ARTICLES. Host-reactive CD8 + memory stem cells in graft-versushost. Yi Zhang, Gerard Joe, Elizabeth Hexner, Jiang Zhu & Stephen G Emerson

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1 Host-retive CD8 + memory stem ells in grft-versushost disese Yi Zhng, Gerrd Joe, Elizeth Hexner, Jing Zhu & Stephen G Emerson Grft-versus-host disese (GVHD) is used y lloretive donor T ells tht trigger host tissue injury. GVHD develops over weeks or months, ut how this immune response is mintined over time is unknown. In mouse models of humn GVHD, we identify new suset of postmitoti lo CD8 + T ells tht generte nd sustin ll llogenei T-ell susets in GVHD retions, inluding entrl memory, effetor memory nd effetor CD8 + T ells, while self-renewing. These ells express S-1, CD122 nd Bl-2, nd indue GVHD upon trnsfer into seondry reipients. The postmitoti lo CD8 + Tells persist throughout the ourse of GVHD, re generted in the initil phse in response to llontigens nd dendriti ells nd require interleukin-15. Thus, their long life, ility to self-renew nd multipotentility define these ells s ndidte memory stem ells. emory stem ells will e importnt trgets for understnding nd influening diverse hroni immune retions, inluding GVHD. A hllmrk of T ell medited immune retions to persistently expressed ntigens, inluding responses to hroni infetions, tumor ells nd utontigens, is the ontinul genertion of ntigen-speifi effetor nd memory T ells 1 9. One importnt linil instne of suh persistent immune retion is GVHD in individuls reeiving llogenei one mrrow trnsplnttion (llo-bt), in whom hostretive donor T ells persist throughout the ourse of the disese Given tht ntigen-speifi effetor T ells re terminlly differentited nd short-lived ells 1,2,6,15,16, there must exist ellulr mehnisms y whih effetor nd memory T ells speifi for persistent ntigens re mintined in the host. It hs een hypothesized tht distint pool of memory stem ells might exist nd ould ount for sustining effetor nd memory T ells 1,2, ut heretofore no suh popultion hs yet een isolted. The immune response of donor T ells ginst host tissues in llo-bt offers unique opportunity to study the development of donor effetor nd memory T-ell responses ginst persistent ntigens 1 14,17. emory T ells derive from proliferting T ells tht re tivted during the primry immune response nd survive the poptoti ontrtion of effetor ells 2,6,16, Although some studies suggest tht T ells with memory phenotype isolted from norml donors re defetive in mediting GVHD 17,26 3, we reently showed in mouse models tht lloretive memory T ells re deteted oinident with the poptoti ontrtion of effetor T ells, nd persist in hosts with GVHD. In vivo dministrtion of these lloretive memory T ells uses severe GVHD in seondry reipients 31. The importne of lloretive memory T ells in mediting host tissue injury hs lso een supported y studies of individuls reeiving humn leukoyte ntigen (HLA)-identil siling or unrelted donor BT 17,32,33. Together, these dt suggest tht oth lloretive effetor nd memory T ells re responsile for persistent host tissue injury. Identifying ell suset ple of persisting in hosts with GVHD nd generting oth host-retive effetor nd memory ells would therefore provide n importnt insight into understnding nd potentilly interditing GVHD retions. Using mouse models of humn GVHD direted ginst minor histoomptiility (H) ntigens, we now identify previously unhrterized popultion of host-retive donor postmitoti lo CD8 + T ells tht express high levels of S-1, CD122 (or interleukin (IL)-2 nd IL-15 reeptor ) nd Bl-2. These postmitoti lo CD8 + T ells re le to selfrenew nd hve greter pity to generte ll popultions of memory nd effetor ells thn either effetor memory or entrl memory T ells, fulfilling the riteri for memory stem ells 1,2,34. Isoltion nd trnsplnttion of these ells into irrdited seondry hosts uses GVHD. Thus, postmitoti lo CD8 + T ells inlude memory stem ells tht hve ruil role in sustining lloretive T ells responsile for persistent host-tissue injury in GVHD. RESULTS Identifition of postmitoti lo CD8 + Tells To determine how lloretive memory nd effetor CD8 + T ells re mintined during the evolution of GVHD, we trnsplnted roxyl fluoresein suinimidyl ester ()-leled donor nive lo CD8 + T ells isolted from C3H.SW (H-2D,CD )mie, together with syngenei B6/SJL T ell depleted one mrrow (TCDB), into lethlly irrdited B6/SJL reipient mie (H-2D, CD45.1) to indue GVHD 26,35 (Supplementry Fig. 1 online). We nlyzed donor CD8 + T ells tht were reovered from the spleens nd livers of B6/SJL reipients with ongoing GVHD on dy 42 fter Deprtments of ediine nd Peditris, University of Pennsylvni Shool of ediine, Room 51, loney, 36 Sprue Street, Phildelphi, Pennsylvni 1914, USA. Correspondene should e ddressed to S.G.E. (emersons@mil.med.upenn.edu). Reeived 2 y; epted 15 Otoer; pulished online 2 Novemer 25; doi:1.138/nm1326 NATURE EDICINE VOLUE 11 [ NUBER 12 [ DECEBER

2 d 85.3 ± ± 2.9 Before BT 2.3 ±.8 hi lo hi lo Reovery rte (output/input, perent) llo-bt (Fig. 1). ost donor dy 42 CD8 + T ells (85.3 ± 6.7%) expressed n effetor nd effetor memory phenotype ( hi lo ); 4.7 ± 2.9% were hi entrl memory ells; nd 2.3 ±.8% of the donor-derived dy 42 CD8 + T ells were lo (Fig. 1), reminisent of nive T ells 6,15,36. But intensity ws very low in these ltter donor dy 42 CD8 + T ells (Fig. 1), inditing tht dy 42 lo CD8 + T ells hd undergone extensive prolifertion in vivo while retining nive T ell like phenotype. Bsed on the intensity of, we termed the ells tht hd diluted s postmitoti ells nd those ells tht hd not diluted s premitoti ells. To test the ility of host-retive donor dy 42 CD8 + T-ell susets to proliferte nd differentite into effetor nd memory ells in response to host minor H ntigens, we seprted eh of these CD8 + T-ell susets using fluoresene-tivted ell sorter, releled them with nd ex vivo stimulted them with llogenei B6 one ,41 Nive CD ,21 hi lo 8 1,1 hi 7 lo lo hi hi Nive Dys fter ex vivo ulture lo lo CD8 + Donor CD8 + T ells/ reipient ( 1 3 ) CD8 + T ells Figure 1 Identifition of postmitoti lo CD8 + T ells. () A low dose of -leled donor C3H.SW lo CD8 + T ells (CD45.2) ws trnsplnted together with B6/SJL TCDB (CD45.1) into B6/SJL reipients (n ¼ 42). Dot plots show perent of donor CD8 + T-ell susets in gted CD CD8 + ells tht were reovered from spleen nd liver of B6/SJL reipients with GVHD 42 d fter trnsplnttion (men ± s.d.). () FACS-sorted donor dy 42 CD8 + T-ell susets were releled with nd stimulted with reipient B6/SJL one mrrow derived DCs + IL-2 + IL-7 + IL-15 for 15 d. Dot plots show the dilution of in eh CD8 + T-ell suset nd perent of lo CD8 + T ells reovered t dy 15 fter ulture. () Perent of ell reovery (output versus initil input numer of CD8 + T ells) of eh dy 42 CD8 + T-ell suset in the ultures t the indited time points. Dt re presented s men ± s.d. from one of three independent experiments. (d) Numer of donor T ells (men ± s.d.) reovered from spleen, lymph nodes, liver nd peripherl lood of seondry B6/SJL reipients (three mie per group) of eh donor dy 42 CD8 + T-ell suset (8 1 4 ells/reipient) nd donor nive lo CD8 + T ells 7 d fter doptive trnsfer. Results re one of two independent experiments. mrrow derived dendriti ells (DCs) + IL-2 + IL-7 + IL-15 or doptively trnsferred them into lethlly irrdited seondry B6/SJL mie. After the ex vivo stimultion for 15 d, we found tht dy 42 lo CD8 + T ells primrily differentited into effetor nd effetor memory ells, while lso produing seondry entrl memory nd lo ells (Fig. 1). In ontrst, dy 42 hi CD8 + entrl memory T ells predominntly generted effetor nd effetor memory with renewl of entrl memory ells ut not lo ells, wheres dy 42 hi lo CD8 + effetor nd effetor memory ells produed only effetor ells (Fig. 1). Overll, dy 42 lo CD8 + T ells expnded in vitro more vigorously thn the hi ell popultions, inresing 11-fold versus 2.6-fold for dy 42 hi ells, nd no net expnsion for dy 42 hi lo ells (Fig. 1). Similrly, we reovered 3.6-fold nd 2.4-fold more donor CD8 + T ells from mie reeiving dy 42 Figure 2 lo CD8 + memory stem T ells indue GVH responses. Donor hi lo, hi nd lo CD8 + T-ell susets (CD45.2) were sorted on dy 42 fter trnsplnttion from B6/SJL mie with GVHD reeiving low dose of donor C3H.SW CD8 + T ells (CD45.2) nd B6/SJL TCDB (CD45.1), nd doptively trnsferred, together with donor C3H.SW TCDB, into lethlly irrdited seondry B6/SJL mie. C3H.SW TCDB lone ws infused s ontrol. Weight hnges () nd survivl rte () of lethlly irrdited seondry B6/SJL mie reeiving donor CD8 + T-ell susets. Dt shown here represent two independent experiments (four to six mie per group). () Histologil exmintion of the livers hrvested from the ontrol reipients (TCDB), nd seondry B6/ SJL mie reeiving donor dy 42 hi lo, dy 42 hi,nddy42 lo CD8 + T-ell susets using H&E nd immunohistohemistry stining with ntiody to CD8 (four mie per group). Originl mgnifition, 4 (left) nd 2 (right). Weight hnges (perent) Perentge of mie tht survived 1 5 TCDB 5 hi Dy42 lo hi lo hi lo Dys fter trnsplnttion TCDB lo hi 4 hi lo Dys fter trnsplnttion TCDB lo hi hi lo H&E CD8 13 VOLUE 11 [ NUBER 12 [ DECEBER 25 NATURE EDICINE

3 Figure 3 Host minor H ntigen H6-driven development of postmitoti lo CD8 + Tells.() Dot plots show perent of H6- speifi lo H6 + nd hi H6 + Tells in gted donor CD CD8 + ells tht were mgnetilly isolted on dy 21 (n ¼ 7) nd dy 35 (n ¼ 12), respetively, fter trnsplnttion from spleen nd liver of BALB.B mie (CD45.2) with GVHD reeiving donor B6/SJL TCDB nd B6/SJL T ells (CD45.1). B6/SJL nive T ells efore BT were stined s ontrols. Dt re representtive of three independent experiments. () Dot plots in left pnel show perentge of eh CD8 + T-ell susets in gted donor CD CD8 + T ells in the spleens nd livers from primry GVHD BALB.B reipients t dy 35 fter trnsplnttion of donor B6/SJL TCDB nd B6/SJL T ells. These dy 35 T-ell susets were sorted, releled with nd doptively trnsferred together with B6 (CD45.2) TCDB into irrdited seondry BALB.B mie (three mie per group). Dot plots in right pnel show perent of lo CD8 +, minor H ntigen H6-speifi CD8 + nd IFN-g produing CD8 + T Before BT ells in gted donor CD CD8 + T ells tht were reovered from these seondry BALB.B reipients of eh donor CD8 + T ell suset t dy 4 fter doptive trnsfer. () Perent of reovered donor CD8 + T ells versus initil input numer of eh dy 35 T-ell suset in the livers nd spleens of these seondry BALB.B reipients (three mie per group) of eh donor T-ell suset. Dt re presented s men ± s.d. of three mie. lo CD8 + ells 7 d fter doptive trnsfer into lethlly irrdited seondry reipients versus mie reeiving dy 42 hi lo ells nd dy 42 hi ells, respetively (Fig. 1d). Furthermore, in vivo dministrtion of these donor dy 42 lo CD8 + ells into lethlly irrdited seondry reipients lso indued linil GVH responses, s mnifested y weight loss, utneous inflmmtion nd deth (Fig. 2,). Pthologi nlysis showed the infiltrtion of mononuler ells nd CD8 + ells in portl res of the liver from mie reeiving donor dy 42 CD CD8 + Dys fter BT H6 (CD CD8 + gted) (CD CD8 + gted) hi lo hi lo Reovery rte of donor CD8 + T ells (output versus totl input, perent) H6 Spleen lo hi lo hi lo lo CD8 + ells nd in those mie reeiving dy 42 hi CD26L lo CD8 + ells, ut not in those reeiving dy 42 hi CD26 hi CD8 + ells (Fig. 2), onfirming the linil findings. These results suggest tht effetor memory nd entrl memory CD8 + ells represent ells tht re not terminlly differentited, ut hve limited ility to self-renew upon persistent ntigeni stimultion. In ontrst, dy 42 lo CD8 + T ells re postmitoti ells tht hve the pity to vigorously proliferte nd hve the ility to self-renew nd to generte oth entrl memory nd effetor IFN-γ hi lo Liver Nive CD8 + FSC hi lo SSC Figure 4 Chrteriztion of postmitoti lo CD8 + Tells.() DonorCD8 + T ells (CD45.2) were mgnetilly seprted on dy 42 fter trnsplnttion from spleen nd liver of GVHD B6/SJL mie (CD45.1, n ¼ 12) reeiving C3H.SW lo CD8 + T ells (CD45.2) + B6/SJL TCDB, nd stined with ntiodies s indited. Nive CD8 + T ells were isolted from norml C3H.SW mie s ontrols (n ¼ 6). The vlues in the histogrms represent the perentges of ells in eh gted CD8 + T-ell suset. The forwrd stter (FSC) nd side stter (SSC) show the properties of the ell size nd density. () RT-PCR results show the expression of hemokine reeptors, perforin nd 18S in eh donor C3H.SW dy 42 hi lo, hi nd lo CD8 + T-ell suset. Nive lo CD8 + T ells were isolted from norml C3H.SW mie (n ¼ 6) s ontrols S-1 CD122 Bl-2 CD25 CD127 IFN-γ Grnzyme-B 18S Prf1 Cr7 Cr5 Cxr3 Cxr4 Cr2 Nive lo 1. lo hi 4. GVHD CD8 + Nive lo hi lo NATURE EDICINE VOLUE 11 [ NUBER 12 [ DECEBER

4 Figure 5 Postmitoti lo CD8 + T ells develop during the initil phse of GVH retions. () Dot plots show perent of eh CD8 + T-ell suset in gted donor CD CD8 + T ells tht were seprted on dy 14 fter trnsplnttion from spleen nd liver of lethlly irrdited B6/SJL mie (CD45.1) reeiving leled C3H.SW lo CD8 + nive T ells (CD45.2) nd C3H.SW TCDB. Histogrm shows perentge of dividing nd nondividing ell popultions in gted donor dy 14 lo CD8 + Tells.(,) Dot plots show dilution in eh donor dy 14 CD CD8 + T-ell suset tht ws sorted y FACS (). Eh sorted donor CD8 + T-ell suset ws releled with nd doptively trnsferred into irrdited seondry B6/SJL mie. Dot plots show perent of lo CD8 + T nd dilution in gted donor-derived CD8 + T ells tht were reovered t dy 7 fter doptive trnsfer from spleen of seondry reipients of eh donor dy 14 CD8 + T-ell suset (). One representtive of two seprte experiments is shown. memory ells. This onlusion is further supported y results in seond mouse GVHD model of donor B6/SJL into reipient BALB.B, in whih host minor H ntigen H6-speifi donor lo H6 + nd postmitoti lo CD8 + T ells omprised 3.6% nd 7.5% of donor CD8 + T ells in BALB.B reipients with GVHD on dy 35 fter llo-bt, respetively (Fig. 3,). Forty dys fter doptive trnsfer into seondry BALB.B reipients, postmitoti lo CD8 + T ells self-renewed nd generted sustntilly more memory nd effetor ells with the ility to ind to H6 nd produe IFN-g thn did trnsferred effetor memory nd entrl memory ells (Fig. 3,). Phenotype of postmitoti lo CD8 + Tells We next sought to hrterize postmitoti lo CD8 + T ells to distinguish them from unstimulted nive CD8 + T ells. Similr to unstimulted nive lo CD8 + T ells isolted from C3H.SW mie, dy 42 postmitoti lo CD8 + ells were smll in size, did not produe IFN-g, expressed low levels of CD25 nd grnzyme B nd equivlent levels of CD127 (IL-7 reeptor ; Fig. 4). Unlike nive lo CD8 + T ells, however, dy 42 postmitoti lo CD8 + T ells were S-1 hi nd expressed high levels of CD122 nd the ntipoptoti moleule Bl-2 (Fig. 4). In ontrst, oth effetor nd effetor memory nd entrl memory CD8 + T ells were heterogeneously lrge in size, produed IFN-g, nd expressed lower levels of CD122, CD127, Bl-2 nd S-1 (Fig. 4). RT-PCR showed tht oth dy 42 postmitoti lo CD8 + nd unstimulted nive lo CD8 + T ells expressed similrly higher levels of Cr7 nd lower levels of Prf1 (whih enodes perforin) when ompred to effetor memory T ells Sorted CD8 + suset 64 Figure 6 Development of postmitoti lo CD8 + T ells depends on oth DCs nd IL-15. () -leled C3H.SW lo CD8 + Tellswere ultured in the presene of B6/SJL one mrrow derived DCs + IL-2 + IL-15. Vlues shown in the histogrm represent the perentge of ells in eh frtion in gted donor lo CD8 + T ell popultion tht ws olleted t dy 6 fter ultures. () -leled C3H.SW lo CD8 + nive T ells were ultured for 14 d in the presene of B6 DCs + IL-2 + IL-15, together with ontrol IgG, CTLA-4, solule IL-15 reeptor or neutrlizing ntiody to CD122. The numers of lo lo nd lo hi CD8 + T ells shown re representtive of three independent experiments. () -leled C3H.SW lo CD8 + nive T ells were ultured in the presene of IL-2 + IL-15, B6 DCs + IL-2 (5 ng/ ml), or B6 DCs + IL-15(12 ng/ml). Dot plots show perent of lo lo ells in gted CD8 + T ells tht were olleted t dy 5 fter ulture lo lo hi hi lo hi lo (Fig. 4). In ontrst, Cr5 nd Cxr3, whose expression ws not deteted in nive lo CD8 + T ells, were indued in postmitoti lo CD8 + ells (Fig. 4). On omprison, ll these tested T-ell susets expressed similr levels of 18S mrna tht ws inluded s internl ontrol (Fig. 4). Thus, dy 42 postmitoti lo CD8 + T ells re S-1 hi CD122 hi Bl-2 hi ells with inresed expression of Cr5 nd Cxr3 mrna, whih re lerly distinguishle from oth nive nd mture memory CD8 + T ells. Genertion of postmitoti lo CD8 + T ells To determine the sequene of the development of nive T ells into postmitoti lo CD8 + ells during GVHD, we nlyzed donor CD8 + T ells reovered from the reipient spleens nd livers on dy 14 fter trnsplnttion (termed dy 14-CD8 +, Fig. 5). All hi lo nd most hi CD8 + ells hd divided more thn seven times, wheres lo CD8 + ells hd undergone vrying numer of divisions nd onsisted of oth 7.8% premitoti ( hi ) nd 92% postmitoti ( lo ) ells Reovered ells ( 1 3 ) IL-2+IL-15.9 Io hi 6 d fter ex vivo ulture 21.4 DC+IL Control IgG CTLA-4F IL-15 reeptor α CD122-speifi ntiody Io Io S DC+IL-15 Nive Io CD S VOLUE 11 [ NUBER 12 [ DECEBER 25 NATURE EDICINE

5 (Fig. 5). When eh of these donor dy 14 CD8 + T ell susets ws sorted (Fig. 5), releled with nd doptively trnsferred into irrdited seondry B6/SJL reipients, eh proliferted in vivo nd differentited into hi lo effetor nd effetor memory ells (Fig. 5). Notly, dy 14 postmitoti lo lo CD8 + ells generted oth 7.6% lo nd 12.% hi T ells in these reipients, wheres dy 14 hi lo nd dy 14 premitoti lo hi CD8 + T ells gve rise to only.3% nd.8% of lo ells, respetively (Fig. 5). Unlike donor dy 42 entrl memory T ells tht did not generte postmitoti lo CD8 + ells (Fig. 1), dy 14 hi CD8 + T ells produed oth lo (12.3%) nd hi (23.7%) ells (Fig. 5). These dt suggest tht lthough postmitoti lo CD8 + T ells re derived from popultion of slowly proliferting lo CD8 + ells, they n lso e generted from hi CD8 + entrl memory phenotype ells during the primry immune response. To explore the ellulr nd moleulr mehnisms y whih donor postmitoti lo CD8 + T ells re produed, we ultured -leled donor nive lo CD8 + T ells in vitro in the presene of host dendriti ells (DCs) supplemented with IL-2 nd IL-15. We identified oth posmitoti S-1 hi lo lo (21.4%) nd premitoti S-1 hi lo hi (78.6%) CD8 + T ells 6 d lter (Fig. 6). Addition of solule IL-15 reeptor, neutrlizing ntiody to CD122 or CTLA-4-IgG mrkedly deresed the numer of postmitoti lo CD8 + ells in these ultures, with neutrlizing ntiody to CD122 eing the most potent regent (Fig. 6). In ontrst, ex vivo stimultion of nive lo CD8 + T ells with host DCs + IL-15 inresed the proportion of postmitoti lo CD8 + ells s ompred to host DCs + IL-2 (Fig. 6). Both host DCs nd donor DCs supported the survivl of donor lo CD8 + T ells irrespetive of ytokines (Supplementry Fig. 2 online). Furthermore, in the sene of host DCs, ex vivo stimultion with IL-2 + IL-15 lone did not indue the genertion of postmitoti lo ells from nive lo CD8 + T ells (Fig. 6). Thus, postmitoti S-1 hi lo CD8 + T ells n e generted in vitro upon stimultion with DCs nd IL-15. DISCUSSION These studies identify nd hrterize new popultion of postmitoti lo CD8 + T ells tht persists in reipients with GVHD. This ell popultion does not produe IFN-g nd ytotoxi moleules, nd requires host DC stimultion for its self-renewl, prolifertion nd differentition into effetor nd memory ells. In ontrst to unstimulted nive CD8 + T ells, lo CD8 + T ells re postmitoti, express Sl-1 hi nd higher levels of CD122 nd Bl-2, nd hve enhned pity to expnd upon reenounter of host minor H ntigens. Although these lo CD8 + T ells represent smll popultion in numer, they re le to expnd to greter extent thn effetor memory nd entrl memory CD8 + T ells to generte seondry effetor nd memory ells, using GVHD. These dt suggest tht postmitoti lo CD8 + T ells re n ntigen-primed ell popultion tht hs n importnt role in sustining lloretive T ells in these GVHD models. In ordne with stem ell model 34, memory stem ells hve the ility to self-renew nd hve the pity of long term prodution of effetor ells nd ll memory ell susets. Although memory stem ell properties hve een suggested to reside within the entrl memory omprtment 1,2, in our experiments, CD8 + T ells with memory stem ell poteny were lerly lolized to the postmitoti lo CD8 + T-ell frtion one the GVHD response ws mture (for exmple, dy 42). Notly, during the first stge of the primry lloimmune response (dy 14), efore the pperne of true memory ells (dy 22) 6, hi s well s lo CD8 + T ells seemed to e the immedite preursors for the postmitoti lo CD8 + T ells tht would eventully form the memory stem ell pool. Thus, it is possile tht CD8 + T ells with hi phenotype ontin two funtionl susets, preursors for memory stem ells tht develop erly in primry immune response nd entrl memory ells fter the quisition of true memory funtion. Postmitoti lo CD8 + T ells were lso identified in GVHD B6/SJL mie reeiving donor C3H.SW CD8 + T ells nd C3H.SW TCDB. But it is diffiult to distinguish whether these memory stem ells develop from trnsplnted donor mture T ells or from engrfted donor TCDB in B6/SJL reipients. To rigorously ssess whether trnsplnted mture T ells re le to generte memory stem ells, in most of our experiments we otrnsplnted donor C3H.SW CD8 + T ells nd syngeni B6/SJL TCDB to estlish ute GVHD in B6/SJL reipients. Although the use of this omintion required slightly lower dose (3 1 5 )ofcd8 + T ells to llow for B6/SJL one mrrow reonstitution, ll B6/SJL reipients developed ute GVHD nd survived over 75 d fter trnsplnttion, permitting isoltion nd hrteriztion of donor mture T ell derived memory stem ells. Identifition of lloretive memory stem ells is importnt for understnding the pthophysiology of GVHD. Our reent studies hve shown tht lloretive effetor memory T ells persist in reipients with GVHD nd ount for the persistent host tissue injury 31.As effetor memory T ells represent pre terminlly differentited ells, onstnt ntigeni stimultion of these ells ould potently elerte the trnsition of effetor memory to end-stge effetor T ells, ultimtely resulting in the exhustion nd/or deletion of ntigenspeifi T ells, s hs een proposed in virl infetions 5,6,15,16.Suh phenomenon ould explin the grdul resolution of GVHD in some individuls reeiving llo-bt. On the other hnd, lloretive T ells n persist during the progression of GVHD. This implies tht there must exist pthologi mehnism(s) y whih lloretive T ells re effetively mintined without exhustion. Our findings indite tht lthough preterminl lloretive effetor memory T ells themselves hve some pity for self-renewl, lo CD8 + memory stem ells hve greter pity to regenerte effetor nd effetor memory T ells, whih ontriutes to the susequent persistene of lloretive T ells in vivo. We hve previously shown tht ex vivo depletion of lo CD8 + T ells (inluding lo CD8 + memory stem ells) from host DC-stimulted T ells efore trnsplnttion prevents GVHD 26. Our results rise the possiility tht it is the loss of these CD8 + memory stem ells tht rogtes the ility of these ex vivo mnipulted T ells to indue severe GVHD. Developing tehniques to deplete lloretive memory stem ells in vivo should prove to e vlule pproh for further evluting their effet on the persistene nd progression of GVHD. IL-15 hs een shown to e one of the ruil ytokines to the genertion nd mintenne of memory CD8 + T ells 8, Our in vitro ulture results show tht IL-15 nd DCs indue the development of memory stem ells nd the susequent differentition into effetor, effetor memory nd entrl memory T ells. In ddition, reent study further onfirms tht donor one mrrow derived IL-15 is ruil for the in vivo genertion of lloretive effetor memory T ells in reipients nd the development of ute GVHD 43. But t wht stge in the indution, mplifition or effetor phse of GVHD IL-15 is involved remins to e determined 43. We found tht NATURE EDICINE VOLUE 11 [ NUBER 12 [ DECEBER

6 ddition of IL-15 enhned the genertion of host DC-indued memory stem ells, wheres ddition of IL-15 ntgonists, suh s solule IL-15 reeptor nd neutrlizing ntiody to CD122, mrkedly deresed the in vitro genertion of memory stem ells nd their prolifertion nd differentition into effetor memory T ells. On the other hnd, in the sene of DCs, the omintion of IL-2 + IL-15 lone did not indue the survivl nd prolifertion of memory stem ells (dt not shown). Thus, oth IL-15 nd DCs re ruil not only for the genertion of memory stem T ells, ut lso the survivl, self-renewl nd differentition of memory stem ells into mture memory ells. Our results rise importnt questions out the role of donor versus host ntigen-presenting ells (APCs) in stimulting the survivl nd funtionl differentition of lloretive memory stem ells. We nd others hve found tht host APCs re essentil to inititing CD8 + T ell medited GVHD 35, Priming donor CD8 + T ells with host APCs for s little s 24 h is suffiient to their susequent funtionl differentition into GVHD effetors 35,45. But more thn 99.% of host DCs re eliminted y 5 d fter llo-bt 35 nd most host B lymphoytes nd mrophges re repled y engrfted donor TCDB y dy 4 fter llo-bt 45,46. On the other hnd, we find tht DCs re le to mintin the homeostti survivl nd prolifertion of syngenei nive T ells in vitro. Therefore, it is possile tht donor-derived APCs re le to support the survivl of lloretive memory stem ells in vivo. Others hve shown tht B6 mie reeiving wild-type C3H.SW CD8 + T ells together with mjor histoomptiility omplex lss I defiient TCDB develop GVHD, ut muh less severe thn B6 reipients of wild-type C3H.SW CD8 + T ells + wild-type TCDB 45. These results suggest tht donor-derived APCs re required for the persistene nd progression of GVHD, proly through regulting the survivl nd funtionl differentition of lloretive memory nd memory stem ell pools tht re generted fter initil priming on host APCs. Cells with the phenotype of CD8 + memory stem ells seem to e involved in humn pthogen-speifi immune responses in whih ntigeni stimultion persists. In smples otined from individuls with HIV, for exmple, one study identified four distint susets of HIV-speifi CD8 + T ells: CD45RA + CCR7 + (2%), CD45RA CCR7 + (5%), CD45RA CCR7 (7%), nd CD45RA + CCR7 (5%) 5. Similrly, ytomeglovirus-speifi, tumor-speifi nd Epstein Brr virus speifi CD45RA + CD8 + T ells hve lso een shown 3 5,7.Inthese humn systems, the ntigen-speifi CD45RA + CCR7 + CD8 + T ells with the nive phenotype showed no diret ytotoxi tivity, ut ould e tivted in vitro to proliferte nd produe effetor funtion nd IFN-g 4,5,7. Thus, lthough the prolifertive, self-renewl nd in vivo funtions of these ells were not tested in detil in these studies, they my represent similr memory stem ell suset to the one we hve identified. However, whether this ntigen-speifi CD45RA + CCR7 + CD8 + T-ell suset is indeed the humn ounterprt of mouse memory stem ells, nd whether lloretive memory stem ells exist in individuls with GVHD fter llo-bt, remin to e determined. In summry, we hve identified new suset of memory stem ells tht persists throughout the ourse of GVHD. The identifition of memory stem ells hs key implitions for understnding the pthophysiology of GVHD nd developing new pprohes for GVHD prophylxis nd therpy. oreover, explortion of the physiology of memory stem ells should yield further insights into the mehnisms of memory T-ell development nd mintenne of T-ell responses ginst tumors nd pthogens, s well s new pprohes for mnipulting hroni immune responses in linil settings, inluding GVHD, grft rejetion nd ner. ETHODS ie. We purhsed B6/SJL (H-2D, CD ), C57BL/6 (B6; H-2D, CD ), C3H.SW (H-2D, CD nd Ly9.1 + ) mie nd BALB.B (H-2D, CD ) from Jkson Lortory. We supplied BT reipients with drinking wter ontining neomyin sulfte nd polymyxin B (Sigm) s previously desried 26,35. The Institutionl Animl Cre nd Use Committee of University of Pennsylvni pproved ll mouse protools. Antiodies, ell lines, ytokines nd flow ytometry nlysis. We otined iotinylted ntiody to mouse nd ll other ntiodies used for immunofluoresene stining from BD Biosiene Phrmingen. We purhsed miroed-onjugted ntiodies nd streptvidin from iltenyi-bioteh, nd ll the reominnt ytokines inluding IL-2, IL-4, IL-7, IL-15, grnuloytemonoyte olony-stimulting ftor (G-CSF), stem ell ftor (SCF) nd tumor nerosis ftor (TNF)- from R&D Systems. NIAID Tetrmer Fility prepred H6/HC-I tetrmer (H2K ). In some experiments, we prepred H6 HC-I dimers y onjugting H6 to HC-I dimers s instruted y the mnufturers (BD Biosiene). We performed immunofluoresene nlyses of ell-surfe phenotypes nd intrellulr ytokines using FACSn (Beton Dikinson) s previously desried 26,35. Cell preprtions. We prepred TCDB y inuting donor one mrrow with miroed-onjugted CD4-speifi ntiody nd CD8-speifi ntiody s previously desried 26,35. We mgnetilly isolted CD8 + nd CD4 + Tells from spleens nd lymph nodes of mie using miroed-onjugted CD8-speifi ntiody nd CD4-speifi ntiody, respetively (iniacs, iltenyi Bioteh). We mgnetilly removed CD11 + ells y CD11speifi ntiody onjugted miroeds efore purifying donor T ells. We further seprted lo CD8 +, lo CD8 +, hi lo CD8 +, hi CD8 + T-ell susets from mgnetilly purified CD8 + Tells tht were stined with APC-onjugted ntiody to nd PE-onjugted ntiody to using fluoresene-tivted ell sorter (oflo, Cytomtion). The purity of eh sorted T-ell suset ws onsistently 495%. We leled donor CD8 + T ells with s previously desried 35. We prepred mture DCs from B6/SJL one mrrow s previously desried 47. Briefly, we sequentilly stined B6/SJL B with iotinylted -kit speifi ntiody nd streptvidin-onjugted miroeds (iltenyi Bioteh). We then mgnetilly sorted -kit + hemtopoieti progenitor ells nd inuted these ells for 6 d in Isove modified Duleo medium (ID; Gio) ontining 1% FBS, G-CSF, SCF nd IL-4. We stimulted immture DCs with G-CSF + TNF- for n dditionl 2 d to indue their mturtion. GVHD indution. ie underwent llo-bt s previously desried 26,35. Briefly, for C3H.SW nti-b6 mouse GVHD, we irrdited B6/SJL reipients t dose of 1. Gy in two frtions from 137 Cs soure. We mixed C3H.SW TCDB (5 1 6 ), with or without C3H.SW lo CD8 + T ells ( ), trnsplnted into B6/SJL reipients through til vein injetion (four to eight mie per group per experiment) fter irrdition. In some experiments, we mixed B6/SJL TCDB ( ) with C3H.SW lo CD8 + Tells ( ) nd trnsplnted into lethlly irrdited B6/SJL reipients. In the donor B6/SJL into reipient BALB.B mouse GVHD model, we irrdited BALB.B reipients t dose of 8.5 Gy in two seprte frtions. We mixed B6/SJL TCDB ells (5 1 6 ), with or without -leled B6/SJL T ells (ontining CD4 + nd CD8 + ), trnsplnted into BALB.B reipients through til vein injetion. We weighed reipient mie twie weekly nd monitored the linil signs of ute GVHD nd survivl s estlished 48. ie were killed nd speimens of liver, skin nd intestine were olleted for histopthologi ssessment of GVHD s previously desried 49. Ex vivo stimultion of CD8 + Tells.We reovered donor nive lo CD8 + T ells from norml C3H.SW mie, or donor CD8 + T-ell suset from GVHD reipients of T ell replete llo-bt, nd ultured these T ells in ID ontining 1% FBS, with or without ddition of IL-2 (5 ng/ml), IL-7 (3 ng/ml) nd IL-15 (3 ng/ml) in 96-well plte, with or without ddition of B6/SJL one mrrow derived mture DCs t DC:T ell rtio of 1:3. In some experiments, CTLA-4, solule IL-15 reeptor, neutrlizing ntiody to CD122 or ontrol IgG ws dded t onentrtion of 1 mg/ml into the ultures. 134 VOLUE 11 [ NUBER 12 [ DECEBER 25 NATURE EDICINE

7 RT-PCR. We isolted totl RNA from highly purified CD8 + T-ell susets using the RNesy min kit (Qigen). We performed RT-PCR using Qigen OneStep RT-PCR kit s instruted y the mnufturer (Qigen). PCR onditions were s follows: 37 yles of denturing t 94 1C for 45 s, nneling t 58 1C for 45 s nd extension t 72 1C for 1 min, followed y one yle of finl 1-min extension t 72 1C. The oligonuleotides used for RT-PCR were s follows: Cr2 forwrd (5 -ATGGAAGACAATAATATGTTACC-3 ) nd reverse (5 -ATGACA AGGCTCACCATC-3 ); Cxr4 forwrd (5 -ATGGAACCGATCAGTGTGAG-3 ) nd reverse (5 -GATGGTGGGCAGGAAGATCC-3 ); Cxr3 forwrd (5 -ATCAG CGCTTCAATGCCAC-3 ) ndreverse(5 -TGGCTTTCTCGACCACAGTT-3 ); Cr5 forwrd (5 -CATCGATTATGGTATGTCAGCACC-3 ) ndreverse(5 -CAG AATGGTAGTGTGAGCAGGAA-3 ); Cr7 forwrd (5 -TGTGCTTCTGCCAAG ATGAGG-3 ) nd reverse(5 -GGAGGAAAAGGATGTCTGCCAC-3 ); nd Prf1 forwrd (5 -CCTCCTATGGCACGCACTTTATC-3 ) nd reverse(5 -CGCGTT CTGTTCTTCCAGTAATGTG-3 ). Housekeeping gene rrna 18S ws mplified y RT-PCR using Certified LUX Primer Sets s ontrols (Invitrogen). To ssess the reltive levels of gene expression in different ells, we ompred the rtio of eh tested mrna to the internl ontrol 18S mrna using Imge Qunt (oleulr Dynmis). Sttistil nlysis. We nlyzed survivl dt using the ntel-peto-cox summry of w 2. We onsidered proility (P) vlueso.5 s signifint. Note: Supplementry informtion is ville on the Nture ediine wesite. ACKNOWLEDGENTS We ppreite the pthology support given y D. Frnk (Deprtment of Pthology nd Lortory ediine, University of Pennsylvni Shool of ediine). This work is supported y grnt from the Speilized Center for Reserh from the Leukemi nd Lymphom Soiety of Ameri nd y grnt RO1 CA from US Ntionl Institutes of Helth. COPETING INTERESTS STATEENT The uthors delre tht they hve no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Feron, D.T., nders, P. & Wgner, S.D. 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Immunol. 161, (1998). 24. Opfermn, J.T., Oer, B.T. & Ashton-Rikrdt, P.G. Liner differentition of ytotoxi effetors into memory T lymphoytes. Siene 283, (1999). 25. Wlzer, T., Arpin, C., Beloeil, L. & rvel, J. Differentil in vivo persistene of two susets of memory phenotype CD8 T ells defined y nd CD122 expression levels. J. Immunol. 168, (22). 26. Zhng, Y. et l. Dendriti ell-tivted hicd8+ T ells re defetive in mediting ute grft-versus-host disese ut retin grft-versus-leukemi tivity. Blood 13, (24). 27. Giver, C.R., Li, J.., Hossin,.S., Lonil, S. & Wller, E.K. Reonstruting immunity fter llogenei trnsplnttion. Immunol. Res. 29, (24). 28. Foster, A.E. et l. Humn - memory T ells re less responsive to llontigen stimultion thn + nive T ells: potentil for doptive immunotherpy. Blood 14, (24). 29. Chen, B.J., Cui, X., Sempowski, G.D., Liu, C. & Cho, N.J. Trnsfer of llogenei - memory T ells without grft-versus-host disese. Blood 13, (24). 3. 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Prevention of grft versus host disese y intivtion of host ntigen- presenting ells. Siene 285, (1999). 45. tte, C.C. et l. Donor APCs re required for mximl GVHD ut not for GVL. Nt. ed. 1, (24). 46. Duffner, U.A. et l. Host dendriti ells lone re suffiient to initite ute grftversus-host disese. J. Immunol. 172, (24). 47. Zhng, Y. et l. Bifurted dendriti ell differentition in vitro from murine linege phenotype-negtive -kit+ one mrrow hemtopoieti progenitor ells. Blood 92, (1998). 48. Cooke, K.R. et l. An experimentl model of idiopthi pneumoni syndrome fter one mrrow trnsplnttion: I. The roles of minor H ntigens nd endotoxin. Blood 88, (1996). 49. del Rosrio,.L., Zuli, J.R. & Ko, K.J. Prevention of grft-versus-host disese y indution of immune tolerne with ultrviolet B-irrdited leukoytes in H-2 disprte one mrrow donor. Blood 93, (1999). NATURE EDICINE VOLUE 11 [ NUBER 12 [ DECEBER