LAG- 3, A NOVEL LYMPHOCYTE ACTI VATI ON GENE Vol ume 171 May 1990 1393- 1405 CLOSELY RELATED TO CD4 BY FREDERI C TRI EBEL, SETSUKOJ I TSUKAWA, ELENA BAI XERAS, SERGI O ROMAN- ROMAN, CATHERI NE GENEVEE, EVANI VI EGAS-PEQUI GNOT, ' AND THI ERRY HERCEND Fr om t he Labor at oi r e d' I mmunol ogi e Cel l ul ai re, I nst i t ut Gust ave- Roussy, 94805 Vi l l ej ui f , Fr ance, and `Uni t e de Recher che 620, St r uct ur e et Mut agenese Chr omosomi que, Sect i on de Bi ol ogi e, I nst i t ut Curi e, 75331 Par i s, Fr ance A number of cel l sur f ace st r uct ur es ar e t hought to bel ong to t he Ig super f amily ( I gSF) t because t hey cont ai n at l east one domai n with a char act er i st i c f ol di ng pat - t er n, cal l ed t he Ig f ol d ( r evi ewed in r ef er ence 1) . Sever al of t hese mol ecul es have cr i t i cal f unct i ons in i mmune r esponses . I n addi t i on to ensur i ng speci f i c ant i gen r ec- ogni t i on ( I g, TCR) , t hey may f unct i on as monomor phi c l i gands cr i t i cal in cel l - cel l i nt er act i ons (e .g ., I CAM, CD4, CD8) , r ecept or s f or vi r uses (e.g ., CD4, I CAM), or l ymphoki ne r ecept or s (e.g ., I L- 1- R, I L- 6- R) . We r epor t her e t he char act er i zat i on of a novel human gene, t ermed l ymphocyt e act i vat i on gene 3 ( LAG- 3) , sel ect i vel y t r anscr i bed in act i vat ed NK and T l ympho- cyt es . It codes f or a membr ane pr ot ei n with f our ext r acel l ul ar I gSF domai ns . The sequence dat a, t he compar ed exon/ i nt r on or gani zat i on, and t he chr omosomal l ocal - i zat i on r eveal ed t hat LAG- 3 is cl osel y r el at ed to CD4. Mat eri al s and Met hods Cel l Li nes . The i sol at i on and gr owt h of t he f et al CD3 - CD2` F55111E5 ( or F5) cl oned cel l l i ne has been descr i bed el sewher e ( 2) . For mas s pr oduct i on, t he cel l suspensi ons wer e pl at ed on a f eeder l ayer composed of i r r adi at ed al l ogenei c PBL pl us t he EBVt r ansf or med B cel l l i ne Laz388 in Vbot t omed 96- wel l pl at es at 3, 000 cel l s per wel l with r I L- 2 and l ymphocyt e- condi t i oned medi um. 200 pl at es wer e har vest ed at a concent r at i on of 3 x 106 cel l s/ ml af t er 12 d in cul t ur e to gi ve 6 x 109 cel l s . For t he Nor t her n bl ot anal yses, si mi l ar cul t ur e condi t i ons wer e used to pr oduce t he r el evant cel l s . I sol at i on of Membr ane- bound ( MB) Cyt opl asmic RNA and Const r uct i on of a c DNA Subl i br ar y. 4 x 109 F5 cel l s wer e washed t wi ce in PBS and al l owed to swel l f or 5 min at a concent r at i on of 5 x 10 8 cel l s/ ml in i ce- col d hypot oni c buf f er medi umRSB (0 . 01 M KCI , 0 . 0015 MMgC12, 0 . 01 MTri s-HCI , pH 7 . 4) ( 3) . The cel l s wer e t hen r upt ur ed mechani cal l y with 10 st r okes of a t i ght - f i t t i ng gl ass homogeni zer ( Dounce) . The homogenat e was di l ut ed f i vef ol d in 2 .5 Msucr ose TKMbuf f er (0 . 15 MKCL, 0 . 005 MMgC12 , 0. 05 M Tri s-HCI , pH 7 . 4) and l ayer ed over 2 vol of 2 .5 Msucr ose TKMbuf f er . Two l ayer s . of sucr ose TKMbuf f er wer e successi vel y added, one with 2 . 05 Msucr ose and a second with 1 .3 Msucr ose . The gr adi ent s Thi s wor k was suppor t ed in par t by Associ at i on pour la Recher che sur le Cancer gr ant s 2110 and 6338 . Addr ess cor r espondence to Fr eder i c Tr i ebel , Labor at oi r e d' I mmunol ogi e Cel l ul ai re, I nst i t ut Gust ave- Roussy, 39, r ue Camille Desmoul i ns, 94805 Vi l l ej ui f , Fr ance . I Abbr evi at i ons used in t hi s paper : aa, ami no aci d ; I gSF, Ig super f amily ; LAG- 3, l ymphocyt e act i vat i on gene 3 ; MB, membr ane bound; TM, t ransmembrane. J. Ex p . MED . © The Rockef el l er Uni ver si t y Pr ess - 0022- 1007/ 90/ 05/ 1393/ 13 $2. 00 1393 Downloaded from http://rupress.org/jem/article-pdf/171/5/1393/1670848/1393.pdf by guest on 26 July 2023