Search

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.

Over the last decades, several studies demonstrated the possibility of lung regeneration through transplantation of various lung progenitor populations. Recently, we showed in mice that fetal or adult lung progenitors could potentially provide donor cells for transplantation, provided that the lung stem cell niche in the recipient is vacated of endogenous lung progenitors by adequate conditioning. Accordingly, marked lung regeneration could be attained following i.v. infusion of a single cell suspension of lung cells into recipient mice conditioned with naphthalene (NA) and 6Gy total body irradiation (TBI). As clinical translation of this approach requires the use of allogenic donors, we more recently developed a novel transplantation modality based on co-infusion of hematopoietic and lung progenitors from the same donor. Thus, by virtue of hematopoietic chimerism, which leads to immune tolerance toward donor antigens, the lung progenitors can be successfully engrafted without any need for post-transplant immune suppression. In the present study, we demonstrate that it is possible to replace NA in the conditioning regimen with Cyclophosphamide (CY), approved for the treatment of many diseases and that a lower dose of 2 GY TBI can successfully enable engraftment of donor-derived hematopoietic and lung progenitors when CY is administered in 2 doses after the stem cell infusion. Taken together, our results suggest a feasible and relatively safe protocol that could potentially be translated to clinical transplantation of lung progenitors across major MHC barriers in patients with terminal lung diseases.

The induction of partial tolerance towards 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 TCR repertoire is associated with a substantial risk for transplant related mortality. Here, we demonstrate in the NOD mouse model for T1DM a safe transplant modality using allogeneic non-myeloablative T cell depleted HSCT in conjunction with donor derived veto cells. Thus, marked chimerism is attained without any transplant-related mortality, and with a very high rate of diabetes prevention. T cell receptor sequencing of transplanted mice shows marked changes in the T cell repertoire and in the prevalence of specific autoimmune T cell clones. Taken together, our results demonstrate a proof of concept for the treatment of T1DM using veto cell-based TD-HSCT.

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.

Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6–8 weeks post‐transplant, with donor derived structures including epithelial, endothelial, and mesenchymal cells. Epithelial cells within these donor‐derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about threefold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Our study provides proof of concept for lung regeneration by adult lung cells after preconditioning to vacate the pulmonary niche. stem cells translational medicine 2018;7:68–77

Summary: Induction of lung regeneration by transplantation of lung progenitor cells is a critical preclinical challenge. Recently, we demonstrated that robust lung regeneration can be achieved if the endogenous stem cell niches in the recipient’s lung are vacated by sub-lethal pre-conditioning. However, overcoming MHC barriers is an additional requirement for clinical application of this attractive approach. We demonstrate here that durable tolerance toward mis-matched lung progenitors and their derivatives can be achieved without any chronic immune suppression, by virtue of co-transplantation with hematopoietic progenitors from the same donor. Initial proof of concept of this approach was attained by transplantation of fetal lung cells comprising both hematopoietic and non-hematopoietic progenitors. Furthermore, an even higher rate of blood and epithelial lung chimerism was attained by using adult lung cells supplemented with bone marrow hematopoietic progenitors. These results lay the foundation for repair of lung injury through a procedure akin to bone marrow transplantation. : Hillel-Karniel et al. demonstrate that co-transplantation of lung and hematopoietic progenitors from the same donor in preconditioned mice allows the induction of durable multi-lineage lung chimerism and lung regeneration across major genetic barriers. These results lay the foundation for lung regeneration through a simple procedure akin to bone marrow transplantation. Keywords: fetal mouse lung, adult mouse lung, mouse bone marrow, allogeneic transplantation, tolerance induction, lung chimerism

Towards ‘off-the-shelf’ genetically modified T cells: prolonging functional engraftment in mice by CD8 veto T cells

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.

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.

Background. Xenogeneic embryonic pancreatic tissue can provide an attractive alternative for organ replacement therapy. However, immunological rejection represents a major obstacle. This study examines the potential of regulatory T cells (Tregs) in the prevention of E42 pancreas rejection. Methods. To develop new approaches to combat rejection, we evaluated engraftment, growth, and development ofE42 pig pancreatic tissue in mice treated with ex vivo expanded Tregs in combination with T-cell debulking and the conventional immunosuppressive drugs, rapamycin and FTY720. Results. Transplantation of E42 pig pancreas into C57BL/6 mice immunosuppressed by this protocol resulted in complete rejection within less than 6 weeks. In contrast, additional treatment with a single infusion of ex vivo expanded third-party Tregs markedly delayed the onset of graft rejection to 10 weeks. The infusion of Tregs was associated with a significant reduction in CD4 + and CD8 + expansion in the lymph nodes and other peripheral organs at the priming stages after implantation. Freezing and thawing of the Tregs did not affect their efficacy, indicating the potential of Tregs banking. Conclusion. Considering the technical difficulties encountered in the generation of Tregs from patients or from specific donors, our results demonstrate the feasibility of using "off-the-shelf" fresh or frozen third-party Tregs to control rejection in organ transplantation.

Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase-labeled MSCs (Luc+ MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune-deficient Balb-Nude or non-obese diabetic severe combined immunodeficiency (NOD-SCID) recipients. Graft rejection in re-challenge experiments infusing Luc+ Fib into mice, which had previously rejected Luc+ MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T-cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re-challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8+ or TEA CD4+ T cells exhibited a memory phenotype (CD122+, CD44+, and CD62Llow). Collectively, these results demonstrate that MSCs are not intrinsically immune-privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (“off the shelf”) MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long-term survival of allogeneic MSCs likely represents a major challenge. Disclosure of potential conflicts of interest is found at the end of this article.

Veto cells have been defined as cells capable of inducing apoptosis of effector CD8 cells recognizing their disparate MHC Ags. Tolerance induced by donor-type veto cells is desirable, because it is restricted to depletion of anti-donor clones without depletion of other immune specificities. It has been shown that anti-third party CTLs exhibit marked veto activity with reduced capacity to induce graft-vs-host disease, when tested on naive effector cells. However, presensitized 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 bone marrow transplantation. To address this question, we compared naive and presensitized TCR transgenic effector CD8 T cells, bearing a TCR against H-2d. Both cell types exhibited similar predisposition to killing by veto CTLs in vitro, and this killing was dependent in both cell types on Fas-FasL signaling as shown by using Fas-deficient CD8 T cells from (lprx2c) F1 mice. When tested in a stringent mouse model, in which bone marrow rejection is mediated by adoptively transferred host type T cells into lethally irradiated recipients, veto CTLs were equally effective in overcoming rejection of naive or presensitized host T cells.

Recently, we have shown that anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate bone marrow (BM) allografting without graft-versus-host disease. We also demonstrated synergism between rapamycin (RAPA) and the veto cells. CD4(+)CD25(+) T-regulatory (Treg) cells are suppressor cells that can enhance alloengraftment. We investigated whether donor Tregs would be synergistic with veto CTLs and RAPA in augmenting alloengraftment or, conversely, would suppress veto CTL effects. Lethally irradiated C3H mice were transplanted at day 2 after irradiation with Balb-nude BM. Graft rejection was induced by purified host-type T cells infused 1 day prior to BMT. The addition of Tregs led to moderate enhancement of engraftment. RAPA at different doses was synergistic with Tregs. The addition of veto CTLs to Tregs enabled reducing the effective RAPA dose fourfold. Combining all three agents was necessary to overcome rejection at low-dose RAPA. Chimerism analysis at 5 to 9 months revealed a significant presence of host-type cells coexisting with the predominant donor T cells, suggesting that tolerance had been attained. The synergistic effects between Tregs, veto CTLs, and RAPA offer an attractive approach for facilitating alloengraftment.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used ("megadose" transplants) overcomes major genetic barriers. In vitro studies suggest that veto cells, within the population of hematopoietic progenitors, facilitate this favorable outcome. Thus, when purified CD34(+) cells were added to bulk mixed-lymphocyte reactions (MLRs) they suppressed CTLs against the donor's stimulators, but not against stimulators from a third party. This tolerizing activity depends on cell contact and can be blocked by the caspase inhibitor BD-FMK, suggesting that the effector host T cells are deleted by apoptosis upon interaction with the CD34(+) cells. Early myeloid CD33(+) cells generated by short-term ex vivo expansion of CD34(+) cells also exhibit veto activity, and these cells can be grown in large numbers. Tolerance induction can be further enhanced by other veto cells. Perhaps the most potent veto cell is the CD8+ CTL. However, this cell is also associated with marked GVHD (graft-versus-host disease. GVHD can be separated from the veto activity by generating anti-third party CTLs under IL2 deprivation. Under such selective pressure only the stimulated clones which make IL2 can survive, while anti-host clones die. In vivo studies show that such anti-third party veto CTLs can be used safely for tolerance induction without GVHD.

Several bone marrow cells and lymphocyte subpopulations, known as “veto cells,” were shown to induce transplantation tolerance across major histocompatibility antigens. Some of the most potent veto cells are of T-cell origin, and in particular a very strong veto activity was documented for cytotoxic T-lymphocyte (CTL) lines or clones. However, these cells also possess marked graft-versus-host (GVH) reactivity. In the present study we evaluated a new approach to deplete CTLs of antihost clones by stimulating the donor T cells against third-party stimulators in the absence of exogenous interleukin 2 (IL-2). We demonstrate that such CTLs are depleted of GVH reactivity while maintaining marked veto activity in vitro. Furthermore, marked synergism was exhibited between the veto CTLs and rapamycin when tested in a murine model, which measures T-cell–mediated bone marrow allograft rejection, or in sublethally irradiated allogeneic hosts.Our results suggest that engraftment of early progenitors could be enhanced by using host-nonreactive anti–third-party CTLs, in conjunction with nonmyeloablative rapamycin-based conditioning protocols, thereby significantly reducing the toxicity of allogeneic transplantation.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used (“megadose” transplants) overcomes major genetic barriers. In vitro studies suggest that veto cells, within the population of hematopoietic progenitors, facilitate this favorable outcome. Thus, when purified CD34+ cells were added to bulk mixed-lymphocyte reactions (MLRs) they suppressed CTLs against the donor's stimulators, but not against stimulators from a third party. This tolerizing activity depends on cell contact and can be blocked by the caspase inhibitor BD-FMK, suggesting that the effector host T cells are deleted by apoptosis upon interaction with the CD34+ cells. Early myeloid CD33+ cells generated by short-term ex vivo expansion of CD34+ cells also exhibit veto activity, and these cells can be grown in large numbers. Tolerance induction can be further enhanced by other veto cells. Perhaps the most potent veto cell is the CD8+ CTL. However, this cell is also associated with marked GVHD (graft-versus-host disease. GVHD can be separated from the veto activity by generating anti-third party CTLs under IL2 deprivation. Under such selective pressure only the stimulated clones which make IL2 can survive, while anti-host clones die. In vivo studies show that such anti-third party veto CTLs can be used safely for tolerance induction without GVHD.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used (“megadose” transplants) overcomes major genetic barriers without induction of graft-versus-host disease. Therefore, transplantation from a human leukocyte antigen mismatched

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

The use of PTCY is associated with reduced risk for GVHD in T cell replete NMA haplo-human stem cell transplants (HSCT); however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. Furthermore, use of immune suppression to ameliorate GVHD likely adversely impacts anti-tumor immunity. Conversely, risk of rejection after NMA conditioning presents a major challenge for graft survival in T cell depleted (TCD) haplo-HSCT. We now show in a total of 66 mice (9 experiments) that combining the power of megadose TCD HSCT with high dose PTCY (Fig.1A), enables marked and durable chimerism following NMA conditioning, while each modality alone was ineffective (Fig1B). Chimerism included all myeloid and lymphoid lineages, and LDA analysis of alloreactive T cells revealed specific immune tolerance towards donor stimulators (Fig.1C), also associated with acceptance of donor but not 3rd party skin. A similar protocol (Fig.2) was therefore developed for clinical use in patients with high risk hematologic malignancies. The first patient, a 54 yr old male with high-risk multiple myeloma (unfavorable cytogenetics, micromolecular disease and compromised renal function at diagnosis) was treated with 4 courses of Velcade-Thalidomide-Dexametasone and autologous HSCT, attaining complete remission with good performance status and normal renal function. Six months later, he received megadose (15.4 x106 CD34+ cells/kg) CD3/CD19 depleted (1.17 x105 CD3+T cells/kg) haloidentical PBPCs. Hematopoietic engraftment was achieved at day +15 with over 96% donor type chimerism during the first 6 months in the myeloid and B cell lineages. T cells during this period were predominantly of host type (10-23% donor type), gradually increasing to 63-72% at 9-12 months post transplant. EliSpot analysis of anti-donor CTLp revealed specific non-responsiveness. The patient overcame CMV (Fig.3A) and subsequently EBV reactivation without any treatment. Dextramer FACS analysis revealed that CMV specific CD8 T cells were exclusively of host origin (Fig3B-C). At 12 months, complete hematological remission and normal Free Light Chain ratio were confirmed. The second patient, a 50 yr old male with MM achieved a partial remission after 4 courses of Lenalidomide-Dexametasone. He subsequently underwent autologous HSCT, and was put on Lenalidomide maintenance until relapse after 3 yrs. Following 8 courses of Bortezomib-Dexametasone he achieved CR. The patient received a megadose (10.8 x106 CD34+ cells/kg) of CD3/CD19 depleted (1.2x105 CD3+T cells/kg) haploidentcal PBPCs. Despite transient engraftment (50% donor cell on day +17), graft failure with autologous recovery (0.04% donor-type chimerism) was documented on day +30. This may be due to the extended treatment (38 months) with lenalidomide, but rejection cannot be excluded. After 5 months, this patient tolerated a second haploidentical HSCT (different donor) after standard myeloablative conditioning (ATG, treosulfan, thiotepa and fludarabine) and alfa/beta TCR/CD19-depleted PBPCs. Two months since the second HSCT, he shows no sign of GvHD, very good immunological reconstitution, excellent quality of life, and remains in complete remission. Collectively, our murine proof of concept data supported by clinical experience in the first high risk MM patient, suggests that combining megadose TCD HSCT with PTCY can enable prompt and durable engraftment with almost complete chimerism in the myeloid and B cell lineages. The marked level of host T cells persisting during the first 6 months after HSCT can provide anti-viral immune protection until thymus-derived donor T cells are generated. Furthermore, our ability to avoid post transplant immune suppression with this GVHD-free protocol, thereby attaining robust anti-viral immunity, could also potentially offer enhanced GVL effect, valuable for high risk MM patients. The rejection experienced by the 2nd patient, although corrected by a 2nd myeloablative TCD HSCT, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient.We are therefore testing, increasing TBI from 2Gy to 3Gy. Further studies will determine the efficacy of this approach in elderly MM patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy. *F.A and E.B.L contributed equally Disclosures BacharLustig: Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Lung diseases are the second leading cause of death worldwide. There is a growing understanding that lung stem cells, rather than being distributed throughout the tissue, are concentrated in specialized niches. Thus, in our previous study we demonstrated that in analogy to bone marrow (BM) transplantation, lung progenitors could navigate through the blood to their appropriate niches, provided that the niches were cleared of endogenous cell populations (Nature Medicine 2015). Lung injury was induced by Naphthalene, and lung progenitors of all lineages were further depleted by 6GY total body irradiation (TBI) 48 hrs after Naphthalene exposure; at this time, endogenous lung progenitors proliferated extensively. In the present study, we further established the feasibility of our approach in an allogeneic setting. To that end, we induced central immune tolerance towards donor cells, with no need for chronic immune suppression, by virtue of hematopoietic stem cells also present at high levels in fetallung alongside epithelial progenitors. The possibility that the E16 lung might contain high levels of hematopoietic progenitors was first suggested by examination of the peripheral blood of C57BL/6 mice transplanted with GFP+ E16 lung cells, or of RAG-SCID mice (H2Kb) receiving C3H (H2Kk) E16 lung cells. Robust chimerism was also documented in the BM and spleen of all transplanted mice. Based on this initial observation, we attempted to define by FACS the level of putative hematopoietic progenitors in E16 lungs. We thus evaluated two commonly used phenotypes, namely, LSK (lineage-, SCA-1+, C-KIT+) and SLAM (lineage-, CD48-, CD150+). Indeed, we found a similar level of LSK and SLAM cells in the fetal liver and lung, representing about 20-40% of the levels found in the adult BM. A competitive chimerism assay in which normal adult BM cells from a CD45.1+ C57BL/6 donor compete with E16 lung cells from a GFP+CD45.2+ C57BL/6 donor, revealed marked capacity of the E16 lung cells to induce robust chimersim following infusion of a 1:1 mixture of these cells into lethally irradiated CD45.1+ C57BL/6 recipients. Thus, at 9 months post-transplant, 4/4 mice exhibited blood cells derived from the lung donor. In 2 out of 4 mice, levels ofchimerism were above 30%, strongly indicating the robust capacity of the lung hematopoietic progenitors for self-renewal. Based on our ability to induce durable hematopoietic chimeras in syngeneic recipients following transplantation of E16 lung cells, we next developed a sub-lethal transplantation protocol enablingchimerism induction of both non-hematopoeitic cells in the lung, and hematopoietic cells in the blood, liver, spleen and thymus ofmis-matched recipients. The protocol used was based on recent work in haploidenticalhematopoietic stem cell transplantation (HSCT), and comprised transient T cell debulkingof host CD4 and CD8 T cells, megadoseT cell depleted HSCT, and post-transplant cyclophosphamide (CY). This conditioning was coupled with NA, and 6GY TBI to vacate lung progenitor niches (Fig.1). T cells, already present at E16 lungs, were removed from the donor lung preparation by magnetic beads. Similar to our results in haploidenticalBMT we found that chimerisminduction required the use of a 'megadose' of stem cells (5x106 compared to 1x106 in the syngeneic model). When tested at 2 months post-transplant, 4 out of 5 mice exhibited substantial hematopoieticchimerism in the BM, liver, thymus, spleen and blood with multi lineage expression, including B cells (B220), T cells (CD4/CD8) and myeloid cells (CD11b) (Fig. 2). Furthermore, 3 months post-transplant, donor-derived lung "patches" were present, exhibiting marked lungchimerism within both functional epithelial lineages (AEC1/2, marked by AQP5/SPC markers) and mesenchymal/endothelial lineages (marked byNestin/CD31 markers) (Fig. 3 a-d), confirming that the hematopoieticchimerisminduced tolerance towards donor non-hematopoietic lung cell lineages. The high level of hematopoietic progenitors with capacity for self-renewal in thefetallung, alongside non-hematopoietic progenitors, offers a novel approach for allogeneic stem cell transplantation without any need for chronic immune suppression. Further fine tuning is needed to replace NA with clinically approved agents and to define the minimal TBI dose required for effective conditioning. *C.H.K and C.R. contributed equally Disclosures Hillel Karniel: Yeda LTD: Patents & Royalties. Rosen:Yeda LTD: Patents & Royalties. Bachar Lustig:Yeda LTD: Patents & Royalties. Shezen:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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 Disclosures Bachar 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.

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-third party CTLs depleted of alloreactivity are endowed with marked veto activity and therefore might potentially facilitate bone marrow allografting without graft versus host disease (GVHD). The veto mechanism is still obscure. While early studies emphasized the role of CD8-mediated apoptosis, more recent evidence indicates a role for Fas-FasL. In the present study we show, by using blocking anti-CD8 antibody, by generating CTLs from FasL or perforin mutated mice, and by gene transfer of FasL, that the veto activity of anti-third party CD8 + CTLs is dependent upon the simultaneous expression of both CD8 and FasL.

Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1+Lin− cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy–irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1+Lin−cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host × donor)F1 T cells, previously shown to enhance T-cell–depleted BM allografts in lethally irradiated mice, synergize with Sca-1+Lin− cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.

Throughout the 1980s, transplantation of unmodified (T cell-replete) bone marrow from full haplotype incompatible family donors was associated with an unsuccessful outcome because of graft failure and severe graft-versus-host disease (GVHD), at times affecting up to 90% of recipients. Although extensive T cell depletion of donor bone marrow was successful in preventing GVHD in children with severe combined immunodeficiency disease (SCID), results were disappointing in leukemic patients because the benefit of preventing GVHD was offset by graft failure. Resistance to engraftment appears to be mediated by host-derived cytotoxic T-lymphocyte precursors that survive supralethal conditioning. In the present paper, we review data that show that these genetic histocompatibility barriers can be overcome in stringent mouse models, employing lethally as well as sublethally irradiated recipients, by two major approaches that are synergistic to each other: escalation of hematopoietic progenitor cell dose and the use of nonalloreactive T cells. The former approach is already being successfully implemented in the treatment of leukemic patients.

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 and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donor-derived central memory CD8+ T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti–third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

Graft-versus-host disease (GVHD) is uniformly lethal in recipients of HLA-mismatched marrow. In patients with severe combined immunodeficiency disease, this major obstacle can be overcome by rigorous T-cell depletion before transplantation. In leukaemia patients, however, the benefit of preventing GVHD is offset by graft rejection or graft failure. Very recently, this problem was overcome by supplementing T cell-depleted bone marrow transplants with megadoses of peripheral blood stem cells collected by leukapheresis after mobilization of the donor stem cells with granulocyte colony-stimulating factor (G-CSF). In the present study, we further demonstrate in a mouse model (C57BL/6--C3H/Hej) that escalation of bone marrow doses by four- to fivefold leads to full donor-type chimerism in sublethally irradiated (6.5 Gy) recipients. Thus, the new source of G-CSF mobilized human haematopoietic stem cells may enable extending the use of mismatched bone marrow transplants to patients with non-malignant diseases for whom supralethal conditioning is not a prerequisite.

Immature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4+ and CD8+ T cells after encountering cognate or noncognate imDCs. Whereas CD4+ T cells were deleted via an MHC-independent mechanism through the NO system, CD8+ T-cell deletion was found to occur through a unique MHC-dependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforin-expressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration.

Enabling engraftment of allogeneic T cell–depleted bone marrow (TDBM) under reduced-intensity conditioning represents a major challenge in bone marrow transplantation (BMT). Anti–third-party cytotoxic T lymphocytes (CTLs) were previously shown to be endowed with marked ability to delete host antidonor T cells in vitro, but were found to be less effective in vivo. This could result from diminished lymph node (LN) homing caused by the prolonged activation, which induces a CD44+CD62L− effector phenotype, and thereby prevents effective colocalization with, and neutralization of, alloreactive host T cells (HTCs). In the present study, LN homing, determined by imaging, was enhanced upon culture conditions that favor the acquisition of CD44+CD62L+ central memory cell (Tcm) phenotype by anti–third-party CD8+ cells. These Tcm-like cells displayed strong proliferation and prolonged persistence in BM transplant recipients. Importantly, adoptively transferred HTCs bearing a transgenic T-cell receptor (TCR) with antidonor specificity were efficiently deleted only by donor-type Tcms. All these attributes were found to be associated with improved efficacy in overcoming T cell–mediated rejection of TDBM, thereby enabling high survival rate and long-term donor chimerism, without causing graft-versus-host disease. In conclusion, anti–third-party Tcms, which home to recipient LNs and effectively delete antidonor T cells, could provide an effective and novel tool for overcoming rejection of BM allografts.

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a β cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.

Objective Recent reports have shown that donor or host CD4 + CD25 + Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. In the present study we investigated the potential of third-party Treg cells compared to donor-type cells to facilitate BM allografting. Methods Graft rejection is assessed in a mouse model of T cell–mediated BM allograft rejection. Lethally irradiated C3H mice are transplanted at day 2 after irradiation with T cell–depleted Balb/Nude BM. Graft rejection is induced by purified host-type T cells infused one day prior to BMT. Cells tested for their facilitating activity are added to the T cell–depleted BM allograft. Results Naive or ex vivo–expanded third-party Treg cells can effectively enhance engraftment of T cell–depleted BM allografts, exhibiting reactivity in vitro and in vivo similar to that found for donor-type Treg cells. Conclusion The use of third-party Treg cells in contrast to donor-type cells could allow advanced preparation of a large bank of Treg cells (off-the-shelf), with all the appropriate quality controls required for cell therapy.

Induction of donor type chimerism in mildly prepared hosts without graft-versus-host disease (GvHD) is a most desirable goal in bone morrow transplantation. We have recently demonstrated in a mouse model that donor veto cytotoxic T lymphocytes (CTLs) can facilitate the induction of donor type chimerism in sublethally irradiated recipients without causing GvHD if they are effectively depleted of alloreactivity against host cells by means of stimulation against a third party. We extend this approach to human cells, by preparing CTLs in two major steps: primary culture in the absence of interleukin 2, leading to death by neglect of antihost clones, and addition of interleukin 2 and subsequent dilution of antihost clones as a consequence of the expansion of the anti-third-party clones. CTLs prepared in this way specifically suppress host cytotoxic T cells directed against antigens of the donor, but not against fourth-party antigens, as demonstrated in a standard (51)Cr release assay. We conclude that human anti-third-party CTLs afford a new source of veto cells that are depleted of potential graft-versus-host-reactive clones. The cells generated by this approach could potentially be used to facilitate engraftment of allogeneic hematopoietic stem cells.

Several bone marrow cells and lymphocyte subpopulations, known as veto cells, were shown to induce transplantation tolerance across major histocompatibility Ags. Due to the low frequency of the effector T cells against which the veto cells inhibitory activity is aimed, the fate of the effector cells was traditionally followed indirectly by functional limiting dilution assays, which are cumbersome and depend on numerous parameters. In the present study the fate of the effector T cells was monitored directly by FACS, using TCR transgenic mouse CD8+ T cells in which the transgene is directed against H-2d (the 2C model). This assay is validated by demonstrating the potency, selectivity, radiation sensitivity, and contact dependency of anti-third-party CTLs previously demonstrated by the limiting dilution assay. In contrast to veto CTLs, nonactivated CD8+ T cells lack veto activity. Comparison by FACS in the 2C model revealed a hierarchy of veto cells, in the order of veto CTLs activated NK cells, activated CD4+ T cells, and activated B cells. The latter cells as well as nonactivated CD4+ or NK cells were shown to be completely devoid of veto activity.

The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8+ T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8+1B2+ effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8+ T cells were from 2C mice, which lack Fas expression ((2CX lpr)F2), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8+1B2+ effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the α3 domain of H2 class 1 on the effector cell.

Previous studies suggest that cells within the CD34+ hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34+ cells.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used (“megadose” transplants) overcomes major genetic barriers. In vitro studies suggest that veto cells, within the population of hematopoietic progenitors, facilitate this favorable outcome. Thus, when purified CD34 + cells were added to bulk mixed-lymphocyte reactions (MLRs), they suppressed CTLs against donor's stimulators but not against stimulators from a third party. This tolerizing activity depends on cell contact and can be blocked by the caspase inhibitor BD-FMK, suggesting that the effector host T cells are deleted by apoptosis upon interaction with the CD34 + cells. Early myeloid CD33 + cells generated by short-term ex vivo expansion of CD34 + cells also exhibit veto activity, and these cells can be grown in large numbers. Tolerance induction can be further enhanced by other veto cells. Perhaps the most potent veto cell is the CD8 + CTL. However, this cell is also associated with marked graft-versus-host disease (GVHD). GVHD can be separated from the veto activity by generating anti-third party CTLs under IL2 deprivation. Under such selective pressure, only the stimulated clones which make IL2 can survive, while anti-host clones die. In vivo studies show that such anti-third party veto CTLs can be used safely for tolerance induction without GVHD.

Achieving allogeneic T-cell depleted BM (TDBM) engraftment under non-myeloablative conditioning represents a potentially safe and attractive approach for generating tolerance as a platform for organ transplantation and for the treatment of non-malignant diseases. However, overcoming the abundant host anti-donor T-cells (HADTC) surviving such protocols remains a major challenge. The potential role of megadose T cell depleted BM transplants in overcoming this barrier was shown when using 6.0Gy sublethal TBI, but chimerism could not be attained upon further reduction of the conditioning. An alternative approach currently in clinical use is based on the administration of high dose cyclophosphamide at days 3-4 after transplantation. However, attempts have been limited to T cell replete transplants containing at least 2x106 T cells/Kg. The latter are clearly critical for achieving engraftment of mis-matched transplants but they are also associated with a significant risk for GVHD. In the present study, we evaluated the potential benefit of combining these two approaches. Initially, regular (5x106) or high dose (25x106) Balb/c-Nude BM cells (fully depleted of T cells by virtue of the 'nude' phenotype) were transplanted (day 0) into fully allogeneic recipients (C3H/Hen). Conditioning included T cell debulking (TCD) with anti-CD4 and anti-CD8 antibodies (300µg each) delivered on day -6, and exposure to 2.0 Gy total body irradiation (TBI) on day -1. High dose Cyclophosphamide (CY) (100mg/kg) was administered on days +3 and +4. Chimerism analysis on days 35, 95, 180 and 225 revealed that none of the BM recipients that were transplanted with a regular dose of 5x106 T depleted BM in the presence or absence of CY, expressed donor type chimerism. In contrast , 4/7 mice receiving 25x106 cells and CY, exhibited (on day 225) more than 47.7±9. % multi-lineage chimerism. The stability of chimerism over more than 7 months indicated that tolerance was likely achieved. Indeed, this was confirmed by subsequent acceptance of donor type Balb/c skin grafts. Thus, 3/4 chimeric mice accepted Balb/c skin and rejected 3rd party C57BL/6 skin, while all mice that were inoculated with regular dose of BM cells in conjunction with CY (7/7), rejected both donor and 3rd party grafts. Encouraged by these results, we attempted to determine the optimal CY and radiation dose, to improve chimerism induction. Thus, mice receiving increasing CY dose of 125 or 150 mg /kg did not exhibit significant enhancement of chimerism. In addition, as shown in Fig1, while most of the recipients that were irradiated with 2Gy (13/15), 2.5Gy (6/6), 3Gy (4/5), or 3.5Gy, TBI (5/5) did not differ significantly in chimerism level (average= 69.1±24.7% at 300 days post transplant), further reduction of irradiation dose to 1.0Gy TBI, resulted in significantly reduced chimerism (average=25.4±26.2%, p≤0.003 ). Importantly, acceptance of donor type skin graft was further manifested when tested in chimeric mice conditioned with 2GY (9/13), while 3rd party skin was completely rejected (Fig.1). Considering previous suggestions by Ildstad et al. that a subset of CD8+ TCR- BM cells is critical for achieving donor type chimerism, we next determined whether such cells are indeed critical for engraftment of megadose T cell depleted BM transplants. To that end, CD8+ cells were depleted from the Balb/c -'nude' mega dose BM preparation, and chimerism induction was compared to control non-depleted 'nude' BM cells. The results of two independent experiments revealed no significant difference (P≥0.9974) in chimerism levels between mice transplanted in the presence (47.3±31, n=16) or absence (47.3±29, n=15) of CD8+TCR- cells, suggesting that such cells are not essential when using megadose T cell depleted transplants in conjunction with CY. Taken together, our results demonstrate that combination of T cell depleted megadose hematopoietic stem cells with CY administration post transplantation can lead to marked chimerism following low intensity conditioning, in contrast to using each approach alone. Importantly, there is no need to preserve CD8+TCR- cells in the transplant preparation. Such transplants, which are completely free of GVHD risk, could potentially pave the way for a safer tolerance induction modality in organ transplantation and in other non-malignant indications in which the risk associated with GVHD is not justified. Disclosures: Reisner: Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Abstract 3726 Induction of specific durable immune tolerance towards transplanted donor cells and tissues, without chronic administration of toxic immunosuppressive drugs, represents a most desirable goal in transplantation medicine. Two major approaches addressing this challenge have shown promise in rodent studies. The first is the use of co-stimulatory blockade to inhibit T cells with anti-donor specificity, and the second uses bone marrow (BM) transplantation to achieve a tolerizing chimeric state in which donor antigens are continuously presented in the host thymus. The exceptional clinical potential of the latter approach has been recently demonstrated in a pilot study of combined renal and bone-marrow transplantation from a single donor. However, the presence of alloreactive T cells in the BM graft imposes a significant risk for GVHD which is unacceptable in patients with non-malignant conditions. While T cell depleted BMT could prevent GVHD, the high rejection rate in the absence of aggressive conditioning treatment, represents a significant obstacle. More than a decade ago a co-stimulatory blockade based on anti CD40L was successfully used to induce chimerism following T depleted BMT. However, anti CD40L was later found to be thrombotic in humans, leading to a search for a safer protocol. In previous studies attempting embryonic pancreas xeno-transplantation (Tchorsh-Yutsis et al. Diabetes 2009), we were able to achieve optimal maintenance of the embryonic graft upon transient treatment with CTLA4-Ig and anti CD48, in conjunction with continuous immune suppression with FTY720. However, durable tolerance was not achieved and rejection ensued upon cessation of immune suppression. Therefore, in the present study we sought to investigate the potential of transient treatment with CTLA4-Ig, anti CD48 and FTY720, the equivalents of which are available for clinical use, combined with transplantation of ‘mega dose' T cell depleted BMT. If successful, such a protocol could achieve the desired durable tolerizing chimeric state. Considering the importance of providing empty niches for successful BM engraftment, we initially determined the minimal myeloablation with busulphan which can induce durable chimerism following infusion of congenic B6-SJL(Ly-5.1) TDBM (25×106) into B6 (Ly-5.2) mice. Testing doses ranging from 10mg/Kg to 100mg/Kg busulphan showed that donor type chimerism above 50% was attained at doses >50mg/Kg (40±26%, 66±7% and 75±2% chimerism at 50, 60 and 100 mg/Kg). Consequently, the sublethal dose of 60mg/Kg was selected for further use in all attempts to induce allogeneic chimerism, in conjunction with transient debulking of host lymphocytes by a single infusion of anti CD4 and anti CD8 depleting antibodies. The well tolerated combined sublethal conditioning presented a formidable barrier for engraftment of allogeneic ‘megadose' T cell depleted BM, and no chimerism was achieved. However, addition of transient post transplant treatment with CTLA4-Ig, anti CD48 and FTY720 (Fig 1A), led in two independent experiments to marked donor type chimerism with a median follow up of 116 days (range: 70 to 163 days) beyond cessation of immune suppression (Fig.1B). Thus, while no chimerism could be detected in mice treated post transplant with FTY720 alone (0/7), transient post transplant immune suppression with CTLA4-Ig, anti CD48 and FTY720 resulted in more than 80% donor type chimerism in 8 of 11 mice. As can be seen in Fig.1C, significant chimerism was attained in both the myeloid and lymphoid lineages. Since agents such as Belatacept (CTLA4-Ig) and Alefacept (blocking the interaction of CD48) are available for clinical use, our results suggest a potentially feasible co-stimulatory blockade approach for the induction of durable hematopoietic chimerism under non myeloablative conditioning, as a platform for cell therapy and organ transplantation. Fig. 1: Non myeloablative conditioning and co-stimulatory blockade for chimerism induction. (A) C3H/Hen recipient mice were conditioned with busulfan (2×30mg/Kg) and T cell debulking (TCD) with 300mg anti CD4 and anti CD8. Post transplant treatment included 200mg CTLA4 Ig, 250mg anti CD48, and 0.1mg FTY720 administered at the indicated time points. (B-C): Long term multilineage chimerism (A) Chimerism level 163 days after cessation of immune suppression. (B) Typical multilineage chimerism in the spleen of a chimeric mouse shown in B. Fig. 1:. Non myeloablative conditioning and co-stimulatory blockade for chimerism induction. (A) C3H/Hen recipient mice were conditioned with busulfan (2×30mg/Kg) and T cell debulking (TCD) with 300mg anti CD4 and anti CD8. Post transplant treatment included 200mg CTLA4 Ig, 250mg anti CD48, and 0.1mg FTY720 administered at the indicated time points. (B-C): Long term multilineage chimerism (A) Chimerism level 163 days after cessation of immune suppression. (B) Typical multilineage chimerism in the spleen of a chimeric mouse shown in B. Disclosures: No relevant conflicts of interest to declare.

Abstract 65 The mechanism underlining tolerance induction by immature myeloid dendritic cells (IMDC) is poorly understood. To address this issue, we generated mouse IMDC from hematopoietic stem cells (HSC) via differentiation to early myeloid cells (10 days with a cytokine cocktail containing: TPO, Flt-3L, SCF, IL-3 and IL-6) and further differentiation to IMDC (10% GM-CSF for an additional 10 days). The phenotype of the cells obtained at the end of this procedure was CD11c+, CD11b+, B220−, CD80+, MHC class II + Low and CD86−. Addition of the IMDC to long term ( 3 days) mixed lymphocyte reaction (MLR) in which TCR transgenic (Tg) CD4+ or CD8+ alloreactive T cells are reacted against their cognate MHC antigens, led to a marked inhibition of proliferation (reaction ratio 1 IMDC: 2 T cells). This suppression was found to be mediated In short term MLR ( 5 hours) through killing of the effector cells. Killing of CD4+ alloreactive T cells was found to be MHC independent; namely, IMDC expressing MHC not recognized by the TCR transgene exhibited similar inhibition as that exhibited by IMDC expressing the cognate MHC (55±3% versus 59±4% killing upon addition of cognate MHC or non-cognate MHC IMDC, respectively). As previously shown, this non-specific killing is likely mediated by the NO system, as it could be completely abrogated by pre-treatment with N (G)-nitro-L- arginine methyl ester (L-NAME ) (5±4% and 4±3% killing upon addition of pre- treated IMDC bearing cognate MHC or non-cognate MHC, respectively). In contrast, killing of CD8+ alloreactive T cells by IMDC was only slightly reversed by L-NAME (74±3% killing with non-treated IMDC bearing cognate MHC, compared to 68±4% after pre treatment with L-NAME), and the remaining killing activity was found to be MHC dependent. Thus, in contrast to cognate IMDC, very low killing of alloreactive TCR transgenic CD8 T cells was exhibited by non-cognate IMDC in the presence of L-NAME (5±2% killing). When tested in a mouse model of T cell mediated bone marrow (BM) allograft rejection, only IMDC of donor, but not of third party origin, could prolong graft survival upon infusion of 3×106 IMDC (median survival of 24 versus 10 days respectively, p Surprisingly, the MHC dependent killing exhibited by IMDC in short term MLR was completely lost when using IMDC generated from perforin KO mice or when IMDC were pre-treated with Bapta-AM or Concanamycin A (6±4%, 5±3% and 3±3% killing, respectively). Thus, killing of the responding alloreactive CD8+ T cells by their cognate IMDC involves a perforin/granzyme mediated mechanism. Indeed, Electron Microscopy (EM) analysis revealed that IMDC were loaded with granules containing perforin and Granzyme A, mostly localized in proximity to the cell membrane. While these granules could barely be detected in early myeloid cells, formation of granules was markedly enhanced upon differentiating into IMDC. Further EM analysis revealed that recognition of the IMDC by alloreactive CD8+ T cells induced granules polarization towards the area of contact. Live video imaging (using perforin and granzyme A immunostaining) further supported the suggestion that this granule polarization leads to specific killing of the responding CD8+ T cell. Furthermore, pre-treatment of IMDC with a blocking peptide for Toll like receptor 7 (TLR7) but not for TLR1, TLR5, TLR6, TLR9, TLR10, or TLR11, completely abolished this granule degranulation, and the eventual killing of alloreactive CD8+ T cells (blocking TLR7 reduced the MLR inhibition from 68±4% to 8±3%, p In conclusion, IMDC can induce immune tolerance through two distinct mechanisms. The first, mediated by the NO system, is MHC independent and is limited to ablation of CD4+ T cell responses. The second, mainly targeting CD8+ T cells, is MHC dependent and is mediated by cytotoxic granules in the IMDC which polarize towards the contact area upon recognition by the T cells. This MHC- specific mechanism is clearly dependent on perforin and is specifically triggered by TLR7 ligation. The latter mechanism was predominant when testing IMDC in a mouse model for T cell mediated BM allograft rejection. Disclosures: No relevant conflicts of interest to declare.

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

Recent reports have shown that donor or host CD4+CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x104 purified host type T cells (HTC) and were transplanted one day later with 2x106 BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4+CD25+ cells. C3H (H2k) recipients received BM from Balb- Nude (H2d) donors and the Treg cells were obtained from Balb/c or FVB (H2q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P>0.05). Considering the low levels of CD4+CD25+ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf’ source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.

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.