Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy

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1 r t i l e s Lethl grft-versus-host isese in mouse moels of T ell reeptor gene therpy Gvin M Benle,6, Crsten Linnemnn,6, Ann I Hooijks, Lur Bies, Moniek A e Witte, Annelies Jorritsm, Anrew D M Kiser, Nine Pouw, Reno Deets, Elis Kiek 3, Wolfgng Ukert 3, Ji-Ying Song 4, John B A G Hnen,5 & Ton N M Shumher Nture Ameri, In. All rights reserve. The trnsfer of T ell reeptor (TCR) genes n e use to inue immune retivity towr efine ntigens to whih enogenous T ells re insuffiiently retive. This pproh, whih is lle TCR gene therpy, is eing evelope to trget tumors n pthogens, n its linil testing hs ommene in ptients with ner. In this stuy we show tht lethl ytokineriven utoimmune pthology n our in mouse moels of TCR gene therpy uner onitions tht losely mimi the linil setting. We show tht the piring of introue n enogenous TCR hins in TCR gene-moifie T ells les to the formtion of self-retive TCRs tht re responsile for the oserve utoimmunity. Furthermore, we emonstrte tht justments in the esign of gene therpy vetors n trget T ell popultions n e use to reue the risk of TCR gene therpy inue utoimmune pthology. Most humn tumor-ssoite ntigens tht re shre etween iniviuls onsist of nonmutte self ntigens. Consequently, the enogenous T ell repertoire tht rets to these ntigens will generlly e smll in size n tivity,3. Given these limittions, it hs een rgue tht infusion of the missing T ell repertoire, whih n reognize suh ntigens with high viity, oul e vlule 4,5. As the ntigen speifiity of T ell is ontrolle y the α n β hins of the TCR, suh tumor-retive T ell repertoire n e generte y the introution of genes enoing tumor-retive TCR αβ hins in peripherl T ells, n pproh lle TCR gene therpy. In ition to its potentil vlue in trgeting tumors, TCR gene therpy oul lso e use to enhne T ell immunity to pthogens suh s HIV 6. Stuies in mie hve shown tht TCR-trnsue T ells n e tive ginst tumors 7 9, n two phse linil trils hve shown tht it is fesile to trnslte TCR gene therpy to the lini,. However, numer of potentil sfety onerns ssoite with TCR gene therpy hve een note 4. A prtiulr onern is the possiility tht the ssemly of enogenous n newly introue TCR hins woul le to the formtion of TCRs with unefine speifiity potentilly yieling self-retive T ells. Previous work, in whih infusion of TCR-trnsue T ells ws omine with ntigen-speifi vintion in mie, showe no eviene of toxiity. However, it hs sine eome ler tht the in vivo tivity of optively trnsferre T ells n e enhne when the enogenous T ell pool hs lrgely een remove y lymphoepleting hemotherpy or totl oy irrition (TBI) 5,9. Furthermore, t from other optive T ell trnsfer stuies suggest tht the use of more highly expresse TCR genes 9, optimize T ell tivtion onitions uring in vitro T ell ulture 3 n in vivo interleukin- (IL-) ministrtion 4,5 n further enhne the in vivo funtion of TCR-trnsue T ells. However, it is unler whether suh improve therpeuti onitions n inrese the likelihoo of TCR gene trnsfer inue toxiity. RESULTS Lethl utoimmune pthology in mie fter TCR gene trnsfer To investigte the effet of more effiient engrftment regimens on the potentil sie effets of TCR-trnsue T ells, we use ohorts of C57BL/6J mie tht h een renere lymphopeni y sulethl TBI. We gve the mie smll numers of T ells tht h een retrovirlly gene moifie with the ovlumin-speifi OT-I TCR ( 6 TCR-trnsue CD8 + T ells, trnslting to the sme ose use in the lini). T ell infusion ws followe y high-ose IL- ministrtion etween ys n fter trnsfer (Fig. ), when sustntil mount of lymphopeni-inue T ell prolifertion hs lrey ourre 9. Aroun 4 fter trnsfer, OT-I TCR trnsue T ell reipients showe mrke hexi, ut reipients of nontrnsue T ells tht h een tivte uner the sme onitions i not (Fig. ). In most mie, hexi progresse rpily, with the mie neeing to e kille within 4 h (Fig. ). At neropsy, the mjor orgns (liver, kiney, spleen et) n loo ppere severely nemi, onsistent with one mrrow filure (t not shown). Histopthologil nlysis showe profoun loss of hemtopoieti tivity in the one mrrow (Fig. ) n spleen (Supplementry Fig. ), s well s epletion of lymphoytes in lymph noes, Peyer s pthes, thymus n spleen (shown for spleen in Supplementry Fig. ). Furthermore, 39% of mie h mrke Division of Immunology, The Netherlns Cner Institute, Amsterm, The Netherlns. Lortory of Experimentl Tumor Immunology, Deprtment of Meil Onology, Ersmus MC-Dniel en Hoe Cner Center, Rotterm, The Netherlns. 3 Mx Delrük Center for Moleulr Meiine, Berlin, Germny. 4 Division of Experimentl Animl Pthology, The Netherlns Cner Institute, Amsterm, The Netherlns. 5 Division of Meil Onology, The Netherlns Cner Institute, Amsterm, The Netherlns. 6 These uthors ontriute eqully to this work. Corresponene shoul e resse to G.M.B. (g.enle@nki.nl) or T.N.M.S. (t.shumher@nki.nl). Reeive 8 Jnury; epte 5 Ferury; pulishe online 8 April ; oi:.38/nm.8 nture meiine VOLUME 6 NUMBER 5 MAY 565

2 A r t i l e s Nture Ameri, In. All rights reserve. Dy 5 Gy TBI Dy ACT Dy High-ose IL- of T ells i.v. i.p. twie ily e 3 Bone mrrow pthology sore Spleen pthology sore pnretitis (Supplementry Fig.,) n 7% of mie h olitis (Supplementry Fig.,). The evelopment of this pthology epene on the infusion of TCR-trnsue T ells, s reipients of nontrnsue T ells tht lso reeive TBI n IL- ministrtion showe no pthology (Fig. f n Supplementry Fig. ). On the sis of the iniene of the vrious pthologies, we onsiere the epletion of lymphoytes in ll lymphoi orgns stuie (% of mie nlyze) long with more generl estrution of the hemtopoieti omprtment (88.% of mie nlyze) to e the efining fetures of the pthology (Fig. e,f n Supplementry Fig. ). We oserve this pthology in seven inepenent experiments using 48 mie tht reeive OT-I TCR trnsue T ells, with n overll mortlity of 87.5% (Fig. ). Given tht we foun pthology in oth Ovlumin-trnsgeni mie (t not shown) n C57BL/6J mie, whih o not express the ognte ntigen reognize y the OT-I TCR (Fig. ), isese evelopment ws eviently not ue to on-trget tivity of the TCR-trnsue T ells. However, this i not rule out the possiility of trivil rossretivity of the OT-I TCR with self ntigen expresse in C57BL/6J mie. To ress this possiility, we trete ohorts of C57BL/6J mie s efore (Fig. ) n optively trnsferre to them OT-I TCR trnsue T ells, OT-I TCR trnsgeni T ells or GFP-trnsue T ells (ll tivte uner the sme onitions). As in previous experiments, reipients of OT-I TCR trnsue T ells evelope ftl utoimmune pthology. By ontrst, neither the reipients of OT-I TCR trnsgeni T ells nor the reipients of GFP-trnsue T ells evelope hexi, n % of these mie survive (Fig. ). Together, these t show tht the oserve utoimmune pthology is not ue to ross-retivity of the introue TCR ut epens on Perentge of initil oy weight f 3 Lymph noes pthology sore Spleen pthology sore 3 5 µm 5 µm 5 µm 5 µm Figure Lethl utoimmune pthology inue y OT-I TCR moifie T ells. () Experimentl setup. () Chexi in reipients of OT-I TCR trnsue (T) T ells. Shown re poole results of six inepenent experiments. Symols represent iniviul mie; rs inite group verges. For non-t versus OT-I TCR-T, P <.. () Kpln-Meyer survivl plot for reipients of OT-I TCR-T T ells (n = 47; T effiieny: 53% 7%) or non-t T ells (n = 33). Shown re poole results of seven inepenent experiments. P vlue of non-t versus OT-I TCR-T: P <.. () Bone mrrow setions from reipients of OT-I TCR-T T ells or non-t T ells showing the mrke ispperne of hemtopoieti ells in reipients of OT-I TCR-T T ells. (e) Histopthologil soring showing reution in hemtopoiesis in one mrrow n spleen of reipients of OT-I TCR-T T ells. P vlues: OT-I TCR-T versus non-t one mrrow: P <.; OT-I TCR-T versus non-t spleen: P <.. (f) Histopthologil soring showing epletion of lymphoytes in lymph noes n spleen of OT-I TCR-T T ell reipients. P vlues: OT-I TCR-T versus non-t lymph noes: P <.; OT-I TCR-T versus non-t spleen: P <.. trnsution of the infuse T ells with TCR genes. Refleting this epeneny on infusion of TCR gene moifie ell grft, we hve lle this pthology TCR gene trnsfer inue grft-versus-host isese (TI-GVHD). TI-GVHD n e expline y mixe TCR imer formtion The ove t re onsistent with the possiility tht the formtion of mixe TCR imers onsisting of enogenous n exogenous TCR hins use the oserve TI-GVHD. To test whether the expression of mixe TCR imers on TCR-trnsue T ells is suffiient to inue TI-GVHD, we trete C57BL/6J mie s efore (Fig. ) n ministere n optive trnsfer of ells trnsue with either the OT-I TCR α-hin or the OT-I TCR β-hin. Notly, we oserve TI-GVHD in reipients of T ells trnsue with only the TCR α-hin n lso in reipients of T ells trnsue with only the TCR β-hin, with slightly ifferent kinetis n iniene (Fig.,). As seon test for the involvement of mixe TCR imer expressing ells, we investigte the effet of in vivo epletion of suh ells just efore IL- ministrtion. We trnsue T ells with moifie TCR α-hin tht rries n extrellulr my tg tht hs een shown to rener trnsue T ells sensitive to in vivo epletion y the ministrtion of tg-speifi monolonl ntioies 6. In vivo epletion of T ells trnsue with the my-tgge OT-I TCR α-hin in peripherl loo ws lmost omplete in monolonl ntioy (ma)-trete mie (Fig. ), n most of these mie i not evelop TI-GVHD (75% survivl; Fig. e). By ontrst, reipients of T ells trnsue with the my-tgge OT-I TCR α-hin tht i not reeive tg-speifi ma n reipients of T ells trnsue with the nonmoifie OT-I TCR α-hin (with or without ma ministrtion) ll evelope ftl TI-GVHD (Fig. e). To etermine the unerlying mehnism ehin TI-GVHD, we nlyze serum ytokine onentrtions in C57BL/6J mie tht were trete s efore (Fig. ) n tht h reeive n optive trnsfer of OT-I TCR trnsue T ells or nontrnsue T ells. This revele moerte elevtion of monoyte hemottrtnt protein- n tumor nerosis ftor onentrtions in reipients of T ells trnsue with the OT-I TCR (Supplementry Fig. ) n sustntil inrese in interferon-γ (IFN-γ) onentrtions (verge 4.7-fol inrese; Fig. 3). To ssess whether inrese serum IFN-γ onentrtions reflete usl role of IFN-γ in the pthogenesis of TI-GVHD, we gve ohorts of C57BL/6J mie either OT-I TCR trnsue C57BL/6J ells or OT-I TCR trnsue Ifng / (lking the gene enoing IFN-γ) C57BL/6J ells. Notly, the mjority of mie tht reeive TCR-trnsue 566 VOLUME 6 NUMBER 5 MAY nture meiine

3 r t i l e s Nture Ameri, In. All rights reserve OT-I TCR Tg GFP T 3 Vα OT-I TCR α-hin T OT-I TCR β-hin T T ells tht were efiient for IFN-γ proution showe no signs of TI-GVHD upon IL- ministrtion, n 87.5% survive (Fig. 3). By ontrst, inution of TI-GVHD ws not signifintly reue when we use TCR-trnsue T ells from Gzm / mie tht re efiient in grnzyme B (Supplementry Fig. ). TI-GVHD with vrious TCRs n juvnt strtegies The ove results inite tht the formtion of mixe TCR imers on T ells trnsue with the OT-I TCR resulte in TI-GVHD in mie tht reeive TBI n high ose of IL-. To unerstn the role of the oserve pthology for the linil evelopment of TCR gene therpy, it ws ruil to estlish whether this oservtion oul e extene to other TCRs n other strtegies tht promote in vivo T ell funtion. To ress whether TI-GVHD is oserve with ifferent TCRs, we gve ohorts of C57BL/6J mie T ells tht were trnsue with one of five mouse TCRs: the melnoyte ifferentition ntigen gp-speifi pmel- TCR 7, the SV4 lrge T onogene speifi SV4 IV TCR 8, the influenz A nuleoprotein speifi F5 TCR 8, the melnoyte ifferentition ntigen tyrosinse relte protein- (TRP)-speifi TRP TCR (esrie in the Online Methos) or the OT-I TCR. Groups of mie tht reeive ells moifie with one of these TCRs, whih re either restrite y mjor histoomptiility omplex (MHC) lss I moleule H-K or H-D, ll showe ftl TI-GVHD (Fig. 4). Notly, the iniene of lethl pthology rnge from low (F5 n pmel-) to very high (OT-I), suggesting tht it will e iffiult to extrpolte lk of toxiity seen in linil trils of one TCR to other TCRs. Mrosopi n histologil nlysis revele the sme pttern of pthology s oserve with OT-I TCR trnsue T ells (t not shown). Hving shown tht TI-GVHD ours in mie reeiving high-ose IL-, we ssesse whether it lso ours in mie reeiving ifferent IL- ose or regimen. We oserve ftl TI-GVHD in mie reeiving OT-I TCR trnsue T ells omine with low-ose IL- (Fig. 5,), with n overll mortlity of 3%. Of note, 58.3% of the 3.7 Vβ OT-I TCR α-hin T OT-I TCR β-hin T 3 Figure TI-GVHD is expline y mixe-tcr imer formtion. () Kpln-Meyer survivl plot for mie reeiving OT-I TCR-T (T effiieny: 53.7%), OT-I TCR-trnsgeni, or GFP-T (T effiieny: 73.53%) T ells. P vlues: OT-I T versus OT-I trnsgeni: P =.7; OT-I T versus GFP T: P =.7. n = 6 mie per group. (,) Lethl TI-GVHD in mie reeiving T ells 65. e surviving mie evelope hroni form of TI-GVHD, hrterize y olitis, irrhe n reution in lymphoytes in the spleen (Supplementry Fig. 3, n Supplementry Tle ). Furthermore, when we restrite high-ose IL- ministrtion to single y, ftl TI-GVHD still ourre (Supplementry Fig. 3). To etermine whether the evelopment of TI-GVHD epens speifilly on IL- ministrtion, we evlute the effet of nother strtegy for promoting the in vivo funtion of TCR-trnsue T ells. Trnsforming growth ftor-β (TGF-β) signling inhiits the prolifertion n effetor funtions of ytotoxi T ells 9, n loke of this signling pthwy in optively trnsferre tumorretive T ells ugments their ntitumor funtion. To ssess the effet of loke of TGF-β signling on the toxiity of optive therpy with TCR-trnsue T ells, we trnsue these ells with ominnt-negtive TGF-β reeptor-ii (ntgfβrii) (Fig. 5). Cohorts of C57BL/6J mie were onitione with TBI n reeive Perentge Vα + (of CD8 + ells) OT-I TCR α-hin my-tg OT-I TCR α-hin my tg speifi ma 3 Dy 7 Dy OT-I TCR α-hin T OT-I TCR α-hin T + ma my tg OT-I TCR α-hin T my tg OT-I TCR α-hin T + ma trnsue with only the OT-I TCR α- or β-hin. () Chrteriztion of T ells efore optive trnsfer. Plots show live-gte CD8 + ells. () Kpln-Meyer survivl plot. P vlues: OT-I T versus non-t: P <.; OT-I α-t versus non-t: P =.4; OT-I β-t versus non-t: P =.7. n = 7 mie per group. (,e) Depletion of OT-I TCR α-hin T T ells limits ftl TI-GVHD. () Depletion of my-tgge OT-I TCR α-hin T T ells in peripherl loo fter tg-speifi ma ministrtion. Flow ytometri t represent men numer of Vα + CD8 + ells per mouse. Error rs inite s.e.m. P vlues: my-tgge OT-I α-t n ma y 7 versus y 4: P <.; OT-I α-t n ma y 7 versus y 4: P =.6. (e) Kpln-Meyer survivl plot for mie reeiving OT-I TCR α-hin T T ells (T effiieny: 65%) or mytgge OT-I TCR α-hin T T ells (T effiieny: 58%). P vlues: OT-I α-t versus non-t: P =.7; my-tgge OT-I α-t versus non-t: P =.; OT-I α-t n ma versus non-t: P =.4; my-tgge OT-I α-t n ma versus non-t: P =.5; OT-I α-t versus my-tgge OT-I α-t: P =.4; OT-I α-t versus OT-I α -T n ma: P >.5; my-tgge OT-I α-t versus my-tgge OT-I α-t n ma: P =.6. n = 6 mie per group exept for the my-tgge OT-I α-t n ma trete group (n = 7). IFN-γ (pg ml serum),, C57BL/6 non-t C57BL/6 lfng / non-t lfng / Figure 3 IFN-γ meite pthogenesis of TI-GVHD. () Inrese serum IFN-γ levels on fter optive ell trnsfer in reipients of OT-I TCR-T T ells (n = 5; T effiieny: 67%) ompre to non-t T ells (n = 6). P vlue of OT-I TCR-T versus non-t: P =.3. Dt re representtive of two experiments. () Kpln-Meyer survivl plot showing reue mortlity in mie reeiving T ells from Ifng / onor mie (T effiieny: 65%) ompre to C57BL/6J onor mie (T effiieny: 69%). P vlues: OT-I TCR-T C57BL/6J versus OT-I TCR-T Ifng / : P =.7; OT-I TCR-T Ifng / versus non-t Ifng / : P =.49; OT-I TCR-T C57BL/6J versus non-t C57BL/6J: P =.8. Numer of mie: non-t C57BL/6J, n = 6; OT-I TCR-T C57BL/6J, n = 6; non-t Ifng /, n = 5; OT-I TCR-T Ifng /, n = 8. nture meiine VOLUME 6 NUMBER 5 MAY 567

4 A r t i l e s Nture Ameri, In. All rights reserve SV4 TCR T F5 TCR T TRP- TCR T pmel- TCR T Figure 4 TI-GVHD is oserve with multiple TCRs. Kpln-Meyer survivl plot showing tht lethl TI-GVHD is oserve in mie reeiving n optive trnsfer of T ells trnsue with OT-I (T effiieny: 53% 56%), SV4 IV (T effiieny: 3% 6%), TRP (T effiieny: 6% 76%), F5 (T effiieny: 33% 37%) or pmel- TCR (T effiieny: 5% 36%). P vlues: OT-I T versus non-t: P <.; SV4 IV T versus non-t: P =.4; TRP T versus non-t: P =.5; F5 T versus non-t: P >.5; pmel- T versus non-t: P >.5. Numer of mie: non-t, n = 9; OT-I T, n = 4; F5 T, n = ; TRP T, n = 5; pmel- T, n = ; SV4 IV T, n = 8. Dt represent umultive results from four inepenent experiments. either T ells trnsue with the OT-I TCR, T ells trnsue with ntgfβrii or T ells trnsue with oth the OT-I TCR n ntgfβrii, in ll ses without IL- ministrtion. One hunre perent of reipients of T ells tht h een trnsue with OT-I TCR n ntgfβrii evelope ftl TI-GVHD (Fig. 5), gin hrterize y nemi, hemtopoieti ell estrution n more generl symptoms of GVHD (t not shown). This pthology ws not ue to n speifi effet of the introue ntgfβrii, s it ws not oserve upon infusion of ntgfβrii-moifie ells tht i not reeive the OT-I enoing trnsgene (Fig. 5). These t show tht TI-GVHD n lso our when strtegy other thn IL- ministrtion is use to promote the in vivo funtion of infuse TCR-trnsue T ells. Limiting the ourrene of TI-GVHD In view of the possile risk tht mixe TCR imer formtion hs een onsiere to pose, numer of strtegies tht im to reue the formtion of potentilly utoggressive mixe TCR imers hve een evelope. One set of strtegies is se on the use of engineere TCR genes tht hve higher propensity to form the intene TCR αβ imer 7. A oneptully unrelte strtegy tht hs een propose is the use of oligolonl or monolonl T ell popultions s reipient ells for TCR gene trnsfer 8,9. In this sitution, ssemly of mixe TCR imers is not influene, ut, euse the iversity of enogenous TCRs is highly limite, the likelihoo of generting utoretive mixe TCR imers is reue. Figure 5 TI-GVHD is oserve with ifferent strtegies tht promote in vivo T ell funtion. () Kpln-Meyer survivl plot showing lethl TI-GVHD in mie reeiving low-ose IL- fter optive trnsfer of OT-I TCR-T T ells (T effiieny: 4% 53%). P vlue of non-t versus OT-I TCR-T: P =.5. Shown re poole results of four inepenent experiments (OT-I TCR-T, n = 6; non-t, n = ). () Chnge in oy weight t y 3 fter optive ell trnsfer of OT-I TCR-T T ells or non-t T ells n tretment with low-ose IL-. Shown re poole results of four inepenent experiments. Symols represent iniviul mie; rs inite group verges. P vlue of non-t versus OT-I TCR-T: P >.5. (,) TI-GVHD is oserve when loke of TGF-β signling is use to promote in vivo T ell funtion. () Chrteriztion of OT-I TCR-T T ells (T effiieny: 53%), ntgfβrii T T ells, OT-I TCR/nTGFβRII o-t T ells (T effiieny: 46%) n non-t T ells efore optive trnsfer. Dot plots n histogrms show live-gte CD8 + ells. () Kpln-Meyer survivl plot. P vlues: OT-I/nTGFβRII o-t versus non-t: P =.6; OT-I/nTGFβRII o-t versus ntgfβrii T: P <.; OT-I/nTGFβRII o-t versus OT-I T: P =.. Numer of mie: non-t, n = 5; OT-I T, n = 6; ntgfβrii T, n = 7; OT-I/nTGFβRII o-t, n = 8. To ssess whether the use of lrgely monolonl T ell popultion s reipient ells for TCR gene trnsfer n reue mixe imer inue toxiity, we trete C57BL/6J mie s efore (Fig. ) n gve them n infusion of either F5 TCR trnsgeni T ells trnsue with the OT-I TCR or polylonl C57BL/6J T ells trnsue with the OT-I TCR (Supplementry Fig. 4). After IL- ministrtion, none of the reipients of F5 TCR trnsgeni T ells trnsue with the OT-I TCR showe signs of hexi, n survivl ws % (Fig. 6), showing tht the use of lrgely monolonl T ell popultion for TCR gene trnsfer forms vile strtegy to prevent TI-GVHD. To etermine whether geneti engineering strtegies n lso e use to limit or prevent the evelopment of TI-GVHD, we first fouse on the introution of n itionl isulfie on etween the TCR α n β onstnt omins s mens to reue mixe imer formtion. To this en, we proue T ells expressing ysteine-moifie vrints (Cys-TCR) of the OT-I n SV4 IV TCRs n evlute them for inution of TI-GVHD in sie-y-sie omprison with ells expressing the orresponing unmoifie TCRs. In reipients of ells trnsue with Cys-TCR vrints, we foun reue iniene of ftl TI-GVHD (Supplementry Fig. 4,; P =.4). However, in line with the oservtion tht ysteine moifition of TCRs n reue mixe imer formtion only prtilly 7,3, sustntil numer of mie reeiving Cys-TCR trnsue T ells still evelope ftl TI-GVHD (3% of ll mie tht reeive T ells trnsue with Cys-TCR ompre to 73% of mie tht reeive T ells trnsue with nonmoifie TCR) (Supplementry Fig. 4,). To further reue the likelihoo of mixe TCR imer formtion, we generte retrovirl vetors in whih the internl riosoml entry site (IRES) sequene tht links the TCR α- n β-hin genes ws reple with the porine teshovirus-erive PA sequene (Supplementry Fig. 4). In ontrst to n IRES sequene, PA sequene ensures equimolr proution of the introue TCR α- n β-hins, therey reuing the likelihoo of mispiring with enogenous TCR hins. Furthermore, the ft tht oth TCR hins re trnslte y the sme riosome my lso inrese the likelihoo of orret piring 3.This replement i not ffet the ntigen sensitivity of these TCRs in vitro or in vivo (t not shown). For the OT-I TCR, Perentge of initil oy weight low-ose IL- regimen + low-ose IL- regimen Low-ose IL- regimen 4 3 Vα ntgfβrii T OT-I TCR + ntgfβrii T Vβ No. ells GFP ntgfβrii T OT-I TCR + ntgfβrii T 568 VOLUME 6 NUMBER 5 MAY nture meiine

5 r t i l e s Nture Ameri, In. All rights reserve. Figure 6 Prevention of TI-GVHD y TCR engineering or y the use of oligolonl T ell popultions. () Kpln-Meyer survivl plot showing tht TI-GVHD is prevente y TCR gene trnsfer into oligolonl F5 TCR trnsgeni T ells. P vlue: OT-I TCR-T C57BL/6J versus OT-I TCR- T F5 Tg: P =.45. Numer of mie: non-t C57BL/6J, n = 4; OT-I TCR-T C57BL/6J, n = 8 (T effiieny: 65%); non-t F5 Tg, n = 5; OT-I TCR-T F5 Tg, n = 7 (T effiieny: 55%). () Kpln-Meyer survivl plot showing reue mortlity with n OT-I Cys-TCR-PA gene expression ssette ompre to the OT-I TCR-IRES gene expression ssette. P vlues: OT-I-IRES T versus OT-I Cys - TCR-PA T: P =.6; OT-I Cys - TCR-PA T versus non-t: P >.5. Numer of mie: non-t, n = ; OT-I-IRES T, n = 4 (T effiieny: 4 63%); OT-I Cys - TCR-PA T, n = 4 (T effiieny: 44 73%). () Chnge in oy weight t y 3 fter optive T ell trnsfer. Symols represent iniviul mie, rs inite group verges. P vlues: non-t versus OT-I-IRES T: P =.3; OT-I Cys - TCR-PA T versus non-t: P >.5; OT-I-IRES T versus OT-I Cys - TCR-PA T: P =.4. Dt shown in n re umultive results from three inepenent experiments. () Kpln-Meyer survivl plot showing tht lethl TI-GVHD is prevente y n SV4 IV Cys- TCR -PA gene expression ssette. P vlue: SV4 IV TCR-IRES T versus whih proue the highest iniene of pthology of the five TCRs teste, the iniene of lethl TI-GVHD ws reue from 8% to 4.3% (Fig. 6), with orresponing reution in hexi (Fig. 6). Furthermore, for the SV4 IV TCR, whih inues n only somewht lower iniene of ftl TI-GVHD, the sme engineering pprohes prevente pthology ltogether (Fig. 6). These t illustrte how strightforwr engineering strtegies n prevent n otherwise ftl utoimmune ttk. DISCUSSION In this stuy, we show tht lethl utoimmune pthology n our in mouse moels of TCR gene therpy uner vrious onitions tht promote the in vivo funtion of TCR-trnsue T ells. This therpyinue utoimmune proess results in ftl estrution of the hemtopoieti omprtment, ompnie y more generl spets of GVHD. The eviene tht this pthology is ue to the formtion of self-ntigen retive mixe TCR imers is ompelling. First, pthology oes not epen on the presene of ognte ntigen, s shown for the OT-I, F5 n SV4 IV TCRs. Seon, pthology epens on the introution of TCR genes ut is not ue to the retivity of the heteroimer itself, s shown y the lk of toxiity seen upon trnsfer of OT-I trnsgeni T ells n y the ft tht the introution of single TCR hins suffies to inue pthology. Thir, pthology n e inue not only y CD8 + T ells moifie with n MHC lss I restrite TCR ut lso y CD4 + T ells (Supplementry Fig. 5), initing tht the MHC restrition of the prentl reeptor is not relevnt. Fourth, pthology n e prevente y the in vivo epletion of T ells expressing mixe TCR imers or y the provision of ell grfts tht lk the pity to proue IFN-γ. Finlly, the most onvining eviene for the involvement of mixe imers in TI-GVHD omes from the oservtion tht pthology n e limite or prevente through the use of systems in whih the piring of enogenous n exogenous TCR hins is reue or in whih the iversity of the enogenous TCR repertoire of the trnsue T ells is highly limite. We note tht the TI-GVHD oserve here ers similrity to trnsfusion-ssoite grft-versus-host isese (TA-GVHD) 3 34, synrome most ommonly oserve when immunoompromise iniviuls reeive fresh loo from reltives. Anlogous to the toxiity oserve here, TA-GVHD is hrterize y the estrution Perentge of initil oy weight OT-I TCR- IRES T C57BL/6 non-t C57BL/6 F5 TCR tg non-t F5 TCR tg of hemtopoieti ells y infuse T ells, is rpily progressive n is nerly lwys ftl. Furthermore, in oth this stuy of TI-GVHD n in mouse moels of TA-GVHD, hemtopoieti ell estrution is t lest prtilly meite y exess proution of type I ytokines 35,36. The key question tht rises from our t is whether the toxiity seen in these mouse moels n e expete to e preitive for the linil trnsltion of TCR gene therpy. No suh toxiity ws oserve in the two phse trils performe so fr,. However, the protool for the preprtion of TCR-trnsue T ell grfts use in these trils n in prtiulr the ft tht ells were grown for ~ oul hve ontriute to the lk of oserve toxiity. In support of this ie, we oserve tht in vitro ulture of mouse TCR-trnsue T ells for lso results in sustntil reution of TI-GVHD (Supplementry Figs. 6 n 7). This oservtion fits well with eviene from erlier optive ell trnsfer stuies tht the in vivo funtion of oler T ells is highly iminishe An lterntive explntion might e provie y the oservtion tht in most ses the ell grft infuse into ptients ontine low frequeny of CD4 + T ells, n our mouse t inite tht CD4 + T ells might hve more ominnt role in the evelopment of TI-GVHD (Supplementry Fig. 5). Further oservtions suggesting tht mixe TCR imer epenent toxiity my lso eome sfety onern in the linil setting hve een me y nother group. Their work emonstrtes tht mixe TCR imers re reily forme fter TCR gene trnsfer into humn T ells, n, ruilly, tht suh mixe TCR imers n show utoretive speifiities in in vitro ssys (M. Heemskerk (Leis Universitir Meish Centrum) personl ommunition). Thus, with proof for mixe imer epenent pthology in mouse moels n eviene for the formtion of self-retive humn T ells in in vitro ssys, it n e expete tht the pthology seen here will our within the lini with the use of more vne T ell growth n engrftment regimens. The evlution n implementtion of tehnologies tht n prevent TI-GVHD shoul therefore e high priority. Methos Methos n ny ssoite referenes re ville in the online version of the pper t 3 OT-I Cys-TCR- PA T OT-I TCR-IRES T OT-I Cys-TCR-PA T 3 4 SV4 IV TCR-IRES T SV4 IV Cys-TCR-PA T 3 4 SV4 IV Cys-TCR-PA T: P <.. Numer of mie: non-t, n = 6; SV4 IV TCR-IRES T, n = 5 (T effiieny: 3% 5%); SV4 IV Cys-TCR-PA T, n = 3 (T effiieny: 37% 4%). Dt shown re umultive results from two inepenent experiments. nture meiine VOLUME 6 NUMBER 5 MAY 569

6 A r t i l e s Nture Ameri, In. All rights reserve. Note: Supplementry informtion is ville on the Nture Meiine wesite. Aknowlegments We thnk the Experimentl Animl Deprtment for niml husnry, H. vn Tinteren for vie on sttistil nlysis n R. Gomez for experimentl ssistne. We re grteful to A. Pfuth n F. vn Diepen for ssistne with flow ytometry. G.M.B. is supporte y Leukemi n Lymphom Reserh Trvel Fellowship (637). C.L. is fellow in the PhD Fellowship Progrm of Boehringer Ingelheim Fons-Fountion for Bsi Reserh in Biomeiine. T.N.M.S. n J.B.A.G.H. re supporte y grnts from The Netherlns Orgniztion for Sientifi Reserh (43--5), the Duth Cner Soiety (NKI 9-48 n 3-86), The Lnsteiner Fountion for Bloo Trnsfusion Reserh (4-8) n FP6 Integrte Projet Aoptive T ell Trgeting to Ativte Cner Killing (ATTACK). Cytotoxi T lymphoyte lone LP9 ws kinly provie y G. Aem (Rou University). AUTHOR CONTRIBUTIONS G.M.B. esigne experiments, performe experiments, nlyze n interprete t n wrote the pper. C.L. esigne experiments, performe experiments, nlyze n interprete t n wrote the pper. A.I.H. esigne experiments, performe experiments, nlyze n interprete t. L.B. performe experiments n nlyze t. M.A..W. n A.J. me the initil oservtion of hexi n rpi eth of mie in mouse moels of TCR gene therpy. A.D.M.K. performe experiments n interprete t. N.P. n R.D. generte n provie the TRP TCR sequenes. E.K. n W.U. provie the Tg-speifi ma n vie on in vivo epletion. J.-Y.S. performe experiments n nlyze t. J.B.A.G.H. interprete t. T.N.M.S. esigne experiments, interprete t n wrote the pper. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Pulishe online t Reprints n permissions informtion is ville online t reprintsnpermissions/.. Rosenerg, S.A. Progress in humn tumour immunology n immunotherpy. Nture 4, ().. Theol, M. et l. Tolerne to p53 y A.-restrite ytotoxi T lymphoytes. J. Exp. Me. 85, (997). 3. Romieu, R. et l. Pssive ut not tive CD8 + T ell se immunotherpy interferes with liver tumor progression in trnsgeni mouse moel. J. Immunol. 6, (998). 4. Shumher, T.N. T-ell-reeptor gene therpy. Nt. Rev. Immunol., 5 59 (). 5. Gttinoni, L., Powell, D.J. Jr., Rosenerg, S.A. & Restifo, N.P. Aoptive immunotherpy for ner: uiling on suess. Nt. Rev. Immunol. 6, (6). 6. Vrel-Rohen, A. et l. Control of HIV- immune espe y CD8 T ells expressing enhne T-ell reeptor. Nt. Me. 4, (8). 7. Morris, E.C., Tsllios, A., Benle, G.M., Xue, S. & Stuss, H.J. A ritil role of T ell ntigen reeptor-trnsue MHC lss I restrite helper T ells in tumour protetion. Pro. Ntl. A. Si. USA, (5). 8. e Witte, M.A. et l. TCR gene therpy of spontneous prostte rinom requires in vivo T ell tivtion. J. Immunol. 8, (8). 9. e Witte, M.A. et l. Requirements for effetive ntitumor responses of TCR trnsue T ells. J. Immunol. 8, (8).. Morgn, R.A. et l. Cner regression in ptients fter trnsfer of genetilly engineere lymphoytes. Siene 34, 6 9 (6).. Johnson, L.A. et l. Gene therpy with humn n mouse T-ell reeptors meites ner regression n trgets norml tissues expressing ognte ntigen. Bloo 4, (9).. e Witte, M.A. et l. Trgeting self-ntigens through llogenei TCR gene trnsfer. Bloo 8, (6). 3. Kneko, S. et l. IL-7 n IL-5 llow the genertion of suiie gene moifie lloretive self-renewing entrl memory humn T lymphoytes. Bloo 3, 6 5 (9). 4. Overwijk, W.W. et l. Tumor regression n utoimmunity fter reversl of funtionlly tolernt stte of self-retive CD8 + T ells. J. Exp. Me. 98, (3). 5. Shriknt, P. & Mesher, M.F. Opposing effets of IL- in tumor immunotherpy: promoting CD8 T ell growth n inuing poptosis. J. Immunol. 69, (). 6. Kiek, E., Chro, J., Sommermeyer, D., Blnkenstein, T. & Ukert, W. A sfegur elimintes T ell reeptor gene moifie utoretive T ells fter optive trnsfer. Pro. Ntl. A. Si. USA 5, (8). 7. Jorritsm, A. et l. Seleting highly ffine n well-expresse TCRs for gene therpy of melnom. Bloo, (7). 8. Kessels, H.W., Wolkers, M.C., vn en Boom, M.D., vn er Vlk, M.A. & Shumher, T.N. Immunotherpy through TCR gene trnsfer. Nt. Immunol., (). 9. Thoms, D.A. & Mssgue, J. TGF-β iretly trgets ytotoxi T ell funtions uring tumor evsion of immune surveillne. Cner Cell 8, (5).. Gorelik, L. & Flvell, R.A. Immune-meite erition of tumors through the loke of trnsforming growth ftor-β signling in T ells. Nt. Me. 7, 8 ().. Benle, G.M., Hnen, J.B. & Shumher, T.N. Prelinil evelopment of T ell reeptor gene therpy. Curr. Opin. Immunol., 9 4 (9).. Seestyén, Z. et l. Humn TCR tht inorporte CD3ζ inue highly preferre piring etween TCRα n β hins following gene trnsfer. J. Immunol. 8, (8). 3. Willemsen, R.A. et l. Grfting primry humn T lymphoytes with ner-speifi himeri single hin n two hin TCR. Gene Ther. 7, (). 4. Voss, R.H. et l. Moleulr esign of the Cαβ interfe fvors speifi piring of introue TCRαβ in humn T ells. J. Immunol. 8, 39 4 (8). 5. Cohen, C.J., Zho, Y., Zheng, Z., Rosenerg, S.A. & Morgn, R.A. Enhne ntitumor tivity of murine-humn hyri T-ell reeptor (TCR) in humn lymphoytes is ssoite with improve piring n TCR/CD3 stility. Cner Res. 66, (6). 6. Cohen, C.J. et l. Enhne ntitumor tivity of T ells engineere to express T-ell reeptors with seon isulfie on. Cner Res. 67, (7). 7. Kull, J. et l. Filitting mthe piring n expression of TCR hins introue into humn T ells. Bloo 9, (7). 8. Heemskerk, M.H. et l. Reprogrmming of virus-speifi T ells into leukemi-retive T ells using T ell reeptor gene trnsfer. J. Exp. Me. 99, (4). 9. Weinhol, M., Sommermeyer, D., Ukert, W. & Blnkenstein, T. Dul T ell reeptor expressing CD8 + T ells with tumor- n self-speifiity n inhiit tumor growth without using severe utoimmunity. J. Immunol. 79, (7). 3. Thoms, S. et l. Trgeting the Wilms tumor ntigen y TCR gene trnsfer: TCR vrints improve tetrmer ining ut not the funtion of gene moifie humn T ells. J. Immunol. 79, (7). 3. Ukert, W. & Shumher, T.N. TCR trnsgenes n trnsgene ssettes for TCR gene therpy: sttus in 8. Cner Immunol. Immunother. 58, 89 8 (9). 3. Greenum, B.H. Trnsfusion-ssoite grft-versus-host isese: historil perspetives, iniene, n urrent use of irrite loo prouts. J. Clin. Onol. 9, (99). 33. Shroeer, M.L. Trnsfusion-ssoite grft-versus-host isese. Br. J. Hemtol. 7, (). 34. Aght, K., Altints, N.D., Topeli, A., Gokoz, O. & Ozee, O. Trnsfusion-ssoite grft-versus-host isese in immunoompetent ptients: se series n review of the literture. Trnsfusion 47, 45 4 (7). 35. Bloom, M.L. et l. A mouse moel of lymphoyte infusion-inue one mrrow filure. Exp. Hemtol. 3, 63 7 (4). 36. Tng, Y., Desierto, M.J., Chen, J. & Young, N.S. The role of the T H trnsription ftor T-et in mouse moel of immune-meite one-mrrow filure. Bloo 5, (). 37. Kolen, S. et l. Bioistriution n retention time of retrovirlly lele T lymphoytes in mie is strongly influene y the ulture perio efore infusion. J. Immunother. 5, (). 38. Gttinoni, L. et l. Aquisition of full effetor funtion in vitro proxilly impirs the in vivo ntitumor effiy of optively trnsferre CD8 + T ells. J. Clin. Invest. 5, (5). 39. Zhou, J. et l. Telomere length of trnsferre lymphoytes orreltes with in vivo persistene n tumor regression in melnom ptients reeiving ell trnsfer therpy. J. Immunol. 75, (5). 57 VOLUME 6 NUMBER 5 MAY nture meiine

7 Nture Ameri, In. All rights reserve. ONLINE METHODS Mie. We otine C57BL/6J mie from Chrles River Lortories n F5 TCR-trnsgeni mie n OT-I TCR-trnsgeni mie from The Experimentl Animl Deprtment of The Netherlns Cner Institute. Ifng / (B6.9S7- IfngtmTs/J) mie n Gzm / (B6.9S-Gzmtmley/J) mie were otine from The Jkson Lortory. We performe ll mouse experiments in orne with institutionl n Duth guielines, n they were pprove y the Experimentl Animl Committee of The Netherlns Cner Institute. Retrovirl onstruts, retrovirl trnsution n optive trnsfer of T ells. We rete pmx retrovirl vetors in pmx-tcrα-ires-tcrβ onfigurtion for OT-I 9, SV4 IV (speifi for the SV4 lrge-t onogene) 8, F5 (speifi for the influenz-a-nuleoprotein) 8, pmel- (speifi for the melnoyteifferentition ntigen gp) 7, TRP (speifi for the TRP-melnoyte ifferentition ntigen), OT-I Cys, SV4 IV Cys n TRP Cys TCRs. We rete pmx retrovirl vetors in pmx-tcrβ-pa-tcrα onfigurtion for the OT-I Cys n SV4 IV Cys TCRs. The OT-I, SV4 IV, pmel-, n TRP TCR genes n erivtives use in this stuy were gene optimize 9,7 y GeneArt (GeneArt). TRP TCR DNA ws erive from the ytotoxi T lymphoyte lone LP9 4 (kinly provie y G. Aem, Rou University). TCR-V enoing DNAs were otine y progressive 5 mplifition retions of DNA ens strting from onserve sequenes of TCR-C omins. Full TCR α- n β-hin sequenes were lone into the retrovirl vetor pbullet n sulone into the retrovirl vetor pmx. We vlite TRP TCR α- n β-hins (sequenes ville upon request) with respet to surfe expression n retivity y flow ytometry. For experiments involving the sole introution of the OT-I TCR α-hin, OT-I TCR β-hin or my-tgge OT-I TCR α-hin, we generte pmx-ot-iα, pmx-ot-iβ n pmx-my tgge OT-Iα onstruts, respetively. We lone the ntgfβrii into the pmx retrovirl vetor to otin pmx-ntgfβrii-ires-gfp onstrut. All retrovirl onstruts were use to trnsfet Phoenix-E pkging ells to generte retrovirus 4. We moifie mouse splenoytes y retrovirl trnsution s esrie 8. One y efore optive ell trnsfer, irrition-inue host onitioning ws hieve y 5 Gy TBI with rioiology onstnt potentil X-ry unit (Pntk HF-3; Pntk Limite). We trete mie with n optive ell trnsfer ontining 6 TCR-trnsue CD8 + T ells on y n, where inite, high-ose (7. 5 IU) or low-ose (.8 4 IU) IL- (Proleukin, Novrtis) intrperitonelly twie ily etween n fter optive ell trnsfer. For the epletion of my-tgge TCR-trnsue T ells, we trete mie with µg of my-speifi ma (9E) on 7, 9 n. Flow ytometry. We mesure surfe TCR expression 4 h fter trnsution y flow ytometry. Cells were stine with the following mas (ll from BD Biosienes): FITC-onjugte ntioy to TCR Vβ5 n phyoerythrin (PE) onjugte ntioy to TCR Vα for the OT-I TCR; FITC-onjugte ntioy to TCR Vβ9 n PE-onjugte ntioies to TCR Vβ, Vβ3, Vβ4, Vβ5, Vβ8, Vβ n Vβ ( SV4 Vβ-pool ntioies) for the SV4 IV TCR; PE-onjugte ntioy to TCR Vβ3 n FITC-onjugte ntioy to TCR Vβ4, Vβ5, Vβ8, Vβ8.3, Vβ9, Vβ, Vβ, Vβ3 n Vβ4 ( TRP Vβ-pool ntioies) for the TRP TCR PE-onjugte ntioy to TCR Vβ n FITC-onjugte ntioy to TCR Vβ4, Vβ5, Vβ8, Vβ8.3, Vβ9, Vβ, Vβ3 n Vβ4 ( F5 Vβ-pool ntioies) for the F5 TCR; FITC-onjugte ntioy to TCR Vβ3 n PE-onjugte ntioy to TCR Vβ, Vβ3, Vβ4, Vβ5., Vβ8., Vβ n Vβ ( pmel- Vβ-pool ntioies) for the pmel- TCR. We stine T ells o-trnsue with the OT-I TCR n ntgfβrii with PE-onjugte ma to TCR Vα, iotin-onjugte ma to TCR Vβ5, PerCP Cy5-onjugte ma to CD8α n llophyoyninonjugte streptviin (BD Biosienes). Propiium ioie (Sigm) ws use to selet for live ells. We quire n nlyze t on FsCliur (BD Biosienes) with FlowJo softwre (Tree Str) or CELLQuest-Pro softwre (BD Biosienes). Cytokine nlysis. We etermine IFN-γ, tumor nerosis ftor, IL-, IL-6, monoyte hemottrtnt protein- n IL-p7 serum onentrtions using the BD Cytometri Be Arry mouse inflmmtion kit (BD Biosienes) oring to the mnufturer s instrutions. Histopthology. We ollete orgns n tissues inluing gstrointestinl trt, liver, kiney, lung, hert, spleen, lymph noes, thymus, Peyer s pthes n one mrrow n smple them in EAF (ethnol eti i formlin) fixtive. Prffin setions were stine with H&E. We efine pthologil ltertions (suh s lymphoyti or inflmmtory infiltrtions n reution or epletion of lymphoi or hemtopoieti omprtments in vrious orgns) semiquntittively s mil (gre ), moerte (gre ) n severe (gre 3). We reviewe setions using Zeiss Axioskop Plus mirosope (Crl Zeiss Mirosopy) equippe with Pln-Apohrom ( 5/.6, /.45, /.6, n 4/.95) n Pln-Neoflur (.5/.75) ojetives. Imges were pture with Zeiss AxioCm HR igitl mer n proesse with AxioVision 4 softwre (oth Crl Zeiss Vision). Sttistil nlysis. We ompre survivl urves using log-rnk (Mntel-Cox) test. We ompre hnges in oy weight n ifferenes in serum ytokine onentrtions using two-tile Stuent s t test. We ompre histopthologil sores using Cohrne-Armitge tren test n ext two-sie P vlues re reporte. P vlues <.5 were onsiere signifint. Aitionl methos. Detile methoology is esrie in the Supplementry Methos. 4. Shreurs, M.W. et l. Denriti ells rek tolerne n inue protetive immunity ginst melnoyte ifferentition ntigen in n utologous melnom moel. Cner Res. 6, (). 4. Kessels, H.W., Shepers, K., vn en Boom, M.D., Tophm, D.J. & Shumher, T.N. Genertion of T ell help through MHC lss I-restrite TCR. J. Immunol. 77, (6). oi:.38/nm.8 nture meiine