Anti-3rd Party Research Articles

Correction of T-Cell Repertoire and Autoimmune Diabetes in NOD Mice by Non-myeloablative T-Cell Depleted Allogeneic HSCT.

The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

Correction of murine sickle cell disease by allogeneic haematopoietic cell transplantation with anti-3rd party veto cells.

Despite advances in gene therapy allogeneic hematopoietic stem cell transplants (HSCT) remains the most effective way to cure sickle cell disease (SCD). However, there are substantial challenges including lack of suitable donors, therapy-related toxicity (TRM) and risk of graft-versus-host disease (GvHD). Perhaps the most critical question is when to do a transplant for SCD. Safer transplant protocols for HLA-disparate HSCT is needed before transplants are widely accepted for SCD. Although risk of GvHD and TRM are less with T-cell-deplete HSCT and reduced-intensity conditioning (RIC), transplant rejection is a challenge. We have reported graft rejection of T cell-depleted non-myeloablative HSCT can be overcome in wild type fully mis-matched recipient mice, using donor-derived anti-3rd party central memory CD8-positive veto cells combined with short-term low-dose rapamycin. Here, we report safety and efficacy of this approach in a murine model for SCD. Durable donor-derived chimerism was achieved using this strategy with reversal of pathological parameters of SCD, including complete conversion to normal donor-derived red cells, and correction of splenomegaly and the levels of circulating reticulocytes, hematocrit, and hemoglobin.

Towards Safer Allogeneic HSCT in Autoimmunity: Proof of Concept in Diabetes-Prone Mice

R.S.K and B.N.L contributed equally to this study. The induction of tolerance towards pancreas autoantigens is a promising approach in the treatment of type 1 diabetes mellitus (T1DM). This goal can be partially attained by immuno-ablation followed by autologous HSCT which mitigates the immune reaction leading to diabetes, at least temporarily, if performed soon after diagnosis. However, most T1DM patients transplanted with autologous HSCT eventually relapse (E Snarski et al., Bone marrow Transplantation; 2016; 51, 398-402). Thus, developing methodologies for allogeneic HSCT, to provide a durable non-autoimmune TCR repertoire, could be a promising treatment approach provided that such protocols can safely achieve donor type chimerism. Accordingly, transplantation of T cell-depleted allogeneic HSCT (TD-HSCT) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option. However, overcoming rejection after reduced conditioning in T1DM represents a major challenge. We previously demonstrated in wild type mice that rejection of TD-HSCT can be prevented using donor-derived veto cells. Here, we show proof of concept of the safety and efficacy of veto cell mediated non-myeloablative mismatched allogeneic TD-HSCT, in the established NOD mouse T1DM model. Veto activity, first defined by Miller (Miller, R. G; Nature; 1980; 287; 544-54), is based on the ability of specific cell populations to attack host CTL-precursors (CTLp) that are directed against the antigens presented by the veto cells themselves. This response spares cells that are not targeted against the veto cells, including those recognizing pathogens. Among different veto cell populations described in the literature, central memory CD8 T cells exhibit the most robust veto activity upon transplantation, but are also endowed with marked GvH activity. We overcame GvH reactivity by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, under culture conditions favoring expression of the central memory phenotype. Such anti-3rd party central memory CD8 T cells (Tcm) are endowed with marked veto activity, while effectively depleted of GVH reactivity in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin. Hematol. 2019; 56(3): 173-182). In this study, Tcm veto cells were generated from splenocytes obtained from Balb/c donors (H2d) cultured against irradiated third-party splenocytes (FVB; H2q), under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators under these conditions leads to selective 'death by neglect' of bystander anti-host T cell clones that could mediate GvHD; such cells are further diluted out by subsequent expansion of anti-3rd party T clones during further culture in the presence of IL15. In addition to causing selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be crucial for robust veto activity in vivo. To evaluate the safety and efficacy of such a transplant for diabetes therapy, 8 week old NOD mice (before diabetes onset) were treated with 4.5 Gy TBI conditioning at day -1, anti-3rd party veto Tcm and megadose nude bone marrow (NuBM) on day 0, and Rapamycin treatment from day -1 to day +4 (Scheme 1). Controls were untreated or received conditioning with no transplant. In four independent experiments (n= 35 NOD mice in the transplanted group), high chimerism from 83.5% to 99.6 % was found in all transplanted mice at 6 months post-transplant. Notably, with a follow-up of 200 days, 72.4% mice in the untreated group, and 96% in the conditioned group died of diabetes, while only 8.5% diabetes-associated mortality (Fig.1A) and no GvHD or other transplant-related mortality was observed in the transplanted mice (Fig.1B). Our results demonstrate a proof of concept for the safety and efficacy of non-myeloablative allogeneic TD-HSCT in type 1 diabetes. A clinical protocol testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is commencing at MD Anderson Cancer Center. If successful, our results support further extension of this platform to autoimmune diabetes and other autoimmune diseases. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Proof of Concept for Repair of Sickle Cell Disease By Non-Myeloablative MHC Disparate T Cell Depleted HSCT Combined with Donor-Derived Veto Cells

Although life extending medical treatments are available for sickle cell disease (SCD), allogenic hematopoietic stem cell transplantation (HSCT) is considered a treatment of choice (Ozdogu et al., Bone Marrow Transplant 2018; 53(7): 880-890). However, HSCT is associated with several limitations, including conditioning-related toxicity and graft-versus-host disease (GvHD), especially when using MHC disparate transplants. Thus, the development of safe transplantation protocols for MHC disparate HSCT in sickle cell disease is vital. While the risk of GvHD and conditioning toxicity can be effectively reduced by the use of T-cell-depleted HSCT (TD-HSCT) under reduced intensity conditioning (RIC), rejection of TD-HSCT remains a challenge. We previously demonstrated in wild type mice that this barrier can be overcome using donor-derived veto cells. Here, we demonstrate the safety and efficacy of this approach in a well-defined murine model for sickle cells disease. Veto activity is based on the ability of certain cells to attack host CTL-precursors (CTLp) which are directed against antigens expressed on the veto cells themselves, sparing cells that are not targeted against the veto cells including T cells needed for defense against pathogens. Central memory CD8 T cells exhibit the most robust veto activity upon transplantation; however, these cells are also endowed with marked GvH activity. We overcame this issue by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, respectively, under culture conditions favoring expression of central memory phenotype. Such anti-3rd party central memoryCD8 T cells (Tcm), which are endowed with marked veto activity, also exhibit reduced risk for GvHD in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin Hematol. 2019; 56(3): 173-182.) To generate Tcm veto cells, splenocytes obtained from Balb/c donors (H2d) were cultured against irradiated third-party splenocytes (FVB; H2q) under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators leads to selective 'death by neglect' of bystander anti-host T cell clones potentialy mediating GvHD, and these are further diluted out by subsequent expansion of anti-3rd party T clones during continued culture in the presence of IL-15. Apart from selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be important for attaining robust veto activity in vivo (Ophir et al., Blood. 2013; 121(7): 1220-8). We first calibrated the optimal irradiation dose for sickle mice (Berkeley model, H2b), comparing 4.5 Gy versus 5 Gy TBI in a conditioning protocol also including short term rapmycin treatment (Scheme 1). Higher levels of engraftment and chimerism were found in the group receiving 5Gy TBI (Fig. 1). All mice of both treatment groups survived >140 days with no evidence of GvHD. To further evaluate this treatment approach, 8 week old sickle mice (N=7) were given bone marrow transplants using the protocol described in Scheme 1, including conditioning with 5 Gy TBI (day -1), rapamycin treatment (day -1 to day 4), and transplantation of NuBM (day 0) plus veto cells (day 7; Scheme 1). Notably, at 44 days post-transplant, 6 out of 7 mice receiving NuBM + TCM + Rapa (85.7%) showed donor chimerism in the peripheral blood, ranging between 77-94% (Fig.2A-B), while no chimerism was detected in mice receiving conditioning alone, or conditioning and transplantation with only NuBM or only veto cells . All mice in all groups survived (N=26) , and no GvHD was detected with a follow up of 77 days, even in the transplanted group which exhibited high donor-derived chimerism. Furthermore, reversal of sickle disease symptoms was observed, including reticulocyte levels (p=0.001;Fig.2C) and expression of wild type hemoglobin ( Fig.2D) in all engrafted mice. Our results offer a proof of concept for the treatment of sickle disease by MHC disparate non-myeloablative T cell depleted HSCT in conjunction with anti-3rd party central memory veto CD8 T cells. A clinical trial testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is currently ongoing at MD Anderson Cancer Center. If successful, our present results support further evaluation of this platform for sickle cell disease. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Next Generation Veto Cells for Non-Myeloablative Haploidentical HSCT: Combining Anti-Viral and Graft Facilitating Activity

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm).When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance.Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2).The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3).In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection.*E.B.L and N.O.G contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBachar Lustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Towards 'Off-the-Shelf ' Universal Chimeric Antigen Receptor (CAR) T Cells: Mouse Anti-3rd Party Central Memory CD8 Veto Cells Prolong Functional Engraftment of Allogeneic Genetically Modified T Cells

We recently demonstrated that mouse anti-3rd party central memory CD8 veto cells (Tcm) enable engraftment of fully mis-matched T cell depleted bone marrow (TDBM) in sublethally irradiated (5.5 Gy TBI) recipients (Eran et al. Blood 2013). Here we found that Tcm generated from (host x donor)F1 or fully allogeneic donors were able to engraft and sustain their presence in such sub-lethally irradiated mice in the absence of TDBM transplants. Limiting dilution analysis revealed that the reactivity of host T cells (HTC) against donor spleen cells was significantly diminished in the chimeric mice treated with Tcm, whereas reactivity against 3rd party was comparable betweenTcm treated and non-treated mice (Fig. 1). This specific immune tolerance indicated that vetoTcm could potentially enable the use of 'off-the-shelf' allogeneic CAR T cells or any other genetically modified T cells from the same donor.To further investigate this possibility, we tested the ability of Tcmveto cells to enable engraftment of CD8 T cells from OT1 mice, expressing a transgenic T cell receptor (TCR) directed againstovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. To that end, 1x106 OT1 CD8 T cells (H2b) were infused into Balb/c (H2Dd) recipients sublethally irradiated with 5.25 Gy TBI in the presence or absence of different doses of veto Tcm. As shown in Fig. 2, when tested at 2 months post-transplant, OT1 cells could not be detected in the peripheral blood of untreated recipient mice, while they were readily detectable in mice receiving 2x106 Tcm (0.78±0.36), reaching a higher level (1.18± 0.61) upon infusion of 5x106Tcm.To evaluate the functionality of the engrafted OT1 T cells in the chimeric mice, we developed and calibrated a new murine model, using a melanoma B16-cell line of C57BL background that expresses the ovalbumin peptide and the tdTomatomarker (B16-OVA-tdTomato) (Fig 3A). In this model, we tried to mimic a state of minimal residual disease by injecting a small number of B16-OVAtdTomato melanoma cells (0.25x106) into syngeneic C57BL mice; we then treated the mice with sublethal irradiation (6Gy TBI) to simulate treatment of the tumor, but also to clear out space for the T-cell adoptive transfer. Finally, we injected 1x106 naïve OT-1 cells generated on a (Balb x-OT1-CD45.1+RAG2-) F1 background in the presence or absence of Balb/c veto Tcm (5x106), and followed the development of the tumor. We found thatBalb/c vetoTcm prolonged the engraftment of the transgenic donor-derived F1 OT1 CD8 T cells inBalb/c recipients, thereby enabling significantly improved control of tumor growth when tested at day 20 post-transplant ( p<0.05). In contrast non OVA-expressing tumor cells grew at the same rate in all treatment groups, thereby demonstrating the specificity of the anti-tumor effect (Fig.3B).Previous studies demonstrated that Tcmare depleted of GVH reactivity by virtue of their culture with 3rd party stimulators under cytokine starvation. Our present results demonstrate that suchTcm veto cells can pave the way for functional engraftment of CD8 T cells with genetically modified specificity. Taken together, these results provide a proof of concept for the potential clinical use of such veto cells to enable therapy with 'off-the-shelf ' allogeneic CAR T cells. The relative ease of growing humanTcm in large numbers over a short period of time (9-12 days) suggests that it could be possible to harvest byleukapheresis from a single donor, a sufficient number of cells, to support CAR therapy for more than 50 allogeneic recipients.* N.O.G and R.G equally contributed [Display omitted] [Display omitted] [Display omitted] DisclosuresOr Geva:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

The Double Benefit of ex-vivo Generated Anti 3rd Party CD8 T Cells With Central Memory Phenotype: Enhanced BM Engraftment Coupled With Marked GVL in the Absence of GVHD

The Double Benefit of ex-vivo Generated Anti 3rd Party CD8 T Cells With Central Memory Phenotype: Enhanced BM Engraftment Coupled With Marked GVL in the Absence of GVHD

Ex Vivo Generated Donor Central Memory CD8 T Cells, Previously Shown to Enhance Engraftment of Allogeneic Bone Marrow, Also Exhibit Significant GVL Activity without Causing Gvhd In An In Vivo b Cell Lymphoma Model

AbstractAbstract 424Generation of T cells endowed with graft versus leukemia (GVL) and depleted of graph versus host (GVH) activity represents a highly desirable goal in BM transplantation (BMT). Donor CTLs directed against 3rd party stimulators were shown to effectively enhance engraftment of BM allografts without causing GVHD. Recently, we demonstrated that activated anti 3rd party CD8 cells with central memory phenotype (Tcm), can further support and improve BM engraftment. This is likely due to the improved lymph node homing of the Tcms, their proliferative capacity and prolonged persistence in BMT recipients. Surprisingly, when tested in vitro, anti 3rd party human CTLs were found to eliminate B CLL and other lymphoma cell types. Investigating the mechanism of the cytotoxicity revealed a unique TCR independent effect mediated against both autologous and allogeneic targets. Inhibition studies using specific blocking Ab revealed initial binding of CTLs to their targets via a rapid ICAM1-LFA1 adhesion, followed by slow induction of apoptosis following interaction of the CD8 on the CTL with MHCI on the tumor cell. The killing did not involve classical CTL death molecules. To establish an appropriate mouse model, we initially verified that anti 3rd party Tcm derived from (B6xBALB/c)F1 exhibit TCR independent killing of A20 lymphoma cells of BALB/c origin (34.8±12.1% in 4 experiments, 5:1 Tcm/lymphoma cell ratio, in comparison to A20 cells incubated without Tcm, p<0.05). Moreover, after 16 hours of incubation with Tcm, AnnexinV staining of A20 cells was enhanced compared to basal staining (14.8±4.5% and 5.2±2.2% respectively, in three experiments, p<0.05), suggesting an apoptosis based mechanism as previously described for the killing of human lymphoma. Using luciferase expressing A20 cells, we were able to follow the fate of the malignant cells in vivo, and study the effect of added donor anti 3rd party Tcm to either syngeneic or allogeneic BMT in a model simulating minimal residual disease. In the syngeneic model, 3×106 Nude BALB/c BM cells were transplanted into lethally irradiated BALB/c mice together with 5000 A20 cells. On the next day, syngeneic Tcm were infused. As can be seen in Fig. 1, none of the untreated mice survived (0/7) 100 days post BMT (23 days median survival). Administration of 1×107 or 2×107 syngeneic Tcms led to significantly diminished tumor burden (Fig. 1A) and improved overall survival of 28% (2/7, P<0.0001) and 40% (2/5, P<0.002) with median survival of 49 and 80 days, respectively (Fig. 1B). As expected, further improvement of tumor eradication was found in the allogeneic setting, in which an additional GVL effect, mediated by residual alloreactivity in the absence of GVHD, occurred in addition to the TCR independent killing. In the allogeneic model, 3×106 allogeneic Nude B6 BM cells were transplanted together with 5000 A20 cells into lethally irradiated BALB/c mice, and mice were treated with donor type Tcm. Similar to the results in the syngeneic model, none of the untreated mice survived 100 days (0/8) (23 days median survival), while administration of 5×106 donor Tcms, led to remarkable overall survival of 100% (7/7) at 100 days post BMT (Fig. 2). Although the allogeneic Tcm displayed enhanced GVL activity compared to syngeneic Tcm, this effect was not associated with any manifestation of GVHD. Thus, as previously described, the weight and overall appearance of mice receiving allogeneic anti 3rd party Tcms were the same as that of mice in the control group, radioprotected with a transplant of Nude BM alone. Collectively, we demonstrate for the first time, by in vivo imaging the GVL reactivity of murine anti 3rd party Tcm. Considering our previous finding that anti 3rd party Tcm markedly enhance BM allografting, these results suggest that anti 3rd party Tcm can provide a ‘double supportive effect’ by promoting BM engraftment, and concurrently inducing GVL reactivity without causing GVHD. Such cell therapy is highly attractive, in particular for elderly patients with B CLL and other B cell malignancies who might not tolerate aggressive conditioning. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Ex-Vivo Acquisition of Central Memory Phenotype Is Critical for Tolerance Induction by Donor Anti-3rd party CD8 T Cells in Allogeneic Bone Marrow Transplantation.

The robust immunity remaining after reduced intensity conditioning represents a major barrier to durable engraftment of MHC disparate T cell depleted BM transplants (TDBMT). One approach to address this challenge could be afforded by using donor CD8 CTLs, endowed with potent veto activity (the ability of a cell to specifically suppress CTL precursors directed against its antigens, while sparing other CTLs capable of affording protection against infections and malignancies). However, their use is limited due to their marked GVH reactivity. This problem can be addressed effectively by stimulation of donor CD8 T cells against 3rd party stimulators under exogenous IL2 deprivation. The anti-3rd party veto CTLs support engraftment of TDBMT without causing GVHD. Nevertheless, although these cells displayed highly efficient veto activity in vitro, they were less efficient in vivo, requiring administration in conjunction with Rapamycin. In our study we found, using in vivo imaging, that veto CTLs display poor homing to the host lymph nodes (LNs). Therefore, we hypothesized that the relatively low efficiency of veto CTLs in vivo could be attributed to their failure to co-localize with the host T cells (HTCs) at the LNs, during the critical time at which the HTCs are triggered against donor antigens. To enhance LNs homing we generated anti-3rd party CD8 T cells under culture conditions favoring the acquisition of central memory (Tcm) phenotype (CD44highCD62Lhigh). In order to monitor CTLs or Tcm cells adoptively transferred into irradiated recipients of BM allografts, we used (Host x Donor)F1 cells co-expressing host and donor MHC that can be distinguished by FACS. When CD62Lhigh Tcm cells, expanded in the presence of IL15, were administered, the number of cells located at the host LNs two days post transplant was 40-fold higher compared to that found for veto CTLs expanded in the presence of IL2, which exhibit a CD62Llow effector phenotype (p<0.05). Moreover, the total number of Tcm cells harvested from various organs was increased by 9 folds between days 2 to 6 post BMT (p<0.05) in sharp contrast to the CTLs which displayed a non significant proliferation, indicating that Tcm cells possess superior proliferative capabilities. In addition, the Tcm cells exhibited marked durable persistence in-vivo when tested 100 days post transplant, comprising 17%±9% of the total CD8 T cell compartment following infusion of 1x106 cells. When evaluated in a stringent mouse model for T cell mediated BM allograft rejection, Tcm cells displayed marked tolerizing activity. In this model, 3x106 Balb-Nude BM is transplanted into lethally irradiated C3H hosts that were previously inoculated with 1.25x104 HTC (a number sufficient to induce BM rejection and lethality). Thus, administration of 5x106 anti-3rd party Tcm cells derived from (C3H x BALB)F1 mice, with no further immunosuppression, led to overall survival of 83% (19/23) at 100 days post BMT, while administration of 107 anti-3rd party CTLs (bearing an effector phenotype) could only afford survival of 19% (3/16) (p<0.05) in the absence of Rapamycin. To evaluate the risk of GVHD, allogeneic Balb/c derived anti-3rd party Tcm cells or naive CD8 T cells were administered in conjunction with 5x106 Balb/c-Nude BM cells into lethally irradiated C3H hosts. As expected, naive CD8 cells caused lethal GVHD and only 6% (1/16) of the mice which received 2x106 naive cells survived 100 days post BMT. In contrast, the mice receiving Tcm cells displayed 83% survival (10/12 or 5/6 for 2x106 or 5x106 Tcm cells, respectively) with no signs of GVHD. Furthermore, when evaluated in the graft rejection model, 5x106 fully allogeneic donor Tcm cells induced survival, accompanied with complete donor chimerism at 100 days post transplant in 73% (11/15) of the recipients. The similar efficacy of overcoming rejection displayed by both fully allogeneic and F1 derived Tcm cells is in accordance with a veto mechanism, as opposed to potential ablation of host resistance by donor alloreactive clones. Collectively, the ability of anti-3rd party CD8 Tcm cells to expand and persist in vivo without GVHD, their marked LN homing and their capacity to overcome rejection of TDBMT in the absence of further immunosuppression, strongly suggest that these cells hold marked potential for tolerance induction in allogeneic hematopoietic stem cell transplantation.

Eradication of B Cell Malignancies by Anti-3rd Party CTLs: A Novel Role for MHC Class I.

Induction of donor type chimerism under reduced intensity conditioning in the absence of GVHD represents a major challenge in transplantation biology. Despite their remarkable potent veto activity, CD8 CTLs could not be used to enhance engraftment due to their GVH reactivity. To address this problem we developed a new approach for the generation of human host-nonreactive CTLs, based on stimulation of donor CD8 T cells against 3rd party stimulators under IL-2 deprivation. A major attribute of such anti-3rd party CTLs reported recently by our group is their ability to eradicate pathological B-CLL cells. This B-CLL killing was shown to be independent of TCR recognition and it was found to be mediated both by autologous and by allogeneic anti-3rd party CTLs. In the present study we demonstrate that anti-3rd party CTLs can also efficiently eradicate primary mantle lymphoma cells and Burkitt and mantle cell-lines. Furthermore, the use of lymphoma cell lines has enabled to define several aspects of the mechanism leading to their apoptosis. Thus, we found that anti-3rd party CTLs avidly bind to target lymphoma cells and form stable aggregates within the first minutes of contact. Conjugate formation as well as the actual killing of the tumor cells were effectively inhibited by blocking antibodies against LFA1 or ICAM1 but not against ICAM3. Moreover, effective inhibition was attained only when applying anti-LFA1 to the CTLs and anti-ICAM1 to the tumor cells (100%±10% and 72%±7% inhibition, respectively) but not vice-versa. Given the role of CD8-MHC class I interaction in veto mechanisms, we sought to evaluate its role in the killing of lymphoma cells by human anti-3rd party CTLs. Indeed, in 10 experiments survival of target lymphoma cells was increased from 50%±5% to 82%±1% upon addition of anti HLA-ABC Ab to the incubation with anti 3rd party CTLs. A similar survival increase to 77.5%±1% was found upon the addition of anti-CD8 Ab, suggesting that CD8 binding to MHC class I constant region is required for lymphoma cell killing. The important role of MHC class I was further established by comparing CTL-mediated killing of Daudi lymphoma cells lacking cell surface MHC class I expression and Daudi cells with reconstituted surface MHC class I due to stable β2-microglobulin overexpression (β2m-Daudi). Thus in contrast to the poor killing of Daudi cells which did not exceed 20%, the β2m-Daudi cell killing was comparable to that exhibited with burkitt lymphoma line expressing MHC class1 (56%±1% and 63%±3%, respectively in 4 experiments). Importantly, FACS analysis showed that, MHC I expression did not influence conjugate formation, but was rather involved in the induction of apoptosis (annexin V). Furthermore, a similar pattern was also found in vivo when β2m-Daudi or Daudi cells were infused into the peritoneum of SCID mice in the presence or absence of a 2.5-fold excess of anti 3rd party CTLs. In 3 experiments, FACS analysis of the cells recovered from the peritoneum showed that the CTLs killed 91% of the β2m-Daudi cells as opposed to 13% killing of the Daudi cells. Taken together, our results suggest that TCR independent killing of B cell malignancies by anti-3rd party CTLs is mediated via a rapid adhesion through ICAM1-LFA1 binding, followed by slow induction of apoptosis upon a critical interaction between CD8 on the CTL and MHC class I on the tumor cell.

The Role of ERK and XIAP in Tolerance Induction by Veto CTLs.

Several bone marrow and lymphocyte subpopulations, known as ' veto cells' were shown to induce transplantation tolerance across major histocompatability antigens. We recently demonstrated, that allogeneic anti-3rd party CTLs depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate allografting of T cell depleted bone marrow without GVHD. Earlier insights indicated that at low veto cell concentrations, both CD8 and FasL on the veto cell might be required to induce specific deletion of the effector cell. Such a mechanism involves initial recognition of the veto cell by the TCR of the effector cell, leading to expression of Fas upon activation of the latter and thereby allowing for Fas-FasL apoptosis. The presence of FasL on the veto cell cannot result in apoptosis of the effector cell unless CD8 on the veto cells is available and can interact with MHC class 1 on the effector cell. Thus, the CD8-MHC interaction seems to be associated with an increased susceptibility of the effector cell to FasL killing. Recently, by using the 2C TCR transgenic mouse model, in which the transgene is directed against H2d, it became possible to monitor the effector cells by staining with an antibody against the TCR transgene (1B2). By using this assay we showed that the marked deletion of 2C CD8 T cells by veto CTLs of DBA origin is markedly inhibited by MEK1/2 inhibitors such as U0126, reducing the veto activity from 86%±7% to 16%±14% in 6 experiments. In the present study we used anti pERK antibody to analyze by FACS the level of ERK phosphorylation in the effctor cells and we demonstrate that upon interaction of 2C effector cells (1B2+CD8+) with veto CTLs of H2d origin as opposed to CTLs of H2s origin, ERK1/2 are significantly more phosphorylated (21%±5.5% Vs.14%±2% phospho ERK1/2 cells out of the total 1B2+CD8+ cells, respectively). The upregulation of ERK1/2 phosphorylation in the 2C effector cells is associated with significant reduction of XIAP levels (36%±4% reduction at 24h and 54%±19% at 48h). This reduction of an apoptosis inhibitor which generally controls death receptor-induced apoptosis in lymphocytes is specifically induced by the veto cells of H2d origin recognized by the transegne TCR and not by non-relevant veto CTLs of H2s origin. Addition of U0126 MEK 1/2 inhibitor reverses this down regulation of XIAP (reduction of 15%±13% at 24h and only 7%±13% at 48h). Blockade of MHC-CD8 interaction with MHC class I α3-blocking Ab, or anti-CD8 (lyt 2.1) Ab partially inhibits the ERK1/2 phosphorylation (average inhibition in 4 experiments was 37%±13% and 28%±8%, respectively) and also prevents XIAP down regulation (18%±9% at 24h and 23%±13% at 48h). Considering that blockade by these antibodies generally leads to partial inhibition of the observed veto activity (44%±21% in 4 experiments), our present results showing partial inhibition of ERK phosphorylation and XIAP down regulation, are clearly relevant. Taken together, these results can explain in part why co-expression of CD8 and FasL on the veto CTLs is required for effective deletion of host anti-donor CD8 T cell clones. [Display omitted] Deletion of host anti-donor CD8 T cell clones by veto CTLs. CD8-MHC engagement induces pERK which is associated with reduction of XIAP levels, thereby enabling Fas-FasL apoptosis.

From ‘megadose’ haploidentical hematopoietic stem cell transplants in acute leukemia to tolerance induction in organ transplantation

The first successful demonstration that effective T cell depletion can enable immune reconstitution without causing graft vs. host disease (GVHD) in SCID patients was achieved in 1980 using lectin-separated haploidentical hematopoietic stem cells. Recipients exhibited immune tolerance towards donor antigens with a follow-up of more than 2 decades. In leukemia patients undergoing supralethal radio- and chemotherapy, T cell-depleted transplants are vigorously rejected by residual host T cells; this barrier was first overcome in 1993 by the use of megadose stem cell transplants. This clinical observation can be explained, in part, by the demonstration that cells within the CD34 compartment, as well as their immediate early myeloid progeny, are endowed with veto activity. Engraftment of mismatched hematopoietic stem cells following reduced intensity conditioning, still represents a major challenge. Progress made recently in murine studies by different approaches including the use of new co-stimulatory blockade agents, as well as by tolerance inducing cells such as anti-3rd party veto CTLs, NK T cells, and T regulatory cells, suggests several promising modalities for clinical translation.

Large-scale generation of human allodepleted anti-3rd party lymphocytes

Although adoptive transfer of donor lymphocytes protects from infections and relapse after allogeneic hematopoietic stem cell transplantation in both mice and in men, it is associated with a high risk of graft versus host disease (GvHD) which rises with HLA mismatching and the number of T lymphocytes that are infused. Elimination/reduction of alloreactive donor T lymphocytes is an appealing approach and several strategies have been proposed. Here we describe generation of anti-3rd party T lymphocytes under conditions of IL-2 deprivation and their effects in a pre-clinical murine model. Our results clearly indicated that anti-3rd party T lymphocytes generated on a large scale by means of IL-2 deprivation maintain a broad T cell repertoire, do not proliferate in a mixed lymphocyte reaction and do not cause GvHD in NOD–SCID mice. These anti-3rd party lymphocytes contain a large adaptive T regulatory cell subset which might contribute to in vitro and in vivo immune modulation.

Hematopoietic stem cell transplantation across major genetic barriers

The first successful demonstration that effective T cell depletion can enable immune reconstitution without causing graft versus host disease (GVHD) was achieved in 1980 using lectin-separated hematopoietic stem cells. In leukemia patients undergoing supralethal radio- and chemotherapy, T cell-depleted transplants are vigorously rejected by residual host T cells; this barrier was first overcome in 1993 by the use of megadose stem cell transplants. This clinical observation can be explained, in part, by the demonstration that cells within the CD34 compartments, as well as their immediate early myeloid progeny, are endowed with veto activity. Engraftment of mismatched hematopoietic stem cells following reduced intensity conditioning, still represents a major challenge. Progress has been made recently by using anti-3rd party veto CTLs and T regulatory cells.

Donor Specific Unresponsiveness Develops Following Loss of Chimerism in Recipients of Haploidentical Combined Bone Marrow/Kidney Transplantation but Not in Recipients of Bone Marrow Transplant Alone.

Pre-clinical data in monkeys receiving non-myeloablative conditioning followed by MHC-mismatched kidney and bone marrow transplantation show that transient chimerism is sufficient to permit achievement of long-term tolerance to a simultaneous donor renal allograft. Our group has recently reported successful induction of tolerance to donor kidneys in patients with advanced multiple myeloma and renal failure through combined bone marrow and kidney transplantation in the HLA-identical setting. On the basis of these results, five end-stage renal failure patients without malignant disease received simultaneous kidney and bone marrow transplantation from haploidentical HLA mismatched related donor after non-myeloablative conditioning with MEDI-507 (anti-CD2 humanized mAb; MedImmune), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. Transplantation of kidney and bone marrow were both performed on Day 0. All patients developed initial mixed chimerism but lost their chimerism by Day 21. We have analyzed three patients who successfully discontinued immunosuppression on Days +240, +422 and +244. At a follow-up of 47, 38, and 17 months, all three patients are off immunosuppression without allograft rejection. T-cell counts exceeded 100 cells/μL by Day +128, +21, +21, respectively, and a high early prevalence of CD4+CD25high cells was detected. Post-transplant in vitro alloreactivity assays (bulk MLR/CML) showed the development of long-lasting donor-specific unresponsiveness in all three patients. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, no killing of donor RTEC was detected post-transplant. We also assessed alloresponses in chemorefractory lymphoma patients receiving haploidentical bone marrow transplantation with a similar conditioning regimen. In contrast to the recipients of combined kidney/bone marrow transplants, these patients showed sustained global hyporesponsiveness in CML and MLR. However, loss of donor chimerism was associated with the eventual appearance of measurable anti-donor CML and/or MLR responses. In contrast, donor-specific and host-specific unresponsiveness with strong anti-3rd party responses developed in a sustained mixed chimera who received haploidentical stem cell transplantation with a modification of this conditioning protocol (i.e. different dose of MEDI-507, Isolex-selected CD34+ cells from G-CSF mobilized PBMC and the addition of fludarabine).In summary, we have obtained proof of principle that durable multilineage mixed chimerism with donor- and host-specific tolerance can be achieved without GVHD in humans receiving haploidentical HCT. In recipients of combined kidney/bone marrow transplants but not in recipients of bone marrow alone, patients who lose chimerism develop donor-specific unresponsiveness. These studies point to a role for the renal allograft in maintaining long-term tolerance following loss of initial donor chimerism.

The Role of ERK5 Signaling in Tolerance Induction by Veto CTLs.

Several bone marrow cells and lymphocyte subpopulations, known as ‘veto cells', were shown to induce transplantation tolerance across major histocompatibility antigens. Recently, it has been suggested that anti-3rd party CTLs depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate bone marrow allografting. The veto mechanism is still obscure. While early studies emphasized the role of CD8 mediated apoptosis, we showed that the veto activity of anti-3rd party CD8+ CTLs is dependent upon the simultaneous expression of both CD8 and FasL. Thus, it seems that although Fas is upregulated on the effector T cells upon engagement of their TCR by class I of the veto cells, the presence of FasL on the veto cells cannot result in apoptosis of the effector cells unless CD8 on the veto cells is available and can interact with class 1 on the effector cells. Thus, the interaction of CD8 on the veto cells with class I on the effector cells seems to be associated with an increased susceptability of the effector cells to FasL killing. To further delineate the mechanism of the veto effect we have now studied the role of different signaling pathways using specific inhibitors. Spleen CD8 T cells from 2C mice (H2b) bearing TCR transgene directed against (H2d) were used as effector cells and anti FVB (H2q, third party) CTLs generated from Balb/c spleen cells (H2d) were used as veto cells. The addition of the latter cells to MLR of 2C against Balb/c (H2d) simulators, leads to deletion of the 2C effector CD8 cells within 72 hrs. Deletion monitored by FACS analysis of cells stained with anti-TCR transgene antibody (1B2+) revealed reduction from 46%±11% 1B2+CD8+ cells to 6%±3% 1B2+CD8+ cells in 6 different experiments. In contrast, veto CTLs generated from SJL (H2s) spleen cells that do not display the H2 recognized by the 2C effector cells, did not result in a significant deletion of the effector cells (42%±12% 1B2+CD8+ cells in 6 different experiments). The specific deletion exhibited by veto CTLs of Balb/c origin, can be inhibited by MEK1/2/5 inhibitors such as U0126, reducing the veto activity from 85.5%±7% to 16%±14% in 6 different experiments. The effective concentration of U0126 was relatively high (10μM), and lower concentration of this drug (1μM) had no response, indicating a potential involvement of the MEK5/ERK5 cascade rather than the MEK1/2-ERK1/2 cascade, in the veto effect. In addition, no inhibition of veto activity could be found with specific inhibitors of other signaling molecules such as JNK, P38, PI3K or PKC. Considering that Fas expression on the effector cells is critical for the veto activity, it is interesting that the ERK inhibitor didn't affect the level of Fas on the effectors (93%±3% of 1B2+CD8+ upregulate FAS in the presence of U0126 in 7 different experiments). Also, this inhibition is not likely mediated by affecting the veto CTLs, as pretreatment of the latter cells with ERK inhibitor didn't diminish the veto effect. The pro-apoptotic effects of MEK5-ERK5 cascade in this system is intriguing because these kinases are usually thought to promote proliferation and survival in most cellular systems. Therefore, the veto cells exhibit a unique signaling system, which utilizes ERK5 cascade to induce apoptosis. Further studies are directed at the potential links between the ERK5 cascade, the Fas system and the rest of the apoptotic machinery.

Anti-Third Party Veto Cells Facilitate Allogeneic Hematopoietic Cell Engraftment in MHC-Mismatched Dogs.

Donor-derived veto cells have the potential to facilitate engraftment of major histocompatibility complex (MHC)-mismatched marrow by neutralizing radioresistant host T and NK cells that mediate rejection of the donor graft. Anti-3rd party cytotoxic T-lymphocyte (CTL) veto activity is CD8 and Fas ligand dependent (Reich-Zeliger et al., Immunity, 2000) and studies in murine models of hematopoietic cell transplantation (HCT) showed that CTL veto cells facilitate engraftment across MHC barriers (Bachar-Lustig et al., Blood, 2003). To translate these results to the well-established pre-clinical dog model of HCT, we had previously demonstrated that donor-derived, anti-3rd party CTL that are not alloreactive against host cells could function as veto cells and block the emergence of host-derived, anti-donor CTL in vitro. Previous studies showed that DLA-nonidentical, unrelated bone marrow grafts were uniformly rejected by host T and NK cells following 920 cGy total body irradiation (TBI) without postgrafting immunosuppression (90% of dogs with graft rejection). In the current study we asked if infusion of donor derived, anti-3rd party CTL veto cells could facilitate engraftment of MHC-mismatched marrow. To date, six recipient dogs have been transplanted. For each hematopoietic cell transplant, four DLA-nonidentical, unrelated dogs were used: one marrow donor, one marrow recipient and two 3rd party dogs. Dendritic cells (DC) were cultured from bone marrow CD34+ cells obtained from 3rd party dogs. Ten days before transplant, donor lymphocytes were collected by leukapheresis and cultured separately with 3rd party DCs to generate anti-3rd party CTL veto cells. On day 0, recipients were irradiated with 920 cGy TBI and transplanted with donor bone marrow (2.3–5.4x108 TNC/kg; 4–12x106 CD34+/kg) and donor derived anti-3rd party CTL veto cells (30–57 x106 cells/kg). Three of six (50%) dogs engrafted 7 to 18 days after HCT (as determined by bone marrow biopsy, complete donor hematopoietic chimerism and recovery of absolute neutrophil count >500/μL) and developed 3-system graft-versus-host disease (GVHD). The other 3 dogs had transient donor engraftment with clinical and histopathological evidence of GVHD. Dogs were euthanized between days 14 and 19 after HCT. In contrast, none of five historical control dogs engrafted after transplantation with DLA-nonidentical, unrelated donor marrow and 30-40x106/kg freshly isolated donor peripheral blood mononuclear cells, p<0.05 (Panse JP et al., Transplantation, 2003). These preliminary data suggest that donor derived, anti-3rd party CTL veto cells eliminate host-anti-donor T and NK cell responses and facilitate engraftment of MHC-mismatched marrow. Future studies will aim to transplant veto cells with T-cell-depleted marrow and administer post-grafting immunosuppression to decrease the intensity of the conditioning regimen needed for MHC-mismatched HCT engraftment without risking graft rejection or GVHD.

Effective Deletion of Anti-Donor Host Memory Effector Cells by Anti-3rd Party Veto CTLs: Implications to Tolerance Induction in Presensitized Bone Marrow Recipients.

Recently we demonstrated that veto CTLs enhance engraftment of mismatched T cell depleted BM in recipient mice following reduced intensity conditioning. This desirable tolerance induction can be further enhanced by combining veto CTLs with CD4+CD25+ cells and Rapamycin. While these results are encouraging, they were largely based on models in which the resistant effector T cells mediating the allorejection are naive CTLp. However, considering that many patients undergoing BMT are presensitized by transfusions of different blood products, memory T cells could play an important role in graft rejection and, therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in BMT. Clearly, memory T cells respond under less stringent conditions to foreign antigens, compared to their naïve counterparts. In particular, they are programmed to be activated promptly, with a reduced requirement for costimulatory signals and therefore they might be more resistant to veto cells. To address this question we used the 2C mouse model, the CD8 T cells of which express a transgenic TCR against H-2d. The CD8 T cells bearing the TCR transgene can be followed by FACS using staining with a clontypic antibody (1B2) against the transgene. In this model, addition of veto CTLs was shown to inhibit expansion of CD8+1B2+ effector cells by induction of apoptosis which can be monitored by annexin V staining. Thus, in a total of 10 experiments the addition of 5% veto cells to 3 day MLR culture of naive 2C effector cells in the presence of H-2d stimulator cells, led to 76%±9% inhibition of expansion. In order to compare the sensitivity of memory cells in the same model, memory cells were established by immunizing 2C transgenic mice with 1x106 irradiated splenocytes from Balb/c donors (H-2d origin). Six weeks later, splenocytes were harvested and after Ficoll separation were shown to be enriched with memory CD8 T cells(CD44+high CD45Rb+ CD62L+, average in 16 different experiments was 73%±11). Upon addition of 5% veto cells to MLR culture of memory 2C spleen cells in the presence of stimulator cells, 78%±7% inhibition of 2C expansion was found. This veto activity was associated with increased apoptosis of allospecific memory CD8 T cells. Thus, in the absence of veto cells the CD8+1B2+ memory cells exhibited a low level of Annexin V (6%±3%) while in the presence of 5% veto cells, a high level of Annexin V (25%±9%) was detected. The deletion of the 2C memory effectors, as previously shown for naive 2C cells, is largely dependent on the presence of Fas-FasL interaction, as indicated by using memory cells from 2C- lpr mice that lack Fas receptor on the cell surface. Upon addition of veto cells to MLR culture with 2C memory spleen cells from lpr mice, only a minor reduction of expansion (5.5%±6% in the presence of 10% veto CTLs) was detected. In conclusion, these results suggest that veto cells can delete memory effector cells as efficiently as exhibited on naive effector cells and by a similar Fas-FasL dependent mechanism. This finding might have significant implications not only for BMT, but also for the treatment of autoimmune diseases in which memory T cells play a major role.

ALLORESPONSES IN TOLERANT RECIPIENTS OF COMBINED KIDNEY/BONE MARROW TRANSPLANTATION WITH NON-MYELOABLATIVE CONDITIONING

P931 Introduction: We analyzed in vitro responses in 6 patients who received combined non-myeloablative HLA-matched sibling bone marrow (BM) and kidney transplantation (Tx) for the treatment of multiple myeloma (MM) with renal failure. Patients 3 - 6 were participants in an Immune Tolerance Network study. Methods: Mixed lymphocyte reaction (MLR), cell-mediated lymphocytotoxicity (CML), and limiting dilution assays (LDA) were performed. Donor chimerism was monitored by microsatellite analysis. T cell recovery was followed by flow cytometry. Results: In Patient 1, initial multilineage mixed chimerism declined to undetectable levels after Day 105. CyA was discontinued on Day 73 and the transplanted kidney has shown no evidence of rejection > 5.5 years. Early CD8 T cell recovery and strong anti-donor cytotoxic T lymphocyte (CTL) responses were seen in CML and LDA, without anti-donor MLR responses. The myeloma is in complete remission (CR) > 5.5 years. In Patient 2, initial mixed chimerism became undetectable after Day 123. CyA was discontinued on Day 77 and the kidney has shown no rejection > 3 years. Early recovery of anti-3rd party responses but not anti-donor responses were seen in MLR and CTL and helper T lymphocyte (HTL) LDA. Urinary light chain was reduced by 90% after Tx, but the myeloma later progressed. In Patient 3, multilineage mixed chimerism converted to donor full chimerism by Day 62. MMF was added to control mild chronic GvHD and CyA was discontinued on Day 379. The patient showed transient anti-donor responses in MLR and CTL-LDA prior to conversion to donor full chimerism, and high anti-host CTLp frequencies following conversion to donor full chimerism. The kidney has shown no rejection. The patient is in CR > 30 months. In Patient 4, initial mixed chimerism declined to undetectable levels by Day 71. CyA was discontinued on Day 60. The kidney has shown no rejection > 21 months. Early recovery of anti-3rd party responses but not anti-donor responses were seen in MLR and CTL and HTL-LDA. Urinary light chain persisted post-Tx. In Patient 5, multilineage mixed chimerism converted to donor full chimerism by Day 90. CyA was discontinued on Day 35. The patient showed transient anti-donor responses in HTL and CTL-LDA prior to conversion to donor full chimerism, and high anti-host CTLp frequencies following conversion to donor full chimerism. The kidney has shown no rejection. In Patient 6, initial mixed chimerism was observed, but became undetectable by Day 98. CyA was discontinued on Day 60. The kidney has shown no rejection > 3 months. No anti-donor responses were seen in MLR and CTL and HTL-LDA, but a high anti-host HTLp frequency was seen on POD70. Conclusions: Combined kidney/BMTx with non-myeloablative conditioning for MM patients is a promising strategy for successful induction of renal allograft tolerance and also provides potent anti-myeloma effects. Donor marrow rejection and anti-donor alloresponses can appear in patients despite maintenance of tolerance to donor kidneys.

Anti-3 rd party veto CTLs depleted of host reactive clones retain a broad TCR repertoire: a potential new source for adoptive immune therapy in bone marrow transplantation

CD8+ cytotoxic T lymphocytes (CTLs) are endowed with a powerful capacity to induce specific tolerance against their histocompatibility antigens (veto). Very recently, we developed a new approach to deplete CTLs of host-reactive clones by stimulating the donor T cells against 3rd party stimulators in the absence of exogenous IL-2. It could be anticipated that this procedure, which leads to a marked frequency reduction of anti-host clones, should also be associated with substantial narrowing of the TCR repertoire, thereby limiting the use of these veto cells for tolerance induction. In the present study we used a new technique (TcLandscape) which enables qualitative and quantitative analysis of Vβ chain usage at the CDR3 length distribution level to compare the repertoire of anti-3rd party CTLs to that of the original donor. Mouse and human anti-3rd party CTLs were prepared as previously described. Mouse splenocytes of Balb/c and FVB were used as effectors and stimulators, respectively. Human PBMC from normal donors were stimulated against EBV transformed cell lines, completely mismatched for HLA class1. cDNA samples obtained from mouse and human CD8+ responder T cells before (day 0) and after initiation of culture (days 22 and 33 respectively) were analyzed for Vβ repertoire. The analysis showed that in CTLs of both human and mice, a significant amplification or reduction was evident only in particular CDR3 lengths of specific Vβ families. Thus, mouse anti-3rd party CTLs exhibit marked clonal reductions in Vβ3, Vβ5, Vβ12, Vβ13 and Vβ16. Changes in the latter one were most dramatic and it is almost oligoclonal. In contrast, only minor alterations were found in all the other Vβ families indicating a preservation of broad repertoire. Analysis of CDR3 lengths of anti-3rd party human CTLs showed similar preservation of broad repertoire along with marked reduction of particular CDR3 length clones in Vβ12, Vβ15 and Vβ21. These findings are of particular relevance to BMT, as it might enable to use anti-3rd party CTLs not only for enhancement of engraftment, but also for adoptive transfer of immunity during the early post transplant period. Furthermore, these results raise new possibilities for molecular probing of the antigens which trigger alloreactivity in mouse and in man.