ALBERT

Description: Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB+ pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC–T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.

Description: Introduction: Providing effective outpatient care to oncology patients is the goal of all programs. There are two potential models of providing this care, a primary physician model which is the model generally employed by large oncology programs, and a team based model which is the model employed by small oncology programs. Medium sized programs (defined as 50-100 newly diagnosed patients per year), face a challenge as to what the best model of oncology outpatient care is to follow given the number of oncologists providing clinical care. We attempted to develop a hybrid model of team based and primary physician model in order to improve care of patients at our medium sized center. Methods: Prior to making any changes from the longstanding team based model of outpatient care, a patient satisfaction survey was conducted. Multiple meetings were held with the physician group to discuss the current model of care (team based model) and the potential ways to change the model given the complexity of patients and protocols. After much discussion it was decided that all patients would have a dedicated oncologist. There would then be two types of weeks of clinical service in the outpatient clinic. The first type was a “Doc of the Day” week where each oncologist would have a specific day in clinic and their assigned patients would be booked to come to clinic on those days. The second type was a “Doc of the Week” week where one physician would be attending in clinic for the week. There would be a 1:1 ratio of the two types of weeks. During vacations or holidays the week would be designated “Doc of the Week”. Results: The patient satisfaction survey done prior to changing the model of care showed that patients were very satisfied with the care they were receiving. A questionnaire to staff 14 months after the change in the model of care showed that the biggest effect was felt to be increased continuity of care to patients, followed by more efficient clinic work flow and increased consistency of care. The responses to what they liked best about the new model of care as members of the health care team, showed that facilitating the planning and delivery of care to patients and having a primary physician assigned to each patient were the most liked, followed by having their patient care questions answered more consistently because they knew which physician to direct the question to and physicians were more aware of their dedicated patients. The patient satisfaction survey post change in model of care showed that patients were still highly satisfied with the care they received. Conclusions: We showed that a model of care with a primary physician for each patient as well as assigned clinic days, alternating with some weeks where one physician covers the outpatient oncology patients for the whole week is a feasible model of care for a medium sized pediatric oncology program. The health care team found this model to be significantly better than a straight team based care model, but in a medium sized program with limited attending physicians, it provided a primary physician model that was felt to be beneficial for patients and other members of the health care team. Disclosures No relevant conflicts of interest to declare.

Description: After lethal irradiation of C57BL mice followed by the injection of 10(7) marrow cells, total cellularity and progenitor cell levels exceeded pretreatment levels within 12 days in the spleen, but regeneration remained incomplete in the marrow. The exceptional regenerative capacity of progenitor populations in the spleen was observed in organ cultures of spleen slices prepared 24 hr after irradiation and transplantation, excluding continuous repopulation from the marrow as a significant factor in splenic regeneration.

Description: Previously, we showed that the co-repressor CoREST (Rcor1) is essential for the maturation of definitive erythroid cells in the mouse fetal liver. To elucidate Rcor1’s function in multilineage hematopoiesis in the adult, we conditionally deleted Rcor1 using Mx1-Cre. The loss of Rcor1 expression in hematopoietic cells led to the rapid onset of a severe anemia due to a complete block of erythropoiesis downstream of committed erythroid progenitors. By contrast, the production of megakaryocyte progenitors, megakaryocyte maturation and platelets were maintained. In the myelomonocytic lineages, although neutrophil differentiation was completely abrogated, the number of monocytic cells was significantly increased, resulting in a peripheral blood leukocytosis and monocytic infiltrations in the liver. Rcor1-deficient monocytes showed a 66% reduction in apoptosis and possessed ~100-fold more CFU-M activity than control cells. The CFU-M derived from Rcor1 deficient bone marrow could be serially replated up to 5 times; however, replating activity was entirely cytokine dependent. Defective myelomonocytic differentiation persisted following transplantation into wild type hosts for up to 12 months but did not progress to leukemia. To begin to understand at the molecular level how Rcor1 regulates monocyte expansion, we evaluated the expression levels of genes whose ectopic expression is associated with myeloid neoplasia. In Rcor1-deficient monocytes, Gata2, Meis1 and Hoxa9 were overexpressed by 8- to 200-fold. Taken together, these data demonstrate that Rcor1 is essential for both erythroid and myelomonocytic differentiation and that its loss of function causes significant myelodysplasia. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3221 Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. Recently, we demonstrated that apart from their pro-inflammatory effects, which are mainly mediated by secretion of interferon-alpha (IFN-a), pDC can also exhibit potent anti-inflammatory functions borne by active secretion of granzyme B (GrB), a serine protease classically known from CTL, NK cells and regulatory T cells. Here, we hypothesized, that commonly used anti-viral vaccines may affect pDC on several levels including their immunophenotype as well as their capacity to secrete either IFN-a or GrB. Using various methods including FACS, ELISpot, ELISA, spinning-disk confocal microscopy and RT-PCR, we could demonstrate that various anti-viral vaccines including vaccines against TBEV, yellow fever, polio, measles, rotavirus, varicella and hepatitis B were able to affect pDC by modulating expression of a series of surface molecules involved in cell adhesion, antigen-presentation and co-stimulation. In addition, major differences between the vaccines were found in terms of their effects on secretion of IFN-a and GrB. Interestingly, while only one vaccine, FSME Immun (TBEV) induced substantial IFN-a responses in pDC, all others tested did not. In contrast, virtually all vaccines tested induced more or less strong suppression of GrB secretion by maturing pDC. Of note, pDC that secreted high amounts of GrB induced by far lower allogeneic T cell proliferation as compared to pDC that secreted little or no GrB. Moreover, suppression of pDC-derived GrB by a substrate-specific GrB inhibitor resulted in significant enhancement of T cell proliferation in co-cultures of GrB-secreting pDC with allogeneic T cells. Our data demonstrate 1) that anti-viral vaccines may have distinct effects on both the immunophenotype and the secretory potential of pDC, and 2) that GrB is an important novel variable affecting the capacity of pDC to either trigger or dampen adaptive T cell responses. Our results may have implications for further study of the role pDC play in the regulation of adaptive immune responses, and for the potential application of this knowledge in the development of novel adjuvants admixed to anti-viral vaccines. Disclosures: No relevant conflicts of interest to declare.

Description: After lethal irradiation of C57BL mice followed by the injection of 10(7) marrow cells, total cellularity and progenitor cell levels exceeded pretreatment levels within 12 days in the spleen, but regeneration remained incomplete in the marrow. The exceptional regenerative capacity of progenitor populations in the spleen was observed in organ cultures of spleen slices prepared 24 hr after irradiation and transplantation, excluding continuous repopulation from the marrow as a significant factor in splenic regeneration.

Description: Background UCBT has proven effective in treating patients with hematologic malignancies (HM),but engraftment is slower and graft failure rates higher compared to other unrelated transplants. We have previously shown that incubation of UCB CD133+ cells with cytokines and the copper chelator TEPA (5µM), inhibits stem cell differentiation and results in a median of 89 and 30 fold expansion of CD34+ and CD34+CD38- cells, respectively (Cytotherapy 2004;6:344). Using this technology (StemEx®) we performed a prospective multicenter myeloablative UCBT trial in patients with HM. Methodology Patients were transplanted with a single CBU of which CD133+ cells from a segregated portion of the CBU (20-50%) were cultured for 21 days with hematopoietic cytokines and TEPA and transplanted along with a minimum of 107nucleated cells (NC)/kg from the un-manipulated (UM) portion of the same unit (NCT00469729). The primary endpoint of this study was 100 day overall survival. Using an intent to treat design, outcome was compared to a 2006-2010 double UCBT (dUCBT) control group (n = 295) collected from and by the CIBMTR and Eurocord registries using identical eligibility criteria to the StemEx® study: lack of a 5/6 or 6/6 matched sibling donor, age 12-55 years with high risk AML or ALL in 1stCR or subsequent, advanced CML or after failing TKI, MDS with Int-2 or high risk features or chemosensitive relapsed lymphoma. GvHD prophylaxis included a calcineurin inhibitor and mycophenolate mofetil. Comparison of the primary endpoint was based on a logistic regression model that adjusts the treatment comparison for imbalance between the groups in important prognostic factors found to impact mortality: age, sex, CMV status and disease risk. Results 25 centers in US, EU and Israel enrolled 101 eligible patients between Oct 2007-Feb 2012 with AML-43, ALL-30, MDS and CML-8 each, and lymphoma-12. Median age was 37 (12.6-55.8); median weight was 68 kg (42.5-128.5). The baseline NC and CD34 cell dose/kg were 3.06 (1.29-11.0) x 107 and 1.64 (0.24-9.23) x 105. 70% of the units were matched at 4/6 loci. Median NC and CD34 fold expansion were 400 (0-764) and 77 (6-280), respectively. StemEx yielded a median of 14-fold increase in the number of CD34+ infused, in comparison to the number of CD34+ cells the patients could have received from the entire UM CBU. In total, patients received a median dose of 2.2 x 107 NC/kg and 9.7 x 105CD34/kg. No significant acute toxicity was seen with the expanded cell infusions. The primary endpoint of this study has been met: 100 day survival was significantly higher in the StemEx® vs control group; 84.2 vs 74.6 % [95% CI 0.5 (0.26-0.95); p = 0.035]. Neutrophil and platelet engraftment rates were faster in the StemEx® vs control group: 21 vs 28 days (p〈 0.0001) and 54 vs 105 days (p = 0.008), respectively. There was a trend in the reduction of engraftment failure from 14.4% in the control to 8.1% in the StemEx® group, p=0.086. Early engraftment (EE) (ANC ≤ day 20 and platelets ≤ day 60) was achieved more frequently in StemEx® (39.4%) than in the control arm (12.4%) , p

Description: Key Points Rcor1 knockout mice show a block in fetal erythropoiesis at the proerythroblast stage. Rcor1 represses expression of HSCs and myeloid genes during erythropoiesis, including Csf2rb, which is important in myeloid function.