Department of Immunology nduction of Immune tolerance Yair Reisner Anna Aronovich, Iby adult Esther Bachar-Lustig, Yaki Edelshtein, Gil Hecht, stem cells and by committed Helena Katchman, Yael Klionsky, Assaf Lask, Oren Milstein, embryonic stem cells Eran Ophir, Shlomit Reich-Zeliger, Induction of tolerance to BM in part, to the failure of these cells to Chava Rozen, Eli Shezen, allografts by central memory- co-localize with the host T cells at the Dalit Tchorsh, Lior Zangi like CD8+ cells LNs during the critical time at which the The induction of immune tolerance latter are stimulated. by specific agents, as opposed to In order to enhance LNs homing we 972 8 934 4023 general immune suppression, is a developed culture conditions that favor most desirable goal in transplantation the acquisition of central memory- FAX 972 8 934 4145 biology. One approach to attain like (Tcm) phenotype by the anti [email protected], this goal is afforded by the use of 3rd-party CD8+ cells, and demonstrated www.weizmann.ac.il/immunology/ veto CTLs. Anti 3rd-party veto CTLs, efficient localization of these cells to depleted of anti host reactivity, were the host LNs. However, we discovered ReisnerPage.html developed in our lab and were shown to that Ag-activated CD8+ cells are not support engraftment of T-depleted BM homogenous in their veto activity, as grafts. However, although these cells anti 3rd-party Tcm cells display poor 6.5 days following the adoptive transfer, displayed highly efficient veto activity veto activity in vitro, in contrast to veto three-fold more Tcm cells than veto in vitro, they were only effective in vivo CTLs. Nevertheless, in vitro, reactivation CTLs could be recovered from various if applied in 1000 veto/effector ratio of the Tcm cells restores their veto organs of the recipient mice and Tcm and only in conjunction with short-term activity by causing them to differentiate cells persisted in vivo in significant treatment with the immunosuppressive into CD62Llow effector cells. When Tcm numbers 80 days post transplant. drug Rapamycin. In the present study cells were adoptively transferred into The high proliferative and persistence we show that the veto CTLs do not lethally irradiated recipients of BM in vivo exhibited by the Tcm cells, in home to the host LNs upon adoptive allografts, they acquired an effector addition to their marked LN homing, was transfer and we hypothesize that the phenotype and a significant amount of found to be associated with enhanced relatively low efficiency of veto CTLs cells with CD62Llow effector phenotype tolerance induction in our stringent in vivo could be attributed, at least were found to be in the LNs. Moreover, mouse model for T cell mediated BM allograft rejection. Thus, while anti-3rd party veto CTLs, bearing an effector memory phonotype, exhibited poor enhancement of engraftment in the absence of treatment with Rapamycin, adoptive transfer of Tcm cells, with no further treatment, led to 94% overall i survival and long term donor type chimerism (Fig. 1).

A Novel mechanism for CTL mediated veto activity: CTLs respond to recognition by alloreactive T cell with activation and cytotoxic granule secretion. The mechanism by which the anti- 3rd party CTLs exert the veto effect is the subject of intense research in our laboratory (Fig. 2). Cytotoxic T

Weizmann Institute of ScienceWeizmann lymphocytes (CTLs) are endowed with ∙ potent veto activity. As such, they induce death of alloreactive T cells directed against them, in a manner that ∙ 2008 does not require engagement of their own TCR with MHC-p of the alloreactive cells. Thus, CTLs are currently being evaluated in patients for their potential Fig. 1 Comparison of tolerance induction by Tcm like cells Vs veto CTLs. Lethally irradiated to induce specific transplantation (10Gy) C3H (H-2K) recipients were inoculated with1.25x104 HTC. Twenty-four hours later, tolerance. CTL veto activity has been these recipients were transplanted with 3x106 Balb/c- Nude BM (H-2d) supplemented shown to be mediated by engagement with 107 anti-3rd party CTL or Tcm like cells that originated from (C3HxBALB/C)F1 (H-2kd) of the CTL CD8 molecule with the donors. Life Science Open Day alloreactive T cell MHC class I3 domain. transgenic CD8 T cell, alloreactive alloreactive cell, H-2towards T CD8 transgenic bev CLalratv CD8 CTL-alloreactive observe to time first the for imaging apply we CTL study, low current the at In and concentrations. points time late at assayed when veto activity FasL in CTL Fas/ for role a shown further have We loetv cl i a C (H-2 2C a is cell the alloreative which in activity veto of model a at level cell-cell early time For points. this we employed the at interactions rgn (H-2 origin released from intracellular stores and PKC is activated, thus inducing granule polarization polarization granule inducing thus activated, is PKC and stores intracellular from released Activation and cytolytic degranulation of CTLs being recognized by cognate T cells: cells: T cognate by recognized being CTLs of degranulation cytolytic and Activation 2 Fig. and degranulation leading to the death of the recognizing T cell. The CTL, on the other other the on CTL, The cell. T recognizing the of death the to leading degranulation and a step-by-step model. (a) Cell-cell interaction is initiated by TCR-dependent recognition of of recognition TCR-dependent by initiated is interaction Cell-cell (a) model. step-by-step a hand, receives a survival signal and is induced to proliferate. (b) A hypothetical model model hypothetical A (b) proliferate. to induced is and signal survival a receives hand, the CTL by the cognate T cell. TCR-mediated signaling in the recognizing cognate T cell cell T cognate recognizing the in signaling TCR-mediated cell. T cognate the by CTL the explaining CTL activation in the absence of TCR ligation. Molecules segregate according according segregate Molecules ligation. TCR of absence the in activation CTL explaining augments integrin-dependent adhesion dramatically, bringing the apposing cell membranes membranes cell apposing the bringing dramatically, adhesion integrin-dependent augments to size at the interface of contacts intitiated by TCR recognition. Thus, in the contacts contacts the in Thus, recognition. TCR by intitiated contacts of interface the at size to to close proximity. This allows the CTL CD8 molecule to bind the recognizing T cell MHC-I3 MHC-I3 cell T recognizing the bind to molecule CD8 CTL the allows This proximity. close to formed by recognition of the CTL by cognate T cell, TCR molecules are segregated from from segregated are molecules TCR cell, T cognate by CTL the of recognition by formed domain, an interaction that normally suffers from low affinity. CD8 engagement initiates initiates engagement CD8 affinity. low from suffers normally that interaction an domain, phophatases with large extra-cellular domains such as CD45. Due to the existance of of existance the to Due CD45. as such domains extra-cellular large with phophatases Src kinase signaling via its association with LcK. Through several signaling steps, Ca steps, signaling several Through LcK. with association its via signaling kinase Src constitutive phosphorylation of CD3 ITAMs, this enables TCR/Src kinase signaling in the the in signaling kinase TCR/Src enables this ITAMs, CD3 of phosphorylation constitutive CTL independent of TCR ligation. CD8-MHC-I ligation may further facilitate this signaling by by signaling this facilitate further may ligation CD8-MHC-I ligation. TCR of independent CTL inducing Lck kinase activity. kinase Lck inducing b X H-2 d , and the CTL is of an F1 an of is CTL the and , d ) and thus readily readily thus and ) + T cell cell T b ) TCR TCR ) completely abolishes killing (2% killing ±2.5). abolishes completely killing is strictly granule mediated as as the intracellular Ca mediated granule strictly is killing when hours at cultured a 1:1 ratio. This of 2C cells are killed by F1 withinCTLs 5 (±1.2)46% that found Accordingly,we killing. mediated granule of indicative was and contacts of 71% in occurred This 2C cells. the with area contact the CTLs F1 that polarize their cytotoxic granules towards revealed Confocal microscopy it. towards alloreactivity void of yet cell, 2C the by recognizable ++ chelator Bapta-AM Bapta-AM chelator ++ is is alternative route to T cell activation. T cell to route alternative relevant physiologically a suggesting Src kinase signaling and PKC activation, yet involving both of engagement, TCR absence the in initiated event signaling wide-ranging a this from that originates killing and them against cells directed T of killing capable mediated are granule of CTLs that these show results Collectively, recruitment. this eliminated completely signaling kinase Src blocking Remarkably, cell. 2C the clearly recruited to the contact area with revealed imaging that in PKC39% CTL of conjugates Confocal studied. was activity veto during localization its CTLs, by killing dependent TCR during site contact cell-cell the to recruited is (PKC) C would be necessary. As PKCactivated kinase θ protein secretion, of granule activation elicit to signaling control CTLs (54% ±5%). For Src kinase ±2.3, p<0.01) to compared PP3 (21%treated killing reduced significantly PP2 veto activity. Pretreatment of with CTLs CTL on PP2 inhibitor kinase Src effect the of the determined we Lck, is cell the into CD8 from signal a relaying for molecule candidate granule primary the CD8 on As polarization. effect detrimental CTL a had the blocking Moreover, ±2.2). (46% Ab. control to ±7.7,(23% cells 2C p<0.01) compared of killing reduce significantly to was found and added was form allelic CD8 CTL the blocks specificaly which 2.1) Toactivity. Ab (Lytthat end, a blocking veto its mediated granule evaluate in to importance us prompted killing, mediated granule dependent TCR for documented domain I3 class MHC cell target the with molecule CD8 of CTL the interaction the of importance The to degranulation is not initiated by TCR. leading event signaling MHC-p,the cell alloreactive F1 the recognize not As do CTLs minutes. 35 of time median a within follows death cell T that alloreactive and contact after rapidly occurs This applied. polarization granule CTL was that revealed imaging cell live death, cell alloreactive with secretion and polarization granule To (±4.2). link 15% directly to killing reduces A concanamycin as perforin on reliant is killing mediated granule Moreover, this is θ is ii Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science iii Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science Correction of hemophilia by implantation of pig E42 tissue. (A) PTT values of of values PTT (A) tissue. spleen E42 pig of implantation by hemophilia of Correction 4 Fig. model. in amouse simulation tissues: precursor pig embryonic of in cells of Treg human shelf’ the serum ‘Off the in levels insulin Pig 3 Fig. wild-type NOD-SCID control mice (n = 10), factor VIII-KO SCID control mice (n = 10), and and 10), = (n mice control SCID VIII-KO factor 10), = (n mice control NOD-SCID wild-type mice. transplanted grow in fully immunocompetent mice mice and immunocompetent fully engraft in grow also can that implants shown such was and It potential immunogenicity. growth teratoma, for risk on based pancreas pig embryonic for choice optimal the as defined was E42 tissues. precursor embryonic pig gestational ‘window’ for transplantation of different optimal the defined have factor VIII-KO SCID mice (n =37) transplanted with E42 pig spleen at different time intervals intervals time different at spleen pig E42 with transplanted =37) (n mice SCID VIII-KO factor after transplantation. Data contain seven independent experiments. (B) Chromogenic Chromogenic (B) experiments. independent seven contain Data transplantation. after determination of factor VIII activity in plasma of transplanted mice (n =21). Comparison Comparison =21). (n mice transplanted of plasma in activity VIII factor of determination of factor VIII-KO SCID control mice (gray), wild-type NOD-SCID control mice (black), and and (black), mice control NOD-SCID wild-type (gray), mice control SCID VIII-KO factor of factor VIII-KO SCID mice transplanted with E42 pig spleen at different time intervals after after intervals time different at spleen pig E42 with transplanted mice SCID VIII-KO factor transplantation. (C) Survival after tail clipping of NOD-SCID control (black) and factor VIII-KO VIII-KO factor and (black) control NOD-SCID of clipping tail after Survival (C) transplantation. SCID (white) mice with and without E42 spleen transplantation. The data represent averages averages represent data The transplantation. spleen E42 without and with mice (white) SCID of three independent experiments (n = 5 in each experiment). each in 5 = (n experiments independent three of uig h ps fw er we years few past the During abc rg, xiie pg insulin in found those pig to similar levels blood exhibited Tregs, Balb/c donor host 4 D C t s o h r o r o n o d t a h t indicated transplantation marrow bone suppressive agents. immune conventional with and CTLA4-Ig or anti-CD40L under suppression immune substantial under of the transplant, of transplant, the of same day the on the addition, the with with protocol treated mice grafts; the rejected FTY720 and Rapamycin protocol a by comprising T cell treated debulking using MoAb, tissue E42 recipients of C57BL immunocompetent fully while Thus, pancreas. pig E42 of engraftment enhance will Tregs party third that possibility a the model in mouse tested further we study present our In induction. tolerance for source ‘off-the-shelf’ rejection, viable new a afford could cells Treg party third that preventing suggesting in type cells donor as Treg effective as party were cells third that we Recently, demonstrated tissue. rejection transplanted the of reduce (Tregs) cells T Furthermore, studies of allogeneic allogeneic of studies Furthermore, ex-vivo + CD25 + expanded expanded regulatory immune suppression protocols. protocols. current suppression of further toxicity enable immune the and outcome might reducing the longer improve of using administration modalities different and studies follow-up ‘off-the-shelf’ individual. Additional mismatched use a possible from Tregs be successfully might to it pancreas pig embryonic of recipients human in that anticipate We implant. pig of growing the rejection prevented donor, mouse mismatched a from Tregs party third minimal pig pancreas, mice embryonic receiving in that suggests with data Our infiltration. elements contained pancreatic 3). and large (Fig. were grafts transplant The after months 2 of period follow-up a over mice SCID spleen transplantation transplantation spleen by fetal diseases monogenic of treatment for concept of proof a as hemophilia of Correction by the growing spleen tissue. growing byspleen the enzyme or a factor that can be replaced an of deficiencies genetic T for modality of treatment novel a appearance provide could cells, the before developmental stage a at obtained spleen, the fetal for a of transplantation principle that concept of results proof These a 4). provide (Fig. levels blood tail VIII factor for assays by by and bleeding demonstrated after as months transplant, of 2–3 alleviation within complete hemophilia to led mice SCID hemophilic into tissue spleen 42 day embryonic of Transplantation VIII. and the mice, growing tissue factor expressed SCID into transplantation upon cells, exhibited optimal growth potential day 42 stage, before the appearance embryonic of the at T harvested spleen pig fetal a that Weshow spleen. donor the mediated by mature disease, T cells derived host from versus graft aggressive with associated were spleen allogeneic or Gaucher disease by transplantation of hemophiliaas such deficiencies genetic correct to attempts clinical Previous

Bachar-Lustig, E.,Bachar-Lustig, Reich-Zeliger, S., publications Selected liver failure. chronic of treatment the for approach A new disease: Wilson of in correction hepatocytes isolated to superior are fragments liver Fetal of chronic liver failure or metabolic metabolic or diseases. failure liver treatment chronic the of in approach curative liver fetal a could offer fragments novel These of transplantation that suggest results transplantation. hepatocytes embryonic liver fragments over isolated of advantage the emphasizing further group, this in noted was tissue hepatic regenerating transplanted by liver host with cirrhotic of replacement complete near transplanted animals Inaddition,liverfragments. embryonic in only noted was in accumulation copper reduction liver significant a hepatocytes. or However, fragments either of recipients in found was levels) normal two of -35% (30 restoration tested serum ceruloplasmin transplantation, When after months cirrhosis. liver copper, decreased of eventually and accumulation hepatic by ceruloplasmin, serum of levels characterized milk), in mice mutated the ATP7B gene (Toxic were tested for their to capacity correct E15 hepatcyts Thus, isolated or E15. fragments tissue at defined was age gestational teratoma-free earliest fragments the liver mouse embryonic of liver disease. of chronic treatment the in hepatocytes isolated to superior of be model mouse can that this approach Wilson’s disease a in demonstrate further we work host present quiescent the In the liver. differentiation of setting the and in to growth leads window, marked time optimal gestational at harvested fragments liver embryonic pig of transplantation that demonstrated have results recent Our liver. host in quiescent the cells hepatic of can enable proliferation transplanted that approaches alternative for need the emphasize hepatocytes isolated of transplantation by failure liver acute of Reisner, Y. (2003) Anti-third-party Based on syngeneic transplantation transplantation syngeneic on Based Disappointing results in the treatment Anna Aronovich, Tchorsh,Dalit Aronovich, Anna Yair Reisner, Shlomit Reich-Zeliger Dalit Eventov-Friedman, Smadar Steiner, D., N., Blazar, Brunicki, BR., S., Eventov-Friedman, Katchman, Reich-Zeliger, S., E., Bachar-Lustig, Reich-Zeliger, S., Gan, J., Bachar-Lustig, monogenic diseases by fetal spleen spleen by fetal diseases monogenic of treatment for concept of proof a as hemophilia of Correction Tal Orna Cohen, Yair and Reisner. Blazar, R. Bruce Sivan Martinowitz, Uri Elias Shezen, Friedman, Eventov- Smadar Katchman, Helena Transplant 11:366-372. 2006. Organ Opin Curr transplantation. marrow in bone Veto of cells role The Bachar–Lustig. Esther and print]. of [Epub ahead 3(7): Med e215. PloS 2006. Diabetes. Treatment the Transplantation for of Tissue Pancreatic Pig Embryonic Yair and Freud, Enrique Reisner. r.Bruce Blazar, Tal, Orna Ilan Feine, Rechavi, Gideon Dekel, Benjamin Hecht, Gil Aronovich, Anna Shezen, Elias Katchman, Tchorsh, Helena Hematol., 34, 66-7.Exp CD4+CD25+ Treg cell. “off-the-shelf” by third-party Tolerance induction E.,Bachar-Lustig, Reisner, Y. (2006) 102,2928-33. USA., Sci Acad Natl Proc windows. time distinct on depends teratoma without organogenesis optimal transplantation for a source as liver, pig lung and pancreas, Reisner, Y. (2005) Embryonic Tchorsh, D., E., B. Freud, Dekel, A., H., E., Shezen, Aronovich, Immunol.. 173, 6654-9. J cell. veto different of reactivity Relative model.I 2C mouse transgenic TCR in the CTLs by veto induction Gan, J., Reisner, Y. (2004) Tolerance 6660-6. J Immunol., 173, apoptosis. ligand by Fas-Fas cells effector of Deletion II model. 2C mouse transgenic TCR in the CTLs by veto induction E., Reisner, Y. (2004) Tolerance 102,1943-50.Blood., dose. cell BM and rapamycin with synergism allografts: hematopoietic of rejection overcome CTLs veto Negri. Mario Farmacologiche, Ricerche Di Instituto Research. Transplantation Biology for Center Rich Gabriella The support INTERNAL grant cure D- 512090 grant Commission European LSHB-CT-2004-503319 Commission European NIH PO1CA100265 grant NIH . CA49369 grant in Immunology. Chair Professorial H. Drake Y.R.Drake. Henry the holds E. Mrs. from received support the For Acknowledgements Katchman H, TalKatchman O, Eventov-Fridman J Immunol. 2007 Nov E, Bar- S, Bachar-Lustig Reich-Zeliger Y.,Reisner stem Hematopoietic Y.,Reisner for organs Growing 2008 Mar 13; Mar 2008 print] of [Epub ahead Liver. Cells. Host Stem Quiescent by the Inhibition Homeostatic in Overcoming Hepatoblasts Isolated Over Implants Liver Intact of Transplantation: Advantage for aSource as Liver Porcine Y. E, Reisner Freude Embryonic G, Hecht A, D, S, Shtabsky Cohen Tchorsh A, E, Aronovich S, Shezen 15;179(10):6389-94. cells. effector memory anti-donor host of deletion effective CTLs: by veto recipients marrow bone in presensitized Y. Reisner Ilan A, Tolerance induction 2007;38(1-3):174-90. Res. Immunol barriers. genetic major across transplantation cell 2007;38(1-3):261-73. Res. Immunol tissues. precursor embryonic from transplantation vol.2006, 103, no. 50, 19075-19080. 12, December PNAS. transplantation.

Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science iv