ALBERT

Description: Crystal Growth & Design DOI: 10.1021/acs.cgd.6b00622

Description: Journal of the American Chemical Society DOI: 10.1021/jacs.5b04840

Description: Journal of the American Chemical Society DOI: 10.1021/jacs.5b10308

Description: Crystal Growth & Design DOI: 10.1021/acs.cgd.8b00604

Description: Journal of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.7b00449

Description: 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.

Description: 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.

Description: Cancer patients have a significantly higher risk of developing a venous thromboembolism (VTE) compared to non-cancer patients. VTE result in reduced quality of life, and increased morbidity and mortality. However, studies suggest VTE risk among ambulatory cancer patients varies widely. Therefore, although several studies demonstrate that prophylaxis is effective, given the cost and concomitant bleeding risk it is not the standard of care to administer prophylaxis. Khorana and colleagues have developed a predictive model for chemotherapy-associated VTE in cancer patients. However, Methods to implement this model into clinical practice have not been developed. The purpose of this study was to create and assess an innovative computerized Care Process Management System(CPMS ) (that would calculate in real-time the risk for VTE by utilizing clinical information and laboratory tests contained in patient electronic records, based on the proposed predictive model, notifying staff to enable patient education about VTE risk and to concomitantly validate the model. Over a four month period new cancer referrals to the Ottawa Regional Cancer Center, the sole provider of cancer care for a region of 1.2 million inhabitants, were assessed by the Khorana tool (Blood 2008;111:4902-7). This model consists of the following five predictive variables: (1) site of cancer (2 points for very high-risk site (brain, stomach, pancreas), 1 point for high-risk site (lung, lymphoma, gynecologic, bladder, and testicular), (2) platelet count ≥350 x 109/L, (3) hemoglobin 11 x 109/L, and (5) body mass index (BMI) ≥35 kg/m2 (1 point each). High risk was defined as a score or ≥2. We approached all new cancer patients but excluded patients if they were: (1) tumours not included as very high risk or high risk according to the prediction model; 2) to be followed up or treated at another facility other than The Ottawa Hospital; 3) had a confirmed VTE/arterial embolism (stroke or peripheral arterial embolism) within the prior 3 months; 4) were receiving continuous anticoagulation; 5) had previously been treated for cancer. Patients with a very high risk tumour site were approached first. Those with other tumour sites had the predictive variables calculated through the CPMS. In all cases the clinic nurse was notified electronically of when patients presented to clinic and of their risk. Patients’ demographics, medical history, malignancy characteristics, diagnostic laboratory features, imaging information, BMI and plan of anti-cancer treatment were documented prior to initiation of their cancer treatment. Educational material was provided to high risk patients regarding the signs and symptoms of VTE and their risk for VTE, in order to prevent delayed diagnosis. Patients were followed-up at 3 months (±7 days) following the initial consult to determine if they were diagnosed with VTE. 699 new referrals were determined to have a cancer diagnosis for the first time as identified by the computer software and qualified for our study and 580 were eligible. On the basis of very high risk tumour site alone, 71 patients were initially deemed at high risk of developing a VTE (score ≥2) and 509 patients were deemed at low risk (score ≤1). Among the initial cohort of patients at low risk, 72 were elevated to high risk based on BMI and pre-chemotherapy blood work. In total 25% had high risk for VTE and during the 3-month follow up period, 16 of the 143 (11%) developed a VTE which further validates the Khorana model for identifying high risk patients. Patients uniformly were receptive to receiving counseling about their VTE risk at enrolment. In summary, we have demonstrated the feasibility of utilizing a computerized CPMS linked to the electronic medical record to populate a predictive tool that is based on clinical information and laboratory data to identify and stratify cancer patients into high and low risk of developing a VTE. Our future aim is to utilize this innovative tool in clinical practice to enable 1) identification of all cancer patients at high risk for VTE, 2) education that will potentially decrease the time between symptom onset, VTE investigation, and treatment, and 3) enrollment in randomized trials of VTE prophylaxis. Disclosures: Wells: bayer healthcare: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Biomerieux: Honoraria.

Description: 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.

Description: 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.