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1/95 1, 2o~3 3 m~. B~'MmT 4page 5): PRff4CIPAL INVESTIGATOR (do not indi¢a~e ar~ oo-P.I, on thi~ page) NAME (LAST, RR~T M.I.) AND Sant, Andrea J., Ph.D. • AP.J~ OR ~oN~Trr,~: Assistant Professor P a t ho I ogy The University of Chicago 5841 S. Maryland Ave., MC 1089 CI-FY, STATE (OR COUNTRY) Z~P CODE: Chicago, IL 60637 MAIUN~ ADDBI~ iF DIPPERE.NT FROM 312/702-9196 I=IIIM4CIAL AOMINISTRATOR ,,, (person to cor~aot for bud~It inform~ior+ Mary Ellen Sheridan Pontoon Tn~E: Ass~smt Vice l~esi(lent: for Director, University Research Admin. 970 E. 58th Street Chicago, IL 60637 ~ONE 312/702-8604 ~~E~S PAYA8~ TO: University of Chicago ~ ~ECK$ TO (~ AND ~ON Chery] Wi I l iams Asst. Dir., Info. Srvcs. MAILING 5801Sou¢h~.l£s Avenue Adm. Room 7 Chicago, IL 60637 (sooel~na let tJ~e In~iWlWn) .., ~mmm~m~:: Director, Ikzfversity Research Ad~tnistra¢ion ~/O E. 58th Strut- 3rd fl. Chicago, ~501740

~.L ~nor~a J. San~, ?h.D. b.~ The University of Chicago c.T~c'ot'~i~" )4He Restricteo Recognition of the P~s Oncog~ne mm ~ts ~ of normal regutstion is though~ to be s k..,'y ~ of its u'ans/'orming activity. Thus, it inmm~ symem, T cell ~ and tumor emdic~.._~ could ~y fotiow. In the experiments 1~ The working h~ to be inve~pmd ~ that ras p~'~enunion iyy Class II may be limited to the minor cytbsolic fc~ms of ms and that presentation may be mediated by specific ~4HC C~tss H mole~nleq snd mrn~ immnni~ In ~ ~ ~ ~une s~t~ ~ el~inam ~~ ~ ~gs) ~ ~de~ ~d ~x ~ major hisw~a~b~ c~plex ~r~) ~m~ ~ t e s~ ~.~n ~~g ~s (~C). ~e ~ of ~enm wi~m ~ A~ ~ m ~e ~~ ~ ~ ~uno~ c~pl~ is t~ ~fi~n ~senmdon. ~C ~v~y.m ~ i~u~W. ~1~on of t~s subset in vivo has ~n ~n to Ic~ ~ d~lay~ ~ a~t ~n~io~c ~ccfion ~.tu~ (I, 2) ~d ~fi~ sm~es have sho~ th~ ~ ~o ex~on ~ ~C O~s ~ mo~o~¢s ~us~s mj~fion ~ a p~¢~u~y pm~i~y ~wing ~~(3). R~nt s~ ~~.~ g~ ~ ~n~g s~li~ ~ sev~ ~l~ ~ m~ mj~. EndoP,~tous anti~en_m'csentation by MH~. Cl~.q.~ II molecnle.q. We have used an experimental mode] syttem to examine t.hc regulation of class II Fresentation of an intraccllular Ag. Studies ~tilizing this .model has revealed a novel pathway of Oass II mstric~ Ag presentation (6). Our ~ s _ugh. ~ ~ mat the .e~. ~ous Ag can b~. In'ocessed early during biosynthesis. This processing pamway ~t m~ amung from the ole~smal exogenous class II presentation pathway. Our ~t~ using c~.lls def.e~tive at .exogenous Ag pt~..~ntation ~ strongly suggest that diffexent . a~s it Ag l~..sen.t~_uon.p.a~, ways ernst, .and that factors c.ddcal for the presentation of exogenous __ tin, rent pm~, .cofaetom revolved m Ag presentation are differentially expressed in diffexent ~ee types,..~ ~ncau~on supported by o~ ~. mr. t ex~ Cla~ H speci~c fllorecognitton fT). SpeCtl~ ~ at A.h'~.~ or- d~e.~ellt l_mca~s display distim peptide& ~~y ~df~ oecogenes, have been identified in a number of minors and are in fact the most cancer (8). oncogene m human been revcmed to be ~ ~ mmma mmme m ~e ~ md line hek ~ aedviff (10.I1).

1. Derive trmm~ectants tltat express MHC Oass II and ras localized to ~ mbceUalar compartments. P.as is pom-~y m~.~fied in several steps m yield a fenn that essocimes with the inner face or" the plasma memla'ane. It is our hypothesis that source. To examine the mechanisms ms ~ we will derF~e uansfecta~ that express a 2. Derive of T cell hybridomas specific for rats pel}tides in the context of MHC Clam il moleeales. To examine the requix~nents for MHC class H restricted recognition of ras, it is ~ to have T cells specifi~ for this pepfide:MI-IC complex. We already have T c~Als raised against muumt ras peptide. To derive T ceils that may differ in fine Sl~...Lfidty, we ,,viii expmui this panel and derive T c~ALS with reaotivity to internally synthesized ra& Mice will be immunized with transfcctants that express mutant ms and a class IT res~u-iction e.lement and ras 3. Determine the requirements for MHC class II restricted presentation of ras. In this aim we will ~ quanfitiatve antigen la'es~ assays to identify the options fro" ras presentation by MHC class II molecules and the intra~-Alular evenm involved in presentation. We -~11 also evaluate the requireanents for MHC encoded protein co-factors for endogenous r-as presentation. 3. SUPPOILTE.~G DATA, EXPERIMENT~ DESIGN and PROC.F.DLTKE-$. Do not attach more than six (6) additional page~ |3a-31). All figu~eSo ¢har~ ~bles and references must fit within pages 3 - 3f. The ability of ms p21 to a£a~.,ss the Class II presentation pathways is currently unknown. It has been possible to demonstrate ras specific CD4 ceils by immunizing animals with peptides b~aring th¢ mutant amiao aeiA (aa 12 or 61 ) (12). Ras protein has been idantified in the urine of tumor bearing ~, suggesting that it may be available f~ pnx:essing by host APC. Intea~stingly, many.of the tumors that have been shown to be transformed by ras express Class II molecules ~tivdy or can be irgluced with ,t-l~. What remains totally unknown however, is whether internally synthesized ras will gala access to MHC Oats II molecules. In this proposal wc will ~ if there is a potential for endogenous presentation of ms, and ff so, the degree of cross v-~ctiviry of T c¢11s speoL~o for endogenous and exo~e~usly rim. In addition, our studies ~ the meelmnism of presentation and whether it is possible to upregulate ras laC.senmtion 50501742

mediau~ upm~ (13-15) or by the proce~ of micro- or macru-autopha~. ~ ianer pmhways axe poorly deUtmed at the pn~ tim~, but a~ thought m alknu impm-t of cytopla~c ~ h~m th~ lysosouml comp~ane~s by ~ vacuo~u and fusim ~n~m (~6,~7). To e~mm~ d~ ~ stud mechanisr~ for class H ~ ~ of ras, it is nex:cssary m derive singl~ amino ~ variant o~ras 21 lh~ lacks in which Cys 186 is su~m~ f~ ~ ~ D. ~ (N~, ~). ~ ~ f~ ~ ~ ~ ~st- ~ (18). We ~ ~ ~ ~ ~ ~ S~ 1~ fo~ ~ ~e ~ ~21 ~~ ~~ ~ ~~ ~ ~ ~ m int~ ~m~es and ~ cy~ol, 186 mu~ ~y is ~m~ ~ ~ ~y~I. A. B. 1 2 3 4 5 6 A. A. B, ~:i_~ure 1. Bx.~giot7 O( WT ~ SER 18~ mutapJe i~ murina ~oblasts. L cells expressing i-Ab were superttansfected with genes encoding WT HaRas or a Ser-186 mutant. Transfected oeils were analyzed by ras specific lr~tracytoplasmic staining, using the mAb Y13-259 (panel A) or by immunopraoip|l~tion (IP) performed on cells b~nthatically labelled with SH-leucine for 16 hrs. (Panel B). Panel B- SDS-t0% PAGE: Ras IP are shown in lanes 1-3 (WT in lane 2 and Ser- 186 in kma 3) and MHC class It IP are shown In lanes 4-6. Lanes 1 and 4 are untransfected ~lls. The difference in post-translational prooessir~g of Ser-186 mutant is apparem by both immuno#u~noe, in which the 8e¢ 186 displays only diffuse cytosolic staining while the WT dkiplay$ ponGtate membrane Iocal'r~Mlon and by SD~ gel mobility, in which the post-translational addlllcm of lipid leads to an incr~med ~;DS-PAGE mobility. 50S0 743

~ exclusively as an sdebte, c3nmeik IXee~ Selxeqeem biochemical and subcdlular des~bed below. Spec/flc Aim 2. Derive of T c~! bybridonms specific for ras peptides in the context of MHC Class I! molecules In order to exa~*~e the req~ for MI-IC c.htss lI resuicxed ~6on of ras, it is necessm'y to have T cells specific f~r this pepfide:MHC complex. We will begin ore" smdie~ with T cells provided by Dr. Hans Schrca'l~, University of ~, with whom we have collaborated our ms stodies. These investigam have generated Class II specific T ~IL~ using ras peptide~. Figme 2 shows the activity of a large pand of ras specific T cell hybridomas that we have tested with t~ peptide and otlr fibrol3la~/m.d.gert ~.ting cells expressing the I-Ab remP~ion element. To inc~..ase the probability of deriving T cells that differ in fine specificity, we will ~ thit panel and derive T cells with reactivity to internally synthesized ras. Utilization of both ex_-ogenous and endogenous ras for immunization will allow us to probe the we...chanistm arid potential for ms presentation lyy MHC Cla~ I[ molecules. To derive T teals specific for endogmtom ms, we will immunize (C57BId10 ~ C3H)F1 mice with Ha ras tram;fecaants that ~ high lev¢£t of the I-Ab re~iction element and accessory molecules sufficient fox T cell actwation. The fibroblasts that we ctm~.~ndy use (DAP-3, C3H derived) express B7 and activate both primm'y T cells and T cell clones (data not shown). Mice will be primed using techniques that have ~ally been used to elicit T cells specific for minor antigens and that we have used to ckaive alkx~-xm,,'~tive CD4 T cells (7). Briefly, mice ,Mll be primed by intraperitoneal injection of the L cell filxoblasta bearing ~yn~ I-Ab and alto hatbo~ng high levels of intracellular Afmx 10 days, CD4 ceils will be ]~m'ified fh3m spleen and from mesenteric lymph nodes, restimulated for 3 days/n viwo with the transfected cells. T cell blasts will be fused with the BW5147 T cell lymphoma (7, 21 ) and selected for fmth.er study b~._~d on their aMlity to be ac~vated by ras-expressing ~tants and an inabi]hy to react in the absence of ras. ROme 2. Ras specific T cells were generated by immunizatton of C.,57BI/10 mice with a Peptide corresponding to a.a. 5-22 of ras p21, containing • the Ar(I sul:stitutlon m position12 (oncogenic mutant Harvey ras). Draining lymph node T coils were resltmulated/n vitro then fuee¢l with BW5147. Hybrids were c~oned by limiting clilu#on, and te~ted with DAP-3 cells transfectsd with genes enooding the ~ II I-Ab molecl~ie (DAP-Ab) in the absence (open bars} or presence (ha~ bars) of Pel~e S-22 HaP, as. 10 ~o~lon (not ~wn). Specilk Aim 3 Determ~ the requirtmenta tot MHC clasl II restricted

~ ~g mmant ras and the I-Ab restriction element to be acfivamd by ms pcpfidc, inta~ ras ptx~dn or ceJB ~ inmmally synthesiz~ ms. Quanritativ~ Ag l~esentation studies will be performed for bo@t types of presentation events as we have deunibcd (6,7,21) ~. If, m inmmally ~~ ~ ~ ~ ~y ~ m ~ T ~ u~g ~ ~d~m ~ s~ul~, ~ ~ T ~ ~ ~y ~y w ~ ~ ~~ b. ~ inlay s~e~ ~ is u~blc ~ ~n ~y ~ ~ eM~c ~~ it ~y ~ly ~ A~ ~ ~~ ~ T c~ may ~ ~t m gen~ ~ ~ T ~ ~ ~ ~~ u~g ~e ms ~gnm but ~ese ~ not ~dv~ ~ ~n~s ~, it ~ sug~t ~at ~ ~ ~@ddes ~n~ by ~e ~do~ous ~d ~~s mum ~ ~sdn~ ~is ~~ ~ i~ ~g ~e ~C Y~ ~~ ~s. Alt~atively, oymsol~ gen~ ~p~s ~ ~t ~mkas ~t ~ ~ up ~Ry Lain ~e ~dos~ ~m~ts ~ cl~s R ~ng. ~ese pos~li~ c~ ~ ~~ ~ Lhe exp~ents ~~ ~low. 2. Is ~ softie ~topl~mic ml~ve ~en~ of ~e c~o~o vs mem~e bo~d f~ of ~ to ~n~tc a cl~ II ~i~, ~es ~g ~ ms.don d~t. We w~ ~d~y um L~- ~s f~ ~c ~~ts ~cau~ ~ey ~ ~y ~~Ic, have ~n~ abiSry ~d~m ~ class ~ ~ a~e~ to l~ze s~ and mu~t ~s m ~c ~ctcd ~Rul~ ~~ ~ I). ff ~n~ B not de.table, wc ~ll cxpbm whe~ ~enmdon d~ on ~ r~es ~n~ ~'~ ~~ ~enm (6,7) eat ~ ~mp~nt ~C. ~lls ~ ~Ment ns ~4A. ~ve ~s ~ ~ ~y~ by su~lu~ ~do~fion ~d ~ ~fic T ~s. a. If the ¢yto~io form of ms is ~tially preseated by class IL it will suggest the acdvity of ~ c~ph .~ic ¢~ V~ ~~ We will ~t for the role o¢ acidic compartments in proces~ng ras by exanamng ~mitivity ~o ly~m~m~q~ rea@~ as we have detmibed (23). If pit depeatd~t events are ~ we will klemify the nmdtaaimn by which the lymamne i~ _t~. I~.. the ~@, 7~nd Im~ slm~ promm (liSP) l~,t lure beea. ~ by Dk~ (13-15). Thi~ HSP (1~p73) ~ pmteim bemi~ ,eqttenm~ mamea to

chased in culture medium (with or withom 5% FCS) comaining a 4 fold ~ of unlabelled leucine for ~g i~iods of time. CelLt wiil be lysed and ras will be isolated by ..... using Y13-259. MHC clard I Kk, isolated with the mAb 16- l- 11 will be used as a conwc/for ~ that is not mediawd by HSP. If ~ov~" ~ r~ is ir~'eased by sorum deprivat then we will verify that the is mediated by lysosomes by testing by SDS-PAC~ H these m~lies ~ that de~radat~ is pl-I ~ v~ will test whether b. If the soluble form of ras is preferentiaily presented by class II, it will also raise t,~e ~tmlt~mmltY that class II presentation of ra~ can be faeilimt~ by compounds that anmgoni~ u~ post Imposing that lead~ to ~ localization. Two recent mlxtrts (26,27) suggest ~ and Imy eaazyamdc step that leads to tmmbrane Iocali~tion of ra_s, effectively block prat-tmmlalional la'Oc~_.~ag of ms, it~ membrane localization and transforming activity. These mma~li~ a~ thus Imy candidatea for anti-tumm thettpy. If, in addition to blocking the trm~orming activity of ras, they could fteilimm immune recognition of the tumor, tumor growth could be stopped and tumor eradication could begin. To examine the ability of the synthetic ~ to facilitate class II Swesentadon of ra$, e,'dls e~ressing WI" Ha ras -,rill be treated with inm-,~in$ doses of the analogues L- 731,734 (27) or BZA-2B (26), both of which have been show~ to block the post-tramlation~ ftrnewylation of ras. Immunoflm.,-a'e-scence, irnmun~tion and subeelhular ftaefionation will be used to verify the efficacy of these rn~tabolites on the post-mmslational processing of r~-~ in our APC. Modulation of epitope generation will then be ~sted. e. If, in the preceding experiments, we obtain the unexpected r~sult, that the me.ml:~ane bound form of ras is the preferred form for presentation, we will ,xplctre the mechanism that leads to this prmentation. The first,xperimemt will teat whether ras is localized to the plasma membrane in the APC or whether it has unexlx,ted localization within eompm~ents of the endocytic pathway. We have been de'¢eloping a method to i~lam endocytic compartments of antigen pre~.nting ceils, in order to study the factc~ that ~gnAate class II targeting to thc~e compm'tment~ (28). The basic procedm'e is to allow cells to ing=t colloidal iron for short periods of time (3-5 h~). Cells are then ~srupted under conditions where the intracellular organulles remain intact. The cellular homogenate is then fractiomtted on a column of ~ mesh that h~ been magnetized with mt elec~tegnet. Endo~Jrnes a_mi lysos~.mms containing the colloidal iron are bound to the colmnn, while other intrecellular organe.ale, and plasma membrane fractions m'e unbound. These sepsrate suboeihflar fractions can then be probed for any cellular component (29). Shown in Figure 3 are ~esults obtained with class II positive L ceils were fra~tionated as described.Wesr~ern blots were ]~obed for MHC class II ~ chain and the major giycoprotein of lysosomes, L&MP- 1. Control e~,ttt~ts (not shown) have indicated that endosomal and lysosomal compartment~ can be ~ in Im~ form, fr~ of #tram mernbram, endoplam~ic redculum and Golgi, and that the yield of lyt..,~omea is greater than 80%. to tl~ #mma ~ or if it c~ Iocali~ to ~ comlmrtment~ C,-als will be lal~lled for MFIC clsss II ~ as a conu~L ~ o/'r~ m ~dosonml/ly~mmml ~

LAMP-1 UB B UB B F~re 3. Endosornal Isolation. Class I! positive L cells were allowed ~o endoc'ftose co~oida~ iron for 4 I~ours. ARer cell homogenization, lysates were fractionated over a maonelized ooh~mn and unbound (UB) and bound (B) fractions were applied to SDS gels and probed for c~ss II ~ chain or the lysosomal membrane protein LAMP-1. This gel shows the bound fraction is enriched for LAMP-1 and mature Class Ii. These fractions can also be used to demonstrate the site of peptide:class Ii complex formation for a typical exogenous antigen (not shown) 3. Is presentation of ras depet,~lent on MtIC Iinked protein co-factors? Recently, protein co-factors that directly pardcipa~ in MHC restricted antigen presentation have bccn idcmificd that ar~ encoded within the MHC. TAP genes encode transmembran¢ channds that control import of cytosolic pepddes for MHC class I rcstricmd antigen presentation (30). Most r~centiy, two proteins that have homology to class II a and [~ chains have been de~'ibcd that regulate class II restrictcd antigen presentation of exogenous antigens (31,32), although their m~chanism of activity r~mains poorly understood at the present time. a. We will test for the possibili~ that soluble ras is introduced into the Class II p~l ent~ion pathway by TAP- 1 and TAP-2 by analyzing the ability of the TAP deficient EE2H3 to present ras. This c~ll line is readily transfcctablc and is an cffec~ve APC for mouse T cells (6). The ge~n¢ encoding soluble ras will be introduced into EE2H3 that is already tmnsfccted with the class II restriction element. Subcloncs of ~ransfectants ~xpr~ssing read~y demctable Icve.ls of ras will be t~sted for their ability to activate the ras spc~fic T cells. If p-r~sentation of ras does not occur with this o011 line,, Ag presentation studies will be pca'fon, n~ using EE2H3 cells that had bccn tranffcoted wi~h TAP gene consa'ucts, which we have derived in our laboratory. If TAP ~= .arc nxluircd for ms presentation, it would be important in sevcxal regards. First, it would ." .m~. lic.at~ t.he C1 .a.~_.I ¢y~_solic p.mt.e~ for ~pitope gent~_tion. Secondly, it would raise ty Cart m¢ ras,,cla~ 11 oomiacx ~s genexat~l in the ER. Finally, sinoe tumor cells have own to down-regulate e, xpnmion of TAP IVr.n~ it would prodd¢ a lXaeatial mech~sm of 50501747

Perg~c~cs and.nonmary Our undersmmting ofela~ rl remieted nndgen p~sentation is largely drayed from ~ involving exogenous protein tati~as. ~y, the~e ~ have doeumemed the l~ely imigalance of pt~ein vofactm~ in the c~ll biology of chtra II antigen ~ DM lmm~(s) encoded by gen~ within the MI-IC The goal of otw work i$ t~ inmamvio~ ~ ofa mmm- tlnvugh the use of a claffit II lmmmmim pathway, and if so, tl~ ~e~lt~kmg term coal.of ~ w~. i~ ~ in.crease our ~g ~y.so that it is possible to Literature Cited 1. Wank, R. and C. Thoms~en. 1991. Nat~re. 352: 723-725. 2. Gxeenberg, P. D., D. E. Kern and M. A. Chec,,er. 1985. Y. Exp. Med. 161:1122-1135. 3. Ostrand-Rosenberg, S., A. Thaknar and V. Clements. 19"90. Jlnmmnol. 144: 4068-4071. 4. Bodme¢, L, $. Tonkn, A. Oza, T. Lister and W. Bodrner. 1989. Lancet. i: 1455-1456. 5. Han, ~_, F. Bteitburd, P. Marche and G. Orth. 1992. Nature. 356: 66-68. 6. Lo~, G. E., C. E. Elias, P. E. Fields, R. IC Ribando, M. MeKisie and A. J. Sam. 1993. Y. Exp. Med. I78: 73-85. 7. K-atz, J. F. and A. L Sant. 1994. J. lmmunol. 152: 162%1640. 8. Bos, J. 1989. Cancer Research. 49: 4682-4689. 9. Shih, T., M. Wee~, P. Gross, R. Dhar, S. Ortxszian and E. Seholnick. 1982. J Virol. 42: 253- 261. 10. Kato, g_, C Der and J. E. Bus~. 1992. Semin. Cane. Biol. 3: 179-188. 1I. I-laneoek, J., H. Pal~rson and C. Marshall 1990. Cell. 63: 133-139. I2. Peace, D., W. Chen, H. Nelson and M. Cb, eever. 1991. J Immunol. 146: 2059-2065. 13. Chiang, H., S. Terleeky, P. CP. and J. Dice. I989. Science. 246: 382-5. l~t. Ahlberg, J., L. Marzella and H. Olaumann. 1982. Laboratory Investigation. 47: 523-532. 15. Ol~on, T., S. Terleeky and J. Dice. I991. Biomcd Biocim Acta. 50: 393-397. 16. $¢glen, P. O. 1983. Methods Enzymol. 96: 737-765. 17. $¢glen, P. O. and P. Bohley. 1992. Experientia. 48: 158-172. 18. Wi'llumsen, B. M., K. Norris, A. t3. Papageorge, N. Hubbert and D. R. and Lowy. 1984. F)dBO X. 3: 2581-2585. 19. Sant, A. J., N. S. Braunsl~rt and R. N. Gexmain. 1987. Proc. Natl. Acad. Sci. 84: 8065. 20. Baleh, W.E. and J~E. Rothman. 1985. Areh.Biochem. Biophy$. 240:413-425. 21. Sant, A. J. 1993. J. lmmunol. 150: 5299-5310. 22. Loss, G., C. G. EHas, P. E. Fields, R. K. Ribaudo, M. MeKisic and A. J. Sant. 1993. J Exp Med. 179: 73-85. 23. Loe~, G. E. and A. J. Sant. 1993. J. Immunol. 150: 3187-3197. 24. Wing, $.,H. Chiang, A. GL-,~Jberg and J. Dice. 1991. Bioche.mJ. 275: 165-169. 25. Dk:e, L 1990. Trends in Biochemical Science. 15(8): 305-309. 26. Iame~ G., L L. Gold~leie, M. $. Brown, T. Raw,n, t. Somer~, R. Mc, Doweli, C. W. Cluwley, B. IC I.ale~, A. L~ L C.. laid J.C lvltt-,,te~ J~. 1993. ,Serrate. 260: 1957-1942. 27. ~ N., $. D. ldm~, |. deSolmt, E. Gdlimi, D. Pemlimo, $. L. Graham, R. L. Smith, ~. ~ A. Cltlff arid J. Gibbs. 1993. Science. 260: 1934. 2~. ~, A.V., Gordon, L. and AJ. Sant. 1994. Int. Inammol. In pre~

L~bor~tery: 600 sq. ft. or ta~ora~ory space aria separate office space are available to me w|thin the [m~unoTogy Quadrangle on ~he 3rd f]~r of Btl]~ngs ~sptt~l. ~ ts a shard cold ro~, dark~ and auLoclave ~ac/lltles and autoc]~ve fac~1~ties and c~n ~u~nt ~ with the I~nology ~uadrang]e In t~ ~par~nt of Patho1~. In addition, there ~s ~ c~le~e f1~ cyt~try fac~1~£y for ~h preparative sorting and analys~s capabl]~£~es, ~1: ~xtens~ve an~e~] ~ac~1~L~es, ]nc]ud~n9 a new]~ developed Pathogen free barrier ~ac~]~y, are available in an~] quar~e~s contiguous Lo ~il]~ngs ~r: F~ depar~n~a] s~ar~-up ~unds, ~ have pu~chase~ ~ Hac~n~osh c~uLer ha~ d~sk dr~ves, ~n~o~s, and a 1~ser printer ~o be used ~rd p~cess]ng and graphics ~or the publications and presentations of work perfo~ed In ~ ~i~: Fu]1 secre~ar~a], xerox and ma~]fng services are ava~]b]e ~n O~R: Co~e ~ac~i~es are ava~lable on campus aL ~he University o~ Chicago ~or pep~de and o]~gonucleo~de s~hes~s and fo~ ~unofluo~escence ava~lab]e £o University of Chicago 5..B_U]~. GET FOS .T~. CATIO:! : Use d~ .space, to, exp~,a~.s~fic n.ee~ ~or, ke, ms ~b~ on budget P~es. I) ~ssue culture ~a~a requ~re~ co aer~ve aria maintain ce~ ~nes ~pr~sing Ras oncogene, antigen, specific T cells, and to perfo~ i~unologic assays. 2) Tissue culture plasticware needed for maintaining cell lines expressing Ras oncogene, antigen, specific T cells, and i~unologic assays. 3) G418 se]ec~ion drug is needed to derive stable transfectants expressing MffC ~lecules and ras oncogene. 4) Protein bioch~istry supplies include subcellular frac~ionation supplies, i~unoadsorbants for isolatlon of MHC and ras molecules, and supplies for Western blotting. 5) Isotopes ~H Leucine is needed for ~tabo]ic labelling of celluIar proteins, ~H-th~idIne i~ needed for assays of T cell activation. I~unofluorescence supplies, ~noclonal antibodies, secondary staining reagents needed to identify subcellu]ar localization of ras. 6) Radiation protection; waste disposal 7) Mice needed to generate additional ras-specific T cells and to identify factors ~eded for tumor rejection. 8) Travel - scientific meeting for P.I. to present results of studies. 9) Publication costs, art~rk, photography. 5O5O1749

Budget J~'fification (contim~) 1) SDS protein gel elecu~hore~ appara~ i~ needed for fractionation- Bio Rad Inc. 2) Ball bearing cell lmmeSeaiz~ is ~ designed for h/gh efficiency and high reproducib'dity of cell homo~nization for tisme culture cel~ that minimizes organelle dama~ Ham W. h~e~, H& Y Enterprtu~.

Andrea O. Sant, Principal Investigator P.LNam~ Andrea J. 5an~ 10% 5,800 Te~lmicai S~pBort Jennifer Laiprasert All Fringe Benefits 80% 18,000 5,831 A. Sdm~s Sutneud 29,631 B. Co~lesal~|ies~ym~orc=tegory) i) Tissue culture media, media, FCS, supplements 5,000 2) Tissue culture plasticware 5,500 3) Selection drugs for transfection 2,500 4) Protein biochemistry supply 3,500 5) Radioactive isotopes, immunofluorescence supplies 4,500 B. ConsaraablesSub~tal. 21,O00 Oth~Expeases(i~mize) ~) Radiation protection and waste disposal 3,500 7) Mice purchase-75 mice/year @ $15/mouse + I0% set-up fee 1,238 Mice-care 10 cages @ $.75/cage/day 2,737 8) Travel-1 mtg, per year 1,000 c. Oth~F~peoses Subto~d .... 10~475 9) Publication costs/photo 2,000 A+B+C Sub~tal 6!+!06 D. 1NDI'RJ~CT COSTS ( 15% of A + B + C). D. Iodi~e~Co~-~ 9~166 SDS protein gel electrophoresis apparatus 4 sets of extra plates Ball bearing cell homogenizer (6) stainless steel ball bearings - i ,095 2OO I, 260 180 E. Penn~ntEq~i._pra~Subtot~t .. 2.~73.5 . I~cate hem a~d on Fage I. F .................................................................................................... ......... TOTAL REQUEST 73,007 64,161 73,785 Year3 67,~ - O* - lO, lOS :]~ ,474

T~le of Prok~ Cell Lineage Dependent Anti- gen Presentation Nolecular Control of MltC Clas.' l! Restricted Antigen Presen- tation Regulation of HHC Class [I [ntrace] I ul ar Transport cost 1 year extension I~u~y aad de~c~be 27 o-P~t~p of this NIH R03 RR08896-1 Arthri ti s FoundaLior NIH RO1 AI-34359 To~ Va~e o~ (dU~ co~) $24,623 $135,000 $475,848 $24,623 $45,000" $100,873 None I~ of 11/30/94 6/30/94* 01131/98 ~udicate the ~ot~t mm~al funds available to you th~s year for all ~e~.ar~ ch projecm • ~der your sup~vision. 170,496 PENDING OR PLANNED MHC ClassII Restricted Presentation of Endogenous Antigens TuBor Antigen P~esentation by Class II Holecules NIH R01 EM51629-01 $751,456 $286,149 (give gram of G~ Amoum a~t~.~) (direct costs) Av~lable to You $t38,423 ACS $93,810 To~ Du~-ation -- 07/01/94- 06/30199 07101/94 - 06/30196