ALBERT

Description: Abstract 2998 Introduction: GvHD remains the most deadly complication of HSCT despite current prevention strategies. To address the unmet need for better GvHD control, we have created a non-human primate (NHP) model with which to rigorously test mechanism and efficacy of novel therapeutics. In this study, we determined whether a novel combination of mTOR inhibition (with sirolimus) and CD28:CD80/86 costimulation blockade (with belatacept) could control GvHD. Here we show for the first time that these two agents combine synergistically to prevent both the clinical and immunologic manifestations of primate aGvHD. Methods: Rhesus macaque recipients were irradiated (9.6 Gy in 2 fractions at 7cGy/min), and then transplanted with G-CSF-mobilized PBSC from a haplo-identical donor (1–5×108 TNC/kg). Recipients were treated with either sirolimus alone (n = 4, troughs targeted at 5–10 ng/mL), belatacept alone (receiving weekly doses of 20 mg/kg), or combination therapy. Clinical GvHD was monitored using our previously described NHP grading scale (Miller et al., Blood 2010), and multiparameter flow cytometric analysis was performed. Results: Untreated controls (n = 5) developed rapid, severe histopathologically-proven aGvHD and succumbed rapidly (MST = 7 days). Recipients treated with either sirolimus or belatacept alone were partially protected from the clinical manifestations of GvHD. Sirolimus-treated recipients (n = 6) developed predominantly GI disease (with diarrhea but no elevation of bilirubin) and had an MST of 14 days (Figure 1). Recipients treated with belatacept alone (n = 3) developed primarily liver aGvHD (bilirubin rapidly rising to 6–30 × normal with histologically-confirmed lymphocytic infiltration) and an MST of 11 days. In striking contrast, recipients treated with combined sirolimus + belatacept (n = 5) demonstrated neither uncontrolled diarrhea nor hyperbilirubinemia at the timed terminal analysis (1 month post-transplant). We employed multiparameter flow cytometry to determine the immunologic consequences of sirolimus and belatacept on T cell proliferation (using Ki-67 expression) and cytotoxity (using granzyme B expression). We found that the clinical synergy observed with combined therapy was recapitulated immunologically. Thus, while untreated aGvHD was associated with rampant CD8+ proliferation (with 83 +/− 14% Ki-67+ CD8+ vs 4.7 +/− 0.6% pre-transplant), sirolimus or belatacept as monotherapy both partially controlled proliferation (35 +/− 3% and 65 +/− 23% Ki-67+ CD8+ with sirolimus or belatacept, respectively). Combined sirolimus + belatacept dramatically reduced proliferation (to 8 +/− 3%, favorably comparing with 13% Ki-67+ CD8+ T cells using standard Calcineurin Inhibitor/Methotrexate (CNI/MTX) prophylaxis). Sirolimus and belatacept both also partially controlled GvHD-related T cell cytotoxicity. Thus, while untreated aGvHD was associated with excessive granzyme B expression in CD8+ T cells (82 +/− 2% granzyme Bvery high CD8+ cells vs 0.3 +/− 0.2% pre-transplant) sirolimus or belatacept monotherapy also partially controlled cytotoxicity (8 +/− 1% and 35 +/− 1% granzyme Bvery high with sirolimus or belatacept, respectively). Combination therapy dramatically reduced the proportion of these cells, to 1.5 +/− 0.8 % granzyme Bvery high, favorably comparing with 4% granzyme Bvery high using CNI/MTX. The ability of sirolimus, belatacept, or the combination to control Ki-67 and Granzyme B expression closely correlated with survival (Figure 2A, B) supporting a pathogenic role for these highly proliferative and cytotoxic cells in aGvHD pathology. Moreover, significant co-expression of granzyme B in the Ki-67+ cells was observed (Figure 2C) suggesting that dual-positive Ki-67/Granzyme B cells may mark a pathogenic population, amenable to tracking in the peripheral blood. Implications: These results reveal a previously undiscovered synergy between sirolimus and belatacept in the control of primate aGvHD, and provide support for future clinical investigation of this novel prevention strategy. They also identify CD8+/Ki-67+/Granzyme Bvery high dual-positive T cells as a potentially sensitive biomarker of GvHD pathogenesis, amenable to monitoring in either the blood or in GvHD target organs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p

Description: Abstract 1888 Introduction: There is a critical unmet need to devise effective strategies to prevent GvHD. However, the best combinatorial therapies remain undetermined, and the identification of new targeted approaches to GvHD prevention remains a challenge. To address this, we have developed a genome-wide approach to studying GvHD, using whole-transcriptome analysis of pathogenic T cells in a clinically-relevant non-human primate (NHP) model. Using computational approaches, we have identified, for the first time, the transcriptional networks that drive primate GvHD, and that lead to its partial control with sirolimus. Methods: CD3+/CD20- T cells were purified flow cytometrically from 4 cohorts: (1) Healthy Controls (“HC” n = 15); (2) Recipients of an autologous HSCT (“Auto” n = 3); (3) Haplo-identical allogeneic HSCT recipients without GvHD prophylaxis, who developed histopathologically confirmed severe aGvHD (“GvHD” n = 4); and (4) Allo-HSCT recipients who received sirolimus alone, and were partially protected from aGvHD (“Sirolimus” n = 4). Purification of T cells after allo-HSCT occurred 1–2 weeks post-transplant. RNA was purified (Qiagen), and rhesus macaque-specific Affymetrix Gene Arrays were performed. Computation: Gene array signals were processed and normalized using the Robust Multichip Averaging Method and ComBat. Principal Component Analysis (PCA) was applied to summarize modes of gene array variance. Importantly, PCA revealed that variation was primarily determined by the experimental cohort (Figure 1). This result was critical, and confirmed that transcriptomics could be applied to identify genes and pathways controlling GvHD. Differentially expressed genes (“DE”, fold change 〉 2) were defined between cohorts, yielding unique and overlapping gene signatures. We found that 775 annotated genes were DE between GvHD and HC and 286 were DE between Sirolimus and HC (Figure 2A, B). Importantly, a subset of the GvHD and Sirolimus DE gene sets were overlapping, indicating incomplete control of T cell activation with sirolimus (Figure 2B), and identifying pathways that could be targeted in combination with sirolimus for improved GvHD control. To further define genes by their individual expression profiles using an unbiased approach, we applied Class Neighbor Analysis (GenePattern, Figure 3A). Finally, using Ingenuity Pathway Analysis (IPA) we characterized gene signatures according to molecular pathways (using right-tailed Fisher's Exact test and FDR correction, Figure 3B). Results: T cells from animals with severe aGvHD demonstrated transcriptional signs of rampant proliferation and cytotoxicity as well as potentially counter-regulatory cell death pathways. IPA identified highly statistically significant upregulation of Cell Cycle and Cellular Movement networks (Figure 3B, p〈 0.001) as well as Cell Trafficking and Inflammatory Response Networks (Figure 3B, p 〈 0.001). These networks contained some expected genes and some surprises. Thus, as previously documented, GvHD was associated with upregulation of JAK and IFN signaling (p 〈 0.001). Unexpectedly, GvHD was also associated with upregulation of the Sonic Hedgehog and Aurora Kinase A Pathways (p 〈 0.01). Both of these represent targetable pathways for which novel therapeutics are currently available. Sirolimus resulted in significantly different gene expression patterns compared to uncontrolled GvHD. This included partial downregulation of the proliferation marker Ki-67 and the cytotoxicity gene, Granzyme B. However, there were many genes, pathways and networks that were shared between the Sirolimus and GvHD cohorts. These prominently included upregulation of the FOXM1 and IRF8 transcription factors, involved in cell cycle progression (p

Description: Here we report the completion of a uniformly calibrated open ocean data product on inorganic carbon and carbon-relevant variables that we call Global Ocean Data Analysis Project version 2 or simply GLODAPv2. The product includes data from approximately one million individual seawater samples collected from over 700 cruises during the years 1972-2013. Extensive quality control and subsequent calibration were carried out for salinity, oxygen, nutrient, carbon dioxide, total alkalinity, pH and chlorofluorocarbon data. Following calibration, the data were used to produce global climatology maps for many of the parameters. In addition to the data products and the mapped distributions, all of the original data files without alteration other than formatting and unification of units are made freely available along with whatever metadata was collected. An on-line cruise summary table provides data access and additional information including references to publications that have used data from specific cruises. The GLODAPv2 database is available free of charge as a numeric data package (NDP-93) from the Carbon Dioxide Information Analysis Center (CDIAC) at http://cdiac.ornl.gov/oceans/GLODAPv2/. The NDP-93 consists of the original cruise data files, adjusted data products, mapped data product and this documentation, which describes the GLODAPv2 project.