.D.Klm1sner,B.R.olllen,R.dlizzmite,W.J. Isaard. 1986. Novel interleukin-2 receptor submit detected by cross-linking under high-affinity conditiors. W R11), R. J., C. H. Rusk, J. Yabi, ard“. C. Gran. 1987. InterlelIanbindingmlealledistinctfruntheTacpretein: analysis of its role in formation of high-affinity receptors. MW Rib, R.J., A.Dllrck, “Khalifil. 1981. Tcellgrcwth factor receptors: quarrtitation, specificity, and biological relevance MM my, 8.41., ard K. A. Snith. 1987. The interleukin 2 receptor: functimal consemenoes of its bimolecllar structure. m W mi, 2., H. E. neirhenz, ard J. J. Ellner. 1986. Defective interlalkianroductionardrespmsiveness inhlmanpllmonary tubercllosis. WW apta, S. 1986. Study of activated T cells in man. II. InterleflanreoeptorardtrarsferrinreoeptorelmressimmT cellsardproductionof interleukinz inpatientswithacquired imme deficiency syndrmle (A113) and ADE-related cmplex. gain. W Prime, H. R., ard J. K. Jam. 1987. Abnormalities of interleukin 2 receptor expression associated with decreased antigen-irriuced lynfilocyte proliferatim in patients with AIIB and related dimmers W Sileglen, 11., R. Ham's, all P. de Bestseller. 1987. Experinental W infectiote selectively sugaress bothinterlelkinzproductimardinterlellcianeoeptor expreeeim- W Peldlan,R.D.,G.w.nmirgtflce,alrlw.Islerdle.1987. fi- adalergic receptoruedlated suppression of interlelkin 2 receptor inmmanlynebocytes W 35. 36. 37. 38. 141 Idlich, R., 8. mi, 8. W, H. Sam, Y. W, J. Yodoi, an! H. “.1987. Impaired eupressim of high affinity interleukinz reoeptorsmactivated lynfilocytes frunpatiarts with systemic lame eryttmtcens M19191 Brodc, J. 11., am! T. minor-Fuller. 1983. The role of irm and transferrin in lynpl'locyte transformation. W m,s.c.,R.B.nlssey,H.Fabi,D.FuK,O.Aalto,K.A. KW, J. C. m, J. P. Prctartis, S. I". Schlcssnan, ard E. L. W. 1984. Analternativepa‘dlwayof T-cell activatimzAftmctionalrolefortheSOdellsheeperythrocyte receptor protein. g1]. 36:821. Nedms,L.ll.,ardJ. Cosmn. 1983. Transferrinreoeptor iniuctiminmitogen—stimlatedhmanlenfilocytesisremired forDlAsynthesisandcell divisionandisregulatedby interleuldnz- W 9*. 142 CHAPTERS lewnmanpmmormmlmmmm MW V ‘. 143 WWTIWmvatimviafleT cell antigen receptor-(1)3 (cm-Ti) calplex. This immosuppression involves a decrease in the proliferative responsiveness of humn peripheral blood moroniclear cells (mic), as well as the expression of interlelkianeceptors(II2R),butmttheproductimofinterlendn2 (112). Inthepresentreport,wehavefcmithat1._gmzialso suppressed Tcell activation through the C132 alternative pathway. Proliferative responses of mic to anti-Tl12 + anti-Tl13 were decreased byco-ciltmewiththeparasiteinadcse-dependentfashim. This decreasewasnotduetodeficient production of IL2, since levels of fllislynfinldrewereacunllyirmeasedinthepresetneofm, butmaybedueinparttoadecreaseintheexpressimoftheIIZR. 1;. midecreasedthepercentageofcellsexpressingtheHZRardthe density of this marker following sinnlation via (:32, although to a lesser degree than following activation by FHA. Both the upregulation of the levels of T112 of the cell surface and the expressial of T113 vereinhibitedbytheparasite. mimsflmfmmsmressT cell proliferative responses by both the (133-Ti and the T11 activation pathways. 144 mm W, theparasiticagentof Chagas' disease, causes a state of immppression during the early, acute plase of infection ofbothhmnansandmice (1-4). misalrpressicn involvesboththe cellularandthetnnnoralarnsoftheimmerespmseininfectedmice (5-8). 0o-ciltme ofmitrypanastigotes with lynphccytes frcn normlmiceortnmansalsodecreasestheproliferativerespmseof _ . ”Ham-1V thesecellstomitogers (9,10) and,inthecaseofhumanperipheral {a bloodmononuclear cells (PBC),toanti-a)3 (Chapter 3),anantl'body 5"”: directedagainstthem-JI‘iTcellantigenreoeptorcamleu (11). The abilityofWitosrppressrewasesofmrmlPflflmylie, at leastinpart,inaredu:tionintheexpressimoftheII2R(dlapter 3). Thisreductionismtacoalpaniedbyadecreaseinthepmduction ofIIZmfleroptimalculmrecalditionsKhapter3). In adiitim to the well-characterized (133-Ti pathway of T lynphocyte activation, several antigen-indeperdent pathways have recentlybeenreported(12-14). Ofthese,thea)2pathwayisbest characterized (reviewedinlS). (1)2[T11,thesheeperythrocyte receptor, lynphocyte function-associated antigen-2 (LEA-2)] is a 50 kD polypeptidethatisexpressedmallthymcytesardnahlreTcells (15). Maxeretal(12)producedm:oolonalantibodieswhidlreacted withthreedistirctepitopesman.'lheT111ardT112epitopesare expressedmalchellsardareupreginateduponactivatim,while '1'113isfcm'rlmlymactivatechellsardthymcytes. Anti-1'11; antibody was also found to rapidly irrluce T113 expressim (within 30 145 min) (12) . 'lhe calbinaticn of anti-T112 ard anti-1113 antibodies ilrilmsrestiqucellstoproduceIIz (16), expressILZR (16), am! divide (12). Since C02 is not associated with (103-Ti m the cell surface (17) , it thus represents an alternative pathway of T cell activatim. Inthepresentsuldy, wehaveexaminedtheeffectofmmT cell activation through the (1132 alternative pathway and carpaled these results with our previous findings with the CD3-Jl‘i pathway. 7"? 146 WWW Parasites. W trypalestigotes ('Iulahuen strain) were isolated frcm the blood of Crl-G)1(ICR) Swiss mice (Charles River laboratories, Portage, MI) infected intraperitoneally with 2 x 105 parasites two weeks previously. me trypcmastigotes were purified by centrifugation (350 x g, 45 min, 20°C) over Ficoll-Hypaque of density 1.077 (18) ard IEAE-cellulose chralatograply (19). After two washings with arm 1640 medium (Gibco, Grand Island, NY) sipplemented with 100 units of palicillinand loougstreptanycin/ml, theparasiteswere resusperdedatthedesiredconoentratimsintheabovemedium cartaining 5% heat-inactivated (56'C, 20 min) fetal bovine serum (Rm+5%F'BS). Perinleralbloodmnlearcells (me). mewere isolated frm the venous blood of normal donors by centrifugatim over Ficoll- Hypaqueasdescribedabove. AfterthreewashingsinRM, them were resuspended at a final concentration of 1.25 x 106 cells/m1. Cell viability, as determined by trypan blue dye exclusim, was always >99%. Proliferatimassay. Pflcwere incubated in96-wel1 plates ina volumeof 0.1ml inthepresenoeorabsenoeofeitherSug/ml phytohemagluttinin (BIA; Sigma Chenical Co., St. Louis, MD), 25 ng/ml 0K1‘3 (Ortho Diagnostics, Raritan, NJ), an antibody reactive with (.133, or a 1:100 dilution of anti-Tug and anti-Tll3 mmoclonal antibodies (reactive with two distinct epitopes of €02; generous gifts of Dr. S. F. Schlossman, Dana-Farber Cancer Institute, Best-m, 1%). Sale wells alsocmtainedmiatcalcentratims rangingfrom2.5tc10x106 147 parasites/m1 , which replaced an eqnal volume of RPM-tam . olltures wereinoubatedat37°c, 58002 for96hrs, withlucicf3nn~thymidine (specific activity = 2.0 Ci/nlnole; New England Nuclear Biotechnology Systenns, Wilmington, IE) beingpresentduringthefinal 24 hrs. Culmreswereterminatedbyautcnatedharvestingardthelevels of incorporated radioactivity were determined in a liquid scintillaticn conmter. Resultswereexpressedasmeancountspermimrte (cpn) :1 standard deviatim of triplicate culmres. 112 Assay. pair: were incubated in 24-well plates in a volume of 1.5ml inthepresenceorabsenceofSugHiA/mloraldOOdilutimof anti-1112 + anti-41113 antibodies with or without 5 x 106 W. At 48 of incubation, cultures were centrifuged (350 X g, 10 min, 4'C) and the supernatant was clarified by filtration through 0.22 nine-pore- size filters and stored at -20‘ C mntil assayed for IL2. IL2 activity was determined using the IL2-dependent cub-2 cell-line as previously described (10). ResultsareeupressedasnmitsIIZ/mlinreferelceto a standard IL2 preparation of 48-hr concanavalin A-stinnlated rat spleen cell supernatants which was assigned a value of 1000 units/m1 (20). Eamressimoffinem 'nlecellpelletoftheabovedescribed clltureswaswashedthreetilneswithnlosplate-buffered saline containing 1% bovine serum albumin (m-EA) and subseqaently incubated for 30minwithafluorescein-conjugatedantibodydirectedagainstthe ILZR, anti-2A3 (Bectm—Dicldnson, Maintain View, CA), following the namfaculrer's instructias. After fixatim in 1% formalin, PEI: (10,000cellspercondition) wereanalyzedinamCSIVflowcytcmeter 148 gatedtoennclnflemiarricelldebris. 'IhepercentageofILZR" cellswascalallatedaftersubtractirqthebadcgrundofmmspecific labeling with fluorescein-cmjugated anti-loayhole linpet henncyanagin ' (Bectm—Dickinsm). 'nnemeandlamelnmberofthelogaritlmofthe fluorescence intensities M31),distrib.rtedcver256 charmels,was usedtocanparetherelativedensityoftheanmthedifferent Demmimtimofflleacprmimofmzarfimr ollturesof Pausetnpin24-wellplatesinthepresanceorabsenceofPHAas describedabovemreinclbatedforperidsoftimerangingfranétofl hrs, washedwith 135-EA, and incubated for 30minwitha 1:150 dilutim of T112 or T113, or with control nmse 196(calbiochen, Ia Jolla,Cm). AftertwowasheswithPBS-EA,cellswereirmbatedfor3O minwith fluorescein-conjugated F(ab')2 goat anti-abuse Igsand analysedbyflcwcytanetryasdescribedabove. 'nnepereentagesofmc expressinnglzarriT113werecalcllatedaftersubtractingthe bacnogmmd of nonspecific labeling obtained with the caltrol mouse :96, andthemhwasusedtcdeterminethedersitiescfthsseepitopes. 149 mi decreased the proliferative capacity of PHI: stinnlated viaeitherthedfifliorthedflpatluaysoleynflnocyteactivatim (Tablel). 'fledecreasedmivenesswasdeperdentupmparasite concentration, withlow levelsof anppressimbeirqobservedwhenZJX 106Wwereused,ardfllea¢entofmppressimirmeasedvhen theparasiteca'ncerrtratimswereraisedto7.5or10X105organisms/ m1. mididmtreduceIIZproductimbyPBCafterstimlatim' withHiAorarnti—CDZ. Irrieed,I121evelsintheanti-CDZ-stimlated cfltneswereircreasedbythepresenoeofWfiatamtstmn). AsmallinzreaseinMcornentratimsisooassimallyalsomtedvmen PHAoranti-Cmisthestinllant(01apter3). 'Iheexpressimofthe n2RuasdecreasedbymfollcwingactivationbyH-1Aoranti-a32 (Table2). 'nlisdecreasetnasseenlbotllintllenrnberofmcells ardinthedersityofthereoeptormthepositivecells.9henanti-CDZ wasusedinactivation,thedecreasewasofalesserextentthanthat dnservedwithPI-IA. ItslmldbenotedthatLMdoesnotbind anti-H2Rantibodies(alapters3ard4),therefore,thedecreaseinthe levelsofIIZRobservedontheMwasmtduetotheparasite reducing the availability of antibodies to these cells. 150 Tablel. WMitsBlastogereeisbyBcultheToellReoeptor arndCDZPatlmaysa 3H-TdR incorporation (cpn x 10'3) in the presence of Stinnlus H-IA 61.7 i- 3.0 20.9 1- 1.1 6.3 i 0.1 3.1 :l; 0.5 1.8 :1- 0.3 anti-m3 37.2 i 2.6 22.1 i- 1.4 12.0 + 0.5 5.6 i- 0.3 2.3 i- 0.4 anti-C02 14.2 i 0.5 6.5 1 0.2 2.4 i 0.1 0.9 1- 0.3 0.4 i 0.1 a PRCwereirnlbatedforQGhrsinthepresenoeorabsenceof PHA, anti-CD3, or anti-0112 + anti-’rl13 (anti-C132 antibodies) with or without W in 96-well plates. one 1401 cf 3H-thymidine (3H-'IdR) waspresentperwell duringthelast 24 hrs. All differencesbetween valmsobtailedforcllmreswifllardwiflnrtmiarestatist- ically signifiant (P50.05, Student's ”t" test). These results are typically representative of three separate repeat experiments. Table 2. 151 The Effect of m an IL2R Dcpression After Stinnlation byeitherHiAoranti-CDZa Stimlus W %II.2R+cells £5 anti-C02 anti-(1)2 - 55.7 + 35.9 - 48.9 + 41.0 161 112 167 124 a PECwereincubatedfor48hrsinthepresenceor-absalceof PHAoranti-Tllz +anti-Tl13 (anti-C132 antibodies) ard/arm: (5X 105organisns/ml). IIZRelpressimwasdeteminedasdescribedmfier Materials and HEthods. Less than 6% of the PEMC incdbated innthe abse'nceoinAoranti-cnenpressedIIZR. 'Iheseresultsaretypimlly representative ofthreeseparate repeat experiments. b m,meanchamelmmbercfthelcgaritmcfthefluorensence intersities of positive cells. 152 DISQBSICN WdecreasedflleproliferativeresponsesofPBCstimlated byeither FHA, anti-CD3, oranti-CDZ (Table 1). This amassimwas d13ervedwithcam1tratimsofLmirargirqflun2£tolOXlOG parasites/mlinadoee-depenrientfashim. Simetheanpatmayhas reoentlybeendaunstratedtobemlyactiveinmenorchellsandmt innaiveTcells(J.A.Byne,J.L.Brtler, E. L.Mlilerz,andu. D. cooper, Abst. Ann. Meet. Fed. Amer. Soc. acper. Biol. 1988,msasa., vol.2, p. A1240.),thedatainTable1arethefirst denanstratian of uneabilityofmtowppreesrespaeesofmewryTlmtes. Anti-@isanantibodydirectedagainstthem-Tiantigen receptor canplex ard triggers T cell activation thralgh this moleclle (11). clzisanarkerfomdmallthymocytesandmaturellymphocytcs whose cell surface expression is upregulated upon lymphocyte activation (21). MarntibodiesdirectedagainsttheTl12ardT113 qnitopes of anareabletoactincmoerttostimlateMpmductim(16),II2R expression (16),andcelldivision(12). 'Iheantibodyto'nlz,an epitopefcnmdmallrestinchells,isab1etoilmnoeacmfirma- tioraldlangeindnwhidlallowstheexpressimofflnem3epitcpe (12). 'nleanbsequentengagenentofm3byantibodythenleadstothe abovenotedevents. Sinceaazisnotassociatedwiththecm-Ti canplexalthecellsurface(17)anisinoeitisoperativeintl'ne stinulatim of (133' thymocytes (16), it thus constitnrtes an alternative pathwayochellactivatim. Sincethispathwaydoesmtreqrirethe presenoeofmalocytes(12),thedatainTable1danastratethatL L! ‘I' 153 miisabletoeuertitssurpressiveeffectdirectlyupmtheT 1W- merevimslyfamdmttodecreasetheproductimor secretionofIIZbyPBCstinulatedbyHiAoranti-afiurrieroptinal c11turecorriitims(€hapter3). 'nnepreserneofminm allunesirrzreasedthelevelsofmfollcwimstinflatimbyanti-wz. 'nlisisoooassionallyalsoseenvmenonranti-anisusedasflle mitogen (Chaptera). 'nneurrierlyingcauseoftheaugmentatiminnz levelsismtclearatthistime,brtmayhaveresultedfrunadecrease intheabflityofthePflIZtointernalizearfidegradefllislynholdne. Alternatively,IL2productionmaybeincreasedbythepresenceof L minuswltures. Inthisregard,itshalldbenotedthat1, midoesnotreleaseIIZnordoeeitirducerestinglynphocytestodo so(datanotshown). Ithasalsobeenfanflthattheparasitedoes causeareductiminIIZproductionmndersupraoptimlstimrlatory conditicrs (5 x 106 mac/ml, 2 25 ugPHA/ml: 10). SimeTcellsqunosedtomidennstrateareducedcapacity toproliferateinthefaceofnormlorabovemrmallevelsofm,we Melaminedtheeffectoftheparasitemtheexpressimofthem. Aswaspreviclslyreported(10),miirhibitedfl1eexpressimof theIIZRmm—stimlatedmcrablem. BoththenunnberoflflR" cellsarrlthedensityofthereoqltormthepositivecellswas decreased. Similarresultswereobtainedwhenanti-dnwasusedasthe stimlant,altlnlghtheleductimvaslessprumcedtlnntlntseanin HIA-stinnlated ciltiires (Table 2). Preliminary results indicate that the upregulation of the T112 154 epitope of C132 ard the expressian of the T113 epitope of this molecule midlooalrdurimthefirst6t012hrsoflynulocyteactivatimwere inhibitedbythepresenoeofm inthecultures (datanotshcwn). Workismrrentlyinprogresstodeterminethekineticsofthe sugnressedexpressimoftheseepitopes. Sincemidecreasesbcth the amber of activated cells bearing T112 ard T113 and the densities of these epitopes on positive cells (data not slum), it is thus possible that these events are at least partially responsible for the sugaressed proliferative respmses of lynphocytes triggered via the C132 activation pathway. 'nleligardfortheTllz epitopeofcmhasreoentlybeen identified as lynphocyte function-associated antigen-3 (us-3) , a glycoprctein present in endothelial, epithelial, ard cannective tissues, as well as (:1 most blood cells (15,22,23). mile resting T cells bird to a HA-3-like moleclle a: sheep erythrocytes, resulting in rosetting (23), only activated T cells with enhanced expression of cm areabletobirdautologouserythrocytesmidlbearlower levels of 1171-3 than their cvine counterparts (23,24). The birding of T lynphocytes to the Im-3-like molecule an sheep erythrocytes allows subsequent activation by anti-Tl13 (25) . 'me putative natural ligard for the T113 epitope awaits identification. 'Ihe role of the C112 alternative pathway of T cell activatim is mtcanpletelywderstoodatthistine. However, thispatlwaymaybe of particllar inportance for the activation of CD3' thymcytes by m- 3+ thymic epithelial cells during artogeny (26-28) . IIhe upregulatim ofCDz andtheexpressimoftheTng epitopemactivatednamreT 155 lymphocytes may enhance activatim via CD3-Ti and increase the avidity cf tr cell interactia'ns with other 1533+ henatopoietic cells (15,29) . milethedB-Ticmplexardanaredistimtentitiesardmt associated on the cell surface (17), these two activatim pathways do havenrtualregulatory interactims. Forexanple, theranovalofcm fren the cell surface blocks activatim via 0132 (12) . Furthermore, stimllatim by the C132 pathway irduces phosphorylation of CD3 (30). It appears, therefore, that the CD3-Ti ard CDZ pathways involve separate signalstransmittedthrouglseparatearddistirctreoeptors, each system, nevertheless, being able to ennert regulatory effects upon the other. Ourfirdings irdicatethatmiisabletoirhibitT lynphocyte activation through both of these pathways. 10. 11. 156 REFERENCES Teixeira, A. R. L., G. Teixeira, V. 0. Macedo, and A. Prata. 1978. W cell-mediated ilnrnmnodepression in acute Chagas' Voltarelli, J. C., E. A. Donadi, and R. P. Falcao. 1987. Dunnlosnlnpressim in hunan acnte Chagas disease. W W152- Hayes, M. M., and F. Kierszenbaum. 1981. mperimantal Gnagas' disease: kinetics of lynrilocyte responses and immunological cmtlel offlnetransitimfranacrtetodumicW infection- W- Kierszenbaum, F. 1981. On evasial of W fran the host imme response. Iynunopmliferative responses to trypanoanelantigensdnmingaartearddnrmicexperinentalclagas' disease W Harland, E. C., and R. E. 1011111. 1978. Suppressim of cellular responses inmioe WWW infection. lust. M- Reed, S. G., C. L. Iarson, and C. A. Sper. 1977. armression of cell-mediated immity in experimental Chagas' disease. A W211].- Rowland, E. C., and R. E. Kuhn. 1978. Suppression of ananmestic cellular responses during American trypanosaniasis. M $3.73.].- Clinta'n, B. A., L. Ortiz-Ortiz, W. Garcia, T. Martinez, and R. Capin. 1975. W: early ilmune respanses in infected mice- MW Maleckar, J. R., and F. Kierszenbaum. Inhibition of mitogen- induced proliferation of nurse T and B lynphocytes by bloodstream Foms of Wm- WM- Beltz, L. A., and F. Kierszenbaum. 1987. Sugaression of tnunnan 1W resporses by W1 W Heller, S. C., K. A. Fitzgerald, R. E. l-hlssey, J. C. W, S. F. Schlossman, and E. L. Reinherz.1983. Clonctypic structures involved in antigen specific hunnan T cell functim: relatim to the CD3 1101691131 00191935 W- Meler, S. C., R. E. Hussey, M. Fabbi, D. Fox, 0. Aorta, K. A. Fltzgerald, J. C. Hodgdan, J. P. Protentis, S. F. Sdllossman, and E. L. Reininerz. 1984. An alternative pathway of T-cell 13. 14. 16. 17 . 18 . 19. 20. 21. 22. 23. 157 activatim: afmctionalrole fortheSOdellsheeperythrocyte Kara, T., S. 24. Fu, and J. A. Kareem. 1985. Human T cell activation. II. A new activation pathway used by a major T cell pcpilatim via a disulfide-bonded dimer of a 44 kilodaltm polypeptide (9.3 antigen). W13- Carrel, S., S. Salvi, L. Giuffre, P. Isler, and J. -C. Cerottini. 1987. A novel 90-ldh polypeptide ('Ip90) pcssibily involved in an magi-independent pathway of T cell activatim. M W- Springer, T. A., M. L. 0min, T. K. Kishimoto, ands. D. Marlin. 1987. The lynphocyte fmctim-associated IFA-l, C112, an! TEA-3 mleciles: cell adhesion receptors of the innine systan. m W- Fox, D. A., R. E. Hussey, K. A. Fitzgerald, A. Betsussan, J. F. mley, S. F. sailossman, and E. L. Reinherz. 1985. Activatim of mmanthymcytsviatl'xeSOKDTllsheeperythrccytebirding iniucestheeiqaressimofinterlelmianeceptorsmboth protein T3+andm‘popu1atims W Meuer, S. C., O. Acuto, R. E. Hussey, J. C. Hodgdcn, K. A. Fitzgerald, S. F. Schlossman, and E. L. W. 1983. Bridence fortre'm-associated9omrxeterodinerastheTcell antigen W- W- Badzko, D. B. and F. Kierszenbaum. 1974. Isolation of W from blood WW Villalta, F., and W. Leon. 1979. Effect of purification by IEAE- cellulose column on infectivity of W blood forms. W- Gillis, S., M. M. Fem, W. Cu, and K. Smith. 1978. T cell gmwth factor. parameters of production and quantitative microassy for activity W Bernard, A., C. Gelin, B. Raynal, D. Em, C. Gosse, and L. Powell. 1982. HammofmmanTcellmsettingwithsheep erythrocytes amlysed with maoclmal antibodies. M 15.511112- Hunig, T. R. 1986. 'meliganioftheerythrccytereceptcrofT : expressim m white blood cells ard possible lynphocytes involvanent in T cell activation. W219}. Selvaraj, P.,M. L.mstin R. Mitnacht T. Hunig, T. A.8pringer andM. L. Plunkett 1987. Rosettimofhumanlemiocyteswith sheepandhtmnerythrccytes: cmparismofmmanarrisheepligand binding using purifiedEreoeptor- W- 24. 25. 26. 27. 28. 29. 30. 158 Makgoba, M. W.,S.Shaw E. D.Gugel andM. E.Sanders 1987. mmnTcellrcse’ctingisnediatedbylfA-3mautologcus WW I-nmig, T., G. Tiefenthaler, K.-H. M. zum msdienfelde, and S. C. Meuer. 1987. Alternative pathway of activatim of T cells by binding of cm to its cell-surface ligard. W. Vollger, L. W., D. T. Tuck, T. A. Sprixger, B. F. Hayes, and K. H. Singer. 1987. 'mymocyte binding to bumn thymic epithelial cells is inhibitedbymmoclonal antibodiestoCDZ andII'A-a antigens. W- Blue, H.-L., J. F. Daley, H. Isvine, K. A. Craig, ard S. F. .. Schlossman. 1987. Activatim of inmature cortical thymocytes thruighthenlsheeperyttmtebixfiingprctein. W 13.83.1123- Elf—‘1‘..- _ .5... Dennirg, S. M., D. T. m, L. W. Vollger, T. A. Sprirger, K. H. Singer, andB. F. Haynes. Lumlmal antibodiestodnard lynphocyte function-associated antigen 3 irhibit hurnan thymic epithelial cell-dependent nature thymcyte activation. .1, W - Shaw, 8., and G. E. G. Ince. 1987. The lynphocyte function associated antigen (LEN-l and CDZ/IEA-B pathways of antigen- iniependent human T cell Mien. W. Breitmeyer, J. B. 1987. How T cells cammicate. m 3.2.9.3169- APPENDIXI 159 AmendixI millediates itsSuppressiveEffectViaaSecretedFactor 'meadditimofmitrypanastigotestociltlmofnorml hmnmnasbeaashcwnintheprecedihgdiapterstowpprossthe proliferativerespmseaswellastheexpressicnoftheIHRbythese cellsmileflleproantimofIIZwasmaltered. Inordertodetermine \hemermisimnmqpressimreqniresdirectcell-to-parasitecartact orwxetherasecnetedwppressivefactorexists,wetestedthe immosurpressiveabilityofLmiinthepreserneorabserneof directcell-tc-parasitecontact. W trypanastigotes were purified fran the blood of infected miceaspreviously described (1) ardresuspendedatafinal cmcmtratim of 5 x 10‘5 parasites/m1 in mm 1640 medium (Gibco, Grand Island, NY) containing 100 units penicillin and 100 ug streptanycinper ml and 55: heat-inactivated (56'C, 20 min) fetal bovine serum (RHII+5%F$). mmanPBCwereisolatedaspreviwslydescribed (1) and resuspendedatafinal canentratim of 1.25x105 cells/mlin RPMI+5%FBS. Inordertotestmetherdirectcelldto-parasiteca'ltact is required for W to inhibit the proliferative respmse of PHI: tomitogers,PBCwereplacedinthewellsof24—wellplatesinthe presenceorabsaneofSug/mlphytohanagglutinin (PHA;SigmaChemica1 ompany, St. Innis, m). Tbeadmwell,aMillicell-HAinser-twasadded (Millipore,Bedford,M). ‘IhisinsertcmtainsaOAS um-pore—size filter which allows mly the passage of solmale material between the 160 twocalpartments. 'Ihevolnmesofmedinmmtheinsideandoutsideof theinser'twereo.4arnd0.5m1,re@ectively. mimpresantin smeofthecfltnmeitheranthewtsideoftheinsertmllcwing directccntactwiththePEC)crwithihtheinsert(mdirectafltact) and replaced an equal volume of RHH+5%F$ . Cultures were pulsed with SuCiof3H-thymidine72hrafterinitiatianardharvestedat78t096 hr. Insaneexperinnents,theinsertswereranovedatvaricustimesof cilmreandreplacedwithnavirsertscmtainingOAmlofRHlnsum andSug/mlPHA. TheabilityofPflttoprcduceMwastestedbymeasnmingthe InactivityinAB-hrsupematantsofmlmressetupasdescribed above. Reactivitywasdeterminedusingthenz-dependentCIHchell line (1). Theeupressionoftheanwasneasuredeacinthepresenceor absence of direct cell-to-parasite contact 48 hr after mitcga'nic stimlatim(cnapter3). manltswereexpressedasthepercentageof Tac+cellsarrineandnamnelnmberofthelcgaritlmcftheflmrescence intensites of the positive cells. 'nnedatapresentedinlableslthrungh3aretypioallyrepresent- ative of two to six separate repeat experiments of similar design. Wmabletoslmessthepmliferativerespasesofm tomAwhetherormtthecellsardparasitesweresqaaratedbya Millicell filter (Table 1). Indeed, the suppressive capacity of L mimethesameinbothcorditims. Itthusappearsthatthe imnnsuppressiveeffectsofmiarenediatedbyasecreted Expressive factor(s) (SSF). Whether this factor(s) originated 161 Table 1. mi Suppresses Him FBI: Proliferation in the Absence of Direct Contact with the Cellsa PHA (5 ug/ml) W 3H-4.'.l'nym.idi.rne incorporation (cpn x 10'3) - - 1.3 .t 0.9 + - 51.6 i 5.9 I + cantactb 21.3 1 4.1 E + no contact 28.7 1 2.3 3*: a Nimty—sixhrmlhnespflsedwithSpCi3H-tlnymidirneat72hr. b "Ca1tact"referstothepresenceofdirectcantactbebdeenmlc alum. "Nocantact"denotesthat1‘_._miwasseparatedfrm the PRC by a Millicell filter. 162 intracellularlyorwasreleasedfruntheparasite's plasnametbraneis rutknown. Next, the suppressive effects of 24 to 96 hr anpernatants ofL mittypanastigcteculturesweretested. mileSXlOGparasites/ml were able to decrease mitoger-induced lynphoproliferation, the wpernatantsofumeanmreasmnasunesupenatantofnxnflg Wdidnoteffecttheihcorporatimof3H—thymidinebyPHA- stimlatedPEC(datanotstnm). SincebothPflCandmAwerealso prosentinthewlmresandmAbirdstoandagglutimtesmia), itisthuspossiblethatthismitogenoracellpmductiniucesthe release of SSF. To examine this possibility, the suppressive activity of24to96hraflturesupernatantscf5x106mi/m1alane,L m1+5nnglmlnm,mi+l.25x105PBC/ml,orL_qmz_1+Pflc +HiAweretasted;mneofthensu;pressedmitogen—irducedpreliferb ationofmmatamtstmm). 'nnus,theSSFappearstobelabileor degraded, duplicating atteIpts to purify and characterize this molewle(s). FurtheranpportforthelabilityofSSFweretheresultsof stuiiesinvanidntheirsertcantainirgmiwasrewvedand replacedwithanewinsertladdng parasites at 24,48,0r72 hr. Thesewlbneswerepflsedwith3H-thynidineat72hrarflharvested6, 12,or24hrlater. Mnentheinsertscantainingmirenainedin the cultures for the duration of the ecperiment, the proliferative respaseeweredecreasedapprmdmtelysacmparedtomflstimlated intheabsenceoftheparasite(Table2). Imentheinsertswere rewvedat72hr(atthetimeofthep.nlse)andharvested6hrlater, 1: -7 '7" Table 2. ‘nne Suppressive Effect of SF is Reversiblea 163 72hr 48hr 24hr flA165 e21m8 (85)° 8A113 (86) n5133 (81) ”314» (37) 4&11m2 (26) 61.7 110.2 8.2 1 1.6 (87) 7.4 1 1.6 (88) 14.9 1 4.6 (76) 50.4 1 6.1 (18) 40.3 1 8.0 (35) 77.4 1 22.2 15.9 1 1.2 (79) 11.6 1 2.4 (85) 36.6 1 2.7 (53) 85.3 1 5.1 (-10) 82.5 1 1.9 ('6) a PBCmreinanbatedwithSW/mlminthepresenceorabsence ofmi. AllculhnesweremlsedwitlnSuCi3I-l-flnymidirne72hr after initiatian. "Corrtact'referstothepresenceofdirectcantact betweenPBCanflmi. "Nocantact"deutesthatmims separated fren the par: by a Millicell filter. b oneneswereharvestedm 12,or24hrafterthep.1lse. C mrcentdecreaseincanpariscntothecorrespa'dimmluxres leddleLmizi- 164 theectertofthesnmessian(81%)msapprcndmatelythesaneasin thosecilturesstillcmtainingm,butasthetineafter parasiterenovalircreased(lZani24hrpflses),thesuppressive effectdecreased (76mfl533t, respectively). misdemeasein awressimovertinewasseentoagreaterectentinthcsewlbnesin midntheinsertscantainimparasitesvererenovedatwcruhrof culture (Table2). SinoeanlyflneparasitesthetselvesandnottheSSF mmed,theseresultsanggestflnatthesuppressimisreversible andthatSSFislabileaninustbecmtinnouslypreducedinordertobe effective. The lability of SSF may either be intrinsic or my be due tomixrternalizixganddegradingthemlecule. InordertodeterminewhethertheimmosuppressiancausedbyL mimfiflneSSFaresimilar,severalotherparanetersoflynrtncyte activatimwereexamined. NodecreaseintheproductianofIIZumer cptimalmltnecanditimswascausedbyeithermiorSSFflable 3). Incantrast,SSFwasabletoirhibitthee¢pressionoftheII.2R (Table 4) ashadbeenpreviouslyshown form (Chapter 3). Insnmnary,1,_mz1appearstosecreteasolublesnmessive factor which is labile andwhoseeffects arereversible. 80mm and its SSF decrease mitogen-induced proliferation and the expressicn oflIZthilenotaffectirgtheprcmctianofIIz. 165 Table3. EvaposureofPBntotheSSFdidthflnibitIIBPmductiona aipern'ntant 3H-thymidine incorporation (qxn x 10'3) by elm-2 tested 1:; 1:4 1:8 1:16 PRC 0.4 1 0.0 0.3 1 0.0 0.3 1- 0.0 0.3 1- 0.0 PEER-FHA 20.6 1 1.7 12.0 1- 1.7 5.1 :1 0.8 1.7 1- 0.4 EDI-HEW 29.3 1- 1.6 16.4 1 0.6 8.7 1 1.8 3.2 1 0.3 maimssr’ 27.0 1 3.2 12.7 1 0.7 7.1 1 2.5 2.6 1 0.5 a Fbrty-eight—hrsupernatantsofthehndicatedoiltureswere tested for IL2 activity using the IL2-dependent CIIIrZ cell line. 3H- thymidine incorporation by GILL-2 cells incubated in RPMI+10%F$ = 0.4 1' 0.2. Similarirmrporationwasproducedbycnlrzirmbatedinthe oilhnesupernatantsofmnstinulatedPBCciltnedinthepresenceof 166 Table4. ILZREbcpressionisDecreasedbythe:[1_gngzj,$81:a Loom. %Tac+ cells and” - 51. 3 140 no contact 46.2 130 ‘1 mummirulbatedforwhrwithPHAGug/mnin thepresenceorabsenceofmpriortostainingfor theTacantigen. "Nocontact"denotesthat1‘_,_mz_iwas separatedfronthePBflbyaMillicellfilter. Iessthan5% ofthemcincubatedaloneorexposedtoSSFintheabsence of PHAwerepositive for Tao. b 'IhedersityoftheTacantigenexpressedasthemean dnamnelmmberofthelogaritrnnofthefluoroscenceinten- sities distributed over 256 channels. 167 REFERENCES Beltz, L.A., and F. Kierszenbaum. 1987. Suppression of lumen 1W responses by Weeni- Immlogy 60:309- Pereira, M.E. A., M. A. Ioures, F. Villalta, and A.P.B. Arrirade. 1980. IectinreceptorsasmarkersforWi. Developnentalstagosarflasmdyoftheinteractionofvmeatgerm agglutinin with sialic acid residues on epinastigote cells. J. Exp. Med. 152:1375. APPENDIXII 168 APPENDIXII WMitstheGrovmofSe/eralmtmtall Imortalizedoelllires Weetivationixmlvesaseriesoftelporallydistinct eventsasthecellsmvefronthecorestingstageintotheGlstageof thecellcyclearxithenceouardtomicleararflcytoplasmicdivisions (1,2). 'nneability ofmtosuppressTcell proliferative resposes to mitogenic stimlation may lie in the inhibition of any one ormoreoftheseevents. Innortalizedcelllines,tnrever,have alreadyenteredthecellcycleandthereforebypassseveralofthe activatimremirenents.1tistrmspossiblethatthesecelllinesare mlongerdepeflentupoltheactivationevent(s)whidn1._mzi inhibits and may subsequently escape the antiproliferative effects of theparasite. Inordertoecplorethispossibility,wehavetestedthe ablilityofLMtodecreasethegrwunofseveralcelllines. WWgotesmreisolatedfronthebloodofmiceat Upkeecspost-infectionaspreviolslydescribedm) andresusperdedat the desired concentration in RPMI 1640 medium (Gibco, Grand Island, NY)containimlOOmnitspenicillinanleOugstreptonycinpermlarfl 10% heat-inactivated (SG‘C, 20 min) fetal bovine serum (macaw) , or supernatants of concanavalin A-stinulated rat queen cells (rat IL2) forthesbxliesusingCl'IL-Zcells. 'nnenn'ineIIz-depe'dentcrurz cell line (American Type oilture Collection), naintained by passage in rat IL2 in a similar fashion to that previously described for Hr-z 169 cells (3),wascentrifugedooepriortouseintheproliferation assay arriwasresmefledatafinalconcentrationonXlO‘cells/mlinrat IL2. 'melnmannonadherantmyelocyticmfl cell line (American'l'ype 011ture collection) and the human T—lynphotmpic virus type 1 (arm-1) - infectedHJTlOZBz cell line(4:prcvidedbyDr.WarrenIeonard, NationlIrstihrtesofHealth,Bethesda,m)weremaintainedbypassage inRHlI+lO%FBSardbrolghttofinalconcentrationsole105arr15X 105 cells/ml, respectively, in the same medium. To test the ability of mi, to inhibit the proliferation of these cell lines, cells were incubated at the previously indicated concentrationsfor24or48hrinthepresenceorabsenceofserial dilutions oftheparasite. CI'lL-Zandm'rcellswereirunbatedin96- well platesinavolume of 0.2ando.l ml, resqnectively, while U937 cultures were set up in 24-well plates containing Millicell filter inserts (Millipore,Bedford,MA) inavclnnnecf0.9m1(asdescribedin AppendixI)toavoidcell infection. outtnrescontainirgcrlIerre pulsedwithluci3H-thymidine (qnecific activity=2.0 Ci/mnnole: New England Nuclear, Wilmington, 1136 hrbefore harvest, while those containimU937animrrcellsreceiveda24hrpulse.01ltnreswere termimtedbyautonetedharvestingardtheamomtsofincorporated3fl- thymidine were determined with the use of a liquid scintillation counter. Reelltswereecpressedasmeanqm1lstamarddeviation. 'nnestainingofmn'cellsfortheecpressionoftheIIZRwas performedasdescribedinChapterBforPflnandthereailtswere expzessedasthepercentageormcellsaratheneandmnelmmer 170 of the logarithm of the fluorescence intensities (Minn) distributed over256channels. Thedatainiablelslmthatmwasabletodecreasethe growth of arm-2 and U937 cells. Proliferation of CI'LD-Z, an IL2- depe'dentnurireTcellline,wasreducedbythepresenceof1Xlo7L Wetter-24hr,while2.5x1051,_mi/m1wereeffectiveat48 hr. GrovthofthehnanmyelocyticU937cellswassuppressedby5X 105Wat 48hr(Table 1),whilelx107 parasites/mlhadan effectafterm1y24hr(datamtslnvn). Simethemflcellsanithe parasiteswereseparatedbyaO.45-m-pore—size filter, then-noted decreasecannotbearesultofinfectionofthismnocyte—likecell line.‘ It should be noted that the suppressive ratio of parasites to cellswaslzsuandsoml for crib-2 at 48and24 hr, respectively,and 50:1ard100:1fortheU937cells. 'nnisisanuchhigherratiothanis required to inhibit the mitqen-induced proliferation of noise spleen cells (1:1:GnapteIZ)orlnmanPHC(4:l:dlapterl). Incontrastto tlneresniltscbtailnedwithtlneCI‘IIrZar'ndm37celllirnes (Tablel)or mrmlrnmenperipneralbloodnumnclearcells(cnapters3arrl4),L m1(5X106parasites/nnl)wasnotabletoaffectthegrodthorthe emessionoftlneMRbyMlOZBZcellsafter48hrsofco—oilture (Table 2). Similar resultswereseenwhentheparasite concentration wasincreasedtoleO7LM/mlwatamtshom). Higher concertrationsofmiwerenottestedduetotherapid acidification of the mlture radium under these conditions (mplblished observation). 'nnefailureofmitoimnibitgrwthorIIZRezpressionof 1'71 Tablel. Wmthemthofcrurzmmflmllmnesa WW 99.11.11.111: 1'. M05) 24 hr 48 hr crib-2 none 13.0 1 0.1 44.4 1 0.6 1.25 14.2 1 0.6 44.5 1 3.3 2.5 15.3 1 0.5 27.6 1 0.1b 5.0 11.9 1 0.4 6.3 1 0.6b 10.0 7.4 1 0.2b 3.1 1 0.2b U937 none NDC 70.9 1 11.1 1.25 ND 78.3 1 4.1 2.5 ND 70.1 1 3.1 5.0 ND 44.5 1 6.3b 10.0 ND 24.8 1 3.7” a 'nneresultswithan-zandupnoellswexeobtainedin separate experiments, eachof whichwas repeated onthree occassions. ’0 p_<_0.05,forthereductionsincpnwithrespecttothe correspondingcontrol which ladcedm, as oalcnlatedbyStudent's "t"test. c Notdetermined. 172 Table 2. m1 does not Affect the Ability of KIT 10232 Cells to Proliferate or Ecpress IIZRa interial 3H—thymidine incorporation 11mm cells log are (M3) HUT 14.6 1- 0.5 66.0 120 W 16.3 1 0.1 69.0 117 a 'nneproliferativerespolseardtheecpressionoftheIIZRwere tested separately in forty-eight hr ciltures of HUT 10282 cells iJnnbatedwithorwitImtSXlosLmi/ml. Theseresultsare representative oftwo separate repeat experiments. 173 1111' 10232 cells may lie in the mechanien of their transformation. This cell lineisinnfectedwithandwas innortalizedbylfl‘IN-l (4,5). The initialstagesofthisimortalizationappeartoutilizeanautocrine mechanismof growth, involvingtheconstiontive production of IL2 and the ILZR, which is inhibitableby antibodies to the IL2R (5,6). later eventsleadtothelcssofIIz-depeflenceandlackofIIZproductionby sonneoftheHl‘lN—l-innfectedlinesueviavedin7). 'nnennechanismof theenhamedtranscriptionofmmndmmamearstorsultfm the interaction of the transactivator gene product (tat-I) of HEN-l withthepronoters ofthese cellulargenes (8,9) whichbearseqnence honology withthe regulatory regions of HI'UV-l (10). Inthe case of theIIZRgene,thepronoterengagedbytat-ldiffersfronthatnnedin thenornalactivationprocess(8). 'nnedifferenoesbebleenthegrowth of normal activated peripneral blood mononuclear cells and HI‘DV-l- infectedcell linnes (loss of 112-dependent growth, constitutive expresionoftheIIZR,arritheuseofadifferentII2Rpronoter)ney etplaintheabflityofmtosuppressgrwthanoHZRetpression intheformerbnntnotthelatteronse. Inennnuary,athighparasitetocellratiosm1isableto suppressthegrowthofalong—termm-deperientlineardamonocyte- like cell line, but not HI'IN-l-infected I-IJT 10282 cells under the conditionstested. ‘nnelatterfindingalsosuggeststhatm- induced growth-inhibition of inrnnortalized cell lines is not merely the resnnltofnntriontconmption. .r-uu 3:." '7 6. 10. 174 Mt, D. T., D. R. innards, and C. L. J. Parfett. 1986. Gene erpression during the nenelian cell cycle. Biochinnica Biqinysica Acta 865:83. Wedner, H. J. 1984. Biochenical events associated with lynphocyte activation. Survey of Inunnrelogic Research 3:295. Beltz, L. A., and F. Kierszebaum. 1987. Suppression of lumen 1W reoponsee by W. homology 60:309- Poiesz, B. J., F. W. Rnsoetti, A. F. Gazdar, P. A. Run, J. D. Minna, and R. C. Gallo. 1980. Detection and isolation of type C retrovirnsparticlesfronfreshardcnlunredlyuphocytesofa patient with cutaneous T-cell lynphone. Proc. Natl. Acad. Sci. USA 77:7415. Gazzolo, L., and M. D. Dodon. 1987. Direct activation of resting T lynrinocytes by lumen T-lyufinotropic virus type I. Nature 326:714. Gootenberg, J. E., F. W. mscetti, J. W. Hier, A. Gadzar, and R. C. Gallo. 1981. Hnmen ontanneons T cell lynphone and leulmia celllinesproduceardreworitoTcellgrowthfactor. J. up. 19d. 154:1403. Greene, W. C., W. J. leoerd, J. M. Dapper, D. L. Nelson, anndT. A. Waldnem. 1986. The lumen interleukin-2 receptor: nnornnel and ahmuelecpressioninTcellsardinleflceniasinducedbythe lunman T-lynunotropic retroviruses. Annals Internal lbd. 105:560. Cross, 8. L., H. B. Feirberg, J. B. Wolf, N. J. Holbrook, F. Wong Staal, and W. J. Ieonard. 1987. Regulation of tlne lumen interleukin-2 receptor a chain pronoter: activation of a nonfunctional pronoter by the transactivator gene of Hl'UI-l. Cell 49:47. Siekivitz, M., H. B. Fein'berg, N. Holbrook, F. W.C. Greene. 1987. Activation of interlenkin 2 and interlonkin 2 receptor (Tao) pronoter expression by the transactivatcr (tat) geneproductoflunmaanlenkeniavirus, typel. Proc. Natl. Acad. Sci. (EA 84:5389. Fujita, T., H. Snibuya, T. dnashi, K. Yamanishi, annd T. Tanigudni. 1986. Regulation of luman interleukin-2 gene: functionalmAseqnencesintheS' flankinngregionforthegene expression in activated T lynphocytes. Cell 46:401. 175 WWWIQB Astateofsugpressedimmereactivityoconrsduringtheearly stagesof infectionwithW. Bothcellularandhnmoral arusoftheimmesysteu,however,areftmctioeldnmingtheinfec- tion's laterstagesandplayavital roleinhostdefense. Itnneythus be hypothesized that the initial state of inmmosuppression enables 1'1 mi to establisln itself inntracellularly and that overoouing this pnenonenonneyallovclearanceoftheparasitebythehost'simme systenbeforetheonsetofpathology. Itwascurgoal,therefore,to deracterizeflneinmmealterationsvdnidnmiinducesinmmanT lymlccyteswiththeultinetegoalofdevelopingmeanstoabrcgate theseeventsanrithesubsequentoconranceofdisease. Priortothismrk,lonovlegeoftheectentoftheparasite—inmnced imnosuppressiveeventsinlumanlymnocytestesectrenelylimited. Ittesslmnhereinthatmiwasabletosuppressthe proliferation of human T cells following activation by either tlne T cell receptor or the CDZ antigen-independent stinulatory patlneys and that the ability of tlne parasite to inhibit the expression of the interlenlanreceptorplayedakeyroleinthisprocess. 'nneadditionofmitryponestigotesorepimastigotesto cultures of noruel lumenperipheral blood monouclear cells (pee) reducedtheabilityofthelattertoproliferateinremtoa variety of mitcgenic lectins in a parasite-dose-dependent manner and cverawiderangeofmitogenconcentrations. 'lhisreductionwasnot 176 duetoconsnmptionofessentialnntrientscrtoalmeringofmitogen concertrationstosuboptinellevelsbymmrtoalossofmc viability after co-onlbnre with the parasite. W was also able to express PHI: proliferative responses after stimlation by anti-CD3, anamloelantibodydirectedagainsttheTcellreceptorconplec,arfl anti-Tllz and anti-T113 antibodies which trigger T cells via the CDZ activationpathway. ‘Ihepresenceofnanocyteswasmtrecpiredforthe decrease in PBoiC responsiveness while parasite viabilty was necessary. Wadiitioellyiflnibitedthegrovthofseveralhntmtall inunortalizedcelllinestested. mwasabletoenertitssnmessiveeffectswhenseparated fronthecellsbyanilliporefilterinsert,denastratingthata soluble factor released by tlne parasite was involved in the suppressive process. Tcellresponsivenessslmedapartialrecoverywithinahr afterrenovaloftheinsertcmtainirgm,denonstratinqthe reversibility of the immune alterations and the lability of the suppressivefactor. lexinelsuppressionvesmtedunenmitesaddedtoonltmes withinthefirst24hrofstimnlationanddecreasedasthetinneof parasiteadditionwasprologed. MWappearedtoaffectan earlystageof lynphocyte activation. Interleukins (IL) land2are prodnedbystimlatedmnocytesanchells,respectively,andare requiredearlydurinngtheTcellgrowthcycle. Underconditionsof optimlstimlatim,mitesmebletoreducetheabilityofmman Pantomoduoeorsecreteeitheroftheseuoleonlesorinterferon—r whilenousewleencellsweredeficientintheproductionofbothnz 177 andinterferm—rafterco—cnlhnrewiththeparasite. Inkeepingwith theseresults, mwasahletorestorethemitogen-induoed prolifer- ativeresponsesofsurpressedmmehntnotlumanlynflecytes. 'nnus, therearerotablediffereoesintheprocessofsnmpressimofmnse spleencellsandlumenPBc. 'nnesereanltsinriicatethatcautimmst beecertedvtnenectrapolatingfinflingscbtainedwiththemmh'enodel systentothelnumandisease. ‘lheinabilityoflumenPflIItorespofltoefiogenousorexogenous IL2 correlates with the ability of mi to inhibit the expressim oftheIIZreceptor(IL2R)mTcells. Boththenmberofcells bearingIIZRardthereceptordensityweredecreasedbymiina nemerwhidnwasdeperientupmtheparasitecmcertratim. This decreasewasobservedwithianhrofstinulatimarflpersisteduntil atleast60hr. Boththelcwarrithehighaffinityformsofthe' receptorwereaffected. Expression of the transferrin receptor, a mleolle required for lynphcproliferatim as well as a late activation nerker, was also imibitedbymimilethelevelsofearlyactivatimantigenl, theearliestreportedactivatimuerkerochells,wereureffectedby theparasiteduringtheinitial6to24hrofstimnlatim. m additionally suppressed the nip-regulatim of the surface ecpressim of theTllzepitopeofCDZaswelltlneecposnmeoftlneTll3epitcpeof thismolecnlewhidnoconrsdurimactivatim. Transmit selectiveinitsininibitimoflunanTcellactivatimeventsanithis specificity may provide the key in overominng the parasite-induced suppressim. 178 'lheeventwhidnamearstobeofthegreatestiuportanceisthe decreasedecpressimofthemsincetriggeringbythismlecnle allowsprogressimfrontheearlytothelateGlstageofthecell cycleanriregnnatesnenyoftheanbsegnenteventsochellactivatim. Acflitioelly,thisistheearliestprocssreportedtobealteredby1‘1 mi. Fuenrewor'kinthisareamightadiressthefollovirgquestims: l. Doechauseanincreaseinthelevelsofthesoluble IlzRasisthecaseinAIlBan-dcertainfonsofmncer? 2. IstheexpressimofthemenbraneformoftheIIZRm activatedBcellsandmnocytesalsoaffected? 3. ArethelevelsofIIZRmRmdecreasedbyWi,arriifso, isthisaneanessimofdecreasedtranscriptimorofdecreasedm stability? 4. Istheecpressimofcellularoncogenesalteredbym? 5. How my the stability of the parasite-induced suppressive factorbeincreasedsoastoallowitspnrificatimarridnaracter— izatim? 'nneanswerstotheseqnestionswillallovagreaternmderstanding oftheprocessofimmosuppressimbymiardneybeofvaluein thestnflyofimnnealteratimscausedbyotherpathogens,in putionlar, the lumen immunodeficiency virus.