ALBERT

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: 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.

Description: Immature dendritic cells (imDCs) can have a tolerizing effect in the steady state or following transplantation. However, due to the significant heterogeneity of this cell population it is difficult to study the mechanisms of their tolerance induction. We previously described the generation of a highly defined population of imDCs expressing perforin and granzyme A (Perf-DCs) from hematopoietic progenitors; using TCR transgenic T cells we monitored their ability to delete cognate CD4 and CD8 T cells. While the former are deleted via an MHC-independent mechanism through the nitric oxide system, CD8+ T cell deletion occurs through a unique MHC-dependent perforin-based killing mechanism. This involves activation of Toll-like receptors 7, and signaling through Triggering Receptor Expressed on Myeloid cells -1. Importantly, this novel subpopulation of Perf-DCs was also detected in various lymphoid tissues in normal animals, and its frequency is markedly enhanced upon GM-CSF administration (Zangi et al, Blood 2012). Here, we investigated the potential regulatory role of Perf-DCs in steady state in-vivo by selectively knocking out the expression of perforin in these cells. To this end, we generated BM chimeras using a 1:1 mixture of BM from perforin KO mice and from BM of mice ablated of CD11chigh DCs using diphtheria toxin expression under the CD11c promoter (Birnberg et al, Immunity 2008). In the resulting PKO-DTA chimeras, perforin expression was completely lost in conventional CD11c+ DCs, while 50% of the T and NK cell populations still expressed perforin. At 6 months post transplant, DTA-PKO chimeric mice spontaneously gained more weight than chimeras created using a mixture of normal BM with BM from perforin KO mice (WT-PKO). The increased weight gain observed in DTA-PKO mice prompted us to test whether this phenomenon was accompanied by other metabolic alterations. Indeed, DTA-PKO mice exhibited elevated serum cholesterol and triglyceride levels compared to control WT-PKO chimeras (140±3.5 vs. 115±8.6, 125±31vs. 88±9.8 mg/dl, N≥5). Total body fat percent as measured by body composition MRI was significantly higher in DTA-PKO mice (30.3%±2.2 vs. 14.5%±2.3), along with highly elevated levels of leptin (37±10.5 vs. 9.8±3 ng/ml). In addition, DTA-PKO chimeric mice exhibited glucose intolerance (p=0.034) and reduced insulin sensitivity (p=6.07x10-6). Immunohistological evaluation revealed a significant reduction in the percentage of insulin expressing pancreatic β cell- tissue (2.2%±0.54 vs. 5.75%±1.98). Importantly, the visceral adipose tissue (VAT) of DTA-PKO chimeras contained more crown-like structures typically formed when macrophages within inflamed AT surround dead adipocytes. Based on these characteristics of metabolic syndrome that develop in DTA-PKO chimeras over 6 months, we tested whether high-fat diet (HFD) enhances the rate of disease development. Indeed, DTA-PKO chimeras maintained on HFD displayed more pronounced weight gain compared to their HFD-maintained WT counterparts when tested 6 weeks after HFD initiation. Likewise, cholesterol and triglycerides as well as leptin and IL-1b in the serum, and TNF-α and IL-6 in the AT were elevated in DTA-PKO mice compared to the WT-PKO animals. Importantly, analysis of immune cell populations in collagenase-digested VAT revealed more CD8+ and CD4+ T cells in DTA-PKO mice compared to control chimeras (78.3x103±17.5x103 vs. 24.9x103±3.2x103 and 113x103±21x103 vs. 43x103±4.4x103respectively). Thus, triggering of inflammation in the AT previously shown to be mediated by T cells (Winer et al, Nat.Med 2009; Nishimura et al, Nat.Med 2009), is not effectively regulated in mice lacking Perf-DCs. Interestingly, a similar enhanced rate of metabolic imbalance was found in regular diet-fed DTA-PKO chimeras using RIP-mOVA mice expressing ovalbumin in the thymus, pancreas and kidneys, and known to be more prone to autoimmunity. Moreover, a significantly larger fraction of dividing cells was observed when CD8 T cells, isolated from AT of DTA-PKO chimeric RIP-mOVA mice were stimulated against splenocytes of mice expressing ovalbumin in all tissues (wOVA mice). Taken together, our results demonstrate that Perf-DCs have a unique immune regulatory role under steady state, controlling unwanted inflammatory processes in adipose tissue. Further studies of the role of Perf-DCs in other models of autoimmunity are warranted. Disclosures: No relevant conflicts of interest to declare.