CA2090689A1 - Regulation of t-cell proliferation via a novel 5htla receptor - Google Patents

Abstract

ABSTRACT

A method of regulating proliferation of activated T cells exhibiting a 5HT1a receptor by introducing a sufficient amount of agonists or antagonists to either increase or decrease T cell proliferation. The basis for regulating cell proliferation may be (1) via the 5HT receptor, (2) via serotonin synthesis inhibition and/or (3) via serotonin stimulation of CD8+ subpopulations of activated T cells.

Description

WO ~04~15 PCT/~591/~J~6 - 1 2 ~ 9 TITLE: Regulation of T-Cell Pro11feration vla a Novel 5HTla Receptor INVENTOR: Thoma~ Mar~in Auna Thi~ iB a continuatlon-in-part application of U.S.
07/57~,710, filed Septe-nber 4, 1990. For purpo.~es o~
clarifica~lon the novel 5HT2-li~e receptox has been 10 ~ede81gnased as a S~Tl~ receptor.
Initlal studie~ demon3trated t~at ~ nov~l 5HT-2-like r~c~ptor wa~ pre~ent on transformed Jur~at cel1~ and that th~ rec~ptor wa~ pre~ent on human T lymphocyte~
(ackivated T-cell~). The re~ults of further experiment~
15 co~firmed that 5HT receptor~ are present on activa~ed but not re~ting T cell~ that thesQ 5HT recep~or~ and ~he 5HT
receptors on Jurkat cell3 have si~ilar ph~rm~cological properti3~, ~hat the 5HT receptor~ regulat~ adenylate cycl~e ~ctivlty and c~n ~uppres~ proliferatlon of CD4~ T
20 C~118 and ~timulate prol1feration o~ CD8~ T cell depending upon condttion~. The characterization of 5HT
sp~cif1c recep~ors on Jurkat C~118 and actlYated ~ cell3 by a combinatlon of pharmacologlcal, biochemical and molecul~r analy~es ha~ con~i.nmed ~he pre~nce o~ 5HT
25 rQceptor~ and 3 udien llnk the re~eptor on Jurk~t cell8 and ac~lYa~ed T c~ o th~ 5HT1a family of receptors, For Jurka~ c~ and actlva~ed T cells thi~ 18 ba~ed on the ollowlng crl~erla: bindlng ~tudle3 wi h the SHTla recsptor ~peclflo agonl~ (80HDP~T), studle~ on ignal 30 transductlon, northern ~naly~is wlth specl~ic human 5HT1a ollgionucleotld~ probe~. Re~ting T c~lls dld not expre~s 5H~la receptor by ~he~e ~ame critesla. The~ flndlngs hav~ provlded the b~sl~ for cont~nuing to explore a varioty of -qtrateg1es to ldent~y compound~ th~t lnteract 35 wlth the recep~or or ~he mechanl~m of cell prollferation.

SUB~;TITUT' Stl':~T

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W~9~ O ~ O ~ ~P~ tUS91/~176 Th~ pre~ent lnv~ntion rel~te~ to regulatlon of cell prolifer~tion b~ad on th~ recognition of a novel serotonln rec~ptor present as a cell surface molecule.
The novel receptor 19 a Bsrotonin r~ceptor linked to the 5 SHT2 f~mily. Th~ 5HT~-like receptor ls pre~ent on neopla~lc or tumor calls and actlva~ed T-cells, a.g.
CD4+ ~nd ~D8+.
The pr~en~ lnvention al~o rela~es to the r~gul~tion oP cell prolifera~ion ba~Q~ on th~ reco~nltion that 10 proll~erat$ng cell~ contaln/expr~s serotonln. Th~
proliferation o neopl~tic or tumor c~ and ~ctlv~tad T-cclls contalning s~rotonln c~n be decrea w d by inhibltion of ~arotonin ~ynth~ls.
Th~ pre~nt lnvsntion al80 r~l~te~ ~o the r~gul~tlDn 15 ~ the proli~eratlon of cell ~xhibitlng th~ nov~l 5HT2 reoeptor by introducing ~n e~fcctiv~ a~oun~ of ~goni~ts or antagonl~ o elther lncrea~e or decre~se c~ll prolif~r~tlon.
A number of neuro~ran~mltter3 have b~en shown either 20 to bind to or to havb c~rtaln e~fectY on cell~ of ths immune ~y~ (for revi~w, 86e r~f. 1~. one such neurotran~mitter, serotonln (5-hydroxytryptamlne, 5-HT), 1~ al~o ~ ma~or product o~ pl~l:el~ts r~leased at slt2s of infla~atlon ~2~~). Scrotonln hA~ b~n ~hown to augment 25 IFN induced ph~gocytosis (5) and ~uppres~ IF~ lndu~ed Ia expression on m~croph~ge~ ~6), to ~ug~ent N~ cell ~ytotoxlcl~y ~7), ~o a~ec~ lon par~ab~lity ln i~ol~ted pre B ly~pho~yt0~ (8), aAd ~o ~uppreQ- ~itogen ~tl~ulated T ce11 prollferstlon ~n ~n v~tro syct~ (9).
30 Addlt~onally, ~ero~on~n ~nd snrotonln recepeor~ h~e been suggested ~o be requlre~ ~or expg~sion o~ dol~y~d type hypersen~l~lvity in ~urlne model~ (lO 11).
The ~unctional, ph~rm~cologlcal ~nd mole~ul&r propertl~s of serotonln receptors h~ve be~n char~teriz~d 3g ln th~ p~rlpher~l and c~ntr~l ner~ou~ sy~s~ (12).
Dl~tinct c~tegori~s of r~ceptors hav~ baen d~lned whlch m~dlat~ dlf~eren~ effect~ of SHT(13). Experl~nt~ uslng SU13~1TUTE SHET

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wos2J~l~ PCT/US9l/~176 2 ~
5HT antagonl~tY have suggest.ed that T lymphocyt~ which can tran~f~r delayed type hyp~rsen~iti~lty re~ponse~ to nalv~ ~eeipiant~ m~y expre~s 5~T xee~ptor~ (11).
Specific 5HT2 antagoni~t~ quch a~ ketan~erln or 5 ritan erin wlll prevent transf~r of thl~ respon~e. Th~
purpo~ o~ the experiments reported hsre W2~ ~0 det~rml~e i 5HT receptor ~ubtype~ could be identlfied on human T
lymphocyt~ and if 5~T rec~ptor~ could med1at~ ~ignal tran~duc~lon event~ -Rlmilar to ~ho~e previou~ly repor~ed 10 for 5HT receptor~ ln the nervou~ gy~t~m. The resul~s ~how ~hat a ~lum~n T lymphocyte lin~ (Jurk~t) expres~
5HT2 llk~-r~ceptor ~ubtype which c~n m~diat~
pho~hatldyllnosltol hydroly~l~ and intrac~llu1~r Ca+~
mobillzatlon.
15 Th~ ~ctivation o~ r~tlng 1' lymphocy~os i3 critic~l to mo~t im~u~ re~pon~e~ CQ c~llulsr ~c~ivation ~llows the~e calls to ~x~rt th~lr regul~tory or effector activltle~. During ~ctivation relatl~ely gul~cent cell3 undergo complcx ch~ng~s lnvol~ing cell dlf~rentlation 20 and prollferntlon. Th~ ac~lvatlon of T lymphocytes is a con~equence of llyand-receptor interactions that occur at the lntorface of the T cell and an ~ntlg~n-pregenting c~ll. A number of diff~r~nt c~ urfac~ mol~cule~ on tha T lymphocyte ~nd the am~ig~n-prQsentlng cell m~y 25 particlpate in tha compl0x c:ell-cell inter~c~1on which oCCUr9 during antlgen pre~en~tlon, ~ntigen-lnduced T
lymphocyt~ ~ctl~a~lon mu~t inYolYe stlmula~ion o~ the T
c~ ntigcn raceptor. S~lmu~ lon of t~ T cell antlgen receptor alone 19 lngufficient to induc~ proll~eratlve 30 response3. Oth~r cell ~ur~ace mol~cule~ expre~ed on T
c~ unctlon ~ acco~sory molecul~. Acce~ory molecul~ ~ay functlon a~ adhe~lon molecule~, modify the ~
Sr~n~membrane signal ~nltlated v~ th~ ~ntlqen receptor and/or lnitl~te thelr own tran~e~br~ne s~gnallng s~nt~.
A number of cell ~urface mslecule~ appear on ~he urface of th~ T cell during ~he ~vent~ a~ociated with th~ ~ctivatlon, dlfferentlation, ~nd proli~ar~tion of T

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' ~, ' , ', ' ~ , , ". ' ~ :' w~ 92/~l~ 2 ~ fi ~ ~ PCr/US91/0~176 cell~. T c~ll proli~eration 1B believed ~co be regulated primarlly ~11rough the ~ctiona of IL-2 on i~ ~peci~lc c~ urf~c0 r~captor. Th~ rol~ of IL-2 lnclud~s both autocrine ~nd paracrln~ effec~ re~ulting '.n the ~; prolieratiorl of other T cell~.
Although IL-2 drlvsn T c~ll prollfer~orl in con~ldered the ma~or ~Dach~nism re~pon3~ble for T c~ll growth, under ~ome c1rcs~ tance~ ~ cell proliferation occur~ dep~nden~ of IL-2.
In addltion 'co determlnirlg that ~ nov~l 5HT2-llke ra~ep~or l~ pre~ent a~ a cell ~urfac:~ mol~cule on activ~ted T cells, lt hMs been de~rmln~d th{~t the nov~l 5HT2-llka c~n be r~gulated by inhlbltlng ~eroton1 n ~ynthe~is.
Thel pr~an~ lnves~tloll also r~ ce~ to the regulatlon of t;he prolif~r~on of cell e~hlbl ing ~he nov~l 51~T2 ant~gonl~'c~ ~o elther incre~e ar dacr~ c~ll prollfer~tlon .
The prQ~ent lrlventlon 2sl80 rell!ltes to r~ulatlon of 20 c~ll prollferatlon exhlbltlng the novel 5}~T2-lllcu receptor~ S~d ~ntlbodiQ~ 3.nclude a plurallty v~ l'types"
Q ~ntibod1Os ha-rlng ~n epltop~ or ~pitope~ ~pes:lflc for the sHrr2~ ce r~ceptor. Such Arltibody "type~" mnr includel polyclonal antiboclle~ I monoclon~l ~ntlbodies, chlmerlc ~ntlbodle~, h~anized or hum~n ~ntibodl~.
Th~ pr~Qrlt inventlon al~o rælates to the r~gul~tlon o~ c~ll proll f 8rl!1tl011 ~xhibltlng th~ nov~l 5HT2- lik~
receptor by gener~tlng lolme~opss, 3m~11 peptlde lig~nd~, that Iblnd to eith~r the 5HT2-lllce rec~ptor or to 30 antlbodle0 to the SH~2-llke r~cep~or. 5uch s~all papt~d~
ligands would funct~on ~ ~g~nl~t~ ~nd~for an~cagonlsts.
Prol~fer~tiorl of norm~l dlploid cell~ gerlarally requlrs~ ~he prasenGel o~ ~ contl nuou~ ~upply of endoganou~ or exogenou~ growth f~ctor~0 Ps:oll~r~ n of 35 tumor c~11 h~ gçlnarally b~aen found no~ to requlre addltlon of ~xogenou~ growth f~:tor-q. This is due to the ~blllty of ~ulzor cel1~ ~o produce ~he~r own growth ~ TE ~

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W092/~15 2 0 9 ~ PCT/US9~ 76 f~ctor~, due to changes in growth f~ctor receptors ~o they are contlnuou~ly actlvated or chang~s ln qignal transduction element~ whlch result in the contlnuou~
acti~ation of tumor cell~. Tumor cells may also e~pre~s unique re~eptor~ or produoe unique hormone~ or grow~h factor~ whlch are not usually as~ociated with the parant ti98Ue. Recently it ha~ been hown that expre~sion of 5HTl~ or 5HT2 receptor3 (by gene tran~fectlon) in normal diploid ~urln~ fibrobla~t~ resul~ in the ~cqulsitlon of a trans~orm~d ph~notype in the~e cells. A~ter ~ntroduclng cDNA~ encodlng ~he~e recep~ors, ~ran~fected cell3 expre~ h1gh af~inity receptor~ ~or 5~T which transduc0 Becond me~Bengsr sign~l~ . Thea~ fibrobla~ts al~o acquirQd ~h~ abllity to form focl in ~i~8U~ ~ulture~
to ~row ln 80~t ~gar and to form ~umor~ in nud~ mlce.
ThiR raisQ~ tha po~sibllltr ~hat synthe31~ of sero~onin and abQrrant expres~ion of 5HT re~eptors may play an important role ln tumorigen~sl~. Normel cell m~y acqulre a tran~ormed phenotype by acqulrlng the abllity to ~yntheslz2 ~erotonin ~nd/or ~erutonin receptor~ which can tr~n~duco second me~ng~r pathway~. Result~
presentod here ~how (1) prollferating cell~ contaln sQrotonln, (2) inhlbltlon of erotonin ~ynth~is lnhibits cell proliferat~on by tumor cell~ but not by nor~al ccll~, and (3) prollferatlon of tumor cell8 but not norm~l c~ lnhibited by c~rtain ~erotonln roc~ptor antagonlst~.
'' SU~MA~Y OF T~E INVENTI0~
A ~ethod o~ down-regulatlng prollferation of cell3 exhlblting ~ 5~T2-llk~ raceptor compri~lng, fu~ctionally d~creaslng the amount of ~rotoni~ availabl~ for bindlng to sald SHT2-llk~ r~ceptor or functionally reduclng the availabill~y of 5HT2-llk~ r~cep~or blnding ~it~B. ThiY
method applle~ to n~opla~lc or tumor ~ell~ and actlvated T-cell3. The acti~ated T-cell~ can be IL-2 dep~nd~nt or IL-2 independent. Th~ proliferatlon of the cells is Sl;~;ST~TU ~ ~: 5'~- .

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W~g~ P~T~US91t~1~6 decra~ed by ~dminlsterlng a ~ufficlent amount of compound to inhibit the activity of th~ enzym~ tryptophan hydroxyl~e th~raby inhibiting ~erotonln ~yn~hesl~. An ex~mple of such a compound 1~ p-chlorophe~ylalanlne.
~ method o~ down-r~gulatLng prolifaration o~ cells exhibltlng a 5HT2-llkY r~ceptor compri~lng funct~onally reduclng the ~vallab111ty of 5~T2-llke rec~ptor blndlng ~i~e~, the prollferatlo~ i~ de~re~sed by lntroducing an a~ount of at le~t on~ ~ntagonl3t ~uffic~ent to b~nd to ~d ~HT2-llk~ recaptor to lnterrupt 0811 prollf~ratlon.
S&id at lea t onQ antagonl~t is ~eleoted from the ~ 8 of 5HT receptor ligand~. Said olas of 5~ rec~ptor ligands in~lude~ ritan~erin, me~ulerglne~ ~lan~erin, spiperone, mimotop~ and antlbodi~s to ~ald 5~2-like r~Optor~
A me~hod of up-r0gul~tlng prollf~r~ion o~ cell~
~xhlblting ~ 5HT2~ e reoep~or compri$1ng ~unetlonally in~re~sing the ~vallabllity of 5~T2-lik~ rec~ptor ~lnding ~lte~. The prolif~ra~lon of a~ld cells i8 incr~a~ed by ntroduclng an ~mount of ~t l~ast on~ ~onl~ ~ufficl~n~
to enh~nce c~ll prolif~r~tlon. Said ~t l~ast one ~gonl~t 1~ s~lect0d from the c1~83 ~f 5NT rec~p~or llg~nd~. S~ld cl~ of SHT rec~ptor ligands include pelansaring ket~n~rin, methyl ~erotonlll, 8-OH-DPAT, and propranolol.
A msthod o~ tre~tlng a 'r-cell depandant di~3~ ~tats ln a ~A~al co~prl~ing down-regulatlng proll~ration of c~ ex~ibitlng ~ 5HT2~ e r~ceptor by functlonally d0cre~1ng the ~oun~ of ~erotonin avallablo ~or blndlng to ~sld 5~2-like rec~p~or or functlon~lly reduclng the 3~ ~v~la~ y o~ 5HT2-llke rQc~ptor blnding sltR~. The proliferatlon of the c811~ 1~ decre~sed by admlnl~terlng ~n effactlv0 a~ount of a compound to inhlbit the ~ctiv~ty o~ the e~zyme tryptophan hydroxyl~e ~hereby inhibl~ng ~rotonl~ synthe~l~. Sueh a compound lnclude~
p-chlorophenylal~nlne.
A ~thod of tre~tlng T-cell depend~nt dls~s~ ~tate in ~ mam~l comprlsing dewn-regul~t~n~ the prollfer~tion of c~ axhlbitlng ~ 5~T~-llke rec~ptor by functionally SlJBSTlTUTE SH~Fr .. : . . . . ........... : ~
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W092J~U1S PCT/U59]/~176 ~ : 7 2~9~
reduclng the availabllity of 5HT2-llke rec~ptor binding ~l~e~ whereln the proliferatlon of sa~d cell~ ~
decraased by introducing an effectlve amount of at 19a8t one antagoni~t ~ufficlent to bind to 3~1d 5HT2-like receptor to tnterrupt cell proliferationO Sald at lea~t on~ antagonls~ i~ selected rom the cla3~ of 5HT receptor ligands. Said cla~ of 5HT receptor ligands include ritanserln, me~ulergin~, pirenperone, 9p~ perone, mlmotope~ and antlbodie~ to ~ald 5HT2-like receptor.
A msthod of treating a nsopla-~tlc disea~e state ln a m~mmal comprl~lng down-regul~ing proll~eration of c~
exhibi~ng a 5HT~-like receptor by functlonally decrea~1ng th~ amount of ~erotonin avallable ~or hlndlng to ~aid 5HT2-llke receptor or ~unctionally reducing th~
avail&blll~y o~ 5~T2~ e receptor blnding si~s. The prol:lfer~tion of ~he c~lls 1~ decr@~d by administerin~
an e~fective amount of a compound ~o lnhiblt the activity of the enz~me tryptoph~n hydroxyla~e ther~by inhibiting ~erotonln ~ynth~ . Such compoundA includ~
p-chloroph~nylalanlne.
A method of treatlng a n~opl~stic di~ea~ ~tate in a mammal compri~lng down-regulatlng prolifer~tion of call~
exhlbitlng a 5HT2-llke receE)tor by functlonally r2ducing tha av~llablllty of 5HT2-llk~ rec~ptor blndlng ~ 8 wh~rein th~ prollferatlon o~ ~id cells ls decse3sed by lntroducing ~n e~fectl~e amount of at lea~t one antagonl~t suP~lcl~nt to blrld to 3aid 5~T2~ e r~ceptor to int0rrupt c~ll prolifer~tion. 5~3d at le~t one ~nt~gon~t 1~ s~l~c~ed fror~ ~he cla39 of 5~T receptor llg~nd~. Sald clas~ o~ SHT receptor ligands include rl~n3exln, me~ulergine, pir~np~ron~, splperone, mimotope~ and an~lbodies to .~aid 5~T2-llke receptors.
A msthod of tr~atlng an lmmun~ d~ficient disea~e sta~e ln a m~mmAl by up-regula~ing prolif~ratlon of T-cell~ exhlbltlng a 5HT2-llke receptor comprl~lng functlon~lly lncrsa~lng ~ha a~ail~blllty of 5~T2-llke rec~ptor blnding sltgs. The prolif~ratlon o~ 3a~d cells .SU5âTlTU ~ E S~

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w092/0~5 P~T~US9l/~t76 8 2 ~

1~ lncr~a~ad by lntroduclng an eff~ctiv~ amoun~ of at least one agonist 3ufficient to ~nhanco cell prolif~ration. 9aid ~t least one agon~t i~ sQl~ct~d ~rom th~ cl~ of 5HT rec~ptor ligands. Sald cla ~ o SHT rac~ptor llgands l~cl~de pelan~e~i~, k~tan~erln, methyl ~Qrotonin~ 8-OH-DPAT, propranolol, and mians~rin.

B~IEF DE5CRIPTION OF T~E DRAWIN~S

l~ Figure 1 i~ 8 graph demonstrat~n~ 5HT blnding ~o Jur~at cQlls. Jurka~ cell9 (l x 106) were lncub~ted wlth the indicated concentratlons of (3H)5HT for 6 mln at 4 C 1n the ab~nc~ (total blndlng) or pr~sence of 50 ~M 5HT
(non~p~cific btnding). Spoclflc binding ( E3 i 18 ~xp~e~8ed a8 the dlffsr~nca ~QtWe~n th~s~ two v~lue~.
Flgur~ how~ the re~ult~ graph~d on a scatch~rd plot.
Figur~ 2 18 a graph demon3~ratlng kinetic~ of a880ciat~0n and di~oclation of (3H)5HT to Jur~at cell~. ~A) ~l~e course of a~oclatlon of (3H)SHT to Juxkat cell~.
2~ Condition~ were a~ d~crib~d in Materlal~ and M~thod~
exc2pt th~t ~lme of lncubation wa~ varl~d (E3). ~) Time cour~o of dis~oclatlon of bound (3H)5HT ~ ~ 3 from Jurka.
c8 2S Figure 3 13 a graph demon~tra~lng comp~rl~on of th~
~ffqct of OKT3 and 51NT sn intracellular ca2~
eon~entr~tlon~ ln Jurkat cell~. Approxim~tely 5 ~ aftsr data collectlon w~ l~lti~t~d. O~T3 (1 ~g/~l) or SHT
(5 yM) w~ added dlr~ctly to cell~ and d~t~ cqu~ on 3~ contlnued ~or a to~al of ~ mln. T~e rel~tlve lntrac~llular calclu~ concentratlon wa3 CAlcul~t~d ~rom th~ ratlo sf violet fluor~cenco Icalciu~ bound i~do-l) to blue f luore~c~nc0 (calc-um ~ree lndv-l) a~d plotted ag a functlon of tlme.

Flgure 4 13 a graph demon~erating conc~ntr~tlon dependenc~ of 5HT medla~ed ~nera~s~s in i~tr~ccllular SUBSTlTl5TE S~5E~

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Wog2~ls P~T/U~91/~176 2 ~
Ca2 . Jurkat cell9, loaded wlth ~ndo-l were incubated wlth 5~ ~t 37C for 2 min. Percent of cell~ with lncraa~ed intracellular CA (~) and tota~ intracellular CA~ cencentr~tlon ~ O ~ wa3 determlned by mea3ur1ng the incr0a~e ln fluorescence with the u~e of ~ACSTAR Plu~ a~
dascrlb~d in Materlal3 and ~ethod~. Percent po iti~e cell3 contaln~d 375 to 425 nM Ca2 and negati~e cell~
contained lSO to 175 nM Ca2 Figure 5 l a graph d~monstr~ting 5~T-medlated increase ~n pho~phatldyllno~itol turnov~rO Jurkat cell~ were labeled wlth (3H)ino~lkol for 48 h. LabalQd cells wer~
incubated wlth 3 ~M 5HT ( 03 Gr 1 ~g~ml OgT3 (~) for the lndlcatad periods of time b~fora harve~t and ~naly~ls of IP level~ by anion exchang~ chromatography.
', Flgur~ 6A is a graph showing speclfic b~nding of 3H-5HT
to T-cell blasts.
Figure 6B ls a graph showing bindlng of ketan~erln to T-cell bl~8ts.
FlgurQ~ 6A-l and 6~-l deplct Sc~tchard analy~is of the binding d~t~ from Flgur~ 5~ and 6B~ respactively.

Flgure 7 18 ~ graph showlng competltion ~xperiments wlth v~rlou~ 5HT receptor llg~nd~.
Figure ~ graph ~howing the ld~n~l~lc~tlon of 5HT ln PBL.

30 Fi~ur~ 9 1 a gr~ph showlng ef~c~ o~ 5HT on :.
proll~ration of PBL.
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Flgur~ 10 1~ a graph ~howing an~logues whl~h blnd to 5~T
r~ceptor~ snd ln~r~a~H or decreAs3 T-cell proLif~ratlon.

Flgure ll l~ ~ gr~ph ~howlng the activa~ion o CD8+
suppres~or ~-cell~ by P~M requir~ 5HT.
S~E31$TITVT~ SH . Et7' .
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WO9~401S lû 2~ 9 P~r/usslto6l76 Fiyure 12 1~ a graph showing the eff~ct~ PBL ~cimulated with O~T3 ln pre~ c~ or ab~ence of ~n~logu~s which bind to SHT.

5 Figure~ 13A ~nd 13B ar~ graphs of propartl~s of the ~erotonln r~ceptor orl tumor c~ . (A) JurX~t cell~ were harvested from log phase growth and incubated at 3xlO6/ml ln a tot~l ~olum~ of 600 ~1 of RPMI 1640 madia at 0-4C
~ n the pre~enc~ o~ 3H-5HT ( ~ ), 1251-LSD ~ 0 3, 3H-ketan-10 serln ( ~ ), 3H-~OH-DPAT ~ ~ or 3H-DO~ ) (NEN) ln the pr~enco or ab . onc~ of 50 M 5HT for 6 min. Bindlng oquillbrlum wa6 reached durlng this period (not ~hown).
Cells w~re coll~ct~d on to nitroc:ellulo~e mombr~n~
(Millipore) and tube~ ~nd fllters w~re wa~lled thre~ tlme~
15 eaGh wl~ch 4ml of cold PBS. Ascorba~ ( 100 ~M) was included in the wa~h solutions to reduce nonspeci~Elc blnd~ng ~o filter~ ( 15) . Sp~ciflc binding 1~ expre~ad ~ the dl~ference betwe~n ~ot~l blnding 2~nd ~lndlng ob~cr~ ~d ln the pre~enco o~ 50 ,uM 5H7~. Figure 13A show~
20 a Scqtchard ~naly~i~ of blnding d~t~ wlth 5HT ~ ~3) and ketan~erin ( ~ ). The ~td was 130 nM. ~3) C~ were lncubat~d at 0C f or 6 mln in ~h~ pr~ence of 100 ~n~q 3H-~HT ~nd the lndlcated eoncentr~tlons of tha followlng 5H~r r~cap~cor llgands: 5HT (E3), k~t~n~erln (~a) 25 a-m~th~l serotonln (~), mesulergine (~D), 80H-DPAT~0 3, ~nd ritansarln ( O ), and bound 3H-SR~ w~ detqrmined a~
de~crlb~d ~bove. Speciflc bind~ng wzt~ de~rmined. ln the pre~nc~ o~ 50 )uM 5HT.

30 Figur~ 14 18 a graph ~howlng r~Yer~al of rit~n~rln mediat~d lnhibl'clon of tu~nor cell prollf~r~lon by 5H'r r~aptor llgarlds. ME180 cell8 were cultured for 48 hr ln tl~e pr~encq or ab~ence of rltanserin ( 20 1~ nd ! n the preJ~nc~ or ~bs~nce of tha ln~lcat~?d conc~ntr~tion3 of 35 the followlng 5HT r~3ceptor ligands: 5HT (~), pel~nserin ~ ~ ), Ketanserln ( Ea ), mlanserin ( O ), aOH-DPAT( ~ ), propranalol ( C~3 . Colls were lab~led with 1 IlCi 3H-TdR

SUE~7 iTUTE ~F~T

W~ ~2/~4015 ~r/us9~/~6a76 11 2~

~or th~ final 6 hr of culture and collQcted to m~a3ur~
incorpor~tion into DNA. Re3ults are expras~ed as the perc~nt of control lncorporat1 on of 3H-TdR lnto ME180 cell cultur~ ln the ab~enca of aitan~rln or other 5 ligand~. Incorporation of 3H-TdR by ME180 cells w~s 40,679 +~~ 2548 in the absence of ritan~erln and 2215 +/-433 in th~ pre~ence of ritan~erin. The~ ligands affect their ~peciflc 5H'r receptor ~lte~ between l-10 nM.

lû Fiqure 15 i~ a graph showing 5HT dependent ch~nges in lntracellular cA~lP content of coll bl~sts~

Flgure 16 is a graph ~howln~ th~ ratQ of change of cA~5P
content ( 0 ) and proll~ratlon ~ ~ ) ln T cell cultur~
15 af ter addition of 5HT O
:.
Flgure 17 1~ a gr~ph ~howlng ~ nhlbltion of antiprollferatlv~ eff~cts on ganona islter~ron (IFN~
ME- 180 cell~ were cul~ured ~or ~hree days with IFN in 20 'che ~bsence (El) or pre~enc~ of 300 ,uM tryptophan (O or 5 yM SHtp (0)- (~) ME-180 cell~ wer~ culturod for three day~ with 100 u~ml XFN in the pre~nce of varying conc~ntration~ o~ 5Htp ~Ç~) or tryptoph~n ( ~ . Control csll prolifor~tlon w~ 88,~65 + 1256 cps~l of 3H-TdR after 25 a 4 hour pul~e wlth 1 ~ICi 3H-TdR.

Figure lB ~ ~ gr~ph ~how~ng ~ cornparlson of the ablllty of 5Htp and met~bolit~ of 5Htp to r~v~rsc antlprolifQr~tlvQ effect~ of g~nma ~FN. P5E-180 cells 30 w~r~ eultur~d wlth 100 u/ml IFN l~ 'che prescnce or absenoe of thç~ indica~d conc~ntr~tlon~ o~ 5Htp ( 3 ), 5HT
(~), melhtonln ( ~9 ), N-methyl serotonir ( O ) or 5-hydro~yindol~ ~cet~c acld (sc) for threQ days. Control proll~eratlon waa 73,~56 ~ 2289 cpm and prollferat1on in 3g the presence of IFN wa~ 16,894 + 1145.

SUBSTlTllTE~ SHE~E~

., - , ,``
` , , .
, , , .;, ,. ,, i ~ . - ,, , wo 92/~ S P~T/U~91/06~76 F~gure 19 i~ a graph showins~ cell~ lo~ tryptophan and 5HT after culture with ganma IFN. ME-laO cell3 were cultur~d for the lndlcated ~umb~r of days in the presance ~ 0 ) or ab~enca ( O ) of 100 uJml IFN , harv~st~d and 5 analyzed for 5~1T cont~n~ and tryptophan contsnt by ~IPLC;
UPPER GR~H: S~l~, LO~ER t;~P~: trypts~ph~n.

Figur~ 2û i8 a graph ~howlng 5~tp doe~ not inhiblt lo~
of ~xtracellular ~cryptopllan ln cultures with gamm~ IFN.
10 ME-1~0 cell~ wer~ cultured in thQ ab ~n~ (0) or pres~nce of 100 u~ml IFN (~,O) and in t31Q preser~ce (19) or absance .
( a ~ of 10 ,uM 5Htp. Culture medi~ WA8 harY~t~d on th~
lndic~ted d~ys ~nd ~n~lyzed f or tryp~oph2lr~ con~:o~s'c by HPLC .

Figure 21 18 B graph showing SH~p rever~4~ lnh~bltion of coll prol~fer~stion ~y low tryp~oph~n. ME-18G cell3 ~erQ
cultured for 48 hr ln RPMI~1640 ~ed1a with the indi~at~d ~mou~ts of tryptophan wl~h (~3 or wlthout ( a ) 3 ~M 5Htp o ~o Ra~ults are Qxpressed as incorpor~lon of 3~-TdR aPter a 6 hr pul~e on d~y 2.

DETAXLED DESCRIPTION OF THE INVENTION
Inltl~l ~tudle~ were conduc~d to d~ter~lne if SHT
r~captor ~ubtyps~ could be ldentified on hum~n T
lymphocyt~ ~nd lf sHT raceptor~ could medlate sl~nal tr~n~duc~ion eYQnt0.
lAbbrevi~tlon~ u~d ln thl~ papsr: 5~T, ~erotonin, 5 hydro~y~ypta~ln~; Da~ bro~o-2~
3~ 5-dimethoxy-a~ph~ta~lne; 80H-DPAT, 8-hydroxy-2-(d~-n-p~opyla~ln~ t3tralln; IP, lnosltol phosphato0; IC50, concentr~tlon required ~o inhlbit re~pon~e by 5Q~; EC50, concen~r~tlon ~equir0d to yield h~lf-m~xlm~l respon~e.

3H-5ht t20 Cl/mmol~, 3~_~e~ rln (65 Ci/mmol)~ 125-I lyserglc acid dlamid~ (LSD, 2200 Cl~m~ol), 3H-80H-DPAT ~12~ Cl/mmol) and T~ S~

' ,:

W092/~1~ PCT/V~91/~176 13 20~Q~

3H-myo-ino~itol ~48 Ci~mmol) were obtained from NQW
England Nucl2ar. FCS, RP~I 1640, and L~glut~mine were obtained from GIBCO. Indo-l wa~ obtalned from Slgma Chem~cal Co .
Cells. Jurkat cells wero obt~inad from the American Type Culture Collection and wer~ mainta~ned ln continuous culture in ~PMI 1640 medium ~upplemen~ed wlth lO~ FCS
without antlbiotlc3 but wlth 1 mM glutamine.
Pr~paration of membranes from Jurkat cell3. Jurk~
call~, 1-2 l cuItur~s, were harvested by c~ntri~ugation, su~pended ln lO mM tri~-HCl, pH 7.5, contalnin~ 1 mM
E~TA, incubated for lO mln on lce, and were homogonlz~d with a Doun~ homogenizer in 20-40 ml tot~l volume.
Homogen~ wer2 centrl~uged at lO,OOOxg for lO min to remove nuclei and cellular d~brl~. The ~up~rnatant was eentrifuged at lOO,OOOxg for l hr. The pellet wa~
su~pended ln tri~-HCl buffer with E~TA at a ~ln~l conc~ntratlon of 7.7 mg/ml pro~ein. This memhrane preparation was stor~d at -70C until u~e in binding as9ays.

Blnding Assay Jurk~t cella 1werQ harve~ted from log phase culture~ and were lncub~ted at 3.0xlO~ml ln a total volum~ of 600 ~l or RPMI l640 ~t 0-4C ln the pras~nce of 3~-5H~ wl h or wl~hout v~rlous 5HT ~gonlst~ or ~ntagoni~s l~ea toxt for dQt~ ) for 6 mln. ~inding equlllbrium W~3 re~ch~d durlng thi~ incub~lon perlod.
Dl~pl~c~m~nt exp~rlment~ were perfor~ed by lncubatlng c~lls with 3HH-5HT, folIowad by lncubation with unlab~led agonl~t~ or antagonl3t~ as de~ribed in the teXt. Cells wer~ collected onto gla~s fib~r filter~ and tubex and filters w~re wa~h~d 3 tlmes each with 4 ml o cold P~S.
Ascorb~tc (lOO ~M) wa~ included ln ~h~ w~h solutlon~
when l~5I-LSD or 3H-~oH-DP~T blndlng wa~ ev~luated to reduce non3p~ciflc b~nding to the ilters (14-15).

Sl)BS~!TU~ SHE~

:

4~5 2 ~ P~9~ 76 ~4 Washing of each f ilt~r took 1~383 ~h~!lll 20 8e~c . Total blnding o H-5~T to ~7urkat c~ll wa~ typically 10,000-15,000 cpm ln the pre~ence of 100 nM 3H-5HT ( Io6 total cp~ added). A~out 1500-3000 cpm remalned bound in ths pre~ence of 100 11M 5~T (non~pecific blnding). For a~sociatlon rate ~tudle~, cell~ w~are incubated for ~arying perlol~ o~ time with 100 ~M 3H-sHT ~ith and wlthout 100 uM 5HT. Blnd~ng wa~ ~topped by addltlon of PBS ~nd filtratlon. For di~oi:iatlon ra~e stud1 Q~ cell~
10 ~r~ lncubated with 100 nM 3H-sH'r for 10 min. at 0-4C.
Ne~ct, 5HT was ~dded to yl6~1d a final conc~ntratlon o~ 100 uM ~nd th~ reac:tlon was ~topped by additlon of cold P~S
and flltratlon of the s~mple~. Blnding as~ay~ w~re p~rf ormed in duplicate a minlmuDl of thre0 tlm~s .
15 Duplica e ~mpl~s wer~ within 55g of each oth~r.
Slmllar procedure~ were employcd when Jurk~t membran~ prf~par~tlon~ w~r~ u~ed in the blndlng a3say~
exc~pt 'ch~t blndlng 888~!1y8 were p~rformed in ~ri~-HCl buffer, p~ 7~5, wlth 500-700 yg/ml total protein.
20 Intracellul~r Ca++ mea~urement~. Mobillzation of intrAc~llul~r Cn~ wa~ lu~ted by th~ indo-l method u~ing a modlfied Becton Dlckin~on F~CSTAR Plu~ (16-17).
~3rlef1y, JurXat cells w~r~ lncubat~d wl~h 2 ,u M lndo-l ln DMEM wlth 10 mM HEPES ~or 4!5 ~aln. a~ 37C, ~ashed thre~
25 tlmes and re~uapend~d at 106cell~ml ln PE~S. Allquot~
ml) wer~ equlllbr~ted to 37C and placed in the modifled ~mple st~tion ~nd dsta ac~ui#ltlon w~s initiat~d.
ApproxiD~t01y 5 8QC. after data collect~oT~ gan, 5E~T or OR~3 (~e~ text ~or concen'cr~tlons) w~s ~dd~d d~r~ctly ~o 30 the ~nmple~ and dclta ~scqul3~tlon co~t~nued for ~ total of 4 minute~. Fluore~cence eml~lon wa~ dl-~ided by a longp~s ~lltelr (455 nm cu~coff) pl~c~d 45 ~o the collectlng len~ and msaEsurç~d ~lmul'c~nQollsly 'chrough two bandpa~a filters, ~P4~5~22 and ~P395f20. ~at~ collected 3~ in ll-~ mode W~13 subsequ~antly proc~Jed to yl~ r~tio of viol~t fluor~cence (395~0 mn) to blu~ fluore~cance (485~22 nm~ ~ a function of t~e. Calciu~ concerltratlon ~U~ST T~E S~

: . . ~

wo~2/~ls PCT/VS9l/~176 . 15 20~a~

wa~ calculated u~ing the foxmula:

[C~ d (R-Rmin) S
tRmax-R) Sb2 where Kd i~ ~he di~sociation constant (250 nm); R, Rmin, and Rmax are ths fluorescence inten3ity ratlos at re~ting, zero, and saturation calcium concentrations, respectively; and Sf2~Sb2 i9 the ratio of fluore~cence in~nslty of the calcium-free and calci~m-bound dye (18).
The efect of concentration of SHT on tha r~pon~o of indlvidual Jurkat c~ versu~ th~ tot~l population wa~ al~o evaluated. C~ were ~quillbrated to 37C, 5HT
was added and the cell~ wer~ incuba~ed for 2 min. prior to collecting lO,OOG event~ in 11st mode. The data were subseguQntly analyz~d for rat~o o~ violet to blue fluorescsnca and perc0nt of cell~ responding about a 2 standard deviatlon thre~hold. The3e experlment~ were performed a mlnlmum of ~hree time-~ wlth similar re3ult~.
Ino itol phoqphate lev~ls. ~Jurkat cells, 2x106/~1, wer~
labeled wlth 10 ~Cl/ml 3H-myo-ino~itol in RPMI 1640 medla with 1~ di~ly2ed FCS for ~8 hrs. C~lls were harve~ted arld wash~d twic~ ln RPMI 1640 media wlthout FCS. Cell~
were susp~nded ~n m~dla without FCS at lxlO~/ml at 37C, 25 treatad with 5-HT a~ desGribed ln the ~ex~, harve~ed at varlous tim~ by centri~ugation. Extracts wera neu~r~lized wlth 7 N ~OH and an~lyzad ~or la~al5 of IP by anion exch~nge chrom~ography a~ previou~ly des~ribed (19). Dupl~ca~s sample~ were within 5~ ~nd experlments 3~ mea~uring ino~itol phosphs~e leval~ wGre per~ormed t~ree tlme~ with simllar re~ult~O
Purif ic~tion o~ P~MC . PB~C were obtained from the buffy ~oats of whole blood from normal human vs~lunteer~.
A~ ter l~Gla~cion on 1301ymph gradientq ~pharmacla~
35 Pi~a~w~y, NJ), PMBS were washed twice in P~S~ suspended in a solution of 10% DMS0 plus 90~ FCS (GI~C0, Grand I~land, NY) and ~ored in l~quld nltrog~n untll u3e.

SUBSTlTlJTE SHEET

wo 92J040~S 2 ~ 3 PCltUS~l/061 C~ urface Ag and prollfer~tive re~ponse~ of P~MC after ~torage in 11 :auld nitrogen were similar to propertl~ of fre~h lyTnphoc~ftss.
PWPI an~ PHA ware cb~aln~d from ~ CO ~OKT3 waB ~rom s ~lte~ of BALB/c mi~e and u~ed at a dilutlon of 1:1000 ), dilut~d in PB5 ~nd used a~c a d~lutlon o~ 1/80 ln cul~cure.
Puri~i~d r~cambln~nt or n~tur~ 2 wara purcha~d from Boehringer 29~nnheim ( Indian~poli~, IN) . Ons uni~c sf IL-2 acti~vity i3 d~flned ~ the amount r~qui3:~d ~o c~uç~e 50~
10 maximal prol lferation o~ tha IL-2 depend~n~ c~ll lina CTI.L ln ~ 24~h e~ y. Purlfied rIL-l, rII.-4, ~nd ~IL-6 war~ obtainffd from Genzym~ (Bo~ton, MA).
Cell culture~. PBMC, CD4+PBMC or CD4+ c~lls wer~
culture~ at sxlO5 c~lls/ml in RP~ 1640 ~edi~
15 ~uppl~ment~d wlth 10~ FC:S w:Lthout ~ntlbiotis~ ~or 3 "co 8 days ln fla~ bottom 96-well mlcrocultllre plate~ (E3e~ton Dickinson) ln 100 to 200 tJl total volu~e i~a a humidlfied atmospher~ of 5% CO ln ~lr. To me~l~ure (3H)TdR
lncorporation~ 1 C1 o~ l3~{)thymidine (2 C1/mmol, 20 Asn~r~ham Internatlonal, Incli~napollF~t IN) w~ Added to cul~ure~ ln dupllcate 1~ to 20 h be~ore cell~ were harv~tod on fllter paper (E~HD cell harvest~r, Cambridgo, ~). DD.t~ are .expr~sJed as ths ~v~r~g~ (311)TdR
lncorporatlon o dupllcate ~rdpl~. Dupllcate ~mple~
25 wera withln 5~ and axp~riment~ w2re p~rformed at le~Dt thre~a tlla~s wlth ~lmll~r ro3u1t~ snd wer~ rep~llt~d ~n ;~
~ub~equsnt exE~erlm~nts ~ po~l~civQ controls. Appl.
~lcroblol . 16 :1706 . B~dlng a~ay~ w~r~ p~srfonnod a~ :~
do~cslbsd on pl5-16 for Jurkat c~
Tab1e 11 .~. .
Inhlbitlon of 5HT ~ynth~sls re~ s ln inhlbltion of prollera~1On by a tumor c~ll 1in~. ME-lBû cervic~l carc lnom~ c~ were obtalned f ro~ A~CC ~nd were 35 3u~1nS~ln~d t!~J d~scrlbed ( ). Coll~ were cultured fo:r 48 hrs 2Ind cul~cure were elther an~lyz~d ~or 5HT con~cen'c or for cell prollferation by addltlon of H-TdE~. Medla SU;: STlTli ' ~ SH.~

., ~ ' . . - ~.`~ ` . '' ' , ::
, ,.
" ;~ `,,. " , : .

" "

wo 9Z/~4015 P~r/uS9~ 176 17 2~0~

and C~ 5 were sepaxat~d, 5ZIT was extraoted and proteln wa~ precipltated wikh 70~ ethanol, and ~lamples were conc~ntrat~d wlth a Sp~d-~ac . Samples w~re ~nalyzed f or 5HT content by HPLC ~Hewlet-Packard) equipped with a fluore~cence de~ector (B~ckman) wlth a 2ao nm ~x~itat~on Pilter and a 360nm emi~lon filt~r. The ~wo mobile phases consi~ted of A: 50mM trlethyla~ne ad~usted to pH
3.0 wlth phosphoric acid and B: SOmM trlethylamlne, pH
3.0 with 40~ methanol. The grad~ent w~3 llnear from 100%
10 A to 100 ~ B over 25 min 551). The r~t~n~lon ti~es of known ~andard~ wer~ a~ follow~: 5HT, 3.50 min;
50H-t~yptophan, 4.77 min; ~ryp~ophan, 9.11 min, SOH-indoleacetic acld, 12~5 min; and melatonln 21.59 min.
Cell~ were al90 pulsed wlth 1 ~C1 3H-~dR for the flnal 4 15 hrs of cultur~ ~nd coll~cted to me2sure incorporation into DNA. Cul~ure were performed ln trlpllcate. Actual synthe~ls of 5~T from tryptoph~n was al-~o demon~tr~ted by culturing c~ wlth 3-H-tryptoph~n, extracting soluble components and analyzlng 3H-5HT by thln layer chroma~tography wlth a ~olvont sy~tem of m2thyl propanol-ethyl acetat~-a~monlum hydroxldo-water (45-30-17-8) (52). Under the~e conditions the Rf for tryptophan wa~ 0.03 and the Rf for 5H~ wa~ 0.50.
A~ a model system, bin~ ng of 3H-s~T ~o transfo:cmed human Jurkat T-cells wa~s exalmined ~o determine wh~ther T-cell~ m~y ~xpre~s sp~cifi.c blndlnq 3itas for this neurotr~namitt~r. The~e cells hav~ b~on used by numerou~
1nv~tlgators to explore ~lgnal tran~uctlon m~ch~nl~m~
following antlgen or mitogen n~imulation (20-21).
Re~ult8 in flgure 1 8how that bindlng of 3H-5HT to Jurkat cell~ wa~ det~ctablQ and that bindlng was ~aturabl~.
Speciflc bindlng w~ dotermlnod by ~ubtracting t~e amount of 3H-5HT bound ln the pre~nc~ o~ 100 ~ unlabeled ~-HT
from the amount o~ 3H-s~T bound ln ~he presence of the lndlcatcd conc~ntratlon~ (figO 11 o~ 5-HT. ~t the~e concentrationq o~ s-HT, ~peci~lc blnding r~presented approxi~tely 70-80~ of total binding and ~axlmum bindlng SUBSTITUTE Slt E~T

., . ' ~, , wo ~ 2 ~ 9 ~ PCr/US91~ 6 of H-5HT waa obtalned wl'chin 5 mln. of ~ncub~ on (fig~
2 ~ . Blndlng O:e 5HT re~ched a m~xlmum at l-2 ~M 511T.
Flgure lh show~ the Scatchard plot of ~hs binding data.
~inding data fEom thre~ ~epal:~t~ experlmerlt3 yl~lded an av~3rage di~ociatlun con~tant (~d) of 90 nM. The slt~
den~i'cy for 3H-5HT binding to ~uskat cell~ wa~ 130 fmol per lO6cell~ or 80 ~ 000 r~ceptor~ per cell . The Hill ~lope value W218 0.8 for ~che~ data (no$ shown3. A
varie~y of blndlllg conditlon~ w~r~ v~lgated t 10 deJtermlne thelr efect on ~fPlnity constants an~ receptor denslty. Inclu lon o~ Mg++ ion~ up ~o 2 mP~, pargyllne up to lO 1IM, E~T~ up to 3 mM or lmipramin~ (bloc:k~ upt~ke o~

5~T) up to lO ~M dld not alt~r ob~erved blnding con~tant~
or rç~ceptor den itle~ (da'sa not shown). All rE~sult~
15 shown her~ wer@ d~rlv~d from log pha~ cultures. Jurkat cell~ from cult:ures e~ceedlng 106 c~lls/ml had ~ignlflc~ntly fewer 5HT receptor~ ~he~se culturs~ alRo fall~d to re3porld to 5H~ by mobllizlnSI Ca++ or increasiny ino~lt~l phosph~t~ concantratlon~.
Isolat~d membr~ne prap~ra~lon~ 80 ~ousld 5HT with simll~r chAract~rlstlcs. ~indirl~ of 5HT reached -~tur~tlon at 1-2 ,u~ 5~3$ and th~ avesage Kd dQtsrmln~ad by S-~a'cchard an~lysl~ wa~ 160 .nM. ~lnetlc~ o~ b~Lnding and dl%sociatlon of 5HT ( ~el3 bl~low) w~re al80 simllar for 25 lnt~ct cell8 and membrane prep~ratlon~. Similnr concQn'cratioll~ of 5~T or !SHT ~gonil3t~ or ~ntagoni t~
prevent~d blndlng of H-5Hr ~o both me~rane pr~3paratlon~
and 'co 1nt!1Ct C3111!1. Th~ or dlffe~ between bls~d1ng o~ SH~ to m~r~n~ prep~r~t~ons or to lsttact 30 c~ waE~ in the c21culat~d numbar of r~e~ptor~cell.
Int~c~ c~ll sxpre~ed 80, 000 ~ceptor~/coll wh~r~
calcula~lorl~ derlved frol4 blnding data ~ro~ olated meJnbr~na prep~ration~ yi~lded 30 J 000 x~cl3ptor~ p~r c~3ll .
Arc thi~ point it 1~ not certaln wh~re thl~ differ~nce 35 ari~e~ from. However, slnce bindlng d~ta are co~p~red to ~he blologlc~l re~pon~Q of lntact c~ to 5HT a~ a function oiE 5Hq~ concentra~ion, only re8ult~3 of b~nding of :. `: ~ ' ' :
'' ~

: ~, wos~/~o~s 2 0 ~ CT/U~9~ 76 . 19 5HT to intact cells will be reported here.
Kinetl cs of binding were investigated. Results in figure 2a ~how that binding of 3H 5HT was complete wlthin 4 min. and half-maximal binding occurred with 30 sec.
5 ~inding wa~ maintained for up to 10 min. From these data polnt~ an a~sociation rate con~nt (kl) of 2.1x101 '-mol 1 min -1 was calculated from the equation:
kl=[l~t(a-b)ln[b(a-x)/a(b-x)~ where a is the lnltial concentratlon of 3H-5HT, b i~ the receptor cuncentratlon and x is th~ amoun~ of receptor-ligand concentration at tlme ~. The receptor-llgand complex readily di sociated in the pre~nc~ of exc~s~ unlab~led SHT. The~e r~ult3 ar~ shown in flgura 2b. The pseudo-first ordar dl 30ciation rate con~tant wa0 1.1 mln 1 calculatad from the follo~lng equation: k2=(lJt)ln(aJx) where a 3H-5HT
bound ~t ti~e t (15). The ratio of k~ dlvided by kl provides a calculated Kd of 180 nM. This value agra~
rea~onably well wi th the Kd calculated by Scatchard analy~
Cell ~urface receptors for 5-HT have been ola~ified lnto several ~ubtype~ on the ~a~is of pharmacologlcal as w~ll as functional propertle~ (12-13). Binding of 5-HT
to 5-H~la or 5-HTlb ~l~e~ re~ults in inhibition of stlmulated ad~nylate cycla~e activity while binding to 5-HTlc or 5-HT2 sltes re~ults in lncrea~e ln ino3itol pho~phate l~vels and ~ntracellular Ca++ concentration.
Jurkat c~lls were incubated wlth indo- 1, a f luorsscent dye scn~it1ve to change~ in intracYllul~r Ca+~, and analyz~d by flow cytome~ry a~ de~cribed ln M~thods after lncubatlon ln the pre~encc or absQnce ~f 5 ~M 5-HT or 1 ~g/ml O~T3. These rssults are ~hown in figure 3.
Cells incubat~d in PBS had a ba~ellne calclum concentratlon o~ 150-175 nM and d1d not chango lntracellu~ar Ca+~ during the 10 min. ob-~er~atlon period.
3S Jurkat cell~ incubated wlth OKT3 increased in~racellular Ca++ to a maximum of ~ ~M wi~hln 2 min. b~fora d~cre~lng to a lev~l of 700 nM. Jurka~ cell~ lncubated wlth 5 ~M

S"B~TITUTE SttEEr , WO9~l3 PCT/USg1/~176 2~ .

5HT in~r~ed Ca~+ conc~ntratlon to 400 nM withln 2 min.
3urkat cell~ treat2d wlth SHT dld not ~how tha larg~
lnl~i~l pe~k ln lCa++] which 1~ characteri~tlc of tha re ponse of Jurkat c~ to O~T3 (20-21~. Fl~ure 4 comp~re~ the increase ln ~ntrac~llul~r Ca++ concentratlon in oulture~ of Jurkat c~ to Ca~+ concentrat1on ln lndlvidual Jurkat cell3 a~ a funotlon of 5HT
oonc~ntra~ion. At the indlcated SHT concentr~tion~ c~
could be dlvld~d into tho~Q wlth low Caf 1 ~150-175 nM) 10 ~nd tho~ with hi~h C~++ (375-425 nM). Thus the response oP the en~lre culture shown ln flgure 3 1~ actually, th~
aver~ge of ~ndivldual cells whLch elther have h~gh or low lntracallular Ca++ conc~ntr~tion~. Incre~a~ in ln~r~c~llular Ca++ concen~ratlon or ~h~ numbor o calls wi~h lncrea~ed lntrac~llular C~++. ~h~ half-maxi~al re~pon~ was batw~en 100-300 nM 5-HT whlch wa~
approximately ~quivalent to the di~so~latlon con~tant ob~ained from the binding d~tA.
Incr~a~ed intr~callular C~ conc~ntr~tion~ can be 70 m~dlat~d by IP3 whlch 1 8 produ~ed by hydroly~is o~
phosphatldylino~ltol biopho~ph~tQ (22). ~lnding o~ 5-~T
to the 5H~2 receptor famlly h~ b~n shown to lncrsa~
levels of lnosltol pho~phatos in neural t1~ue. ~urk~t T-cell~ w~ra l~beled for ~IB hr~. wlth 3H-lno~itol and stimul~t~d wlth 3 ~M 5-HT or with 1 ~g/ml O~T3. Cells wer~ h~v~ted at v~rlou~ tlm~ after add~ion of 5HT or O~T3 and ~n~lyzed for 1GVe1~ 0~ IP by ~n~on exchangQ
ch~o~atog~phy. Results ln fig. 5 show that IP lev~l~
~ncrs~ a~r ~ddltlo~ o~ 5~ or OXT3 and r0~che~
~æxi~u~ wlthln 1 ~in. 1e~ o~ IP decre~ad over th~
n~xt ten mlnu~s ~ppro~ching ba~ellne le~el~. ~oth 5~T
and O~T3 ylelded slmll~r lner~a~es ln IP 1~V~1R undQr these ~ondltlons.
A numb~r o~ 5-HT agoni~t~ or ~ntagoni~t~ have b~en employ~d to h~lp dl~crimln~te HmOng the d1fferent 5-HT
receptor ~ubtype~. ~etan~erlnf ~ 5-HT2 raceptor antagonl~t, slpha-methyl ~ro~on~n, a 5H~2 receptor ~'.'SSTITlJ~ SH-~T

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' W092/~ PCT~US91/~176 21 2~9~;3g~

agoni~t, and 5HT inhlbited binding of 3H-5HT to Jurkat cell~. Concentratlons which inhlbited 50~ o~ ~peclflc blnd~ng of 3H-5HT (XC50) were 20 ~M, 3 ~M and 0. a ~M for ketanserln, alpha methyl ~erotonin and 5~T, respectively.
80H-DPAT~ a speciflc agonlst for the 5HTla xeceptor ~nd ICS-205930, a ~pecific antagonist of the 5}1T3 receptor failed to prevent 3~-5HT blnding to Jurka cells. Two other 5HTlcJ2 receptor antagonl~t~, mianserin and mesulergin~r failed to inhlblt 5~T blnding to Jurkat cells. The~4 result are ~hown ln table l. Other 5HT2 receptor an~agonists quch a~ rltanserin and pelan~erin al~o failed to inhiblt 5HT binding ~not ~hown).
l25I-LSD, 3H-ketan~erin and 3~- W B have been u~ed to label 5~Tlc or 5~T2 sites ln the central nervous sy~tem.
3H-80HDPAT h~ be~ used ~o label 5HTla ~ltes ln the cen~ral nervous qystem. Tabl~ 2 comp~r~3 binding o~
the~e lig~nd~ to Jurkat cell~ as reflected by the ~d determined by Scatchard analysl~. Speci~ic binding wa~
determined in the prese~ce of lOO~M 5HT. Both 3H-k~anserln and 3H-sHT la~aled Jurkat cells wherea~
l25I-L5D and 3H-DoB failed to label Jurkat cell6 under the candltion~ employed. 3~-80HDPAT also fa~l~d to label Jurkat cell~. The blnding con~tant for keta~erin wa~
100 nM. Theae S~T rec~ptor ~gonists ~nd antagoni~s have afflnlty con~t~nts in the range o~ 1-5 nM for their specl1c receptor cla~e~.
Ths 5HT2 agonlst, alpha methyl ~erotonln, was al~o tested for i~ abllity to ~t~mulate Ca~+ mobiliz~tion (table 3). M~xlmum CA~+ mobiliz~tlon wa3 obtain~d wlth 3~ M alpha meShyl serotonln comp~red to 3 yM 5HT and the conc~ntratlon required ~o yleld half-maxlmal 3timulation (EC50) w~ M for ~lpha m~thyl 3erotonin compared to 200 nM 5HT. Value~ for dlsplacement of bound ~HT were ~lmilar. The IC50 for di~pl~cement of bound 5HT w~ 300 n~ for 5~T and 3 ~M for alph~ methyl serotcnin. ~ble 4 compare~ ths ablllty of ~veral 5HT2 antagonls~s ~o inhlbit 5HT-m~diated Ca++ mobllizat~on in Jurkst cell6.

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wo 92~s 2 ~ PCr/USs1/0~1~6 Of th~ antagonls~c~ t~t~d, only ketan~erin inhlhitsd 5HT-m~diat~d Ca~+ moblliza~ion. ThE~ IC50 wa~ l0 l M.

T~ble 1 Inhibltiorl of 5Hr blndlng by 5HT reoeptor Agonl~t/Antagonist Raceptor Subtype IC50 .
5HT ~ndog~nou~0 . 8 ~IM
ket~n~erln 5HT2 20 ~IM
~lph~-methyl ~erotonirl 5H~r2 3 15 8-OH DPA~ 5HT1a ~50 ~M
IC3-205-930 5HT3 ~20 ~( me~uleE~aine 5Hr~ >5û ,uM
mians4rln 5HTlc/2 >.50 ~M
._ .
aBindlng experlm~nt~ were perform~d ~ d~cribad in the method~ ~ectlon wlth 2xl06 Jurkat s~ ln the prE~aence of l00 nM 3H-5~T and v~riou~ conc~rltr~tiorl~ of agonlst3 ~nd ~!1ntagOnl8t8. -:

~ur~ T~ Si~

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WO92~015 2 ~ 9 ~ PCT/USsl/~176 Table 2 5HT receptor ligand ~da Kdb 5HT site (Jurkat~ (publi hed value~) 3H-ketan3erin 100 nM 1 nM 5HT2 125I-~SD~10 nM 1 nM 5HTlc 3H-DoB >100 nM 1 nM SHT2a aJuxkat cell~ were incubated with 3H-ketanserin (1-500 nM~ 5I-LSD (0.3-10 nM), or 3H-DoB (0.5-200 ~M) for 10 mln. at 0~4C. Specifie blnding wa3 determined in the pre~ence of 100 yM 5HT.

Publi3hed value~ were obtalned from reference~
12 and 13.

T~ble 3 Ca~+ mobilizatlon by_5HT2 agoni~t-~
Agonl~tDi~placem~nt of 3H-SHT Ca+~ Mobilization IcSOa EC50b 5HT 800 nM 200 nM
alpha-methyl serotQnln 3 y~ 1 ~M

aData for determinatlon of dlsplacement of 3H-5HT
were ob~ain~d fxom flgure 5.

SUBSTI~IJTE SHEET

.. . .
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: , wo 92/O~OIS 2 D ~ ~ ~ 8 ~ P~/VS91/06~76 2~

bEC:50 ' g were determlned by incubating 3urk~t c~lls wlth v~rious concentra~ion~ of 5HT2 agonist~ and following change in ra+~ concentration as a fun~:tion of time. The r~te o~ chany~ of Ca~ 18 lin~r ~or tha ~irst 60 ~ec.
5 and ~ ~ complete 2-3 mLn. after nddLtlon of 5HT agonlst.~ .

Tabl~ 4 Inhlbi~ion of Ca++ mobiliz~tlon by 5HT2 ~nt~onl~ts Ca~+ Mobilizatlvn [Ca~+3, nM IC50 5HT ( 1 11~) 425 --+ ~atansQrin 28g 10 ~
15 ~ Pelan~erln 435 >10 ~IM
+ Mlan~ris~440 alO ~
~ Mesulerglne 460 >10 ~JM
_ _ _ aJurkat ~:~118(1x~L06/ml) were ~ncub~ed for 5 min. in the 20 pr~8enc~? of 10 1uM of the indlc~t~d ~nt~gonlst~ before~
addition o~ SHT. Ca++ mob`lllza'cion ln Jurk~t c~118 W~
detenn1ned by f low cyl:ome~ry a~ des~ribed ln tho mathod~
section 2 mila. aft~3r addl~cion of 5HT. At thls ~lme the r~spon0e ~o 5Hl` 18 stlll lln~r wlth re~p~ct to time 25 ( Flg . 4 ) .
b~C50 1~ th0 concen~r~tlolt of ~gonl~t whlch lnhlbltæ SOi~
of C~ t+ ~oblllza~lon by Jurkat cell~ in ~he pr~8ence oî
1 ,uM 511T.
Th~ neurotrans~lt~er, 5Hl~ relaa~ed durin~
lnf lammatlon and has be~n sugg~?s~ed to play an l~port~nt role ln delayed typ~ hyp~rsQnsltlvlty r~pon~e~ ~ 3, 4, lO ) .
Antagonist~s o~ 5HT, ~uc:h a~ ketan~rln or rlt~ns~rin, hav~ b~en shown to prev~nt DTH responses ~n ~nl~l mod~
35 ( lû-ll) . Furth~r, tre~tmen~ of cartain antlg~n spæs:i~lc T-CQ11 C10ne9 W1~h 5HT antagonl~t~ bloclc~ th~lr ~bi~ity to tran~fer D~l re3pon~e~ to nalv~ reclp~ent8. However, SlJBSTlTUT~ SHEFr . ' . ;.; . . ~;

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W092/~ 25 2 0 ~ ~ ~ 8 ~ PcT/ussl~Q6l76 functlonal 5HT receptors which transduce intracellular ~ignal~ through ~econd mes~engers have not been defined on T-cell~. The purpo~e of experiments described here was to determine by both biochemical and pharmacologlcal analysis whether functional receptor~ for 5HT could be iden~lfied on human T-cell~ and whe~her they were slm~lar to subtypes already defined on other cell types present in the central orperipheral nervous system.
R~ceptor Rubtyp~s for SHT have been char~cterlzed functionally, pharmacologloally and molecul~rly (12-13).
The followlng de~igna~ions have been employed; the 5HTl family con~i~tR of SHTla, 5HTlb, and 5HTld; the S~T2 family consist~ of SHTlc, SHT2a and 5HT2b, and the 5HT3 family pre~ently con~ s of only one member. cDNA's encod~ng 5HTla, 5HTlc and 5HT~ receptors have been identifled and all encoda proteins whlch are G
protein-linked receptors wlth soven membrane ~panning unit~ (23-25). Functionslly, binding of 5HT to 5HTla Rltes result~ in modulation of adenylate cy~lase acti~ity (26), alteration3 in potassium ion channels, and inhibition of nerve cell transmissions (12-13). Specific agonlsts have al~o been ident:ified which exclusively bind to 5HTla sita~. Two of these are 80H-DPAT a~d lp3aplrone. ~ndlng of 5HT ~o SHTlc or SHT2 sites ~5 results ln lncreased phoqphatldyllno~itol turnover and intracQllular Ca+~ mobll'z4tion. A n~mber of 5HT2 antagonlstR h~ve b~n ldentlfied which interact wlth 5H~lc and 5HT2 slta~ wlth equivalent a~flnitie~ and ~xamples lnclud~ m~sulerglne and mianserin. Other 5HT2 antagonlst~ lnclude ketanserln, rltan~erin, and pelan~erin. The~ ligands have been used to label 5HT2 sites, po~es nanomolar afflnltie for 5HT2 sites and inhibit SHT-mediated pho~phatldyl-inosi~ol turnover ln target tl89ue9 (12-13). The 5~T~ fam11y may con~aln additlonal heterogenelty. 5HT site~ which medlate phosphatldyl lno~ltol turnov~r have also beell identlfled ln th~ hippocampuq region of the brain as well a~ in the SUBSTITUTE SHEE~T

WO92~040l5 2 ~ ~ Q b ~ ~ Pcr/lJS9l/o6176 limblc forebr~in. In the3e ti~U~9, phosph~tidylinoslt~l turnover i8 only lnhlblted by v~ry high concantr~tlon o~
ketan~erln ~compara 1-10 ~M to the normal affinlty of ket~n~erin ~or 5HT2 3it~ o~ 1-10 nM (27-2B). 5HT3 rac~ptor~ app~ar to bQ locallzed prlmarlly in th~
periph~ral n~rvou~ ~y~em. These r2ceptors ar~ not afec~d by SHTl and/or 5HT2 electlve lig~nd~. Howevsr, speclfic ligands, such a~ ICS 205903, h~v~ b~en identl~lad whlch can blocX the ~fect~ o~ 5HT in the p~rlph~ry (12-13).
Th~ Kd for`5HT blnding to Jurkat ~ell~ wa~ 90 nM
when d~ermlned by Scatchard analy~iR ~nd wa~ 180 nM when determined by klnatlc ~naly~l~. Th~ ~lt~ d~n~ity ~or 5HT
r~coptos~ ~a9 130 fmol par mil1~on Jurkat cell~ or 80,000 1$ sltc~ per cell. 5~T stimulAt~d pho~phatldylinositsl turns::ver and lncrea~2~ ln i.ntr~cellular Cl~+ in the~a c~118. S~turntion of 5HT binding A~ well ~ x$~um C~+
re~pon3e w~s ob0erved at 1-3 ~M 5HT. H~ sximal Ca~+
respon~e3 wers ~oen at 200 nM 5Hr which iY c103~ to th~
Rd for 5HT blnding to 3ur~at cell~. Blndlng of 3H-5HT
~nd 5HT modl~t~d C~++ mobil$z~tlon w~re not ~ff~cted by the specific 5HTla agonl~t 80H-DPAT or by the ~peclftc 5HT3 llg~nd ICS-20S930. Furt:her, 5HT or 80H-DPA~ did not modul~te cAMP concentratlon~l in Jurkat cell~ ~datn ~ot ~hown). The~ da~ ~re con8l~tent with binding of 5HT to the 5~r2 receptor fa~ily. Addl~lon~lly, ~lpha m~thyl surQtonin ~al~t~, ~ 5HT2 ~gonist, dl~plac~d ~ound 5H~
~nd ~tlmula~d Ca++ moblliza~ion in Ju~kat c~
HowQver, ketansQrln ~ound eO Jur~at c~ with only w~a~
affinity (~d of 100 nM~ nnd no ~pecific blndlng of 125I-LSD (dlspl~c~d by 5HT) to Jurk~t ~ells could be d~teCtQd. ~180, mi~n~er~n, me~ulergi~; p~l~n~rln, ~nd rltanserln (~11 5~T2 ~nt~gonl~ts) dld not inhlblt 5HT
bindlng to Jurkat cell~ at conc~ntraSlon~ 20 ~ and dld not inhlblt 5H~ madi~ed Ca~+ moblllzation~ nserin only di~plac~d 5HT ~nd inhiblted Ca+~ ~oblllz~tlon at hlyh conc~ntratlon~ (50% at 10 ~M. ~ur~her, 3H-Do~, a - . ; : ,, ,-,, "

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WO92J~o1s PCT/US91/~176 ~peclfic 5HT2a ligand, did not label Jurkat cells.
Effects of 5HT antagoni~t~ were equivalent when 5HT
binding was compared to Ca~+ mobilizatlon st~dies. Taken together the~e data sugge~ that the 5HT receptor on Jurkat cell~ l~ no~ a 5~Tlc, 5HT2 or 5HT2b receptor. It appsar~ to be mors simll~r to the one found on the hippocampu~ whi~h binds 5HT ~nd mediate~
pho~phatidyl-~no~itol turnover but l~ only lnhibited by ketan~erin a~ micromolar concentration~ (27-28). Thi~
5ugge~t~ that addltional heterogeneity exists ln the 5HT2 family.
Da~a presented here d~scribe SHT receptor~ on human Jurk~t T-cell and show that the recep~or stlmulates phosph~tidyl-lno~ltol turnover and increa~es in intracellular Ca++ concentration in these cell3. The increa~e in levels of inoRitol phosphate caused by 5HT
wa~ ~imilar to ~he increase caused by O~T3. The lncrease in Ca++ concentratlon by 5HT wa from 175 nM to 400 nM
wlthln 2 min. The increasa in Ca++ ~oncentra~ion caused by OKT3 wa~ to a maxlmum of 2 ~M wlthin 2 mln. Th~s increa~a rapidly decrea~sed to 700 nM wlthin an additional min. The~e dAta do not a1ddres~ the biological or ~mmunological consequences of interaction of 5HT with lts receptors on Jurkat cell~. However, pho~phatldylino~itol 25 turnov~r reprsRents a critlc~l elom~nt of ~lgnal tran~ductlon ln T-cell actlvls~ion t for review 3e~ ref .
29 ) and stimul~tion of thls path~ay by 5H~ ~hould have imporSant lmmunological conge~allence~ . Suf f ici~nt 5HT may exist at 31tes o~ lnf lalT~atlon to modulate T-cell ~nct~on. S~T l~ a m~or 3tor~ge product of platelets and lq released upon platelet aggregatlon whlch oc~ur at sites of inflammatlon (2-4). Interestingly, 5~T levels ln pl~telet~ are depr~sed 1n humans with autolmmune disease ~uch a~ rheumatold arthr~tl~ and ~ystemlc lupu5 erythema~osus (30). 5~T2 antagoni~t~ have been ~hown to exacerbate inflammatory and arthritlc re3pona*s to s~raptococcal cell wall extracts ln normally reglstant SIJ~TITU~ SHFET

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W092J~ 2o9a~ p~uss~ 76 rat~ ( 3i ) . How~ver, in this ln~ nce lt 1~ not clear that the~e ~1~fect~ ar~ medlated through 5HT receptor~ on T cell~ ~lth the avail~bility of ~peclflc 5HT
an~agoni~t~ s7r agonlst~ lt ~hould b~ po~lble to defin~
5 the lLmmunological consequ~nceQ of in~eraction batween 511T
and 5H~ receptor~ on actlvated T cell~ and bett~r unders~and th~ rol~ of 5HT receptors ~ T c~ ln lm~un~
and ~nfl~3mmatory re~pon~ea in vl vo. Flnally, ~imllar types o~ 5HT ret:eptor~ ar~s pre~ent on human p~rlpheral T
10 cQll bla~ts but are ab~e:lt on res'cing human peripher~l T
cell8 ( ln prepa:ration) .
Following th~ demonstratlon ~hat a nov~l 5HT-2-llke receptor w~ pre~nt on tr~n~formed cells ~Jurkat c~
f urth~r ~xperlm~nts wer~ conduc~ced to demon~tr~te th~t 15 the rec~ptor was pre ent on hum~n T ly~phoc:yt~R
~ ~ctlvat~d T cell~
5HT r~ceptor~ on actlva~d T c~lla-. ~he 5~T r~ceptor on Jurk~t cell~ ha~ ~ waak ~ffln~ty ~or 3~-5~T l 100 nM) and for lcetArl~rln ~100 nM) when compar~d to clas~lc 5HT2 20 recYptor~. ~pscl1c blndlns of 3H-5HT to q~ cell bl~st~
wa~ det~ct~ble ~nd satur~ble. A~out 809~ of 3H-5~r bound wn~ displaced by 100 IIM SHT. Binding 3aturat~d a~t 1-3 ~M
5HT. At ~aturatlon, 200-250 fmol 5}~ bound per 10 T
cell~. Bindlng o~ 5~ q~tur~ltecl betwe~n 1-3 Jl~ 5HT. The 25 Xd, datorminQd by Scatchard analy~l3 wa~ lBO nM (aversge of three sep~r~t~ expe~ri~nt~ c~ll bla~tA bound 224 ~mol SHT p~r 106 c~ whlch raps~s~nt~ a ~it~ d~ns ~ ty o 120, OGO r~ceptor~3 psr c~ll . T~es~ r~ult~ ~r~ ~hown in Flgur~ 6A. Flgur~ how~ the Scatchard an~ly~is of 30 th~ bindlng data. E~ndlng data ~ron~ thr~o sep~ra~
~xp~r1 ~ent~ ylelded ~n aver~ge dl~soc:latlon con~tant of 200 nM. Flgure bE3 ~hvw~ blns~lng o~ ketan~erill to T cell bl~tsO Blndlng wa~ ~a~ur~bl~ ~nd T c~ll bla~ts expres~od ~lmll~r number~ of ketan~ç!rln ~nd SHT
35 receptor~ . Spi~c:1 f ic blnding o ketz~nserln ~d~8 determlned ln th~ pre~ence or ~bsence o~ 100 ~lM ~HT.
Flgur~ 6a~1 ~how~ the Scatchard an~ly~l~ of SUBSTITUl~ SHE~

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W0~2~15 P~T/US91/~176 ket~nserln binding data whlch yield~ ~n average diR ociation constant of 400 nM. Taken together, these data support the notlon that their is a slngle receptor on T call bla~ts whlch binds both 5HT and ketanserln wlthin these concentration ranges. ~ompetlt~on experlment~ wi~h variou 5HT receptor ligand~ showed tha~
SHT, a-methyl serotonln and ketan~erln could compete with H-5HT for blnding to T cells, Flgure 7. IC50'~ were O.l ~M, 0.06 ~M and 20 ~M, respectively. Other 5HT
receptor ligands ~uch as ~OH-DPAT (~pecific for 5HTla rec~p~ors), ICS-205930 (3peclfic for 5HT3 receptors), mlan~erin ~peci~ic for 5~1Tlc and 5HT2 receptor~) and spipQrone (specific for 5HT2 receptors) did not compete with 5HT for blndlng to T cell bla3t~. The properties o~
the 5HT receptor on T cell bla t-~ are comparabl~ to the properties of the 5HT receptor found on Jurka~ cells and appear distinct from the well characterized receptor~
found in nervou~ ti~3ue.
A number of dlfferent T cell populatlon~ were evaluated for the pre~ence of 5HT receptors. Resting lymphocyte~ expre~sed low levels of receptors (~4,000/cell3 when compared to T cell blast3 (l50,000/cell). Both C~4+ and CD8+ T cell llnes expre~sed elevated levels of SHT receptors ~l43,000 and 17l,000 receptor~/cell, respectlvely wh1le CD3~,4-,8- T
call llnes only expressod 53,000 receptors/cell whlch 18 only ~lightly more than ~:haS ob~erved on re~ting lymphocy~e~. In other exper~ment~, expre~sion o~ 5HT
r~ceptor was examlned a~ a function of ~lme after ~o stimula~ion wlth dlfferent T cell mltogens. Increa~e ln the number of receptor~ was llnear for 72 hr after stimulation. Three T cell mltogens, P~, PWM, or OKT3, did not yleld 3ubstantlally dlfferent increa~es in the 5HT rece~tor n~mber on T cell3 (Table 8).

SUBSTITUTE SHE~ET

WO 92/04~lS 2 ~ p~/US9~ 76 T~ble 8 Colls 5 HT Blndlng Fmol~106 c~lls R~ceptor~call PBL 40 ~ 3 24, 000 T Cell Bla~t~ 250 ~ 11 lS0,000 CD4+ ~ C~1l L~ne 238 + 9 143,Q00 CD8+ T Cell Lln~ 285 + 5 171,000 CD3~ 4 ~-T Cell Line ~8~ 6 52, 800 In lymphocyt2~, elevatlon o~ intr~cellular c~5P i8 g~n~rally a~soc1 ated with ~he lnhibitlon o~ phocyte proli~er~tlon ~nd lymphocyto ~ffe~ctor furlction (57)~
20 Conversoly, elevo,tion of intr~cellular Ca+~ i~ gonerally found ~o be requir~d for ~c~lvation of lylophocyte3 by antlg~nic or mltog~nlc ~lgnal~ and sub~equealt lymphocyt~
proll~eratlon and e1'~ector functiclll (53~. Result3 pre~ented here ~how th~t the 5H~1a recep~or c~n mediata 25 ~n incr~æ ln intr~cellular Ca++ in Jurk~t cella. Thl~
F~hould Gl~rly entl~nce lymphocyte prolifs~tion ~nd ef0ctur gunctio~n. The 5Hr1a rec~p~or ~l~e r~gul~tes aâenyl~t~ cy~la~u and lev~l~ of cAMP in ly~phocytes.
Dep0ndlng upon the ac~civatlon ~ta~e oi~ ~donyl~t~ cy~laYe 3Q 5Hq!1a agonl~t~ may elth~r eleva~e or 13UpprQ~I~! lev~l8 CA~qP
end n~ay elther enh~nce or 8uppre88 lymphocyte prolif~ra~lon 2and functlon.
'.
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3s SlJ~ITUTE SHEE~T

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w092/~01~ 2 0 ~ P~T~US91/06~76 Blological reqpon~e of Jurkat cells and T cell blasts to 5HT, the ~pecific 5~Tla agonlst, 80HDPAT, and the 5HTla antagonist, ~piperone.

JURKAT T CELL BLASTS
Ca+~ cAMP Ca~ cAMP
Conditions (nM) (pmol) (nM) (Pmol) -- 20~ 4.7 260 1.8 5~+ 610 4.9 260 0.6 lU 5HT+ splperone 225 4.7 250 1.4 80HDPAT 470 4.~ 2S5 0.5 80HDPAr+ spiperone 280 4.6 265 1.6 For Ca+~ measur~ments, cells wer~ labeled with Indo-1 and intracellular Ca~ concentration~ were monitoxed for 5 min at ln the presence or absence of 3 ~ 5HT, 10 nM
80HDPAT with or wlthout 100 nM spiperone. Result~ are expressed a~ the concentration of intracellular Ca+~.
cAMP was extracted from cells after variou~ treatment~
described above and quantitative as de~crlbed. All cells were pretreated wl~h 10 ~M forskolln to activate adenylate cycla~e. Reqult~ are expressed as pmol cAMP/
million cells. Compar~on of the biological and pharm~cological re~pon~e~ o~ T and the 5HTla specific agoni~t, 80HDPAT~ ln T cell~.

13IOLOGICAL PHA~COLOGICAL

Liqand~ C2~+ CAMP Ca~+cAMP (bindln~ data) 9oHDpAr 5 nM 3 nM 1. 5 nM
5HT 200 nM 400 nM 3.0 nM
s iDerone 60 nM 70 nM 50.0 nM
P . _ . ..... _ Mea~urement~ of lntracellular Ca++ were performed with Jurkat cell~ and mea~urements of ~AMP were performed with actlvated T cells. Jurkat membrane3 were labeled SUF3STITUTE SH~T

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: ` , . , Wo 92/U~OlS 32 2 ~ Pcr/VSS~ 76 wi'ch 1 n~q ~OHDPAT ~nd displ~:ement exp~rim~nt~ werç
par~ormed in th~ pr~enoe of varying ::oncentr~'Llon3 of 'ch~ lndlc~t~d ligands.
Id~3ntif lc:~stlon o~ 5HT ln P~L . Re~tlng PBL were 5 cultured ln the pre~nce or ab~nc~ of P~, P~M or OXT3 ~or ~arylng perlod~ of tim~, harv~ted and ~nalyz~d for 5H~ conten~ e~ r~ult~ ~r~ ~hown ln ~lgure 8. PBL
cont~ir~d 4 p~nol 5HT/19 cell~ at culture inltiatlon.
Srhi~ l~vel wa~ maint~ln~d f or ~ d but was dlD~lnlgh~d by 10 day 7. E3y contr~t, ~tlmul~tion of P8L. wlth ~lth~r OKT3 or P~A r~sulted ln 10~8 of over 75~ o~ 5H~r conte~e wlthln 72 hr, about the tlmQ 5HT r~ceptor h~ve achl~ved m~atlmum levels. Stimulation of PBL wlth PWM resulted in ~n lnltlal lncr~a~e ln 5XT lev6~1~ by 50% ov~r the ~lr~t 48 15 hr of culture ~nd then a gr~dual d~cse~ o. ~he~e cell~
~till ~lnt~sln~d about 2 pmcl 5HT/10 cell~ Oll d~y 7.

Prolifaratlon of PBL ~lmul~tcd by P}L~ partlally lnhib~ed by rel~tlv01y h~gh concentri~tlons of 5Hl~.
20 ReEIult~ ln F151ure 9 r~produce this ~lndlng and also show the ~ffect of 5~1T on prolif~ration of PBL to two oth~r T
cell mltogen~, O~tT3 nnd P~M. Prollferatlon of P~I, in th~
pree~rlce of O~T3 wa~ unaf fecteld by addltion of 5HT. E~y co~tr~0t, additlon of 5HT to cultures stimul~t~d with PWM
resulted ~n a thr~e-~old lnc:cea~e in lncorporntion of H-TdR. Hal~-ma~lmal lncre~l~e ln inc~rpor~tion W1~18 obtalned wn~ 15 ~JM s~rO Thi~ baerved whon SIIT wa~
addud on dsyls 3-5 o~ the culture period, th~ t$me nt wh~Ch 5HT raceptor~ ~re ~axi~ally eJtpr~ d ( ~ee ~ble 30 V). Adtil~lon of 5~{T earlier in th~ cul~uro p~rlod ~l~o resulted ln ~ thr~e-fold enh~nc~ment o~ proli~r~tion but more s~r ( loo ~IM) wa~ r~quirQd to achl0v~ thl~ e fect.
One possl41l~ty to axp~aln ~his differ~nce 1~ that the actlvlty of sHT 18 medlated through 5HT recE~ptor~ whlch 35 don ~ t appe~r untll day 3 ~nd metaboll~ or oxldatlon of 511T ~rly ln the culture period r~duc~ the ef ~ctive concentr3tlon of 5HT ln the culture m~dium by day 3.

SUB8TITUTE SHE~

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W~s2/~U1~ 2 0 ~ ~ ~ 8 ~ PCT/~S9l/~176 Several analogues whlch blnd to 5HT receptors and which interact with the 5HT re~eptor ~n acti~ated T cells were te~ted for their ability to stimulate or inhlbit prollferatlon of PBL Qtimulated w1th PWM. Of ~ho~e te~ted, 5HT, ketan~erin (5HT2 receptor antagonist) and ~methyl ~erotonin ~5HT2 receptor agonist) stimulated T
cell proliferatlon by about three-fold at concentration~
between 10-30 ~M. Two other 5HT2 rec2ptor antagonists, ritan~erin (5HT2 ~lective) and mian~erin (5HT2 and 5HTlc ~elect~ve) falled to stimulate T cell proliferation. In fact ritanserin was somewhat inhibitory at the hlgh~r concentration~. These re~ults are shown in figure 10.
Both CD4~ and CD8~ T c~lls prollferate in culturQs of PBL
~timulated with PWM. In mlxed cultures of CD4~ and CD8 lymphocyteq, the CD8+ lymphocyte makes up the ma~or percentage ~f proliferating cells on day 7. Re~ult~ in figure 5 al~o show that the ability to s~imulate prol1feratlon of PBL cultures with 5HT or 5HT re~eptor ligancl~ is lost lf CDB+ l~mphocy~es are eliminated by ant1body and complement treatment.
Re~ult~ presented above indicated th~t T cell blasts expre~ 5HT receptor~, tha~ 5HT enhanGes T cell prollferation ~timulated by PWM, and that T c011~ contain 5HT. Thi~ raise~ the pos!3ibillty that 5HT may be impor~ant to the normal proliferation of T cells activated by PWM. Inhlbltor~ of tryptophan hydroxyla~e, the rate limltln~ s~ep in S~T synthesls, haYe been u~ed to depl~te store~ of 5~T ~n3ide ~ells. Results in table 9 ~how that lnhlbi~ion of 5HT 3ynthe is by p-chlorophenylalanlne (pCPA~ alqo pa~tlally inhLbited proliferation of cellY ~tlmul~ted by P~M. Addltion of 5HT
or ke~anserln to theqe culture3 reversed the inhibltory effect~ of pCPA.

STITUTE SllEET

~.' ' wo ~ 2 0 3 0 ~ ~? ~ pCr/lJS91/06176 T~b:le 9 Inhlbit1 on of 5HT ~yn'Ghesis ~uppr~s~
T Cel1 preliferation P~L PW~ pC~ SEIT lRet~n~erln3H-~dR

10 + - - - - 1,728 +210 11, 95~ ~ 1022 + + + - -4, 30~ ~688 + + - ~,726 +16~4 :
+ + ~ - +10,06~ ~4~i9 ~
5H5'. CD8~ ~uppre~sor T cell~ ~ke up ~h~ largest perc~nt~ge of prollferatlns~ ccll~ ln cultureQ o~ PBL
3timulat~d wl~h PWM. Slnc0 inhlbltlQn of 5HT synthe~1~
~y pCPA lnhlbl~ced prollfQr~tlon ln PWM ~tlmulated cultur~, lt ~ugge~ted that pCPA m~y al~o lnhibit ~pre;3~ion of ~uppre~or cell funct~oll. The~a re~ult~
~r~ shown ln ~1~7ure 11. Cultur~ of PBL w~ro ~timul~ted w~th P~ in th~ pr~ellcE? or ~baerlc~ o~ pCPA to rcduc~
SHl~ co~ . So{oe cu~tur~ wer~ ailYo ~crea~ed wl~h k~tan~rl n to provlde ~n agolli0t for the 5HT rec~ptor to po~lbly rever~o ~he effact~ of pCP~. After 7 d, culture were w~hed, treæt~sl wlth ~lto~ycin C ~sld add~d to fr~sh cultures of CD4+ T cell~ 3tlmulatE~d wlth PWM to t~t for ~upprQsslon cell acti~tity. The re~ults ~how 3~ that c~118 for PWM ~tlmul~t~d cultur~ suppr~ ed proll~er~lon of CD4~ T cslls ln Sh~ pre-QsnCa of P~M by a maxlmum of 70% at a 1: 2 ratlo of 8uppre9802: C1311~ to SL813Sll~ITIJTE SHlg~T

., , WO9~015 2 Q 9 ~ b 8 ~ PCT/~S9l/06l76 ~esponding cells. SuppreRslon of proliferation by sQ~
wa~ obtained w~th 7 x 10 cells or at a ratio of 7 re3ponding cell to 1 suppre~or cell. The presence of pCPA ln the primary culture used to produce suppressor cells completely blocked generation of suppre~so~ cell activity. When ketanserin was included in cultures wlth pCPA, ~uppres~or cell activity was normal when compared to control~.
Other T cell mitogen3 such as PHA or OXT3 stlmulate prollferatlon of both CD4+ and CD8+ T cells but do not result ln the expre~sion of ~uppressor cell activity by CD8+ T cell withln the f ir5~ week of cult~re. PBL
stimulated with either O~T3 or PHA lose their 5HT contsnt within 72 hrs whlle cultures ~tlmulateded with PWM xetain 5HT content fo~ at lea~t 7 d. Addlt~on of pCPA to cultures stimulated with either PHA or OKT3 did not inhib~t proliferatlon (not shown). To determlne whether 5HT content may be r~lated to ~uppre~or cell actlvation, PBL w~re ~timulated wlth ORT3 in the presence or absence of the T cell 5HT receptor agonl~t, ketanserin ~5HT
receptor antagoni~t), and t~ted for suppre~or cell activity after 5 days of culture. T cell blaYts, har~e~ ed from culture~ stimulated with OKT3 lacked detectable suppressor cell actlvity. By contrast, T
cell bla3t3 from culture~ ~timulated wlth both OXT3 and ketanserln expre~ed suppres~or cell activity when tested on s~cond culture~. These re~ults are shown in figure 12.
5HT a~ B co-mltoqen for CD8+ T ly~hocy~e~.
Prollferation of CD~ T lymphocytes in re~ponse to PWM
re~ulre~ CD4+ T cell3. The three-fold enhancement of proliferatlon of PBL cultures caused by 5HT or 5HT
receptor ligand~ was lost after ellminating CD8+ T cells.
Therefore, lt ~eemed posslble that 5HT may ser~e as a co-mltogen ~or CD8+ ~ cell~ and stlmulate prollferation in the pre~ence of PWM. These results are shown in F.igur~ 9. Cultures of C~8+ T cells failed to pr~lifera~e SU13~iTlTUTE SHEE7 .
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WO 92/0401S PC~/US91/06176 i~ th~ pre~enc~ o~ PWM or ln th~ presenc~ o~ P~ and IL2.
E3y contra~t, cultur~s contalnlng CD8~ T cell~ ~nd PWM
proll:Eeratcd ln tha presence of either 5HT or ketanserin.
Th~ l*v~l of prol~ fzra~lon wa~ cDmparable to that wh~?n 5 CD4+ T cell~ w~re ~ddedl to cultur~s of CD8+ T cQllsO
Addltlon of 5HT or ke~n3erln alon~ did not stlmulata proli~rRItion of CD8~ T c~ . T~ken together, thes~
data ~ugg~t th~t 5HT i~ a s:o-mltogen for CD8~ ~uppr~s~or ~ celll3.
Modul~ 1 c _ Isvels in_~c~l~rat~}d T c~ll0 by 5HT. Two ~lgnal tran~ductlon p~thways whlch ha~e, b~en linked to ~pecif ~ c 5HT rsc~ptor subtypes ar~ ~odul~tlon of ~denylate cycl~e ~nd actlvation of pho~phol Lp~e C .
Actlv~tion of phospholipa~0 C: rQ~ul~c~ ln el~ tlon of 15 ino9itol pho~ph~t~8 and incre~e in lntr~c~llul~r Ca++
concan~r~tlon. In Jurk~t cell~, 5HT stlmulnt~s ~l~vation o~ inosltol phosph~t~ and intracellul~r C~+
ooncerltr~tlon. Thexefore, ch~nge~ ln ino~ltol phosphate levQl~ ~nd in~racellul~r C~++ wer0 me~sured in T cell 20 bla8t~ ~PHA) ln re~ponse to 5HT ~nd, ~ ~ po~l~ivo control, in re~pon~e ~o 01C~r3. R~ul~cs ln Tabl~ 7 ~umm~r~ ze the~e experlment~. In Contra8t to Jurlcat coll~, 5HT dld not cau~ a oh~ng~ in lno~ltol pho~ph~te levels or ln intr~scellular Ca++ c:onc~ntr~tlon., A9 a 25 po~ltlv0 control, O~T3 stlmul~ted ~n lncr~as~ in both inoæl~col pho~phate concentrn~lon~ and in lnt~¢~llular t:~++ concent~ation ln T cQll bl~t~. Slnce T c~3ll bl~t~
dld no~ re~pond to 5HT by ~ er~rlg islo~itol phogph~te le~ nd lntr~c~llular C~+ t:oncentr~tlons, 30 lntra~ellul~r cAMP levlal8 w~rc colDp~r~d in th6a præ~ence and ~b~en~e o 5~To Thrse c~ll typ~3 wer~ comp~r~d:
r~Y'clng T c~ , T c811 bl~t~ ~nd Jurka~ lls, and cAMP
lev~ls w~re comp~red ln ~h~ pr0~nce elnd ~b~erlc~ of for~kolln. The~Q re~ul~s ar~ shown ln Tabl~ 8. R~tlng 35 T ; ~ d~d not ch~nge ~ntr~cellular cAMP 18~ 3 ln r~pons~ eo 5HT where as ~ctlvated T cell~ lncre~d cAMP
by over 2 fold in reYpon e to S~T (5 ~IM~. Ju~k~t cells - ~;UB~ T~

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wos2/~o~s ~ PCT/U59l/06l~6 dld not alter cAMP levels in re~ponse to 5HT. Forskolln activates adenylate cyclase and causeq an increase in intracellular cAMP. Forskolin increased cAMP
concentratlon in resting T cells, activated T cells and Jurkat cells. Forskolin-dependent lncrea~e in cAMP
levels w~s lnhibited by addition of 5HT to activated T
cell~ but not to restlng T cells or to Jurkat cells.
Flgure 2 shows the concentration of 5HT required to cause an incr~qe in cAMP lev~ls in actlvated T cells, or in the pre~ence of for~kolin, a decrea~e ln cAMP levels ln actlvated T cells. Change~ ln cAMP levels were proportlonal ~o 5HT concentratlon~ between 30 nM and 3 ~M. The half-maxlmal re~ponse was between 200-800 nM
5HT which was similar to the Rd obtained from the blndlng data. Addltion of PMA al90 results in an increase ln cAMP levels in T cell blasts or in Jurkat cell.q. In contra~t to the caRe with forskolin, 5HT ~alled to inhlblt the PMA-dependent increase in cAMP in elther cell type. The~e results are shown in Table 9.
Stimulation of proliferatlon of CD~+ I' cells_by_~T.
Changes in ~ntracellular cAMP concentrations are known to affect the function and proliferatlons of activated T
cell~ (53). Based on the above results, 5HT may regulate lntrac~llul~r c~MP level~ differently in dlfferent ~ubpopulations on actlvated T cell~, or ln activated T
cells expo~d to different ~I.muli. Results in Table VII
co~pare change~ ln c~MP conc~ntration3 in T cell blasts inltially ~timulated wlth PHA, OKT3 or PW~. T cells were harv~ted durlng peak proliferatlve re~pon~es and assayed for c~MP cont~nt ln tha pre~ence or absence of 5HT.
Cultures were also lncub~ted wlth 5HT for the ~inal 48 hr of culture to determlne any affect on proliferation. 5HT
cauqed a 2-old increa~e ln cAMP levels in T cells ~timulated wlth PH~, a sllqht increa9e ~n e~MP in cells cultured wlth OKT3, but caused a 60~ decrea~e in cAMP
levels in T cells stlmulated with PWM. Prollferation responses of the T cell cultures wlth the dlfferent SlJB~;T1~TE g~

WC~ 92~ 38 ~ ~ Pcr/US9l/06176 ~ti~nuli in th~ pr~ence or ~b~nce of SH~ appeared to reiElect their 1eYe1 sf intracellular c~MP. Thua, 5~T
htly inhlbited prs71ifer~tion of T cell~ in r~ponse to PHA but stimulated p~ liferation o~ ~ cells ln 5 re~pon~ to P~ by ov~r 3-fold. Addltion~lly, ~liminAtion oP CD8~ T csll~s b~fore ~tlmulation with PW~
ellminated th~ co-mitogellic e~ect o~ 5HT ln these ;:ulture~. Thu~, SHT c~n eith~r enhance or ~uppre~ T
c~ll prollfs~ratlon, apparently by x~qulating cAMP level~.
~he diraction of th~ re pon~ 98Q103 ~C0 d~psnd upon th~
mltog~nl~: ~klmulu~ And the T cell subpopu~altlon re~pondlng to ~hat 3timulu80 Rssults in Flgure 3 show th~ th~ prolifera~ive re~pon~ of PWM ~c~ivat~d T cell~
to 5HT i8 fairly r~pid. Incra~ae~ in prollf~ratlon ln 15 the pr~s2nc~ of 5HT ware detectable wlthin hours ~nd r~ach~d maximussl levels with ~0 hour~. Lsvel~ of cAMP
were d~creased tc~ 60~ of control.with 30 mln ~nd ~co 401t of cont:col over thQ naxt 40 hr o~ culture. R~sult~ ln Figu~e 4 ~how ll:he c:on~entration depç?ndenc~ of the 20 co mltog6nlc effect of 5~ on PS~ ctlvated T c:ell~. 5HT
W~ dded to cul~ur~ 48 hr before a~ ay of proli~r~tlon. M~xlm~l ~tlmul~tlon of prollferation wa~
ob~rvad ~t 30 ~u~t ;HT ~nd th~l IC50 was 10 ~IM 5HT. .
The con~cquence~ of additlon of 5Hl! to T cell~
25 dap~nd8 upon the phenotyp~ of the respond~ng cells.
Stl~ul~lon of T c~ll prol~eration Wit~l OR~3 or PH~ ;
yleld~ T ccll blA3t~ o~ botl~ C~4 and CD8 phQnotype~ whlle stl~ul~tion.of cultura~ of T c~lls with P~M yl~ld~ T c~
b1~t~ prlmarlly o~ the CD8 ph~no~ype. R~pon~e~ of the~e two type~ o~ cul~ur~ ~o 5HT ~9 al~o dif~rent.
Addltlo~ of SHT ~o culture~ Rtlmulated with PHA or OKT3 r~8ult8 ~n elev2t~0n of cA~P and slight lnhlbl~ion of T
c~ll prollferation. By contra~t, ~ddltion of 5HT ~o ~ulture~ 3tlmul~t~d wlth PWM ~U8S~ a 60~ decr~a~e in 35 CA~P ~2vel8 ~nd a 3-4 fold incr~a~e ln T c~
pro1~feratlon. C~119 pr31ifer~ing ln the~e ~ulture8 are prlm~rlly CD8+ T c~lls a~ mea~ured by FMS (not ~hown) and SUBSTlTlJ~ SJ~EE~

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W0~2/~01~ PC~/~S~1/06176 ' 39 2~9~

exhlblt ~uppre~sor cell but not cy~otoxlc activity.
Elim~nation of CD8+ T eells elim~nates the co-mitogenlc effect of 5HT in these cultures. 5HT ls al~o a growth facter for certaln other typed of cells such as smooth muscle cells or certain types of flbroblasts (46,47~.
This suggest~ that the ~rowth factor propertieq of 5HT
ar~ not llmlted to cells of the immune system.
Re~ults presented cleaxly show that T cells expres~
receptors for 5HT upcn actlvation and that 5HT can influence tha prollferation and function of activated T
cells. It i~ well e~tabll~hed that 5HT is a ma~or comp-onent of the qecretory granules of platelet~ and should be released at site~ of lnflammation (2-4). Thu~ 5HT
should be pr~ent at SitQS of inflammation to in~luence T
cell function. More recently it has been ~hown that purlfied resting T cell~ al~o contaln 5~T (~0). T cells relea~e 5HT lnto the media in respons~a to IFN gamma.
Under the~e conditions, the concentra~lon achieved in medla i~ approxlmately ~00 nM/ml/lO T cells~ Thls is very similar to ~he Kd oÇ the T cell SHT receptor. If 5HT
ls released by T cells, it could play an important role in regulatlon of T cell functlon by T cell~O It ls well establish~d that CD8+ T c~alls do not proliferate in respon~a to PWM ln the absence of CD4~ ~ cell~ t54~56).
25 Thus it i8 lnterestlng tu hypothe~lze that 5HT could be released by CD4~T cell8 in r~spon~e to IFN gamm~ and act a~ a growth ~8ctor for CD8+ T cell~. Rheumatoid arthrltis l~ a ma~or diseasa of a larse group of rheumatic dise~e~. Rheumatlc dlsea~es wlth- ~n a~tolmmune component lnclude rheumatald arthrltl~, sy~temlc lupu~
erythemato~u~, S~ogren's ~yndrome, ~clerodsrma, m1~ed connectlve tlssue dlsea~e, denmatomyo3ltls, polymyositis, Reiter' 8 ~yndrome and Behcet'~ dl~ea~e. The ar~hritis of rheum~toid arthritls c~n re~ult in de3truct~0n o the ~olnt with con~equent ~eformlty. The disea~e is not con~lned to ~olnts; va~culitis, caused by immune complexes, Can involve the skln, the eye, and th~ lung.

Sl)BSTlTUTE~ SHEEr , `` . . .
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;r,i, wo 92/0401~ 9 pcr/us9~ 76 The arthrlti~ re~ult~ fro~ ~ comple~ tsractivn of ~ynovlal c~ w~ th variou~ cel1ular elem~nt3 ~ and th~lr solubla product~ ) th~t lnfiltr~te from th~ clrculatlon into the synovial llnlng of ~oint~. Thl~ le~ds to 5 m~lvQ proliferat10n and activa~ien of ~yno~ial cells.
The propertleE~ of ~ynovi~l call8 in ti~u~ cultl~re have been lllcened to thosc of tran~iEormed cell~. For ~ mora det~ d dl~cussion ora the ~qu~nce3 of events leadlng ~o ~oint le~ionR, ~ Fundamental Immunology 20d., Paul, 10 (19a9~ pp 84~-849.
A for~ of exp~rlm~ntal ~rthxitls i8 ad~u.v~nt arthrltls, whlch 1~ a pur~ly T cell-medl~ted autoimmune dlsea~e . Thi~ f orm ef ar~hrl~ can be induced in ~3lusc~3ptlblQ 8tral118 o~E rats (e.g. Lewl~ r~ts) by ln~ectlon o~ Mycsbatarlum tub~rculo~l~ in oil (complete Fr~und~ ad~uvant). ~her~ i~ a structur~l r~mblance batw~en mycob~ct~rial p~ptldoglycanY and proteo~ly~n~ in ~olnt c~rtllag~. A nonp~ptlde of ~ ~h~3~ E19 tuù~rculo~l~ antigen conkain~ ~he epltope recognlz~d by T
c~llA m~d1~tlng ad~u~ent arthritls. In addi lon, T c311~
from p~lent~ wlth rh~umatold arthriti~ re~pond to thls shar~ ~pltop~. P~ul~ supra.
Proto~ol: Adluvant Arthr~ti~
Co~plete Fr~und'8 Ad~uvant (CFA) 1~ m~da by 2~ supple~n~lng extra he~vy mln~lral oil with lO ~g~l hoat klllod ~y5~ E~B ~ 31~ or Mycob~ct~rlum tub~rculo~l- H37R~. ~n day 0, ~e~l~ Lswls r~t~
~200-22S ~3 ~r~ ~lv~n 0.1 ml ln~sctlon o~ thl~ ad~uvant (100 ~g/~nl~ ubcut~neou~ly lnto the rlght hlnd 3~ foo~pad (ln~ecte~ paw~. ~ prl~ry infla~ato~y re~ction oc~ur ln the ln~ect~d foot. Thl~ r~pon e ~ub~ldes by day 5 and 1~ ~ollow~d on days 9-12 by ~ ~econd~ry, ~hronlc inflammatory/~r~hrltl~ re~pon~e in both the ln~cted foot an~ the contral~t~r~l non-ln~ected left 3S f oot. ~easurement~ of both footp~d a~d anklo di&me~er of the ~y 2 primary re ponse and ths days 12 and 16 ~econd~y re~pon~e (both feet~ are m~ds u~ing ~ hand-held SUBSTITU ~ c s~_ .r . . . ~
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W092/04015 PCr/VSsl/~1~6 41 2 Q ~ O ~ ~ ~

engineer' 8 caliper. Results are expre ~ed ~g th~ mean change ~well1ng + or - S.E. from the day 0 unin~ected paw diameters.
Drug~ are dlssolved or suspended ln water, phy~iolo~ic salin~ or a mixture of el~her of the latter in 2~ polyethylene glycol 400/0.1% Tw~en 80 and adminl~tered by normal route.q lorally, intravenously or intlamu~cularly) elther prlor to or after eqtablishment of dl~aqe.
Adjuvant arthritis i clearly mediated by ~ cells and activated T cells clones have been isolated whlch can transfer the diq~ase to a nalve animal. Rltan~erln inhlbit~ development of the dl~ease at 10 or 30 ~g~kg.
Keta~erin dee0 not prevent development of the dl~ea~e.
Tabl~ 10 ~itanserin Data - Ad1uvant Arthritis Exp #1.
2 Re~pon~e - Dav 16 Mean Ankle Diam % Inhib Status + S.E. (mm) Control 2.55 + .44 Con~rol Extrm.
+ Rltan8erin ~.93 + ,31 63.5 Actlvc 30 mg/~g l.p.
(Day~ - 1,0,1,5,7,9,12) + ~ot~n~erln 2.09 + .46 23.7 Inaotive Exp, ~Z
Control 2.35 ~ .56 Control + Rit~n~erln 1.39 + ~30 40.3~ Actlve 30 mg~kp p.o.
3~(Day~ - 1,0,3,6,8,10,13) ~ Pelan~erin 2.44 + .34 4.3Inactive .. . .

SUB~;TIT~TIE SHEET

~;rJ
wo ~ 2 ~ ~) Q ~ ~ ~ PCT~U~9~/~17fi Exp. ~3 ;:
Co~ol 3.28 ~ ,44 Control + R~tan~erln 2.33 ~ .43~9.0 InactlYe 3.0 mg/kg p.o.
+ Rltan~erin 0.89 + .2172.9 Extrm.
10 mg/kg p.o. A~ti~e ~ .
~Dlff~rence ln activlty may be expl~ined by slightly dif~eren~ doslng ~cAedulQ.
MaterL~L~. Pur~fied re~ombinant human IFN wa~ purc~ased from Amg~n (Thous~nd Oaks, CA) ~nd dilutsd in medl~
b~ore u~o. S-O~-~ryptoph~n, 5~T, tryptophan, 5-OH-indol~catlc acid, ~nd mela~nln were obt~lne~ fro~
IS Si~mG and p-chlorophenylal~nine (pCPA) w~ obtalned ~ro~
Rsso~rch Biochemlc~ls, Inc. ~Natick, ~). RPNI 16~0 medl~, f~t~l c~lf ~Qru~ (FCS), ~odium pyruva~e ~nd ~odlum glutam~te wer~ from GI~CO. FlAt-bottom ~ulti-well tl~ue cul~ure dishe were f~om Becton ~icklnson or from Co~t~r.
Solvent~ ~or HP~C war~ from Aldr~ch ~nd were HPLC grade.
Cell Culture~. Ths hum~n c~rvlca1 earclnoma cell llnQ, ~-180, was obtained frorn A~eric~n Typa Cultur~
Collection ~Roc~vllla, MD) ~nd w~a o~ln~alnod ln tl~sue cultur~ fl~ks in RP~I 16~0 medlu~ wlth 10~ FCS wlthout ~ntibiotic~ in a hu~idlfled atmo~phera o~ 5~ C02 ln Rlr ~t 37~.
L31~t~ tL~lt~9~L~ E-180 cel1~ w~r* pl~ted ln co~pl~te ~dl~ ~t lx105 cell~/ml, 100 ~1/w~11 ln 96 w~ll pl2teo ~nd ~r~ cultur~d in the px~nc~ or ~baenc~ o~
~rylng ~mount~ of IFN for 3 d. On th~ thlrd d~y, c~lture0 were pul~ed wlth 1 ~Ci 3~-TdR for 5 hr ~nd wera harv~ted on fi1tYr p~per. Culture~ w~ro p~rfermed ln dupll~a~e a minlmum o~ ~hree tl~e~ wl~h sl~llar re~ult~;
mnny were rep~rted a~ po~ltive ~on~rol3 for sub~equen~
~5 exp~rl~n~s. Dupllc~tes wer~ wlthln 10% of ~ch oth~r.
Incorporatlon of 3H-TdR wa~ dete~mlned wlth a liquld ~cintlllatlon ~pec~rometer.

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WO92~015 2 o ~ PCT/US9~ 6 E traction and analysls _of try and 5HT. After various treatments for var1Ou~ periods of time a~ deqcribed in the text, cells and med~a were ~eparated and mlxed with ethanol to achieve a flnal concen~ra~ion of 70%. Samples ~ere stored at 0-4C
overnight before removing preclp~tates and cell debris by centrlfugation. Samples were concentrated to dryness with a Speed-vac and dis~olved in 20 ~l of buffer A
b~for analy~ by HPh~ (Hewlet-PacXard) (13). The HPLC
ln~trument was equipped with a fluorescence detector (Gllson) ~ith a 280 nm excltatlon filter and a 360 nm emlq~ion filter. The two mobile pha~es con~i~ted of A;
50 nM triethylamine adjusted to pH 3.0 with pho~phoric ~cld ~nd ~: buf~er A wlth 40~ methanol. The gradlent 15 wa3 line~r from lO0~ A to 100% ~ over 25 mln. The retention times of known standards were as follows: SHT, 3.50 min; 5~0H-tryptopAan, 4.77 min; tryptophan, 9.ll mln; 5-OH-~ndoleacetlc acld, 12.5l min; and ~elatonin, 2l.69 mln. Identlf~catlon of peaks ~n cell extracts wlth Xnown tandard~ wa~ achieved by mixlng known amount_ of ~tandard3 wlth cell extract~ and ~howing identity of peak~ on the chromatograms. Recovery of tryptophan and :~
5~T from c~ll extracts was greater ~han 90~ as determlned by mixing known amoun~s of standard~ wlth cell before inltiatlng the extractlon procedure. Experiments quantitating level~ of 5~T and ~ryptophan were performed :~
three ti~0~ e~ch with ~imllar re3ults.
~ev~r~al of IFN-mediated qrowth inhlbitlon_b~ tr~ptophan or 5Htp. Inhibltlon of cell prollferatlon by IFN ls cau~ed by lo ~ of ~ryptophan catalyzed by indolaamlne 2,3-dloxygenA~e in certa~n tumor cell llne4. Addltlon of exogenou3 tryptoph~n reduce~ lnhibition of cell prollferatlon by IFN (Ref 6-9 and Flgure l).
Tryptophan, an es~entlal amlno acld, ls required for protein ~ynthesl~ and for synthe~1~ of the neurotran~mittars, 5~T and melatonln. R~ults ln Flgure 1 -~how that tryptophan, an w~ll as SH p, reduces IFN

SUBSTITIF~ E SHE~ `~

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, ' I` `' , - wo 92/04015 2 0 ~ pcr/us9~ 76 4~1 -madi~ted inhlbition of c811 prollf~r~tlon. On a molnr ba~ 5~Jcp i~ gr~ater th~n 5û0-fold ~or~ o~fectlve th~n coph~n ~t Lnhlbltlng IFN activity ag~lnst ME-180 cells (Figure 17B). 5Htp i8 a precur~or for 5HT
5 syn'chç~si ~nd i~ llot u~ed ~or protein synt~e 18. ThLs rais~s the po~ibility that lnhlbltiorl of SHT ~ynth~
may be an import~nt reslalt oiE tryptoph~n depletion ln cult1lr~ of cell8 eXpO5Qd to IFPlo Results in Flgure 2 compsre IFN -medlated inhibltloll o~ cell prolif~ratlon.
10 Optlmal re~ult~ were obt~insd wh~n compound~ wer~ added on d~y one ~nd two of tha thre~ d~y culture. Und~r th~se condltlon~ only 5Htp blocked IFN ac~lvlty. Oth*r 5Htp ~slet~bollte~, 5~'r, melatonln, N-Ac-5HT or 5-OH-indlole~c~tlc acid, nl o products o~ 5Htp me~aboll~
15 f~ilad to block I~ -m~diated inhlbitlon o~ cell prollf~r~tisn.

and ~r. Ta3c~n together, the abc~v~ re~ult~ r~i~e the posslblllty th2lt 10~8 of 'cryptoph~n in cultur~3 medi~
re~ult~ ln 109~ of SHtp or it~ metsbollt~R, such at 5HT, and ~hlæ leada to lnh~bltion of c:ell prolifer~tlorl.
I~lgure 3 shows the loYel of lntrac~llul~r tryptophan and 5HT l r~ a f t~r vr~rying ip~rlods o~ tlnie l rl cul tur~
wlth IFN. CE311~ cont~lned bol:h tryptophan and 5F~r; bo~h 25 W~r~ lo~t upon cultur~ wlth IFN. SHtp wa~ undet~ctable ln s:211 ~xtract~. Synth~01~ o~ 51~tp 1~ t~l~ r~t0 ll~iting 8tGp in 5H~ syTIthe~ls ~nd 5H~p doe~ not slanerally a~::cu~ulat~ ln cQlls.

30 ere~ or c~ w~r~ cultured with inhlbltory amounta o~ IFN ln ~ch~ pr~erlce or ~b~enoe of 5Htp to detnrmlne i~ 5~tp ra~torQd 5HT lev~ T~bl~ 5 ) .
Treel~cmea~t wlth IFN re ulted irl ~ d~:r~a~ ~n 5HT to unde~sct~bln 1eYe1~. In the preel~nc~B o~ 5Htp, level~ of 3g 5HT were ~alnt~ ed. In ~ai:~c, culture~ o c~ wl~h I~N
and 5Htp cont~ln~d f our- old h~ghær lev~l~ o~ 5HT th~n untreatad cul'cure~ Ad~l~lon ~f 5H~p ~l~o rastor~d cell proll~eratlon ln thQse culture~.

SvB!~ U~E~

w~ g2~040ls 2 ~ 9 ~ PCT/VS91/~176 . ~5 5Htp doe~ not prevent he lo ~ of tryptophan from media Two posqible explanation~ for the ability of 5Htp to prevent IFN-mediated inhlbition of cell prollferation are th~t it prevents the lo~5 of tryptophan from med~a or that it restores intracellular SHT le~els. Re~ult~ in Flgure 4 ~how that 5Htp doe~ not lnhlbit lo95 of extracellular tryptophan in cultures of cell~ expo~ed to IFN. Control cultures of ME-l80 oellY
did not con~ume slgnlfic~nt a~ounts of tryptophan in media over the 72 hr culture pe~iod. By contra~t, cultures o~ c~ treated wlth IFN coneum2d 50% of tryp~ophan ~n 24 hr and Y5% wlthin 48 hr. The ra~e of lo~ of tryp~ophan in medla for IFN treated culture~ wa~
not inhiblted by additlon of 5Htp. Under the~e condLtlon~, 1QVe1~ o SHT and cell prollferation were re~tored to control value~. :
Inhi~ition vf cell proliferation by low concentr~tlon~ of tryptoph~n_i~ r~duced bY _5Ht~ Prollferation o tumor cells in the pre~ence of varying ~oncentrat1ons of zo meth1onlne or l~uclne re~che~ maximum rate~ between 10-20 ~M amino acld. By contra~t, prollfer~tion of tumor cells ln the presence of varying concentr~ion~ o~
tryptoph~n reaches maxl~um ra1:e~ between 50-lO0 ~M amino acld (6~. Flgure 5 ~omp~res proliferatlon o~ ME-l80 cell~ ln the pr~ence of varylng concen~rations of tryptoph~n with or wlthout 3 luM SHtp. In th~ ab~ence of 5Htp3 m~xlmum prollfer~tion wa3 obtained with 50 ~M
tryptopha~. In the pr~ence of 5Htp, maxlmum proliferation wa~ obtaln~d wlth lO ~M t~yptophan. Under there conditlon~, addltlon of S~tp dld not a~fect the ability of ce11~ ~o lncorporate 3H-tryptophan into proteina. Thi~ lndlcate~ that 5Htp is not converted to tryptophan by ME-180 c~ and used for proteln YyntheRls (not ~hown).
Inhibltlon of_5~T 3ynthasi~ inhibits pro1ifer~tion by ME-180 ce11s. The above result~ 3ugge~t that lowering intrac~llular 5HT should lnhlblt prollferatlon of M~ 0 SUE3STlrl)TE~ SHEE~T

, 1;-4~ ' -W~:3 92J~15 PC3/llS9l/06~76 ce11æ. ThereforeO IIE~1870 csll~ wera cultur~d wlth pCPA, a speciic lnhlb1tor of tryptoph~ll hydro~ylase, to de~ermine if ~hl~ re~ulted ln 10~3 o intraca11u1Ar 5HT
and inhi3:~ition of cell prolif~3ratlon. The result~ are shown ln Talbl~ 6. Culture of ME-180 cell~ for 48 hr wlth pCPA resultsd in 10~B of t~r and lnhibltion of cell proliPer~tion but dld not C~U9~ a 108~3 ln extr~collular tryptoph~n. Los~ o~ 5~r and lnhibition of oell prolifQr~tion wa~ prev~nted by addltlon of ~Htp. Thu~, d~pletion o~ 5Hq! by two indep~ndent mech~ni~
lndoleultln~ 2, 3 dioxygen~0 cat~lzy~3d oxid~ion . of tryptophan or lnhibition of ~ryptoph~n hydro~sylase, re~ultad ln inhibition of tumor ceLl prollfara~ion. In both ln~t2mces, tumor cell prollferation wa~ recsv~r~d by restorlng 5HT levels with 5~1tp.
Additional ~cumor c~ll line~, a~ normal dlplold cell~ ,. wer~ e3caminad to de~erml ne lf thoy ~l~o cont~lned SHT, whether pCP~ lower~d intracellul~r 5HT
concen~ratlons and inhlbi~ed prolifer~ lon and whether 29 51~tp would ro3tore 5~r level~ ~nd r~Qrse inhlbition of prollfara~lon. The~e resul~c~ are ~hown in Table 7~ All tumor cell lln~ 2x~m1n~d con~ined 5H~r and prollfer~tlon of ~o~ur of f ive ~11 llne~l wa~ inhlblted by pCPA.
Inhibltion o prol~ f~r~tion by, ~umor ~:oll lln~ W219 2S largely ~ver~od by ~ddltlon of 5Htp. Thi~ suyge~ts ~chat ~ny tumor c~ n~y r~quire 5~r or ~netnbollt~ of 5~r for prolleratlon. ~y ~on~ra~, prollf~r~tl3n of ral dl~rent typoa of normAl dlplo~ Cell8 W~5 not inhlblt~d by pCPA.

. .

3~

WO92/~01~ PCT~US91/~176 . . ~7 Table 5 5Htp re~tore~ 5HT levels and proli~eratlon ln IFN-traated ME-180 cells.

Intracellular Levels~

Culture SHT Proliferatlon Conditlon~ (pmol/mg protein) (3H-TdR) ME-180 Cells18 98,069 ~ 1025 +IF~ l l9,7a9 ~ 874 +IFN +5Htp 80 99,672 + 1984 ~5~ p 32 96,331 + 1593 _ _ _ 15 ~ME-180 cell culture~ wer~ for ~hree day~ and wera ;:~
inltlated at 2xlO cell~tml. IFN (300 ~/ml) was added at lnltiat1On, and 5H~p (3 ~M final concentration3 wa3 added ~ve~y day. Sample~ were harvested ~nd analyzed for 5HT by HPLCo Table 6 Inhlbltion of tr~ptophan hydroxyla~e depressos 5HT lev~l~ and lnhib~t~ cell Prollferation.

Culturs Intra- Ex~ra- Pro-Co~dltlons oellular~ cellular llf~r-atlon 5HT Tryp (3~-~dR~
(pmol/mg protein) (um) ~
ME-l80 C~ 16.5 12 44443 + 2244 +pCPA 3.l 25 l6642 + 1723 +pCPA
+SH~p 37.l n.d. 42221 ~ 4128 3~ ~SHtp 44.6 n.d. 42982 ~ 2529 -- .

..
: . ~ , ., :, . :
; :/;, ".

. , :,: -wo 92~04015 2 a 9 ~ b ~ ~ ~/US91~C6176 0 e~lla ware cultursd for 4B hr in the presQnc of 2~9 pCP~ andJor l0 uM 5Htp. C~l~ur~ medla er c~lls wera harv~sted and analyzed fox 'cryp'coph~n and 5BT by HPLC.

Table 7 Inhib1tion of SHT ~ynthe~ nhlblts prolifer~tion b~y tumor cell~.
Proll f ex~tlc~n C~ll 5t~'r ( cpmxl000 ) 10 E~opulatlcn* Content (mol/mg protein) Control ~pCPA +pCPA
+5 -OH-l:ryp Tr n~ormlad Csll Lln ME-181~ 13 + 244.7 12.6 4232 ~29 24 * 356.7 14.1 ~7.4 Jurkat 31 + 385.6 24.0 40.2 ~t:lLT-4 22 + 534.3 36.5 31.5 :-U937 39 ~ 6l~a.0 71.8 lS5.6 E3T20 N.D. 29.0 6.5 29.7 20 Norm~ll Diplold Cells T cell 13la~ts 25 + 2 45.9 37.1 41.4 Fibroblast~ 29 ~ 4 l0 . 3 9 . 3l0 .2 ~ lunS~ ) Flbroblast~ N.D. 4.B 3.9 4.5 ( ~y~ovlal ) Endoth~l~al tl . D O _ 7 . _~ ~
*Dl~e~en~c hu~an c0ll llne~ wer~ obt~ln~d ~rom .~l~CC.
Th~lr culture c:ondlt.ion~ h~v~ b~en d~crlb~d ( 6 ) . Sera WeE~ dlalyzed before uae to r~olov~ 5HT~ 180 (Cervic~l 30 carclrlomel), BT20 (breaat carc1no~), HT29 (colon adenocarclnoma), U937 (hl~tocytic l~mpho~, monocyt~
linea9e), Jurk~ (T cell ly~phom~), MOLT-4 (acut~
ly~phobla ~ic leuk~ ), endothellal cello (pri~ry cultura~ ynov~l f~brobla~t~ (prlm~y cultur~), lung flbroblastq (~RC-5; long ~er~ lln~) or prlm~ry culture~
of T c~ll blasts were cul~ur~d for 48 hr.

SIJBSTITI)TE S;~

WO92/~ 2 D 9 ~ PCT/US9,/~l76 Serotonin was found to be present in n20plastic or tumor cell~ and the 5HT2-like re~eptor wa~ found to be pre3ent on tumor cells. It ha3 been demonstrated that like activated T cells, tumor cell prollferation can be regulated by ~erotonin receptor agonists and ~ntagonists as well as by i~hibit1On of serotonin synthe~ls.
In nervouR tl~sue, serotonin (5-hydroxytryptamlne, 5HT) i8 ~ynthe~l~ed by hydroxylation and decarboxylatlon of tryptophan and ~ stored in granule~ ~35-36). It i5 rele~sed in re~pon~e to appropriate stimull and blnd~ to speciflc receptors on neighboring c~ actlva~lng ~econd me~seng~r pathw~y~ ( 37-39 ) . The rate llmltlng step ln erotonin ~ynthesi~ i~ th~ l~vel of tryptophan hydroxyl~s~ activity. A 8pecific enzyme lnhibltor of tryptoph~n hydroxyla3e, p-chloroph~nylalanlne (pCPA), ha~
been employed to deplets serotonin level~ within cell8 ~40-4ll. Human cervical carcinoma cell~ (ME-180) were ~rea~ed with pCPA ~or 4~ hr ln the pre~ence or sb~enoe of S-OH-tryptoph~n and analyzed for 5~T content or for rate~
Of cell proliferation. Cultu.red cells contalned 13 + 2 pmol/mg protein 5HT whlch wa~ reduced to 4 ~ 1 pmol/mg proteln SBT af~er treatmont w:Lth pCPA. Slmilarly, med~a from ~ultured ~lls contained 44 nM 5H~ which was reduced to 10 nM by culturlng wlth pCP~. Addl~lon of 5-OH-tryptophan re~tored 5HT level~ to that o~ control cell~. Tr~atmen~ of ME-180 cell~ with pCPA al~o reduce~
H-TdR incorporatlon by 70~. Inh1bltion wa~ al~o largely rever~ed ~y 5-OH-tryptophan tTabl~ VIII). Several add1~1Onal tumor cell~ lln29 and no~mal dlploid cell llne~ were al90 an~lyzed for 5~T content and cultured for 48 hr ln the presencs of pCPA ~o reduce 5HT levelR (table IX). Th~ concentrat1On of SHT in cell ex~r3ct~ ra~ged b~w~en 10-40 pmol/mg prot~ln. 5-O~-tryptophan, the precur~of of 5HT~ wa~ not detectable in c~ll extract~. :
Culturlng c~lls ln the presence of pCP~ lnhibited cell prollferatlon in 5 of ~ tumor cell l1nos ~xamined but dld not significantly affec~ proliferation o nor~.al diploid ~.I. 'QS I I ~ E~T

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wo 92/0401~ ~ Pt~r/VS91/06176 cell~. Addltion of 5-OH-~ryptophan to coll cultura~
tr~t~d wlSh pCP~ larg~ly rever~ed inhlbltlon of prollf~ration ln tumor c811 cultures but dld not ~ppr~clably ~ff~ct prolleration in culture~ of s~ormal 5 dlploid c~ll Thus, dopletion of 5HT ra~ulted in inhibition of tumor cell proliferatlon but not normal cell prolifera~ en ~d restor~tion of sHT le~el~ by ~ddltion of 5-0~-tryptoph~h r~e~tored proli~r~t~on ln the23Q s~
S~rotorlln ~ccumulated ln medla ~t concesltlation~
which can tr~n~duc6~ s~cond massen~!aer path~ely~ through 5H~
r~c:eptor~ ~ 37-38 ) ~nd 5H'r receptor~ which tran~du~e ~cond me~ng~r pathway~ ( ino~i~ol phosphate rele~ e and chang~ ln intr~cellul~r C~ ) h~ve been :Identlflad on 15 Jurkat ccll~ but no'c on Molt 4 cell~ (42). Therefsre a nu~ber of 5HT recepeor agonls~ and ~nt~gonl~t~ wer~
te~ted to determln~ if th~y would ~l~o inhlblt tumor cell prol~fer2lt~0n in a 5H~r rev~r~lbl~ m~nn~r. Of tho~e t~ed, rit~ns~rln, ~ 5~11r2 r~ceptor 2r~t~gonl~t 40, 20 inhlb~ted tu~or c811 prollferation by 70-g5~ (fl~e of ~ix llne~ ~0stad) ~It conc~ntratlon betwa3n 10-50 ,u~ ~ut did not lnhiblt proliY~rAt~on by normal dlplold coll~. Lower concqntratlon~ of rltAn~erln ( <10 ~JM) did no~ inhlbit prollferatlon by tuunor c~ . Addltion o~ 5H~r p~rtlally 25 r~VerJed th~ lnhlblton ef ~ec~1~ of rltan~erln (T~ble X) .
Proll~era'clon by the one tu~or c311 llne, ~olt-4, whioh was not lnlalblt~ by pCP~, W1!18 al~o not inhiblted by rltanserin. Addltlonally~ whlle pCI~ lo~ered conc~n~ratlons o 5HIr ln oQll extr~cta, rlt~nsqrln 30 actu~lly lnorea~0d 5HT content ln ~çlll extr~c'c~. For example, extr~ct~ fro~ ME-lB0 s:9118 contalned 15.5 ol/mg protaln of 5HT whlle ~xtr~l:ts from cu1tur~s tr~ated with rltanserln con~ln~d 58 . 8 pmol/mg pro~eln of 5HT. Thus, bo~h lnhibitors o~ sHT ~ynthR~ as wall as 35 3HT r~csptor antagoni~ts ls~hlblted prollfer~lon by tumor c~ but not by normal dlploid cell~ A number o~
llgand~ whlch blnd 5~T r~ptorg w~3r~ ~es~Ged f or thelr SU13STITUTE SHE~T

~ ; .
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.

WO92/~ 2 ~ PCTt~S91/~176 ` 51 ablllty to prevent ritanserln-mediated inhibltlon of tumor cell prollferatlon. The~e ~e~ultR are ~hown in figure 14. ~etan~erin and pelanserin, alsD 5HT2 receptor an~gonlst~, mlan~erin, ~ 5HTlc receptor antagoniet and 8-O~-DPAT and propranolol, a 5HTl raceptor agonlst and antago~lst, respectively, partially prevented lnhibitlon of prolier~tion by rltan~erin ln a concentrat~on depend~nt m~nner. These data which larg~ly parallel binding data on Jurket cells hava failad to cla~eify these SHT receptor~ into on~ of the w811 characterized 5HT raceptor 3~btype (42). Interestingly, ritan3~rin, ketan~erln and pelanaerln are all 5H~2 r~ceptor antagonists with simllar phaxm~cologlcal and blochemlcal propertie but rlt3nserln inhlbit~ tumor c~ll lS prollfer~tion and katan~exln and pelanserln provent inhibltion by ritan~erin. This i~ not to lmply th~t these data are sufflcient to dafina a new 5HT receptor subtype, Analysl~ o these r~ceptor~ in much greatQr det~ll will be required te compl~tely de~ine these SHT
receptor typB8. Taken to~ether the3a data rai~e the po~sibillty th~t 5HT ~nd 5HT r~cQptors may be required for prollfaration of certain t~mor cell lin~ in tl~sue culture. Soma of ~he charactaristlc~ of this prollferatlon is consl~tent wlth an ~utocrlne pa~hway of ~5 growth factor actlon wher~ a cell relea~e~ its own growth f~c~or~ whlch blnd to c~ ur~ace receptor8 and ~tlmul~te cell function (43~4). ~ata presanted here ~how that 5H~ i produced by tu~or cell~ ~nd that it i5 raqulr~d for prol~feratlGn. Studies with llgand3 known to bind to 5HT recep~or~ support the notlon that 5HT may act ~hrough c~ urface receptors to s~mulate prollf~r~tion. In thl~ regard lt ~8 important to note that 5HT h~s be~n ghown to be a grow~h factox for both qule~cent flbrobla~t~ a~ well ~ smooth mu3cle cells (45~47). In tha~e lnstance3 exog~nous a8 opposed to endogenous 5HT was employed to stimulate DNA ~y~the~
The3e re8ult~ are conqlstent with m~ny current notion~ of SUBSTITU I E S~tE~T

, : . . .; , . :
. ~ :
... .. . .

WO ~2J04015 2 0 ~ PCll~US~l/0617S

tumorlgenQ~is wh~ra normal diploid o~ may becomQ
tr~nsfQrmecl by ac~ulr~ng the ability to produc~ th21r own growth factor or ~row~h fac~or rQc:~ptors (48-49).
Produc~lon oi' 5HT ~nd 5H~ receptor~ may ba one way which S norm~l oE~118 aequ1re a pe~manent transformed phenotypa.
Whether tumor oallR ~yntheslze serotonln and requ~re ~rotonln for proli~sr~tlQn ln vlvo is ~n lmportant qua~tion whlch ls un~n~w~rod.

0Cultur0~ S~T Prollferatlon lntr~callul~r extracellul~r ~ cpm) (pmol/mg protein (n~q) 15 ~!IE-180 Cells13 +2 44 ~3 44,700 +5~0 +pC?A 4 +1 lD +2 12, 600 +1220 ~pCPAtS C)H-tryp 16 +4 56 ~ 420200 ~4220 __ Ta~le 12 Inhlbltlon of 5~r aynthe~iEI lnhlblt~ prollfer~tlon by tumor cells but not by norm,~l dlplold c:ell~ Dlf ferent hum~n cell llneE~ were obt~ined from ATCC. Their cultur~
oonditlon~ h~v~ b~an d3acrib~d ( 50 ~ 0 . ~er~ were dialyzed 25 }7~or~ u#e to remo~2 5HT . ME180 l c~rvlcal carcino~a ), ~T20 (br~lst carclno~), ~r29 (colon ~ano~ar~ino~) y U937 (hlstocy~ic lymphom~l, monc~ te llne~g~) O Jurkat (T cQll ly~pho~, MOLT-4 ( acute lyraphoblastic l~uke~~
~ndoth011al coll~ ( prlloary cultur~s ), ~ynovial 30 fibrobln~t~ ~srlll3asy culture~), luny flbrobl~t~ ~IRC-5;
long t~rm llne) or prl~ary culture~ of T cell bla~t~ were cultur~d for 48 hr. O~h~r condlt~ on~ were a~ ln Ta~le SU8ST~ 5HE~

wo 92/04015 ~ r/US91tO5176 5HT Csntent Prolif eration Cell (pmol/mg (cpm x lOOQ) Populatlon Pro'celn~
Corltrol ~pCPA +pCPA
+pCPA ~5-0~-tryp Tran~_ormed Cell Llnes ME180 13 +24~ . 7 12 . 6 42 .
HT29 24 ~356 . 7 l4 .1 47 . 4 Jurkat 31 +385 . 6 24 . 0 40 . 2 MOLT~4 22 +534 . 3 36 . 5 31. 5 1~937 39 +6l9d, . 07 1 . û155 . 6 BT20 N.D. 29.0 6.5 2g.7 Normal Diploid Cells T cell Blas~cs 25 +245.9 37.1 41.4 Flbro-bl~st~ 29 +410.3 9.3 l0.2 ( lung ~
Flbro-bl~st~N . D . 4 . 8 3 . 9 4 . 5 ( 8yl'10vi~
Endothellal N.D. 7.5 7.9 7.5 '`1'5S

: . ~ .. . ..

WO 92~û4B~ PCIJUS91/06176 5~

Table 13 Inhibition of prol~feration of tumor ~119 by a SHT
r~Qptor antagonlst and ra-tQr~al by 5~T. ~umor c~ll l~n~ and norm2l1 diploid cells WQa:e clllturad ~or 4a hr~ ~ n ~ch~ pr~3en~e or ab~ance of 30 7uM retan erln ( a 5HT2 receptor antagoni~t whlch 1~ ~CtiVQ ag~in6t a 5HT2 ~ite a~ concen~ra~ion of 1-10 nM) with or w1 thout 1 y29 5;~T. Addltlon of s}~r dld not af f~ct control proli$Qratlorl. Culture~ were ~et up ln trlpllca~e and thQ ~tandard error wa~ th~n 109t (d~t~ not 3hown).

C~ll Population 3H-TdR Incorpor~tlon ( cpmx lDOO ~
Con~rol +rl~anserin ~rlt~ ln Tr~nsformed Call Line~
. .
ME180 4û.7 2.2 ~1.4 : Jurlcat: 75.8 0.4 51,6 HT29 159 . 525 . 3 58 . 4 ; U937 6~.g 1502 45.8 0I.~-4 34.5 32.S 55.5 ~T20 27.3 3.1 19.1 Normal Dlploid C~
2S Flbroblast~ 10 . 3 8 . 7 8 . 0 T Cell Bla~-t~ 43.9 40.5 45-5 F1 brD~la~tlY
( 8yrl0~r~L81 ) 10 - 9 14 . 4 :' El~doth~llsl cells 7.4 7-4 ~-4 In &~dl~on ~o agonl~t~ and antagoniLst~ that clm be che~lcally synth~zed, it 1~ w~khin the seope of thl~ lnv~ntlon to genernu~ mimot~p~ ~nd/or antlbodle~ ~o the 5~T2-llke receptor.

'll'LIT~ ~IF~T

WO 92J0401~5 2 ~ PCJ'tU~;91/06176 Th~ 1dantlflcation of the novel 5HT2-lika recep~or allow~ for the generatlon of antibodies to the r~ceptor.
The antibodl~ have application as a probe for re~ear~h and a8 a ther~peutic. Technlques for generatlng antibodies are well known in th~ art. For reference to mQthods ~ Fundamen~al Immunology, Second Edition, ~upra Chapter 12. It i~ recognlæed that a plurallty of "types"
of antibodie~ can be generated including polyclonal, monoGlonal, chimeric, humani~ed and human. What the~e antib4dl~s "typ~" h~e in common i.~ ~h~ ability to recognize ~n ep~OpQ OX ep~tope speclflc to the novel 5H~2-likQ receptor~
~ lth ~he ldentificatlvn of the novel 5HT~ e receptor, purlfiad receptor c~n be u~ed a~ a prob~ for lS ~creening llbrarie0 of random peptide sequenc~ to identlfy peptide that 8p~ i flcally blnd ~o protein~.
Alternatlvely, antibodies gen~rated aga$n~ the purif1ed roceptor c~n be used as a probe. ~or technique~ on constructlng a llbr~y of p~ptldes, see Cwirla et ~1., Peptid~a en p~ge: A va t llbr~ry o~ pep~ides ~or lden~lfying llgand~, Proc.N~ . AcDd. Sci~ USA, Vol. 87, 63~8-6382, August 19~0. For methods of 3cr~ening seQ
Devlln Qt ~1., Random P~ptide Llbraries: A gourcQ of Speci~ic Proteln Blndlng Molscule~, Sclencs, Vol. 249, 404-406, 27 3uly 1990 ~nd S~ott and Smlth, 5earching for Pep~lde ~lgand~ wlth an Epltope Llbrary Science, Vol.
249, 386-390, 27 July 1990.

3~ :

~;IJE3STlTlJTE SHEr wo ~ 2 0 ~ p~/US9l,06,76 BIBLI~3R~PHY
1. Pl~ut, M. 1987. ArmuO Rev. Immunol. 5s62l.
2. Hen~on, P.M. 1970. Mechani~ms of ~eloa~e of eonstitue31ts from rabbit platelet~ by antiçlen-~ntlbody 5 oompl~xe~ asld comple~Dent . J. Imsnunol . 105 ~ 476 .
3. Es~man, W.~3. 197û. S~ro~onin ln tl~sue~ and fluid~.
In Serotonln in ~lealth ~nd Dlsea~. W.B. Es~m~n, ed.
Sp~ctrum, New York. p. 15.
4. 0180n, P.S., U. L~uns~vi~t, and S. E. Bergsntz. 1974.
10 An~ysi~ of pl~tel~t, red cell and flbrln cont~nt ln exp~rimelltal art~rlal ~nd venou~ thro~bi. Thro~. Re~.
5~
5 . Sternberg , E . M ., H . J . Wedner ~ M . R . Leun~ and C . W .
Parker. 1997~ l~ffect of ~rotc?nln (5~ ndl o~her 15 mono~mlne~ on murln~? mac:rophage~: ~qodul~tiorl of Interferorl-gamm~l lnduced phagocyto3ig. J. Immunol. 13~:
4360.

6. Sternberg, E.M., J. Tri~l ~nd C:.~. Park~r. 1986.
EffQ of ~srotonin on ~urlne m~crophJ~g~ uppra~Blon of I~ e~pre3~ion by ~erotonln and it~ rever~al by 5~HT2 ~oroton~rglc r~ceptor ~ntagonlst~. J. Immunol. 137: 276.

7~ Hell~trand, K., and ~. ~er~od3~0n. 1987. Role of serotonln ln the regul~lon of hum~n n~tural kill~r cell cyto~oxlci~y. J. Immunol. 139: 86g.
25 8. Choquet, D., ~nd H. ~orn. 1988. Dual e~ect~ of ~eroton~n on a volthge-gatod conductance in ly~pho~yt~
Proc. Natl. Acdd. Scl. U~A. 85: 4557.
9. Bonn~t, M. 9 G. La~plnat~, and C. ~urtln. 1984.
Hls~a~ln~ and ~erotonin suppr~ion of ly3pho~yte ra~pon~e to phytohemaglutlnln and antlgen. Cell.
Im~unol. 83: ~80.
10. ~or~hon, ~.~., P.W. A~ken~, and ~.D. ~or~hon, 1975.
Requlrement for vasoac~lve amlnes for productlon of del~yed-typQ hyper~ensitivity ~kin reactlons. J. E~p.
35 Med- 142: 732.
11. Amei~n, J.- C., R. M~ade, and P.W. Ask~n~e. 1989.
- A new interpratation of the involvem~n~ of ~arotonln ln âl~BSTi I lf ~

,: :
, .

w~g2,~i5 ~ ~ 9 ~ PCT/US91t~76 delayed-typ2 hypersensltivlty: S~rotonill-2 receptor ant~gonists lnhibit con~act ~nsitivi~y by an ~ffect of T
c~ . J. Immunol. 1989:. 3171.
12. Schmldt, A.W., ~nd S.J. Peroutka. 1989~
5-hydroxytrypta~ln~ r~cep~or familles. F~b J. 3:
2242.
13. Peroutka, S.J. 1~80 5-~ydroxy~ryptamine recQptor ~ubtypes. Ann. Re~. Neuro~cl. 11: 45.
14. Kadan, M.J., A.M. ~rohn, M.J. Ev~n~, ~.L. Waltz, and P.R. Hartlg~ 1984. Ch~xacterlzatlon o 125I-lysQrgic ac~d dlethyl~mlde binding to ~rotonin r~ceptor~ ln r~t frontal cortex. J. Neuroche~. 43: 601.
15. Morettl-Ro~a~, I., E.G. Ezrfiilson, L. ~irnbaumQr, M.~. Entm~n, and A.J. ~arber. 1983. Seroton~rgic and adr2n~rglc regul~tion of ~eletal mu~cle ln the r~t: II.
The ug~ of l125I] lodolyserglc acid ~lethyl~mlne ~nd ~125I3 lodopindolol ~ probe~ o ~arcola~mal receptor function ~nd sp~cificlty. J. ~lol. Chem. 258: 124g9.
16. R~binovltch, P.S., C.H. Jun~, A. Gro~ n, and J.A.
Ledbett~r. 198fi. Het~rogeneity among T cells in intracellul~r freo calcium re~ponses aftsr mitogen ~timul~tlon with PHA or ~nti-CD3. Slmultaneous u~e of IND0-1 and 1mmunofluor~cence wlth flow cytom~try. J.
Im~unol. 137: 952.
17. Xelley, X.A. 1989. A ~pl~ statlon ~odl~icatlon provldlng on~llne re~gont alddltion ~nd reduced sample translt ~lme for ~low cytometers. Cyto~stry. 10 (ln press).
18. ~ryn~ewlcz, ~ . Po~nle ~nd R.Y. T~i~n. 1985. A
~w g~n~ratlon of Ca~+ lndlcator~ with gro~tly impro~d ~ luore~cence proper~l~0. J. ~lol. Chem. ~60: 3440.
19. ~mith, C.D., and R. Snyderman. 1987. Guanln~
nucleotide regul~ory pro~sin~ in receptor-mediated polypho~phoino~l~lde hydrolysl3 in human leukocyt~
Method~ in Enzymology. 141s 261.
20. Roso~f, P.M., N. S~v~ge ~nd C.A. Dinarello, 1988.
Intarleukin-l stlmul~t3~ dla~ylglycerol production in T

SUB$TITIJTE S~E~ET

: ~ -.
.

,~ ~

wo 92~ 2 ~ 9 ~ PC~/U~9~ 76 lymphocytes by a novel meoh~nism. Cell. 54: 73.
21. W~1~38~ A., R.L. Wi~ooll and J. Stobo. l984. The rola of T3 Qusfac~ molecule~ in the act~vation of humzln T
call~: a two-~imulus raquireDI~nt for IL-2 production 5 ref lect~ event~ oc~urrirlg a~ a pretran lat 1 onal level . J .
- Immunol . 133: 1~3 .
22. B~:rridge, M.J. 1987. . Ino~its~ ri~pho~phat~ and dia~ylglycerol: ~wo lnt~rac~clng second m~ nger~. Ann.
Rev . Blochom . 56: 15 9 .
23. Fargln, ~., J.~. R~ymond~ M.J. Lohs~ . Xobll;ca, M.~:. Caron, and R.J. L~ft~owl~z. l988. The genomic ~lone G-21 which resembleq ~ beta-adrenerglc receptclr sequ~rlce encosies ~he 5-HTla receptor. Natur~ (London) 335- 358.
24~ Jullus, D., A.B. MacD~ot~, R. ~x21 and T. Je~el.
19E18. ~olecular charac arizatiorl of a ~uncti-lal c3N~
encodlng the ~rotonin lc recaptor. Sclenc0 2~1: 558.
25. Prltche'ct, D.B. r A.W. ~nch, M. Wozny, o. T~leb, R.
D~l Toso, J.C. Shlh and P.H. Sesburg~ 1988. St~ucture and functional axpre3~lon of cloned rat ~ero~nin 5HT2 recep~or. E~B0. J. 7: 4135.
26. DeVlvo, M., and S. Maay~nl. 1986. ~haracterization of the 5HT1a receptor-medi~t~d inhibltlon of forskolln-~imulated adanyl~t:~ cycl~Ye actl~lty in guine~
pig ~nd rat hlppoc~mpal m~br~na~. J. Pharm~col. Exp.
~h~r. 238: 248.
27. Conn, P.J., ~nd E. S~nders-Bu0h. 198~.
~ero~onln-~tl~ula~ed phospholno~ltid~ turnover:
medlAtlon by t~ 52 blnding slte in ra~ c~rebr~l cor~ex but not ln sub~ortlc~l reglon~. J. ~ha~. ~nd Exp.
Th~rAp. 234: l95.
28. Conn, P.~ nd E. San~ers-Bu~h. l98~. Selectivo 5HT-2 ~nt~gonlst~ inhibl~ serotonin stlmulsted phosphotldyl lnosltol met~bo~ls~ ln cer~br~l cortex.
~europh~rm. 23: 993.
29. aerrld~e, M.J., and R. F. Irvin2. l9~9 Ino~i~ol pho~phat~s and ~ell sign~ling. ~kure. 34l: l97.
30. Zellar, J., ~. Wels~bsr~h, ~. Baruth, H. Ml~lke and ~;~JF;;~ I ~T~ r~

wo 92/~15 2 0 ~ o n PCT/US91/~176 Ht Deichar. 1983. Serotonin content of platelat~ in lnf la~atory rheumatic disa~e3: Corr~lation w~ th cllnical ~c~ivlty. Arthriti~ and Rheum~tl~m. 26 s 532 .
31. Sternberg, E.M., J.M. Hlll, G.P. Chrouso~, T.
5 KamllariQ/ S. ~. Listwak, P. W. Gold, and R.L. Wilder.
1989. Inflam}natory mediator-lnduced hypo~halmic-pltui~sry-adrenal axi~ activation i~
dQ~ectiv~ in ~treptococcal ~ell wall ~-arthrltis-~u~eptible Lewl~ rats. Proc. Natl. Acad. Scl.
10 US~. 86: 2374.
32. Baylln~ S.B., and Msnde~ohn, G. Endo~rln~ ReviQw~ 1, 45-77 (1980).
: 33. Jullu~, D., Livelli, T.J., Jes~ell, T.M. and Ax~l, R.
sciQnc~ 244, 1057-1062 (19~9~.
34. Jul~us, D., Huang, K.N., LiY~lli, T.J., AXQ1~ R., and Je~ell, T.M. Proc Na~ c~d. Sci. 87, 928~932.
35. Buc-Caron, M.H., Launay, J.M., L~mblin, Do~ and Rellermann, O. Proc. Natl., Ac~d. Sci. 87, 192~-1926 t 1990) .
; 20 36. Laub~chor, A. ~nd Plet~ch~r, A.J. Pharm. Pharm~col.
31, Z84-289 (1979).
37. Peroutk~, S.J~ Ann. Rev. N~uro~c~. 11, ~5-60 (1988).
38. Schmidt, A.W., and Peroll'cka, ~.J. Fed. AmO Soc. Exp.
Biol. J. 3, 2242-224g ( 1989) .
25 39. ¢lennon, R.A. J. M~d. Chem. 30, 1-1~ 51987).
40. Hamon, ~., Bourgoin, S., H~ry, S., and Sl~onnet, ~.
Mol. Phnrm~col . 14, 9g-110 ( 1978) .
41. E~oe, B.~., and We~s~man, A. J. Pharra~col. Exp. Sol.
154~ 4~9-506 ~1966~.
30 42. Aune, T.M., Kelley, ~.A., Ranqos, G.E., ~nd Bombara, .P. J. Immunol. in pre~ (1990).
43. D~Larco, J.E., ~nd Todaro, G.J. Proc. N~tl. Acad.
Sci. U.S.A. 75, 4001-4005 (1~78).
44. Todaro, G.J., DeLarco, ~.~., and Fryling, C.~. FASEB
. 35 J. 41, 2996-3003 (l~a~
: 45. Seuwen, X., Magnaldo, I., and Pouys~egur, J. Natur~.
335, 254-256 (198~.

Sl.J~ 1TUTE~ SHEET

:

, - W~92~ Q~Q3 ~ P~/U~1/06176 61) 41i. ~suwen, ~, and Pouy~egur, J. ~ioeh~lD. Pharmasol.
39, ~8~-9gO (1990).
4 7 . Nem~c~J~ 9 G . M ., Cou~hlln , S . R ., H~ndley , D . A ., and Iqo~kowitz, M . A . Proc . Na tl . Acad . ~el . 8 3, 6 7 4 - 6 7 ~ 1~86 ~ .
48. Bi~hop, J.M. Annu. R~v. ~lochem. 52, 301-359 ~1983).
49. va~u~, ~.E. Annu. R0v. ~çlnet. 18, 553-587 ~l~B4).
- 50. A~ e, T.~ nd Pogue, S.L. J.Clirl. Inve~t. 84, 863-~75 ~198~).
51. Finocchiaro, L.M.E. ~ c, E.S., Fernl~nde~-Ca~telo, S. ~ Crl~cuolo, M., Finlcielm~n, S., and ~hmod~ V.E. J.
Int~rferon Re~. 8, 705-716.
52. ~alker, R.F., Frled~n, r).w., and Jlm~n~z, A. Lif~
Sci. 33, 1915-1924 ( 19~3) .
53 . Alt~n, A., ~ . Mu~t~lln arld ~ . Cogqe~hall , 1990 ly~phoey~ff activ~tion. ~ biologlc~l model o~ nal tranE~duetlon . Crltic{ll Rev . ~ n I~ unol . 10 s 347 .
54. Puck, J.M., and R.R. ~ls3-. 1984. Regulatory intar~etlon~ governlng ~ch~ prolif eratlon of T eell ub~et~ 3tlmul~t0d wlth pokeweted mltogen. 3. Immunol.
132: 1106.
55. Aun~3, T.M. ~nd S.L. Pogue. 1989. Gell~r~lon and char~cterlz~tlon of contirlou~ llne~ of CD8~ ~uppre~or T
lympho~y~es. J. Immunol. 1g.2: 3731.
25 56. Fox~ E.J., R.G. Cook, D.E. L~wi~, ~nd R.R. Rlch, 19~6~ Proll~er~tlve ~lgn~ or 3uppre~0r T ~ell~:
h~l~er cel1~ 8tlmul~ted wlth pokewa~d ~itogen ~ n vltro produGe a suppr~sor cell growth factor. J. C11n.
Tnv~t. 78 . 214 .
30 S7. ~er~ G.M., 19~8. ~rhe ~d~nylata cyel~ cAMP-proteln k~na~e. ~ pathw~y ~nd regll1atlon of th~ une r~pon~e. Immunology Today, 9, 222-229.

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Claims (15)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:-

1. A method of regulating the functions of activated T
cells exhibiting a 5HT1a receptor comprising reducing the availability of 5HT1a receptor binding sites.

2. The method of claim 1 wherein the availability of 5HT1a receptor binding sites is reduced by introducing an effective amount of at least one antagonist selected from the class of 5HT receptors ligands.

3. The method of claim 2 wherein said class of 5HT
receptor ligands includes spiperone, mimotopes and antibodies to said 5HT1a receptor.

4. A method of regulating the function of activated T
cells exhibiting a 5HT1a receptor comprising functionally decreasing the amount of serotonin available for binding to said 5HT1a receptor.

5. The method of claim 4 wherein the function of activated T cells is decreased by administering an effective amount of a compound to inhibit the activity of the enzyme trypothan hydroxylase thereby inhibiting serotonin synthesis.

6. A method of up-regulating the function of activated T
cells exhibiting 5HT1a receptors comprising functionally increasing the availability of 5HT1a receptor binding sites.

7. The method of claim 6 wherein the funtion of activated T cells is increased by introducing an amount of at least one agonist selected from the class of 5HT1a receptor ligands including 9-hydroxydipropylaminotetralin HBr.

8. A method of treating a T-cell dependent disease state in a mammal comprising down-regulating the function of cells exhibiting 5HT1a receptors by reducing the availability of 5HT1a receptor binding sites.

9. The method of claim 8 wherein the function of cells is reduced by introducing an effective amount of at least one antagionist selected from the class of 5HT receptor ligands, sufficient to bind to said 5HT receptor to interrupt cell function.

10. The method of claim 9 wherein said class of 5HT
receptor ligands includes spirerone, mimotopes and antibodies to said 5HT receptors.

11. A method of treating a T-cell dependent disease state in a mammal comprising down-regulating proliferation of cells exhibiting 5HT1a receptors by functionally binding to said 5HT receptor.

12. The method of claim 11 wherein the proliferation of activated T cells is decreased by administering an effective amount of a compound to inhibit the activity of the enzyme trypothan hydroxylase thereby inhibiting serotonin synthesis.

13. The method of claim 12 wherein the proliferation of activated T cells is decreased by introducing an effective amount of at least one antagonist selected from the class of 5HT receptor ligands including ritanserin, mianserin, spiperin, mimotope and antibodies to said 5HT1a receptor.

14. A method of treating an immune deficient disease state in a mammal comprising up-regulating the function of activated T cells exhibiting 5HT1a receptors by functionally increasing the availability of 5HT1a receptor binding sites by introducing and effective amount of at least one agonist selected from the class of 5HT1a receptor ligands including 5HT, ketansein, and L-methyl 5HT said effective amount sufficient to enhance cell function.

15. A method of up-regulating the function of the subpopulation CD8+ of activated T cells exhibiting a 5HT1a receptor comprising an effective amount of serotonin sufficient to increase cell function.